key: cord-342312-rnq1hfsj authors: liu, bingfeng; shi, yaling; zhang, wanying; li, rong; he, zhangping; yang, xiaofan; pan, yuejun; deng, xilong; tan, mingkai; zhao, lingzhai; zou, fan; zhang, yiwen; pan, ting; zhang, junsong; zhang, xu; xiao, fei; li, fang; deng, kai; zhang, hui title: recovered covid-19 patients with recurrent viral rna exhibit lower levels of anti-rbd antibodies date: 2020-09-16 journal: cell mol immunol doi: 10.1038/s41423-020-00528-0 sha: doc_id: 342312 cord_uid: rnq1hfsj nan plasma samples were collected from these 47 patients with covid-19 at the time of convalescence and assessed for antibodies against the following sars-cov-2 proteins: the spike glycoprotein (s); the receptor-binding domain (rbd); conserved heptad repeats (hr1-hr2) in the s2 domain; and the nucleocapsid (n), membrane (m), and envelope (e) proteins. the concentrations of igg secreted in response to these sars-cov-2 proteins varied in different patients, with detection rates of 100.0% (47/47), 83.0% (39/47), 97.9% (46/47), 100.0% (47/47), 55.3% (26/47), and 21.3% (10/47) for the s, rbd, hr1-hr2, n, m, and e proteins, respectively ( fig. 1a; fig. s2 ). the detection rates of igm to the s, rbd, hr1-hr2, and n proteins were 100.0% (47/47), 95.7% (45/47), 83.0% (39/47), and 100% (47/47), respectively ( fig. 1b; fig. s2 ). notably, significantly higher levels of sars-cov-2-specific igg and igm developed to the s and n proteins (fig. 1a, b) . to evaluate the effect of specific antibodies on rp status, we compared the levels of anti-sars-cov-2 igg to the s, rbd, hr1-hr2, n, and m proteins in these patients during their convalescent period ( fig. 1c; fig. s3 ). the results showed that rp patients induced significantly lower levels of anti-rbd igg than prn patients (p = 0.013) (fig. 1c) . as all of these rp patients were in a moderate condition before recovery, the prn patients were further classified as moderate (28 patients) or severe (11 patients) according to their symptoms before recovery. the levels of anti-rbd igg in rp patients were still significantly lower than those of either prn-severe or prn-moderate patients (p = 0.012 and p = 0.040, respectively; fig. 1d ). in addition, the patients with severe symptoms within the prn group were more likely to induce higher levels of anti-rbd igg (p = 0.012; fig. 1d ), which is consistent with previous reports. 12 in contrast, there were no significant differences either in igg to other viral proteins or in igm between prn and rp patients (fig. 1c, e; figs. s4, s5) , suggesting that the humoral response to rbd rather than to other regions of the s protein or the full-length s protein might have played an important role in preventing viral rebound during recovery. furthermore, we observed that the titers of igg to rbd among these recovered patients positively correlated with the spikebinding antibodies targeting the s, hr1-hr2, and n proteins (r = 0.71, p < 0.0001; r = 0.53, p < 0.0001; and r = 0.33, p = 0.022, respectively) but not with the m or e proteins (fig. s6a) . moreover, the y-axis represents optical density units at od450 nm, and the x-axis represents reciprocal plasma dilutions. c normalized od450 nm values of the anti-sars-cov-2 igg to the rbd, s, hr1-hr2, n, m, and e proteins are compared between prn and rp patients. the p value was calculated using a two-tailed mann-whitney u test or unpaired student's t test. d normalized od450 nm values of the anti-rbd igg were compared between prn-severe, prn-moderate, and rp patients. the p value was calculated using a two-tailed mann-whitney u test or unpaired student's t test. e normalized od450 nm values of the anti-rbd igm were compared between prn and rp patients. the p value was calculated using a two-tailed mann-whitney u test the level of igm to the rbd protein among these recovered patients also correlated with the s, hr1-hr2, and n proteins (r = 0.67, p < 0.0001; r = 0.56, p < 0.0001; and r = 0.60, p < 0.0001, respectively) (fig. s6b) . in addition, a positive correlation was also observed between age and igg level to the rbd, s, hr1-hr2, and n proteins (r = 0.38, p = 0.0077; r = 0.40, p = 0.0055; r = 0.45, p = 0.0017; and r = 0.44, p = 0.0021, respectively; fig. s7 ), indicating the important role of age in the generation of specific binding antibodies. 13 because of the lack of clinical characteristics and the unknown significance of rp patients, it is critical to provide comprehensive serological profiling to guide the management of recovered covid-19 patients after discharge. an important feature of the rp patients was their younger age than that of the prn patients, and the ages of these recovered patients positively correlated with titers of igg to the rbd protein. 1, 2, 13 these observations are consistent with the conclusion that the level of igg to the rbd protein in rp patients is significantly lower than that in the prn group. based on our findings, the anti-rbd igg level could serve as an indicator of rp status. to minimize the risk of possible viral rebound and retransmission during the current pandemic, close monitoring of anti-rbd igg levels at viral shedding and a longterm follow-up of patients with lower levels of rbd antibodies is needed. moreover, the relationship between anti-sars-cov-2 igg titers and rp status suggests that the interplay between the virus and the host immune response in coronavirus infections should be further investigated for the development of more accurate diagnostic technologies and effective vaccines against viral infection. positive rt-pcr test results in patients recovered from covid-19 clinical characteristics of the recovered covid-19 patients with redetectable positive rna test recurrence of positive sars-cov-2 in patients recovered from covid-19 clinical course and risk factors for recurrence of positive sars-cov-2 rna: a retrospective cohort study from wuhan prolonged sars-cov-2 rna detection in anal/rectal swabs and stool specimens in covid-19 patients after negative conversion in nasopharyngeal rt-pcr test recurrence of sars-cov-2 pcr positivity in covid-19 patients: a single center experience and potential implications assessment of patients who tested positive for covid-19 after recovery recurrence of covid-19 after recovery: a case report from virological assessment of hospitalized patients with covid-2019 detection of sars-cov-2-specific humoral and cellular immunity in covid-19 convalescent individuals coronavirus disease 2019 test results after clinical recovery and hospital discharge among patients in china viral kinetics and antibody responses in patients with covid-19 neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient cohort and their implications the online version of this article (https://doi.org/10.1038/s41423-020-00528-0) contains supplementary material.competing interests: the authors declare no competing interests. key: cord-332185-a96r1k7a authors: zhang, shuyuan; qiao, shuyuan; yu, jinfang; zeng, jianwei; shan, sisi; lan, jun; tian, long; zhang, linqi; wang, xinquan title: bat and pangolin coronavirus spike glycoprotein structures provide insights into sars-cov-2 evolution date: 2020-09-22 journal: biorxiv doi: 10.1101/2020.09.21.307439 sha: doc_id: 332185 cord_uid: a96r1k7a in recognizing the host cellular receptor and mediating fusion of virus and cell membranes, the spike (s) glycoprotein of coronaviruses is the most critical viral protein for cross-species transmission and infection. here we determined the cryo-em structures of the spikes from bat (ratg13) and pangolin (pcov_gx) coronaviruses, which are closely related to sars-cov-2. all three receptor-binding domains (rbds) of these two spike trimers are in the “down” conformation, indicating they are more prone to adopt this receptor-binding inactive state. however, we found that the pcov_gx, but not the ratg13, spike is comparable to the sars-cov-2 spike in binding the human ace2 receptor and supporting pseudovirus cell entry. through structure and sequence comparisons, we identified critical residues in the rbd that underlie the different activities of the ratg13 and pcov_gx/sars-cov-2 spikes and propose that n-linked glycans serve as conformational control elements of the rbd. these results collectively indicate that strong rbd-ace2 binding and efficient rbd conformational sampling are required for the evolution of sars-cov-2 to gain highly efficient infection. studies revealed that the sars-cov-2 s trimer, similar to that of sars-cov, needs 58 to have at least one rbd in an "up" conformation to bind hace2 17-23 . therefore, a 59 spike trimer with all three rbds "down" is in a receptor-binding inactive state, and 60 the conformational change of at least one rbd from "down" to "up" switches the 61 4 spike trimer to a receptor-binding active state 18 overall structures of ratg13 and pcov_gx spikes 106 the overall structures of homotrimeric ratg13 and pcov_gx spikes resemble the 107 previously reported pre-fusion structures of coronavirus spikes (fig. 1a ). both spikes 108 have a mushroom-like shape (~150 å in height and~120 å in width), consisting of a 109 cap mainly formed by β-strands and a stalk mainly formed by α-helices (fig. 1a) . 110 like other coronaviruses, the ratg13 and pcov_gx spike monomers are composed 111 of the s1 and s2 subunits with a protease cleavage site between them (fig. 1b,1c) . 112 the structural components of the spike include the n-terminal domain (ntd), rbd 113 (also called the c-terminal domain, ctd), subdomain 1 (sd1) and subdomain 2 (sd2) 114 in the s1 subunit; and the upstream helix (uh), fusion peptide (fp), connecting 115 region (cr), heptad repeat 1 (hr1), central helix (ch), β-hairpin (bh), subdomain 3 116 (sd3) and heptad repeat 2 (hr2) in the s2 subunit (fig. 1d, fig. s5 ). table s1 . 419 a novel coronavirus associated with severe acute 421 respiratory syndrome isolation of a novel coronavirus from a man with pneumonia 425 in saudi arabia a new coronavirus associated with human respiratory disease in 428 a novel coronavirus from patients with pneumonia in china a pneumonia outbreak associated with a new coronavirus of 432 probable bat origin origin and evolution of pathogenic coronaviruses identifying sars-cov-2-related coronaviruses in malayan 437 pangolins isolation of sars-cov-2-related coronavirus from malayan 439 pangolins are pangolins the intermediate host of the 2019 novel 441 coronavirus (sars-cov-2)? associated with the covid-19 outbreak recombination, reservoirs, and the modular 447 spike: mechanisms of coronavirus cross-species transmission sars-cov-2 cell entry depends on ace2 and 450 tmprss2 and is blocked by a clinically proven protease inhibitor structure of the sars-cov-2 spike receptor-binding domain 453 bound to the ace2 receptor structural basis for the recognition of sars-cov-2 by 456 full-length human ace2 structural and functional basis of sars-cov-2 entry by 459 18 using human ace2 structural basis of receptor recognition by sars-cov-2 cryo-electron microscopy structures of the sars-cov spike 464 glycoprotein reveal a prerequisite conformational state for receptor binding cryo-em structure of the sars 467 coronavirus spike glycoprotein in complex with its host cell receptor ace2 cryo-em structure of the 2019-ncov spike in the prefusion 473 conformation sars-cov-2 and bat ratg13 spike glycoprotein 475 structures inform on virus evolution and furin-cleavage effects a ph-dependent switch mediates conformational masking of 478 sars-cov-2 spike. biorxiv receptor binding and priming of the spike protein of 480 sars-cov-2 for membrane fusion sars-cov-2 and three related coronaviruses utilize multiple 483 ace2 orthologs and are potently blocked by an improved ace2-ig structural insights into coronavirus entry adaptation of sars-cov-2 in balb/c mice for testing vaccine 488 efficacy a mouse-adapted model of sars-cov-2 to test covid-19 countermeasures functional and genetic analysis of viral receptor ace2 cryo-em structures of mers-cov and sars-cov spike 495 glycoproteins reveal the dynamic receptor binding domains glycans on the sars-cov-2 spike control the receptor 498 immunogenicity and structures of a rationally designed 501 prefusion mers-cov spike antigen eman2: an extensible image processing suite for electron 504 microscopy new tools for automated high-resolution cryo-em structure 506 determination in relion-3 motioncor2: anisotropic correction of beam-induced 508 motion for improved cryo-electron microscopy real-time ctf determination and correction quantifying the local 513 resolution of cryo-em density maps swiss-model: homology modelling of protein 516 structures and complexes ucsf chimera--a visualization system for exploratory 519 research and analysis coot: model-building tools for molecular graphics phenix: a comprehensive python-based system for 525 macromolecular structure solution molprobity: all-atom structure validation for 528 macromolecular crystallography emringer: side chain-directed model and map validation 531 for 3d cryo-electron microscopy 6vxx) rbd in wheat，and sars-cov-2 (pdb id: 6zge) rbd in marine; 602 remaining regions shown in gray. (b) detailed structures of the rbd-glycans 603 interface are shown 6zge/6vxx) rbds are colored the same as in a. glycans are shown as red sticks 605 and asn-linked glycans are labeled. sequence alignment of the sars-cov-2 ratg13 and pcov_gx rbd-interacting glycosylation sites is shown in the bottom 607 panel. some sequences between the three sites are omitted and indicated by black dots amino acid positions of asparagines are indicated above the sequences according to 609 asparagines (n) are colored red and threonines (t) are colored blue binding affinities and cell entr y of the differ ent spikes. (a) binding curves 612 of immobilized hace2 with the sars-cov-2, pcov_gx or ratg13 spike. data are 613 shown as different colored lines and the best fit of the data to a 1:1 binding model is 614 shown in black. (b) the cell entry efficiencies of pseudotyped viruses as measured by 615 luciferase activity. sars-cov-2 c) the representative micrographs 617 and 2d classification results of negative-staining em. both spikes were incubated 618 with 4-fold molar ratio of hace2. the red box shows the complex of the pcov_gx 619 spike with hace2 534 we thank the tsinghua university branch of china national center for protein fig.4 the r esidues and glycans inter acting with one rbd of the differ ent spikes. 599 (a) the residues and glycans interacting with one rbd are shown as salmon spheres. 600 the ratg13 rbd is colored in magenta, pcov_gx rbd in green, sars-cov-2 601 key: cord-328578-9qzo18v3 authors: wang, yunfei; wang, lichun; cao, han; liu, cunbao title: sars‐cov‐2 s1 is superior to the rbd as a covid‐19 subunit vaccine antigen date: 2020-07-21 journal: j med virol doi: 10.1002/jmv.26320 sha: doc_id: 328578 cord_uid: 9qzo18v3 since its emergence in december 2019, severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2) has developed into a global pandemic within a matter of months. while subunit vaccines are one of the prominent options for combating coronavirus disease 2019 (covid‐19), the immunogenicity of spike protein‐based antigens remains unknown. when immunized in mice, the s1 domain induced much higher igg and iga antibody levels than the rbd and more efficiently neutralized sars‐cov‐2 when adjuvanted with alum. it is inferred that a large proportion of these neutralization epitopes are located in the s1 domain but outside the rbd and that some of these are spatial epitopes. this finding indicates that expression systems with posttranslational modification abilities are important to maintain the natural configurations of recombinant spike protein antigens and are critical for effective covid‐19 vaccines. further, adjuvants prone to a th1 response should be considered for s1‐based subunit covid‐19 vaccines to reduce the potential risk of antibody‐dependent enhancement (ade) of infection. this article is protected by copyright. all rights reserved. biology, chinese academy of medical sciences & peking union medical college (imb, cams). animals were randomly divided into 5 groups with 6 mice in each group (n=6). antigens were diluted to 10 μg/mouse/dose in 25 μl of pbs and mixed with the same volume of alum adjuvant (thermofisher scientific) prior to immunization. thus, 50 μl of immunogens were administered intramuscularly into the thigh muscle three times at 2 week intervals. 2 weeks after the final immunization, mice were anesthetized with ketamine, and blood was collected via cardiac puncture. after clotting at 4 °c overnight, serum was collected by centrifugation at 3 000 rpm for 10 min and pooled by group. all experiments were performed in compliance with the guiding principles for the care and use of laboratory animals of the animal ethics committee of the imb, cams (permit number: scxk (dian) k2017-0002). ninety-six-well plates were coated with 2 μg/ml hek293k cell-expressed recombinant sars-cov-2 s1 or rbd proteins overnight at 4 °c. plates were washed one time with wash buffer (pbs containing 0.05% (v/v) polysorbate 20) and then blocked with 5% (w/v) skim milk dissolved in wash buffer for 1 hour at 37 °c. plates were then washed 4 times and incubated with serially diluted mouse sera for 1 hour at 37 °c. next, plates were washed 5 times and incubated with goat anti-mouse igg/iga/igg1/igg2a hrp-conjugated secondary antibodies this article is protected by copyright. all rights reserved. article (thermofisher scientific) for 1 hour at 37 °c. following 5 additional washes, 3,3',5,5'-tetramethylbenzidine (tmb, bd bioscience) substrate was added. the plate was incubated at room temperature in the dark for 10 min, and reactions were stopped by the addition of 2 m sulfuric acid. absorbance (450 nm) was detected using a microplate reader (bio-tek instruments, inc). antibody titers were defined by end-point dilution with a cut-off signal of od450=0.1. sera samples that did not produce an od>0.1 at 1:500 were determined as 0. the igg1-to-igg2a titer ratio was calculated to evaluate th1-th2 balance 28,29 . for neutralization, mouse sera were diluted with dmem in a two-fold series. then, 50 μl of sars-cov-2 diluted with dmem to 3.3 lg ccid 50 was added to 50 μl of diluted serum, incubated at 37 °c for 1 hour, and then added to 100 μl of this article is protected by copyright. all rights reserved. article co 2 for 4 days, the neutralization titer was reported as the serum dilution at which sars-cov-2 infection was inhibited by 50%. all sars-cov-2 manipulations were carried out in a biosafety level 3 (bsl-3) laboratory at imb, cams. data are shown as the mean and standard deviation. graphpad prism 7.0 (san diego, ca, usa) was used for statistical analyses. here, we fused s1 and rbd to the carboxyl terminus of the norovirus shell domain, which has been reported to present recombinant expressed proteins on the surface of virus-like particles to enhance the immunity of recombinant proteins 23, 24 . while both rbd and s1 were expressed well with the norovirus shell domain (s-rbd and s-s1, respectively), as certified by the corresponding band (~52 kda for s-rbd and ~108 kda for s-s1) by sds-page ( figure 1a ) and western blot ( figure 1b ), both were expressed as inclusion bodies. following sonication, washing and dialysis, while s-rbd showed quite high purity (lane s-rbd in figure 1a ), s-s1 showed only approximately 60% purity by sds-page (lane s-s1 in figure 1a ). transmission electron microscopy showed that after dialysis, only a small portion of the s-rbd and s-s1 fusion proteins formed similar but not identical virus-like particles with diameters of approximately 30-60 nm (showed by arrows in figure 1c & 1d) , while the majority of these recombinant proteins formed irregular aggregates ( figure 1c & 1d) . this article is protected by copyright. all rights reserved. article sars-cov-2 s1 induces higher igg and iga titers than rbd 2 weeks after the third intramuscular immunization (figure 2a) , both s1-specific (s1-coated plate in figure 2 ) and rbd-specific (rbd-coated plate in figure 2 ) antibodies were analyzed. hek293k cell-expressed recombinant s1 (s1 immunized) and e.coli-expressed norovirus shell domain-s1 fusion protein (s-s1 immunized) induced similar s1-specific igg titers (64 000) and similar rbd-specific titers (8 000) ( figure 2b ). hek293k cell-expressed recombinant rbd (rbd immunized) induced low s1-specific igg titers (8 000) and rbd-specific igg titers (6 000), implying low immunogenicity of the rbd alone. unlike s1 (similar igg titers between s1 and s-s1 may be attributed to low purity and thus low s1 content in s-s1), fusion of the rbd with the norovirus shell domain (s-rbd immunized) elevated both rbd-specific igg titers (from 6 000 to 32 000) and s1-specific igg titers (from 8 000 to 32 000). as sars-cov-2 is a respiratory virus, mucosal immunity is important to fight infection and we therefore detected iga titers ( figure 2c ). while both hek293k cell-expressed recombinant s1 (s1 immunized) and e.coli-expressed norovirus shell domain-s1 fusion protein (s-s1 immunized) induced equivalent levels of s1-specific iga titers and igg titers (64 000), both hek293k cell-expressed recombinant rbd (rbd immunized) and e.coli-expressed norovirus shell domain-rbd fusion proteins (s-rbd immunized) induced half the level of s1-specific iga titers as igg titers (4000 vs 8000 for rbd and 16 000 vs 32 000 for s-rbd, respectively). while rbd-specific iga titers were the lowest of all the igg and iga titers tested, there was a tendency for norovirus shell domain to elevate rbd immunogenicity on rbd-specific iga titers (rbd coated, s-rbd this article is protected by copyright. all rights reserved. article immunized vs rbd immunized in figure 2c ), as was observed for the s1-specific iga titers (s1-coated, s-rbd immunized vs rbd immunized in figure 2c ). sars-cov-2 s1 induced more balanced th1-th2 responses than the rbd similar to s1-specific total igg titers ( figure 2b , s1-coated), both hek293k cell-expressed recombinant s1 (s1 immunized) and e.coli-expressed norovirus shell domain-s1 fusion protein (s-s1 immunized) induced the highest s1-specific igg1 ( figure 3a , s1 coated) and igg2a ( figure 3b , s1 coated) titers and comparably low rbd-specific igg1 ( figure 3a , rbd coated) and igg2a ( figure 3b , rbd coated) titers. hek293k cell-expressed recombinant rbd (rbd immunized) and e.coli-expressed norovirus shell domain-rbd fusion proteins (s-rbd immunized) induced low levels of igg1 and igg2a titers specific to both s1 (s1 coated in figure 3a&b ) and the rbd (rbd coated in figure 3a&b ). notably, the igg1 titers in each group in figure 3a are considerably higher than the igg2a titers in figure 3b . to enable direct comparisons between groups, we compared the igg1/igg2a ratios in each group induced by their own antigens ( figure 3c ). while both hek293k cell-expressed recombinant s1 (s1 immunized) and e.coli-expressed norovirus shell domain-s1 fusion proteins (s-s1 immunized) induced an igg1/igg2a ratio of 4, hek293k-expressed recombinant rbd (rbd immunized) induced an igg1/igg2a ratio as high as 16. this ratio could be lowered by ligation to the e.coli-expressed norovirus shell domain (the s-rbd immunized igg1/igg2a ratio was 8) but was still higher than in that in the s1 and s-s1 immunized groups. higher igg1-to-igg2a ratios, including the those for the s1 and s1-rbd groups, imply a th2-biased immune response for these antigens. igg2a subtype antibodies, which are present in neutralizing sera 49 . this result is consistent with the observed eosinophilic infiltration following vaccination and virus exposure, a typical characteristic of th2 immune responses with elevated igg1/igg2a proportions 28,29 . unfortunately, both sars-cov-2 s1 and rbd showed a th2-like immune response with high proportions of igg1 when immunized with alum as adjuvant (figure 3) , implying a similar immunopathological risk to those reported for other coronaviruses. though no ade has been reported in animal models re-exposed to sars-cov-2 or exposed following vaccine immunization, special attention should be paid to th1-biased adjuvants, as reported in the development of sars-cov-1 and mers-cov vaccines 31,32 . in conclusion, the sars-cov-2 s1 domain is more immunogenic than the rbd domain, inducing higher igg and iga antibodies and also efficient virus neutralization antibodies. we infer that a large proportion of these neutralization epitopes exist within the s1 domain but outside of the rbd and that some of these are spatial epitopes. while s1 induced a more balanced th1/th2 response than the rbd when adjuvanted with alum, increased levels of igg1 antibodies still indicate a potential risk of ade, and adjuvants prone to a th1 response should be considered for s1 subunit-based covid-19 vaccines. no potential conflicts of interest. genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding a new coronavirus associated with human respiratory disease in covid-19 in remdesivir in adults with severe covid-19: a randomised, double-blind, placebo-controlled, multicentre trial clinical benefit of remdesivir in rhesus macaques infected with sars-cov-2 vaccine development and therapeutic design for 2019-ncov/sars-cov-2: challenges and chances the early landscape of covid-19 vaccine development in the uk and rest of the world molecular basis of coronavirus virulence and vaccine development sars vaccine development prospects for a mers-cov spike vaccine sars immunity and vaccination antigenic and immunogenic characterization of recombinant baculovirus-expressed severe acute respiratory syndrome coronavirus spike protein: implication for vaccine design effects of toll-like receptor stimulation on eosinophilic infiltration in lungs of balb/c mice immunized with uv-inactivated severe acute respiratory syndrome-related coronavirus vaccine immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus gold nanoparticle-adjuvanted s protein induces a strong antigen-specific igg response against severe acute respiratory syndrome-related coronavirus infection, but fails to induce protective antibodies and limit eosinophilic infiltration in lungs the spike protein of sars-cov--a target for vaccine and therapeutic development mers-cov spike protein: a key target for antivirals severe acute respiratory syndrome (sars) coronavirus: application of monoclonal antibodies and development of an effective vaccine receptor-binding domain of sars-cov spike protein induces long-term protective immunity in an animal model recombinant receptor binding domain protein induces partial protective immunity in rhesus macaques against middle east respiratory syndrome coronavirus challenge receptor-binding domain of mers-cov with optimal immunogen dosage and immunization interval protects human transgenic mice from mers-cov infection key: cord-352934-ypls4zau authors: wan, jinkai; xing, shenghui; ding, longfei; wang, yongheng; gu, chenjian; wu, yanling; rong, bowen; li, cheng; wang, siqing; chen, kun; he, chenxi; zhu, dandan; yuan, songhua; qiu, chengli; zhao, chen; nie, lei; gao, zhangzhao; jiao, jingyu; zhang, xiaoyan; wang, xiangxi; ying, tianlei; wang, haibin; xie, youhua; lu, yanan; xu, jianqing; lan, fei title: human igg neutralizing monoclonal antibodies block sars-cov-2 infection date: 2020-07-03 journal: cell rep doi: 10.1016/j.celrep.2020.107918 sha: doc_id: 352934 cord_uid: ypls4zau summary covid-19 has become a worldwide threat to humans, and neutralizing antibodies have therapeutic potential. we have purified more than one thousand memory b cells specific to sars-cov-2 s1 or rbd (receptor binding domain), and obtain 729 paired heavy and light chain fragments. among these, 178 antibodies test positive for antigen binding, and the majority of the top 17 binders with ec50 below 1 nm are rbd binders. furthermore, we identify 11 neutralizing antibodies, 8 of which show an ic50 within 10 nm, and the best one, 414-1, with ic50 of 1.75 nm. through epitope mapping, we find 3 main epitopes in rbd recognized by these antibodies, and epitope b antibody 553-15 could substantially enhance the neutralizing abilities of most of the other antibodies. we also find that 515-5 could cross-neutralize the sars-cov pseudovirus. altogether, our study provides 11 potent human neutralizing antibodies for covid-19 as therapeutic candidates. covid-19 has become a worldwide threat to humans, and neutralizing antibodies 35 have therapeutic potential. we have purified more than one thousand memory b cells 36 specific to sars-cov-2 s1 or rbd (receptor binding domain), and obtain 729 paired 37 heavy and light chain fragments. among these, 178 antibodies test positive for 38 antigen binding, and the majority of the top 17 binders with ec 50 below 1 nm are rbd 39 binders. furthermore, we identify 11 neutralizing antibodies, 8 of which show an ic 50 40 within 10 nm, and the best one, 414-1, with ic 50 of 1.75 nm. through epitope 41 mapping, we find 3 main epitopes in rbd recognized by these antibodies, and 42 epitope b antibody 553-15 could substantially enhance the neutralizing abilities of 43 most of the other antibodies. we also find that 515-5 could cross-neutralize the 44 we screened sera samples from 11 patients recently recovered from covid-19, and 119 found all individuals showed certain levels of serological responses, with #507 and 120 #501 being the weakest, to sars-cov-2 spike rbd and s1 proteins ( figure 1a ). we 121 also found that 10 sera, except for 507, showed neutralization abilities against 122 sars-cov-2 pseudoviral infection of hek293t cells stably expressing human ace2 123 ( figure 1b ). such observations, i.e. the sera from different individuals displayed a 124 wide range of antibody responses, were consistent with a recent report (wu et al., 125 2020a). of note, no.509 blood sample was obtained at the second day after 126 hospitalization (table s2) , the sera already showed weak s1 antigen response and 127 pseudoviral neutralizing activities. 128 the rbd domain in the s1 region of sars-cov-2 spike protein is the critical region 129 mediating viral entry through host receptor ace2. using recombinant rbd and s1 130 antigens, we then isolated rbd and s1 bound memory b cells for antibody 131 identification using the pbmcs (peripheral blood mononuclear cells) from the 11 132 individuals by fluorescence activated cell sorting ( figure s1a ). 133 sequences encoding immunoglobulin heavy (igh) and light (igl) chains were 134 amplified from single b cell complementary dna samples after reverse transcription 135 and then cloned through homologous recombination into mammalian expressing 136 vectors (robbiani et al., 2017) . overall, 729 naturally paired igh and igl clones were 137 obtained, and the numbers of clones derived from each individual were listed in table 138 s1. 139 140 in order to screen for sars-cov-2 spike antigen specific monoclonal antibodies, we 143 used two primary assays based on elisa (enzyme linked immunosorbent assay) and 144 fca (flow cytometry assay), respectively. among the 729 candidate antibodies expressed in hek293e cells, 178 were positive for rbd or s1 binding ( figure 2a ). all 146 the positive clones were then sequenced. notably, almost all (98.6%) of the 147 sequences obtained were unique ones ( figure 2b) , similarly as what were previously 148 reported in ebola zhang et al., 2016) and yellow fever (calvert et 149 al., 2016) studies. 150 based on the ranking of elisa values and fca positivities, we focused on 29 151 antibodies for further characterization. we first measured the precise values of ec 50 152 by elisa, and identified 17 strong binders for s-ecd (extracellular domain) and rbd 153 with ec 50 below 1 nm, and the most potent one showing ec 50 at 0.057 nm (8.55 154 ng/ml) ( figure 2c and figure s2a ). of note, among these 29 antibodies, 4 antibodies 155 (413-3, 414-4, 105-28 and 105-41) were fca negative but bound recombinant rbd 156 relatively well in elisa ( figure 2c ). on the other hand, we also identified another 4 157 antibodies (505-8, 414-3-1, 505-17 and 515-5) showed strong fca positivity but with 158 barely detectable elisa signals ( figure 2c ). these findings indicated certain 159 conformational differences might exist between the recombinant and 160 we also noticed that the majority of the 17 strong binders were rbd binders, except 162 for 413-2, 553-13 and 553-18 ( figure 2c ). and we further confirmed that 553-13 and to identify neutralizing antibodies, we first employed pseudoviral infection assays 170 using hek293t-ace2 cells. from all the antibodies tested, we found a total of 16 171 pseudoviral neutralizing antibodies ( figure 2c , and figure 3a , column 3). among 172 these, 11 could neutralize authentic virus entry into vero-e6 cells, and 8 of them 173 showed potent ic 50 within 10 nm ( figure 3a , column 1). we next characterized the 174 best one, 414-1, which was able to effectively block authentic viral entry at ic 50 of 1.75 nm ( figure 3b , left). we also tested 414-1 expressing in cho cells, and found it could 176 achieve ~ 300 mg/l without any optimization suggesting for a great potential in 177 therapeutic development. furthermore, the rbd binding affinity of 414-1 was also 178 tested by bli (bio-layer interferometry assay), and showed comparable k d of 0.413 179 nm ( figure 3b, right) . 180 since cdr3 are the most critical region for antibody diversity, we then aligned the 181 cdr3 sequences of the heavy (cdr3 h ) and light (cdr3 l ) chains of the 11 authentic 182 viral neutralizing antibodies and found 9 unique ones ( figure 3a to understand the neutralizing mechanism of the 11 antibodies, we performed 191 epitope mapping experiments for the 8 rbd binders (note, 505-8, 413-2 and 515-5 192 were non-rbd binders, figure 2c and figure 3a ). in order to do so, we first utilized 193 rbd-ace2 blocking elisa, and found that 6 of them could effectively compete ace2 194 binding to rbd with ic 50 below 3 nm ( figure 3a , column 6), indicating their binding 195 epitopes overlapping with ace2 binding surface ( figure 3c , middle left). 196 we then carried out bli competition and mutagenesis assays for further analyses. 197 due to the identical cdr3 sequence and high similarities among 505-3, 515-1 and 198 505-5 mentioned above, we only chose 505-3 as the representative in these assays. 199 based on bli competition results, the 8 rbd binders could be classified into 3 groups, replaced in the non-ace2 binding surface ( figure 3c , left) to locate these epitopes. 203 all epitope a antibodies were largely unaffected by these mutations ( figure s3c, left) , 204 indicating that epitope a is limited within ace2 binding surface ( figure 3c , middle left) 205 considering that they competed ace2 binding in blocking elisa mentioned above. 206 epitope b antibody, 553-15, was sensitive to f374a, a372t and c379a mutations 207 ( figure s3c , middle) and also competed ace2 in blocking elisa ( figure 3a , column 208 6), therefore we speculated that epitope b should include these residues and partially 209 overlap with ace2 binding surface ( figure 3c , middle right). finally, residues critical 210 for epitope c antibody binding were shown in figure 3c , right panel. since they did 211 not compete ace2 in blocking elisa ( figure 3a , column 6), we proposed epitope c 212 at the indicated area of rbd ( figure 3c, right) . 213 notable, despite that the epitope c and non-rbd binders could not block ace2 214 binding for rbd ( figure 3a the spike proteins of sars-cov-2 share 76% and 35% of amino acid identities with 231 sars-cov and mers-cov, respectively. therefore, we wondered whether our 232 antibodies could cross-react with the s proteins of these two other coronaviruses. in 233 order to do so, we overexpressed the s proteins of sars-cov-2, sars-cov and mers-cov in hek293t, and tested the cross-reactivities by flow cytometry analyses. 235 from this exercise, we found 3 antibodies, 415-5, 415-6 and 515-5, cross-recognizing 236 sars-cov s, but not mers-cov s ( figure 4a and 4b). 415-5 and 515-5 shared 237 similar s protein affinities between sars-cov-2 and sars-cov, but 415-6 had 238 much lower affinity towards sars-cov s compared to sars-cov-2 s ( figure 4c ). elisa signals towards both rbd and s, but could robustly bind freshly expressed s 281 protein in a549 membrane ( figure 2c, column 4, 6) . these included two neutralizing 282 antibodies 505-8 (mentioned above) and 515-5, indicating that the recombinant rbd 283 or s protein may differ from the membrane bound s in terms of 3d conformation. we 284 would have missed these antibodies if we only had used elisa for antibody triage. 285 therefore, future antibody study should consider multiple approaches for the initial 286 identification and quality control. the fluorescently labeled s1 bait was previously prepared by incubating 5 µg of his 422 tag-s1 protein with anti his tag antibody-pe (phycoerythrin) for at least 1 hr at 4 °c in 423 the dark, rbd bait performed as before. pbmcs were stained using 7aad, cocktail neutralization assay was performed with 2 antibodies by 1:1 (n:n), and 514 calculated ic 50 by total antibodies concentrate ion. 515 all experiments related to authentic virus were done in bsl-3. monoclonal antibodies 516 were incubated with 200 pfu sars-cov-2 sh01 at 37 °c for 1 hour before added 517 into vero-e6 cell culture (96-well plate, 4 x 10 4 cells per well), and the cells were 518 continued for 48 hr before microscopic analyses for cpe (cytopathic effect). 519 520 the descriptive statistics mean ± sem or mean ± sd were determined for continuous 522 variables as noted. ec50 and ic50 values in this study were determined after log10 523 transformation of antibody concentration using a 4-parameters nonlinear fit analysis 524 positive cells (%) assessing the pandemic potential of mers-cov. the 529 neutralizing epitopes of the sars-cov s-protein cluster independent of repertoire, 532 antigen structure or mab technology potent neutralizing 535 antibodies from covid-19 patients define multiple targets of vulnerability. biorxiv the coronavirus 538 pandemic in five powerful charts a humanized monoclonal antibody neutralizes yellow fever virus 541 strain 17d-204 in vitro but does not protect a mouse model from disease role of the ebola membrane in the protection 544 conferred by the three-mab cocktail mil77 potent neutralizing antibodies against sars-cov-2 identified by high-throughput 547 single-cell sequencing of convalescent patients' b cells genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a 550 patient with atypical pneumonia after visiting wuhan. emerging microbes & infections 9 the zika outbreak of the 21st 553 century convalescent plasma as a potential therapy 555 for covid-19 epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus 558 pneumonia in wuhan, china: a descriptive study human monoclonal antibodies block the binding of sars-cov-2 spike protein to angiotensin 561 converting enzyme 2 receptor a potent neutralizing human antibody reveals the n-terminal domain of the 564 spike protein of sars-cov-2 as a site of vulnerability the 566 species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and 567 naming it sars-cov-2 potent neutralizing monoclonal antibodies against ebola virus isolated 570 from vaccinated donors a randomized controlled trial of zmapp for ebola virus infection refined protocol for generating monoclonal antibodies from single 576 human and murine b cells sars-cov-2 cell entry 579 depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor clinical features of patients infected with 2019 novel coronavirus in wuhan, china. 583 the lancet 585 characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune 586 cross-reactivity with sars-cov clinical progression and viral load in a community outbreak of 589 coronavirus-associated sars pneumonia: a prospective study cross-neutralization of sars-cov-2 by a human 592 monoclonal sars-cov antibody safe pseudovirus-based assay for neutralization antibodies against influenza 595 a(h7n9) virus recurrent potent 598 human neutralizing antibodies to zika virus in brazil and mexico rapid generation of fully human monoclonal antibodies specific to a vaccinating 601 antigen chimeric 604 camel/human heavy-chain antibodies protect against mers-cov infection 607 characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: 608 implication for development of rbd protein as a viral attachment inhibitor and vaccine potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific 612 human monoclonal antibody function, and antigenicity of the sars-cov-2 spike glycoprotein a human monoclonal antibody 618 blocking sars-cov-2 infection cryo-em structure of the 2019-ncov spike in the prefusion 621 conformation neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient 624 cohort and their implications. medrxiv a new coronavirus associated with human respiratory disease in china identification of human single-domain antibodies against sars-cov-2 a noncompeting pair of human neutralizing antibodies block covid-19 virus binding to its 633 receptor ace2 cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic 636 receptor binding domains potent neutralizing monoclonal antibodies against ebola virus 639 infection a pneumonia outbreak associated with a new coronavirus of probable bat 642 origin key: cord-332134-88wfcc3y authors: li, tingting; cai, hongmin; yao, hebang; zhou, bingjie; zhang, ning; gong, yuhuan; zhao, yapei; shen, quan; qin, wenming; hutter, cedric a.j.; lai, yanling; kuo, shu-ming; bao, juan; lan, jiaming; seeger, markus a.; wong, gary; bi, yuhai; lavillette, dimitri; li, dianfan title: a potent synthetic nanobody targets rbd and protects mice from sars-cov-2 infection date: 2020-09-24 journal: biorxiv doi: 10.1101/2020.06.09.143438 sha: doc_id: 332134 cord_uid: 88wfcc3y sars-cov-2, the causative agent of covid-191, recognizes host cells by attaching its receptor-binding domain (rbd) to the host receptor ace22–7. neutralizing antibodies that block rbd-ace2 interaction have been a major focus for therapeutic development8–18. llama-derived single-domain antibodies (nanobodies, ∼15 kda) offer advantages including ease of production and possibility for direct delivery to the lungs by nebulization19, which are attractive features for bio-drugs against the global respiratory disease. here, we generated 99 synthetic nanobodies (sybodies) by in vitro selection using three libraries. the best sybody, mr3 bound to rbd with high affinity (kd = 1.0 nm) and showed high neutralization activity against sars-cov-2 pseudoviruses (ic50 = 0.40 μg ml−1). structural, biochemical, and biological characterization of sybodies suggest a common neutralizing mechanism, in which the rbd-ace2 interaction is competitively inhibited by sybodies. various forms of sybodies with improved potency were generated by structure-based design, biparatopic construction, and divalent engineering. among these, a divalent mr3 conjugated with the albumin-binding domain for prolonged half-life displayed highest potency (ic50 = 12 ng ml−1) and protected mice from live sars-cov-2 challenge. our results pave the way to the development of therapeutic nanobodies against covid-19 and present a strategy for rapid responses for future outbreaks. strategy for rapid responses for future outbreaks. the binding through hydrophobic interactions and h-bonding that involves both side 142 chains and main chains (fig. 1d ). in addition, tyr37, a framework residue, also 143 participated binding by forming an h-bond with the rbd gly447 backbone. 144 145 mr17 also binds to the rbd at the 'seat' and 'backrest' regions but approaches 146 the rbd at an almost perfect opposite direction of sr4 (fig. 1c, 1e) , indicating 147 divergent binding mode for these sybodies. the binding of mr17 to the rbd occurred 148 on an 853.94 å 2 surface area with noticeable electrostatic complementarity (extended 149 data fig. 5b) . interestingly, this surface was largely shared with the sr4 binding surface 150 (fig. 1f) . the interactions between mr17 and the rbd were mainly mediated by h151 bonding. apart from the three cdrs, two framework residues, lys65 and tyr60, 152 interacted with the same rbd residue glu484, via a salt bridge with its side chain, and 153 an h-bond with its main chain (fig. 1g) . molecular mechanism for neutralization 157 structure alignment of sr4-, mr17-and ace2-rbd 4 showed that both sybodies 158 engage with rbd at the receptor-binding motif (rbm) ( fig. 2a, 2b) . superposing sr4 159 and mr17 to the s trimer showed both sybodies could bind to the 'up' conformation 2 160 of rbd with no steric clashes (fig. 2c, 2d) , and to the 'down' conformation with only 161 minor clashes (extended data fig. 6 ) owing to their minute sizes. consistent with the 162 structure observation, both sr4 and mr17 inhibited the binding of ace2 to rbd, as 163 revealed by bio-layer interferometry (bli) assays (fig. 2e, 2f) . 164 165 to probe the epitope for mr3 without a structure, competitive bli binding assays 166 were carried out. the results showed that mr3 could block ace2 (fig. 2g) , and sr4 167 and mr17 (fig. 2h, 2i) , suggesting it also binds to at least part of the rbm, although 168 the possibility of allosteric inhibition remains to be investigated. taken together, sr4 169 and mr17, and probably mr3, neutralize sars-cov-2 by competitively blocking the for biparatopic fusion, we first identified two sybodies, namely lr1 and lr5 (fig. 208 3a, 3b), that could bind rbd in addition to mr3 using the bli assay. as lr5 showed 209 higher affinity and neutralization activity than lr1 (fig. 1a) , we fused this non-210 competing sybody to the n-terminal of mr3 with various length of gs linkers ranging 211 from 13 to 34 amino acids (extended data table s1 ). interestingly, the linker length 212 had little effect on neutralization activity and these biparatopic lr5-mr3 sybodies 213 were more potent than either sybodies alone ( fig. 1a) with an ic50 of 0.11 g ml -1 (fig. 214 3c). lr5-mr3 may be more tolerant to escape mutants 34-37 owing to its ability to 215 recognize two distinct epitopes. this decreased ic50 by 10 folds for fc-mr3 (39 ng ml -1 ) and 25 folds fc-mr17 (0.48 g 219 ml -1 ), respectively (fig. 3d, 3e) . consistently, the fc fusion increased the apparent 220 binding affinity for both sybodies, with a kd of 0.22 nm for fc-mr3 and less than 1 pm 221 for fc-mr17 (extended data fig. 4h, 4i) . note, however, fc-mr17 did not gain as much 222 neutralization potency as for the apparent binding affinity. table 228 1). the optimal construct for mr17m-mr17m had the shortest linker (13-gs) (fig. 3d , 229 3e). by contrast, optimal neutralization activity was observed with the longest linker 230 (34-gs) for mr3-mr3 (fig. 3d, 3e) . again, mr3-mr3 was superior compared to 231 mr17m-mr17m, showing a 2-fold higher neutralization activity with an ic50 of 12 ng 232 ml -1 (fig. 3e) . compared to the monovalent mr3 (ic50 of 0.40 g ml -1 ), the divalent the most potent divalent sybody (mr3-mr3) was chosen to investigate the 240 potential of nanobodies to protect mice from sars-cov-2 infection. nanobodies have 241 very short serum half-lives of several minutes due to their minute size 38 . to circumvent 242 this, we fused mr3-mr3 to the n-terminus of an albumin-binding domain (abd) 39 243 which has been known to extend the circulating half-life of its fusion partners by 244 increase in size and preventing intracellular degradation 31 . conveniently, we expressed 245 mr3-mr3-abd in pichia pastoris, which is the preferred host to express nanobody 246 therapeutics owing to its robustness and its endotoxin-free production. small-scale 247 expression of mr3-mr3-abd showed a secretion level of ~250 mg l -1 with an apparent 248 purity of >80% without purification (fig. 4a) . note, this experiment was carried out 249 using a shaker which gave cell density of od600 of 16. given its ability to grow to od600 250 of 500 without compromising yield, the expression level of mr3-mr3-abd may reach 251 7.5 g l -1 in fermenters. the potential for simple and high-yield production is especially 252 attractive for the pandemic at a global scale. 353 the construct for the rbd with an avi-tag for biotinylation was made by fusing 354 dna, from 5'-to 3'-end, of the encoding sequence for the honey bee melittin signal neutralization assay 718 results for sars-cov-2 pseudovirus. (b) neutralization assay results for sars-cov 719 pseudovirus. veroe6-hace2 cells were infected with a premix of pseudotypes and 720 sybodies at two concentrations (1 m and 100 nm) a-i) biotinylated rbd 724 immobilized on a streptavidin-coated sensor was titrated with various concentrations 725 (nm) of sybodies as indicated open-book' view of molecular electrical potential surfaces of the 730 interface between the rbd and sr4 (a) and between the rbd and mr17 (b). the 731 electrical potential maps were calculated by adaptive poisson-boltzmann solver (apbs) 732 52 built-in in pymol structure-based design of a mr17 mutant (mr17m) with 743 improved affinity and potency. (a,b) neutralization assay for sars-cov-2 (a) or sybody concentrations were used at 1 m (green) and 746 100 nm (magenta) concentrations. data are from three independent experiments electrostatic repel and hydrophobic mismatch would make lys99 750 unfavorable at this position. according to the original library design, lys99 was 751 unvaried 26 , meaning that lys99 was not selected and hence opportunities for 752 optimization. (e) the k99y mutation fits the hydrophobic microenvironment well, as 753 revealed by the crystal structure of mr17m (extended data table 2). (f) binding 754 kinetics of mr17m binding to rbd. bli signals were recorded under ic50 values (g ml -1 ) for sars-cov-2 are indicated in brackets. data for mr17 are from 757 data are from three independent experiments extended data fig. 8. evaluation of in vivo stability and toxicity of nanobodies. (a) for neutralization assay, sera were preincubated with sars-cov-763 2 pseudovirus for 1 h before infection at 1/200 dilution. the infection rates on veroe6-764 hace2 were measure by facs 3 days post infection. (b) body weight changes. the body 765 weight data are presented as means  the sd of mice in each group (n= 4). no 766 significant differences are observed. (c) representative histopathology of the lungs, 767 heart, liver, spleen, lungs, kidney, and thymus for the different sybodies injected the images and areas of interest are magnified 100 ×. bars indicate 100 m a novel coronavirus outbreak of global health 773 concern structure, function, and antigenicity of the sars-cov-2 spike glycoprotein cryo-em structure of the 2019-ncov spike in the prefusion conformation structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 779 receptor structural basis of receptor recognition by sars-cov-2 structural and functional basis of sars-cov-2 entry by using human ace2 structural basis for the recognition of sars-cov-2 by full-length human ace2 a noncompeting pair of human neutralizing antibodies block covid-19 virus 787 binding to its receptor ace2 potent neutralizing antibodies against sars-cov-2 identified by high-throughput 789 single-cell sequencing of convalescent patients' b cells human neutralizing antibodies elicited by sars-cov-2 infection general strategy to humanize a camelid single-domain antibody and 794 identification of a universal humanized nanobody scaffold isolation of potent sars-cov-2 neutralizing antibodies and protection from 797 disease in a small animal model studies in humanized mice and convalescent humans yield a sars-cov-2 799 antibody cocktail an alpaca nanobody neutralizes sars-cov-2 by blocking receptor interaction. 801 biorxiv neutralizing nanobodies bind sars-cov-2 spike rbd and block interaction with 803 selection, biophysical and structural analysis of synthetic nanobodies that 805 effectively neutralize sars-cov-2 synthetic nanobodies targeting the sars-cov-2 receptor-binding domain an ultra-high affinity synthetic nanobody blocks sars-cov-2 infection by 809 locking spike into an inactive conformation. biorxiv nanobodies® as inhaled biotherapeutics for lung diseases the socio-economic implications of the coronavirus pandemic (covid-19): a 814 review cell entry mechanisms of sars-cov-2 sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a 818 clinically proven protease inhibitor convergent antibody responses to sars-cov-2 in convalescent individuals natural single-domain antibodies yeast surface display platform for rapid discovery of conformationally 825 selective nanobodies synthetic single domain antibodies for the conformational trapping of 827 membrane proteins generation of synthetic nanobodies against delicate proteins nali-h1: a universal synthetic library of humanized nanobodies providing 831 highly functional antibodies and intrabodies an improved yeast surface display platform for the screening of nanobody 833 immune libraries selection, biophysical and structural analysis of synthetic nanobodies that 835 effectively neutralize sars-cov-2. biorxiv the therapeutic potential of nanobodies inference of macromolecular assemblies from crystalline state tracking changes in sars-cov-2 spike: evidence that d614g increases molecular cancer therapeutics fusion to a highly stable consensus albumin binding domain allows for tunable 860 pharmacokinetics. protein engineering, design & selection : peds 28 generation of a broadly useful model for covid-19 pathogenesis, vaccination, 863 and treatment a human neutralizing antibody targets the receptor-binding site of sars-cov-2 a sars-cov-2 infection model in mice demonstrates protection by 867 neutralizing antibodies enzymatic assembly of dna molecules up to several hundred kilobases a fluorescence-detection size-exclusion chromatography-872 based thermostability assay for membrane protein precrystallization screening the protein complex crystallography beamline (bl19u1) at the shanghai 875 synchrotron radiation facility how good are my data and what is the resolution? phaser crystallographic software features and development of coot phenix: a comprehensive python-based system for macromolecular 885 structure solution the pymol molecular graphics system, version 2.0 schrödinger, llc electrostatics of nanosystems: 889 application to microtubules and the ribosome key: cord-328189-jpkxjn6e authors: brielle, esther s.; schneidman-duhovny, dina; linial, michal title: the sars-cov-2 exerts a distinctive strategy for interacting with the ace2 human receptor date: 2020-03-12 journal: biorxiv doi: 10.1101/2020.03.10.986398 sha: doc_id: 328189 cord_uid: jpkxjn6e the covid-19 disease has plagued over 110 countries and has resulted in over 4,000 deaths within 10 weeks. we compare the interaction between the human ace2 receptor and the sars-cov-2 spike protein with that of other pathogenic coronaviruses using molecular dynamics simulations. sars-cov, sars-cov-2, and hcov-nl63 recognize ace2 as the natural receptor but present a distinct binding interface to ace2 and a different network of residue-residue contacts. sars-cov and sars-cov-2 have comparable binding affinities achieved by balancing energetics and dynamics. the sars-cov-2–ace2 complex contains a higher number of contacts, a larger interface area, and decreased interface residue fluctuations relative to sars-cov. these findings expose an exceptional evolutionary exploration exerted by coronaviruses toward host recognition. we postulate that the versatility of cell receptor binding strategies has immediate implications on therapeutic strategies. one sentence summary molecular dynamics simulations reveal a temporal dimension of coronaviruses interactions with the host receptor. to gain access to host cells, coronaviruses rely on spike proteins, which are membrane-anchored trimers containing a receptor-binding s1 segment and a membrane-fusion s2 segment (6) . the s1 segment contains a receptor binding domain (rbd) that recognizes and binds to a host cell receptor. the angiotensin-converting enzyme 2 (ace2) was identified as the critical receptor for mediating sars-2002 entry into host cells (7, 8) . binding of the spike protein to the receptor is a critical phase where the levels of the ace2 expressed on the cell membrane correlates with viral infectivity, and govern clinical outcomes (9) . consistent with the clinical pulmonary manifestation, ace2 is widely expressed in almost all tissues, with the highest expression levels in the epithelium of the lung (10) . similar to the sars-2002 virus, the covid-19 virus enters the host cell by rbd binding to the host cell ace2 receptor (7, 11, 12) . host receptor recognition for cell entry is, however, not specified by the cov genus classification. mers-cov is a member of the bcov genus but does not recognize the ace2 receptor. in contrast, hcov-nl63 is a member of the acov genus and does recognize the ace2 receptor (13) . herein, we analyze the binding of several cov rbds to ace2 with molecular dynamics (md) simulations and compare the stability, relative interaction strength, and dynamics of the interaction between the viral spike protein and the human ace2 receptor. the covid-19 rbd (residues 319-529) shares a 72.8% sequence identity and high structural similarity with the sars-2002 rbd ( table 1 ). in contrast, the rbd of hcov-nl63 is only 17.1% identical to that of covid-19 and there are no significant structural similarities between them (fig. s1) . remarkably, the rbd of mers-cov, which is structurally similar to that of covid-19 (20.1% sequence identity, 65% structure similarity) recognizes a different host receptor (dpp4) for its cell entry and does not bind ace2 (14) . we ran 100ns molecular dynamic (md) simulations of ace2 in complex with the rbds of the covid-19, sars-2002, and hcov-nl63 viruses to quantify the energetics and the dynamics of the different rbd-ace2 interactions. the simulation trajectory snapshots at 10 ps intervals (10,000 frames) were analyzed by a statistical potential to assess the probability of the rbd-ace2 interaction (soap score, (16)), with lower values corresponding to higher probabilities and thus higher affinities. the interaction scores for covid-19 rbd-ace2 were comparable to those of sars-2002, median of -1865.9 and -1929.5, respectively (fig. 1a) . hcov-nl63 has rbd-ace2 interaction scores are higher than both of the sars-covs (median of -941.6). mers, which is structurally similar to covid-19 ( table 1) does not bind ace2. mers virus which binds dipeptidyl peptidase-4 (dpp4, also known as cd26 (14)), has rbd-ace2 interaction scores that indicate extremely weak affinity (median of -692.6), as expected from a non-cognate receptor interaction. covid-19 has the largest buried surface area at the interface (1204å 2 ), followed by the interface area for sars-2002 (998å 2 ) and hcov-nl63 (973å 2 ). the number of ace2 contacting residues maintains the same order, with 30, 24, and 23 for covid-19, sars-2002, and hcov-nl63, respectively (fig. 1c) . the three rbds exploit specific binding sites on ace2 based on the analysis of the md trajectories ( fig. 1 , c and d; movie s1). there is a significant overlap of ace2 interacting residues between covid-19 and sars-2002 (at least 73%), while hcov-nl63 shares only 17% and 36% of contacts with sars-2002 and covid-19, respectively. these findings suggest that the coronaviruses exert different interaction strategies with their cognate receptors to achieve the affinity that is required for effective cell entry. an ace2 residue is considered as part of the interface if one of its atoms is within 4å from any rbd atom in at least 10% of the 10,000 md simulation frames. (d) overlay of 50 snapshots for each of the three rbds. the ace2 is in surface representation (gray). the frames were aligned using the n-terminal fragment of ace2 that contains the two helices participating in the rbds binding. while the sequence identity between the rbds of covid-19 and sars-2002 is 73% (table 1) , we observe a significantly higher residue substitution rate at the interaction interface with the ace2 receptor. out of 29 rbd interface residues, only 10 residues (34%) in covid-19 are conserved with respect to sars-2002 ( fig. 2a , table s1 , fig. s1 ). similarly, only 12 residues (40%) in sars-2002 are conserved with respect to covid-19. to investigate these interface residues, we construct and overlay the contact maps for the rbd-ace2 interfaces for covid-19 and sars-2002 (fig. 2b) . we define a residue-residue contact frequency (cf) as the fraction of md trajectory frames in which the contact appears. remarkably, only 8 out of the total 72 residue-residue interface contacts have comparable (<50% difference) contact frequencies between the covid-19-ace2 and sars-2002-ace2 interfaces ( fig. 2b , colored gray). furthermore, we find two interaction patches unique to covid-19 ( fig. 2b , patches 1 and 3) and another patch unique to sars-2002 ( fig. 2b , patch 2). covid-19 has a significant and unique contact site between residues 500-505 of the rbd and residues 353-357 of ace2 (fig. 2 , b and c). covid-19 also creates a new interaction patch with the middle of the n-terminal ace2 helix (fig. 2 , b and c), while sars-2002 has a unique interaction patch with the end of the same helix (fig. 2, b and c) . the rest of the changes in the interface contact frequencies are due to the different interface loop conformations (covid-19 residue numbers 474-498, sars-2002 residue numbers 461-484) (fig. 2, a and b , table s1 ). covid-19 has a significantly higher number of well-defined contact pairs compared to sars-2002: 52 vs. 28 contacts (with 44 and 20 unique pairs, excluding the ones with similar cfs) were found for rbd-ace2 of the covid-19 and sars-2002, respectively (fig. 2b) . results from fig. 2 expose the accelerated evolution among the key anchoring residues of the rbd-ace2 interface. this comparison raises the following question: how does sars-2002 rbd reach an ace2 binding affinity that is comparable to that of covid-19 but with fewer contact pairs and a smaller interface area? the distribution of soap scores throughout the simulation trajectory has a larger fluctuation range for sars-2002, relative to covid-19 ( fig. 1, a and b; fig. s2a ) suggesting that sars-2002-ace2 interaction is fluctuating between several structural states. moreover, analysis of contact frequencies along the entire trajectory reveals that none of the sars-2002 contacts are maintained over 90% of the frames while covid-19 still maintains about half of its contacts at 90% of the trajectory (fig. s2b) . to investigate the dynamics of covid-19 binding compared to sars-2002, we calculate the root-mean-square fluctuation (rmsf) of each residue with respect to the lowest energy snapshot from their respective 100ns md simulation trajectory. the interface region in the rbd contains two loops (loop1: residues 474-489, loop2: residues 498-505; using covid-19 numbering, fig. 3d ) that bind to the ace2 n-terminal helix on both of its ends. these two loops are highly flexible in the sars-2002 rbd (fig. 3, a and d) . while loop1 is also fluctuating in the covid-19 rbd, albeit much less, loop2 remains relatively rigid in the covid-19 rbd. in addition, we find that in the covid-19-rbd, a region centered around k417 leads to further stability relative to the corresponding region in sars-2002. we attribute this difference to the unique interaction of covid-19 at position k417 with the middle of the n-terminal ace2 helix, thus serving as an anchor site to the receptor ( fig. 2c and fig. 3a) . the contribution of k417 to ace2 binding is observed in a recent cryoem structure of the covid-19 spike protein bound to ace2 (17) . overall, covid-19 is more rigid compared to sars-2002 (fig. 3, a and d) . we investigate the dynamics of a designed sars (sars-des) variant (11) table s1 ). the l486f mutation is of special interest for the covid-19 rbd as well because it has this same substitution. our md simulation analysis reveals that the sars-des has a substantially lower interaction scores with ace2 (median of -2199.2, fig. s2) , as expected for an optimized human ace2-binding rbd design. we observed that these two mutations not only enhance the binding affinity to ace2, but also lead to a substantial stabilization of the interaction interface. the fluctuation signatures along the rbd of sars-des are surprisingly similar to those recorded for covid-19 (fig. 3, b and c) . thus, the switch from a flexible binding mode (for sars-2002) to a stable one (covid-19 and sarsdes, fig. 3b ) highlights the remarkable capacity of the rbd to adopt alternative receptor binding strategies driven by a minimal number of amino acid substitutions. this analysis reveals the critical role of l486f (sars-des residue f472) for stabilizing the covid-19-ace2 interface and a reduction in the number of states of the covid-19 spike protein bound to an ace2 receptor. experimental affinity measurements (e.g. surface plasmon resonance, spr) confirm the high affinity of sars-2002 rbd-ace2 binding, with an equilibrium dissociation constant (kd) of ~15 mm (18) (19) (20) (21) , similar to the binding affinity of ace2 and the covid-19 rbd (22, 23) . our md based calculation is consistent with sars-2002 displaying a similar but slightly higher affinity relative to covid-19 (fig. 1a, fig. s2 and table s2 ). binding affinity is achieved through a combination of interface contact optimization and protein stability (fig. 3e) . while the rbd-ace2 complex can be resolved at high-resolution by cryo-em (17, 23) , md simulations provide orthogonal information about the interaction dynamics on a nanosecond timescale. in the case of covs, md simulations reveal an exceptional versatility of viral receptor binding strategies (fig. 3e) . covid-19 adopted a different strategy for achieving comparable affinity to sars-2002: the interface of covid-19 is significantly larger than that of sars-2002 (1204å vs. 998å) with a remarkable number of interacting residues (ace2: 30 vs. 24, fig. 1c) . in contrast, sars-2002 is more flexible in its interaction with ace2, interacting through fewer contacts that serve as "hot spots". therefore, we predict that sars-2002 rbd neutralizing antibodies will not be effective for covid-19. the failure of several of these antibodies to neutralize the binding of covid-19 rbd to its receptor is consistent with our findings (20, 23) . the fluctuation from high-to low-affinity conformations in sars-2002 leads to an increased efficacy for inhibiting peptides (24) and high-affinity antibodies (25) compared to covid-19. this implies a therapeutic challenge is attributed to the enhanced rigidity of the covid-19 rbd relative to that of the sars-2002. the geometric and physicochemical properties of rbd-ace2 interfaces resemble those of antibody-antigen interactions. in both cases the interface benefits from long loop plasticity, bulky aromatic side chains as anchoring sites, and the stabilization of the complex by distributed electrostatic interactions (26) . both covid-19 and sars-2002 interfaces contain long flexible loops and nine aromatic residues (tyr, trp, phe) in the interface with ace2 ( fig. 2a) . moreover, in the sars designed variant (sars-des (11)), the addition of an aromatic residue (l486f substitution) significantly improved the interaction scores and interface stability (fig. 3, b and d) . our findings shed light on the accelerated evolution of spike protein binding to the ace2 receptor similar to the rapid evolution along the antibody-antigen affinity maturation process. structural modeling the structural model of the covid-19 spike protein receptor binding domain (rbd) in complex with ace2 was generated by comparative modeling using modeller 9.18 (27) with the covid-19 sequence (refseq: yp_009724390.1). we relied on the crystal structure of the spike protein receptor-binding domain from a sars coronavirus designed human strain complexed with the human receptor ace2 (pdb 3sci, resolution 2.9å) as a template for comparative modeling. the sars-2002 spike protein rbd and hcov-nl63 in complex with ace2 were taken from pdb 2ajf (resolution 2.9å) and 3kbh (resolution 3.3å), respectively. missing residues were added in modeller. mers rbd structure was taken from the complex with the neutralizing antibody cdc2-c2 (pdb 6c6z, resolution 2.1å) and structurally aligned onto sars-2002 rbd in complex with ace2 receptor. the designed variant is from pdb 3sci. the md simulations were performed with gromacs 2020 software (28) using the charmm36m force field (29) . each of the complexes was solvated in transferable intermolecular potential with 3 points (tip3p) water molecules and ions were added to equalize the total system charge. the steepest descent algorithm was used for initial energy minimization until the system converged at fmax < 1,000 kj/(mol · nm). then water and ions were allowed to equilibrate around the protein in a two-step equilibration process. the first part of equilibration was at a constant number of particles, volume, and temperature (nvt). the second part of equilibration was at a constant number of particles, pressure, and temperature (npt). for both md equilibration parts, positional restraints of k = 1,000 kj/(mol · nm 2 ) were applied to heavy atoms of the protein, and the system was allowed to equilibrate at a reference temperature of 300 k, or reference pressure of 1 bar for 100 ps at a time step of 2 fs. following equilibration, the production simulation duration was 100 nanoseconds with 2 fs time intervals. altogether 10,000 frames were saved for the analysis at intervals of 10 ps. we superimposed several md snapshots on the recently submitted to the pdb x-ray structure (6vw1, resolution 2.7å) of covid-19-ace2 complex. the average rmsd over the interface ca atoms is ~1å. interaction scores between the virus spike rbd and ace2 were calculated for each frame of the trajectory using the soap statistical potential (16) . in the interface contact analysis, a residue-residue contact was defined based on the inter-atomic distance, with a cutoff of 4å. table s2 . rbd-ace2 interface evaluated by several methods for analysis of protein-protein interactions movie s1. overlay of 50 random snapshots from the md trajectories of covid-19-ace2, sars-2002-ace2, and hcov-nl63-ace2 complexes. for clarity only one copy of ace2 is shown (gray), covid-19, sars-2002, and hcov-nl63 are colored blue, red, and green, respectively. characterization of a novel coronavirus associated with severe acute respiratory syndrome return of the coronavirus: 2019-ncov coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus epidemiology and clinical characteristics of human coronaviruses oc43, 229e, nl63, and hku1: a study of hospitalized children with acute respiratory tract infection in guangzhou, china a new coronavirus associated with human respiratory disease in china structural basis for human coronavirus attachment to sialic acid receptors a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirusinduced lung injury susceptibility to sars coronavirus s protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor exogenous ace2 expression allows refractory cell lines to support severe acute respiratory syndrome coronavirus replication tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry the s proteins of human coronavirus nl63 and severe acute respiratory syndrome coronavirus bind overlapping regions of ace2 structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 tm-align: a protein structure alignment algorithm based on the tm-score optimized atomic statistical potentials: assessment of protein interfaces and loops structural basis for the recognition of the 2019-ncov by human ace2. biorxiv computational characterization and design of sars coronavirus receptor recognition and antibody neutralization receptor and viral determinants of sars-coronavirus adaptation to human ace2 potent binding of 2019 novel coronavirus spike protein by a sars coronavirusspecific human monoclonal antibody crystal structure of nl63 respiratory coronavirus receptor-binding domain complexed with its human receptor structure, function and antigenicity of the sars-cov-2 spike glycoprotein. biorxiv cryo-em structure of the 2019-ncov spike in the prefusion conformation a hexapeptide of the receptorbinding domain of sars corona virus spike protein blocks viral entry into host cells via the human receptor ace2 the spike protein of sars-cov--a target for vaccine and therapeutic development the indistinguishability of epitopes from protein surface is explained by the distinct binding preferences of each of the six antigen-binding loops comparative protein structure modeling using modeller gromacs 4.5: a high-throughput and highly parallel open source molecular simulation toolkit charmm36m: an improved force field for folded and intrinsically disordered proteins acknowledgments. the authors gratefully acknowledge barak raveh for useful suggestions. tables s1-s2movie s1references (27) (28) (29) residue key: cord-326282-uxn64olw authors: lu, maolin; uchil, pradeep d.; li, wenwei; zheng, desheng; terry, daniel s.; gorman, jason; shi, wei; zhang, baoshan; zhou, tongqing; ding, shilei; gasser, romain; prévost, jérémie; beaudoin-bussières, guillaume; anand, sai priya; laumaea, annemarie; grover, jonathan r.; liu, lihong; ho, david d.; mascola, john r.; finzi, andrés; kwong, peter d.; blanchard, scott c.; mothes, walther title: real-time conformational dynamics of sars-cov-2 spikes on virus particles date: 2020-09-13 journal: biorxiv doi: 10.1101/2020.09.10.286948 sha: doc_id: 326282 cord_uid: uxn64olw sars-cov-2 spike (s) mediates entry into cells and is critical for vaccine development against covid-19. structural studies have revealed distinct conformations of s, but real-time information that connects these structures, is lacking. here we apply single-molecule förster resonance energy transfer (smfret) imaging to observe conformational dynamics of s on virus particles. virus-associated s dynamically samples at least four distinct conformational states. in response to hace2, s opens sequentially into the hace2-bound s conformation through at least one on-path intermediate. conformational preferences of convalescent plasma and antibodies suggest mechanisms of neutralization involving either competition with hace2 for binding to rbd or allosteric interference with conformational changes required for entry. our findings inform on mechanisms of s recognition and conformations for immunogen design. against the virus(1-4). s is synthesized as a precursor, processed into s1 and s2 by furin 15 proteases, and activated for fusion when human angiotensin-converting enzyme 2 (hace2) engages the receptor-binding domain (rbd) and when the n-terminus of s2 is proteolytically processed (5) (6) (7) . structures of soluble ectodomains and native virus particles have revealed distinct conformations of s, including a closed trimer with all rbd oriented downward, trimers with one or two rbds up, and hace2-stabilized conformations with up to three rbd oriented 20 up (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) ). real-time information that connects these structures, however, has been lacking. smfret is well suited to inform on conformational dynamics of proteins reporting domain movements in the millisecond to second range, and has previously been applied to study hiv-1, influenza a, and ebola spike glycoproteins, via measurements of the distance-dependent energy transfer from an excited donor to a nearby acceptor fluorophores in real-time(19-23). to probe dynamics of sars-cov-2 spikes, we used available high-resolution structures of the sars-cov-2 s trimer to identify sites of fluorophore pair labeling that have the potential to 5 inform on distance changes expected to accompany conformational changes between the rbddown and receptor hace2-induced rbd-up trimer structures(14, 17) (fig. s1 ). accordingly, we engineered a4 and q3 labeling peptides before and after the receptor-binding motif (rbm) to allow site-specific introduction of donor and acceptor fluorophores at these positions ( fig. 1, a, b, and fig. s1 ). we optimized retroviral and lentiviral pseudoviral particles carrying the sars10 cov-2 s protein (fig. s2) to test the impact of these peptides on infectivity, and found that they were well tolerated, both individually and in combination (fig. s1d ). to measure conformational dynamics of the sars-cov-2 s trimer on the surface of virus particles, we established two types of particles, lentiviral pseudoparticles carrying s, as well as coronaviruslike particles generated by expression of s, membrane (m), envelope (e) and nucleocapsid (n) 15 protein (s-men)(24, 25) (fig. 1, a and b ). s-men particles co-express coronavirus surface proteins m and e. particle quality and the presence of the corona-like s proteins on both particle surfaces were confirmed by cryo-electron microscopy ( fig. 1, c and d) . for smfret, lentivirus particles and s-men particles were generated (see materials and 20 methods) by transfecting hek293t cells with an excess of plasmid-encoding wild-type, doped with trace amounts of plasmid expressing labeling peptide-carrying s to ensure the production of virus particles that contain, on average, only a single engineered s protein. as for analogous investigations of hiv-1 envelope protein(19, 21), donor (cy3b(3s)) and acceptor fluorophore (ld650) were enzymatically conjugated to the engineered s proteins presented on the virus particle surface in situ (see materials and methods). a biotinylated lipid was then incorporated into the virus particle membrane to allow their immobilization within passivated microfluidic devices coated with streptavidin to enable imaging by total internal reflection microscopy 5 (fig.1a) . donor fluorophores on single, immobilized virus particles were excited by a singlefrequency 532 nm laser and fluorescence emission from both donor and acceptor fluorophores were recorded at 25 hz ( fig. 2a) . from the recorded movies, we computationally extracted hundreds of smfret traces exhibiting anti-correlated donor and acceptor fluorescence intensities, the telltale signature of conformational changes within the s protein on individual 10 virus particles. analyses of smfret data from ligand-free s protein on lentiviral particles revealed that the sars-cov-2 s protein is dynamic, sampling at least four distinct conformational states to identify the receptor-bound conformation of the sars-cov-2 s protein by smfret, we measured the conformational consequences of soluble, monomeric hace2 binding. addition of 5 the monomeric hace2 receptor to surface-immobilized virus particles lead to increased occupancy of the low-(~0.1) fret s protein conformation (fig. 2e) , which was observed at both the single-molecule and population level (fig. 2f ). similar hace2 receptor impacts on the sars-cov-2 s protein were observed in both lentiviral particle and s-men coronavirus-like particle contexts (fig. 2 , e to g). dimeric hace2, a more potent ligand (fig. s4a )(26), 10 stabilized the low-(~0.1) fret s protein conformation more efficiently (fig. s4 , b and c), suggesting that the observed low-fret state likely represents the receptor-bound state in which all three rbd domains are oriented upwards (rbd-up conformation). a unique strength of single-molecule imaging is its capacity to reveal directly both the structural 15 and kinetic features that define biological function (27, 28) . to extract such information for the sars-cov-2 s protein, we employed hidden markov modeling (hmm)(29) to idealize individual smfret traces. these data allowed quantitative analyses of the thousands of discrete hace2-binding -could be achieved spontaneously. 15 as expected, the binding of the hace2 receptor modified the dynamic s protein conformational landscape towards the rbd-up conformation (~0.1 fret), rendering it the most populated ( fig. 2 , b, c, f, g). this change resulted from an increase in the transition rate from the rbd-down conformation (~0.5 fret) towards the intermediate-(~0.3) fret state and subsequently the rbd-up (~0.1 fret) conformation, which was also modestly stabilized. the energy barriers for 20 reverse transitions towards the rbd-down conformation (~0.5 fret), were also elevated, explaining receptor-bound conformation accumulation over time (figs. s5). these analyses lead to a qualitative model for hace2 activation of the sars-cov-2 s protein from the ground state to the receptor-bound state through at least one intermediate conformation (fig. 2h ). the summary of relative state occupancies, transition rates among conformations and errors are listed in tables s1 and s2, respectively. in most cell types, the serine protease tmprss2 is required for ph-independent sars-cov-2 5 entry (5, 30, 31) . in vitro, the effect of tmprss2 is mimicked by the serine protease trypsin, which has similar cleavage specificity (5, 31) . smfret analysis of trypsin-treated s protein on lentiviral particles in the absence of receptor revealed a clear shift towards activation (fig. 3 , a, b). after trypsin treatment, the addition of hace2 receptor was more effective at stabilizing the s protein in the rbd-up (~0.1 fret) conformation (fig. 3 , c and d, fig. s6 ). to further 10 validate this finding, we measured the effects of trypsin pre-treatment in virus-cell and cell-cell fusion assays using split nanoluc system consisting of lgbit and hibit (fig. 3, e and f, fig. s7 ). here, membrane fusion restored luciferase activity between lentiviral particles carrying the s protein as well as a vpr-hibit fusion protein with cells expressing the lgbit counterpart fused to a ph domain. this assay revealed fusion to be strictly dependent on the hace2 receptor 15 and to be stimulated by trypsin treatment (fig. 3 , e and f). nearly identical results were observed for cell-cell fusion between donor cells expressing s and target cells expressing hace2 ( fig. s7) , confirming the activating role of trypsin treatment. we next explored the suitability of the smfret assay to characterize the conformational 20 consequences of antibody binding to the sars-cov-2 s protein. multiple studies on antibodies generated from covid-19 patients have shown that one type of antibody often dominates immune responses(32-37). this prompted us to screen plasma from convalescent patients with 8 neutralizing activity that can bind to the s protein on lentiviral particles(38) using a modified virus-capture assay (vca)(39). cross-reactive cr3022(40), one of the very first reported antibodies from sars-cov-1 that also bind to sars-cov-2 spike rbd domain, served as a good indicator of rbd binding (fig. 4a) . we identified two plasma samples (s002 and s006) able to specifically bind the rbd, recognize s expressed at the cell surface and to neutralize 5 viral particles (fig. 4 , a to c, and fig. s8 ). smfret analysis of antibody-bound s revealed that both cr3022 and plasma from patient s006, stabilized s in the rbd-up (~0.1 fret) conformation, in a similar fashion as receptor hace2 (fig. 4 , d and e). these data point to the presence of rbd-directed antibodies in patient s006. by contrast, smfret indicated that plasma from patient s002 contained an activity that stabilized the rbd-down (~0.5 fret) 10 conformation (fig. 4f ). plasma s002 antagonized hace2 binding, but rbd competition did not affect its recognition of s, suggesting that its neutralization activity does not solely rely on blocking the receptor interface we then assessed the conformational preference of four rbddirected antibodies: the potently neutralizing antibodies h4, 2-4 and 2-43, and the neutralization nanobody vhh72, each of which binds rbd in a different way(41-43). antibody h4 and 15 nanobody vhh72 stabilized the s protein in an rbd-up (~0.1 fret) conformation similar to hace2, cr3022, and s006, whereas antibody 2-4 shifted the conformational landscape towards rbd-down (~0.5 fret) conformation, similar to s002 (fig. 4 , g to j). the very potent neutralizing antibody 2-43(43), meanwhile, showed a partial shift to the rbd-up (~0.1 fret) conformation (fig. s9) . the absence or presence of hace2 did not appear to affect the rbd-up 20 stabilization evidenced for antibodies cr3022, s006, vhh72, or h4 (fig. s9) . however, plasma s002, and to a lesser extent antibody 2-4, reduced the hace2-dependent stabilization of the rbd-up (~0.1 fret) conformation, suggesting that they may interfere with hace2 receptor binding via an allosteric mechanism. these findings indicate that sars-cov-2 neutralization can be achieved in two ways: 1) antibodies that conformationally mimic hace2 and directly compete with hace2 receptor binding, or 2) by allosterically stabilizing the s protein in its rbd-down conformation. 5 the strength of the presented smfret approach is revealed by the capacity to examine the dynamic properties of the s protein in real time, including: 1) the distinct conformational states that it spontaneously transits under physiological conditions; 2) the impact of sequence alterations on s protein dynamics; and 3) the responses of the s protein to cognate hace2 receptor and antibody recognition. 10 the present analyses of dynamic s protein molecules provides three lines of evidence that indicate that the intermediate-(~0.5) fret state observed represents the rbd-down, ground state conformation of the s protein, in which all three rbd domains are oriented towards the viral particle membrane. first, in line with previous electron microscopy (em) investigations (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) 16), the rbd-down state is the most populated. in further agreement with recent em studies, both the disulfide bridge (s383c, d985c)(9, 11) and antibody 2-4 stabilized the s protein in a conformation with all three rbd oriented down 42 . while our smfret observations highlight considerable conformational flexibility in these contexts compared to em of soluble trimers, we attribute these distinctions to a tendency of our analysis approach to over-emphasize dynamic 20 features, while em may over-emphasize static conformations rigidified by cryogenic temperatures that may be more readily identified and classified(44). multiple lines of evidence also facilitated assignment of the rbd-up (~0.1 fret) conformation of the s protein with all three rbd domains oriented away from the virus particle membrane. for instance, this conformation was stabilized by soluble monomeric hace2 receptor, and even further stabilized in the presence of soluble, dimeric hace2 receptor as well as rbd-targeting antibodies, such as cr3022, that are known to access their epitopes when the s protein is in an activated, rbd-up conformation(40-42). the structure of the on-path (~0.3 fret) intermediate observed during s1 5 opening is likely similar to the all-down ground state; cryo-em structures of soluble sars-cov-2 s trimers(17) that engage one or two hace2 molecules receptors(18) reveal that the distance between the two labeling sites increases in the ligand-free protomers adjacent to a protomer engaged to hace2 (fig. s1a ). the additional, highly compacted s conformation (~0.8 fret) evidenced, which is also depopulated by activating ligands, remains unknown. 10 these smfret analyses are in global agreement with the conformational states observed at the single particle em and cryoet level (6, 8, 9, 11-17, 40-42, 45, 46) . the observed fret changes are also are in good agreement with expected increase in the distance between the labeling peptide insertion sites that carry the fluorophores in the rbd-down and rbd-up conformations 15 of the s trimer. the capacity to examine the conformational preferences of rbd-directed antibodies to the s protein enabled us to identify conformational signatures of antibodies in patient plasma. this approach identified patients with antibody activities that either mimicked ace2 (indicating anti-rbd activity) or stabilized the ground state of s, thereby interfering with data and materials availability: all data is available in the main text or the supplementary materials. the data that support the findings of this study are available from the corresponding authors upon reasonable request. the full source code of spartan, which was used for analysis of smfret data, is publicly available. (http://www.scottcblanchardlab.com/software). some small customized matlab scripts are available upon reasonable requests. a full-length wild-type pcmv3-sars-cov-2 spike (s1+s2)-long (termed as pcmv-s, codon-optimized, sino biological, cat # vg40589-ut) plasmid was used as a template to generate tagged pcmv-s. the translated amino acid sequence of pcmv-s is identical to each pair of inserted tags did not compromise s-dependent lentivirus infectivity. 15 infectivity measurements the infectivity of lentivirus particles carrying sars-cov-2 spike proteins was cryo-electron tomography 6 nm gold tracer was added to the concentrated s-decorated hiv-1 lentivirus and s-men particles viruses at 1:3 ratio, and 5 µl of the mixture was placed onto freshly glow discharged holey carbon grids for 1 min. grids were blotted with filter paper, and rapidly frozen in liquid 10 ethane using a homemade gravity-driven plunger apparatus. cryo-grids were imaged on a cryo-transmission electron microscope (titan krios, thermo fisher scientific) that was operated at 300 kv, using a gatan k3 direct electron detector in counting mode with a 20 ev energy slit. tomographic tilt series between −51° and +51° were collected by using serialem(55) in a dose-symmetric scheme(56) with increments of 3°. the 15 nominal magnification was 64,000 x, giving a pixel size of 1.346 å on the specimen. the raw images were collected from single-axis tilt series with accumulative dose of ~50 e− per å 2 . the defocus was -3 µm and 8 frames were saved for each tilt angle. frames were motion-corrected using motioncorr2(57) to generate drift-corrected stack files, which were aligned using gold fiducial makers by imod/etomo(58). tomograms were 20 reconstructed by weighted back projection and tomographic slices were visualized with imod. virus particles were labeled through site-specifically enzymatic labeling, as previously the sars-cov-2 rbd elisa assay used was recently described (38, 53) introduction of fluorophores (cy3b, green; ld650, red) was guided by conformational changes in s1 induced by binding of the cellular receptor human angiotensin-converting enzyme 2 (hace2) from the "rbd-down" to the "rbd-up" conformation (fig. s1) . rbd, receptorbinding domain; ntd, n-terminal domain. structures were adapted from rcsb protein data bank accessories 6vsb ('down' s1/s2 protomer: s1, light cyan; s2, dark blue) and 15 6vyb/6m0j ('up' protomer s1/s2 engaged with hace2: hace2, magenta). table s1 . (h) relative free energy model of conformational landscapes of sars-cov-2 spikes in response to the hace2 binding. the differences in free energies between states were roughly scaled based upon relative state occupancies of each state. (fig. 2b) . fret histograms represent mean ± s.e.m., determined from three randomly assigned populations of fret traces. for state occupancies see table s1 . fret histograms represent mean ± s.e.m., determined from three randomly-assigned populations of all fret traces. evaluated state occupancies see table s1 . table s1 . table s1 . relative state-occupancy and fitting parameters in each of four fret-defined conformational states of sars-cov-2 spike protein on the surface of virus particles. the fret efficiency histograms were fitted into the sum of four gaussian distributions (µ, the mean or expectation of the gaussian distribution; σ, s.d. of the gaussian distribution) for each conformational state. parameters were based upon the observation of original fret efficiency 5 data and were further determined using hidden markov modelling. relative conformational stateoccupancy of sars-cov-2 spike protein on viral particles are presented as mean ± s.e.m., determined from three independent measurements. r-squared values were evaluated to indicate the goodness of fit. ligand-free 0.9886 26% +/-6% 23% +/-11% 39% +/-12% 12% +/-7% + hace2 0.9977 49% +/-8% 24% +/-11% 20% +/-11% 7% +/-6% table s2 . transition rates between observed conformational states of sars-cov-2 spike on virus particles. the survival probability plots (figs. s5 and s6) were derived from distributions of dwell times for each state-to-state transitions determined through hidden markov modeling (hmm). then plots were fitted by double-exponential distributions: y(t) = a 1 exp -k1t + a 2 exp -k2t ), where y(t) is the probability and t is the dwell time. the presented rates were the 5 weighted average of two rates derived from double-exponential decays. rates were finally presented in the table as (weighted average +/-95% confidence intervals). development of an inactivated vaccine candidate for sars-cov-2 an mrna vaccine against sars-cov-2 -preliminary report dna vaccine protection against sars-cov-2 in rhesus macaques single-shot ad26 vaccine protects against sars-cov-2 in rhesus macaques sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor structure, function, and antigenicity of the sars-cov sars-cov-2 and bat ratg13 spike glycoprotein structures inform on virus evolution and furin-cleavage effects distinct conformational states of sars-cov-2 spike protein controlling the sars-cov-2 spike glycoprotein conformation structure-based design of prefusion-stabilized sars-cov-2 spikes closing coronavirus spike 30 glycoproteins by structure-guided design. biorxiv structural basis of receptor recognition by sars-cov-2 structural and functional basis of sars-cov-2 entry by using human ace2 structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor molecular architecture of the sars-cov-2 virus. biorxiv structures and distributions of sars-cov-2 spike proteins on intact virions key: cord-300784-4jeaqqn9 authors: ma, huan; zeng, weihong; he, hongliang; zhao, dan; yang, yunru; jiang, dehua; zhou, peigen; qi, yingjie; he, weihuang; zhao, changcheng; yi, ruting; wang, xiaofang; wang, bo; xu, yuanhong; yang, yun; kombe kombe, arnaud john; ding, chengchao; xie, jiajia; gao, yong; cheng, linzhao; li, yajuan; ma, xiaoling; jin, tengchuan title: covid-19 diagnosis and study of serum sars-cov-2 specific iga, igm and igg by a quantitative and sensitive immunoassay date: 2020-04-22 journal: nan doi: 10.1101/2020.04.17.20064907 sha: doc_id: 300784 cord_uid: 4jeaqqn9 background the current pandemic of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has caused a great loss in lives and economy. detecting viral rnas on nasopharyngeal and throat swabs is the standard approach for sars-cov-2 diagnosis with variable success. currently, there are only a few studies describing the serological diagnostic methods that involve the detection of sars-cov-2-specific igm and igg. here, we aimed to develop a more quantitative and sensitive serological test for covid-19 diagnosis, monitoring and clinical investigation, based on the detection of antigen-specific iga as well as igm and igg in blood in response to sars-cov-2 infection. methods in this investigation, we report the development of a set of validated diagnostic kits for detecting serum iga, igm, and igg specific to sars-cov-2 nucleocapsid protein (np) and receptor-binding domain (rbd) of the spike protein by chemi-luminescence immuno-analysis. the kits were tested with a cohort of 216 sera from 87 laboratory-confirmed covid-19 patients, and 483 sera from sars-cov-2 negative or healthy individuals as negative controls. a standard receiver operating characteristic (roc) analysis was conducted to evaluate the diagnostic accuracy. using the kits, serum levels of iga, igm, and igg were analyzed, in response to sars-cov-2 infection and covid-19 pathogenesis. findings the diagnostic kits based on the rbd antigen outperformed those based on the np. rbd-specific iga, igm, and igg detection kits showed sensitivities of 98.6%, 96.8%, and 96.8%, and specificities of 98.1%, 92.3%, and 99.8%, respectively. in addition, using purified rbd-specific immunoglobulins from a serum pool of covid-19 patients as standards, the serum concentrations of rbd-specific iga, igm, and igg proteins were determined. the concentrations varied widely among different patients. median concentration of iga and igm reached peaks at 16-20 days after illness onset at 8.84 μg/ml and 7.25 μg/ml, respectively, while median concentration of igg peaked during 21-25 days after illness onset at 16.47 μg/ml. furthermore, the serum iga level positively correlates with covid-19 severity. interpretation our immunoassay of measuring sars-cov-2 specific antibodies iga, igm, and igg in serum provides a better serological testing with improved sensitivity and specificity. data of iga, igm, and igg responses in blood of covid-19 patients may provide novel insight for the monitoring and treatments of covid-19. the kits are also suitable for epidemiological studies and vaccine validations. the diagnostic kits based on the rbd antigen outperformed those based on the np. rbd-specific iga, 70 igm, and igg detection kits showed sensitivities of 98· 6%, 96· 8%, and 96· 8%, and specificities of 98· 1%, 71 92· 3%, and 99· 8%, respectively. in addition, using purified rbd-specific immunoglobulins from a cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april 22, 2020. . https://doi.org/10.1101/2020.04.17.20064907 doi: medrxiv preprint a sensitivity below 70%. 9-13 therefore, there is an urgent need for more reliable and rapid diagnostic 126 methods to screen sars-cov-2 infected people including those who do not have overt symptoms. a 127 serological test of virus-induced antibody production has unique advantages in clinical diagnostics, 128 especially for identifying people who acquired immunity against pathogens without noticeable 129 symptoms. 14 when the virus invades host, the body produces large amounts of immunoglobulin (ig) by 130 the immune system and released into blood, among them, igg, igm, and iga isotypes. 15 it has been 131 widely believed that igm is the first antibody to be transiently synthesized in response to the virus 132 invasion. 15 igg is a major class of immunoglobulins found in the blood, comprising 75% of total serum 133 immunoglobulins and has long-term immunity and immunological memory. 15, 16 therefore, a 134 combination of igm and igg has been used in various serological tests for detecting infection of sarscov-2 as previously used for sars and other coronaviruses. 10, 11, 14, [17] [18] [19] [20] [21] in contrast, iga, which is mainly 136 produced in mucosal tissues to hinder virus invasion and replication but also detected in blood (~15% of 137 total immunoglobulins in blood), 22 has not been widely used in serological tests for detecting coronavirus 138 infection. iga's production kinetics and roles in anti-viral immunity of iga are even less known. currently, only a few published studies reported diagnosis of covid-19 by using elisa or "flow 140 immunoassay" for detection of serum igm and igg with limited accuracy, 10, 11, 17-20 although sars-cov-141 2 specific iga in serum was also detected in recent papers or a preprint. 12, 23, 24 the kinetics of antibody 142 responses in covid-19 remains undefined, specifically for iga production. in this study, we designed and evaluated a set of sensitive and quantitative kits to measure serum iga, cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april 22, 2020. . https://doi.org/10.1101/2020.04.17.20064907 doi: medrxiv preprint underline illnesses; the most common one was hypertension in eighteen patients (20· 7%). a total 216 168 serum samples were taken from the 87 covid-19 patients. negative controls and potentially interfering non-covid-19 patient serum samples were collected in 170 order to evaluate the reliability of the kits. this cohort contains 330 sera from obviously healthy people, 171 fifteen sera from once suspected cases (rt-qpcr negative but had typical manifestation of pneumonia) 172 and 138 sera from other patients with different underlying diseases. all sera were stored at -20°c. diagnostic kit preparation and testing 186 briefly, the purified np or rbd viral antigens were coated to magnetic particles to catch sars-cov-187 2 specific iga, igm, and igg in patient sera. then a second antibody that recognizes iga, igm, or igg is . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 22, 2020. . https://doi.org/10.1101/2020.04.17.20064907 doi: medrxiv preprint based on the clinical rt-qpcr diagnosis results of sars-cov-2 infection, receiver operating 208 characteristic (roc) analysis was conducted using medcalc software to determine the optimal cut-off 209 value (criterion) and evaluate the diagnostic value of np-or rbd-specific iga, igm, and igg kits. the specificity and sensitivity of the antibody kits were calculated according to the following formulas: in order to analyze the correlation of serum antibody levels and age with disease severity, we first used 215 the kruskal wallis test 26 to test if there is any significant difference of iga among the three groups (mild, moderate, severe). then dunn's test 27 was used to perform a pair-wise test between each group, and 217 benjamini-hochberg procedure 28 was used to adjust p-values. all the above analyses use r software 218 version 3.6.1 29 . a p value less than 0· 05 was judged statistically significant. results 221 highly purified sars-cov-2 np and rbd proteins (supplementary figure 1) were employed to 222 develop a series of serological test kits, to detect the presence of np-and rbd-specific iga, igm, and 223 igg, respectively (hereinafter referred to as "np kit" and "rbd kit"). a cohort of 216 sera from 87 224 sars-cov-2 infected patients was tested with both np and rbd kits, together with 20 sera from 20 225 non-sars-cov-2 infected patients as negative controls initially. the np kits for iga, igm, and igg 226 showed diagnostic sensitivities of 89· 8%, 78· 2%, and 95· 8%, and specificities of 85· 0%, 95· 0%, and 227 100% respectively (supplementary figure 2a-c). however, the rbd kits for detecting iga, igm, and 228 igg showed higher diagnostic sensitivities of 97· 2%, 93· 1%, and 96· 8%, and specificities of 100%, 229 90· 0%, and 100%, respectively (supplementary figure 2d-f). we conclude that the rbd based kits igg kits, the sensitivity, specificity and overall agreement elevate to 99· 1%, 100%, and 99· 7%, 243 respectively. this is much better than when igm and igg were combined. when iga, igm, or igg 244 individual kit was used, we observed a total of 9 (0.61% to 6.67%), 37 (5.54% to 40.0%), and 1 (0 to cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 22, 2020. . https://doi.org/10.1101/2020.04.17.20064907 doi: medrxiv preprint and 153 non-covid-19 patients also indicate that our rbd-based detection kits did not cross-interact 250 with antibodies raised against other human coronaviruses presenting in ~15% of common cold cases and 251 also causing pneumonia. taken together, our detection systems are highly specific to sars-cov-2 252 rbd. we attempted to analyze the kinetics of all the three isotypes of antibodies when multiple serum were significantly older (median age of 62· 5) than those patients with moderate (median age of 46) and 287 mild symptoms (median age of 30). remarkably, we found that iga concentrations in severe cases were 288 significantly higher than mild or moderate cases (figure 3a). igg levels in moderate and severe covid-289 19 patients were also higher than mild cases (p < 0· 0001) ( figure 3c ). the observation that serum igg 290 levels were higher in severe and moderate than mild covid-19 patients have been previously reported 291 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 22, 2020. . https://doi.org/10.1101/2020.04.17.20064907 doi: medrxiv preprint 10, 21 . we also provided here a novel observation that serum iga levels correlate with covid-19 severity 292 (figure 3a), how the levels and roles of different types of antibodies as related to covid-19 severity 293 remain to be determined. discussion 296 compared to sampling of nasopharyngeal or throat swabs, blood extraction is more convenient and 297 reliable. furthermore, serum antibody test is more convenient, fast and accurate, and with other 298 advantages over the detection of viral rna. 9, 17 we report here an improved serological kit that can 299 sensitively and quantitatively detect serum levels of iga as well as igm and igg. together with recent 300 reports by others 9-12, 17-20, 23, 24 , the serological data that we obtained from 216 serum samples of 87 the nucleocapsid protein (np) is the most abundant protein in coronaviruses, which was reported to 307 be highly immunogenic and often used as a diagnostic marker for coronaviruses such as sars-cov 31 . the rbd of the spike protein on viral surface is the ligand binding to the major host receptor ace2; 309 therefore rbd could be a main target for neutralization antibodies. 32, 33 in this study, we explored the 310 possibility of using either np or rbd as an immobilized antigen in for developing a clinical covid-19 311 diagnostic kit. our data (supplementary figure 2) showed rbd-based diagnostic kits were better 312 performed than that of np in detecting all the three types of antibodies. a few previous studies reported 313 that rbd-based igm and igg detection is better than np once a comparison was made 19, 23 , and the 314 measurement is agreeable with the titers measured by virus neutralization assays 23, 24 . we provided here 315 the evidence that rbd as an immobilized antigen is also better than np in detection serum iga from 316 covid-19 patients. the exact mechanisms of difference between the use of two types of viral antigens 317 remain to be resolved. it could be that the np as a highly basic protein interacts with acidic residues in 318 complementarity determining region in antibodies is less specific. it could also be due to the fact that the 319 np antigen is expressed in bacteria as most investigators do, and the rbd protein we used is expressed 320 in a human cell line enabling critical glycosylation and high-affinity binding to antibodies raised in 321 covid-19 patients. nonetheless, we showed that our serological kits based on sars-cov-2 spike 322 protein rbd as an immobilized antigen provide a high sensitivity and specificity for detecting iga, igm, 323 and igg in a quantitative manner. our serological kits have overall good performance 326 our kits have much higher accuracy than rt-qpcr (sensitivity less than 70%) for detecting viral 327 rna 9-13 , and published immune-assays such as " flow immunoassay" and elisa in earlier studies 10-12, 328 17-21, 23, 24 . when we combined rbd-specific iga and igg kits together, the sensitivity, specificity and 329 overall agreement elevate to 99· 1%, 100%, and 99· 7%, respectively (table 1). in addition, this rbd-330 based detection kit may also help to screen and detect neutralization antibodies targeting sars-cov-2 331 rbd, because this peptide domain is exposed on viral surface and functions as a ligand binding to the 332 host cell surface receptor ace2. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 22, 2020. although one serum collected at the 4th day after illness onset was diagnosed as positive by our igm 336 kit (not iga or igg kits in this study), the igm kit overall showed a lower diagnostic specificity of 92· 3% 337 compared to that of igg and iga (figure 1). igm is known to have relatively lower affinity toward 338 antigens compared with that of igg or iga. in addition, igm often causes false positive signals as we 339 also observed (supplemental table 2), due to its pentameric structure 34 . to the contrary, iga or igg 340 antibody does not have this problem. our rbd-specific igg kit showed high specificity of 99· 8% (figure 341 1) but relatively low sensitivity of 96· 8%. this is expected, because that most (6/7) false negative cases 342 were samples collected at 4-10 days after illness onset when igg production is likely very low. our rbd-based iga kit showed high sensitivity and specificity of 98· 6% and 98· 1%, except two sera 344 collected at the 4th day after illness onset. all other sera (2 at the 6th day, 3 at the 7th day, 1 at the 8th we observed the presence of high-level of rbd-specific iga in covid-19 patients' sera. it is widely 368 believed that mucosal plasma cells are a major production source of iga, which is rapidly transported . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 22, 2020. . https://doi.org/10.1101/2020.04.17.20064907 doi: medrxiv preprint iga is traditionally recognized to play an anti-inflammatory role and prevent tissue damage at mucosal 375 sites. however, recent reports also demonstrated that serum iga is involved in the formation of immune 376 complexes to amplify inflammatory responses. 38 serum iga induced proinflammatory cytokine 377 production by macrophages, monocytes and kupffer cells in non-mucosal tissues including liver, skin 378 and peripheral blood. 39 in this study, we observed that iga was present in covid-19 patients' serum, 379 and its levels positively correlated with covid-19 severity ( figure 3a) . in our cohort, we also observed 380 that igg levels were associated with worse clinical outcomes (figure 3c), as previously described 10, 21 . the latter phenomena has been suggestive of possible antibody-dependent enhancement (ade) of these observations suggest that gut may be an important place for anti-viral response to coronaviruses, 394 and large amounts of secretory iga could be detected in these mucosal tissues in addition to that in blood. weakness of this study the current study at the present form has several limitations. we used 216 serum samples from 87 398 confirmed covid-19 patients in this study, and serum samples were not available every day for each 399 patient. the earliest collected serum is at the 4th day after self-reported illness onset, and the last one there were 17 severe and five critical cases, respectively, accounting for 19· 5% and 5· 75% respectively. there were also few cases of covid-19 patients whose symptoms remained mild and serum samples 407 were collected during hospitalization. therefore, this study of the correlation between antibody levels 408 and disease severity needs further verification. in summary, this study reports a novel sensitive and quantitative serological testing kit of detecting 410 iga as well as igm and igg, for the diagnostics of covid-19. due to its high specificity and sensitivity, 411 this kit could sensitively and quantitatively measure levels of iga in blood and other tissues. the serological study also provides valuable information for monitoring and understanding of covid-19. acknowledgements 415 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 22, 2020. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 22, 2020. 544 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april 22, 2020. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april 22, 2020. . https://doi.org/10.1101/2020.04.17.20064907 doi: medrxiv preprint coronavirus disease 2019 a novel coronavirus from patients with pneumonia in china covid-19 and the cardiovascular system a novel coronavirus associated with severe acute 442 respiratory syndrome isolation of a novel 444 coronavirus from a man with pneumonia in saudi arabia diabetes and covid-19 detection of 2019 novel coronavirus (2019-ncov) by real-447 time rt-pcr clinical features of patients infected with the laboratory diagnosis of covid-19 infection: current issues and challenges a preliminary study on serological assay for severe acute 457 respiratory syndrome coronavirus 2 (sars-cov-2) in 238 admitted hospital patients profiling early humoral response to diagnose novel coronavirus disease 460 (covid-19). clinical infectious diseases 2020 comparison of throat swabs and sputum specimens 462 for viral nucleic acid detection in 52 cases of novel coronavirus (sars-cov-2) infected pneumonia 463 (covid-19) detection of specific antibodies to severe acute respiratory 465 syndrome (sars) coronavirus nucleocapsid protein for serodiagnosis of sars coronavirus pneumonia structure and function of immunoglobulins role of natural and immune igm antibodies in immune responses development and clinical application of a rapid igm-igg combined 472 antibody test for sars-cov-2 infection diagnosis temporal profiles of viral load in posterior 477 oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an 478 observational cohort study. the lancet infectious diseases 2020 antibody responses to sars-cov-2 in covid-19 patients: the 481 perspective application of serological tests in clinical practice immune phenotyping based on neutrophil-to-lymphocyte ratio and 484 igg predicts disease severity and outcome for patients with covid-19 interaction of antigens and antibodies at mucosal surfaces. annual review of 487 microbiology sars-cov-2 specific antibody responses in covid-19 patients a serological assay to detect sars-cov-2 491 seroconversion in humans the use of solvent/detergent treatment in pathogen reduction of 493 use of ranks in one-criterion variance analysis key: cord-342557-a7q8vp8m authors: chowdhury, surid mohammad; talukder, shafi ahmad; khan, akib mahmud; afrin, nadia; ali, md ackas; islam, rajib; parves, rimon; al mamun, abdulla; sufian, md. abu; hossain, md nayeem; hossain, mohammed akhter; halim, mohammad a. title: antiviral peptides as promising therapeutics against sars-cov-2 date: 2020-10-23 journal: j phys chem b doi: 10.1021/acs.jpcb.0c05621 sha: doc_id: 342557 cord_uid: a7q8vp8m [image: see text] over 50 peptides, which were known to inhibit sars-cov-1, were computationally screened against the receptor-binding domain (rbd) of the spike protein of sars-cov-2. based on the binding affinity and interaction, 15 peptides were selected, which showed higher affinity compared to the α-helix of the human ace2 receptor. molecular dynamics simulation demonstrated that two peptides, s2p25 and s2p26, were the most promising candidates, which could potentially block the entry of sars-cov-2. tyr489 and tyr505 residues present in the “finger-like” projections of the rbd were found to be critical for peptide interaction. hydrogen bonding and hydrophobic interactions played important roles in prompting peptide–protein binding and interaction. structure–activity relationship indicated that peptides containing aromatic (tyr and phe), nonpolar (pro, gly, leu, and ala), and polar (asn, gln, and cys) residues were the most significant contributors. these findings can facilitate the rational design of selective peptide inhibitors targeting the spike protein of sars-cov-2. a new type of coronavirus was first detected in december 2019 at wuhan city, the capital of the hubei province of china. this virus is designated as severe acute respiratory syndrome-related coronavirus-2 (sars-cov-2). 1 the pneumonia-like disease caused by the virus is globally known as covid-19. apart from china, covid-19 has spread to 213 countries and killed over 441,000 people in total as of today (17 june, 2020) . with the case count and death toll rising each day, there is an urgent need for antiviral drugs or vaccines against sars-cov-2. the sars-cov-2 is a positive-sense single-stranded rna virus. it is a member of the same family belonging to sars-cov and middle east respiratory syndrome (mers-cov). the sars-cov-2 virion has a diameter of 50−200 nm. 2 like other coronaviruses, sars-cov-2 has four structural and many nonstructural proteins. the structural proteins are called spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins. s, e, and m proteins perform together to form the viral envelope. 3 the spike protein has a crown-like (corona) appearance. the spike (s) protein allows the virus to be attached into the host surface by interacting with human angiotensin-converting enzyme-2 (hace2) receptors present in the upper and lower respiratory system. 4, 5 hace2 receptors are expressed in many organs including the lung, small intestine, testis, and kidney. ace2, which acts as an exopeptidase, catalyzes the conversion of angiotensin i to angiotensin i−ix and angiotensin ii to angiotensin i−vii. 6−8 cryo-electron microscopy analysis has indicated that unlike other coronaviruses, the s protein of sars-cov-2 has 10−20 times greater affinity to the hace2 receptors, resulting in greater transmissibility than others. 9,10 upon performing sequence alignment and homology modeling, it is evident that the s protein of sars-cov and sars-cov-2 share 76% sequence identity. 10, 11 the s protein comprises s1 and s2 domains. the s1 domain is responsible for binding to ace2 receptors via its receptorbinding domain (rbd), whereas the s2 domain performs the fusion, enabling viral genome entry. 12 electron microscope imaging revealed that the s glycoprotein forms a clove-shaped spike with three s1 heads and a s2 trimeric stalk. 13 the rbd has greater variability. six amino acids (l455, f486, q493, s494, n501, and y505) in the rbd are extensively responsible for the efficient binding. 12 the s protein of sars-cov-2 is glycosylated containing 22 predicted n-glycosylation in the sequence, having one site less than the sars-cov-1 at n370. 13 the rbd of sars-cov-2 shares 72% sequence identity with that of sars-cov at the protein level. 13 the interaction between the s protein and the ace2 receptor is the critical route of entry for the virus. therefore, the s protein is a potential target for drug or vaccine development. small molecules or peptides can be designed as therapeutics that will disrupt the interaction between the s protein and the ace receptor; however, small molecules are not ideal for targeting large protein−protein interactions (ppis). peptides, on the other hand, can disrupt the ppis effectively as they possess a larger surface compared to small molecules and thus specifically bind to the interface-binding region. 14 in this context, a team from the massachusetts institute of technology (mit) developed 23-mer peptide against the spike protein. 15 a research group from the university of illinois at chicago designed four ace2-based peptide inhibitors of sars-cov-2. 2 while this early stage of peptide inhibitor development shows great promise, only few ace-2-based peptides were screened and proposed. in this work, we, therefore, computationally screened 51 antiviral peptides that were known to work against sars-cov-1, targeting the rbd of the s protein of sars-cov-2. peptides that showed higher s protein-binding affinity compared to the α-helix (ah) of the ace2 peptidase were further analyzed with molecular dynamics (md) simulation and the structure− activity relationship (sar) in order to achieve a high-affinity binder for the s protein. ■ methods molecular docking. a total of 51 peptides were selected from the antiviral peptide database avpdb, 16 which were experimentally verified to be effective against the sars-cov-1. all the peptides were modeled by the cabs-fold. 17 the crystal structure 6m0j of the rbd was retrieved from the rcsb protein data bank (pdb). 18 peptides were docked to the rbd using patchdock, 19 and initial 1000 peptide−rbd complexes obtained from patchdock were then refined by firedock. 20 peptides were further docked using cluspro 21 and haddock 2.4, 22 with an aim to reach a consensus score. peptides that exhibited better binding scores in all three docking modes were subsequently analyzed by md simulation. md simulations. a 150 ns md simulation was conducted for apo-rbd, ah-rbd, s2p1-rbd, s2p3-rbd, s2p25-rbd, s2p26-rbd, s2p28-rbd, and s2p30-rbd complexes to evaluate the peptide−protein conformational dynamics and interaction. md simulation was performed three times for each case. yasara dynamics software was used, and amber 14 force field was considered for all calculations. 23, 24 water molecules (0.998 g/cm 3 density) were added, and the system was neutralized by adding nacl salt at 0.9% concentration at 310 k temperature. the particle-mesh ewald method 25 was used for long-range electrostatic interaction calculation. a berendsen thermostat was used to control the simulation temperature. periodic boundary condition was employed for performing the simulation, and the cell size was 20 å larger than the protein−peptide complex in all cases. a simulated annealing method was used for the initial energy minimization process of each simulation system, using the steepest gradient approach (5000 cycles). a 1.25 fs time step was used for the overall simulations. finally, 150 ns md simulation was performed for each system, and the snapshots were saved at every 100 ps. bond distance, bond angle, dihedral angles, solvent-accessible surface area (sasa), coulombic and van der waals (vdw) interactions, root-mean-square-deviation (rmsd), root-mean-square-fluctuation (rmsf), and values for backbone, alpha carbon, and heavy atoms were analyzed from md simulation. md snapshots were collected to evaluate the interactions in peptide−protein complexes over 150 ns. a total of 150 md snapshots were selected for the binding free energy calculations by prodigy server, 26 which measures the free energy based on intermolecular contacts and properties derived from the noninterface surface. different multivariate energy factors were analyzed by employing the principle component analysis (pca) method to understand the structural and energetic changes of proteins in the presence of the peptide during md simulation. the structural and energy information including bond distances, bond angles, dihedral angles, planarity, vdw energies, and electrostatic energies were considered. pca analysis can disclose the hidden structural and energy profile among different groups. 27, 28 the last 50 ns of the md trajectory data for both apo-rbd and peptide−protein complexes were considered for pca analysis. data were preprocessed using centering and scaling prior to this analysis. the multivariate factors were arranged in the x matrix and reduced into a product of two new matrices by using the following equation. here, t k is the matrix of scores, which signifies the relation of samples with each other, p k is the matrix of loadings, carrying information about the relation of variables to each other, k is the number of factors into the model, and e is the unmodeled variance. for performing all the calculations, r 29,30 -based inhouse-developed codes were used. peptide sar analysis. peptide sar was performed considering the best 15 peptides. relevant peptide properties including acidic (a), basic (b), aromatic (ar), polar (p), nonpolar (np) amino acids, net charge at ph 7, molecular weight, and approximate volume (table s5 ) are calculated using the protparam tool. 30 initially, stepwise multiple linear regression (mlr) was performed considering these properties as variables to predict the calculated binding affinity of the test peptides with the rbd of the sars cov-2 spike protein. subsequently, pca was performed taking the five most important peptide properties to cluster the test peptides in a biplot to explore the structural variance. peptide-binding affinity and interaction. the amino acid sequence, length, and in vitro inhibition efficiency (these data are collected from the peptide database avpdb 16 ) against sars-cov-1 of 51 peptides are summarized in table s1 . all 51 peptides were docked to the rbd of the sars cov-2 spike protein using patchdock. the binding pockets of the rbd were specified during the molecular docking. the best 1000 peptide−protein complexes obtained from patchdock were submitted to firedock for subsequent refinement. only the complexes that showed the higher binding affinity and the expected binding interaction were chosen as the best candidates ( table 1 ). the ah of the ace2 peptidase domain (pd) is considered as a control peptide. the binding affinity of all peptides is tabulated in out of 51 peptides, 27 peptides showed satisfactory binding interaction when docked using cluspro2.0 (table s2) . moreover, strong binding affinity was also observed for s2p3 and s2p26, which agreed with firedock results. although s2p5, s2p10, s2p35, and s2p49 exhibited better affinity, they shifted away from the binding pocket. in haddock results, nine peptides displayed more favorable docking scores, namely, s2p1, s2p3, s2p9, s2p25, s2p26, s2p28, s2p30, s2p34, and s2p43. however, none of these peptides exceeded the control ah in terms of binding affinity (table s2) . various residues including glu484, tyr449, and tyr505 present in the ace2 binding site of the rbd were involved in noncovalent interaction with the antiviral peptides ( figure 1a) . notably, glu484 and tyr449 exhibited multiple interactions with several antiviral peptides, indicating that these residues might be crucial for attachment with peptides ( figure 1c) . other important residues that also interacted with the antiviral peptides are gln493, leu455, tyr453, and tyr489. hydrogen bonding played a crucial role in peptide−rbd interaction, contributing 53% of all interactions (figure 1b) . besides hydrogen bonding, hydrophobic interactions contributed to 42%, while electrostatic interactions were involved in only 5% of the total interactions. md simulation. md simulations of apo-rbd and complexes of ah, s2p1, s2p3, s2p25, s2p26, s2p28, and s2p30 were performed. s2p1 and s2p3 showed significant changes in rmsd (figure 2a) . when md snapshots were analyzed, it became clear that such changes in rmsd were not due to the change in protein conformation, rather it could be attributed to the movement of the peptide. both peptides, s2p1 and s2p3, were found to be deviated from their binding interface, although s2p1 was more deviated than s2p3. the ah-rbd complex remained stable over the simulation period, as indicated by its rmsd profile and respective snapshots (figure 3a ). although s2p25, s2p26, s2p28, and s2p30 complexes exhibited a slightly greater rmsd, their respective md snapshots (figure 3b ,c) revealed that these peptides occupied the binding interface and remained as stable complexes throughout the simulation period. the s2p28-rbd complex exhibited lower radius of gyration (rg) values compared to the ah-rbd complex, indicating that this peptide induced the compactness in the rbd upon binding (figure 2b ). overall, a trend of reduction in the sasa was detected for all complexes (figure 2c) . nonetheless, the most prominent downgrading in the sasa was observed in the case of s2p28-rbd complexes, thus confirming the induction of (figure 2d) . a high rmsf value illustrated the s2p1 displacement from its binding site. however, high fluctuations were observed in regions spanning residue numbers 474−487 and 517−521 in the rbd for all complexes. this is not unexpected because these regions correspond to loops which lack any definite geometry. binding free energies of ah-rbd, s2p25-rbd, s2p26-rbd, s2p28-rbd, and s2p30-rbd complexes were calculated, for which ah showed an average binding energy of −11.13 ± 0.03 kcal/ mol which was the highest compared to other peptides ( figure 4a ). the average binding affinities of s2p25 and s2p26 were found to be better than those of s2p28 and s2p30. overall, the journal of physical chemistry b pubs.acs.org/jpcb article md simulation suggests that s2p25 and s2p26 could be our potential candidates. a pca model including eight training sets (apo-rbd and seven peptide−rbd complexes) is generated to understand structural and energy changes in the peptide−protein complexes relative to apo-rbd during md simulation. here, the first two pcs explain 93.1% of variance, where pc1 explains 68% and pc2 explains 25.1% of variance. in the score plot of pc1 and pc2 (figure 4c ), apo-rbd shows a major rightward shift relative to all the peptide−protein complexes along pc1. this clustering pattern is usual because majority of the variables, that is, coulomb energy, angle, bond distance, and vdw energies (figure 4d ) have largely influenced the variance along pc1. the rbd-s2p28 complex is at the farthest interaction of s2p26, s2p25, and ah with the rbd obtained from md simulation. in the s2p26-rbd complex, tyr505 and tyr489 in the rbd exhibited remarkable interactions over the 150 ns simulation period (figure 5a ), whereas asn487, leu455, tyr473, gln493, and phe456 also interacted frequently. most notable residues found in the s2p26 peptide were pro7, tyr21, cys4, and tyr5 (figure 5b) . interaction between s2p26 and the rbd was elevated by both hydrogen bonding and hydrophobic interactions (figure 5c ). tyr505 in the rbd formed hydrophobic interaction and hydrogen bonds with gly20 and tyr21 in s2p26 (figure 5d ). however, tyr489 showed hydrophobic interaction only with tyr5 and pro7 in the s2p26 peptide. hydrophobic interaction (49%) and hydrogen bonding (47%) contributed to most of the interactions between s2p26 and the rbd. in the s2p25-rbd complex, ala475 and arg403 present in the rbd were involved in multiple interactions during the simulation period ( figure s1a ). other residues such as tyr489, tyr473, tyr505, and phe456 were also detected. in the s2p25 peptide, tyr29, cys10, his13, cys12, and phe1 residues were involved in such interactions ( figure s1b ). figure s1c illustrates that the interactions between s2p25 and the rbd were mostly governed by hydrogen bonds covering around 52% of the total interactions. arg403 in the rbd showed major interaction with glu18 in s2p25 through electrostatic and hydrogen bonding ( figure s1d ). in the ah-rbd complex, arg403 and lys417 residues in the rbd showed significant interactions during the 150 ns md simulation. the rbd residue arg403 interacted with the glu18 in ah through electrostatic and hydrogen bonding, whereas lys417 interacted with asp11 and his15 by electrostatic, hydrophobic, and hydrogen bonding ( figure s2 ). md simulation results suggested that the arg403 and lys417 in the rbd were essential for strong binding of ace2 with the rbd of the spike protein. besides these residues, asn501, gln493, tyr505, tyr489, gln498, gly496, and leu455 were involved in interaction with ah residues including glu18, his15, gln23, lys12, gln5, tyr22, and asp11 ( figure s2 ). overall, tyr489 and tyr505 residues in the rbd commonly participated in all stable peptide−protein complexes ( figure 4b ). these residues are present in the "finger-like" projections of the rbd (involved in ace2 receptor binding), which suggests that these projections and specifically these residues are crucial in peptide−rbd binding. structure−activity relationship. mlr analysis is executed with the most relevant peptide properties to sort out the significant predictors of the binding affinity of the test peptides (table s3) . aromatic, nonpolar, and polar residues are found to be the most significant predictors, which explains the observed dominance of hydrogen and hydrophobic interactions (95% in total) of the peptide−rbd complexes. in other words, tyrosine and polar residues stabilize the peptide−protein complexes by hydrogen bonding interactions, whereas nonpolar and other aromatic residues stabilize the complexes by hydrophobic interactions. this regression model also holds true in the peptide−rbd dynamic interactions in 150 ns md simulation as 10 high-frequency residues of the best-performing peptides (s2p25 and s2p26) in contact with the rbd are predominantly aromatic, nonpolar, or polar (table s4 ). in addition, the clustering behavior of the top 15 peptides based on the most significant five peptide properties are analyzed to get an insight into their structural variance. in the generated biplot, the clustering pattern of s2p1, s2p3, s2p25, s2p26, and ah replicated the energy score plot of their complexes with the rbd, except that s2p26 is close to ah ( figure 6 ). besides, nonpolar, polar, and aromatic residues play a significant role in the clustering pattern of the test peptides as nonpolar and polar residues are heavily loaded onto pc1 and the aromatic residue onto pc2, which altogether explains 76.34% structural variance. designing and developing high-affinity antiviral peptides represent a promising therapeutic strategy for covid-19 treatment. encompassing the extended protein contact interface, high-affinity antiviral peptides can strongly inhibit the rbd of the spike protein, thus blocking the sars-cov-2 from entering cells and subsequent replication. although ace2based peptide inhibitors are suggested, our results demonstrated that over 15 peptides can show better binding affinity than the ah of ace2. details from md simulation indicate that s2p25 and s2p26 could be the most promising antiviral peptide for sars-cov-2. some critical residues of both the rbd and peptides are also observed by analyzing the residuespecific contact maps of these peptides. sar reveals that by combing aromatic, polar, and nonpolar residues, one can further optimize these peptides to improve their binding affinity for the s protein. we anticipate that these peptides can serve as the next-generation antiviral therapeutics for the treatment of the covid-19 disease. in addition, these antiviral peptides can be conjugated to gold nanoparticles that are expected to act as potent nanoinhibitors enhancing the antiviral activity. our study provides valuable information for the rational design and development of peptide inhibitors against sars-cov-2 that can show high in vitro and in vivo efficacy. the supporting information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpcb.0c05621. sequence, length, inhibition efficiency, binding affinity, stepwise mlr analysis, interaction residues, and distribution of noncovalent interactions (pdf) coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 computational design of ace2-based peptide inhibitors of sars-cov-2 analysis of therapeutic targets for sars-cov-2 and discovery of potential drugs by computational methods the spike protein of sars-cov -a target for vaccine and therapeutic development cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor structure, function, and evolution of coronavirus spike proteins angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus expression and functional characterization rare driver mutations in head and neck squamous cell carcinomas converge on notch signaling genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding learning from the past: possible urgent prevention and treatment options for severe acute respiratory infections caused by 2019-ncov the proximal origin of sars-cov-2 structure analysis of the receptor binding of 2019-ncov future directions for peptide therapeutics development. drug discovery today the first-in-class peptide binder to the sars-cov-2 spike protein avpdb: a database of experimentally validated antiviral peptides targeting medically important viruses cabs-fold: server for the de novo and consensus-based prediction of protein structure structural basis of receptor recognition by sars-cov-2 patchdock and symmdock: servers for rigid and symmetric docking fast interaction refinement in molecular docking the cluspro web server for proteinprotein docking the haddock2.2 web server: user-friendly integrative modeling of biomolecular complexes making optimal use of empirical energy functions: force-field parameterization in crystal space ff14sb: improving the accuracy of protein side chain and backbone parameters from ff99sb particle mesh ewald: an n· log(n) method for ewald sums in large systems prodigy: a web server for predicting the binding affinity of protein−protein complexes a molecular modeling approach to identify effective antiviral phytochemicals against the main protease of sars-cov-2 prediction of deleterious non-synonymous snps of human stk11 gene by combining algorithms protein identification and analysis tools on the expasy server. the proteomics protocols handbook the authors declare no competing financial interest. we are grateful to our donors who supported to build a computational platform (http://grc-bd.org/donate/). the authors like to acknowledge the world academy of science (twas) to purchase the high-performance computer for performing md simulation. the journal of physical chemistry b pubs.acs.org/jpcb article key: cord-323514-jaom3p6s authors: he, yuxian; li, jingjing; jiang, shibo title: a single amino acid substitution (r441a) in the receptor-binding domain of sars coronavirus spike protein disrupts the antigenic structure and binding activity date: 2006-05-26 journal: biochemical and biophysical research communications doi: 10.1016/j.bbrc.2006.03.139 sha: doc_id: 323514 cord_uid: jaom3p6s abstract the spike (s) protein of severe acute respiratory syndrome coronavirus (sars-cov) has two major functions: interacting with the receptor to mediate virus entry and inducing protective immunity. coincidently, the receptor-binding domain (rbd, residues 318–510) of sar-cov s protein is a major antigenic site to induce neutralizing antibodies. here, we used rbd-fc, a fusion protein containing the rbd and human igg1 fc, as a model in the studies and found that a single amino acid substitution in the rbd (r441a) could abolish the immunogenicity of rbd to induce neutralizing antibodies in immunized mice and rabbits. with a panel of anti-rbd mabs as probes, we observed that r441a substitution was able to disrupt the majority of neutralizing epitopes in the rbd, suggesting that this residue is critical for the antigenic structure responsible for inducing protective immune responses. we also demonstrated that the rbd-fc bearing r441a mutation could not bind to soluble and cell-associated angiotensin-converting enzyme 2 (ace2), the functional receptor for sars-cov and failed to block s protein-mediated pseudovirus entry, indicating that this point mutation also disrupted the receptor-binding motif (rbm) in the rbd. taken together, these data provide direct evidence to show that a single amino acid residue at key position in the rbd can determine the major function of sars-cov s protein and imply for designing sars vaccines and therapeutics. the spike (s) protein of severe acute respiratory syndrome coronavirus (sars-cov), similar to those of other coronaviruses, is a large type i transmembrane glycoprotein, which is incorporated into the viral envelope and provides the virion with a corona-like appearance [1, 2] . unlike those of many other cornaviruses, the s protein of sars-cov may not be cleaved in the virus-producing cells [3, 4] ; however, two domains corresponding to the n-terminal s1 subunit and the c-terminal s2 subunit of processed coronaviruses can be defined by sequence alignment [5, 6] . the s1 subunit of coronavirus s protein forms the surface knob-like structure, whereas the s2 subunit is membraneanchored and forms the stem-like structure beneath the knob [7, 8] . the binding of coronavirus to its specific receptor on the target cell is an initial step of infection [7] [8] [9] . angiotensinconverting enzyme 2 (ace2) is a functional receptor for sars-cov [10] [11] [12] . the s protein of sars-cov can bind to ace2 with high affinity and mediates viral entry. a 193residue fragment within s1 domain (residues 318-510) has been defined as a minimal receptor-binding domain (rbd) (fig. 1) [4, 13, 14] . crystal structure of an independently folded rbd bound to human ace2 reveals that residues 424-494 constitute the receptor-binding motif (rbm) [15] . the s2 domain of sars-cov s protein contains a putative fusion peptide and two heptad repeat regions (hr1 and hr2), which can associate to form a six helix bundle comprised of three helices from hr1 that run antiparallel to three helices from hr2 [16] [17] [18] . the second major function of coronavirus s protein is its capacity to elicit neutralizing antibodies and sterilizing immunity, and is thereby considered as a critical immunogen for vaccine development [19, 20] . similarly, it has been shown that the s protein of sars-cov is a major protective antigen among four structural proteins [21] . several live virus and dna vaccines expressing the s protein have been tested in preclinical studies [22] [23] [24] . coincidently, the rbd of sars-cov s protein is a major target of neutralizing antibodies induced in patients infected with sars-cov and in animals immunized with inactivated viruses or s proteins [25] [26] [27] . we previously demonstrated that rbd-fc, a fusion protein containing the rbd linked to fc portion of human igg1, is a potent inducer of neutralizing antibodies and has potential to be developed as a subunit vaccine [28, 29] . several conformation-dependent neutralizing epitopes (conf i-vi) were identified in the rbd [29] . although the rbd is a major neutralizing domain of sars-cov, the s protein also contains neutralizing epitopes in other regions [30] [31] [32] . interestingly, it was recently reported that the major function of full-length s protein, i.e., mediating viral entry and inducing neutralizing antibodies, could be abolished by single amino acid substitutions in the rbd (e.g., r441a) [33] . in this study, we used the rbd-fc as a model molecule to further investigate the impact of r441a substitution on the immunogenicity and receptor-binding activity of independently folded rbd. our data are important for understanding the mechanism how a single amino acid residue determines the major function of sars-cov s protein. recombinant s proteins and mabs. the plasmid encoding the receptorbinding domain (residues 318-510) of sars-cov s protein tor2 (accession no. ay274119), fused with the fc portion of human igg1 (rbd-fc), was previously described [28, 29] . rbd-fc bearing r441a substitution (designated as rbd-r441a) was generated by mutagenesis using the quickchange xl kit (stratagene) and verified by dna sequencing. each of the recombinant fusion proteins was expressed in 293t cells transfected with the plasmid using fugene 6 reagents (boehringer mannheim, indianapolis, in) according to the manufacturer's protocol and purified by protein a-sepharose 4 fast flow (amersham biosciences, piscataway, nj). the full-length s protein (fl-s) of sars-cov urbani (accession no. ay278741) was expressed in expressf +ò insect cells with recombinant baculovirus d3252 by the protein sciences corporation (bridgeport, ct). a panel of 26 mabs specific for the rbd of sars-cov s protein was prepared in our laboratory, which include 14 mabs isolated from mice immunized with rbd-fc [29] , 6 mabs isolated from mice immunized with fl-s, and 6 mabs isolated from mice immunized with inactivated sars-cov. immunization of mice and rabbits. rbd-fc and its mutant rbd-r441a were, respectively, used to immunize mice and rabbits. four female balb/c mice (6 weeks old) per group were subcutaneously immunized with 20 lg of purified proteins re-suspended in pbs plus mlp + tdm adjuvant (sigma, saint louis, mi) and boosted with 10 lg of the same antigen plus the mlp + tdm adjuvant at 3-week intervals. four nzw rabbits (12 weeks old) per group were immunized intradermally with 150 lg purified proteins re-suspended in phosphate-buffered solution (pbs, ph 7.2) in the presence of freund's complete adjuvant (fca), and boosted three times with freshly prepared emulsion of 150 lg immunogen and freund's incomplete adjuvant (fia) at 3-week intervals. pre-immune sera were collected before starting the immunization and antisera were collected 7 days after each boost. sera were kept at 4°c before use. enzyme-linked immunosorbent assay. the reactivity of mouse and rabbit antisera or anti-rbd mabs with s proteins (rbd-fc, rbd-r441a or fl-s) was determined by enzyme-linked immunosorbent assay (elisa). briefly, 1 lg/ml recombinant protein was used to coat 96-well microtiter plates (corning costar, acton, ma) in 0.1 m carbonate buffer (ph 9.6) at 4°c overnight. after blocking with 2% non-fat milk, serially diluted antisera or mabs were added and incubated at 37°c for 1 h, followed by four washes with pbs containing 0.1% tween 20. bound antibodies were detected with hrp-conjugated goat anti-mouse igg or goat anti-rabbit igg (zymed) at 37°c for 1 h, followed by washes. the reaction was visualized by addition of the substrate 3,3 0 ,5,5 0 -tetramethylbenzidine (tmb) and absorbance at 450 nm was measured by an elisa plate reader (tecan us, research triangle park, nc). generation of sars pseudovirus and neutralization assay. sars-cov pseudovirus system was developed in our laboratory as previously described [27, 28] . in brief, hek293t cells were co-transfected with a plasmid encoding the s protein corresponding to sars-cov tor2 isolate and a plasmid encoding env-defective, luciferase-expressing hiv-1 genome (pnl4-3.luc.re) by using fugene 6 reagents (boehringer mannheim). supernatants containing sars pseudovirus were harvested 48 h post-transfection and used for single-cycle infection of human ace2transfected 293t (293t/ace2) cells. briefly, 293t/ace2 cells were plated at 10 4 cells/well in 96-well tissue-culture plates and grown overnight. the supernatants containing pseudovirus were pre-incubated with serially diluted mouse or rabbit antisera at 37°c for 1 h before addition to cells. the culture was re-fed with fresh medium 24 h later and incubated for an additional 48 h. cells were washed with pbs and lysed using lysis reagent included in a luciferase kit (promega, madison, wi). aliquots of cell lysates were transferred to 96-well costar flat-bottomed luminometer plates (corning costar, corning, ny), followed by addition of luciferase substrate (promega). relative light units (rlu) were determined immediately in the ultra 384 luminometer (tecan us). receptor-binding assays. binding of rbd-fc or rbd-r441a protein to soluble ace2 was measured by elisa. briefly, recombinant soluble ace2 (r&d systems, inc., minneapolis, mn) at 2 lg/ml was coated onto 96-well elisa plates (corning costar) in 0.1 m carbonate buffer (ph 9.6) at 4°c overnight. after blocking with 2% non-fat milk, serially diluted rbd-fc or r441a was added to the wells and incubated at 37°c for 1 h. after washing, the hrp-conjugated goat anti-human igg (zymed) was added and incubated an additional 1 h. after washing, the substrate tmb was used for detection. binding of rbd-fc or rbd-r441a to ace2expressing cells was measured by flow cytometry. briefly, 10 amino acid residue 441 (arginine) is essential for the immunogenicity of rbd to induce neutralizing antibodies it was recently reported that the full-length s protein of sars-cov bearing r441a substitution failed to induce neutralizing antibodies against s protein-pseudotyped viruses [33] . we are interested in investigating whether this point mutation can also affect the immunogenicity of rbd-fc, a potent inducer of neutralizing antibodies [28, 29] . the rbd-r441a mutant was generated by mutagenesis as described. both wild-type rbd-fc and rbd-r441a were expressed in 293t cells and purified to homogenicity by protein a chromatography (fig. 1c) . for comparison, both the wild-type and mutant proteins were, respectively, used to immunize mice and rabbits. as shown in fig. 2 , both mice and rabbits developed robust antibody responses against the corresponding immunogens after the third boosting immunizations. relatively, rbd-fc induced higher titers of antibodies in both mice (mean end-point titer was 1/656,100) and rabbits (mean end-point titer was 1/5,904,900), whereas rbd-r441a induced antibodies with mean end-point titers at 1/145,800 in mice and at 1/328,050 in rabbits. we used a recombinant full-length s protein (fl-s) as a coating antigen to measure the titers of antibodies specific for the rbd in the antisera collected after the third boost (fig. 3) . surprisingly, while the rbd-fc could induce high titers of rbd-specific antibodies in mice and rabbits (mean endpoint titers were 1/656,100 and 1/437,400, respectively), the r441a mutant only induced rbd-specific antibody titers at 1/2700 in mice and 1/1800 in rabbits, respectively. we then tested whether mouse and rabbit antisera had neutralizing activities against s protein-mediated viral entry by using sars pseudovirus. consistent with our previous findings [28, 29] , both mouse and rabbit antibodies induced by rbd-fc could potently neutralize sars pseudovirus with mean 50% neutralizing titers at 1/58,320 and 1/19,440, respectively (fig. 4) . however, the antibodies induced by rbd-r441a mutant in both mice and rabbits could not significantly inhibit sars pseudovirus at 1/40 or higher dilutions. these results indicate that r441a substitution severely impairs the immunogenicity of rbd-fc to elicit rbd-specific neutralizing antibodies in immunized animals. we previously identified six groups of conformationdependent neutralizing epitopes (conf i-vi) with a panel of 27 anti-rbd mabs isolated from the mice immunized with rbd-fc [29] . we have recently isolated a set of novel anti-rbd mabs from the mice immunized with inactivated sars-cov or recombinant full-length s protein, and their epitopes have been initially grouped by binding competition (designated as group a-c and group a-c, respectively) (fig. 5) . consistently, these novel anti-rbd mabs possess potent neutralizing activity, but they may target different epitopes as shown by their unique specificity to block receptor binding (data not shown). to probe antigenic structure in the rbd bearing r441a mutation, the representative anti-rbd mabs from each epitopic groups were used in elisa to measure their reactivity with both wild-type and mutant proteins. as shown in fig. 5 , all anti-rbd mabs strongly reacted with rbd-fc, but only three conformation-dependent mabs (24f4, 33g4, and s25) were able to recognize the rbd-r441. we have known that the epitope for s25 (group d) differs from those of the conf v mabs (24f4 and 33g4) as shown by their binding competition and their capacity to block receptor binding (data not shown). it was of interest to note that the reactivity of one mab (4d5) targeting the mouse antisera (1/dilution) linear epitope (residues 435-451) was significantly reduced by r441a substitution, while the mab 17h9 targeting the residues 458-465 reacted with both proteins equally. these results suggest that r441a substitution can disrupt the majority of conformation-dependent neutralizing epitopes in the rbd, and that the conf v and group d epitopes are relatively stable and conserved. to further characterize the antigenicity of rbd-r441a mutant, we compared it with wild-type rbd-fc for the reactivity with the mouse antisera induced against the fl-s by elisa. as expected, the rbd-fc reacted strongly with the mouse anti-s sera (mean end-point titer at 1/218,700), comparable with the ff-s protein (1/328,050) (fig. 6) . however, rbd-r441a mutant did not significantly react with mouse anti-fl-s sera, confirming that r441a mutation disrupts the antigenic structure in the rbd. the s protein of sars-cov is responsible for binding with the receptor and mediates viral entry into target cell [7] . however, pseudovirus expressing the full-length s protein with r441a substitution completely loses infectivity [33] . we hypothesized that the substitution of r441 might disrupt the receptor-binding motif in the rbd and thereby the pseudovirus was unable to bind to the receptor. first, we used an elisa-based assay to compare the binding activities of both rbd-fc and rbd-r441a to soluble ace2. as shown in fig. 7 , rbd-fc could bind to the soluble ace2 in a dose-dependent manner, whereas rbd-r441a had no binding activity at a concentration up to 10 lg/ml, at which the wild-type rbd-fc reached a reactive plateau. we then measured whether rbd-fc and rbd-r441a bind to cell-associated ace2 by flow cytometry. consistent with our previous report, rbd-fc bound to ace2-expressing 293t cells efficiently; however, the rbd-r441a could not bind to ace2 expressed on the cells (fig. 8) . rbd-fc itself had inhibitory activity against pseudovirus entry, suggesting its potential application as an antiviral therapeutic [14] . in parallel, we tested the inhibitory activity of both rbd-fc and rbd-r441a on sars pseudovirus. as expected, rbd-fc was able to inhibit sars pseudovirus infection with an ic 50 of 1.12 lg/ml rabbit antisera (1/dilution) ( fig. 9) , whereas the rbd-r441a had no inhibitory effect at a concentration as high as 100 lg/ml, suggesting that the rbd mutant could not compete with the virion to bind to the receptor. taken together, these data suggest that r441a substitution may damage the receptor-binding motif in the rbd. sars-cov emerged in the winter of 2002-2003 and killed approximately 800 people, 10% of those infected [1, 2, [34] [35] [36] . although there are no recent sars outbreaks, the need to develop effective vaccines remains of high importance to prevent future epidemic caused by the sars-cov, which may re-emerge from animal reservoirs [37] [38] [39] [40] [41] . to this end, structural and functional characterization of sars-cov is one of the highest priorities. yi et al. [33] recently reported that single amino acid substitutions in the sars-cov s protein determine the viral entry and immunogenicity to induce neutralizing antibodies, but the mechanisms that caused these phenotypes remain to be elucidated. in this study, we used the rbd-fc as a model to study how a single residue mutation in the rbd can abolish the major function of full-length s protein, since this molecule can efficiently bind to the receptor ace2 and contains multiple conformation-dependent epitopes (conf i-vi) capable of inducing highly potent neutralizing antibodies [29] . we converted arginine 441 to alanine (r441a), which was shown to disrupt the immunogenicity of full length to induce neutralizing antibodies and s protein-mediated viral entry [33] , and evaluated its effect on the antigenic structure and binding function of the rbd. first, we found that r441a substitution could completely abolish the ability of rbd to induce neutralizing antibodies in the immunized mice and rabbits. we then probed the antigenic epitopes in the rbd bearing r441a by a panel of anti-rbd mabs recognizing different epitopes in the rbd and found that this mutation could disrupt the major neutralizing epitopes. these results provide direct evidence to explain why rbd-r441a mutant could not induce neutralizing antibodies. although conf v and group d neutralizing epitopes in the rbd-r441a retained partial reactivity with the corresponding mabs, they failed to elicit functional antibodies in either mice or rabbits. these data indicate that the residue r441 is essential for maintaining the antigenic structure in the rbd, which confers the immunogenicity to induce neutralizing antibodies. it was understandable that a single residue change in the rbd (e.g., r441a) could abolish its ability to induce functional antibodies through disrupting its major conformation-dependent neutralizing epitopes, but the mechanism how r441a substitution was able to determine the immunogenicity of full-length s protein is poorly understood [15] . although the rbd of sars-cov is a major target of neutralizing antibodies, the s protein also contains some neutralizing epitopes in the other domains. for example, it was reported that the linear epitopes in the hr2 region of the s2 subunit, which is far away from the rbd, could induce antibodies with moderate neutralizing activity [30] . we have recently found that the n-terminal region (residues 13-327) of s protein also contains neutralizing epitopes (data not shown). therefore, further structural characterization on the s protein may provide important information for understanding why a single point mutation in rbd affects the immunogenicity of the entire s protein. we subsequently documented that r441a mutation was able to completely abolish rbd-mediated binding activity to the receptor ace2. the rbd-r441a molecule could not bind to either soluble or cell-associated ace2 as shown by elisa and flow cytometry-based assays, respectively. moreover, the rbd with r441a substitution also lost its inhibitory ability against viral entry. therefore, our data indicate that the residue r441 is not only essential for the antigenic structure in the rbd, but also critical for the receptor-binding motif (rbm). crystal structure of the rbd in complex human ace2 reveals that only a few of the many contacting residues in the large interface between the s protein and receptor determine the efficiency of virus binding and infection [29] . the ace2 is bound by an extended loop in the s protein that projects from a compact core formed by residues 424-494, the rbm. in particular, a methyl group from a threonine residue at position 487 of the s protein at the interface extends into a hydrophobic pocket in ace2 that contains a lysine residue at position 353. although the r441 is not one of 14 residues on the loop that contact 18 residues on human ace2 [29] , its substitution might change the configuration of rbm and thereby disrupts interaction of the residues between rbm and ace2. subsequently, the conformational change of the rbd might result in a dramatic alteration in its antigenic structure. therefore, retaining the critical residues and proper antigenic conformations in rbd is important for developing sars vaccines. the sites in rbd containing critical residues, e.g., r441, can be used as targets for rational design of therapeutics. characterization of a novel coronavirus associated with severe acute respiratory syndrome retroviruses pseudotyped with the severe acute respiratory syndrome coronavirus spike protein efficiently infect cells expressing angiotensin-converting enzyme 2 the sars-cov s glycoprotein: expression and functional characterization coronavirus spike proteins in viral entry and pathogenesis molecular modelling of s1 and s2 subunits of sars coronavirus spike glycoprotein cellular entry of the sars coronavirus sars-associated coronavirus coronavirus spike proteins in viral entry and pathogenesis angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus a model of the ace2 structure and function as a sars-cov receptor expression cloning of functional receptor used by sars coronavirus amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor a 193-amino acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme 2 structure of sars coronavirus spike receptor-binding domain complexed with receptor structural characterization of the fusion-active complex of severe acute respiratory syndrome (sars) coronavirus interaction between heptad repeat 1 and 2 regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors structural characterization of the sars-coronavirus spike s fusion protein core severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus coronavirus immunogens contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice mucosal immunisation of african green monkeys (cercopithecus aethiops) with an attenuated parainfluenza virus expressing the sars coronavirus spike protein for the prevention of sars a dna vaccine induces sars coronavirus neutralization and protective immunity in mice recombinant modified vaccinia virus ankara expressing the spike glycoprotein of severe acute respiratory syndrome coronavirus induces protective neutralizing antibodies primarily targeting the receptor binding region inactivated sars-cov vaccine elicits high titers of spike protein-specific antibodies that block receptor binding and virus entry identification of a critical neutralization determinant of severe acute respiratory syndrome (sars)-associated coronavirus: importance for designing sars vaccines receptor-binding domain of sars-cov spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine receptor-binding domain of sars coronavirus spike protein contains multiple conformational-dependant epitopes that induce highly potent neutralizing antibodies monoclonal antibodies targeting the hr2 domain and the region immediately upstream of the hr2 of the s protein neutralize in vitro infection of severe acute respiratory syndrome coronavirus b-cell responses in patients who have recovered from severe acute respiratory syndrome target a dominant site in the s2 domain of the surface spike glycoprotein an exposed domain in the severe acute respiratory syndrome coronavirus spike protein induces neutralizing antibodies single amino acid substitutions in the severe acute respiratory syndrome coronavirus spike glycoprotein determine viral entry and immunogenicity of a major neutralizing domain identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome virus detectives seek source of sars in china's wild animals isolation and characterization of viruses related to the sars coronavirus from animals in southern china cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human bats are natural reservoirs of sars-like coronaviruses severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats key: cord-309898-sju15hev authors: hu, yiwen; buehler, markus j. title: comparative analysis of nanomechanical features of coronavirus spike proteins and correlation with lethality and infection rate date: 2020-11-02 journal: matter doi: 10.1016/j.matt.2020.10.032 sha: doc_id: 309898 cord_uid: sju15hev the novel coronavirus disease, covid-19, has spread rapidly around the world. its causative virus, sars-cov-2, enters human cells through the physical interaction between the receptor-binding domain (rbd) of its spike protein and the human cell receptor ace2. here, we provide a novel way in understanding coronavirus spike proteins, connecting their nanomechanical features – specifically its vibrational spectrum and quantitative measures of mobility – with virus lethality and infection rate. the key result of our work is that both, the overall flexibility of upward rbd and the mobility ratio of rbds in different conformations, represent two significant factors that show a positive scaling with virus lethality and an inverse correlation with the infection rate. our analysis shows that epidemiological virus properties can be linked directly to pure nanomechanical, vibrational aspects, offering an alternative way of screening new viruses and mutations, and potentially exploring novel ways to prevent infections from occurring. the novel coronavirus disease, covid-19, has spread rapidly around the world [1] [2] [3] [4] [5] . its causative virus, sars-cov-2, enters human cells through the interaction between the receptor-binding domain (rbd) of its spike protein and the cell receptor ace2 [6] [7] [8] . due to the significant role that coronavirus spike protein plays in receptor recognition, viral fusion and cell entry, it is a promising target for drug and vaccine development. here, we provide a novel way towards better understanding the coronavirus spike proteins, connecting its nanomechanical features -especially their vibrational patterns -with virus lethality and infection rate 6 . in a broader context, the mechanics of proteins has long been a subject of interest, and this study shows how it can be a useful tool to help us understand complex disease etiology by connecting nanoscopic physical features with epidemiological data [9] [10] [11] [12] [13] [14] [15] . to provide a comparative analysis -specifically focused on how nano-level features relate with macroscopic epidemiolocal observables -we focus on different coronavirus types within the same family of pathogens. over the past decades, several types of coronaviruses have emerged. the virus types hcov-nl63 and hcov-hku1 are often reported to cause lower respiratory tract infections, while hcov-oc43 and hcov-229e are usually associated with comparatively mild symptoms similar to the common cold 5, 16, 17 . the ones that threaten public health more seriously are three highly pathogenic human coronaviruses -namely: sars-cov, mers-cov and sars-cov-2. sars-cov was first reported in china in november 2002, then quickly spread globally, resulting in over 8,000 infections with about 800 deaths 18 . mers-cov was first identified in saudi arabia in june 2012, featuring limited transmission with case fatality rate as high as 35% 19, 20 . sars-cov-2 was first reported in china in december 2019 3, 21, 22 . it can easily transmit from human to human, resulting in more than 40 million global cases as of october 2020 23, 24 . the spike protein of the coronavirus plays an essential role in receptor recognition, viral fusion and cell entry 6, 25, 26 . the process represents a complex mechano-chemical process, whereby during the entry into the host cell, the spike protein first binds to a cell receptor through the receptor-binding domain (rbd) and then begins the fusion process. it is believed that the rbds of different coronaviruses recognize different cell receptors 3, 27 . sars-cov, sars-cov-2 and hcov-nl63 recognize angiotensinconverting enzyme 2 (ace2) as their receptor in the human body, while mers-cov recognizes dipeptidyl peptidase 4 (dpp4) as its receptor 1, 7, 8, 28 . in order to successfully bind to the receptor, the spike protein of a specific coronavirus must maintain a receptor-accessible state with at least one rbd in upward conformation. this is because otherwise there would be steric clashes hindering the binding process 4 . in experimental work, this specific type of receptor-accessible state has been captured for mers-cov, sars-cov and sars-cov-2 6, 29 . while the structure of coronavirus spike protein is well studied, much less attention has been focused on the connection between the mechanical features of the virus with virus lethality as well as infection rate. the structural basis of sars-cov viral infectivity has been explored to some extent, pointing out that the trp-rich region of s protein is essential 30 . it has also been observed that cleavage of the spike protein of sars-cov is associated with viral infectivity 31 . however, there has been no lateral comparative study between similar coronaviruses on this type of connection. it remains a question which kind of mechanical and structural properties could possibly relate to the mortality and infection rate of the virus. if successful, this approach may provide an alternative or complementary way to screen viruses or mutations against large-scale epidemiological data, provide additional mechanistic insights into disease etiology, and offer potential targets for therapies or preventive measures. in protein science, normal mode analysis (nma) has long been one of the most comprehensive yet efficient methods to calculate vibrational normal modes and analyze protein flexibility, which provides the rationale for use in this study 32 . another reason behind the broad use of nma is that the lowfrequency modes elucidated by nma could often describe the real-world motions of a protein, and often bear important functional significance 33 . in nma, the atoms are modeled as point masses, which are connected by springs that represents the interatomic force fields. after constructing the hessian matrix based on the second-order partial derivatives of the potential energy function, the normal modes and corresponding frequencies can be directly obtained by diagonalizing the matrix and computing its eigenvalues 34 (further details see experimental procedures). sars-cov-2 enters human cells through the interaction between the receptor-binding domain (rbd) of its spike protein and the cell receptor ace2, as is depicted in the schematic shown in figure 1 . our study hence focuses on the spike protein, which is essential for the infection to take place. the spike protein of coronavirus is composed of an amino (n)-terminal s1 subunit and a carboxyl (c)-terminal s2 subunit. the s1 subunit, which consists of an n-terminal domain (ntd) and three c-terminal domains (ctd), is responsible for recognizing and binding to the host cell receptor. it has been reported that for the betacoronavirus that utilizes ctd1 of its s1 subunit as rbd, there is a prerequisite conformational state for receptor binding where at least one rbd should be upward 4, 6 . in this paper, we refer to this receptoraccessible state as "open state" and receptor-inaccessible state as "closed state". as outlined in figure 1 , we conduct a normal mode analysis (nma) 33 , our open-state analysis is limited to these three highly pathogenic beta-coronaviruses. for closed-state exploration, since there have been no reports about closed conformational state of the mers-cov spike protein, we choose the hcov-nl63 spike protein because it shares the same relevant cell receptor ace2 with sars-cov and sars-cov-2 spike proteins. figure 2 depicts data that shows that the lowest-frequency normal modes of mers-cov, sars-cov and sars-cov-2 spike proteins are all associated with a swing motion of upward receptor-binding domain (rbd) to different extents. this type of global movements corresponds well with the molecular motions directly reported in experiments 4, 6 . considering the required receptor-accessible state for receptor binding, the swing of rbd is of functional significance since it is the likely way by which a spike protein changes from closed state to open state to facilitate the binding of target receptors. from a lateral aspect, this observed shared type of lowest-frequency normal mode movements indicates the structural similarity of mers-cov, sars-cov and sars-cov-2 spike proteins. according to our analysis, while the sequence identity of sars-cov s and sars-cov-2 s is as high as 78.8%, mers-cov s has only about 30% sequence identity with sars-cov s and sars-cov-2 s. this indicates that a small portion of the whole sequence of beta-coronavirus would largely determine the general structure topology and thus the overall global motion (details see supplementary material figure s1 ). figure s1 depicts a sequence alignment of mers-cov s, sars-cov s and sars-cov-2 s, where identical residues are denoted by *. the analysis reported in 36 reveals that the percentage identity in ntd and rbd, which reflect the major parts participating in the lowest normal mode movement, is about 22%. compared with 30% sequence identity of the whole spike protein, this partial percentage identity is lower and confirms the concept that likely only a few sequence pieces ultimately determine the shared global movement of coronavirus spike protein in open state. we further note that the sequence similarity in the s2 subunit could play an important role, as it may contributes to the comparatively higher rigidness of the s2 subunit. while mers-cov, sars-cov and sars-cov-2 spike proteins share the same type of lowestfrequency normal mode movements, their fluctuation profiles differ dramatically, as is illustrated by figure 3 . generally speaking, the s1 subunit of coronavirus spike protein is much more flexible than its s2 subunit. among these beta-coronaviruses, the active rbd of mers-cov spike enjoys super mobility and only sars-cov-2 spike protein and its d614g mutant exhibit a comparatively flexible downward rbd. interestingly, when we focus on fluctuations over the upward rbd, the figure reveals that for all these spike proteins the fluctuations first slowly build up to its maximum and then decrease sharply, which demonstrates that the appearance of large flexibility of upward rbd is based on some common detailed structures of these beta-coronaviruses. notably, panel (d) of figure 3 shows that the d614g mutation decreases the general flexibility of upward rbd and significantly enhances the mobility of a limited region in one downward rbd in the spike protein. it is also shown that the d614g mutation results in a general slight flexibility decrease in the ntds of all three chains, and importantly, no noticeable differences in other areas. using the fluctuation profile data, two significant mechanical factors are identified, which are: (1) the overall flexibility of upward rbd and (2) the mobility ratio of rbds in different conformations. figure 4 provides a correlation diagram for mers-cov, sars-cov and sars-cov-2 spike protein, where the overall flexibility of upward rbd is evaluated by the average fluctuation of open-state rbd and the mobility ratio is quantified as the ratio of maximum fluctuations over upward and downward rbds. the data shows that both factors have positive correlation with case fatality rate and inverse relationship with the virus infectivity. we find that for the flexibility ratio, the smaller it is, the larger the mobility of downward rbd is compared to upward one, which could indicate a larger possibility toward a second standing rbd. this would make it even easier for the spike protein to bind to the host cell receptor and thereby increasing the virus infectivity. on the other hand, if the flexibility of downward rbd is not large enough to generate conformational change, as flexibility ratio decreases, it becomes more difficult for the j o u r n a l p r e -p r o o f receptor to bind with the right rbd since the downward one is quite active. this may provide an explanation for the positive correlation between flexibility ratio and virus lethality that we see in the epidemiological data. one possible reason for the positive relationship between overall flexibility of upward rbd and the mortality rate could be that the flexible upward rbd is more active when binding to the receptor, and may hence benefit the subsequent membrane fusion process. even though there is limited available empirical data at this point, there is actually an intrinsic negative relationship between the mortality rate and infection rate of mers-cov, sars-cov and sars-cov-2, which could help explain why the infectivity is inversely correlated with the general flexibility of upward rbd. while there are many other factors situated between nanomechanical and epidemiological aspects, such as binding affinities 37 and dysregulation of type i interferon responses 38 , the influence of which on different epidemiological characteristics of coronavirus has not been fully explained, and our analysis points out the direct correlation between nanomechanical features and the lethality and infection rate of coronavirus. the goal is to attempt to improve our understanding of the direct relationship between the nanoscale and epidemiological level, not considering internal relationships. as the results show, this perspective provides useful insights into the mechanics of disease relationships ( figure s2 ). figure 5 show different flexibility variations in sars-cov-2, sars-cov and hcov-nl63 spike protein, which share the same human receptor ace2. among them, there exists a sharp increase and large variation in flexibility in rbd of sars-cov-2 s, while sars-cov s appears to have more flexible structural regions. hcov-nl63, the only one in this comparative study that is classified as alpha-coronavirus and cause only mild symptoms, has a different s1 subunit topology, bringing more flexibility to ntd rather than ctd1, where its rbd is situated. thus, our suggestion about the importance of the general flexibility of upward rbd in open-state analysis needs to be expanded. as for closed states, the overall flexibility of rbd in a single protomer shows positive relationship with virus lethality, since the disease severity is regressive in order of sars-cov, sars-cov-2 and hcov-nl63. notably, for these three coronaviruses in closed states, the overall flexibility of rbd of a single protomer is positively associated with their disease severity. panels (e) to (g) in figure 5 provide detailed structures of the rbd of the above virus complexed with their shared receptor human ace2. the ctd1 in s1 subunit of coronavirus often contains a core structure, which is composed of several antiparallel βsheet and short connecting α-helices, and one or more extended loops. theses extended loops, referred to as receptor binding motif (rbm), is located at the edge of the core structure and is usually responsible for realizing the interactions with the receptor if the virus uses ctd1 as its rbd. beta-coronaviruses such as sars-cov and sars-cov-2 have a unique long-extended loop as their rbm, accounting for the most flexible region of their rbd in both open and closed states. as for hcov-nl63, there are three separated short rbms, which are quite restricted and unable to generated large mobility. these insights may also explain its lower-affinity interaction with ace2, at least to some extent. we reported an analysis linking key nanomechanical vibrational features of various coronavirus spike proteins with epidemiological data. as shown in figure 2 , structural similarity and major movement associated with the swing of upward rbd is seen throughout this family of viruses, representing a sort of universal feature of this class of pathogens. the molecular modeling results show that the general motion corresponds with experimental observation and have functional importance. we find that the active rbd of mers-cov enjoys super mobility, whereas only sars-cov-2 and its d614g variant show a comparatively flexible downward rbd. the more recently occurring d614g mutation decreases the general flexibility of upward rbd and largely enhances the mobility of some small region in one downward rbd in the spike protein. as shown in figure 4 , the key result of this study, the general fluctuation profiles of upward rbd and the associated fluctuation ratio have a positive correlation with case fatality rate and inverse relationship with the virus infectivity. our results offer two different explanations for the effects of the flexible downward rbd: (1), the possibility towards a second standing rbd if the mobility is large enough, and (2) that it could indicate difficulty for the receptor to bind with the right rbd if no conformational change happens. we hypothesize that there may be a possible threshold between these two effects, which could be studied in future research. these insights offer several possible applications, including a search for inhibitors that could bind to downward rbd may provide a viable strategy. we find a sharp increase and large variation in flexibility in sars-cov-2 s, whereas more flexible structural regions are present in the closed-state sars-cov s. the long-extended loop is unique for beta-coronaviruses, and also accounts for the most flexible region in open states. hcov-nl63 s has three separated short rbms, which are unable to generate large mobility. this may also explain its lower-affinity interaction with ace2. as for possible applications, we may target the rbm to identify new inhibitors that lock the closed s-protein conformation. this opens a question whether perhaps, we can we utilize the significant flexibility difference for potential inhibitor screening for the development of novel treatment methods or design future experiments for gain-offunction research 39, 40 . future work may address additional influences of temperature dependence (to explore whether seasonal variations of temperature and other environmental factors can be linked to nanoscopic phenomena). other aspects may include a more detailed analysis of intermediate steps in the mechanistic hierarchical progression as schematically outlined in figure s2 , including aspects of the strong age-dependence of covid-19 disease progression. please contact prof. markus buehler via mbuehler@mit.edu. no new materials were generated in this work. all data are available upon request to the lead contact author. to assess the molecular mechanics from an atom-by-atom perspective, we conduct normal mode analyses (nma) of coronavirus spike proteins in receptor-accessible state with one upward rbd as well as receptor-inaccessible state where all three rbds are in downward position. we access the desired threedimensional protein structures from the protein data bank 41 and prepare the atomistic models with visual molecular dynamics (vmd) 2, 42 . before normal mode analysis, 10,000 steps of conjugate gradient energy minimization are performed using nanoscale molecular dynamics (namd) 43 in order to relax the protein structure 44 . no further md simulation is performed since the protein structure from protein data bank is already experimentally equilibrated. a coarse-grained elastic network model (enm) available in the bio3d package in r is employed to analyze the normal modes of coronavirus spike protein structures [45] [46] [47] . this model uses n, ca, c atoms to represent the protein backbone and selects 0 to 2 significant side chains based on their size and distance to ca atoms, proved to have comparable accuracy with all-atom enm. here, the atomic displacements are scaled for temperature 300k. for the sars-cov-2 d614g mutant, we implement the mutation to the open-state spike protein (pdb id: 6vsb) and carry out 10,000 steps of energy minimization and md simulation for 50 ns with namd. we then compute the average residue mean square derivations (rmsd) based on the last 25 ns equilibrium period and pick out 10 frames with the nearest rmsd from the trajectory file so that we could conduct normal mode analysis on them. the fluctuation profile of d614g mutant is calculated as the average of the normal mode fluctuations of these 10 configurations. we notice that some local unfolding occurs at the end of each chain, which induces abnormally high fluctuations in the fluctuation profile. since these local events are far from the rbd, we do not consider them in the analysis and set the fluctuations of the terminal 5-8 residues to zero. for consistency, the same approach is used for the fluctuation difference of sars-cov-2 spike protein and the d614g mutant. the global confirmed case numbers and the case fatality rate presented in figure 4 are updated as of sep 1, 2020. according to the analysis based on gisaid sars-cov-2 sequence database, by aug 31, 2020, 80% of the global sequence database were identified with d614g mutation (71242 sequences counted in total) 48, 49 . based on this estimate we use 80% of the covid-19 global case number as the infection number of sars-cov-2 with g614 and the remaining 20% as the case number of the original sars-cov-2 virus type. since there has been little evidence assessing an association between d614g mutation and disease severity, the same case fatality rate is applied to the original sars-cov-2 spike protein and its d614g variant. the two factors depicted in figure the causative virus of the covid-19, sars-cov-2, enters human cells through the physical interaction between the receptor-binding domain (rbd) of its spike protein and the human cell receptor ace2. while the structure of coronavirus spike protein is well studied, it remains unclear how those mechanical features of the virus affect its epidemiological characteristics. here, we report that both, the overall flexibility of upward rbd and the mobility ratio of rbds in different conformations, represent two significant factors that show a positive scaling with virus lethality and an inverse correlation with the infection rate. our analysis shows that epidemiological virus properties can potentially be linked directly to pure nanomechanical, vibrational aspects, offering an alternative way of screening new viruses and mutations, and perhaps even novel ways to prevent infections from occurring. • this work provides a novel way in understanding coronavirus pathology using mechanics • reports major movement associated with the swing of upward rbd for open-state viruses • links key nanomechanical vibrational features directly to the epidemiological data • provides possibility of screening new viruses or mutations from a mechanical aspect we provide a novel way towards understanding coronavirus spike proteins, connecting their nanomechanical features -specifically its vibrational spectrum and quantitative measures of mobilitywith virus lethality and infection rate. our study shows that the nanomechanics of proteins -captured in their continuous motions -can be a useful tool to help us understand complex disease etiology by connecting nanoscopic physical features with epidemiological data. potential application 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100,000 protein structures and application in sonification dynamut: predicting the impact of mutations on protein conformation, flexibility and stability the embl-ebi search and sequence analysis tools apis in 2019 cell entry mechanisms of sars-cov-2 dysregulation of type i interferon responses in covid-19 might sars-cov-2 have arisen via serial passage through an animal host or cell culture?: a potential explanation for much of the novel coronavirus' distinctive genome ethical and philosophical considerations for gain-of-function policy: the importance of alternate experiments the protein data bank the protein data bank namd: a parallel, object-oriented molecular dynamics program scalable molecular dynamics with namd harmonicity in slow protein dynamics a new approach for determining lowfrequency normal modes in macromolecules building-block approach for determining low-frequency normal modes of macromolecules tracking changes in sars-cov-2 spike: evidence that d614g increases infectivity of the covid-19 virus covid-19 and cardiovascular disease: from basic mechanisms to clinical perspectives we acknowledge support from the mit-ibm ai lab, onr (n000141612333 and n000141912375), afosr (fate muri fa9550-15-1-0514), nih (u01 eb014976), as well as aro (w911nf1920098). the authors declare no competing interests. key: cord-355728-wivk0bm0 authors: schoof, michael; faust, bryan; saunders, reuben a.; sangwan, smriti; rezelj, veronica; hoppe, nick; boone, morgane; billesbølle, christian b.; puchades, cristina; azumaya, caleigh m.; kratochvil, huong t.; zimanyi, marcell; deshpande, ishan; liang, jiahao; dickinson, sasha; nguyen, henry c.; chio, cynthia m.; merz, gregory e.; thompson, michael c.; diwanji, devan; schaefer, kaitlin; anand, aditya a.; dobzinski, niv; zha, beth shoshana; simoneau, camille r.; leon, kristoffer; white, kris m.; chio, un seng; gupta, meghna; jin, mingliang; li, fei; liu, yanxin; zhang, kaihua; bulkley, david; sun, ming; smith, amber m.; rizo, alexandrea n.; moss, frank; brilot, axel f.; pourmal, sergei; trenker, raphael; pospiech, thomas; gupta, sayan; barsi-rhyne, benjamin; belyy, vladislav; barile-hill, andrew w.; nock, silke; liu, yuwei; krogan, nevan j.; ralston, corie y.; swaney, danielle l.; garcía-sastre, adolfo; ott, melanie; vignuzzi, marco; walter, peter; manglik, aashish title: an ultra-potent synthetic nanobody neutralizes sars-cov-2 by locking spike into an inactive conformation date: 2020-08-17 journal: biorxiv doi: 10.1101/2020.08.08.238469 sha: doc_id: 355728 cord_uid: wivk0bm0 without an effective prophylactic solution, infections from sars-cov-2 continue to rise worldwide with devastating health and economic costs. sars-cov-2 gains entry into host cells via an interaction between its spike protein and the host cell receptor angiotensin converting enzyme 2 (ace2). disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the sars-cov-2 spike and ace2. by screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on spike and block ace2 interaction via two distinct mechanisms. cryogenic electron microscopy (cryo-em) revealed that one exceptionally stable nanobody, nb6, binds spike in a fully inactive conformation with its receptor binding domains (rbds) locked into their inaccessible down-state, incapable of binding ace2. affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mnb6-tri, with femtomolar affinity for sars-cov-2 spike and picomolar neutralization of sars-cov-2 infection. mnb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. these properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century. monoclonal antibodies disclosed to date. our lead neutralizing molecule, mnb6-tri, blocks 106 sars-cov-2 entry in human cells at picomolar efficacy and withstands aerosolization, 107 lyophilization, and elevated temperatures. mnb6-tri provides a promising approach to deliver a 108 potent sars-cov-2 neutralizing molecule directly to the airways for prophylaxis or therapy. 109 110 synthetic nanobodies that disrupt spike-ace2 interaction 112 to isolate nanobodies that neutralize sars-cov-2, we screened a yeast surface-displayed 113 library of >2x10 9 synthetic nanobody sequences. our strategy was to screen for binders to the 114 full spike protein ectodomain, in order to capture not only those nanobodies that would compete 115 by binding to the ace2-binding site on the rbd directly but also those that might bind 116 elsewhere on spike and block ace2 interaction through indirect mechanisms. we used a 117 mutant form of sars-cov-2 spike (spike*,) as the antigen (15). spike* lacks one of the two 118 activating proteolytic cleavage sites between the s1 and s2 domains and introduces two 119 mutations to stabilize the pre-fusion conformation. spike* expressed in mammalian cells binds 120 ace2 with a kd = 44 nm ( supplementary fig. 1) , consistent with previous reports (17). next, we 121 labeled spike* with biotin or with fluorescent dyes and selected nanobody-displaying yeast over 122 multiple rounds, first by magnetic bead binding and then by fluorescence-activated cell sorting 123 (fig. 1a) . 124 125 three rounds of selection yielded 21 unique nanobodies that bound spike* and showed 126 decreased spike* binding in the presence of ace2. closer inspection of their binding properties 127 revealed that these nanobodies fall into two distinct classes. one group (class i) binds the rbd 128 and competes with ace2 (fig. 1b) . a prototypical example of this class is nanobody nb6, which 129 binds to spike* and to rbd alone with a kd of 210 nm and 41 nm, respectively ( fig. 1c ; table 130 1). another group (class ii), exemplified by nanobody nb3, binds to spike* (kd = 61 nm), but 131 displays no binding to rbd alone (fig. 1c, table 1 ). in the presence of excess ace2, binding of 132 nb6 and other class i nanobodies is blocked entirely, whereas binding of nb3 and other class ii 133 nanobodies is decreased only moderately (fig. 1b) . these results suggest that class i 134 nanobodies target the rbd to block ace2 binding, whereas class ii nanobodies target other 135 epitopes and decrease ace2 interaction with spike allosterically or through steric interference. 136 indeed, surface plasmon resonance (spr) experiments demonstrate that class i and class ii 137 nanobodies can bind spike* simultaneously (fig. 1d) . 138 139 analysis of the kinetic rate constants for class i nanobodies revealed a consistently greater 140 association rate constant (ka) for nanobody binding to the isolated rbd than to full-length spike* 141 (table 1) , which suggests that rbd accessibility influences the kd. we next tested the efficacy 142 of our nanobodies, both class i and class ii, to inhibit binding of fluorescently labeled spike* to 143 ace2-expressing hek293 cells (table 1, fig. 1e ). class i nanobodies emerged with highly 144 variable activity in this assay with nb6 and nb11 as two of the most potent clones with ic50 145 values of 370 and 540 nm, respectively (table 1) to define the binding sites of nb6 and nb11, we determined their cryogenic electron 156 microscopy (cryo-em) structures bound to spike* ( fig. 2a state rbds only contacts a single rbd (fig. 2d) . 175 176 nb3 interacts with the spike s1 domain external to the rbd 177 our attempts to determine the binding site of nb3 by cryo-em proved unsuccessful. we 178 therefore turned to radiolytic hydroxyl radical footprinting to determine potential binding sites for 179 nb3. spike*, either apo or bound to nb3, was exposed to 5-50 milliseconds of synchrotron x-ray 180 radiation to label solvent-exposed amino acids with hydroxyl radicals. radical-labeled amino 181 acids were subsequently identified and quantified by mass spectrometry of trypsin/lys-c or glu-182 c protease digested spike*(18). two neighboring surface residues on the s1 domain of spike 183 (m177 and h207) emerged as highly protected sites in the presence of nb3 we assessed multivalent nb6 binding to spike* by spr. both bivalent nb6 with a 15 amino acid 204 linker (nb6-bi) and trivalent nb6 with two 20 amino acid linkers (nb6-tri) dissociate from spike* 205 in a biphasic manner. the dissociation phase can be fitted to two components: a fast phase with 206 kinetic rate constants kd1 of 2.7x10 -2 s -1 for nb6-bi and 2.9x10 -2 s -1 for nb6-tri, which are of the 207 same magnitude as that observed for monovalent nb6 (kd = 5.6x10 -2 s -1 ) and a slow phase that 208 is dependent on avidity (kd2 = 3.1x10 -4 for nb6-bi and kd2 < 1.0x10 -6 s -1 for nb6-tri, respectively) 209 ( fig. 3a) . the relatively similar kd for the fast phase suggests that a fraction of the observed 210 binding for the multivalent constructs is nanobody binding to a single spike* rbd. by contrast, 211 the slow dissociation phase of nb6-bi and nb6-tri indicates engagement of two or three rbds. 212 we observed no dissociation for the slow phase of nb6-tri over 10 minutes, indicating an upper 213 boundary for kd2 of 1x10 -6 s -1 and subpicomolar affinity. this measurement remains an upper-214 bound estimate rather than an accurate measurement because the technique is limited by the 215 intrinsic dissociation rate of spike* from the chip imposed by the chemistry used to immobilize 216 spike*. 217 we reasoned that the biphasic dissociation behavior could be explained by a slow 219 interconversion between up-and down-state rbds, with conversion to the more stable down-220 state required for full trivalent binding. according to this view, a single domain of nb6-tri 221 engaged with an up-state rbd would dissociate rapidly. the system would then re-equilibrate 222 as the rbd flips into the down-state, eventually allowing nb6-tri to trap all rbds in closed 223 spike*. to test this notion directly, we varied the time allowed for nb6-tri binding to spike*. 224 indeed, we observed an exponential decrease in the percent fast-phase with a t1/2 of 65 s ( table 1 ). nb6-tri shows a 2000-fold enhancement of inhibitory activity, with 237 an ic50 of 1.2 nm, whereas trimerization of nb11 and nb3 resulted in more modest gains of 40-238 and 10-fold (51 nm and 400 nm), respectively (fig. 3c) . 239 240 we next confirmed these neutralization activities with a viral plaque assay using live sars-241 nb6-tri proved exceptionally potent, neutralizing sars-cov-2 with an average ic50 of 160 pm 243 (fig. 3d ). nb3-tri neutralized sars-cov-2 with an average ic50 of 140 nm (fig. 3d) . 244 245 we further optimized the potency of nb6 by selecting high-affinity variants. to this end, we 247 prepared a new library, starting with the nb6 coding sequence, in which we varied each amino 248 acid position of all three cdrs by saturation mutagenesis (fig. 4a) . after two rounds of 249 magnetic bead-based selection, we isolated a population of high-affinity clones. sequencing 250 revealed two highly penetrant mutations: i27y in cdr1 and p105y in cdr3. we incorporated 251 these two mutations into nb6 to generate matured nb6 (mnb6), which binds with 500-fold 252 increased affinity to spike* as measured by spr (fig. 4b) . as a monomer, mnb6 inhibits both 253 pseudovirus and live sars-cov-2 infection with low nanomolar potency, a ~200-fold 254 improvement compared to nb6 ( fig. 4i -j, table 1 ). 255 256 a 2.9 å cryo-em structure of mnb6 bound to spike* shows that, like the parent nanobody nb6, 257 mnb6 binds to closed spike (fig. 4c, supplementary fig. 7) . the higher resolution map allowed 258 us to build a model with high confidence and determine the effects of the i27y and p105y 259 substitutions. mnb6 induces a slight rearrangement of the down-state rbds as compared to 260 both previously determined structures of apo-spike* and spike* bound to nb6, inducing a 9° 261 rotation of the rbd away from the central three-fold symmetry axis (fig. 4h) (14, 15) . this 262 deviation likely arises from a different interaction between cdr3 and spike*, which nudges the 263 rbds into a new resting position. while the i27y substitution optimizes local contacts between 264 cdr1 in its original binding site on the rbd, the p105y substitution leads to a marked 265 rearrangement of cdr3 in mnb6 (fig. 4f-g) . this conformational change yields a different set 266 of contacts between mnb6 cdr3 and the adjacent rbd (fig. 4d) . remarkably, an x-ray crystal 267 structure of mnb6 alone revealed dramatic conformational differences in cdr1 and cdr3 268 between free and spike*-bound mnb6, suggestive of significant conformational heterogeneity 269 for the unbound nanobodies and induced-fit rearrangements upon binding to spike* (fig. 4e) . 270 the binding orientation of mnb6 is similar to that of nb6, supporting the notion that our 272 multivalent design would likewise enhance binding affinity. unlike nb6-tri, trivalent mnb6 273 (mnb6-tri) bound to spike with no observable fast-phase dissociation and no measurable 274 dissociation over ten minutes, yielding an upper bound for the dissociation rate constant kd of 275 1.0x10 -6 s -1 (t1/2 > 8 days) and a kd of <1 pm (fig. 4b) . as above, more precise measurements 276 of the dissociation rate are precluded by the surface chemistry used to immobilize spike*. 277 278 mnb6-tri displays further gains in potency in both pseudovirus and live sars-cov-2 infection 279 assays with ic50 values of 120 pm (5.0 ng/ml) and 54 pm (2.3 ng/ml), respectively (fig. 4h-i, 280 table 1). given the sub-picomolar affinity observed by spr, it is likely that these viral 281 neutralization potencies reflect the lower limit of the assays. mnb6-tri is therefore an 282 exceptionally potent sars-cov-2 neutralizing antibody, among the most potent molecules 283 disclosed to date. 284 285 nb6, nb6-tri, mnb6, and mnb6-tri are robust proteins 286 one of the most attractive properties that distinguishes nanobodies from traditional monoclonal 287 antibodies is their extreme stability (21). we therefore tested nb6, nb6-tri, mnb6, and mnb6-tri 288 for stability regarding temperature, lyophilization, and aerosolization. temperature denaturation 289 experiments using circular dichroism measurements to assess protein unfolding revealed 290 melting temperatures of 66.9, 62.0, 67.6, and 61.4 °c for nb6, nb6-tri, mnb6 and mnb6-tri, 291 respectively ( fig 5a) . aerosolization and prolonged heating of nb6, mnb6, and mnb6-tri for 1 292 hour at 50°c induced no loss of activity (fig 5b) . moreover, mnb6 and mnb6-tri were stable to 293 lyophilization and to aerosolization using a mesh nebulizer, showing no aggregation by size 294 exclusion chromatography and preserved high affinity binding to spike* (fig. 5c-d) . 295 there is a pressing need for prophylactics and therapeutics against sars-cov-2 infection. 298 most recent strategies to prevent sars-cov-2 entry into the host cell aim at blocking the 299 ace2-rbd interaction. high-affinity monoclonal antibodies, many identified from convalescent 300 patients, are leading the way as potential therapeutics (22-29). while highly effective in vitro, 301 these agents are expensive to produce by mammalian cell expression and need to be 302 intravenously administered by healthcare professionals (30). moreover, large doses are likely to 303 be required for prophylactic viral neutralization, as only a small fraction of systemically 304 circulating antibodies cross the epithelial cell layers that line the airways (31). by contrast, single 305 domain antibodies (nanobodies) provide significant advantages in terms of production and 306 deliverability. they can be inexpensively produced at scale in bacteria (e. coli) or yeast (p. 307 pastoris). furthermore, their inherent stability enables aerosolized delivery directly to the nasal 308 and lung epithelia by self-administered inhalation (32). 309 310 monomeric mnb6 is among the most potent single domain antibodies neutralizing sars-cov-2 311 discovered to date. multimerization of single domain antibodies has been shown to improve 312 target affinity by avidity (32, 33) . in the case of nb6 and mnb6, however, our design strategy 313 enabled a multimeric construct that simultaneously engages all three rbds, yielding profound 314 gains in potency. furthermore, because rbds must be in the up-state to engage with ace2, 315 conformational control of rbd accessibility can serve as an added neutralization mechanism. 316 indeed, our nb6-tri and mnb6-tri molecules were designed with this functionality in mind. sars-cov-2 seroconversion in humans: a detailed protocol for a 435 serological assay, antigen production, and test setup trimeric sars-cov-2 spike interacts with dimeric ace2 with limited intra-438 spike avidity. biorxiv yeast surface display platform for rapid discovery of conformationally 440 selective nanobodies automated electron microscope tomography using robust prediction 442 of specimen movements motioncor2: anisotropic correction of beam-induced motion for 444 improved cryo-electron microscopy cryosparc: algorithms for 446 rapid unsupervised cryo-em structure determination new tools for automated high-resolution cryo-em structure 448 determination in relion-3 grigorieff, cistem, user-friendly software for single-particle image 450 processing structure of the sars-cov-2 spike receptor-binding domain bound to the 452 ace2 receptor structure of a nanobody-stabilized active state of the β(2) 454 adrenoceptor rosettaes: a sampling 456 strategy enabling automated interpretation of difficult cryo-em maps coot: model-building tools for molecular graphics isolde: a physically realistic environment for model building into low-461 resolution electron-density maps phenix: a comprehensive python-based system for macromolecular 463 structure solution allosteric nanobodies reveal the dynamic range and diverse 465 mechanisms of g-protein-coupled receptor activation the beamline x28c of the 467 center for synchrotron biosciences: a national resource for biomolecular structure and 468 dynamics experiments using synchrotron footprinting fast quantitative analysis of timstof pasef data with msfragger and 471 ionquant msstats: an r package for statistical analysis of quantitative mass 473 spectrometry-based proteomic experiments automatic processing of rotation diffraction data from crystals of initially 475 unknown symmetry and cell constants phaser crystallographic software buster version 1.10.0. . cambridge, united 481 kingdom: global phasing ltd figure 2. cryo-em structures of nb6 and nb11 bound to spike. a, cryo-em maps of spike*-601 nb6 complex in either closed (left) or open (right) spike* conformation. b, cryo-em maps of 602 spike*-nb11 complex in either closed (left) or open (right) spike* conformation. the top views 603 show receptor binding domain (rbd) up-or down-states. c, nb6 straddles the interface of two 604 down-state rbds, with cdr3 reaching over to an adjacent rbd. d, nb11 binds a single rbd in 605 the down-state (displayed) or similarly in the up-state nb11 in either rbd up-or down-state. e, comparison of rbd epitopes engaged by ace2 607 (purple), nb6 (red), or nb11 (green) multivalency improves nanobody affinity and inhibitory efficacy. a, spr of nb6 611 and multivalent variants. red traces show raw data and black lines show global kinetic fit for nb6 and independent fits for association and dissociation phases for nb6-bi and nb6-tri dissociation phase spr traces for nb6-tri after variable association time ranging from curves were normalized to maximal signal at the beginning of the dissociation phase. percent 615 fast phase is plotted as a function of association time (right) with a single exponential fit. n = 3 616 independent biological replicates. c, inhibition of pseudotyped lentivirus infection of ace2 expressing hek293t cells. n = 3 biological replicates for all but nb11-tri (n = 2) d, inhibition of 618 live sars-cov-2 virus. representative biological replicate with n = 3 (right panel) or 4 (left 619 panel) technical replicates per concentration. n = 3 biological replicates for all but nb3 and nb3-620 tri (n = 2) dissociation was observed for mnb6-tri over 10 minutes. c, cryo-em structure of spike*-mnb6 comparison of receptor binding domain (rbd) engagement by nb6 and mnb6 demonstrating changes in nb6 and 630 mnb6 position and the adjacent rbd. e, comparison of mnb6 complementarity determining 631 regions in either the cryo-em structure of the spike*-mnb6 complex or an x-ray crystal structure 632 of mnb6 alone. f, cdr1 of nb6 and mnb6 binding to the rbd. as compared to i27 in nb6 nb6 and mnb6 binding to the rbd demonstrating a large conformational 635 rearrangement of the entire loop in mnb6. h, comparison of closed spike* bound to mnb6 and 636 rotational axis for rbd movement is highlighted. i, inhibition of pseudotyped lentivirus 637 infection of ace2 expressing hek293t cells by mnb6 and mnb6-tri. n = 3 biological replicates 638 j, mnb6 and mnb6-tri inhibit sars-cov-2 infection of veroe6 cells in a plaque assay representative biological replicate with n = 4 technical replicates per concentration. n = 3 640 biological replicates for all samples average values from n = 2 biological replicates for nb12, nb17, and nb11-tri are presented c average values from n = 2 biological replicates for nb3, nb3-bi, and nb3-tri. n = 3 biological 658 replicates for all others nb -no binding 660 nc -no competition 661 np -not performed 662 we thank the entire walter and manglik labs for facilitating the development and rapid execution 485 of this large-scale collaborative effort. we thank sebastian bernales and tony de fougerolles 486 for advice and helpful discussion, and jonathan weissman for input into the project and reagent 487 and machine use. we thank jim wells for providing the ace2 ecd-fc construct, jason 488mclellan for providing spike, rbd, and ace2 constructs, and florian krammer for providing an 489 rbd construct. we thank jesse bloom for providing the ace2 expressing hek293t i 9.0x10 5 5.3x10 -1 5.9x10 -7 1.0x10 6 9.9x10 -1 9.7x10 -7 8.3x10 -6 (1.7x10 -6 ) np np nb3 ii 1.8x10 6 1.1x10 -1 6.1x10 -8 nb nc 3.9x10 -6 (7.9x10 -7 )3.0x10 -6 (3.2x10 -7 )nb6 i 2.7x10 5 5.6x10 -2 2.1x10 -7 2.1x10 6 8.7x10 -2 4.1x10 -8 3.7x10 -7 (4.9x10 -8 ) 2.0x10 -6 (3.5x10 -7 )3.3x10 -6 (7.2x10 -7 )nb8 i 1.4x10 5 8.1x10 -1 5.8x10 -6 6.6x10 5 3.3x10 -1 5.1x10 -7 4.8x10 -6 (4.9x10 -7 ) np np nb11 i 1.2x10 6 1.6x10 -1 1.4x10 -7 3.2x10 6 2.4x10 -1 7.6x10 -8 5.4x10 -7 (1.2x10 -7 )2.4x10 -6 (5.4x10 -7 ) np nb12 i 1.2x10 2 2.0x10 -4 1.6x10 -6 biphasic biphasic biphasic 2.5x10 -7 (5.5x10 -8 )1.2x10 -6 (9.0x10 -7 ) np nb15 i 1.7x10 5 2.3x10 -1 1.3x10 -6 6.0x10 5 2.2x10 -1 3.6x10 -7 2.2x10 -6 (2.5x10 -7 )6.7x10 -6 (3.6x10 -6 ) np nb16 i 1.1x10 5 1.3x10 -1 1.3x10 -6 np 9.5x10 -7 (1.1x10 -7 ) np np nb17 ii 7.3x10 5 2.0x10 -1 2.7x10 -7 nb nc 7.6x10 -6 (1.0x10 -6 ) np nb18 ii 1.4x10 5 6.4x10 -3 4.5x10 -8 nb 5.2x10 -5 (1.5x10 -5 ) np np nb19 i 2.4x10 4 1.1x10 -1 4.5x10 -6 1.0x10 5 8.9x10 -2 8.8x10 -7 4.1x10 -6 (4.9x10 -7 )2.4x10 -5 (7.7x10 -6 ) np nb24 i 9.3x10 5 2.7x10 -1 2.9x10 -7 2.4x10 6 3.5x10 -1 1.5x10 -7 7.5x10 -7 (1.0x10 -7 ) np np ace2 n/a 2.7x10 5 1.2x10 -2 4.4x10 -8 np np np 1.7x10 -7 (6.6x10 -8 ) 6.2x10 -7 (1.7x10 -7 ) np mnb6 i 1.0x10 6 4.5x10 -4 4.5x10 -10 1.1x10 6 6.4x10 -4 5.6x10 -10 1.3x10 -9 (4.1x10 -10 ) 6.3x10 -9 (1.6x10 -9 ) key: cord-333465-cha7ndv5 authors: horspool, a. m.; kieffer, t.; russ, b. p.; dejong, m. a.; wolf, m. a.; karakiozis, j. m.; hickey, b. j.; fagone, p.; tacker, d. h.; bevere, j. r.; martinez, i.; barbier, m.; perrotta, p. l.; damron, f. h. title: interplay of antibody and cytokine production reveals cxcl-13 as a potential novel biomarker of lethal sars-cov-2 infection date: 2020-08-31 journal: nan doi: 10.1101/2020.08.24.20180877 sha: doc_id: 333465 cord_uid: cha7ndv5 the sars-cov-2 pandemic is continuing to impact the global population. this study was designed to assess the interplay of antibodies with the systemic cytokine response in sars-cov-2 patients. we demonstrate that significant anti-sars-cov-2 antibody production to receptor binding domain (rbd), nucleocapsid (n), and spike s1 subunit (s1) of sars-cov-2 develops over the first 10 to 20 days of infection. the majority of patients produced antibodies against all three antigens (219/255 sars-cov-2 positive patient specimens, 86%) suggesting a broad response to viral proteins. patient mortality, sex, blood type, and age were all associated with differences in antibody production to sars-cov-2 antigens which may help explain variation in immunity between these populations. to better understand the systemic immune response, we analyzed the production of 20 cytokines by sars-cov-2 patients over the course of infection. cytokine analysis of sars-cov-2 positive patients exhibited increases in proinflammatory markers (il-6, il-8, il-18) and chemotactic markers (ip-10, sdf-1, mip-1{beta}, mcp-1, and eotaxin) relative to healthy individuals. patients who succumbed to infection produced decreased il-2, il-4, il-12, il-13, rantes, tnf-, gro-, and mip-1 relative to patients who survived infection. we also observed that the chemokine cxcl13 was particularly elevated in patients that succumbed to infection. cxcl13 is involved in b cell activation, germinal center development, and antibody maturation, and we observed that cxcl13 levels in blood trended with anti-sars-cov-2 antibody production. furthermore, patients that succumbed to infection produced high cxcl13 and also tended to have high ratio of nucleocapsid to rbd antibodies. this study provides insights into sars-cov-2 immunity implicating the magnitude and specificity of response in relation to patient outcomes. loading, plates were incubated for 10 minutes at room temperature shaking at 60rpm. 135 plates were then washed four times with pbs-t. secondary antibody buffer (100µl of 1% 136 milk diluted in pbs-t containing 1:500 goat anti-human-igg-hrp; invitrogen part #: 137 31410) was added immediately following the washing procedure. the plates were 138 incubated for 10 minutes at room temperature shaking at 60rpm. plates were washed five 139 times with pbs-t. sigmafast opd substrate (sigma part#: p9187) was prepared in 140 milliq (18.2mωxcm) water and 100µl was aliquoted into each well. ten minutes after 141 loading the substrate, 25µl of stop solution (3n hcl) was added to end colorimetric 142 development. the absorbance of the substrate in each well was measured on a synergy 143 h1 (biotek) spectrophotomer at 492nm. antibody concentration was calculated based on 144 area under the curve analyses of a492 vs. dilution factor plots for each sample. prepared for analysis by heating at 56 o c for 1 hour. samples were then centrifuged at 152 13,000 x g for 2 mins to pellet aggregates. samples (25µl) were diluted 1:2 with universal 153 assay buffer and incubated at room temperature on an orbital shaker at 500 rpm for 1 154 hour. select samples (based on sample quantity) were diluted 1:4 or 1:5 with the universal 155 assay buffer, which was taken into account during analysis. a standard curve was 156 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 31, 2020. . https://doi.org/10.1101/2020.08.24.20180877 doi: medrxiv preprint generated using antigen standards provided by the manufacturer. samples were 157 resuspended in 120 µl wash buffer prior to running on a magpix (luminex) instrument, 158 and 35 µl was analyzed per samples. bead cytokine production and antibody production were pooled into microsoft excel and 177 imported to clustvis 17 . data were transformed by the ln(x) transformation provided in the 178 webtool and grouped with a 95% confidence interval. groups were based on patient 179 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted august 31, 2020. in-patient anti-sars-cov-2 antibody production: antibody binding target and the timing 192 of the antibody response are critical factors in mediating immunity. we evaluated anti-193 sars-cov-2 antibody production to 3 antigens (rbd, n, and s1) in 82 in-patients 194 table 1 ) by developing a novel rapid-elisa technique. our rapid-elisa 195 technology evaluates igg antibody production to the sars-cov-2 rbd, n, and s1 196 proteins in approximately 1 hour with greater than 99% accuracy (supplementary table 197 2). our survey of sars-cov-2 positive patients demonstrated that antibody (igg) 198 production to rbd, n, and s1 proteins developed over the first 10 to 20 days post-199 symptom onset (figure 1a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted august 31, 2020. . https://doi.org/10.1101/2020.08.24.20180877 doi: medrxiv preprint supplementary data file). to better understand the kinetics of the antibody response, we 203 plotted igg production of every patient over time to rbd, n, or s1. patients produced igg 204 against rbd rapidly after symptom onset with the peak igg response occurring 10 days 205 after symptom onset (figure 1g ). anti-s1 igg production escalated over a slightly larger 206 period (13 days, figure 1i ) and anti-n igg production was slower than either anti-rbd or 207 anti-s1 antibody production (22 days, figure 1h ). taken together, these data describe 208 the breadth and timing of the igg response to sars-cov-2 antigens. populations, we analyzed patient groups based on sex, patient mortality, blood type, and 214 age against anti-rbd, anti-n, or anti-s1 antibody production. as igg production is more 215 consistently detectable after ten days post-symptom onset 18,19 , we assessed differences 216 in igg production beyond ten days post symptom onset. limiting sample analysis to those 217 greater than ten days post symptom onset did not significantly impact the mean antibody 218 production of the patients (supplementary figure 1) . patients who did not survive sars-219 cov-2 hospitalization produced significantly more antibodies to sars-cov-2 n than 220 patients that survived infection ( figure 2a) . furthermore, patients that did not survive 221 sars-cov-2 infection did not produce different quantities of anti-n antibodies than 222 surviving patients during early infection (supplementary figure 2) . to accurately assess 223 differences in antibody production independently of disease outcome, we quantified anti-224 sars-cov-2 igg production in patients who survived infection grouped by biological sex, 225 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 31, 2020. . https://doi.org/10.1101/2020.08.24.20180877 doi: medrxiv preprint blood type, and age. we determined that, in our cohort, females significantly produced 226 more anti-s1 igg than males ( figure 2b ). we also observed that blood type was 227 significantly associated with anti-sars-cov-2 igg production ( figure 2c ). blood type b+ 228 patients produced significantly more igg to rbd and s1 than a+ or o+ patients ( figure 229 2c) and a+ patients produced the lowest quantities of anti-rbd and anti-s1 igg. o+ 230 patients produced reduced anti-n igg relative to a+ or b+ patients. previous studies have 231 identified that age impacts antibody production to sars-cov-2 20,21 . our study 232 demonstrates that antibody production against rbd or s1 antigens increased with age 233 ( figure 2d ). in contrast, antibody production to n increased in patients over 50 years old 234 but did not continue to increase with age after 80 years of age. this is particularly evident 235 when examining pearson correlations between age and anti-sars-cov-2 igg production 236 for each antigen (supplementary figure 3) . overall, these data document a significant 237 impact of patient demographics on anti-sars-cov-2 antibody production. 238 changes in sars-cov-2 patient cytokine responses correlate with disease severity: 240 antibody production represents the antigen-specific response to pathogens but is only 241 one facet of immunity. we examined the broader immunological response to sars-cov-242 2 infection by quantifying the production of cytokines involved in a representative subset 243 of sars-cov-2 or healthy patients. sars-cov-2 patients exhibited significant increased 244 pro-inflammatory cytokine production (il-6, il-8, il-18) and increased chemotactic 245 cytokine production (ip-10, sdf-1, mip-1 , mcp-1 and eotaxin) relative to non-infected 246 individuals (figure 3 ). of the sars-cov-2-infected patients, mortality was associated 247 with increased il-6, il-8, il-18, ip-10, and mcp-1 production. patients who succumbed 248 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 31, 2020. this response is critical for eradicating many pathogens. as many sars-cov-2 patients 263 produced robust antibody responses to multiple antigens, we hypothesized that germinal 264 center formation would be increased in these patients. to quantify this, we measured the 265 serum concentration of cxcl13, a critical mediator of germinal center formation and a 266 biomarker of this immunological response 8,10,22,23 . we observed that cxcl13 production 267 primarily correlated with peak antibody production to rbd and s1 antigens across sars-268 cov-2 infected patients (figure 4a-c) . additionally, we observed that there was a 269 significant increase in average production of cxcl13 in positive patients relative to 270 negative sars-cov-2 patients. in addition, we discovered that cxcl13 production was 271 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 31, 2020. . https://doi.org/10.1101/2020.08.24.20180877 doi: medrxiv preprint significantly increased in patients that did not survive sars-cov-2 infection compared to 272 those that did (figure 4d ). when we compared antibody and cxcl13 production based 273 on patient survival over time, we observed that patients who did not survive sars-cov-274 2 infection exhibited a sustained increase in antibody and cxcl13 production relative to 275 surviving patients (figure 4ef) to n increased over a longer period than antibodies against rbd, or the s1 domain. this 288 could be due to a variety of factors including antigen immunodominance 29,30 , incongruent 289 antigen processing and availability 31,32 , differences in antibody utility and turnover, or prior 290 exposure to similar rbd/s1 antigens of other coronaviruses. theoretically, as n is not 291 expressed on the viral surface, b cells producing antibodies against this antigen may not 292 be selected for as rapidly as those that are specific to the rbd or s1 antigens and may 293 not possess neutralizing function. as infection worsens, more cells lyse. this may 294 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 31, 2020. . https://doi.org/10.1101/2020.08.24.20180877 doi: medrxiv preprint increase the local concentration of free nucleocapsid available for antigen processing and 295 presentation, particularly in lymphoid tissue 33 . in this respect, a more robust antibody 296 response to nucleocapsid later in infection may be due to increased cellular damage. this 297 may initiate a positive feedback loop where infected cells lyse and release nucleocapsid, 298 which induces a less functional anti-nucleocapsid antibody response that fails to alleviate 299 the cell lysis. more evidence is required to support these hypotheses, but these are 300 interesting paradigms to consider in the context of anti-sars-cov-2 immunity. 301 lethal sars-cov-2 infection is significantly correlated with higher antibody 302 production 19,20,26 and is described further in this study. in analyzing antibody production 303 between patient demographics, it was important to eliminate increased antibody 304 production due to lethal infection as a source of bias. as such, our analyses presented 305 here describe igg production of sars-cov-2 survivors grouped by demographic. there 306 are a multitude of studies reporting differences in igg production between demographics 307 including: trends in anti-sars-cov-2 antibody production between sexes 20,21,34-36 , a 308 correlation of genetically encoded blood type with sars-cov-2 immunity 37 , and 309 variability in antibody production in the aging population 20,21 . from these prior studies and 310 others 38,39 it is known that biological sex can impact antibody production during infection. 311 we observed this phenomenon when quantifying sex specific anti-s1 igg production. the 312 anti-viral response is mediated in part by toll-like receptors which are differentially 313 regulated between the sexes 40,41 . a higher frequency of anti-s1 igg in females would 314 suggest an increased neutralizing response to the virus which has not been thoroughly 315 evaluated to-date. our data exhibited a modest difference in antibody production between 316 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 31, 2020. . https://doi.org/10.1101/2020.08.24.20180877 doi: medrxiv preprint sexes. as a result, we do not consider biological sex to be a major contributor to anti-317 it is documented that red blood cell phenotypes can influence microbial 319 pathogenesis as antigens can function as receptors and/or co-receptors for pathogenic 320 organisms 42 . historically, an association was identified between abo type and pathogen cov spike protein binding to ace2 44 . although the underlying mechanism relating blood 326 type to sars-cov-2 pathogenesis remains unclear, it appears there may be a 327 relationship between abo blood type and coronavirus infection. recent data identified 328 the 9q34.2 locus (abo blood group locus) as potentially involved in susceptibility to 329 covid-19 respiratory failure with evidence that type a phenotypes are at higher risk while 330 type o phenotypes are partially protected 45 . the data generated in these studies show an 331 interesting pattern that may reinforce blood type related outcomes in severe disease due 332 to a previously unreported association to the level and type of antibody response. as seen 333 in figure 2c , the relative quantity of anti-rbd and anti-s1 antibodies was highest among 334 anti-n antibodies. this is further accentuated by evaluating the ratio of anti-rbd or anti-336 s1 versus anti-n in our patient cohort which shows that higher n:rbd or n:s1 ratios are 337 associated with poor prognosis (supplementary figure 5) . it is plausible that type-a 338 individuals may have a misdirected humoral response due to antigenic homology 339 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 31, 2020. . https://doi.org/10.1101/2020.08.24.20180877 doi: medrxiv preprint between n-acetyl-galactosamine sugar moieties on the a antigen and spike protein 340 resulting in molecular mimicry. this would result in type-o and -b individuals registering 341 more spike protein epitopes as foreign and eliciting a more robust humoral response; in 342 turn, this putative mechanism could reduce infectious dose and decrease the risk of 343 mortality. further studies evaluating physiologic modifications of spike protein and its 344 antigenic moieties would help support or disprove this theory. as the conclusions from 345 these observations are currently theoretical, a more extensive review of comorbid 346 conditionswith a multivariate analysis and estimations of associated odds ratiosmay 347 reveal other associations outside of blood type. 348 the aging process is associated with decreased t-cell functionality 46 , resulting in 349 hyperactive b-cell proliferation that does not confer immunity 47 . we discovered that older 350 patients typically produced more antibodies to rbd and s1 than younger patients. the 351 lack of increase in antibody production to nucleocapsid in the elderly may be a function 352 of antigen availability. to speculate, if elderly patients have higher viral loads due to 353 decreased remediation of virus this would increase the relative abundance of surface 354 exposed antigens (rbd and s1), but not necessarily hidden antigens (n). increased 355 antibody production would therefore predominantly occur to rbd and s1, and not n. 356 other challenges are associated with studying this population including co-presentations 357 of multiple diseases which complicates this analysis. regardless, our study has identified 358 several patient demographics associated with differences in the anti-sars-cov-2 359 antibody response. 360 the anti-viral immune response depends on a variety of signaling pathways 361 mediated by cytokines and chemokines. many of the pro-inflammatory cytokines 362 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 31, 2020. of cardiac distress. separately, we discovered increased eotaxin production in sars-384 cov-2 patients. eotaxin was increased or similar to healthy patients during sars-cov-2 385 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 31, 2020. antibody maturation signaling has not been investigated in the context of sars-406 cov-2. we assessed the activity of the antibody maturation pathway by measuring 407 cxcl13 concentrations in the serum of sars-cov-2 patients. increased cxcl13 in 408 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 31, 2020. . https://doi.org/10.1101/2020.08.24.20180877 doi: medrxiv preprint . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted august 31, 2020. . https://doi.org/10.1101/2020.08.24.20180877 doi: medrxiv preprint to rbd (a), n (b), or s1 (c). correlation of antibody production to rbd vs. n (d) or s1 (e). 628 correlation of antibody production to n vs. s1 (f). antibody production of anti-rbd (g), 629 anti-n (h), or anti-s1 (i) antibodies by sars-cov-2 positive patients vs. days post sars-630 cov-2 disease onset. 631 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted august 31, 2020. . https://doi.org/10.1101/2020.08.24.20180877 doi: medrxiv preprint cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted august 31, 2020. . https://doi.org/10.1101/2020.08.24.20180877 doi: medrxiv preprint . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted august 31, 2020. . https://doi.org/10.1101/2020.08.24.20180877 doi: medrxiv preprint analysis of serum cytokines in patients with severe acute 588 respiratory syndrome the immunobiology of sars candidate genes associated with susceptibility for sars-592 cxcl10/ip-10 in infectious diseases pathogenesis and potential 594 therapeutic implications highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 597 (sdf-1) sdf-1α and cxcr4 as therapeutic 599 targets in cardiovascular disease stromal cell-derived factor-1α is cardioprotective after 601 myocardial infarction stromal derived factor 1α: a 603 chemokine that delivers a two-pronged defence of the myocardium sars-cov-2 and cardiovascular complications: from molecular 606 mechanisms to pharmaceutical management outcomes of cardiovascular magnetic resonance imaging 608 in patients recently recovered from coronavirus disease clinical features of patients infected with 2019 novel coronavirus 611 in wuhan role of eotaxin-1 (ccl11) and cc chemokine receptor in bleomycin-induced lung injury and fibrosis influenza virus a stimulates expression of eotaxin by nasal 616 epithelial cells the β-chemokine receptors ccr3 and ccr5 facilitate infection by 618 primary hiv-1 isolates t cell exhaustion networking at the level 621 of host immunity: immune cell interactions during persistent viral infections international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity maturation of anti-sars-cov-2 antibodies. the significant increase of cxcl13 in 410 patients with lethal disease suggests this may be an emergency response to uncontrolled 411 infection. it is possible that sustained infection stimulates increased antibody affinity 412 maturation that is unable to keep pace with viral replication and the cytokine storm. in this 413 sense, cxcl13 could be used as a marker of sars-cov-2 disease severity. there is a 414 precedent for the utility of cxcl13 as a biomarker that is predictive of immune activation 415 during hiv exposure 8, 9, 24 . this adds credibility and feasibility for this utility, but further 416 studies are required to validate this approach. we have provided a schematic of how the 417 cxcl13 response interplays with our other observations of sars-cov-2 immunity in 418 figure 6 . 419to summarize, this study provides insight into the breadth of the immunological 420 response against sars-cov-2. we demonstrated increasing antibody production to 421 multiple sars-cov-2 antigens over the first ten days of infection using a rapid-elisa 422 assay. our results exhibit that patient mortality, sex, blood type, and age impact antibody 423 production to sars-cov-2, adding to what is known about sars-cov-2 pathogenesis. 424furthermore, lethal sars-cov-2 infection triggers a pro-inflammatory cytokine response, 425 in combination with the secretion of several chemotactic agents. interestingly, patients 426 with lethal sars-cov-2 disease exhibited divergent cytokine production compared to 427 patients with non-lethal disease. finally, we discovered that a marker of germinal center 428 activity (cxcl13) is upregulated in sars-cov-2 patients, and that this upregulation is 429 amplified in lethal disease. ultimately, these studies help to elucidate the interplay 430 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review)the copyright holder for this preprint this version posted august 31, 2020. 20-plex cytokine assays. pf produced rbd used in this study. amh analyzed and 445 compiled assay data and figures. all authors took part in writing and editing the 446 manuscript. we would like to thank bei resources for providing the following reagents 447 (nr-52422). we would finally like to express our gratitude to drs. laura gibson and clay 448marsh for enabling this research during the global pandemic. 449 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review)the copyright holder for this preprint this version posted august 31, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 31, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 31, 2020. biol. 3, 1-12 (2020) . is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 31, 2020. . https://doi.org/10.1101/2020.08.24.20180877 doi: medrxiv preprint production by sars-cov-2 production is compared to anti-rbd (a), anti-n (b), or anti-s1 655 (c) igg quantity over the course of patient disease. red arrows represent cxcl13 656 maxima, and green arrows represent local igg maxima. cxcl13 production was 657 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review)the copyright holder for this preprint this version posted august 31, 2020. . https://doi.org/10.1101/2020.08.24.20180877 doi: medrxiv preprint compared between sars-cov-2 negative (-) and positive (+) patients, and sars-cov-658 2 positive survivors (s), or non-survivors (d) (d). examples of a surviving patient 659 producing low cxcl13 and low anti-rbd igg response (e) or deceased patient producing 660 high cxcl13 and high anti-rbd igg response (f). statistical significance was assessed 661 with a brown forsyth and welch's one-way anova followed by tukey's multiple 662 comparison test. **** = p<0.0001, n.s. = not significant. 663 664 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review)the copyright holder for this preprint this version posted august 31, 2020. . https://doi.org/10.1101/2020.08.24.20180877 doi: medrxiv preprint key: cord-287205-k64svq6n authors: pollet, jeroen; chen, wen-hsiang; versteeg, leroy; keegan, brian; zhan, bin; wei, junfei; liu, zhuyun; lee, jungsoon; kundu, rahki; adhikari, rakesh; poveda, cristina; mondragon, maria-jose villar; de araujo leao, ana carolina; rivera, joanne altieri; gillespie, portia m.; strych, ulrich; hotez, peter j.; bottazzi, maria elena title: sars-cov-2 rbd219-n1c1: a yeast-expressed sars-cov-2 recombinant receptor-binding domain candidate vaccine stimulates virus neutralizing antibodies and t-cell immunity in mice date: 2020-11-05 journal: biorxiv doi: 10.1101/2020.11.04.367359 sha: doc_id: 287205 cord_uid: k64svq6n there is an urgent need for an accessible and low-cost covid-19 vaccine suitable for lowand middle-income countries. here we report on the development of a sars-cov-2 receptor-binding domain (rbd) protein, expressed at high levels in yeast (pichia pastoris), as a suitable vaccine candidate against covid-19. after introducing two modifications into the wild-type rbd gene to reduce yeast-derived hyperglycosylation and improve stability during protein expression, we show that the recombinant protein, rbd219-n1c1, is equivalent to the wild-type rbd recombinant protein (rbd219-wt) in an in vitro ace-2 binding assay. immunogenicity studies of rbd219-n1c1 and rbd219-wt proteins formulated with alhydrogel® were conducted in mice, and, after two doses, both the rbd219-wt and rbd219-n1c1 vaccines induced high levels of binding igg antibodies. using a sars-cov-2 pseudovirus, we further showed that sera obtained after a two-dose immunization schedule of the vaccines were sufficient to elicit strong neutralizing antibody titers in the 1:1,000 to 1:10,000 range, for both antigens tested. the vaccines induced ifn-γ, il-6, and il-10 secretion, among other cytokines. overall, these data suggest that the rbd219-n1c1 recombinant protein, produced in yeast, is suitable for further evaluation as a human covid-19 vaccine, in particular, in an alhydrogel® containing formulation and possibly in combination with other immunostimulants. introduction 34 the number of coronavirus disease 19 (covid-19) cases globally is readily approaching the 50-35 million-person mark, with over 1.2 million deaths. in response to the pandemic, an international 36 enterprise to develop effective and safe vaccines is underway. there are many ways to categorize the 37 more than 100 potential covid-19 vaccine candidates 1 , but one approach is to divide them as those 38 employing new technologies for production, but that have not yet been licensed for use, versus terms of production, scale-up, potential efficacy and safety, and delivery. 47 we have previously reported on recombinant protein-based coronavirus vaccine candidates, 48 formulated with alhydrogel ® to prevent severe acute respiratory syndrome (sars) 9-11 and middle 49 east respiratory syndrome (mers) 12 . in both cases, the receptor-binding domain (rbd) of the 50 sars or mers spike proteins was used as the target vaccine antigen. in a mouse model, the sars-51 cov rbd219-n1/alhydrogel ® vaccine induced high titers of virus-neutralizing antibodies and 52 protective immunity against a mouse-adapted sars-cov virus challenge. it was also found to 53 minimize or prevent eosinophilic immune enhancement compared to the full spike protein 9 . 54 the rbd of sars-cov-2 has likewise attracted interest from several groups now entering 55 clinical trials with rbd-based vaccines 7,13-17 . our approach was to apply the lessons learned from the 56 development of the sars-cov vaccine candidate and accelerate the covid-19 vaccine induction temperature was set to 25 °c and the ph to 6.5 and, the methanol feed rate was between 1-95 15 ml/l/hr. the fermentation supernatant (fs) was filtered (0.45 m pes filter) and stored at -80 °c 96 before purification. 97 a hexahistidine-tagged sars-cov-2 rbd219-wt was purified from fermentation 98 supernatant (fs) by immobilized metal affinity chromatography followed by size exclusion 99 chromatography (sec). the fs was concentrated and buffer exchanged to buffer a (20 mm tris-100 hcl ph 7.5 and 0.5 m nacl) using a pellicon 2 cassette with a 10 kda mwco membrane 101 to evaluate the size of rbd219-wt and rbd219-n1c1, 2 μg of these two proteins were loaded 118 onto a 4-20% tris-glycine gel under non-reduced and reduced conditions. these two proteins were 119 also treated with pngase-f (neb, ipswitch, ma, usa) under the reduced condition to remove n-120 glycans and loaded on the gel to assess the impact of the glycans on the protein size. gels were 121 stained using coomassie blue and analyzed using a bio-rad g900 densitometer with image alhydrogel ® formulations were centrifuged at 13,000 x g for 5 min, and the supernatant was 131 removed. the protein in the supernatant fraction and the pellet fraction were quantified using a micro 132 bca assay (thermofisher, waltham, ma, usa). 133 134 for the ace-2 binding study, the alhydrogel ® -rbd vaccine formulations were blocked overnight 136 with 0.1% bsa. after hace-2-fc (lakepharma, san carlos, ca, usa) was added, the samples 137 were incubated for 2 hours at rt. after incubation, the alhydrogel ® was spun down at 13,000 x g for washed once with 300 l pbst using a biotek 405ts plate washer and diluted mouse serum 156 samples were added to the plate in duplicate, 100 l/well. as negative controls, pooled naïve mouse 157 serum (1:200 diluted) and blanks (0.1% bsa pbst) were added as well. plates were incubated for 2 158 hours at room temperature, before being were washed four times with pbst. subsequently, 1:6,000 159 diluted goat anti-mouse igg hrp antibody (100 l/well) was added in 0.1% bsa in pbst. plates 160 were incubated 1 hour at room temperature, before washing five times with pbst, followed by the 161 addition of 100 l/well tmb substrate. plates were incubated for 15 min at room temperature while 162 protected from light. after incubation, the reaction was stopped by adding 100 l/well 1 m hcl. the absorbance at a wavelength of 450 nm was measured using a biotek epoch 2 spectrophotometer. 164 duplicate values of raw data from the od450 were averaged. the titer cutoff value was calculated 165 using the following formula: titer cutoff = 3 x average of negative control + 3 x standard deviation 166 of the negative control. for each sample, the titer was determined as the lowest dilution of each 167 mouse sample with an average od450 value above the titer cutoff. when a serum sample did not 168 show any signal at all and a titer could not be calculated, an arbitrary baseline titer value of 67 was 169 assigned to that sample (baseline). sample/ rlu of negative control) x 100. serum from vaccinated mice was also characterized by the 186 ic50-value, defined as the serum dilution at which the virus infection was reduced to 50% compared with the negative control (virus + cells). when a serum sample did not neutralize 50% of the virus 188 when added at a 1:10 dilution, the ic50 titer could not be calculated and an arbitrary baseline titer 189 value of 10 was assigned to that sample (baseline). as a control, human convalescent sera for sarsfor the re-stimulation assays, splenocyte suspensions were diluted to 8x10 6 live cells/ml in a 206 2-ml deep-well dilution plate and 125 l of each sample was seeded in two 96-well tissue culture 207 treated culture plates. splenocytes were re-stimulated with 10 g/ml rbd219-wt, 20 ng/ml pma 208 + 1 g/ml ionomycin or just media (unstimulated). for the flow cytometry plate, the pma/i was not added until the next day. 125 l (2x concentration) of each stimulant was mixed with the 125 l 210 splenocytes suspension in the designated wells. after all the wells were prepared, the plates were 211 incubated at 37 °c 5% co2. one plate was used for the cytokine release assay, while the other plate 212 was used for flow cytometry. for flow cytometry, another plate was prepared with splenocytes, 213 which would be later used as fluorescence minus onecontrols (fmos). 214 after 48 hours in the incubator, splenocytes were briefly mixed by pipetting. then plates were 216 centrifuged for 5 min at 400 x g at rt. without disturbing the pellet 50 l supernatant was 217 transferred to two skirted pcr plates and frozen at -20 °c until use. 218 for the in vitro cytokine release assay, splenocytes were seeded in a 96-well culture plate at 219 1x10 6 live cells in 250 µl crpmi. splenocytes were then (re-)stimulated with either 10 µg/ml 220 rbd219-wt protein, 10 µg/ml rbd219-n1c1 protein, pma/iomycin (positive control), or nothing 221 (negative control) for 48 hours at 37 °c 5% co2. after incubation, 96-well plates were centrifuged to 222 pellet the splenocytes down and supernatant was transferred to a new 96-well plate. the supernatant 223 was stored at -20°c until assayed. a milliplex mouse th17 luminex kit (md millipore) with 224 analytes il-1β, il-2, il-4, il-6, il-10, il-12(p70), il-13, il-17a, il-23, ifn-γ, and tnf-α was 225 used to quantify the cytokines secreted in the supernatant by the re-stimulated splenocytes. an 226 adjusted protocol based on the manufacturers' recommendations was used with adjustments to use 227 less sample and kit materials 23 . the readout was performed using a magpix luminex instrument. 228 raw data was analyzed using bio-plex manager software, and further analysis was done with excel 229 and prism. surface staining and intracellular cytokine staining followed by flow cytometry was performed to 232 measure the amount of activated (cd44=) cd4+ and cd8+ t cells producing ifn-, il-2, tnf-, 233 and il-4 upon re-stimulation with s2rbd219 wt. 234 five hours before the 24-hour re-stimulation incubation, brefeldin a was added to block 235 cytokines from secretion. pma/i was also added to designated wells as a positive control. after the 236 incubation, splenocytes were stained for the relevant markers. a viability dye and an fc block were 237 also used to remove dead cells in the analysis and to minimize non-specific staining, respectively. results 251 here we report on the expression of a modified, recombinant rbd of the sars-cov-2 spike protein 252 using the yeast (p. pastoris) expression system. the candidate antigen selection, modifications, and 253 production processes were based on eight years of process development, manufacture, and preclinical 254 prior experience with a sars-cov recombinant protein-based receptor-binding domain (rbd) 9-11 . 255 the rbds of the sars-cov-2 and sars-cov share significant amino acid sequence similarity 256 (>75% identity, >80% homology) and both use the human angiotensin-converting enzyme 2 (ace2) 257 receptor for cell entry 25,26 . process development using the same procedures and strategies used for the 258 production, scale-up, and manufacture of the sars-cov recombinant protein allowed for a rapid 259 acceleration in the development of a scalable and reproducible production process for the 260 sars-cov-2 rbd219-n1c1 protein, suitable for its technological transfer to a manufacturer. 261 we found that the modifications used to minimize yeast-derived hyperglycosylation and 262 optimize the yield, purity, and stability of the sars-cov rbd219-n1 protein were also relevant to 263 the sars-cov-2 rbd expression and production process. the modified sars-cov-2 antigen, 264 rbd219-n1c1, when formulated on alhydrogel ® , was shown to induce virus-neutralizing antibodies 265 in mice, equivalent to those levels elicited by the wild-type (rbd219-wt) recombinant protein 266 counterpart. 267 the wild-type sars-cov-2 rbd amino acid sequence comprises residues 331-549 of the spike (s) 269 protein (genbank: qhd43416.1) of the wuhan-hu-1 isolate (genbank: mn908947.3) (figure 1) . 270 in the rbd-219-wt construct, the gene fragment was expressed in p. pastoris. after fermentation at 271 the 5 l scale, the hexahistidine-tagged protein was purified by immobilized metal affinity 272 chromatography, followed by size-exclusion chromatography. we observed glycosylation and 273 aggregation during these initial expression and purification studies, and therefore, similar to our previous strategy 10 , we generated a modified construct, the rbd219-n1c1, by deleting the n331 275 residue and mutating the c538 residue to alanine. the additional mutation of c538 to a538 was done 276 because we observed that in the wild-type sequence nine cysteine residues likely would form four 277 disulfide bonds. therefore, the c538 residue was likely available for intermolecular cross-linking, 278 leading to aggregation. as a result, in the rbd219-n1c1 construct, and based on the modifications, 279 the pichia-derived hyperglycosylation, as well as aggregation via intermolecular disulfide bridging, 280 were greatly reduced. we note that the deleted and mutated residues are structurally far from the 281 immunogenic epitopes and specifically the receptor-binding motif (rbm) of the rbd (figure 1) when mixing 25 µg of either rbd219-wt or rbd219-n1c1 proteins to 500 µg of alhydrogel ® , we 300 observed that >98% of the proteins bind to alhydrogel ® after 15 min of incubation. only when the 301 alhydrogel ® was reduced to less than 100 µg (alhydrogel ® /rbd219 ratio <4), the alhydrogel ® 302 surface was saturated, and protein started to be detected in the supernatant (figure 2a) . it is known 303 that unbound protein may impact the immunogenicity of the vaccine formulation, therefore we 304 proceeded to only evaluate formulations with alhydrogel ® /rbd219 ratios higher than 4. 305 figure 2b shows that hace-2-fc, a recombinant version of the human receptor used by the 306 virus to enter the host cells, can bind with the rbd proteins that are adsorbed on the surface of the 307 alhydrogel ® . this demonstrates that bound rbd proteins are structurally and possibly functionally 308 active and that after adsorption the protein does not undergo any significant conformational changes 309 that could result in the loss of possible key epitopes around the receptor-binding motif (rbm). 310 we saw no statistical differences between the binding of hace-2-fc to rbd219-wt (red, 311 figure 2b ) or rbd219-n1c1 (green, figure 2b ) proteins, based on an unpaired t-test (p=0.670). 312 likewise, we saw no relation between the amount of alhydrogel ® to which the rbd was bound and 313 the interaction with hace-2-fc, indicating that the surface density of the rbd proteins on the 314 alhydrogel ® plays no role in the presentation of ace binding sites. alhydrogel ® , produced a lower igg response, albeit slightly higher than the negative control that had 338 been immunized with 500 g alhydrogel ® alone (figure 3b, supplemental table 1) . importantly, 339 based on a mann-whitney test, we determined that there was no statistical difference between the 340 groups vaccinated with the modified and the wild-type version of the rbd protein (p=0.3497). the 341 average neutralizing antibody titers observed on day 35 (ic50 range: 5.0x10 3 to 9.4x10 3 , 342 supplemental (figure 3c) . 345 on day 43, 22 days after receiving the boost vaccination, half of the mice in each group 346 (n=4), those with the highest igg titers, were sacrificed to determine the total igg, the igg subtypes, 347 and the neutralizing antibody titers. as we observed on day 35, all animals that had received the 348 vaccine produced strong antibody titers, with the groups receiving >200 g alhydrogel ® eliciting a 349 higher titer than those that received only 100 g of alhydrogel ® , albeit no statistical significance was 350 detected (figures 3b) . for all animals, as typical for vaccine formulations containing aluminum, the 351 igg2a:igg1 titer ratio was <0.1 (supplemental figure 3) . in the pseudovirus neutralization assay 352 for the day 43 samples (figure 3c) , all vaccines containing >200 g alhydrogel ® elicited ic50 titers 353 that, on average, were several-fold higher than on day 35 (ic50 range: 1.1x10 4 to 1.2x10 5 , 354 supplemental table 2 ). there again was no difference between the rbd219-wt and rbd-n1c1 355 vaccines. on day 57, all remaining animals were sacrificed. in contrast to the animals studied on days 357 35 and 43, these animals had received a second boost vaccination. a robust immune response in all 358 vaccinated mice, including those immunized with the protein adsorbed to 100 g alhydrogel ® 359 achieved high average igg titers. the total igg titers in the mice sacrificed on day 57, had increased 360 after the third vaccination, compared to the titers seen on day 35. likewise, we observed a 361 corresponding increase in the average ic50 values (ic50 range: 3.8x10 2 to 1.1x10 4 , supplemental 362 table 2 ) for all animals, including those immunized with the protein adsorbed to 100 g 363 alhydrogel ® . interestingly, for this time point, the cohort receiving 25 g rbd219-n1c1 with 500 364 g alhydrogel ® appeared to show higher neutralizing antibody titers than the corresponding 365 for all samples, we employed flow cytometry to quantify intracellular cytokines in cd4+ and cd8 + 379 cells after restimulation ( figure 4a) . on day 43, high percentages of cd4 + -il-4 and, to a slightly 380 lesser extent cd4 + -tnf producing cells were detected. conversely, as expected for an 381 alhydrogel ® -adjuvanted vaccine, low levels of il-2 producing cd4 + cells were seen. in a cytokine 382 release assay, strong ifn-, il-6, and il-10 secretion was observed independent of whether the 383 animals had received two or three immunizations, whereas low amounts of secreted th1-typical 384 cytokines such as il-2 or il-12 were seen ( figure 4b ). cytokine concentrations of non-stimulated controls were subtracted from re-stimulated samples. discussion 396 here we report on a yeast-expressed sars-cov-2 rbd219-n1c1 protein and its potential as a 397 vaccine candidate antigen for preventing covid-19. building on extensive prior experience 398 developing vaccines against sars-cov and mers-cov 10-12 , we initially selected and compared the 399 sars-cov-2 rbd219-wt and the sars-cov-2 rbd219-n1c1 proteins for their potential to 400 induce high titers of virus-neutralizing antibodies, t-cell responses, and protective immunity. 401 previously we observed that the sars-cov rbd219-n1 antigen, formulation with 402 alhydrogel ® elicited high levels of neutralizing antibodies without evidence of eosinophilic immune 403 enhancement. that rbd-based vaccine was even superior to the full-length spike protein in inducing 404 specific antibodies and fully protected mice from sars-cov infection while preventing eosinophilic 405 pulmonary infiltrates in the lungs upon challenge 9 . 406 in this work, using the sars-cov-2 rbd219 protein analog, we observed that, just like in 407 the case of the sars-cov rbd antigen, the deletion of the n-terminal asparagine residue reduced 408 hyperglycosylation, thus allowing for easier purification of the antigen obtained from the yeast 409 expression system. moreover, mutagenesis of a free cysteine residue further improved protein 410 production through the reduction of aggregation. based on the predicted structure of the rbd, no 411 impact on the functionality of the rbd219-n1c1 antigen was expected, and using an ace-2 in vitro 412 binding assay we indeed showed similarity to the rbd219-wt antigen. in addition, we showed that, 413 in mice, the modified rbd219-n1c1 antigen triggered an equivalent immune response to the 414 rbd219-wt protein when both proteins were adjuvanted with alhydrogel ® . 415 similar to our previous findings with the sars-cov rbd antigen 9 , we show the rbd219-416 n1c1 protein when formulated with alhydrogel ® elicits a robust neutralizing antibody response with 417 ic50 values up to 4.3x10 5 in mice, as well as an expected t-cell immunological profile. some of the titers of virus-neutralizing antibodies exceed the titer, 2.4x10 4 , measured in-house with human 419 convalescent serum research reagent for sars-cov-2 (nibsc 20/130, national institute for biological 420 standards and control, uk). 421 in a mouse virus challenge model for the sars cov rbd recombinant protein vaccine, we 422 found that alhydrogel ® formulations induced high levels of protective immunity but did not 423 stimulate eosinophilic immune enhancement, suggesting that alhydrogel ® may even reduce immune the selection of the p. pastoris expression platform for the production of the rbd antigen 442 was motivated by the intent to develop a low-cost production process that could easily be transferred 443 to manufacturers in lmics. currently, there are several types of covid-19 vaccine candidates in 444 advanced clinical trials 6,40-45 . the focus of some of the initiatives behind these vaccines is to provide 445 vaccines for the developed world that might struggle to be successful without advanced 446 infrastructure. being able to match the existing experience in lmics with the production of other 447 biologics in yeast increases the probability of successful technology transfer 20 . for example, 448 currently, the recombinant hepatitis b vaccine is produced in yeast by several members of the 449 development country vaccine manufacturers network (dcvmn), and we foresee that, given the 450 existing infrastructure and expertise, those facilities could be repurposed to produce a yeast-produced 451 covid-19 vaccine 46 . recently, the research cell bank and production process for the rbd219-n1c1 452 antigen was technologically transferred to a vaccine manufacturer in india and produced under cgmp 453 conditions with the intent to enter into clinical development. in addition, preclinical studies using the 454 rbd219-wt and rbd219-n1c1 antigens are ongoing to further optimize and evaluate other novel 455 formulations, including a challenge study in a non-human primate model. 456 the covid-19 vaccine-development multiverse. the new england journal of 488 medicine the sars-cov-2 vaccine pipeline: 490 an overview developing safe and effective covid vaccines -operation warp 493 speed's strategy and approach. the new england journal of medicine who. access to covid-19 tools (act) accelerator development of an inactivated vaccine candidate for sars-cov-2 phase 1-2 trial of a sars-cov-2 recombinant spike protein nanoparticle 501 a vaccine targeting the rbd of the s protein of sars-cov-2 induces 503 protective immunity sars-cov-2 spike produced in insect cells elicits high neutralization titres in 505 non-human primates. emerging microbes & infections 9 yeast-expressed sars-cov recombinant receptor-binding domain 508 (rbd219-n1) formulated with alum induces protective immunity and reduces immune 509 yeast-expressed recombinant protein of the receptor-binding domain in 511 sars-cov spike protein with deglycosylated forms as a sars vaccine candidate optimization of the production process and characterization of the yeast-514 expressed sars-cov recombinant receptor-binding domain (rbd219-n1) vaccine candidate engineering a stable cho cell line for the expression of a mers-518 coronavirus vaccine antigen randomized double blind, placebo controlled phase i trial for anti novel 521 coronavirus pneumonia (covid-19) recombinant vaccine (sf9) kbp-201 covid-19 vaccine trial in healthy volunteers a study to evaluate the safety and immunogenicity of covid-19 527 (adimrsc-2f) vaccine clinical study of recombinant novel coronavirus vaccine soberano 01 -estudio fase i/ii, aleatorizado, controlado, adaptativo, a doble ciego 531 y multicéntrico para evaluar la seguridad, reactogenicidad e inmunogenicidad del candidato 532 vacunal profiláctico finlay-fr-1 anti sars -cov -2 en un esquema de dos dosis developing a low-cost and accessible covid-19 vaccine for 535 global health will covid-19 538 become the next neglected tropical disease? plos neglected tropical diseases 14 developing a low-cost and accessible covid-19 vaccine for 541 murine leukemia virus (mlv)-based coronavirus spike-543 pseudotyped particle production and infection vaccine-linked chemotherapy improves benznidazole efficacy for acute 546 transferring luminex(r) cytokine assays to a wall-less plate technology: 548 validation and comparison study with plasma and cell culture supernatants differences in cd44 551 surface expression levels and function discriminates il-17 and ifn-gamma producing 552 a pneumonia outbreak associated with a new coronavirus of probable bat 554 origin sars-cov-2 cell entry depends on ace2 and tmprss2 and is 556 blocked by a clinically proven protease inhibitor covid-19 vaccines: neutralizing 559 antibodies and the alum advantage the potential role of th17 immune responses in 562 coronavirus immunopathology and vaccine-induced immune enhancement. microbes and 563 infection prospects for a safe covid-19 vaccine draft landscape of covid-19 candidate vaccines s-trimer, a covid-19 subunit vaccine candidate, induces protective 570 immunity in nonhuman primates scb-2019 as covid-19 vaccine development of cpg-adjuvanted stable prefusion sars-cov-2 spike 575 antigen as a subunit vaccine against covid-19. biorxiv a study to evaluate the safety and immunogenicity of mvc-cov1901 study of the safety, reactogenicity and immunogenicity of 582 "epivaccorona" vaccine for the prevention of covid-19 (epivaccorona a study to evaluate the safety, tolerability, and immunogenicity of ub-585 612 covid-19 vaccine enhancing blood-stage malaria subunit vaccine immunogenicity in rhesus 587 macaques by combining adenovirus, poxvirus, and protein-in-adjuvant vaccines combining viral vectored and protein-in-adjuvant vaccines against the 590 blood-stage malaria antigen ama1: report on a phase 1a clinical trial. molecular therapy : 591 the journal of the sars-cov-2 mrna vaccine development enabled by prototype 594 pathogen preparedness. biorxiv chadox1 ncov-19 vaccine prevents sars-cov-2 pneumonia in 596 rhesus macaques safety and immunogenicity of the ad26.rsv.pref investigational vaccine coadministered with an influenza vaccine in older adults safety and immunogenicity of the chadox1 ncov-19 vaccine against 601 sars-cov-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial an mrna vaccine against sars-cov-2 -preliminary report. the new 604 england journal of medicine rna-based covid-19 vaccine bnt162b2 selected for a pivotal 606 efficacy study. medrxiv : the preprint server for health sciences prequalified vaccines the authors declare that baylor college of medicine recently licensed the rbd219-n1c1 482 technology to an indian manufacturer for further development. the research conducted in this paper 483 was performed in the absence of any commercial or financial relationships that could be construed as 484 a potential conflict of interest. 485 key: cord-340960-abanr641 authors: brigger, d.; horn, m.p.; pennington, l.f.; powell, a.e.; siegrist, d.; weber, b.; engler, o.; piezzi, v.; damonti, l.; iseli, p.; hauser, c.; froehlich, t.k.; villiger, p.m.; bachmann, m.f.; leib, s.l.; bittel, p.; fiedler, m.; largiadèr, c.; marschall, j.; stalder, h.; kim, p.s.; jardetzky, t.s.; eggel, a.; nagler, m. title: accuracy of serological testing for sars‐cov‐2 antibodies: first results of a large mixed‐method evaluation study date: 2020-09-30 journal: allergy doi: 10.1111/all.14608 sha: doc_id: 340960 cord_uid: abanr641 background: serological immunoassays that can identify protective immunity against sars‐cov‐2 are needed to adapt quarantine measures, assess vaccination responses, and evaluate donor plasma. to date, however, the utility of such immunoassays remains unclear. in a mixed‐design evaluation study, we compared the diagnostic accuracy of serological immunoassays that are based on various sars‐cov‐2 proteins and assessed the neutralizing activity of antibodies in patient sera. methods: consecutive patients admitted with confirmed sars‐cov‐2 infection were prospectively followed alongside medical staff and biobank samples from winter 2018/2019. an in‐house enzyme‐linked immunosorbent assay utilizing recombinant receptor‐binding domain (rbd) of the sars‐cov‐2 spike protein was developed and compared to three commercially available enzyme‐linked immunosorbent assays (elisas) targeting the nucleoprotein (n), the s1 domain of the spike protein (s1) and a lateral flow immunoassay (lfi) based on full‐length spike protein. neutralization assays with live sars‐cov‐2 were performed. results: one‐thousand four‐hundred and seventy‐seven individuals were included comprising 112 sars‐cov‐2 positives (defined as a positive real‐time pcr result; prevalence 7.6%). igg seroconversion occurred between day 0 and day 21. while the elisas showed sensitivities of 88.4% for rbd, 89.3% for s1, and 72.9% for n protein, the specificity was above 94% for all tests. out of 54 sars‐cov‐2 positive individuals, 96.3% showed full neutralization of live sars‐cov‐2 at serum dilutions ≥1:16, while none of the 6 sars‐cov‐2 negative sera revealed neutralizing activity. conclusions: elisas targeting rbd and s1 protein of sars‐cov‐2 are promising immunoassays which shall be further evaluated in studies verifying diagnostic accuracy and protective immunity against sars‐cov‐2. governments worldwide are facing a unique challenge: to save thousands of lives threatened by coronavirus disease 2019 (covid19) , while minimising economic and social damage caused by lockdown and other strict measures. serological immunoassays will play a central role in addressing these challenges for the following reasons 1 . first, serological tests might improve the rate of diagnosis as real-time rt-pcr is associated with a high number of false-negative results due to pre-analytical and other issues 2 . second, antibody assays may support intensive surveillance measures such as universal testing, active case-finding, contact tracing, and linking clusters and thereby may facilitate an exit strategy from lockdown [3] [4] [5] [6] 9, 10 . in patients with severe disease extensive activation of cytokine-secreting cells from the innate and adaptive immune system has been reported to result in a cytokine storm contributing to acute respiratory distress syndrome and multiorgan failure [11] [12] [13] [14] [15] . antibody responses against different sars-cov-2 antigens have been described in serological samples of infected patients. few patients with anti-viral antibodies have been identified in the first 5 days following symptom onset but the positive rate rapidly increases thereafter 16, 17 . to date, antibody testing has focused primarily on two highly abundant structural antigens of sars-cov-2, specifically the nucleoprotein (n) protein this article is protected by copyright. all rights reserved and the spike (s) protein 18 . while the n phosphoprotein ensures the linkage of the viral rna to the membrane 19 , the s glycoprotein binds to ace2 and thereby initiates viral entry into the host cell 13, [20] [21] [22] . neutralizing antibodies (nab) are typically generated against the s protein and often target the receptor binding domain (rbd) 23, 24 . as demonstrated in a vaccination approach using inactivated virus, the rbd represents an immunodominant viral antigen since at least half of the detectable anti-s igg antibodies were directed against the rbd 25 . in contrast, the amount of anti-n antibodies was 30-fold lower. lateral flow immunoassays (lfi) 26, 27 as well as enzyme linked immunosorbent assays (elisa) 28, 29 have been developed but not yet adequately evaluated. while lfis are remarkably fast and only require minutes to perform, significant concern regarding their sensitivity and specificity has been raised 30 . elisas are considered more robust but require highly specialized laboratories with the capacity to run automated high-throughput measurements. at the time of compiling this paper, the diagnostic performance of different immunoassays as well as their predictive value for protective immunity remains unclear. before a broad implementation of immunoassays can be justified, the following points need to be carefully assessed in adequately powered and designed diagnostic studies: (1) diagnostic accuracy (or sensitivity/ specificity respectively) in the acute and subacute phase of the disease, (2) antibody kinetics over time in patients with confirmed covid-19, (3) extent of cross-reactivity with other pathogens and patients with autoimmune disorders, (4) reliability between different assay settings and material characteristics, as well as (5) correlate of protective immunity 3 . with the present study, we aimed to comprehensively establish the utility and diagnostic accuracy of serological immunoassays for sars-cov-2 infection and to explore protective immunity 12 as predicted by such immunoassays in a mixed-method observational study of hospital inpatients as well as medical personnel. this article is protected by copyright. all rights reserved international guidelines on study design were strictly followed 31 and cross-sectional, prospective observational, as well as case-control designs were used. participants were recruited via three different routes: (i) inpatients with a sars-cov-2 test result (real-time pcr; rt-pcr), (ii) medical personnel of the inselspital, and (iii) residual material from patients stored at the liquid biobank bern (www.biobankbern.ch). inclusion criteria of inpatients are (i) hospitalisation in inselspital, (ii) tested positive for sars-cov-2 using rt-pcr (nasopharyngeal swab), (iii) aged 18 or older and (iv) signed general consent (exemption was granted for a few patients). for this manuscript, only inpatients who had tested positive for sars-cov-2 with more than 4 days of residual material available were considered. the temporal pattern of antibody response and seroconversion rate was assessed in a subgroup of inpatients; the first 25 consecutive patients were selected. inclusion criteria of medical personnel were (i) medical staff at inselspital since february 2020, (ii) aged 18 or older, and (iii) signed informed consent. the personnel were recruited via mailing lists. a limited number of fully anonymized, residual biobank samples were also used for the purpose of this study with the inclusion criterion of having been collected from inpatients between december 2018 and february 2019. a total of 54 randomly selected sera from individuals who were tested positive in either of the three elisa immunoassays as well as 6 negative controls were assessed in a live sars-cov-2 neutralization assay (all collected in april 2020). the university hospital bern (inselspital) is one of the largest tertiary hospitals in switzerland covering a catchment area of more than 1 million inhabitants. with several associated smaller hospitals, it provides the full spectrum of general as well as highly specialised medical services. more than 10,000 employees work at the insel gruppe ag. the study was supported by the local covid-19 task force. the study protocol was approved by the appropriate ethics committee and the authorities of the university hospital and conducted in accordance with the declaration of helsinki. the manuscript was prepared according to the standards for reporting diagnostic accuracy studies (stard) guideline 32 . this article is protected by copyright. all rights reserved blood was taken following an established in-house protocol to ensure adequate preanalytical conditions and samples were collected using plastic syringes (serum or lithium heparin respectively, s-monovette®, sarstedt, nümbrecht, germany). only residual material was used in the case of inpatients. two tubes (serum and lithium heparin respectively) were drawn in the case of medical personnel. samples were immediately transported to the central laboratory, processed using a glp laboratory track and centrifuged within 30 minutes with an established protocol 33 . with regard to inpatients, pseudonymized demographical, clinical as well as laboratory data were extracted and transferred by the insel data science center (idsc) from electronic patient documentation. limited data were collected for the purpose of this substudy: age, gender, time interval since rt-pcr (nasopharyngeal swab). a positive sars-cov-2 rt-pcr result was used as additional inclusion criterion. with regard to medical personnel, a redcap database survey was constructed collecting demographical data, covid-19 symptoms (presence, extent and date), comorbidities and risk factors, professional exposure, and date of rt-pcr. the s1 protein and rbd are regarded as ideal candidates for the development of diagnostic tests and vaccines targeting sars-cov-2 34 . the pcaggs plasmid containing the human codon-optimized sequence of the sars cov-2 s protein receptor binding domain (rbd, amino acids r319-f541) with native s signal sequence (amino acids m1-s14) and a c-terminal hexahistidine tag was kindly provided by prof. florian krammer. plasmid dna was prepared using the gene elute hp plasmid maxiprep kit (sigma-aldrich). prior to transfection expi293f cells (thermo-fisher) were grown to a density of 3.0 x 10 6 cells/ml in culture medium (a mixture of 33% expi293 and 66% freestyle-293 media from thermo-fisher). for each liter of transfection, 0.5mg of plasmid dna was diluted in 100ml of culture medium, mixed with 1.3ml fectopro transfection reagent (polyplus), and incubated for 10 minutes at room temperature prior to addition to cells. immediately following transfection cells were supplemented with 100x d-glucose (400g/l) this article is protected by copyright. all rights reserved and 100 x valproic acid (300mm) boost solutions. three days post transfection the cell culture supernatants were harvested by centrifugation at 7,000 x g for 15min. supernatants were passed through a 0.22µm filter and 1:1 diluted with pbs containing 10 mm imidazole. for purification of his-tagged rbd protein 5 ml ni-nta resin (hispur ninta thermofisher) was washed three times with washing buffer (pbs with 10mm imidazole) and incubated on a stir plate at 4°c for 1 hour. subsequently, the mixture was poured into a glass column with a frit and washed 3 times with 5 column volumes of washing buffer. the protein was then eluted three times with 15 ml pbs containing 250mm imidazole. elutions were pooled and dialyzed overnight against pbs using 3.5 kda cutoff snakeskin dialysis tubing. the final protein concentration was determined by nanodrop measurement at a 280. the quality of recombinant rbd protein was analyzed by sds-page and analytical size-exclusion chromatography. all elisa assays were performed on a dsx automated elisa system device (dynex technologies). the in-house assay was prepared as follows: 96-well plates were coated overnight at 4°c with 100µl of 1µg/ml rbd protein in pbs. the following day, each well was blocked with 300µl of pbs/0.15% casein at 4°c until use and at least overnight. subsequently plates were washed twice with pbs and 100µl sera were added at a 1:100 dilution in pbs/0.15% casein for 1 hour at rt. after five washes with 300µl pbs/0.1% tween 100µl of hrp-labeled secondary polyclonal anti-human igm (sigma, a0420) and anti-human igg (sigma, a0170) antibodies were added in a 1:10'000 dilution for 30 minutes at rt. again, the plates were washed 5 times with pbs/0.1% tween and 100µl of tmb substrate solution (sigma, t4444) was added for 15 minutes at rt. the development was stopped by adding 100µl of 0.5m h 2 so 4 and results were measured at od450-620nm. all samples with an od > 0.5 were assigned as positive. several commercial tests were conducted according to the manufacturers' instructions. an elisa produced by euroimmun ag, lübeck, germany targeting the s1 protein as the this article is protected by copyright. all rights reserved immobilized antigen for the detection of igg antibodies was employed. briefly, samples were diluted 1:100 in sample buffer and 100l of diluted samples, pre-diluted positive and negative controls, as well as pre-diluted calibrator were added for 1 hour at 37°c. after three wash steps with 300µl wash buffer, 100µl of hrp-labeled secondary anti-human igg antibodies were added for 30 minutes at 37°c. the plates were washed again three times with wash buffer and 100µl of tmb solution was added for 20 minutes at rt. the development was stopped by adding 100µl of 0.5m h 2 so 4 and results were measured at od450-620nm. antibody values were expressed as a ratio (od sample /od calibrator ). all samples with a ratio >1.1 were assigned as positive. comorbidities and risk factors, which will be used as covariables in subsequent phases of this study, will be extracted from electronic patient records and asked in the redcap this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved at university hospital bern we first established a carefully designed mixed-method diagnostic accuracy study (fig. 1) . this article is protected by copyright. all rights reserved breathlessness, coughing, or loss of smell recombinantly expressed rbd has been used to establish an in-house elisa for the detection of igm and igg anti-sars-cov-2 antibodies in human serum samples (supplementary fig. 1a,b) . optimal serum dilutions were determined by titration of sera derived from six sars-cov-2+ and six sars-cov-2-individuals. the serum dilution of 1:100 allowed efficient discrimination between positive and negative outcome (supplementary fig. 2) . after automatization on a dynex dsx device, the intra-assay (within-run) and inter-assay (day-to-day) precisions of the in-house rbd elisa was assessed (supplementary fig. 3a3e,f) . overall, the in-house rbd elisa assay showed high intra-and inter-assay reproducibility and demonstrated a high degree of agreement between plasma and serum samples. among a subgroup of 25 sars-cov-2+ inpatients, seroconversion for igm and igg antibodies was observed between day 0 and day 21 after the rt-pcr result and between day 2 and day 21 after the start of symptoms (fig. 2) . interestingly, igm and igg antibody responses against rbd and s1 were substantially more pronounced as compared to n. assessment of the longitudinal dynamics of patient sera revealed a marked and consistent increase of igg antibodies for rbd and s1 (fig. 3a) . igm antibodies were measured in the rbd and n elisa and detectable at least for two weeks after seroconversion (fig. 3b) . interestingly, the individual temporal igg and igm patterns showed a high degree of inter-individual variability with one group of patients this article is protected by copyright. all rights reserved of these samples, all were negative for anti-rbd igm and igg, as well as anti-s1 igg. however, two biobank samples tested positive for anti-n igg (elisa; 6.2%), and one tested positive for anti-n igm (elisa; 3%). all samples were negative for anti-s igg and igm (100%) as tested by lfi. the pooled study population consisted of 1477 individuals, 112 of whom tested as rt-pcr positive (prevalence 7.6%). sera from all individuals were tested in the three different elisa setups for igg and igm anti-sars-cov-2 antibodies (fig. 4a) . a subgroup of samples (n=159) was additionally assessed on lfi (fig. 4b) . both assay formats showed high specificity above 94% for igg and igm measurements (supplementary table 4 ). however, the sensitivity between assays and formats varied considerably. the highest sensitivities were reached for igg measurements with the s1 (89.3%) and rbd (88.4%) elisa, followed by igg measurements on n (72.9%) elisa. sensitivities for igm measurements were all considerably lower for both elisa and lfi formats, which could be due to the more transient detectability of igm upon infection. to detect potential sources of variability, we additionally studied the antibody response in salient subgroups of rt-pcr positive individuals (fig. 4c) this article is protected by copyright. all rights reserved in the tested inpatient population, we observed three "false-negative" (negative in s1 elisa despite positive rt-pcr) outcomes. among three false-negative inpatients (p07, p041, and p042), two were measured at an early time-point (patient 7 and 41), and one patient (p042) might have experienced seroconversion at a very late time-point because of a significant increase of antibody titers at day 24 (supplementary fig. 3 and supplementary fig. 4) . in the assessed hospital staff, seven were classified as "falsenegative". all of these reported mild diseases and had symptoms clearly associated with covid-19 (fever, breathlessness, cough, and loss of taste or smell). twenty-two individuals in the hospital staff group tested "false-positive" (positive s1 elisa results despite negative rt-pcr). fourteen of them experienced one or more symptoms clearly associated with covid-19. the remaining eight individuals were clearly positive in at least three assays. all other individuals were either classified as "true-positive" (positive in s1 elisa, and positive in rt-pcr), or as "true-negative" (negative in s1 elisa, and negative in rt-pcr). in terms of performance, the calculated area under the receiver operating characteristic (fig. 5b) . a total of 54 randomly selected sera from individuals who were tested positive in either of the three elisa immunoassays as well as 6 negative controls were assessed in a live sars-cov-2 neutralization assay using ace2-expressing vero-e6 cells (34 inpatient samples, and 26 samples of medical personnel). full neutralization of viral infection has been determined based on 100% inhibition of the cytopathic effect in a serial dilution of the sera (supplementary fig. 6) . the means of highest serum dilutions at which full neutralization was observed correlated remarkably well with the measured antibody responses in the elisa immunoassays (fig. 6a-c) . importantly, 96.3% of the sera from elisa positive individuals showed full inhibition at serum dilutions ≥1:16. the two sera that did not show neutralization (p037 and p042) were drawn at an early time point this article is protected by copyright. all rights reserved where the patients did not yet show antiviral antibodies. both patients, however, fully neutralized the virus after seroconversion at a later time point (fig. 6d) . further, all 6 sera from elisa negative individuals showed no neutralizing activity. of note, one or two elisa assays were negative in 17 samples with full neutralization. we report first results of a large, mixed-design evaluation study which was implemented to compare the diagnostic accuracy of serological immunoassays for sars-cov-2 antibodies. while the time to seroconversion varied substantially between infected individuals, the mounted igg responses were robust and stable over time in all assays relying on rbd, s1 as well as n. with regards to the elisa assays, the overall diagnostic accuracy was adequate with a high specificity. some "false-positive" results are likely due to a rather narrow diagnostic window and limited sensitivity of the rt-pcr as well as asymptomatic disease course 36 . "false-negative" results may be caused by a long seroconversion period observed in some patients and mild disease course in other individuals. the accuracy measures of lfi and n were inferior compared to elisa targeting s1 and rbd. strikingly, there is a high degree of correlation between antibody responses to these viral surface proteins and the neutralizing activity against live sarsa few other studies have previously assessed the diagnostic accuracy of serological immunoassays. recently, long and colleagues studied the antibody response in 285 patients with covid-19 using a magnetic chemiluminescent immunoassay. 37 in accordance with their results, we observed high inter-individual variation in the time to seroconversion. in contrast to their study, we confirmed these findings with an appropriate diagnostic accuracy protocol using different serological immunoassays. in another case-control study, infantino et al. analyzed 61 covid-19 inpatient samples and 64 selected patients collected before 2020 using a magnetic chemiluminescent immunoassay 38 . in agreement with their results, we found limited sensitivity but high specificity of the serological sars-cov-2 immunoassays. in further study conducted at the geneva university hospital, 181 samples of covid-19 patients were included as well as 176 controls collected before 2020, and analyzed with the same s1 elisa that we this article is protected by copyright. all rights reserved used in our study. similar to our results they report a high specificity for igg, particularly with an adjusted cut-off value 39 . in line with other studies the accuracy and performance lfis was rather weak 40,41 . the study presented here adds important value to previous reports as it (i) was designed as a comprehensive diagnostic accuracy study combining different research methods, (ii) directly compares major assay approaches, (iii) was fully approved by all appropriate authorities, (iv) was independently conducted at a university hospital, (v and indicate that such serological tests might even be used to predict protective immunity in near future 45, 46 . to draw further conclusions, however, sars-cov-2 positive patients have to be followed over an extended time period in future studies. this article is protected by copyright. all rights reserved in line with previous studies 47 , we observed that the antibody response is more pronounced in patients with severe disease than patients without (figure 4 , panel c; inpatients, hospitalized patients, older patients). however, the response was similar in patients with mechanical ventilation and hospitalized patients. this is most likely due to limitations in sensitivity, which does not contradict our general observations. in summary, we report the first results of a large, mixed-design evaluation study that has been conducted in an independent academic setting at the university hospital bern to assess the diagnostic accuracy of various immunoassays to determine antibody responses against sars-cov-2. while antibody responses of individual covid-19 patients against rbd and s1 protein were similar, a weaker reactivity against n protein became apparent. the time to seroconversion varied substantially between covid-19 patients but the igg response was robust and stable in all three elisa setups. their overall diagnostic accuracy was adequate with a high specificity but limited sensitivity. the antibody responses measured in these elisas correlated remarkably well with sars-cov-2 neutralizing activity of the sera. on the other hand, accuracy measures of s protein based lfis were poor. together, our results emphasize that appropriate serological immunoassays represent a valuable tool to identify a good portion of patients with previous sars-cov-2 infection, will help to facilitate exit strategies from lockdown and might even be used to predict immunity to sars-cov-2 in near future. this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved accepted article serology assays to manage covid-19 the laboratory diagnosis of covid-19 infection: current issues and challenges the important role of serology for covid-19 control connecting clusters of covid-19: an epidemiological and serological investigation investigation of three clusters of covid-19 in singapore: implications for surveillance and response measures universal weekly testing as the uk covid-19 lockdown exit strategy immunology of covid-19: mechanisms, clinical outcome, diagnostics and perspectives -a report of the european academy of allergy and clinical immunology (eaaci) distribution of ace2, cd147, cd26, and other sars-cov-2 associated molecules in tissues and immune cells 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all rights reserved 27 cross-sectional pilot study exploring the feasibility of a rapid sars-cov-2 immunization test in health and nonhealthcare workers a serological assay to detect sars-cov-2 seroconversion in humans. medrxiv sars-cov-2 seroconversion in humans: a detailed protocol for a serological assay, antigen production, and test setup evaluation of antibody testing for sars-cov-2 using elisa and lateral flow immunoassays studies for evaluating diagnostic and prognostic accuracy stard 2015: updated reporting guidelines for all diagnostic accuracy studies rapid centrifugation in the routine hemostasis laboratory sars-cov-2 immunogenicity at the crossroads detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr distinct characteristics of covid-19 patients with initial rrt-pcr-positive and rrt-pcr-negative results for sars-cov-2 antibody responses to sars-cov-2 in patients with covid-19 diagnostic accuracy of an automated chemiluminescent immunoassay for 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the spike all authors declare that there is no conflict of interests. this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved key: cord-356264-q0yqnlyl authors: armijos-jaramillo, vinicio; yeager, justin; muslin, claire; perez-castillo, yunierkis title: sars-cov-2, an evolutionary perspective of interaction with human ace2 reveals undiscovered amino acids necessary for complex stability date: 2020-03-23 journal: biorxiv doi: 10.1101/2020.03.21.001933 sha: doc_id: 356264 cord_uid: q0yqnlyl the emergence of sars-cov-2 has resulted in more than 200,000 infections and nearly 9,000 deaths globally so far. this novel virus is thought to have originated from an animal reservoir, and acquired the ability to infect human cells using the sars-cov cell receptor hace2. in the wake of a global pandemic it is essential to improve our understanding of the evolutionary dynamics surrounding the origin and spread of a novel infectious disease. one way theory predicts selection pressures should shape viral evolution is to enhance binding with host cells. we first assessed evolutionary dynamics in select betacoronavirus spike protein genes to predict where these genomic regions are under directional or purifying selection between divergent viral lineages at various scales of relatedness. with this analysis, we determine a region inside the receptor-binding domain with putative sites under positive selection interspersed among highly conserved sites, which are implicated in structural stability of the viral spike protein and its union with human receptor hace2. next, to gain further insights into factors associated with coronaviruses recognition of the human host receptor, we performed modeling studies of five different coronaviruses and their potential binding to hace2. modeling results indicate that interfering with the salt bridges at hot spot 353 could be an effective strategy for inhibiting binding, and hence for the prevention of coronavirus infections. we also propose that a glycine residue at the receptor binding domain of the spike glycoprotein can have a critical role in permitting bat variants of the coronaviruses to infect human cells. the recent emergence of the novel sars coronavirus 2 (sars-cov-2) marked the third introduction of a highly pathogenic coronavirus into the human population in the twenty-first century, following the severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov). the first, sars-cov emerged in november 2002 in the guangdong province of china and spread globally during 2002-03, infecting more than 8000 people and causing 774 deaths (drosten et al., 2003; who, 2004) . mers-cov was the second emergence and was first detected in saudi arabia in 2012 and resulted in nearly 2500 human infections and 858 deaths in 27 countries (fehr et al., 2017; zaki et al., 2012) . in december 2019, sars-cov-2, a previously unknown coronavirus capable of infecting humans was discovered in the chinese city of wuhan, in the hubei province zhu et al., 2020) . sars-cov-2 is associated with an ongoing pandemic of atypical pneumonia, now termed coronavirus disease 19 (covid-2019) that has affected over 209,000 people with 8778 fatalities as of march 19, 2020 (who, 2020 . both sars-cov and mers-cov are thought to have originated in colonies of bats, eventually transmitted to humans, putatively facilitated by intermediate hosts such as palm civets and dromedary camels, respectively (cui et al., 2019) . the genome of sars-cov-2 shares about 80% nucleotide identity with that of sars-cov and is 96% identical to the bat coronavirus batcov ratg13 genome, reinforcing the probable bat origin of the virus . however, better assessing the evolutionary dynamics of sars-cov-2 is an active research priority worldwide. sars-cov, mers-cov and sars-cov-2 belong to the genus betacoronavirus within the subfamily coronavirinae of the family coronaviridae. members of this family are enveloped viruses containing a single positive-strand rna genome of 27-32 kb in length, the largest known rna virus genome. the coronavirus spherical virion consists of four structural proteins: the spike glycoprotein (s-protein), the envelope protein, membrane protein and nucleocapsid. the transmembrane trimeric s-protein plays a critical role in virus entry into host cells (gallagher & buchmeier, 2001; tortorici & veesler, 2019) . it comprises two functional subunits: s1 subunit, where the receptor-binding domain (rbd) is found, is responsible for binding host cell surface receptors and s2 subunit mediates subsequent fusion between the viral and cellular membranes (kirchdoerfer et al., 2016; yuan et al., 2017) . both sars-cov and sars-cov-2 interact directly with angiotensin-converting enzyme 2 (ace2) to enter host target cells (hoffmann et al., 2020; li et al., 2003; walls et al., 2020; yan et al., 2020) . in the case of sars-cov, ace2 binding was found to be a critical determinant for the virus host range and key amino acid residues in the rbd were identified to be essential for ace2-mediated sars-cov infection and adaptation to humans (li et al., 2006; li et al., 2005) . understanding the dynamics that permits a virus to shift hosts is of considerable interest, and further be an essential preliminary step towards facilitating the development of vaccines and the discovery of specific drug therapies. we employ a multidisciplinary approach to look for evidence of diversifying selection on the s-protein gene, and model the interactions between human ace2 (hace2) and the rbd of selected coronavirus strains, which ultimately afforded us novel insights detailing virus and host cell interactions. given the rapid pace of discovery we aim to add clarity to evolutionary dynamics of diseases strains by more precisely understand the dynamics at the s-protein and its interaction with hace2. the most similar genomes to sars-cov-2 mn908947 were retrieved using blastp (altschul et al., 1997) vs the nr database of genbank (table 1) . genomes were then aligned using mauve (darling et al., 2004) and the s-protein gene was trimmed. the extracted genomic sections were aligned using a translation align option of geneious (kearse et al., 2012) with a mafft plugin (katoh & standley, 2013) . the phylogenetic reconstruction of s-protein genes was performed with phyml (guindon et al., 2010) , using a gtr+i+g model, using 100 non-parametric bootstrap replicates. both, the alignment and the tree were used as input for paml codeml (yang, 2007) . the presence of sites under positive selection was tested by the comparison of m2 (it allows a proportion of positive, neutral and negative selection sites in the alignment) vs m1 (it allows a proportion of neutral and negative selection sites in the alignment) and m8 (ω follows a beta distribution plus a proportion of sites with ω>1) vs m7 (ω follows a beta distribution) models using the ete toolkit 3.0 (huerta-cepas et al., 2010) . the presence of tree nodes under positive selection was obtained with the free branch model and then tested by the comparison of branch free (different ω for each selected branches) vs m0 (negative selection for all sites and branchesnull model) and branch free vs branch neutral (ω=1 for selected branches) models. the presence of sites with positive selection under specific branches of the tree was tested with bsa (proportion of sites with positive selection in a specific branch of the tree) vs bsa1 (proportion of sites with neutral and purifying selection in a specific branch of the tree) models. likelihood ratio test (lrt) was performed (p≤ 0.05) to compare the hypothesis contrasted by each model. we used the set of programs available in hyphy (kosakovsky pond et al., 2020) , fast unconstrained bayesian approximation (fubar) to detect overall sites under positive selection, and fixed effects likelihood (fel) to detect specific sites under positive selection in specific branches. we used mixed effects model of evolution (meme) to detect episodic positive/diversifying selection and adaptive branch site rel (absrel) to detect branches in the tree under positive selection. the web server datamonkey (weaver et al., 2018) was used to perform the hyphy analyses. finally, treesaap 3.2 (woolley et al., 2003) was used to detect sites under adaptation (in terms of physicochemical properties). the same alignment and tree described above were used for this analysis. all these experiments were performed again using the s-protein genes of a shorter list of accessions and more distantly related (broad dataset) to sars-cov-2 (ay304488, ay395003, dq412043, fj882957, ky417144, mg772933, mg772934, mn908947, nc_004718) to test the reproducibility of the predicted branches and sites under positive selection. the crystal structure of the sars-cov s-protein rbd ( genebank id nc_004718) in complex with hace2 was retrieved from the protein data bank (code 2ajf) (berman et al., 2000) . homology models were constructed using this structure as template for the rbds of sars-cov-2 (sars2, genebank id mn908947), the bat sars-like coronavirus isolate rm1 (rm1, genebank id dq412043) and the bat sars-like coronavirus isolate rs4231 ( rs4231, genebank id ky417146). one additional homology model for the g496d mutant of the sars-cov-2 rbd (sars2-mut) was constructed. homology models were built with modeller v. 9 (webb & sali, 2016) using its ucsf chimera interface (pettersen et al., 2004) . five models were constructed for each target sequence and the one with the lowest dope score was selected for the final model. all non-amino acidic residues were removed from the sars-cov rbd-hace2 complex to obtain a clean complex. the homology models of the sars2, rm1, rs4231 rbds and sars2-mut were superimposed into the sars-cov rbd to obtain their initial complexes with hace2. these complexes were then subject to molecular dynamics (md) simulations and estimation of their free energies of binding using amber 18 (case et al., 2018) . for the later, ace2 was considered as the receptor and the rbds as ligands. the protocol described below was employed for all complexes and otherwise noted default software parameters were employed. systems preparation was performed with the tleap program of the amber 18 suite. each complex was enclosed in a truncated octahedron box extending 10 å from any atom. next, the boxes were solvated with tip3p water molecules and na+ ions were added to neutralize the excess charge. systems were minimized in two steps, the first of which consisted in 500 steps of the steepest descent algorithm followed by 500 cycles of conjugate gradient with protein atoms restrained using a force constant of 500 kcal/mol.å 2 . the pme method with a cutoff of 12 å was used to treat long range electrostatic interactions. during the second minimization step the pme cutoff was set to 10 å and it proceeded for 1500 steps of the steepest descent algorithm followed by 1000 cycles of conjugate gradient with no restrains. the same pme cutoff of 10 å was used in all simulation steps from here on. both minimization stages were performed at constant volume. the minimized systems were heated from 0 to 300 k at constant volume constraining all protein atoms with a force constant of 10 kcal/mol.å 2 . the shake algorithm was used to constrain all bonds involving hydrogens and their interactions were omitted from this step on. heating took place for 10000 steps, with a time step of 2 fs and a langevin thermostat with a collision frequency of 1.0 ps -1 was employed. all subsequent md steps utilized the same thermostat settings. afterward, the systems were equilibrated for 100 ps at a constant temperature of 300 k and a constant pressure of 1 bar. pressure was controlled with isotropic position scaling with a relaxation time of 2 ps. the equilibrated systems were used as input for 10 ns length production md simulations. the free energies of binding were computed under the mm-pbsa approach implemented in ambertools 18 (case et al., 2018) . a total of 100 md snapshots were evenly selected, one every 50 ps, from the last 5 ns of the production run for mm-pbsa calculations. the ionic strength was set to 100 mm and the solute dielectric factor was set to 4 for all systems. in order to detect branches and sites under positive/negative selection, two datasets were explored. the first ('closer' dataset) harbors the most similar genomes to wuhan-hu-1 coronavirus (sars-cov-2) (mn908947). for this dataset, several genomes were excluded from the analysis because they showed minimal variation to other sequences. we used a preliminary phylogeny to select a representative isolate of each clade (table 1 ) in order to exclude highly similar sequences. the second dataset ('broad' dataset) includes some accessions of the first dataset plus isolates less related to sars-cov-2, like sars-like coronavirus isolates from different countries (see methods). we compare the results of two dataset because the phylogenetic distance between orthologues in a given dataset has been demonstrated to alter the ability to detect selection in paml and meme (mcbee et al., 2015) . in both datasets, we observed evidence of purifying selection in the majority of nodes of the tree. specifically, in the 'closer' dataset we identified 38 nodes with evidence of negative selection, and 4 under positive selection when free ratios model of codeml model was applied. to confirm the four nodes under positive selection we use ltr test for contrasting hypothesis using branch free, branch neutral and m0 models of codeml. using these approximations, any node predicted by free ratios model with ω>1 was significantly different to the purifying (ω<1) or neutral (ω=1) models. an equivalent analysis was performed using absrel of hyphy, observing episodic diversifying selection in at least 8 of 41 nodes of the phylogenetic tree reconstructed with the 'closer' dataset ( figure 1 ). interestingly, one of the divisions detected with diversifying selection was the branch that contains sars-cov-2, pangolin coronavirus isolate mp789 and bat coronavirus ratg13 (called sars-cov-2 group) but not the specific branch that contains sars-cov-2. under positive selection in sars-cov-2 using the closer dataset without pangolin coronavirus isolate mp789. it is interesting despite the influence of the dataset in the results, because site f486 is directly involved in hace2-rbd interaction , explaining at least in part strong selection at this site. moreover, the branch-site model bsa (positive selection) vs bsa1 (relaxation) of codeml were compared to find evidence of sites under positive selection in branch of sars-cov-2 using the 'closer' dataset, but bsa does not show significant differences with bsa1 (p>0.05) indicating selection cannot be confidently implicated, but it was when other datasets were used (including f486). in summary, we do find evidence of sites under positive/episodic selection in branches of close related strains of wuhan-hu-1 isolate coronavirus. however, there is not strong evidence of specific sites under positive selection in sars-cov-2 using the tools mentioned in this work. this result does not disregard the presence of positive selection sites in sars-cov-2, nonetheless, it shows the limitation of the methods to identify with precision specific sites under positive selection in a precise taxon of a phylogenetic tree. we further warn researchers need to be conservative with interpretations of studies utilizing these methodologies, given the equivocal results can be generated by datasets varying in genetic similarity. to complement our analyses looking for evidence of selection among lineages, we specifically analyzed for patterns of selection across sites in the s-protein genes, we used the sites models available in codeml and hyphy. model m2 of codeml detected 0.133 % of sites under positive selection (ω>1) and models m1 and m2 detected 85% of sites under purifying selection (ω<1). model m2 explains the significant data better (p=7e-4) than m1 model, that takes in account only sites with neutral and purifying selection. to resolve these ambiguities in positive selection sites we calculate putative selection sites with codeml (using bayes empirical bayes from m2 and m8 models) and fubar with different datasets reflecting the addition of novel sequences to online repositories (broad, closer, closer without mn996532 and mt084071 and closer without mt084071) and we obtain different results. it is becoming increasingly clear that predictions of positive selected sites are highly influenced simply by the diversity of the individual sequences included in the datasets. in any case, the majority of predicted sites converge in the region between 439 to 508, a section of the rbd. additionally, we used treesaap to detect important biochemical amino acid properties changes over regions and/or sites along betacoronavirus s-protein. using a sliding window size of 20 (increasing by 1) we detect that the region between 466 to 500 (using sars-cov-2 s-protein as a reference) have drastic amino acid changes for alpha-helical tendencies. in addition, the section between 448 to 485 residues registers radical changes in amino acids implicated in the equilibrium constant (ionization of cooh). in the structural analysis we performed, the section between 472 to 486 forms a loop that is not present in certain s-proteins of coronavirus isolated in bats. this loop extends the interaction area between rbd of s-protein and human ace2, in fact, the lack of these loop decreases the negative energy of interaction (increasing the binding) among these two molecules (see table 2 ). these results obtained from independent analysis strongly highlight the importance of 439 to 508 section. additionally, important hace2-binding residues in the rbd from sars-cov-2 obtained from the crystallography and structure determination performed by shang et al. (2020) are also present in the section we highlight here. we propose that this region is the most probable to contain the sites under positive selection due to predictions by our codeml and fubar models. in that sense, we refer to this section as region under positive selection (rps). it is important to additionally clarify that even inside the rps we found at least 20 aa highly conserved between coronaviruses, several of them are predicted as sites under purifying selection. this shows that it is necessary to maintain sites without change around polymorphic sites, probably to conserve the protein structure and at the same time to have the ability to colonize more than one host. interestingly, the rps of the pangolin coronavirus isolate mp789 differs only in one amino acid with the homologous region of sars-cov-2, whereas in contrast the bat coronavirus ratg13 (the overall most similar isolate to sars-cov-2 sequenced at the moment) shows 17 differences in the same region. several explanations could derive from this observation. the hypothesis of recombination inside the pangolin between a native coronavirus strain and a bat coronavirus (like ratg13) is congruent with our observation. this scenario was proposed and discussed as the origin of sars-cov-2 by (lam et al., 2020; wong et al., 2020; xiao et al., 2020) , however, other explanations are possible. if the sars-cov-2, ratg13 and pangolin coronavirus mp789 isolate are closely related as shown in the tree of the figure 1 , we are observing the ancestral sequence of rps in human and pangolin coronaviruses, and a mutated version in bat virus. elucidating the origin of sars-cov-2 is beyond the scope of this work, nevertheless sequencing of new coronavirus isolates in the near future could resolve this question. with a list of broader observations related to the role of selection across viral genomes we aimed to specifically understand how these regions could affect virus/host interactions. to understand more in deep the importance of rps in the evolution of sars-cov-2, we quantified the relative importance of this region in the interaction between rbd and hace2. in that sense, md simulations were run for five complexes (listed in methods). in all cases the systems were stable with root mean square deviations (rmsd) of their backbones between 1.11 å and 3.30 å relative to the initial complexes structures during the last 5 ns of the production run. we first investigated the network of contacts between the ligands (coronaviruses rdb) with the receptor (hace2). overall, all complexes present a large number of contacts between the ligands and the receptor in at least 50% of the md snapshots selected for mm-pbsa calculations. common interactions with t27, f28, k31, h34, y41, k353, g354, d355 and r357 of the receptor are observed in all systems. the full networks of interactions between the coronaviruses and the hace2 receptor are provided as supporting information. next we estimated the free energies of binding of the coronaviruses' rbds to hace2 and the results of these evaluations are summarized in table 2 . these calculations show that the sars2, sars and rs4231 viruses are predicted to favorably bind to the human hace2 receptor, while the rm1 and sars2-mut variants present unfavorable free energies of binding. the fact that the bat's coronavirus rs4231, in addition to sars and sars2, presents favorable interaction with hace2 is in accordance with the previous observation that it is able to infect human cells expressing this protein (hu et al., 2017) . to get more insights into the contribution of the receptor and the rbds to the binding process, we performed energy decomposition experiments. the contribution of each residue in the studied coronaviruses that interact with the hace2 receptor are shown in table 3 . rows are presented in such a way that each of them contains the residues occupying the same position in the viruses rbds structures as in the sar2 rbd structure. from here on, residues numeration will take that of sars2 as reference. in general, most rbds residues show negative values of contribution to the free energies of binding to the human receptor. all studied rbds, except that of the rm1 coronavirus, have amino acids with large favorable contributions to the free energies of binding that directly interact with hace2: k417 of sars2 and sars2-mut, r426 in sars and r480 in rs4231. on the other hand, the g476d mutation (d463 present in bat coronavirus strains) have a negative contribution to the binding of the rdb to hace2. this site was predicted to be under purifying selection by fubar analyses, and is located within the rps. strikingly, the g496d mutation (sars2 numeration) has a large negative influence in the free energy of binding in the two complexes that contain it. it is also worth noting that the three aspartic acid substitutions present in all systems negatively contribute to the systems stability. taking into account that the only difference between sars2 and sars2-mut is the g496d mutation, we postulate that this rbd position is critical for the human receptor recognition by coronaviruses. to the best of our knowledge, no coronavirus having aspartic acid at this position is able to infect human cells. this result supports the prediction from fubar analyses indicating that the site g496d is under purifying selection. combined, our results strongly suggest that the mutation of the d496 residue present in the coronaviruses from bats is critical for their rbds to recognize the human hace2 receptor. additionally, it shows the importance of sites under purifying selection in rps for the rbd evolution. to better interpret the influence of the key interactions between the coronaviruses rbds and their hace2 receptor, their interactions were analyzed. to select the representative structure of each system the md snapshots employed for mm-pbsa calculations were clustered. then, the representative structure of a system was selected as the centroid of the most populated cluster. the predicted rbd-hace2 complexes for sars2, sars and sars2-mut are depicted in figure 2 . many studies have focused on coronaviruses mutations that favor adaptations for human hosts infections. for example, it has been shown that specific substitutions at positions 455, 486, 483, 494 and 501 (442, 472, 479, 480 and 487 in sars) of the rbd of sars favors the interaction between the rbd of sars and hace2 (cui et al., 2019) . likewise, homology modeling studies found favorable interactions between the residues occupying these positions in the sars2 rbd and the human receptor . the cornerstone of these favorable interactions is the complementarity of the rbds with hot spots 31 and 353. these are salt bridges between k31 and e35 and between d38 and k353 of ace2 which are buried in a hydrophobic environment (see figure. 2). in the cases of sars2 and sars, q493 (n479 in sars) and n501 (t487 in sars) add support to the hot spots according to these previous studies. these observations should also hold for the rs4231 strain, however the n501a change in the later compared to sars2 (a488 in rs4231) add little support to hot spot 353. in this case, to continue permitting human infection, the large favorable contribution of r480 in rs4231 to the free energy of binding could compensate the weak support provided by a488 to hot spot 353. interestingly, k353 is the residue forming the largest network of contacts with the analyzed rbds among those belonging to both hot spots. our simulations also show that in sars2 and sars the rbd amino acids with the largest contribution to the free energy of binding, k417 and r426 (see table 3 ) respectively, do not interact with any hot spot residue. instead, they interact with d30 of hace2 in the sars2 complex and with e329 of the human receptor in the sars complex. this could indicate that interactions additional to those previously identified with the hace2 hotspots could be critical for the stabilization of the rdb-human receptor complexes. finally, we analyzed the possible reasons for the predicted negative impact that the g496d mutation has on the predicted free energies of binding of the rbd to hace2. as depicted in figure 2 , g496 directly interacts with k353 in hot spot 353 and its mutation interferes with the d38-k353 salt bridge. specifically, d496 of the rdb point to d38 of hace yields a high electric repulsion between these amino acids. consequently, this portion of the rbd is pushed to a position further from hace2 than that observed in the wild type receptor, resulting in the reduction of its network of contacts with k353. as a result, the binding of the rbd to hace2 is considerably inhibited and unlikely to occur. a priority in ongoing research is to better understand coronavirus evolution, with specific interests in understanding the role of selection pressures in viral evolution, and clarifying how viral strains can infect novel hosts. our experiments suggest that there are sites under positive selection in the s-protein gene of sars-cov-2 and other betacoronaviruses, particularly in a region that we called rps (region under positive selection) inside of the rbd. however, we have identified that by in large, sites in this region (and overall, in the s-protein gene) are under purifying selection. particularly, for the site d496g, the presence of aspartic acid seems indispensable for the interaction with the hace2. additionally, we performed md simulations and free energies of binding predictions for five different complexes of coronaviruses that do and do not infect human cells. our results suggest that as long as no disrupting interference occur with both salt bridges at hot spots 31 and 353 coronaviruses are able to bind with hace2. modeling results suggest that interference with the hot spot 353 could be and effective strategy for inhibiting the recognition of the rbd of the sars-cov-2 spike protein by its human host receptor ace2 and hence prevent infections. although additional simulations and experiments are required, all evidence suggests that the mutation of d496 in the bat variants of the coronaviruses permit infection of human cells. giving the large contribution of sars2 k417 to the free energy of binding of the rbd to hace2 we propose that blocking its interaction with the receptor d30 could be a promising strategy for future drug discovery efforts. gapped blast and psi-blast: a new generation of protein database search programs the 2019-new coronavirus epidemic: evidence for virus evolution the protein data bank origin and evolution of pathogenic coronaviruses mauve: multiple alignment of conserved genomic sequence with rearrangements identification of a novel coronavirus in patients with severe acute respiratory syndrome middle east respiratory syndrome: emergence of a pathogenic human coronavirus coronavirus spike proteins in viral entry and pathogenesis new algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of phyml 3.0. systematic biology sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor discovery of a rich gene pool of bat sars-related coronaviruses provides new insights into the origin of sars coronavirus clinical features of patients infected with 2019 novel coronavirus in ete: a python environment for tree exploration more effective purifying selection on rna viruses than in dna viruses mafft multiple sequence alignment software version 7: improvements in performance and usability geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data pre-fusion structure of a human coronavirus spike protein hyphy 2.5-a customizable platform for evolutionary hypothesis testing using phylogenies identification of 2019-ncov related coronaviruses in malayan pangolins in southern china animal origins of the severe acute respiratory syndrome coronavirus: insight from ace2-s-protein interactions angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus receptor and viral determinants of sars-coronavirus adaptation to human ace2 the effect of species representation on the detection of positive selection in primate gene data sets ucsf chimera-a visualization system for exploratory research and analysis structural basis for receptor recognition by the novel coronavirus from wuhan on the origin and continuing evolution of sars-cov-2 structural insights into coronavirus entry structure, function, and antigenicity of the sars-cov-2 spike glycoprotein receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars datamonkey 2.0: a modern web application for characterizing selective and other evolutionary processes comparative protein structure modeling using modeller summary of probable sars cases with onset of illness from 1 evidence of recombination in coronaviruses implicating pangolin origins of ncov-2019 treesaap: selection on amino acid properties using phylogenetic trees isolation and characterization of 2019-ncov-like coronavirus from malayan pangolins structural basis for the recognition of the sars-cov-2 by full-length human ace2 paml 4: phylogenetic analysis by maximum likelihood cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains a pneumonia outbreak associated with a new coronavirus of probable bat origin a novel coronavirus from patients with pneumonia in china the authors declare that they have no conflicts of interest. key: cord-339724-roj8ksvc authors: lan, jiaming; deng, yao; chen, hong; lu, guangwen; wang, wen; guo, xiaojuan; lu, zhuozhuang; gao, george f.; tan, wenjie title: tailoring subunit vaccine immunity with adjuvant combinations and delivery routes using the middle east respiratory coronavirus (mers-cov) receptor-binding domain as an antigen date: 2014-11-18 journal: plos one doi: 10.1371/journal.pone.0112602 sha: doc_id: 339724 cord_uid: roj8ksvc the development of an effective vaccine is critical for prevention of a middle east respiratory syndrome coronavirus (mers-cov) pandemic. some studies have indicated the receptor-binding domain (rbd) protein of mers-cov spike (s) is a good candidate antigen for a mers-cov subunit vaccine. however, highly purified proteins are typically not inherently immunogenic. we hypothesised that humoral and cell-mediated immunity would be improved with a modification of the vaccination regimen. therefore, the immunogenicity of a novel mers-cov rbd-based subunit vaccine was tested in mice using different adjuvant formulations and delivery routes. different vaccination regimens were compared in balb/c mice immunized 3 times intramuscularly (i.m.) with a vaccine containing 10 µg of recombinant mers-cov rbd in combination with either aluminium hydroxide (alum) alone, alum and polyriboinosinic acid (poly i:c) or alum and cysteine-phosphate-guanine (cpg) oligodeoxynucleotides (odn). the immune responses of mice vaccinated with rbd, incomplete freund’s adjuvant (ifa) and cpg odn by a subcutaneous (s.c.) route were also investigated. we evaluated the induction of rbd-specific humoral immunity (total igg and neutralizing antibodies) and cellular immunity (elispot assay for ifn-γ spot-forming cells and splenocyte cytokine production). our findings indicated that the combination of alum and cpg odn optimized the development of rbd-specific humoral and cellular immunity following subunit vaccination. interestingly, robust rbd-specific antibody and t-cell responses were induced in mice immunized with the rrbd protein in combination with ifa and cpg odn, but low level of neutralizing antibodies were elicited. our data suggest that murine immunity following subunit vaccination can be tailored using adjuvant combinations and delivery routes. the vaccination regimen used in this study is promising and could improve the protection offered by the mers-cov subunit vaccine by eliciting effective humoral and cellular immune responses. in 2012 a novel human coronavirus, middle east respiratory syndrome coronavirus (mers-cov), caused outbreaks of a sars-like illness in the middle east, and is now considered a threat to global public health [1, 2] . as of july 23, 2014, the world health organization (who) reported 837 confirmed cases of mers-cov infection, including 291 deaths (a case fatality rate of 34.8%) [3] . now, studies show that camels are a likely primary source of the mers-cov that is infecting humans [4, 5, 6] . but the routes of transmission between camels and people which is the key point to stop transmission of the virus, is far from clearly understood. the continued threat of mers-cov necessitates the development of an effective vaccine. some studies have indicated that recombinant receptor-binding domain (rrbd) protein of mers-cov spike (s) is a good candidate antigen for a mers-cov subunit vaccine [7, 8, 9, 10] . however, highly purified proteins are typically not inherently immunogenic, as they usually lack the means to directly stimulate the innate immune system [11] . besides, they are often prone to degradation. hence, they call for efficient delivery systems and potent immunostimulants, jointly denoted as adjuvant(s) to evoke the desired antigen-specific immune response phenotype enabling successful vaccination [12] . aluminium is one of the most common adjuvant in non-living vaccines, has a record of successful use in human vaccination where it promotes antibody-mediated protective immunity [13] . another classic adjuvant is that based on a water-in-oil-emulsion formulation, such as incomplete freund's adjuvant (ifa). recently, researches have focused on adjuvants that signal through pattern recognition receptors (prrs), such as toll-like receptors (tlrs) [14] . cysteine-phosphate-guanine (cpg) oligodeoxynucleotides (odns), which activate b cells and plasmacytoid dendritic cells via tlr9 and induce both innate and adaptive immunity, are currently being developed as a vaccine adjuvant [15] . another frequently used adjuvant is polyriboinosinic acid (poly(i:c)), a synthetic dsrna that mimics the effects of naturally occurring dsrna, a tlr3 agonist [16, 17] . beside of enhancing the immune response, adjuvant(s) can tailor-make the polarization immune response. for example, ppolarized th1-type immunity can be achieved by the addition of freund's adjuvant or cpg dna to an antigen. on the other hand, th2 antibody responses can be induced by the alum, as indicated by increased igg1 relative to igg2a [18, 19] . however, in situations where both th1 and th2 responses are required for protection, the choice of one regimen over another might be counter effective. this has led to additional research for alternative adjuvants or adjuvant combinations that promote balanced mixed th1/th2 responses [18] . in recent years, the combination of antigens with more than one adjuvant, called the adjuvant system approach has produced vaccines with the ability to generate effective immune responses adapted to both the pathogen and the target population [20] . by using multiple adjuvants in combination, antigen presenting cell (apc) activation is influenced at more than one level, guiding the subsequent adaptive pathways and ultimately inducing a more robust immune response [20] . the induction of a robust humoral, including potent neutralizing antibodies, and cellular immune response is likely essential for immediate and sustained protective immunity in a mers-cov vaccine design. in this study, different adjuvants combination regimens including alum, ifa, cpg and poly(i:c) were compared in an effort to promote balance between th1 and th2 immune response to bystander rrbd antigen spanning residues 367-606 of mers-cov s in a murine model to develop an effective vaccine against mers-cov infection. animal studies were carried out in strict compliance with the guide for the care and use of laboratory animals of the people's republic of china. the study protocol was approved by the committee on the ethics of animal experiments of the chinese centre for diseases control and prevention. all procedures were performed under ethylether anesthesia and all efforts were made to minimize suffering. mers-cov rrbd protein, containing a 240-amino-acid fragment spanning residues 367-606 ( figure 1a ) of genbank number jx869059, was prepared using a bac-to-bac baculovirus expression system as described in detail previously [21] . the required rrbd was measured by sds-page ( figure 1b) and western blot ( figure 1c ) with a mice polyclonal antibody against spike of mers-cov ( figure 1c ). before vaccination, the rrbd protein was quantified by bradford method. the rrbd protein was combined with different adjuvants immediately prior to immunisation. aluminium hydroxide was kindly provided by the north china pharmaceutical group corporation genetech biotechnology development company. the odn motif containing unmethylated cpg (59-tccat-gacgttcctgacgtt-39) was synthesised by takara bio inc. poly(i:c) and ifa were purchased from sigma (st. louis, mo). a single dose (10 mg) of rrbd protein (100 ml) was combined with either 100 mg of alum alone (rbd/a), alum plus 10 mg of cpg (rbd/a+c), alum plus 50 mg of poly(i:c) (rbd/ a+p) or 10 mg of cpg and 100 ml of ifa (rbd/i+c). six-to-eight-week-old female balb/c mice (animal care centre, chinese academy of medical science, beijing, china) were randomly distributed into eight groups. eight mice of each group were vaccinated three times with rrbd proteins at 3-week intervals by either an intramuscular (i.m.) or a subcutaneous (s.c.) route (table 1 ). sera were collected 2 weeks after each vaccination and heat-inactivated at 56uc for 30 min before detection of rbdspecific and neutralizing antibodies. mice were scarified 2 weeks after the last immunisation, and their lungs and spleens were harvested for detection. a schematic of the vaccination and analysis timeline is shown in figure 2 . elisa was used to detect the mers-cov rbd-specific antibody response in immunised mice. briefly, 96-well elisa plates were pre-coated with rrbd protein (100 ng/well) overnight at 4uc and blocked with 2% non-fat milk for 2 h at 37uc. serially diluted sera of eight mice in each group were added to the plates and incubated at 37uc for 1 h, followed by four washes with phosphate-buffered saline (pbs) containing 0.1% tween 80 (pbst). bound antibodies were incubated with hrp-conjugated anti-mouse igg, igg1, igg2a or igg2b (1:5,000, sigma) for 1 h at 37uc. the reaction was visualised by using 3, 39, 5, 59tetramethylbenzidine (tmb) peroxidase substrate solution (invitrogen) and stopped by addition of 2 m h 2 so 4 . absorbance at 450 nm was measured using an elisa plate reader (wellscan mk 3). the cut-off value was set 2.1-fold above that of the negative control. antibody avidity was determined using the elisa method described by vermont et al [22] . briefly, sera were diluted to a titre of 1:100, and an ascending concentration of the chaotropic agent nascn (0-7 m) was added to the plate. plates were incubated for 15 min at room temperature (rt) before washing and development to determine total igg. as a control for antibody specificity, elisa was used to measure the total anti-mers-cov igg titres of pre-and post-vaccination sera samples. the conventional neutralization assay using live mers-cov is cumbersome and has to be performed in biosafety level-3 facilities. therefore, we adapted a mers-cov pseudovirus system which is sensitive and quantitative, and can be conducted in biosafety level-2 facilities as reported by zhao et al [23] . in brief, 293t cells were co-transfected with a plasmid encoding codon-optimized mers-cov s protein and a plasmid encoding env-defective, luciferaseexpressing, hiv-1 genome (pnl4-3r-e-luc) using fugene hd reagents (roche, basel, switzerland). supernatants containing mers-cov pseudovirus were harvested 48 h post-transfection and used for single-cycle infection. huh7.5 cells were plated at 10 4 cells/well in 96-well tissue-culture plates and grown overnight. the supernatants containing pseudovirus were pre-incubated with 2-fold serially diluted mouse sera at 37uc for 1 h before addition to cells. the culture was refed with fresh medium 24 h later and incubated for an additional 48 h. cells were washed with pbs and lysed using lysis reagent included in a luciferase kit (promega). aliquots of cell lysates were transferred to 96-well costar flatbottom luminometer plates (corning costar), followed by addition of luciferase substrate (promega). relative light units were determined immediately in the gaomax luminometer (promega). all experiments were carried out in triplicate. pseudovirus inhibition (pi) rate was calculated as: (relative luciferase units of mock sera -relative luciferase units of immune serum for a given dilution)/relative luciferase units of mock sera. to evaluate the antigen-specific t-cell response induced by the vaccination regimes, an ifn-c elispot assay was performed as described previously [17] . briefly, 96-well plates were coated with 100 ml per well of 5 mg/ml anti-mouse ifn-c antibody (bd pharmingen) overnight at 4uc and then blocked for 2 h at rt. freshly harvested splenocytes (5610 5 per well) or lung lymphocytes of eight mice in each group were isolated as described previously [22] . then, 4 mg/ml of a synthesised 18-mer peptide library, which overlapped the mers-cov s rbd by 10 amino acids, was added to the wells in triplicate. next, a biotinylated detection antibody (bd pharmingen) and streptavidin-horseradish peroxidase were added. blots were developed by the addition of an aec (3-amino-9-ethylcarbazole) substrate solution, which produced a coloured spot after 5-min rt incubation in the dark. finally, ifn-c spot-forming cells (sfcs) were counted. phorbol 12-myristate 13-acetate (pma) and ionomycin were added to the positive-control group, whereas the negative-control group received no stimuli. the number of peptide-specific ifn-c secreting t cells was calculated by subtracting the negativecontrol value from the sfc count. a cba analysis was conducted to investigate the levels of th1and th2-type cytokine secretion [18] in mice after three times' immunization. in brief, splenocytes (5610 5 per well) of eight mice in each group were distributed in 96-well plates and stimulated with 4 mg/ml of pooled rbd peptide. plates were incubated for 24 h at 37uc and supernatants were harvested. the concentrations of cytokines, including il-2, il-4, il-6, il-10, tnf-a, il-17a and ifn-c, were measured using a mouse th1/th2/th17 cytokine kit (bd biosciences) and a facs calibur flow cytometer (becton dickinson). data were analysed using the fcap array software (becton dickinson). statistical analysees were conducted using the one-way anova function in the spss 17.0 software package. a p-value less than 0.05 were considered to indicate statistical significance. the vaccination regime affects the rbd-specific igg response in mice to assess the humoral immune response to different immunisation regimens, mice were immunised with rrbd protein combined with different adjuvants three times at 3-week intervals. serum samples were collected 2 weeks after each vaccination and total anti-mers-cov rbd igg antibody titres were determined by elisa. the results indicated that rrbd protein combined with any adjuvant, including alum, ifa, cpg or poly(i:c), could induce a rbd-specific igg antibody response in the majority of mice after the second immunisation. in a few vaccinated mice, rbd-specific igg antibodies could be detected even after the first immunisation. the seroconversion rates of the different groups following the first and the second immunisations are shown in table 1 . as shown in figure 3a , there was no discernible increase in igg titres after the third immunisation compared to the second immunisation. among the vaccination regimes, rbd/a+c and rbd/i+c elicited the highest total igg titres (p,0.05, figure 3a) . besides, the difference of igg titer in these two groups was not significant (p$0.05, figure 3a ). similarly shown in figure 3a , the difference of igg titer in rbd/a and rbd/a+p groups had no significance. the rbd-specific antibodies were lower than 1:10 in the adjuvant control groups at each of the three vaccinations. the responses to the various vaccination regimes were investigated using nascn antibody-displacement elisa to measure antibody avidity ( figure 3b ). mice received the rbd/ a+c or rbd/i+c regimes had higher antibody avidity 2 weeks after the final vaccination than those received the rbd/a or rbd/a+p regimes. it was also noteworthy that high antibody avidity correlated with a high igg titre in mice. to further characterise the immune response to the different vaccination regimes, igg isotype analyses were performed 2 weeks after the final vaccination using secondary antibodies against igg1, igg2a and igg2b. as shown in figures 3c, d , e and f, mice immunised with rbd/a+c or rbd/i+c produced higher igg1 and igg2a titres than mice immunised with rbd/a or rbd/a+p. also, the igg1 to igg2a ratio revealed a th1 skewed response in mice that received the rbd/a+c or rbd/i+c regimes. in contrast, the rbd/a and rbd/a+p regimes produced a higher igg1/igg2 ratio, indicating a th2 response. the titres of rbd-specific igg2b antibodies, however, were not significantly different among the vaccination groups (p$0.05). nneutralizing antibodies in the sera of mice immunised with different vaccination regimes were evaluated with a pseudovirusbased neutralization assay. a low level of neutralizing antibodies were detected 2 weeks after the first or second vaccination in aall of the sera tested, although the total igg antibody levels had almost peaked after the second vaccination. the highest level of neutralizing antibodies was induced after the last vaccination ( figure 4 ). the pseudovirus inhibition (pi) rates are shown in figure 4 . as shown, the rbd/a+c regime had the highest neutralizing antibody activity (p,0.01). surprisingly, there was low level of detectable neutralizing antibody in the sera of mice immunised s.c. with the rbd/i+c regime, although sera from this to characterise the cellular immune responses elicited by the vaccination regimes, single ifn-c-producing cells were quantified by elispot. both systemic and local cellular immune responses were assessed using lymphocytes from the spleen and lungs of immunised mice. the peptide library used to stimulate the lymphocytes was described in the materials and methods section. results are expressed as the number of sfcs per 10 6 input cells. adjuvants without rrbd did not elicit a clear cellular response in the spleen 2 weeks after the third immunisation ( figure 5a ). neither rbd/a nor rbd/a+p induced a significant cellular immune response. in contrast, rbd/a+c and rbd/i+c regimen enhanced a detectable systemic cellular immune response. furthermore, the rbd/i+c regimen induced the greatest cellular immune response with the greatest number of ifn-c-producing cells in the spleen (p,0.05). a significant cellular immune response in the lung was induced only by the rbd/i+c regime, although a few ifn-c producing cells were detected in all immunised mice ( figure 5b ). we therefore concluded that while both the rbd/a+ c and rbd/i+c regimes could induce a systematic cellular immune response in mice, only the rbd/i+c regime could elicit a significant local cellular immune response in the lung. a higher frequency of rbd-specific, tnf-a-and il-4producing t cells were induced with alum and cpg via an i.m. route the cytokine profiles of spleen cells from immunised mice were analysed after stimulation with rbd-specific peptides. during cba, splenocytes from mice immunised with rbd/a+c or rbd/i+c produced ifn-c ( figure 6a ). in contrast, il-2 was produced by splenocytes following immunisation with rrbd combination of any adjuvants ( figure 6b ). but the differences in ifn-c and il-2 production among the groups were not significant (p$0.05). compared with other groups, splenocytes from mice immunised with rbd/a+c induced significantly higher levels of tnf-a ( figure 6c ) and il-4 ( figure 6d ) (p,0.01). all these indicated that the adjuvants of alum and cpg combination could induce a th1 and th2 mixed immune responses in the rrbd antigen model of mers-cov, though the responses revealed a th1 polarization in the isotype elisa and elispot detection. similarly, the high levels of ifn-c, il-2 and il-6 also indicated a th1 and th2 mixed immune responses could be induced by the rbd/i+c regimes. (figure 6a, 6b, 6e) . different from all of these, as shown in figure 6f , the rbd/a+p regimes induce the highest level of il-10 (p,0.01), which indicated a th2 response inclination consistent with the results of igg isotype. however, il-17a was not detected in any of the vaccination groups (data not shown). coronaviruses can adapt rapidly to new hosts, and an adaptation of mers-cov that allowed the virus to efficiently replicate in humans would be a major public health concern, since such an adaptation could trigger a pandemic [24] . the development of an effective vaccine is critical to prevent a potential mers-cov pandemic. previous studies have shown that vaccination with the sars-cov rbd induces highly potent neutralizing antibodies and significantly inhibits sars-cov infection [25, 26] . therefore it was proposed that vaccination with the rbd of mers-cov, which belongs to the same betacoronavirus genus as sars-cov [27, 28] , might also inhibit mers-cov infection and induce a neutralizing antibody response against mers-cov. du et al [8] identified a recombinant protein containing a 212-amino acid fragment (residues 377-588) in the truncated rbd of mers-cov spike protein fused with human igg fc fragment (s377-588-fc) was able to induce in the vaccinated mice strong mers-cov sspecific antibodies, which blocks the binding of rbd to dipeptidyl peptidase 4 (dpp4), the human mers-cov receptor [29] and effectively neutralizes mers-cov infection. besides, they [9] showed that residues 377 to 662 in the s protein of mers-cov induced significant neutralizing antibody responses, suggesting that this region had a potential to be developed as a mers-cov vaccine. mou et al [10] showed the polyclonal antibodies in rabbits against the rbd in the s protein to a 231-amino-acid fragment (residues 358 to 588) efficiently neutralized virus infectivity. however, none of the studies evaluated the immunogenicity of rrbd protein systematically in an animal model. recently, ma et al [7] ssuggested the possibility of developing a recombinant rbd protein containing residues 377-662 into an effective and safe mucosal mers vaccine through the intranasal route in the presence of the only poly(i:c) adjuvant in a mouse model. while the need for vaccines with the ability to generate an effective immune response has led to the combination of antigens with more than one adjuvant, the 'adjuvant system' approaches. the adjuvant system approach aids in the development of vaccines that generate effective immune responses [11, 30] . in this study, the roles of three adjuvants-alum, ifa, cpg and poly (i:c)-in rrbd subunit vaccination were investigated aimed at inducing an effective immune response through use of tailored adjuvant combinations and delivery routes. consistent with above studies, all vaccination regimes containing rrbd induced an rbd-specific cellular and humoral immune response. however, a more robust immune response was elicited when mice were immunised with the rbd/a+c and rbd/i+c regimes. an unexpected result was the absence of neutralizing antibodies in the sera of rbd/i+c immunised mice, despite anti-rbd specific igg titres being similar for the rbd/i+c and rbd/ a+c regimes. to further understand the riddle, we detected the aantibody avidity of different vaccination regimes by aavidity elisa. however, the results showed the high antibody avidity correlated with a high igg titre in mice of rbd/i+c and rbd/ a+c groups. so, we speculated maybe the adjuvants of destroyed the conformation of rrbd and covered the antigen binding sites. another probable cause of the low titer of neutralizing antibodies in the sera of rbd/i+c immunised mice was the delivery route of subcutaneous. as known, the subcutaneous may be associated with degradation at injection site, which leads to decreased bioavailability [31] . whatever, further studies are in process. compared with other studies, the regimes in this study induced lower titres of neutralization antibodies. for example, the pi 50 of alum plus cpg, the group showing the highest titer of neutralizing antibody in all immunization groups was 1:500. while the rrbd protein in the above studies acquired a 1:1000 in mice neutralization antibody titre. the differences may be caused by the detection methods of neutralization antibody. as showed in the materials and methods parts, the neutralization antibodies in this study were detected by a pseudovirus system which can be conducted in biosafety level-2 facilities. while the differences of induced neutralization antibodies among different groups can be shown clearly. the subclass of immunoglobulin induced after immunization is an indirect measure of the relative contribution of th1-type cytokines vs. th2-type cytokines [18] . to characterise the immune response of the different vaccination regimes, igg isotype including igg1, igg2a and igg2b analyses were performed. as expected, the rbd/a regimes produced a th2 response with high igg1/igg2 ratio. in contrast, mice received the rbd/a+c or rbd/i+c regimes revealed a th1 skewed response. consistently, the rbd/a+c or rbd/i+c regimes induced a systematic cellular immune response in mice by elispot analysis. the high level of ifn-c and il-2 in the cba was also a proof of the cellular immune response in mice. besides, the mice in the rbd/a+c group had a high level of il-4 and il-10, which were an index of th2 skewed response. taken together, the rbd/a+c induced a th1 and th2 mixed immune responses, though the responses had a th1 inclination. it was our original intention of mixed th1/th2 responses for better protection. similarly, the rbd/i+c regimes induced a mixed th1 and th2 responses. however, it was a pity that the rbd/i+c regimes could not induce an effective neutralization antibody, which was the most important factor of a prophylactic vaccine. above all, in this study, mers-cov s rrbd combined with the adjuvants alum and cpg produced the most robust immune response. it indicates that the combination of alum and cpg was the optimal strategy for i.m. rrbd antigen delivery in a murine model. this result will facilitate future mers-cov vaccine design. the results of the present study also support the importance of the adjuvant system approach, although adjuvant combinations do not always produce the desired response, as seen with rbd/i+c. consistent with the results of the present study, cpg plus alum was found to induce protective humoral, as well as cellular immunity, in mice immunised with a recombinant haemagglutinin vaccine that protected against influenza virus challenge [32] . the ideal immunity of the cpg and alum combination may be the result of mutual complementation of these two adjuvants. it is well known that alum can promote antibody-mediated protective immunity. however, alum is a poor inducer of cellular immune responses [12] . recently, adjuvants including oil-in-water emulsions have shown improved efficacy for avian influenza protection suggesting that even for diseases where humoral immunity can confer protection, cellular immune responses may be necessary in vaccine design [33] . the key features of cpg-odn used as a vaccine adjuvant, include the ability to elicit th1 cell, but only under certain conditions, cd8+ cytotoxic t cell responses and an additional ability to divert the pre-existing th2 response in neonates and elderly mice toward a th1 phenotype [34] . thus, we expect that the combination of alum and cpg will prove applicable in a range of infectious diseases that have defeated current immunisation strategies. except for a choice of adjuvants in combination with optimal protective antigen, practical items such as the antigen: adjuvant ratio, dose, vaccination regimen and often route of administration will strongly impact on both the effectiveness and safety of the vaccine formulation. in most cases, an experimental vaccine will be initially tested in an animal model [29] . to evaluate the immunogenicity of rrbd protein thoroughly, it is necessary to test the protective effects of rrbd subunit immunisation in an animal model of mers-cov infection. to date, rhesus macaques have been reported to generate pneumonia-like symptoms within 24 h of mers-cov infection [35] , and we are testing the effects of rrbd immunisation in rhesus macaques. considerable efforts are being made to establish a small animal model of mers-cov infection. though the lung cells of the syrian hamster express the receptor for mers-cov, they are not susceptible to mers-cov infection [35, 36] . recently, a mouse model of mers-cov infection was reportedly generated by transduction of mice with adenoviral vectors expressing dpp4 [24] . in the future, we expect the protective effect of the rbd/a+c vaccination should be investigated in this murine model of mers-cov infection. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus 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syrian hamsters key: cord-344180-v8xs5ej8 authors: vadlamani, bhaskar s.; uppal, timsy; verma, subhash c.; misra, mano title: functionalized tio(2) nanotube-based electrochemical biosensor for rapid detection of sars-cov-2 date: 2020-10-17 journal: sensors (basel) doi: 10.3390/s20205871 sha: doc_id: 344180 cord_uid: v8xs5ej8 the coronavirus disease (covid-19) is a newly emerging viral disease caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2). rapid increase in the number of covid-19 cases worldwide led the who to declare a pandemic within a few months after the first case of infection. due to the lack of a prophylactic measure to control the virus infection and spread, early diagnosis and quarantining of infected as well as the asymptomatic individuals are necessary for the containment of this pandemic. however, the current methods for sars-cov-2 diagnosis are expensive and time consuming, although some promising and inexpensive technologies are becoming available for emergency use. in this work, we report the synthesis of a cheap, yet highly sensitive, cobalt-functionalized tio(2) nanotubes (co-tnts)-based electrochemical sensor for rapid detection of sars-cov-2 through sensing the spike (receptor binding domain (rbd)) present on the surface of the virus. a simple, low-cost, and one-step electrochemical anodization route was used for synthesizing tnts, followed by an incipient wetting method for cobalt functionalization of the tnts platform, which was connected to a potentiostat for data collection. this sensor specifically detected the s-rbd protein of sars-cov-2 even at very low concentration (range of 14 to 1400 nm (nano molar)). additionally, our sensor showed a linear response in the detection of viral protein over the concentration range. thus, our co-tnt sensor is highly effective in detecting sars-cov-2 s-rbd protein in approximately 30 s, which can be explored for developing a point of care diagnostics for rapid detection of sars-cov-2 in nasal secretions and saliva samples. the current outbreak of novel coronavirus (ncov-2019 or sars-cov-2), was first detected in wuhan, china in december 2019, but quickly spread to other parts of china as well as to the entire world, causing a pandemic [1] . according to the who, as of 16 august, 2020, around 21,294,845 people are infected, and 761,779 people have died due to sars-cov-2 infection [2] . sars-cov-2 infection causes a variety of symptoms including fever, cough and respiratory distress, which are collectively called the coronavirus disease or covid-19 [3] . the spreading of sars-cov-2 primarily occurs from person-to-person transmission through close contact or via small droplets produced during coughing, sneezing, and talking [4, 5] . the incubation period for sars-cov-2 is around 2-7 days, with no noticeable symptoms; however, the viral transmission from an infected person to a non-infected person is still possible during this asymptomatic period [6] . under the current scenario, with no vaccines in the market, global lockdown regulations are in place in order to minimize the viral spread. consequently, this pandemic has caused a severe socio-economic impact on the world economy and raised fears of a global recession [7] . currently, the real-time reverse-transcriptase polymerase chain reaction (rt-pcr) technique is the most common and reliable laboratory testing method for qualitative/quantitative sars-cov-2 detection [8, 9] followed by serum virus neutralization assay (svna) for the determination of antibody neutralization [10] and enzyme-linked immunoassays (elisa) for the detection of antibodies against sars-cov-2 [11] . however, the major limitations of these laboratory-based diagnostic tests are the invasive nature of the tests that often require trained personal for nasopharyngeal sample collection, along with the requirement of highly sophisticated machines, cross-reactivity with other viruses, and longer duration of testing. in order to contain the viral spread, surveillance of even asymptomatic individuals is needed, which is feasible only after the development of a simple, portable and rapid point-of-use sensor for the detection of sars-cov-2. sars-cov-2 has a positive-sense, single-stranded rna (~30k bp) genome with 14 orfs that encode for structural, replication and non-structural proteins [12] . similar to its genetic cousin, human sars-cov, sars-cov-2 consists of four structural proteins viz. spike (s), envelope (e), membrane (m), and nucleocapsid (n). coronaviruses are named for the crown like spike glycoprotein, s (composed of two subunits: the s1 subunit and the s2 subunit) on the surface/envelope [13] . the s1 subunit of the s protein consists of a receptor binding domain (rbd) that has a high binding affinity towards the host angiotensin-converting enzyme ii (ace2) receptor present on the human cells; the s2 subunit mediates virus-host cell fusion and entry [14] . importantly, the s protein is highly immunogenic and induces immune response to produce neutralizing antibodies as well as t-cell responses in sars-cov-2 infected individuals [15] . functionally, binding of s-rbd to the hace2 receptor is crucial for the entry of sars-cov-2 into human cells. interestingly, sars-cov-2 s-rbd shares only 70% sequence identity with sars-cov s-rbd, which has been evaluated for vaccines and therapeutic drug development [16] . hence, the s-rbd of sars-cov-2 is an excellent target for diagnostic and therapeutic interventions. electrochemical biosensors are advantageous for sensing biomolecules because of their ability to detect biomarkers with accuracy, specificity and high sensitivity [17] . electrochemical biosensors have been successfully used in medical diagnostics for the detection of viruses such as the middle east respiratory syndrome coronavirus (mers-cov) [18] , the human enterovirus 71 (ev71) [19] , the human influenza a virus h9n2 [20] , and the avian influenza virus (aiv) h5n1 [21] . lahyquah et al. [18] used an array of carbon electrodes modified with gold nanoparticles for the detection of mers-cov. very recently, a biosensor using gold nanoparticle decorated fto glass immobilized with ncovid-19 monoclonal antibody was reported for the detection of sars-cov-2 [22] . the functionality of the electrochemical biosensor can be further improved by nanostructuring the electrode as it increases the electrochemical reaction rate due to an increased electrode surface area to volume ratio, thereby increasing the electrode surface area to analyte fluid volume. in the work by chin et al. on the encephalitis virus, it was found that nanostructuring of carbon electrodes with carbon nanoparticles increased the current response by 63% due to an enhanced electron charge transfer kinetics [23] . similarly, we have reported that co functionalized tio 2 nanotubes (ni-tnts) with higher surface-to-volume ratio can detect the biomarkers associated with tuberculosis [24, 25] . the proposed sensing mechanism involves the formation of a complex between co and the biomarker at a specific bias voltage, due to the reduction of co ions and oxidation of the biomarker. similarly, we hypothesized that s-rbd or sars-cov-2 can be detected through complexing of functionalized nanoparticles with the s-rbd protein. a schematic of viral detection directly from a patient sample is shown in figure 1 . in the current work, we have determined the potential of co-functionalized tio 2 nanotubes (co-tnts) for the electrochemical detection of s-rbd protein of sars-cov-2. tnts were synthesized by a simple, cost-effective, one-step electrochemical anodization route, and co functionalization was carried out using the incipient wetting method. our data shows that cobalt functionalized tnts can selectively detect the s-rbd protein of sars-cov-2 using the amperometry electrochemical technique in~30 s. in the current work, we have determined the potential of co-functionalized tio2 nanotubes (co-tnts) for the electrochemical detection of s-rbd protein of sars-cov-2. tnts were synthesized by a simple, cost-effective, one-step electrochemical anodization route, and co functionalization was carried out using the incipient wetting method. our data shows that cobalt functionalized tnts can selectively detect the s-rbd protein of sars-cov-2 using the amperometry electrochemical technique in ~30 s. tnts were synthesized by electrochemical anodization of the ti sheet. a ti sheet of size 1.5 × 1.5 cm, with a tab 2 mm in width, was cut out of a g1 grade ti sheet (thickness 0.01016 mm). one side of the coupon was polished with 600 grit polishing paper for 4 min to remove any surface metal oxide layer. the coupon was ultrasonicated in a 1:1 solution of ethanol and acetone for 2 min. the unpolished side was masked with kapton tape to avoid any exposure to electrolyte during anodization. the electrochemical anodization was performed in a standard two-electrode configuration, using ti foil as a working electrode and platinum foil as a counter electrode with a 3 cm gap between them. the anodization was carried out using an electrolyte of composition 96.5 ml (ch2oh)2, 3 ml di h2o, and 0.505 g nh4f, in a teflon beaker. the electrolyte was maintained at a subzero temperature and was continuously stirred using a magnetic stirrer at a speed of 140 rpm. the anodization was carried out by maintaining a constant voltage of 30 v across both the electrodes for 50 min. after anodization, the sample was rinsed in di h2o and baked in an oven at 120 °c for 4 hrs. the kapton tape was removed from the sample after baking, and the sample was annealed in a tube furnace at 500 °c for 3 h in a continuous flow of oxygen. the annealed tnts obtained from the furnace were functionalized with cobalt using an incipient wetting method, i.e., a wet ion exchange process. the same side of the sample that was masked earlier was again masked with kapton tape. the sample was ultrasonicated in a solution containing 2.306 g of cocl2.6h2o in 20 ml ethanol for 35 min. the sample was baked in an oven at 120 °c for 4 h to obtain cobalt functionalized tnts. tnts were synthesized by electrochemical anodization of the ti sheet. a ti sheet of size 1.5 × 1.5 cm, with a tab 2 mm in width, was cut out of a g1 grade ti sheet (thickness 0.01016 mm). one side of the coupon was polished with 600 grit polishing paper for 4 min to remove any surface metal oxide layer. the coupon was ultrasonicated in a 1:1 solution of ethanol and acetone for 2 min. the unpolished side was masked with kapton tape to avoid any exposure to electrolyte during anodization. the electrochemical anodization was performed in a standard two-electrode configuration, using ti foil as a working electrode and platinum foil as a counter electrode with a 3 cm gap between them. the anodization was carried out using an electrolyte of composition 96.5 ml (ch 2 oh) 2 , 3 ml di h 2 o, and 0.505 g nh 4 f, in a teflon beaker. the electrolyte was maintained at a subzero temperature and was continuously stirred using a magnetic stirrer at a speed of 140 rpm. the anodization was carried out by maintaining a constant voltage of 30 v across both the electrodes for 50 min. after anodization, the sample was rinsed in di h 2 o and baked in an oven at 120 • c for 4 h. the kapton tape was removed from the sample after baking, and the sample was annealed in a tube furnace at 500 • c for 3 h in a continuous flow of oxygen. the annealed tnts obtained from the furnace were functionalized with cobalt using an incipient wetting method, i.e., a wet ion exchange process. the same side of the sample that was masked earlier was again masked with kapton tape. the sample was ultrasonicated in a solution containing 2.306 g of cocl 2 .6h 2 o in 20 ml ethanol for 35 min. the sample was baked in an oven at 120 • c for 4 h to obtain cobalt functionalized tnts. the morphology of the tnts and co-tnts were examined using dual beam scanning electron microscopy (sem, thermofisher scientific). the cobalt content in the co-tnt sample was analyzed using the eds detector attached to sem. the sem micrographs were analyzed using imagej software. the pcaggs vector containing sars-cov-2 wuhan-hu-1 spike glycoprotein receptor binding domain (rbd) with a c-terminal hexa-histidine tag was obtained from bei resources (niaid, nih, nr-52309). his 6 -tagged s-rbd containing pcaggs plasmid was expressed in hek293t (human embryonic kidney) cells obtained from the american type culture collection (atcc) and maintained in dulbecco's modified eagle medium (dmem), supplemented with 10% fetal bovine serum (fbs, atlanta biologicals), 2 mm l-glutamine, 25 u/ml penicillin, and 25 µg/ml streptomycin. cells were grown at 37 • c in a humidified chamber supplemented with 5% co 2 . for his 6 -tagged s-rbd protein generation, hek293t cells were transfected with recombinant plasmid using neon transfection system (thermo scientific) according to the manufacturer's instructions. supernatants from transfected cells were harvested on day 3 post-transfection and the cell debris was removed by centrifugation (4000 rpm, 20 min at 4 • c). supernatants were then incubated with 1 ml of ni-nta agarose (qiagen) for every 10 ml of supernatant, for 2 h at 4 • c with rotation. for s-rbd purification, gravity flow columns were used to load the ni-nta agarose bound his 6 -tagged spike-rbd protein, followed by washing with wash buffer (20mm sodium phosphate, 500 mm nacl, 8 m urea, 20 mm imidazole, ph 6.0) and eluting with elution buffer (20 mm sodium phosphate, 500 mm nacl, 8m urea, 200 mm imidazole, ph 4.0). the eluted protein was concentrated using protein concentrators (thermo scientific, 87,748 and 87772), quantified using bradford assay and nanodrop (thermo scientific) and further analyzed by sds-page. the electrochemical sensing of s-rbd protein was carried out using a custom-built co-tnt packaged printed circuit board setup. the sensor response was measured with the help of gamry reference 600+ potentiostat attached to the printed circuit board. the custom-made printed circuit board consists of a copper clamp that holds the co-tnt grown over the ti sheet. the upward-facing co-tnt side acts as a working electrode, and the bottom-facing ti side acts as a counter electrode, to which electrical connections were made via copper lines running on the top and bottom of the custom-built chip, respectively. the detailed schematic of the printed circuit board was reported in our earlier work [26] . the schematic of the whole sensing set up along with the detection methodology is shown in figure 1 . amperometry is an electrochemical technique where a constant voltage is applied across the electrodes and response current is monitored as a function of time [27] . the technique uses response current to determine the concentration of the analyte in the electrolyte solution between the electrodes. the s-rbd protein in the elution buffer (20 mm sodium phosphate, 500 mm nacl, 8 m urea, 200 mm imidazole, ph 4.0) was transferred onto the surface of co-tnt using a micropipette. the sensor response with various s-rbd protein in concentrations was determined using the amperometry technique, at a bias voltage of −0.8 v. the bias voltage was determined by conducting the cyclic voltammetry experiments in the voltage window −2 to +2 v. all the experiments were carried out at room temperature. the scanning electron microscopy (sem) micrographs of the tnts, prepared by electrochemical anodization, are shown in figure 2a . the inset shows the side view of the tnts (figure 2a) . the outer diameter and wall thickness of tnts were~60 and~10 nm, respectively. the average length of tnts was found to be~1.1 µm. in our earlier work, tnts synthesized under similar conditions were found to show the crystalline anatase phase predominantly [25] . the surface morphology of the co-tnts examined under sem is shown in figure 2b . the sem micrograph reveals the presence of precipitates on top of the tnt surface. eds analysis confirmed the uniform distribution of co on top of tnts (figure 2c) , and the co content was found to be~4 wt% (figure 2d) . we have previously shown using sensors 2020, 20, 5871 5 of 10 detailed xps studies that co exists in the co +2 state (2p 3/2 peak at 781.5 ev) and also co(oh) 2 is the predominant phase present on the surface of co-tnts [28] . therefore, the morphology of tnts can be visualized as having a very large surface area, uniformly decorated with co +2 ions. sensors 2020, 20, x 5 of 10 to show the crystalline anatase phase predominantly [25] . the surface morphology of the co-tnts examined under sem is shown in figure 2b . the sem micrograph reveals the presence of precipitates on top of the tnt surface. eds analysis confirmed the uniform distribution of co on top of tnts (figure 2c) , and the co content was found to be ~4 wt% (figure 2d) . we have previously shown using detailed xps studies that co exists in the co +2 state (2p3/2 peak at 781.5 ev) and also co(oh)2 is the predominant phase present on the surface of co-tnts [28] . therefore, the morphology of tnts can be visualized as having a very large surface area, uniformly decorated with co +2 ions. the receptor binding domain of the spike glycoprotein (s-rbd), present as a crown on the surface of the virus is an easily accessible target for the detection of sars-cov-2. the rbd domain comprises of amino acids 329-521, which is a ~25 kda protein with potential n-glycosylation sites. as shown in figure 3a , b, the sds-page gel of his6-tagged s-rbd protein, either stained with simplyblue safestain ( figure 3a ) or immunologically detected with mouse anti-his monoclonal antibody ( figure 3b ) showed the presence of specific protein in our viral protein preparation. immunoblot detected sars-cov-2 s-rbd protein at approximately 35kda, as expected, but also at 70kda, representing the dimeric forms of s-rbd protein ( figure 3b ). detection of a slightly higher molecular weight (~35kda) s-rbd protein as compared to the calculated size was possibly because of post-translational modifications, including glycosylation on the protein. importantly, the s-rbd purified protein from the human embryonic kidney cells were of high purity; it was used for quantitation and detection on co-tnt sensors. the ability of co-tnt to sense the s-rbd protein of sars-cov-2 was determined by performing an amperometry experiment at a bias voltage of −0.8 v. the amperometry curves obtained at various concentrations of protein are shown in figure 4 . the sensor was exposed to protein 30 s after the the receptor binding domain of the spike glycoprotein (s-rbd), present as a crown on the surface of the virus is an easily accessible target for the detection of sars-cov-2. the rbd domain comprises of amino acids 329-521, which is a~25 kda protein with potential n-glycosylation sites. as shown in figure 3a , b, the sds-page gel of his 6 -tagged s-rbd protein, either stained with simplyblue safestain ( figure 3a ) or immunologically detected with mouse anti-his monoclonal antibody ( figure 3b ) showed the presence of specific protein in our viral protein preparation. immunoblot detected sars-cov-2 s-rbd protein at approximately 35 kda, as expected, but also at 70 kda, representing the dimeric forms of s-rbd protein ( figure 3b ). detection of a slightly higher molecular weight (~35kda) s-rbd protein as compared to the calculated size was possibly because of post-translational modifications, including glycosylation on the protein. importantly, the s-rbd purified protein from the human embryonic kidney cells were of high purity; it was used for quantitation and detection on co-tnt sensors. the ability of co-tnt to sense the s-rbd protein of sars-cov-2 was determined by performing an amperometry experiment at a bias voltage of −0.8 v. the amperometry curves obtained at various concentrations of protein are shown in figure 4 . the sensor was exposed to protein 30 s after the beginning of the experiment (marked by an arrow). the sensor response current increases sharply and rapidly as the sensor was exposed to the protein. at a protein concentration of 1400 nm (nano molar), the peak sensor current output was found to be~0.74 µa (micro ampere). the peak current decreases to~0.45 µa at a protein concentration of 140 nm and further decreases to~0.23 µa at a protein sensors 2020, 20, 5871 6 of 10 concentration of 14 nm. the sensor detection time was~30 s over the concentration range of 14 to 1400 nm. it is hypothesized that the rapid increase in sensor response current could be attributed to the electrochemically triggered unfolding of protein that exposes its interior [29] [30] [31] and subsequent complex formation between co and the protein [32, 33] . each s-rbd protein monomeric unit contains 15 tyrosine, 2 tryptophan and 9 cysteine amino acid residues [14] , and all of them were reported to underdo electrochemical oxidation under application of potential [29, 32] . the electrochemical oxidation process involves deprotonation, where the -oh functional group in the protein is converted to -o − . we envisage that the complex formation occurs between the co +2 ion in co-tnt and the -o − radical in the protein. a very similar mechanism was reported earlier, where methyl nicotinate biomarker was exposed to co-tnt [28] . attributed to the electrochemically triggered unfolding of protein that exposes its interior [29] [30] [31] and subsequent complex formation between co and the protein [32, 33] . each s-rbd protein monomeric unit contains 15 tyrosine, 2 tryptophan and 9 cysteine amino acid residues [14] , and all of them were reported to underdo electrochemical oxidation under application of potential [29, 32] . the electrochemical oxidation process involves deprotonation, where the -oh functional group in the protein is converted to -o -. we envisage that the complex formation occurs between the co +2 ion in co-tnt and the -oradical in the protein. a very similar mechanism was reported earlier, where methyl nicotinate biomarker was exposed to co-tnt [28] . the average sensor response time, which is defined as the time taken to reach the peak current, was found to be ~2 sec. it is very short compared to our earlier studies on the sensor for colorectal cancer, where a sensor response time of ~200 sec was documented [33] . the shorter sensor response time indicates higher kinetics of the reaction between co-tnt and the protein molecules. at all the protein concentrations, it was observed that the sensor current did not recover to the initial baseline current within the experimental timeframe. therefore, the sensor recovery time, defined as the sensor's time to recover to the initial baseline current value, could not be reported. the sensor's strange behavior could be due to the change in the surface chemistry of co-tnt after interaction with the protein. the sensor response (sr) was calculated at various protein concentrations based on the following equation: i , − i , the average sensor response time, which is defined as the time taken to reach the peak current, was found to be~2 s. it is very short compared to our earlier studies on the sensor for colorectal cancer, where a sensor response time of~200 s was documented [33] . the shorter sensor response time indicates higher kinetics of the reaction between co-tnt and the protein molecules. at all the protein concentrations, it was observed that the sensor current did not recover to the initial baseline current within the experimental timeframe. therefore, the sensor recovery time, defined as the sensor's time to recover to the initial baseline current value, could not be reported. the sensor's strange behavior could be due to the change in the surface chemistry of co-tnt after interaction with the protein. the sensor response (sr) was calculated at various protein concentrations based on the following equation: sensor resposne (sr) = i max, protein − i max, base line i max,base line where i max,protein is the maximum current obtained when the sensor is exposed to sars-cov-2 s-rbd protein and i max,base line is the maximum current obtained when the sensor is not exposed to the protein. the value of i max,base line , which is the current obtained when the sensor is not exposed to the protein, was found to be~10 pa ( figure 4 ). the sensor responses measured at different protein concentrations are shown in figure 5 . the sensor response was found to increase with an increase in the concentration of protein. moreover, the sensor response exhibited excellent linearity over the concentration range 14 to 1400 nm with a correlation coefficient of r 2 = 0.99. the regressed linear calibration curve for sensor response was obtained as follows: where sr is the sensor response, and c is the concentration of protein in nm. according to statistical analysis [34] , the detection limit fof measurements using the sensor was determined to be 0.7 nm. sensors 2020, 20, x 8 of 10 the limit of detection can be further improved by the use of (i) co-tnt synthesized by an insitu anodization technique and (ii) co-tnts of even higher length. previously, we found that co-tnt synthesized by in-situ anodization with higher sensor sensitivity compared to co-tnt synthesized by the incipient wetting route, towards the detection of tuberculosis biomarkers [25] . a higher sensor sensitivity corresponds to a better limit of detection and sensitivity of quantitation. the increased sensitivity was attributed to the presence of co (oh)2 precipitate sites in direct contact with the parent tio2, due to which direct conduction is possible. the sensor sensitivity can also be improved by using longer co-tnts as higher surface area results in a higher reaction rate; thereby, higher sensor response current can be obtained even at lower protein concentrations. the limit of detection can be further improved by the use of (i) co-tnt synthesized by an in-situ anodization technique and (ii) co-tnts of even higher length. previously, we found that co-tnt synthesized by in-situ anodization with higher sensor sensitivity compared to co-tnt synthesized by the incipient wetting route, towards the detection of tuberculosis biomarkers [25] . a higher sensor sensitivity corresponds to a better limit of detection and sensitivity of quantitation. the increased sensors 2020, 20, 5871 8 of 10 sensitivity was attributed to the presence of co (oh) 2 precipitate sites in direct contact with the parent tio 2 , due to which direct conduction is possible. the sensor sensitivity can also be improved by using longer co-tnts as higher surface area results in a higher reaction rate; thereby, higher sensor response current can be obtained even at lower protein concentrations. in this study, we developed a co-metal functionalized tnt as a sensing material for electrochemical detection of sars-cov-2 infection through the detection of the receptor binding domain (rbd) of spike glycoprotein. we confirmed the biosensor's potential for clinical application by analyzing the rbd of the spike glycoprotein on our sensor. amperometry electrochemical studies indicated that the sensor could detect the protein in the concentration range 14 to 1400 nm. the relationship between sensor response and protein concentration was found to be linear with the limit of detection as low as~0.7 nm levels. importantly, our sensor detected sars cov-2 s-rbd protein in a very short time (~30 s), confirming its implication in developing a rapid diagnostic assay. our report thereby demonstrates the development of a simple, inexpensive, rapid and non-invasive diagnostic platform that has the potential of detecting sars-cov-2 on clinical specimens, including nasal, nasopharyngeal swabs or saliva. moreover, the developed approach has the potential for diagnosis of other respiratory viral diseases by identifying appropriate metallic elements to functionalize tnts. author contributions: formal analysis, methodology, writing-original draft preparation, writing-review and editing, b.s.v.; methodology, writing-original draft preparation, writing-review and editing, t.u.; conceptualization, methodology, project administration, funding, writing-review and editing, m.m.; conceptualization, methodology, project administration, funding, writing-review and editing, s.c.v. all authors have read and agreed to the published version of the manuscript. funding: this work was supported by the departmental and institutional funds. who. novel coronavirus (2019-ncov) situation report-1. available online who. coronavirus disease 2019 (covid-19): situation report-69 the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 cluster of sars among medical students exposed to single patient identification of severe acute respiratory syndrome in canada the 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arrays determination of the lower limit of detection the s-rbd expression vector was obtained through bei resources, niaid, nih: vector pcaggs containing the sars-cov-2, wuhan-hu-1 spike glycoprotein gene rbd with c-terminal hexa-histidine tag, nr-52309. the authors declare no conflict of interest. key: cord-327654-9g8zcxaa authors: chi, xiaojing; liu, xiuying; wang, conghui; zhang, xinhui; li, xiang; hou, jianhua; ren, lili; jin, qi; wang, jianwei; yang, wei title: humanized single domain antibodies neutralize sars-cov-2 by targeting the spike receptor binding domain date: 2020-09-10 journal: nat commun doi: 10.1038/s41467-020-18387-8 sha: doc_id: 327654 cord_uid: 9g8zcxaa severe acute respiratory syndrome coronavirus 2 (sars-cov-2) spreads worldwide and leads to an unprecedented medical burden and lives lost. neutralizing antibodies provide efficient blockade for viral infection and are a promising category of biological therapies. here, using sars-cov-2 spike receptor-binding domain (rbd) as a bait, we generate a panel of humanized single domain antibodies (sdabs) from a synthetic library. these sdabs reveal binding kinetics with the equilibrium dissociation constant (k(d)) of 0.99–35.5 nm. the monomeric sdabs show half maximal neutralization concentration (ec(50)) of 0.0009–0.07 µg/ml and 0.13–0.51 µg/ml against sars-cov-2 pseudotypes, and authentic sars-cov-2, respectively. competitive ligand-binding experiments suggest that the sdabs either completely block or significantly inhibit the association between sars-cov-2 rbd and viral entry receptor ace2. fusion of the human igg1 fc to sdabs improve their neutralization activity by up to ten times. these results support neutralizing sdabs as a potential alternative for antiviral therapies. c oronavirus disease 2019 is caused by infection of emerging severe acute respiratory syndromeassociated coronavirus 2 (sars-cov-2) and had been declared by world health organization as the first coronavirus pandemic in human history 1 . the severity of covid-19 symptoms can range from asymptomatic or mild to severe with an estimated mortality rate from less than 2% to up to 10% of patients depending on various factors 2 . sars-cov-2 is spreading rapidly and sustainably around the world, urging prompt global actions to develop vaccines and antiviral therapeutics. sars-cov-2 polyprotein shares~86.15% identity with sars-cov (genbank id: aas00002.1) and is classified into the genus betacoronavirus in the family coronaviridae 3 . sars-cov-2 is an enveloped, positive-sense, single-stranded rna virus with a large genome of approximately 30,000 nucleotides in length. the virusencoded membrane (m), spike (s), and envelope (e) proteins constitute the majority of the protein that is incorporated into sars-cov-2 envelope lipid bilayer. the s protein can form homotrimers and protrudes from envelope to show the coronal appearance, invading susceptible cells by binding potential sars-cov-2 entry receptor angiotensin converting enzyme 2 (ace2) 3 . recently, researchers have figured out the molecular structure of sars-cov-2 s protein 4 . it is composed of 1273 amino acids and structurally belongs to the type i membrane fusion protein with two areas s1 and s2. the s1 region mainly includes the receptor binding domain (rbd), while the s2 region is necessary for membrane fusion. the rbd structure determines its binding efficiency with ace2 and provides an important target for neutralizing antibody recognition. single domain antibodies (sdabs), namely nanobodies, were initially identified from camelids or cartilaginous fish heavy-chain only antibodies devoid of light chains, where antigen-binding is mediated exclusively by a single variable domain (vhh) 5 . therefore, sdabs are the smallest fragments that retain the full antigen-binding capacity of the antibody with advantageous properties as drugs, imaging probes and diagnostic reagents 6 . the advantages of short development time, flexible formatting and robust production efficiency make sdab a powerful means to defeat infectious disease pandemics. for therapeutic purpose, relatively sophisticated humanization techniques have been adopted to modify the camelid-specific amino acid sequences in the framework to their human heavy chain variable domain equivalent, without altering sdab's biological and physical properties and reducing species heterogeneity 7 . as sars-cov-2 is an emerging human virus, the whole population is susceptible due to the lack of protective antibodies. the existing neutralizing antibodies in convalescent plasma have been adopted as powerful therapeutic alternatives for covid-19 patients. in this study, using a synthetic humanized sdabs discovery platform, we obtain several high-affinity sars-cov-2 rbd targeting sdabs with desired neutralization activities. the results illustrate the potential of synthetic sdab library as a resource for antiviral molecules that can be rapidly accessed in a pandemic. these sdabs offer a potential hope for future anti-sars-cov-2 antibody cocktails. identification of sars-cov-2 rbd binding sdabs. sars-cov-2 makes use its envelope s glycoprotein to gain entry into host cells through binding ace2. recent cryo-em research revealed that the s protein shows an asymmetrical homotrimer with a single rbd in the "up" confirmation and the other two "down" 4 . antibodies may take advantage of this rbd structure to block virus entry. to enrich for sars-cov-2 rbd binding sdabs, we performed four rounds of biopanning using a lab owned, full synthetic, humanized phage display library with recombinant rbd protein. after phage elisa identification of 480 clones, a number of sdabs exhibited an excellent affinity for sars-cov-2 rbd (supplementary table 1 ). five distinctive sdad sequences (1e2, 2f2, 3f11, 4d8, and 5f8) were cloned into a prokaryotic expression vector and recombinant sdab proteins were purified by nickel-charged sepharose affinity chromatography (fig. 1a) . humanized sdabs obtained in this study are about 125 amino acids with a single vhh domain in average molecular weight less than 15kda (fig. 1a) . the sdabs consist of three complementarity determining regions (cdrs), as well as four framework regions (frs). the amino acids in the frameworks have been maximally humanized, except for residues phe-42 and ala-52 (numbers refer to the international immunogenetics information system amino acid numbering (imgt.cines.fr)) in framework-2 to maintain proper antigen affinity and best stability 7 . framework residues are illustrated in supplementary fig. 1 . surface plasmon resonance (spr) technology is widely accepted as a golden standard for characterizing antibodyantigen interactions. to determine the kinetic rate and affinity constants, detailed analysis of spike rbd-binding to purified sdab proteins was carried out by spr. the sars-cov-2 or sarc-cov rbd protein was immobilized on the surface of biacore chip cm5, respectively. then, various concentrations of purified sdabs were prepared and injected to pass over the surface. the sensorgram data were fitted to a 1:1 steady-state binding model. spr results demonstrated that the equilibrium dissociation constant (k d ) for the sars-cov-2 rbd protein against sdabs 1e2, 2f2, 3f11, 4d8, and 5f8 were 35.52 nm, 5.175 nm, 3.349 nm, 6.028 nm, and 0.996 nm, respectively ( fig. 1b-f, h) . however, the sdabs showed no binding with sars-cov rbd, except for the clone 5f8 demonstrating a relatively low affinity with k d = 239.2 nm (fig. 1g, h) . overall, as monovalent antibody fragment, the sdabs identified in this study reveals a satisfactory binding performance in a sars-cov-2 specific manner. neutralization of sars-cov-2 by rbd-specific sdabs. to further evaluate the neutralization activity of these sdabs, sars-cov-2 spike-pseudotyped particle (sars-cov-2pp) infectivity assay was first established. pseudotyped particles are chimeric virions that consist of a surrogate viral core with a heterologous viral envelope protein at their surface, which can be operated in biosafety level 2 (bsl-2) and frequently used tool for studying virus entry mechanism and neutralizing antibodies 8 . we observed that all five sdabs showed inhibition potency of sars-cov-2pp infection with ec 50 (half maximal neutralization concentration) ranging from 0.0009 to 0.069 µg/ml (fig. 2a) . we next tested the neutralization activity of the sdabs with sars-cov-2 live virus (fig. 2b) . the copy number of viral rna that was present in the cell culture supernatant was used as a proxy for viral replication. similarly, these sdabs showed comparable neutralization efficiency, with ec 50 at approximately 0.13-0.51 µg/ml. totally, these monovalent sdabs demonstrated encouraging neutralization activity against both pseudotyped and authentic virus, although the neutralization potency is not completely matched (fig. 2c) . this phenomenon was normally reported in middle east respiratory syndrome coronavirus (mers-cov) neutralizing antibodies and may be likely explained by the difference in sdab recognized rbd spatial epitope or the steric hindrance formed by antigen-antibody complex 9, 10 . interference of the ace2-rbd interaction by the sdabs. within sars-cov-2 rbd, the receptor binding motif (rbm) directly contacts ace2. recent report demonstrating that sars-article nature communications | https://doi.org/10.1038/s41467-020-18387-8 cov-2 uses ace2 as its receptor with a much stronger affinity (10-fold to 20-fold higher) than sars-cov 4 . to determine whether sdabs targeted different antigenic regions on the sars-cov-2 rbd surface, we performed a competition-binding assay using a real-time biosensor (fig. 3) . we tested all five sdabs in a competition-binding assay in which human ace2 was attached to a cm5 biosensor. compared with a non-related isotype control sdab (fig. 3a) , addition of 1e2 and 4d8 completely prevent binding of sars-cov-2 rbd to ace2 (fig. 3b, e) . whereas, sdabs 2f2, 3f11, and 5f8 could partially compete the rbd/ receptor association (fig. 3c , d, f). these data suggested that these sdabs can be divided into rbm targeting or non-rbm targeting groups though it is not directly associated with either affinity or virus neutralization activity, which has laid a solid foundation for further development of bispecific neutralizing antibodies to overcome potential virus mutation in the future. inhibition of sars-cov-2 entry by fc-fused sdabs. sdabs can be readily fused to human igg fc-domain to overcome the limitations of the monovalent sdabs, such as the short blood residential time and lacking antibody-dependent cell-mediated cytotoxicity and complement dependent cytotoxicity 11 . in addition, bivalent sdabs can be obtained via the disulfide bond formation in fc hinge area, which was reported to increase sdab's activity 12 . to further explore the possibility of sdab-based antiviral therapeutics and enhance neutralization activity, we constructed human heavy chain antibodies by fusing the human igg1 fc region to the c-terminus of sdabs ( fig. 4a, b) . these fc fusion sdabs were produced in mammalian cells with supernatant yields around 25-50 µg per milliliter in shaking flask. fc fusion sdabs in culture supernatants were affinity purified with hitrap protein a hp antibody purification columns ( supplementary fig. 2 ) and analyzed in both reducing and non-reducing conditions in western blot using an anti-human igg to detect fc. as shown in fig. 4c , the size of the constructed intact sdab-fc is around 80 kda in the non-reducing condition, but a 40 kda monomer was observed by prior treatment in reducing condition to break disulfide bonds. this suggests a correct expression and secretion of heavy chain antibodies in consistence with our design. neutralization assay results showed that genetic fusion of human fc could maintain or increase the neutralization activity of these sdabs for up to 10-fold in molar concentration of ec 50 using the sars-cov-2pp entry assay ( fig. 4d and supplementary fig. 3 ). importantly, all fc-fused sdabs demonstrated potency with ec 50 at sub-nanomolar level (fig. 4d) . finally, we showed that some of the sdabs are suitable for immunofluorescence staining (supplementary fig. 4) and western blot to detect ectopically expressed sars-cov-2 s protein ( supplementary fig. 5 ). given the disease severity and rapid global spread of covid-19, there is an urgent need for development of vaccines, monoclonal antibodies, and small-molecule direct-acting antiviral medications. neutralizing antibodies directly target viral envelope protein, precisely block the virus-receptor association, and inhibit virus entry through a variety of molecular mechanisms. in this study, we isolated and characterized several humanized neutralizing sdabs that exhibit one-digit to two-digit nanomolar or even subnanomolar ec 50 against sars-cov-2 using both pseudotyped and infectious viruses. sdabs have been investigated as important therapeutic alternatives against viral infection because of their high yield, low cost and intrinsic stability. for mers-cov, neutralizing sdabs were isolated from immunized dromedary camels or llamas and demonstrated ec 50 value between 0.001 and 0.003 µg/ml with low k d values (0.1-1 nm) 13, 14 . comparable inhibition efficiency on sars-cov-2pp and affinity kinetics were obtained for the sdabs identified in this study using a nonimmune library, which can speed up the discovery of neutralizing antibodies in an emergent outbreak. with further optimization and increase of library size and diversity, the synthetic sdab library technology will promote the discovery speed of powerful therapeutic antibodies 15, 16 . fda approved the first sdab-based medicine for adults with acquired thrombotic thrombocytopenic purpura in 2019 [17] [18] [19] [20] . considering the cost and potential risks of full human antibody in some viral diseases, such as dengue virus infection, sdab fragments are a novel category of therapeutic molecules and can be readily reconstructed in a tandemly linked way to increase their blood residential time, biological activity, and eliminate underlying concerns about antibody-dependent enhancement (ade) of viral infection 21 . in addition to being used as an injectable drug, the stable sdabs can be also developed into aerosolized inhalations and disinfection products for the prevention of covid-19. besides, prior to the success of covid-19 vaccines, the construction of sdab-based adenovirus or adeno-associated virus gene therapy might provide long-term passive immune protection in vulnerable population, health care workers, or in severely affected areas. since the mature covid-19 animal models have not been developed, this study did not involve in vivo studies. as a next step, the crystal structure analysis of antigen-antibody complexes will be put on the agenda. in conclusion, the discovered neutralizing antibodies in this study could lead to new specific antiviral treatments and shed light on the design and optimization of covid-19 vaccines. library design and construction. a synthetic sdab phage display library was used for the screening of sars-cov-2 neutralizing antibodies. to minimize a possible antigenic effect from camelid sequences, sdab frameworks (frs) for library construction were determined according to a universal humanized scaffold architecture 7 , and the sequences of the frs were illustrated in supplementary fig. 1 . briefly, residues in frs 1, 3, and 4 were mutated based on human heavy chain vh in maximum. in fr 2, humanization of residues at positions 49 and 50 was adopted to increase stability of sdabs, whereas residues 42 and 52 are maintained in camelid due to their critical impact on antigen affinity and/or stability (supplementary fig. 1 ). for the design of variable regions, we analyzed a robust cdr repertoire from immune or naïve llama vhh clones. a synthetic diversity was introduced in the three cdrs by the positioned nucleotide assembly with cysteine and stop codon avoided. a constant length of 8 amino acids was selected for cdr1 and cdr2, and 18 amino acids for cdr3 (supplementary fig. 1 ). frameworks and cdrs were assembled using only 8 cycles of overlapping polymerase chain reaction (pcr) to prevent drift during amplification. diversified sdab mixture was cloned in phagemid vector fadl-1 (antibody design labs, san diego, ca, usa) using sfii/bgli sites with the pelb peptide leader sequence fused with the sdabs at nterminus. the ligation product was purified and used to transform electrocompetent e. coli tg1 cells. a total 50 electroporations was performed in the condition of 1800 v, 10 mf, 600 w. each electroporation was resuspended with 2 ×yt and incubated with a shaking agitation for 1 h at 37°c, and then combined and plated onto more than a thousand agar petri dishes (140 mm) to ensure enough size of the library. library size was calculated by plating serial dilution aliquots and at least 1.2 × 10 10 individual recombinant clones were obtained. quality control was carried out by sequencing more than 1000 clones. more than 950 clones are full length and unique sdabs and less than 50 clones show various errors, such as vector self-ligation, reading frame shift and fragment deletion. antibody selection by phage display. screening for sars-cov-2 rbd targeting antibodies was performed by panning in both immunotubes and native condition using a proprietary full-synthetic library of humanized sdabs with high-diversity, according to a standard protocol. briefly, for the 2nd and 4th panning rounds, the purified sars-cov-2 rbd protein fused with mouse fc was coated on nunc maxisorp immuno tubes (thermofisher) at around 5 µg/ml in pbs overnight. for the 1st and 3rd panning rounds, rbd protein was first biotinylated with ez-link™ sulfo-nhs-lc-biotin (thermofisher) and then selected with streptavidin-coated magnetic dynabeads™ m-280 (thermofisher). the tubes or beads were blocked using 2% w/v skimmed milk powder in pbs (mpbs). after rinsing with pbs, about 1 × 10 13 flowed over the chip surface. after each cycle, the sensor surface was regenerated with10 mm glycine-hcl ph 2.5. the data were fitted to a 1:1 interaction steadystate binding model using the biaevaluation 1.0 software. for competition-binding assays, the ace2 protein was diluted in 10 mm sodium acetate buffer, ph4.5, and was immobilized on the chip at about 650 response units. for the analyses, the his-tagged sars-cov-2 rbd protein was diluted in hbs-ep buffer or hbs-ep buffer with 100 nm antibody (1e2, 2f2, 3f11, 4d8, or 5f8). the rbd in different buffer at gradient concentrations (0, 6.25 nm, 25 nm, 100 nm and, 400 nm) was flowed over the chip surface. after each cycle, the sensor surface was regenerated with 10 mm glycine-hcl ph 2.5. the binding kinetics was analyzed with the software of biaevaluation using a 1:1 binding model. sars-cov-2 spike pseudotyped particle (sars-cov-2pp). to produce sars-cov-2pp, hek293t cells were seeded 1 day prior to transfection at 2.5 × 10 6 cells in a 10-cm plate. the next day, cells were transfected using lipofectamine 2000 (thermofisher). the plasmid dna transfection mixture (1 ml) was composed of 15 µg of pnl-4.3-luc-e − r − and 15 µg of pcdna-sars-cov-2-s that was purchased from sino biologicals and reconstructed by deletion of 18 amino acid cytoplasmic tail. a nonenveloped lentivirus particle (bald virus) was also generated as negative control. 16 h after transfection, the media was replaced with fresh media supplemented with 2% fbs. supernatants containing sars-cov-2pp were typically harvested at 36-48 h after transfection and then filtered through a syringe filter (0.22 µm) to remove any cell debris. sars-cov-2pp was freshly used or allocated and frozen at −80°c. to conduct the virus entry assay, 293t cells were transiently transfected with human ace2 expression plasmid and 1 × 10 4 cells and seeded in each well of a 96-well plate at 1 day prior to transduction. the next day, 100 µl of supernatant containing sars-cov-2pp was added into each well in the absence or presence of serially diluted sdabs or human igg1 fc-fused sdabs. forty-eight hours after transduction, the cells were lysed in 100 µl of passive lysis buffer and 50 µl lysate was incubated with 100 µl of luciferase assay substrate according to the manufacturer's instructions (promega, madison, wi, usa). ethics statement and virus isolation. sars-cov-2 was isolated from bronchoalveolar lavage fluid (balf) from a covid-19 patient in the jin yin-tan hospital of wuhan as reported previously 22 . briefly, the patient was a 65-year-old man who reported a high fever and cough, with little sputum production, at the onset of illness. he had a continuous fever and developed severe shortness of breath 16 days later. balf sample was collected from this hospitalized patient by nurses according to a standard procedure in which a bronchoscope is passed through the mouth into the lungs to obtain cells and other components from bronchial and alveolar spaces. a clinical protocol was conducted in accordance with the declaration of helsinki and was approved by the national health commission of china and ethics commission of the jin yin-tan hospital of wuhan (no. ky-2020-01.01). written informed consent was waived by the ethics commission of the designated hospital for emerging infectious diseases. clearing the airway and collection of balf were as standard of care and for clinical etiological diagnosis. therefore, the requirement for written informed consent was waived given the context of emerging infectious diseases. for the isolation and identification of potential pathogens, the balf specimens were filtered and inoculated onto vero cells. all cultures were observed daily for a cytopathic effect (cpe). cpe were observed in 30% of vero cells after two passages. the viral particles in culture supernatants were characterized by negative staining electron microscope. the isolated sars-cov-2 was obtained from the patient by dr. lili ren and the virus full length sequence was deposited in gisaid database with accession id of epi_isl_402123, which is completely as same as genbank accession number mn908947. gisaid is a globally recognized virus database and more than 56,000 viral genomic sequences of hcov-19 have been shared via gisaid since the start of the covid-19 outbreak. sars-cov-2 neutralization assay. the 50% tissue culture infectious dose (tcid 50 ) assay was performed for sars-cov-2 in vero cells. briefly, cells were seeded 24 h before infection in a 24-well plate at a density of 8 × 10 4 cells/well. viruses were serially diluted at 1:10 dilution. after 72 h of incubation, the media were removed, and cells were fixed and stained with crystal violet. the tcid 50 /ml titer was determined. for antibody neutralization assay, vero cells were seeded in 96-well plates at 1 day prior to infection. serially diluted sdabs were mixed with sars-cov-2 at 100 tcid 50 per well and incubated at 37°c for 1 h. the antibodyvirus mixture was incubated on vero cells at 37°c for 1 h. unbound sars-cov-2 virions were removed by washing cells with fresh medium, then incubated for 24 h at 37°c. the culture supernatants were collected for viral nucleic acid quantification. viral rna quantification was carried out by taqman real-time rt-pcr as reported with plotted standard curves using in vitro transcribed rna. briefly, the viral rna was isolated using trizol ls reagent (invitrogen, carlsbad, ca) according to the manufacturer's protocol. rna was extracted from 100 µl culture supernatants and eluted in 50 µl dnase/rnase-free water. the viral nucleocapsid gene-based quantification assay was developed using the taqman production of human igg1 fc fusion sdabs. the sequences of selected sdabs were cloned into a mammalian expression vector under the control of hef1-htlv promotor and fused with n-terminal interleukin-2 signal peptide and c-terminal fc region, comprising the ch2 and ch3 domains of human igg1 heavy chain and the hinge region. maxiprepped plasmids were transiently transfected into 293-f cells (thermofisher) and the cells were further cultured in suspension for 6 days before harvesting antibody-containing supernatant. fc-fused sdabs were prepared with prepacked hitrap® protein a hp column (ge healthcare). the produced fcfusion protein was analyzed by sds-page and the western blot using standard protocols for dimerization, yield and purity measurement. the primary antibody used for western blot was a horseradish peroxidase conjugated goat anti-human igg (sigma-aldrich, st. louis, mo, usa). immunofluorescence microscopy and western blot. cultured 293t cells on coverslips were transfected with either sars-cov-2 s expression plasmid or empty vector for 24 h and then fixed using 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.1% triton x-100 (sigma-aldrich) in pbs for 10 min. the cells were then incubated with each sdab overnight at 4°c. after three washes with pbs, the cells were incubated with alexa fluor 488-conjugated 6x-his tag monoclonal antibody (his.h8) (thermofisher, ma1-21315-a488, 1:1000) for fig. 4 inhibition of sars-cov-2 entry by fc-fused sdabs. a representation of the human igg1 fc-fused sdabs in this study. sdab-fc fusion construction generates a bivalent molecule with an approximate molecular weight of 80 kda. b homology modeling of the bivalent 5f8-fc molecule with swiss-model server (https://swissmodel.expasy.org) 23 . the template structure for 5f8 modeling was based on a humanized camelid sdab in the pdb database (3eak). the structure is depicted as cartoons and colored with secondary structure. three cdrs, hinge region and fc were indicated. c five fc-fused sdabs were analyzed by western blot with gradient sds-page in reducing (with β-mercaptoethanol) or nonreducing (without β-mercaptoethanol) condition. d summary of ec 50 value of fc-fused sdabs neutralization against sars-cov-2pp. ec 50 fold increases versus the corresponding monovalent sdabs were calculated. 1 h at room temperature. the nuclei were stained with dapi (1:10,000) diluted in pbs for 5 min and mounted with an antifade reagent (thermofisher). images were acquired with a leica tcs sp5 confocal microscope system. for western blot, 293t cells in 6-well plate were transfected with sars-cov-2 s, sars-cov-2 s or empty vector individually. twenty-four h post transfection, cell lysates were prepared, and the samples were boiled with 2× sds loading buffer and loaded onto a 10% polyacrylamide gel. after electrophoresis, the separated proteins were transferred onto a nitrocellulose membrane (bio-rad, hercules, ca, usa). the resulting blots were probed with a sdab as primary antibody and an hrp-linked 6x-his tag antibody (thermofisher, his.h8, ma1-21315-hrp, 1:1000) as the secondary antibody. antibody against β-actin is from sigma-aldrich (a1978, 1:8000). the ecl reagent (amersham biosciences, piscataway, nj, usa) was used as the substrate for detection. statistics and reproducibility. data were analyzed using graphpad prism 6.01 (graphpad software, san diego, ca, usa). the values shown in the graphs are presented as means ± sd. one representative result from at least two independent experiments was shown. antibody neutralization experiments usually use two to four duplicated wells for each treatment. for sars-cov-2pp entry assay and sars-cov-2 infection, the infectivity data were first inversed to neutralization activity. each neutralization data set was normalized by the background control (no virus) to define the real value for 100% neutralization. after transformation to neutralization, the lowest concentration point of antibody treatment was set to 0% neutralization. then, a 4-parameters neutralization nonlinear regression model was fitted to report ec 50 values. all experiments were performed 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caplacizumab treatment for acquired thrombotic thrombocytopenic purpura molecular mechanism for antibody-dependent enhancement of coronavirus entry identification of a novel coronavirus causing severe pneumonia in human: a descriptive study swiss-model: homology modelling of protein structures and complexes reporting summary. further information on research design is available in the nature research reporting summary linked to this article. the sequences of sdabs have been deposited in genbank with the accession numbers mt813428-mt813432. the isolated sars-cov-2 full length sequence was deposited in gisaid database with accession id of epi_isl_402123, which is completely as same as genbank accession number mn908947. all other data are available from the corresponding author upon reasonable requests. source data are provided with this paper.received: 29 march 2020; accepted: 12 august 2020; this work was supported by cams initiative for innovative medicine grant 2020-i2m-2-010 and 2016-i2m-3-020. w.y., x.c., l.r. q.j., and j.w. designed experiments and interpreted the data. w.y., x.c., x.l., c.w., x.l., j.h., and x.z. performed experiments and analyzed the data. w.y. conceived the study, supervised the work, and wrote the paper. all authors read and approved the final manuscript. a patent application has been filed on 17 march 2020 on single domain antibodies targeting sars-cov-2 (china patent application no. 202010185593.9). supplementary information is available for this paper at https://doi.org/10.1038/s41467-020-18387-8.correspondence and requests for materials should be addressed to q.j., j.w. or w.y.peer review information nature communications thanks the anonymous reviewers for their contribution to the peer review of this work. peer reviewer reports are available.reprints and permission information is available at http://www.nature.com/reprintspublisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. key: cord-343185-lbmbp9ca authors: hansen, c. b.; jarlhelt, i.; perez-alos, l.; hummelshoj landsy, l.; loftager, m.; rosbjerg, a.; helgstrand, c.; bjelke, j. r.; egebjerg, t.; jardine, j. g.; svaerke jorgensen, c.; iversen, k.; bayarri-olmos, r.; garred, p.; skjoedt, m.-o. title: sars-cov-2 antibody responses determine disease severity in covid-19 infected individuals date: 2020-07-29 journal: nan doi: 10.1101/2020.07.27.20162321 sha: doc_id: 343185 cord_uid: lbmbp9ca globally, the covid-19 pandemic has had extreme consequences for the healthcare system and calls for diagnostic tools to monitor and understand the transmission, pathogenesis and epidemiology, as well as to evaluate future vaccination strategies. here we have developed novel flexible elisa-based assays for specific detection of sars-cov-2 antibodies against the receptor-binding domain (rbd): an antigen sandwich-elisa relevant for large population screening and three isotype-specific assays for in-depth diagnostics. their performance was evaluated in a cohort of 350 convalescent participants with previous covid-19 infection, ranging from asymptomatic to critical cases. we mapped the antibody responses to different areas on protein n and s and showed that the igm, a and g antibody responses against rbd are significantly correlated to the disease severity. these assays-and the data generated from them-are highly relevant for diagnostics and prognostics and contribute to the understanding of long-term covid-19 immunity. coronaviruses (covs) are zoonotic pathogens primarily targeting the human respiratory system (1) . while most human cov infections are mild, three coronaviruses have appeared in the past two decades that cause deadly pneumonia. severe acute respiratory syndrome coronavirus (sars-cov-1), first observed in china in 2002, spread rapidly to 26 countries infecting more than 8,000 people and causing 774 deaths before being contained in 2003 (2) . barely a decade later in 2012, middle east respiratory syndrome coronavirus (mers-cov) was identified on the arabian peninsula and has caused more than 2,500 cases and 866 deaths (3) (4) (5) . at the end of 2019, a novel sars-cov strain (sars-cov-2) emerged in wuhan (china) and has been spreading at an unprecedented speed around the world ever since. the disease, named coronavirus disease 2019 (covid19) , accounts for more than 15 million confirmed cases and 630,000 related deaths at the time of manuscript preparation (6) . the clinical features of the covid-19 infection are diverse, ranging from asymptomatic carriers of the infection to acute respiratory distress syndrome and multiple organ dysfunction (7) . sars-cov-2 can infect people of all ages; however, elderly and people suffering from co-morbidities such as diabetes, cardiovascular disease, chronic respiratory disease or cancer are more inclined to suffer from a severe disease progression (8) . sars-cov-2 is an enveloped rna virus with a diameter of 65-125 nm to accommodate one of the largest genomes of all known rna viruses (29.8-29.9 kb) (9) . one-third of its genome encodes four structural proteins: spike (s), membrane (m), envelope (e) and nucleocapsid (n) proteins. the protein s that extends as homotrimers on the outer viral membrane binds to the host angiotensin-converting enzyme 2 (ace2) receptor and allows the virus to enter and infect the host target cell (10) . host proteases process the protein s into the s1 and s2 subunits: s1 is responsible for receptor recognition and is comprised of an n-terminal and a c-terminal domain, the later containing the receptorbinding domain (rbd) (11) ; while the s2 mediates the fusion of the viral envelope with the membrane of the host cell. the protein s of sars-cov-2 shares 76% homology at the amino acid level with the protein s of sars-cov-1 and while both interact with the ace2 receptor, sars-cov-2 does so with a 10-20 times higher affinity (12) , which may explain the higher transmission rate of covid-19. precise diagnosis of covid-19 with rt-pcr detection of sars-cov-2 nucleic acids remains crucial to identify symptomatic carriers to secure correct treatment and as a tool in quarantine strategies to limit the infection rate in asymptomatic carriers. serological detection of specific sars-cov-2 antibodies is a useful tool to identify . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 29, 2020. . https://doi.org/10.1101/2020.07.27.20162321 doi: medrxiv preprint convalescent individuals that have developed immunity and thereby might be protected against reinfection, although this issue is still not resolved (13) . moreover, serological testing is necessary to understand the transmission, pathogenesis and epidemiology of sars-cov-2, providing critical data to inform public health authorities for controlling the spread of covid-19 and eventually re-opening societies. another question that has arisen is whether an overwhelming antibody response may even aggravate covid-19 infection in patients (14) . due to urgency and demand, many serological tests have been developed rapidly and made commercially available with only limited validation on clinical samples (15) . we have developed a flexible elisa-based platform for rapid and specific detection of sars-cov-2 antibodies. the platform includes an indirect rbd sandwich elisa (s-elisa) for pan immunoglobulin (ig) detection suitable for large scale antibody surveillance and direct elisas for in-depth analyses of the igm, iga and igg isotype antibody responses towards rbd and protein n. moreover, we set out to characterize the antibody response levels in relation to symptom characteristics and disease severity, to elucidate the immunological response in covid-19 convalescent individuals. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july 29, 2020. . https://doi.org/10.1101/2020.07.27.20162321 doi: medrxiv preprint the assays were optimized with a specific focus on diminishing nonspecific binding and the final setup was chosen based on the optimal intensity (od) of the absorbance signal and signal-to-noise (s/n) ratio between the positive sample and the negative quality control. the developed elisa-based assays proved to be suitable for automatization and were used in high-throughput setups with both 96-well and 384-well formats, correlating significantly a total of 350 convalescence plasma samples from previously infected individuals with sars-cov-2 (positive samples) and 580 plasma samples from healthy individuals collected before the pandemic outbreak (negative controls) were subjected to antibody measurement in the rbd s-elisa ( figure 1a ) and in the direct elisa setups ( figure 1b -d). s/n ratio between the od of a positive sample and the od of the negative quality control and receiver operating characteristic (roc) were assessed to calculate the best fit cut off to estimate the performance of the assay. the rbd s-elisa performed with a 94.9% sensitivity and 99.7% specificity ( figure 1a ). the sensitivities and specificities of the direct elisas were 61.4%, 99.1% for igm ( figure 1b) , 63.4%, 99.3% for iga ( figure 1c ) and 94.3%, 99.5% for igg ( figure 1d ), respectively. the intra-and inter-assay variation were found to be acceptable (< 10%) for all four assays ( table 1 ). the limit of detection was determined by interpolating the cut off od value and converting it into antibody concentrations. the resulting values indicate an estimated 10-fold higher sensitivity of the direct elisa setup (0.406 ng/ml) compared to the s-elisa (4.046 ng/ml) ( table 1) . detection of igm, iga and igg antibodies against sars-cov-2 protein n was evaluated by analyzing 136 positive samples and 174 negative controls and roc curve analyses were assessed to estimate the assay performance . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july 29, 2020. . https://doi.org/10.1101/2020.07.27.20162321 doi: medrxiv preprint to provide a better insight into antibody seroconversion during sars-cov-2 infection and reactivity against different locations on protein s and protein n, we conducted igm, iga and igg detection in 90 positive samples against 14 protein fragments and short peptides located on the protein s and protein n structures, full-length rbd, protein s and protein n (figure 2a ). the heatmap shown in figure 2b indicates a different reactivity of igm, iga and igg towards the 17 proteins/peptides analyzed. figure 2c represents a simplified overview of the % of individuals with antibodies recognizing each protein/fragment. a clear tendency towards a higher prevalence of igg responses against the s1 subunit part of protein s and the middle part of protein n was observed. parts of the s2 subunit and most of the n terminal part of rbd showed little immunogenicity. the cut off was calculated as the average of the negative controls plus three times the standard deviation. levels of antibodies against sars-cov-2 were measured from plasma samples of 350 recovered individuals with covid-19. the rbd s-elisa measures total anti-rbd igs present in the samples using a single dilution ( figure 3a figure 3b ). in comparison, responses of igm and iga isotypes were detected in 215 and 222 of the individuals, respectively ( figure 3c and figure 3d ). a dynamic range of antibody titers expressed as arbitrary units (a.u.)/ml was obtained when od values were interpolated by regression analysis using a four-parameter logistic curve fitting. the convalescent individuals were classified according to disease severity ( figure 4a -c) and symptom onset calculated as the time from the first selfperceived symptom related to covid-19 to the moment of blood sampling ( figure 4d -f). we observed a highly significant difference in the antibody titers between the disease severity groups ( figure 4a -c) (p < 0.0001), with a clear association between increasing antibody levels and more severe disease symptomatology for all three antibody isotypes. igg shows the most significant increase between the severity groups ( figure 4c ). in contrast, asymptomatic . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july 29, 2020. . https://doi.org/10.1101/2020.07.27.20162321 doi: medrxiv preprint individuals appeared not to follow the same severity tendency. this could be explained by the low number of individuals in this group, thus not being representative. when we assessed the difference in antibody titers between groups based on the time of symptom onset ( figure 4d -f), we observed a significant increase in igg levels continuously over the time of sampling ( figure 4f ), while igm ( figure 4d ) and iga levels ( figure 4e ) did not change significantly. table 2 depicts the correlation between self-perceived covid-19 symptomatology and the igm, iga and igg titers, as well as the disease severity, sex and age. symptoms such as fever, shortness of breath and lack of appetite were significantly and positively correlated with igm and igg levels. in contrast, iga levels were significantly negatively correlated with the loss of sense of smell/taste and headache. both age, sex (male) and disease severity were significantly positively correlated to the level of all three antibody isotypes. when adjusting the analysis between antibody levels and disease severity for age and sex, there was no longer a significant correlation between iga and severity. whereas it remained highly significantly correlated for igm and igg. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 29, 2020. . https://doi.org/10.1101/2020.07.27.20162321 doi: medrxiv preprint we have developed an elisa-based platform for detection sars-cov-2 antibodies comprising an indirect rbd s-elisa for pan ig detection and direct elisas for in-depth analyses of the igm, iga and igg isotype responses towards rbd and protein n. rbd was chosen as the primary antigen for the screening and estimation of antibody titers for several reasons. it is regarded to be a sufficient representative part of protein s to induce an immunogenicity response (16) and we did not detect additional sensitivity improvements by employing the full protein s as a ligand-target in the direct elisa (to be described in detail elsewhere). in a recent phase 1 trial, antibody responses against a vaccine candidate (s-2p antigen) and the rbd were assessed, finding similar ig responses in pattern and magnitude between both antigens (17) . one of the advantages of using rbd instead of full-length protein s is the more efficient production and higher stability of recombinant rbd due to its reduced size and simple tertiary structure. the relative unique primary sequence of the sars-cov-2 rbd (18) also reduces the risk of cross-reactive antibody signals derived from prior b cell responses against other types of coronaviruses. it is; however, important to annotate that this setup is highly flexible, making it possible to substitute the antigen upon a change in demand or in case of viral mutations. the use of protein n as a target antigen for serological screening has several theoretical pitfalls: the location of protein n makes it less accessible for b cell receptor interaction on the naïve b cell and probably requires a viral membrane degradation. furthermore, protein n shows higher sequence conservation (90.5%) and thereby increase the risk of false-positive detection (19) . nevertheless, large commercial providers have chosen to use protein n as the serological target in different types of sars-cov-2 antibody assays (20) . in our study, the detection of igm antibodies towards protein n or its fragments was weaker than igg, suggesting a fast seroconversion of igm into igg. furthermore, around 5-10% of the convalescent individuals did not mature any detectable antibody response, which is in good agreement with a previous study (21) . the direct elisa setup allows the use of different sars-cov-2 proteins in their full-length, shortened variants or fragmented immunogens, offering a useful tool to study the different reactivity patterns of igm, iga and igg towards specific exposed areas on the viral antigens. we measured several different antigen areas on protein n and s to establish a heatmap of the antibody landscape. based on the results, we could demonstrate a tendency towards immune dominating areas in the s1 unit and the central part of protein n. however, it is important to highlight that the heatmap does not represent the full sequences of the cov antigens and that the dissection of antigens into shorter fragments could have a significant impact on the antibody reactivity. it could; however, in a more extensive setup, provide valuable information towards a targeted . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 29, 2020. we show that igg levels increases during the first 11 weeks after symptom onset, while the igm and iga levels remain stable for the same period of time. this was surprising as we would have expected a more pronounced decrease in igm levels over time. this observation could indicate an importance of the two isotypes, reinforced by the fact that severity is correlated with high levels of antibodies of all three isotypes. it has previously been shown that both igg independently and total antibody levels correlate with disease severity in patients during hospitalization (13) , but to our knowledge, the prolonged clear correlation between iga, igm and igg titers and disease severity have not been reported before. the data illustrates that individuals with mild symptoms during infection with sars-cov-2, in general, will mount a lower antibody response compared to individuals with moderate and severe symptoms several months after recovering from a covid-19 infection. this observation gives an essential insight into the immunological response regarding clinical disease presentation, which further highlights the demand for more quantitative assays. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 29, 2020. . https://doi.org/10.1101/2020.07. 27.20162321 doi: medrxiv preprint in this study, all three antibody isotypes correlate positively with age and sex (male), which can be explained by the fact that severe covid-19 infection is more pronounced in the elderly male population (22) . we show that the antibody titers, especially igg levels, are correlated with specific symptom characteristics, including fever, pharyngalgia, shortness of breath and nausea, which again shows the link between clinical manifestations and the immunological response. the iga level, on the other hand, was negatively correlated with loss of sense of taste/smell and surprisingly showed no other correlation to the symptomatology of the upper respiratory tract in this study. the role of iga, which is considered the predominant antibody involved in mucosal immunity, remains to be fully understood. iga is suggested to mediate anti-viral defense functions at different anatomic levels in relation to mucosal epithelium (23) . however, the mechanisms behind this remain unknown and often gain limited attention during infectious studies. in this respect, it could be interesting to examine the iga levels in mucosal tissue during sars-cov-2 infection and determine whether the mucosa-associated iga plays a significant role during sars-cov-2 infection. our findings provide support to the notion that antibodies towards sars-cov-2 represent a double-edged sword. antibodies are important in viral neutralization, but also in fc receptor-mediated phagocytosis, antibody-dependent cellular cytotoxicity (adcc) and complement-dependent cellular cytotoxicity (cdcc) and subsequent elimination of pathogens. however, it is known that particularly adcc and cdcc can drive harmful and systemic pro-inflammatory responses that can have severe pathophysiological consequences. thus, based on our findings and others, it may be suggested that an unwanted immune response towards sars-cov-2 may be one of the mechanisms causing hyperactivation of macrophages and monocytes, leading to the deadly cytokine storm, which seems to be a hallmark of covid-19 (24) . whether a previously infected individual can expect stable long-term protection against reinfection with sars-cov-2 remains unknown. a recent study found a correlation between the production of neutralizing antibodies against rbd and elevated igg titers in convalescence covid-19 individuals (25), reinforcing the use of rbd as the candidate to analyze for neutralizing antibodies in these individuals. the durability of neutralizing antibodies (primarily igg) against sars-cov-2 has yet to be defined, but persistence for up to 40 days from symptom onset has been described (13, 26) . in comparison, when following infection with sars-cov-1, concentrations of igg remained high for approximately 4 to 5 months before subsequently declining slowly during the next 2 to 3 years (27) . it is uncertain whether an individual with low antibodies titers, mainly igg, has a higher risk of reinfection compared to an individual with high levels of . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 29, 2020. . https://doi.org/10.1101/2020.07.27.20162321 doi: medrxiv preprint antibodies post disease. it has recently been suggested that in the absence of antibody levels, an individual could be protected against reinfections by the presence of memory t cells (28) . furthermore, the major proportion of convalescent individuals, included in this study, indeed show a dominating igg response, suggesting that both affinity maturation, isotype class switching and b cell memory response has occurred. these b cell populations could, together with the memory cd4+ and cd8+ cells, secure a fast and efficient response to a secondary exposure of sars-cov-2. this study is limited by relying on participants' retrospectively self-reported symptomatology and symptom debut, which allows for an unknown amount of misclassification. moreover, with this design, we could not monitor the antibody response concerning survival. however, because of the detailed analysis of the antibody responses, and the clear associations despite the retrospective design, we assume that the associations would be even stronger in a carefully conducted prospective designed study. in conclusion, we have established robust, flexible and specific elisa-based platforms for detection sars-cov-2 antibodies and presented novel insight into the link between antibody responses and covid-19 disease severity. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 29, 2020. . https://doi.org/10.1101/2020.07.27.20162321 doi: medrxiv preprint the following buffers were used: pbs (10.1 mm na2hpo4, 1. the coding sequence for protein s rbd (qic53204.1, aa r319-s593) was synthesized by genscript and cloned into a pcdna3.4 expression vector with an n-terminal human vh1-2 signal peptide and a c-terminal 10xhis tag followed by an avitag (-gsg-hhhhhhhhhh-gsg-glndifeaqkiewhe). the sequence of protein n (qld29192.1) was optimized as described elsewhere (29) and synthesized by geneart (thermo fisher scientific, waltham, ma, usa) into a pcdna3.4 vector with the human serum albumin signal peptide and a c-terminal 8xhis tag (-gs-hhhhhhhh). both constructs were expressed using a mammalian transient expression system. on day five after transfection, the supernatants were harvested by centrifugation and sterile filtered. the recombinant proteins were purified using a 2-step automated purification method setup on an äkta express chromatography system with an immobilized metal affinity excel histrap column and a size exclusion superdex200 column (chromatography system and columns cytiva, marlborough, ma, usa). the purified proteins were stored in a buffer composed of 20 mm hepes, 150 mm nacl, ph 7.4. a portion of the rbd was specifically biotinylated in the avitag sequence using a bira500 kit (avidity llc, co, usa). a total of 350 recovered individuals previously tested rt-pcr positive for sars-cov-2 were included in the study. the department of cardiology at herlev university hospital in denmark recruited the participants. the rt-pcr positive participants are comprised of males and females aged from 18-86 and course of disease ranged from asymptomatic to critically ill. all participants were invited to complete an electronic self-report questionnaire providing additional information about symptom onset, characteristics and disease severity divided into the following groups: asymptomatic, mild, moderate, severe or critical. the mild disease was defined as having few symptoms and generally feeling well, moderate disease as being bedridden at home, severe disease as the need for hospitalization and critical disease as need of admission to the intensive care unit (icu) for mechanical ventilation. characteristics of the study participants are detailed in table 3 . serum and edta plasma samples were stored in aliquots and kept frozen at −80 °c . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 29, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 29, 2020. . https://doi.org/10.1101/2020.07.27.20162321 doi: medrxiv preprint usa). plates were washed four times with pbs-t between the steps mentioned above. all 384-well plates were handled by the biomek fx automated workstation (beckman coulter, brea, ca, usa). development and validation: specificity of the signal, limit of detection, variation, parallelism and clinical performance the rbd s-elisa and the direct elisas was subjected to optimization with regards to dilution range, the use of blocking buffers and variations in incubation times. the final conditions were chosen based on s/n ratios. specificity and sensitivity were calculated based on roc curve representation and selection of the most appropriate cut off by prioritizing the specificity. assay sensitivity regarding the limit of detection was determined by the concentration given by interpolating the od value of the cut off. the calibrator was prepared by spiking 100 µg/ml of recombinant human monoclonal igg antibody against sars-cov-2 spike1 (a02038, genscript, piscataway, nj, usa) into normal human serum and diluting in serum into a 2-fold dilution. samples were treated as patient samples and further diluted 1:100 in the s-elisa and 1:1000 in the direct elisa in pbs-t followed by incubated as stated above. intra-assay variation was evaluated by calculating the coefficient of variation (cv) of an individual cvs for all the duplicates in a total of 40 samples. inter-assay variation was evaluated by calculating the cv of two samples run in duplicates in 8 separate plates on three different days. the parallelism between serum and plasma samples was evaluated by comparing 137 pairs of serum and plasma samples using spearman rank correlation tests. to evaluate whether the antibody levels correlated with the disease severity and/or the days after symptom onset, sample absorbances were logistically transformed and a four-parameter logistic curve fitting was applied to calibrate the antibody levels into a.u./ml. the appropriate dilution for each sample was chosen based on the best fit in the linear range of the calibrator. the interpolated value in a.u./ml was corrected by the dilution factor (400, 1200 or 3600). a sample od value below 0.06 was automatically given the value of 1 a.u./ml. a total of 12 different sars-cov-2 protein fragments on the protein s1 and s2 and protein n coupled via an nterminal cysteine and maleimide conjugation to recombinant-human serum albumin (rhsa) (albix, novozymes, bagsvaerd, denmark), 2 short proteins from protein s and full-length protein s, protein n and rbd were analyzed for immunogenicity capacity on the direct elisa. proteins details are illustrated in figure 2a . nunc™ maxisorp flat-bottom plates nonsterile 384-well plates were coated with 1 µg/ml of the proteins in pbs on at 4°c. a total of 90 rt-. cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 29, 2020. . https://doi.org/10.1101/2020.07.27.20162321 doi: medrxiv preprint pcr positive samples and six negative controls were diluted 1:20 in dilution buffer and incubated as mention above. detection and development procedures were followed, as described in subsection 4.2. statistical analyses were performed using graphpad prism version 8 statistical differences between disease severity and symptoms onset groups were analyzed using one-way anova (kruskal-wallis test) with dunn's multiple comparison test. spearman rank correlation tests were used to determine the correlation between different experimental parameters. data are represented as the average of sample duplicate and the median. significance levels: * = p < 0.05, ** = p < 0.01, *** = p < 0.001 **** = p < 0.0001 and p < 0.05 was considered statistically significant. all procedures involving the handling of human samples are in accordance with the principles described in the declaration of helsinki and ethically approved by the regional ethical committee of the capital region of denmark (h-20028627) . . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 29, 2020. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 29, 2020. . https://doi.org/10.1101/2020.07.27.20162321 doi: medrxiv preprint . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 29, 2020. . https://doi.org/10.1101/2020.07.27.20162321 doi: medrxiv preprint . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 29, 2020. . https://doi.org/10.1101/2020.07.27.20162321 doi: medrxiv preprint kruskal-wallis test was performed. p value < 0.05 was considered significant. * = p < 0.05, ** = p < 0.01, *** = p < 0.001 **** = p < 0.0001. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 29, 2020. . https://doi.org/10.1101/2020.07.27.20162321 doi: medrxiv preprint tables table 1 . intra-and inter-assay variation and limit of detection for the s-elisa setup and the direct elisa setup. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 29, 2020. spearman rank correlation analysis was performed. p value < 0.05 was considered significant. * = p < 0.05, ** = p < 0.01, *** = p < 0.001 **** = p < 0.0001. a n = 349 participants. b n = 348 participants. c partial correlation adjusted for age and sex. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july 29, 2020. . https://doi.org/10.1101/2020.07.27.20162321 doi: medrxiv preprint . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july 29, 2020. . https://doi.org/10.1101/2020.07.27.20162321 doi: medrxiv preprint origin and evolution of pathogenic coronaviruses sars -beginning to understand a new virus identification of a novel coronavirus in patients with severe acute respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group sars and mers: recent insights into emerging coronaviruses coronavirus disease (covid-19) pandemic clinical features of patients infected with 2019 novel coronavirus in wuhan, china estimates of the severity of coronavirus disease 2019: a model-based analysis covid-19 infection: origin, transmission, and characteristics of human coronaviruses the covid-19 pandemic: biological evolution, treatment options and consequences structural and functional basis of sars-cov-2 entry by using human ace2 cryo-em structure of the 2019-ncov spike in the prefusion conformation. science (80-. ) antibody responses to sars-cov-2 in patients of novel coronavirus disease dissecting antibody-mediated protection against sars-cov-2 the receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in sars-cov-2 patients an mrna vaccine against sars-cov-2 -preliminary report the coronavirus nucleocapsid is a multifunctional protein biochemical characterization of sars-cov-2 nucleocapsid protein diagnostic accuracy of serological tests for covid-19: systematic review and meta-analysis seroprevalence of antibodies against sars-cov-2 among health care workers in a large spanish reference hospital gender differences in patients with covid-19: focus on severity and mortality multiple functions of immunoglobulin a in mucosal defense against viruses: an in vitro measles virus model dysregulation of immune response in patients with covid-19 in wuhan, china detection of sars-cov-2-specific humoral and cellular immunity in covid-19 convalescent individuals immune phenotyping based on neutrophil-to-lymphocyte ratio and igg predicts disease severity and outcome for patients with covid-19 duration of antibody responses after severe acute respiratory syndrome robust t cell immunity in convalescent individuals with asymptomatic or mild covid-19 chimeric proteins containing map-1 and functional domains of c4b-binding protein reveal strong complement inhibitory capacities the authors thank jytte bryde clausen and bettina eide holm for excellent technical assistance and josé juan almagro armenteros (university of copenhagen, copenhagen, denmark) for his statistical advice. this work was financially supported by the carlsberg foundation (cf20-0045) and the novo nordisk foundation (205a0063505). the authors have declared that no conflict of interest exists. the assays were developed in a non-commercial collaboration between rigshospitalet and novo nordisk a/s. key: cord-303868-aes92l6s authors: steffen, tara l.; stone, e. taylor; hassert, mariah; geerling, elizabeth; grimberg, brian t.; espino, ana m.; pantoja, petraleigh; climent, consuelo; hoft, daniel f.; george, sarah l.; sariol, carlos a.; pinto, amelia k.; brien, james d. title: the receptor binding domain of sars-cov-2 spike is the key target of neutralizing antibody in human polyclonal sera date: 2020-08-22 journal: biorxiv doi: 10.1101/2020.08.21.261727 sha: doc_id: 303868 cord_uid: aes92l6s natural infection of sars-cov-2 in humans leads to the development of a strong neutralizing antibody response, however the immunodominant targets of the polyclonal neutralizing antibody response are still unknown. here, we functionally define the role sars-cov-2 spike plays as a target of the human neutralizing antibody response. in this study, we identify the spike protein subunits that contain antigenic determinants and examine the neutralization capacity of polyclonal sera from a cohort of patients that tested qrt-pcr-positive for sars-cov-2. using an elisa format, we assessed binding of human sera to spike subunit 1 (s1), spike subunit 2 (s2) and the receptor binding domain (rbd) of spike. to functionally identify the key target of neutralizing antibody, we depleted sera of subunit-specific antibodies to determine the contribution of these individual subunits to the antigen-specific neutralizing antibody response. we show that epitopes within rbd are the target of a majority of the neutralizing antibodies in the human polyclonal antibody response. these data provide critical information for vaccine development and development of sensitive and specific serological testing. natural infection of sars-cov-2 in humans leads to the development of a strong neutralizing antibody response, however the immunodominant targets of the polyclonal neutralizing antibody response are still unknown. here, we functionally define the role sars-cov-2 spike plays as a target of the human neutralizing antibody response. in this study, we identify the spike protein subunits that contain antigenic determinants and examine the neutralization capacity of polyclonal sera from a cohort of patients that tested qrt-pcr-positive for sars-cov-2. using an elisa format, we assessed binding of human sera to spike subunit 1 (s1), spike subunit 2 (s2) and the receptor binding domain (rbd) of spike. to functionally identify the key target of neutralizing antibody, we depleted sera of subunit-specific antibodies to determine the contribution of these individual subunits to the antigen-specific neutralizing antibody response. we show that epitopes within rbd are the target of a majority of the neutralizing antibodies in the human polyclonal antibody response. these data provide critical information for vaccine development and development of sensitive and specific serological testing. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was initially identified in patients with severe pneumonia in wuhan, china in december of 2019. due to its initial zoonotic transmission and human to human spread within an immunologically naïve population, it has since caused over 4 million confirmed cases and over 790,000 deaths worldwide (who 2020), with approximately 30% of all cases occurring in the united states as of july 15 th 1-5 . infection with sars-cov-2 can result in a range of states from asymptomatic to symptomatic, with symptomatic cases ranging from mild non-specific symptoms, like malaise, to severe pneumonia and multiple organ failure 1-3,5,6 . sars-cov-2 is a positive sense, single stranded, enveloped rna virus with a ~29 kb genome that is virologically similar to the enzoonotic beta-coronaviruses sars-cov and mers-cov. the sars-cov-2 genome encodes 16 non-structural proteins and 4 structural proteins: spike (s), nucleocapsid (n), envelope (e), and membrane (m). the coronavirus n protein functions by interacting with viral rna to form the ribonucleoprotein, while e and m function in virion assembly and budding [7] [8] [9] [10] . spike is a homotrimeric transmembrane protein that is comprised of two subunits per monomer, s1 and s2 that are responsible for binding the host cell receptor and viral fusion, respectively. similarly to the human coronavirus nl-63 and sars-cov, sars-cov-2 spike uses human angiotensin converting enzyme 2 (ace2) to gain entry into target cells [8] [9] [10] . specifically, the s1 subunit of sars-cov-2 contains the receptor binding motif (rbm) within the receptor binding domain (rbd) that makes direct contact with the ace2 receptor for receptor-mediated entry [9] [10] [11] . important to note for antibody structural determinants, the pre-fusion confirmation of the trimeric spike has a range of states that are described as "up" or "down" based on the angle of rbd within s1. for a virion to be able to interact with ace2 and gain entry into host cells, rbd must be in the "up" conformation between 50° and 70° that represents a receptorbinding active state 10, 12 . when the interaction between rbd and ace2 is disrupted, the entry of sars-cov-2 into susceptible cells is blocked 13, 14 . spike is known to be a major antibody antigenic determinant for both mers-cov and sars-cov 15 that leads to the generation of protective immune responses including the production of highly neutralizing antibodies [16] [17] [18] . targets for these antibodies within spike include both conformation dependent and linear epitopes of rbd and the s2 fusion peptide. these neutralizing antibodies are proposed to block rbd-ace2 receptor interactions or prevent s2 fusion with host membranes 19, 20 . spike being a major antigenic determinant for the antibody response against closely related beta-coronaviruses contributes to our hypothesis that the neutralizing polyclonal antibody response to sars-cov-2 will target spike and its sub-domains. to determine the current antigenic variation and display that variation within the structure of sars-cov-2 spike we interrogated 34,756 sars-cov-2 genomes derived from human samples available from gisaid on june 15, 2020. the spike homotrimer contains multiple subunits, including s1 and s2, both of which contain a total of 22 glycosylated residues which can affect spike protein folding, receptor interactions and potentially block antibody recognition and are represented as lollipops in the schematic ( figure 1a ). the s1 subunit (residues 13-303) of spike contains the n terminal domain (ntd), c terminal domain (ctd), the receptor binding domain (rbd, residues 319-541), and the receptor binding motif (rbm, residues 437-508). the s2 subunit contains the fusion peptide (fp, residues 788-806), and heptad repeat 1 and 2 (hr1, residues 920-970, hr2, residues 1163-1202), the transmembrane domain and cytoplasmic tail. in our analysis of naturally occurring amino acid (aa) variation, low quality sequences determined by gaps or ambiguous nucleotides >50nt were removed (-497 sequences). the 34,259 remaining sequences were translated and aligned using muscle (supplemental file 1), then duplicate sequences were removed. this resulted in a multiple sequence alignment of 1273 amino acids (aa), with 4,065 unique aa sequences and an overall pairwise identity of 97.5%. the prevalence of aa variation per site and aa conservation was determined using the sequence variation tool (viprbrc) 21 and the consurf server 22 , respectively. the level of aa variation was measured by calculating the aa frequency at each position within the multiple sequence alignment, then shannon entropy was used to define aa conservation using data on the 20 potential aa present. these conservation scores were broken down into 6 discrete color coded categories with a score of 6 being most variable, representing >1,000 mutations at that site, and 1 being most highly conserved with 0-1 mutations per site. the aa conservation was then displayed in the context of the spike pre-fusion trimer (pdb:6vsb) 23 to represent exposure to the human antibody response, where chain a displays the rbd within the up position and the b and c chains display the rbd in the down position ( figure 1b) . the spike trimer color coded for aa variation is located next to the spike protein where the subunits are color coded with s1 ntd in cyan, rbd in dark green, and s2 in light green. from the aa sequence variation analyses, we observed the well documented g614d variation, which may have a fitness advantage. we also observed an additional 422 positions that contained a range of variation from 51/4065 (1.25%) to 2/4065 (0.05%). once the aa variation was mapped onto the trimer structure, we observed that the greatest level of aa variation is found within the s1 ntd (94.2% identity), while the lowest level of aa variation is within the rbd (96.7% identity) and s2 (99.6%). the low level of aa variation within rbd was also recently described by starr et al 24 . our data, in addition to that of starr et al, indicate that overall the rbd and s2 domains are highly conserved and are currently genetically stable targets for vaccine and therapeutic intervention. we next wanted to investigate the specificity and immunodominance of the polyclonal antibody response to the subunits of spike due to its potential as a target for the development of vaccines and therapeutics. this concept is based upon vaccines developed against sars-cov and mers-cov 15, 25, 26 and the strongly neutralizing monoclonal antibodies that have been identified as spike specific [27] [28] [29] [30] [31] [32] . to address specificity and immunodominance, we analyzed serum samples from 16 laboratory confirmed sars-cov-2 infection cases as determined by qrt-pcr, with 11 of the 16 subjects being admitted to the hospital. the sars-cov-2 positive serum samples analyzed were obtained from patients in saint louis, mo, cleveland, oh, and san juan, pr, with no subjects succumbing to infection. the median age of the individuals is 59.1 years, with 7 males and 9 females. specific demographics regarding the subjects are listed in table 1 . the cohort controls were collected prior to the emergence of sars-cov-2 (2015-2018) from previous studies conducted at saint louis, mo, cleveland, oh, and san juan, pr, and had a similar age and sex distribution. to investigate and quantify the igg response to sars-cov-2, we performed elisa assays using serum from 16 sars-cov-2 + subjects and 28 sars-cov-2 negative control subjects. we serially diluted sera from 1:50 to 1:64,000 as four-fold dilutions and evaluated binding to recombinant s1, s2, and rbd (figure 2a -c). polyclonal sera from all 16 sars-cov-2 subjects showed igg binding to each spike subunit by elisa. igg reactivity to the s1 subunit, which contains the rbd, ranged in optical density (od) from 1.4 to 3.4, while the 28 control subjects had an od range from 0.13 to 2.2 at the highest concentration tested. four subjects were responsible for the majority of the elisa binding to s1 within the control group ( figure 2a ). the overlap of these four negative subject samples with the sars-cov-2 + subjects suggests that there is antibody cross-reactivity to the sars-cov-2 s1 protein, most likely due to prior human coronavirus (hcov) infections (nl63, hku1, oc43, 229e). however, the focus of our study is to functionally define the key targets of the neutralizing antibody response to sars-cov-2, and further studies would need to be completed to define the nature of the cross-reactive response. we next interrogated the antibody response to the s2 subunit, where at a 1:50 dilution the sars-cov-2 positive subjects had an od range of 1.3 to 3.1, while control subjects had an od range of 0.1 to 1.07 ( figure 2b ). only one control subject had antibody binding that overlapped with the lower range of the sars-cov-2 subjects. we used an identical approach to evaluate the antibody response to rbd ( figure 2c ). here we saw a robust antibody response with a range in od from 0.9 to 3.5 at a serum dilution of 1:50, and we observed no antibody binding above background in the control group. multiple groups have observed a similar responses to the rbd subunit, indicating the specificity of the rbd antibody response 33, 34 , which could in part be due to the low level of conservation of the rbd amino acid sequences between sars-cov-2 and the hcovs, which cause the common cold 24, 34 . to further quantify differences in binding to the individual spike subunits we calculated the area under the curve (auc) of the s1, s2, and rbd elisa assays for each subject ( figure 2d -f, table 3 ). quantification of the auc measures the antibody binding at multiple antibody concentrations quantifying a combination of avidity and specificity of the sera for each subject. when assessing binding to s1, the mean auc of sars-cov-2 subjects was 2.61 +/-1.38 and the mean auc of controls was 0.71+/-0.53 (p<0.0001; figure 2d ). upon comparing the auc for antibody binding to s2, we observed a mean auc of 2.6 +/-1.0 and 0.47 +/-0.22 (p<0.0001) sars-cov-2 + patients and controls, respectively ( figure 2e ). interestingly, when we assess binding to rbd, we observed a mean auc of 3.8 +/-1.7 and 0.3 +/-0.07 (p<0.0001) showing minimal cross-reactivity from the negative subjects ( figure 2f ). the rbd binding data matches recently described results 33, 34 , which show that the antibody response to rbd is specific to sars-cov-2 infection, with no known cross-reactivity from antibodies derived from endemic hcov infection. overall, we show that s1, s2, and rbd from spike are targeted by the human polyclonal response in all individuals from our cohort. additionally, we observe potential cross-reactivity within the control group to the s1 domain outside of the rbd. this cross-reactivity is important to note for serological and vaccine evaluation, as using rbd as a target antigen may provide the most specific and sensitive test that results with fewer false-positives. interestingly, this also highlights the potential for cross-reactive s1 antibodies to play a role in either protection or exacerbation of sars-cov-2 disease. it has been recently demonstrated that human mabs generated after sars-cov infection were shown to cross-react and neutralize sars-cov-2 ( 35 ), while sars-cov infection generates a polyclonal antibody response that is able to bind spike from sars-cov-2 while not able to neutralize the virus 36 . furthermore, mechanisms aside from neutralization that are dependent on the fc region of the antibody are capable of limiting viral infection 15, 37, 38 . as there was a broad dynamic range of antibody binding to spike subunits from our sars-cov-2 + subjects, we stratified the samples based upon days post qrt-pcr positive test, because days post symptoms was unavailable, to evaluate the potential role of time post infection on antibody binding and specificity. samples were stratified into three groups: prior to day 18, day 21-26, and day 26-40 post qrt-pcr + test. we compared the auc values from the elisa binding curves to s1, s2, and rbd over this time period and did not observe a role for time post infection on antibody binding and specificity with our limited sample set (supplemental figure 1 a-c). the heterogeneity of antibody binding has been observed in other patient cohorts 33, 34, 39 . additionally, we quantified the relative binding of the polyclonal antibody response between different spike subunits to determine if the subunits were equally targeted by the antibody response. to this end, we evaluated the correlation of auc between the s1, s2 and rbd subunits (figure 2 g-i). when we compared the auc of s1 and s2 we observed significant correlation (p=0.0055, r=0.6265) ( figure 2g ). as expected, based on the location of rbd within s1, s2 auc significantly correlated with rbd auc (p=0.0100, r=0.5824), which may suggest epitopes for binding within s2 as well as rbd ( figure 2h ). based on the location of rbd within s1 we would anticipate correlation between their auc values ( figure 2i ), and indeed there is a significant correlation (p=0.0120, r=0.5676) that would suggest that either the majority of binding to s1 occurs within rbd, or that there are antibody epitopes throughout s1 that drive a robust antibody response. the elisa antibody binding results indicate that all sars-cov-2 + patients within our cohort had antibodies which bound to each subdomain of the spike protein. antibody neutralization is one mechanism of protection from severe viral disease. the mechanism of action of neutralizing antibodies often include the targeting of viral proteins that interact with the host receptor for entry or viral proteins required for fusion with host cell membranes (reviewed in 40 ). for sars-cov-2, the multifunctional spike protein is required for entry and fusion. specifically, the s1 domain contains rbd, which is responsible for binding the human ace2 protein mediating entry 41, 42 , while s2 contains the fusion peptide 43 . it has been shown by other groups that monoclonal antibodies targeting spike can block infection with sars-cov-2 and that natural infection of humans often produces neutralizing antibodies 29-32,34,44,45 , which is thought to prevent subsequent covid-19. however, the specificity of human polyclonal neutralizing antibodies against infectious sars-cov-2 is only now beginning to be understood. to begin to understand the human polyclonal neutralizing antibody response we utilized a focus reduction neutralization tests (frnt) ( figure 3a ) based upon the assay we had developed for multiple emerging infectious diseases [46] [47] [48] and for sars-cov-2 49 ( figure 3a ). there are multiple advantages to the frnt assay over pseudotype-virus assays and plaque assays, including the use of infectious virus that may better reflect heterogeneity in the conformational structure of the virion, quantitative measurement of the reduction of viral replication and spread as each foci diameter measured represents multiple cells, and finally the use of 96 well plates allowing for titers to be quantified using multiple technical and biological replicates. overall, this assay allows for a rigorous and quantitative determination of antibody neutralization potential. using the frnt assay, we determined the concentration of patient sera required to neutralize sars-cov-2 infection. based upon the antibody neutralization curve ( figure 3b ), the serum dilution necessary to neutralize 50% of the virus (frnt50) ranged from 1/53 to 1/4168 with a mean of 1/768 ( figure 3c ). the serum dilution necessary to neutralize 90% of the virus (frnt90) ranged from 1/50-1/995 with a mean of 1/200 ( figure 3c) . notably, the sera from sars-cov-2 patients in our cohort were capable of neutralizing infectious virus independent of day post positive test ( figure 3b -c, table 2 ); while, sera from the majority of control subjects had no demonstrated antibody neutralization. one control subject, whose sera was cross-reactive in the s1/s2 elisa binding assay demonstrated 10% sars-cov-2 neutralization potential at a 1/50 dilution, but further investigation of cross-neutralization is beyond the scope of this current study. based upon the ability of the sars-cov-2 subjects to neutralize at least 90% of the virus, we show that the polyclonal antibody response has the breadth and specificity to completely neutralize sars-cov-2 infection. this would suggest that natural infection would be capable of controlling viral infection and limiting the potential of disease and transmission at the timepoints we assessed ( figure 3d ). in animal model studies, hamsters have demonstrated that immune sera can protect from challenge 50 , although currently the mechanisms of that protection are unknown. to functionally determine which of the spike subunits are the main target of neutralizing antibodies, we performed a functional assay developed by the de silva lab for use in flaviviruses 51 . in this approach individual spike subdomains are linked to beads and are used to depleted sera in an antigen specific manor. in our studies his-tagged proteins are conjugated to cobalt coated magnetic beads and serum from sars-cov-2 subjects are incubated with the conjugated proteins. this allows a complex of antibody:antigen:bead to form and be pulled down by a magnet, leaving the serum depleted of that particular antibody specificity ( figure 4a ). to understand the contribution of antibodies specific to each individual subunits, antibodies specific to each spike subunit, s1, s2, and rbd, were depleted from human polyclonal sera, and the antibody binding and neutralization potential of polyclonal sera after depletion was determined by elisa and frnt, respectively. using the bead-based approach, sera from 10 patients were depleted for s1, s2, and rbd individually by sequentially incubating serum two times with protein coated beads. to quantify the effects of the antigen-specific antibody depletions, the auc from elisa binding curves pre and post depletion (supplemental figure 2 , table 3 ) were quantified, and the values were paired per subject ( figure 4b ). after antigen specific depletions we observed significant reduction in spike subunit antibodies represented by a 3.4 (p=0.0005), 3.6 (p<0.0001) and 4.7 (p<0.0001) fold reduction in auc binding to s1, s2, and rbd respectively ( figure 4b ). moreover, to confirm that depletion protocol did not impact sars-cov-2 neutralization we performed depletions with an irrelevant protein, vacv a33r ( figure 4b ). the subunit depletion protocol significantly reduced the level of subunit specific antibody, which allowed us to evaluate the contribution of each individual subunit to the neutralizing antibody response. to measure the functional effect of s1, s2, and rbd antibody depletion on virus specific neutralization we evaluated post-depletion neutralization activity by frnt (supplemental figure 3 ). to confirm that the depletion protocol itself had no off-target effects on sars-cov-2 neutralization, a control depletion with vacv a33r was completed and neutralization pre and post depletion was measured. the control depletion had a minimal effect on the ability of the polyclonal sera to neutralize sars-cov-2 ( figure 4c : frnt50:1.3 fold decrease; frnt90:1.8 fold decrease). we then measured the antibody neutralization curves after depleting serum with s1, rbd or s2, and determined the serum dilution required to reduce infection by 50% (frnt50) and 90% (frnt90) ( table 4 ). to take into account the effects of the antibody depletion protocol, we compared the frnt50 of the control depleted serum with the subunit depleted serum, and observed a 3.7, 4.2, and 1.2 fold reduction after s1, rbd, and s2 depletion, respectively ( figure 4c ). based upon the frnt50 and frnt90 values, the depletion of s1 and rbd significantly reduced virus neutralization (p=0.0020 and p=0.0020). this suggests that polyclonal antibody binding to the rbd domain of the spike protein represents the key target of neutralizing antibody to sars-cov-2 after natural infection. since we observed a similar fold reduction after s1 and rbd depletion, it is likely that the majority of the neutralizing response is found within the rbd domain of s1. however, this is the average neutralizing antibody response, which is applicable to our cohort. when we evaluate changes in individuals, there are two patients that have a strong rbd neutralizing response, but also have a s2 specific neutralizing antibody response with 1.47, and 1.21 fold change after s2 depletion. overall, these data demonstrate natural sars-cov-2 infection generates a robust anti-rbd polyclonal neutralizing antibody response with some individuals mounting a neutralizing antibody response to s2. we conclude that the polyclonal neutralizing antibody response to sars-cov-2 primarily targets receptor interactions (s1/rbd) in the majority of individuals. to compare the relative neutralizing differences between spike domains, we normalized the data based upon frnt50 values and represented the data as % subunit specific neutralizing antibodies. this allows us to calculate the percentage of neutralizing antibodies that bind to s1, s2, or rbd, while taking into account the impact of the depletion protocol, based on our control and subunit specific depletions ( figure 4d ). further confirming the paired frnt data, 70% +/-19 and 68% +/-11% the highest percentage of neutralizing antibodies indeed bind to rbd and s1, suggesting a prevention in virus interaction with viral receptor maybe the dominant mechanism for antibody neutralization of sars-cov-2 after natural infection. additionally, s2 has the lowest percentage 19% +/-13% of s2 binding antibodies capable of neutralization, suggesting that viral fusion with host membranes is not a dominant target of the neutralizing antibody response to sars-cov-2 after natural infection, with our cohort of patients ( figure 4d ). this data has been further represented as % binding neutralizing antibodies based on the pre depletion frnt50 values (supplemental figure 3d) . overall, these data further confirm that a majority of neutralizing antibodies are targeted against the rbd within s1. in this study we examined the antigenic targets of the sars-cov-2 igg neutralizing antibody response that develop during natural infection. we quantified the immunodominance of anti-spike subdomain antibodies for binding by elisa and neutralization activity by antigen specific depletion followed by a sars-cov-2 neutralization assay. to define the specificity of the antibody response during natural infection, we needed to understand the amino acid variation present in the currently circulating sars-cov-2 human isolates. human sars-cov-2 isolates has a low frequency of amino acid variation within the spike protein, with the exception of the d614g mutation, allowing us to estimate that the majority of known isolates permit effective polyclonal antibody binding and neutralization. the human polyclonal antibody response recognizes three subdomains (s1, s2, and rbd) of the spike protein as evidenced by elisa. interestingly, we identified cross-reactive sera from sars-cov-2 naïve subjects to s1 suggesting conserved sequences in the s1 subunit of spike may impact non-neutralizing responses to sars-cov-2 as well as serological tests for sars-cov-2. most importantly, our antigen-specific antibody depletion approach demonstrated that the rbd domain of the spike protein is responsible for 70% +/-18.9% of the human polyclonal neutralizing antibody activity to spike after natural sars-cov-2 infection. although our study shows that the dominant target of igg neutralizing antibody response after natural sars-cov-2 infection is the rbd domain of the spike protein, we have evaluated a limited number (n=10) of patients by antigen-specific antibody depletion. there is the potential that immunodominance of the neutralizing antibody response may vary based upon a number of variables including viral load, co-morbidities including age and obesity, as well as genetic background. additionally, we have only focused on the igg response and it has been recently determined that the iga antibody response can neutralize sars-cov-2 virus and the antigen specificity of that response could be different than the igg response 54 . importantly, it has also been recently described that more than 90% of individuals who seroconvert generate detectible neutralizing antibody responses and that these igg responses are indeed sustained for up to three months 39, 55 , which has the potential to protect against re-infection. to begin to evaluate the correlates of protection beyond antibody neutralization, and investigate additional antibody mechanisms such as antibody dependent cellular cytotoxicity. as we detected antibodies targeted against s2 that are non-neutralizing these could provide a different mechanism of protection that may be valuable when considering vaccine design. there is also a strong t cell response established during natural infection [56] [57] [58] [59] , as well as a cross-reactive t cell response from potentially prior hcov infection 60, 61 . currently the role of the human t cell response to sars-cov-2 has only begun to be dissected. overall our study describes the polyclonal igg response to sars-cov-2 from sera obtained from patients in a range of 14-40 days post positive qrt-pcr test. we focused on the relationship between antibody binding to the subdomains of spike and the neutralization capacity against infectious virus. we demonstrate that infection with sars-cov-2 results in an antibody response that results in a similar amount of igg that targets spike subunits s1, s2, and rbd regardless of time post infection (supplemental figure 1) . furthermore, we show that this response results in a neutralizing antibody response by 14 days post positive qrt-pcr, as determined by frnt ( figure 3b) . finally, using a bead-based immune depletion approach, we show that the highest percentage of neutralizing antibodies against sars-cov-2 bind to the receptor binding domain (rbd) ( figure 4d ) that directly interacts with human ace2. these findings are important in the further development and prioritization of therapeutics and vaccine development. plates were coated with 50ul of a 1ug/ml mixture of recombinant protein in carbonate buffer (0.1m na2co3 0.1m nahco3 ph 9.3) overnight at 4°c. the next day plates were blocked with blocking buffer (pbs + 5%bsa + 0.5% tween) for 2 hours at room temperature and washed 4x with wash buffer prior to plating of serially diluted polyclonal sera. sera was incubated for 1 hour at room temperature in the elisa plate, washed 4x with wash buffer, followed by addition of goat-anti-human igg hrp (sigma) conjugated secondary (1:5000) for 1 hour at room temperature. the plate was washed again 4x with wash buffer and the elisa was visualized with 100ul of tmb enhanced substrate (neogen diagnostics) and placed in a dark space for 15 minutes. the reaction was quenched with 1n hcl and the plate was read for an optical density of 450 nanometers on a biotek epoch plate reader. total peak area under the curve (auc) was calculated using grappad prism 8. antigen specific antibody depletions. antigen specific antibodies were depleted in a beadbased approach using ni-nta magnetic beads (thermo scientific) as described ( 62 ). sars-cov2 his tagged proteins or vacv his tagged protein (control depletion) were conjugated to the hisspecific magnetic beads as suggested by manufacturer's protocol. briefly, 1mg of beads were washed with equilibration buffer followed by addition of 50ug of protein diluted in equilibration buffer. after addition of protein, the tube was rotated end over end for 1 hour at 4°c. the beads were collected on a magnetic stand and washed twice with wash buffer followed by separation into two tubes of 200µl each. next, the human sera were diluted in tissue culture sterile pbs and placed into the first tube of beads and incubated end-over end at 37°c for 1 hour. once again, the beads were collected with a magnetic stand, the supernatant was removed and transferred into the second tube for another end-over-end incubation at 37°c for 1 hour. after incubation the beads were collected, and the supernatant was removed and placed at 4°c for subsequent elisas and (a)schematic of the full-length sars-cov-2 spike protein with the s1 and s2 highlighted. s1 is divided into the n-terminal domain, and the c-terminal domain which contains the receptor binding domain (rbd) subunit in dark green with the receptor binding motif displayed using black hashed lines. the separation between s1and s2 is represented by a slash line. s2 contains the fusion peptide (fp) and heptad repeat one and two (hr1 and hr2) . the spike protein is where the surface reconstruction is colored according to 6 discrete groups, with a score of 1 being highly conserved (0-1 mutation per position) to being highly diverse with a score of 6 (>1000 mutations per position). the color coded bar describes the corresponding color for each range of mutations. next to each aa variation coded structure is the cryoem trimer structure with the individual trimers color coded to allow orientation. the forward facing trimer for the rbd up is color coded by subdomain, with rbd up being dark cyan, s1 as cyan, and s2 as pale green. the rbd down trimer is color coded with rbd down as brown, s1 as gold and s2 as pale yellow. we observed naturally occurring aa variations are less within the rbd as noted by the high level of purple colored ca residues, and greatest aa variations within the s1-ntd as indicated by the white and green color residues. the epidemiology and clinical information about covid-19 clinical characteristics of coronavirus disease 2019 in china clinical features of 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humans sars-cov-2-reactive t cells in healthy donors and patients with covid-19 oligomeric state of the zikv e protein defines protective immune responses key: cord-309182-t9ywnshj authors: premkumar, lakshmanane; segovia-chumbez, bruno; jadi, ramesh; martinez, david r.; raut, rajendra; markmann, alena; cornaby, caleb; bartelt, luther; weiss, susan; park, yara; edwards, caitlin e.; weimer, eric; scherer, erin m.; rouphael, nadine; edupuganti, srilatha; weiskopf, daniela; tse, longping v.; hou, yixuan j.; margolis, david; sette, alessandro; collins, matthew h.; schmitz, john; baric, ralph s.; de silva, aravinda m. title: the receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in sars-cov-2 patients date: 2020-06-11 journal: sci immunol doi: 10.1126/sciimmunol.abc8413 sha: doc_id: 309182 cord_uid: t9ywnshj the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) that first emerged in late 2019 is responsible for a pandemic of severe respiratory illness. people infected with this highly contagious virus can present with clinically inapparent, mild, or severe disease. currently, the virus infection in individuals and at the population level is being monitored by pcr testing of symptomatic patients for the presence of viral rna. there is an urgent need for sars-cov-2 serologic tests to identify all infected individuals, irrespective of clinical symptoms, to conduct surveillance and implement strategies to contain spread. as the receptor binding domain (rbd) of the spike protein is poorly conserved between sars-covs and other pathogenic human coronaviruses, the rbd represents a promising antigen for detecting cov-specific antibodies in people. here we use a large panel of human sera (63 sars-cov-2 patients and 71 control subjects) and hyperimmune sera from animals exposed to zoonotic covs to evaluate rbd's performance as an antigen for reliable detection of sars-cov-2-specific antibodies. by day 9 after the onset of symptoms, the recombinant sars-cov-2 rbd antigen was highly sensitive (98%) and specific (100%) for antibodies induced by sars-covs. we observed a strong correlation between levels of rbd binding antibodies and sars-cov-2 neutralizing antibodies in patients. our results, which reveal the early kinetics of sars-cov-2 antibody responses, support using the rbd antigen in serological diagnostic assays and rbd-specific antibody levels as a correlate of sars-cov-2 neutralizing antibodies in people. the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is responsible for an ongoing pandemic that has already killed over 320,000 people and paralyzed the global economy (1) . currently, the main method for laboratory diagnosis of sars-cov-2 is pcr testing of nasopharyngeal swabs. there is an urgent need for highly specific and sensitive antibody detection assays to answer fundamental questions about the epidemiology and pathogenesis of sars-cov-2 and to implement and evaluate population-level control programs (2) . efforts to understand the pathogenesis and define risk factors for severe sars-cov-2 disease have been hampered by our inability to identify all infected individuals, irrespective of clinical symptoms. to contain the pandemic, many countries resorted to the widespread quarantine of cities and regions. by deploying reliable antibody assays for population-level testing, it will be possible to obtain the highresolution spatial data needed to implement policies for containing the epidemic and informing strategies for re-opening communities and cities. studies with sars-cov-2 and other human covs demonstrate that people rarely develop specific antibodies within the first 7 days after onset of symptoms (3) (4) (5) (6) (7) . by 10-11 days after onset of symptoms, greater than 90% of sars-cov-2 the receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in sars-cov-2 patients (page numbers not final at time of first release) 2 patients develop specific igg and igm (3) (4) (5) (6) . for sars-cov-1 and the more distantly related mers-cov, igg antibodies have been observed to persist for at least one year after infection (8, 9) . these observations strongly support the feasibility of using antibody assays for identifying recent and remote sars-cov-2 infections and for conducting population-level surveillance. sars-cov-2 is a β-coronavirus, a subgroup that includes the closely related sars-cov-1 and the more distantly related mers-cov and the common-cold human covs (hcov-oc43 and hcov-hku1) (10) . many companies have quickly developed tests for sars-cov-2 antibody detection. these assays utilize the inactivated whole virion, viral nucleocapsid protein or viral spike protein as antigens in elisa, lateral flow or other testing platforms. while the performance of these assays has not been fully evaluated, some assays appear quite sensitive when used 10 days or more after the onset of symptoms (6, 11) . the specificity of sars-cov-2 antibody assays has not been adequately addressed. humans are frequently infected with hcov-oc43 and hcov-hku1 and most adults have antibodies to these viruses (10) . any antibody cross-reactivity between common hcovs and sars-cov-2 would result in false-positive results interfering with antibody-based testing and surveillance for sars-cov-2. sars-cov-1 and hcov oc43 elicit antibodies that crossreact against related covs (12, 13) . following the sars-cov-1 outbreak in 2003, the overall specificity of serological assays utilizing the nucleocapsid protein of sars-cov-1 was poor, whereas assays based on the spike protein were more specific (14) (15) (16) . in recent studies, the receptor binding domain (rbd) of the spike protein of sars-cov-2 has shown promise as an antigen for specific antibody detection (4, 17, 18) . here we report the production of properly folded recombinant receptor binding domains (rbds) from the spike proteins of sars and common-cold hcovs in mammalian cells. we use these recombinant antigens and a large diverse panel of human and animal sera to evaluate the rbd as an antigen for sars-cov-2 serology. we demonstrate that the recombinant sars-cov-2 rbd antigen is highly sensitive and specific for detection of antibodies induced by sars-covs. we also observed a strong correlation between the levels of rbd-binding antibodies and levels of sars-cov-2 neutralizing antibodies in patients. our results support the use of rbd-based antibody assays for serology and as a correlate of neutralizing antibody levels in symptomatic people who have recovered from sars-cov-2 infections. the s1 and s2 subunits of the spike (s) protein of coronaviruses are required for viral entry. the surface accessible receptor binding domain (rbd) on the s1 subunit binds to receptors on target cells, whereas the exposure of the fusion loop in the s2 subunit induces fusion of the viral envelope to the host cellular membranes (19) . the rbds of sars-covs, which bind to angiotensin-converting enzyme 2 (ace2) receptor on the host cells, are also a major target of human antibodies ( fig. 1a and b) . as the rbd is a common target of human antibodies and poorly conserved between sars-covs and other pathogenic human coronaviruses (fig. 1c) , this domain is a promising candidate for use in antibody-based diagnostic assays. we expressed the rbd of 2003 and 2019 sars-co-vs and four common human coronaviruses (hcov-hku-1, -oc43, -nl63 and -229e) as fusion proteins that were secreted from human cells. the recombinant rbds were purified from the cell culture medium by affinity chromatography and purity was confirmed by sds-page (fig. 1d ). we used sera and monoclonal antibodies from animals immunized with sars-cov-1 or -2 spike proteins to assess the structural integrity of the purified recombinant rbd antigens. pooled serum from mice immunized with sars-cov-2 spike protein had antibodies that bound well to the rbd of sars-cov-2 and poorly to the rbds of sars-cov-1 and other common hcovs (fig. 1e ). sera from mice or rabbits immunized with sars-cov-1 or cross-reactive monoclonal antibody 240c reacted with the rbds of sars cov-1 and -2 but not common human covs (fig. 1e ). human serum collected before sars-cov-2 emerged contained antibodies to common α-and β-hcovs (nl63 and hku-1) but not to sars-cov rbd antigens (fig. 1e) . these results suggest that the purified recombinant rbd antigens retain native structures required for specific antibody binding. to evaluate the specificity of the recombinant sars-cov-2 rbd in serology, we used human sera collected from different populations before the current pandemic. the sera were tested at a high concentration (1:20 dilution) for binding to the recombinant rbds from sars-cov-1, sars-cov-2 and common α-and β-hcovs (fig. 2) . sera collected from healthy american adults (n = 20) before the sars-cov-2 pandemic frequently had high levels of antibodies to the recombinant rbds of nl63 and hku-1 covs but not to sars-covs ( fig. 2a) . we also tested archived pre-sars-cov-2 pandemic sera collected from individuals in south asia, the caribbean and central america who had recently recovered from arbovirus infections. as in the case of healthy adults from the usa, most of the subjects from different parts of the world had high levels of antibodies to the rbd of common hcovs but no antibodies to the rbd of sars-covs (fig. 2b ). to assess if other human respiratory viruses stimulated antibodies that cross-reacted with the recombinant sars-cov rbd, we tested early convalescent sera from people with laboratory the known pathogenic human covs are members of the α-coronavirus and β-coronavirus genera (fig. 3a) . hcov-nl63 and 229e are two α-coronaviruses that frequently infect and cause a mild common-cold-like illness in most people. hcov-oc43 and hku-1 are two group 2a βcoronaviruses that also commonly infect people and cause mild disease. most adults (>90%) have antibodies to these common-cold hcovs. sars-cov-1 and -2 and mers-cov are group 2b and 2c zoonotic β-coronaviruses that have recently crossed into humans and caused severe illness. the α-and βcoronavirus genera also contain a large number of zoonotic viruses that infect different animal hosts, which have not been implicated in human disease to date. to further assess the specificity of sars-cov-2 rbd for serology, we obtained and tested sera from people who had recently recovered from a laboratory-confirmed common-cold hcov infection and sera from guinea pigs immunized with different animal covs ( fig. 3 b and c) . none of the immune sera from people exposed to recent hcov infections cross-reacted with the recombinant rbd of sars-covs. none of the guinea pigs vaccinated with different zoonotic covs had antibodies that cross-reacted with the recombinant sars-cov rbds ( fig. 3b and c). these results establish that most individuals, including people who have been recently exposed to acute common hcov infections, do not have detectable levels of cross-reactive antibodies to the recombinant rbd of sars-covs. to evaluate the sensitivity of the rbd of sars-cov-2 for identifying infected individuals, we obtained a total of 77 serum samples from 63 patients with laboratory-confirmed (i.e., pcr positive) sars-cov-2 infections collected at different times after the onset of symptoms. all the samples were tested for binding of total immunoglobulin (ig) and igm antibodies to recombinant rbd antigens from sars-covs and common-cold hcovs. the sensitivity of the assay was high (98% and 81% respectively for ig and igm) for specimens collected 9 days or more after onset of symptoms (fig. 4a ). as expected, overall sensitivity was lower (57% and 43% respectively for ig and igm) for specimens collected between 7 and 8 days after onset of symptoms (fig. 4a ). with samples collected 9 days or more after onset of symptoms, we observed some ig and igm antibody cross reactivity with the rbd of sars-cov-1 (67% and 30% respectively for ig and igm), which was anticipated as these viruses are closely related group 2b β-coronaviruses (20, 21) . when the specimens were further analyzed to estimate the timing of seroconversion, we observed a marked transition from seronegative to seropositive for both ig and igm about 9 days after the onset of symptoms ( fig. 4a and b ). by day 9 after onset of symptoms, most patients had high end-point titers in the rbd ig elisa (fig. s1 ). to analyze the kinetics of all three of the major isotypes of serum antibodies within the first 6 weeks after the onset of symptoms, we separately measured igg, iga, and igm in 49 serum samples obtained from sars-cov-2 infected patients at >9 days after onset of symptoms. most individuals (46/49) developed igg responses (fig. 4c ). iga and igm responses were observed less frequently (iga = 38/49, igm =34/49) than igg (fig. 4c ). for 14 individuals with laboratory-confirmed sars-cov-2 infection, we had two specimens collected at different times early in the infection (fig. 4d ). two subjects (p70 and p50) were seronegative within the first 4 days and seropositive for both ig and igm 9 or more days after onset (fig. 4d ). for three subjects (p58, p56, p52) the acute samples were collected after 9 days and the convalescent samples were collected 21 days or more after onset. in these individuals both acute and convalescent samples were positive, and we observed an increase in ig and igm levels in the second specimen. for the remaining 9 subjects, the acute specimen was collected on day 7 after onset and the convalescent specimen was collected >9 days after onset. six out of the 9 subjects already had specific ig, igm or both in the acute specimen collected on day 7. all the subjects except one (p54) seroconverted or had elevated levels of antibody in the convalescent sample collected >9 days after onset of symptoms. these results indicate that most people seroconvert between days 7 and 9 after onset of symptoms. subject p54 was an outlier and did not develop specific ig or igm antibodies. all the individuals with documented sars-cov-2 had ig but not igm antibodies that bound to the rbd of common hcovs, which is consistent with their high prevalence in humans (fig. 4a ). these results demonstrate that the rbd of sars-cov-2 is a highly sensitive antigen for antibody detection in patients 9 days or more after onset of symptoms. the administration of convalescent plasma containing antibodies to sars-cov-2 is being evaluated for patients with severe disease. while the fda has not approved convalescent plasma therapy, on may 1, 2020, the fda recommended that sars-cov-2 neutralizing titers of at least 1:160 should be used for human passive immunization studies. further, the fda also recommended that a titer of 1:80 may be acceptable if an alternative matched unit is not available. as the rbd domain of s protein is critical for viral entry, antibodies targeting this domain of sars-cov-2 are likely to be neutralizing and potentially protective, as is seen in cell culture and animal models for other pathogenic covs (19, 22) . to assess the relationship between the rbd-binding activity and the neutralizing antibody response, we tested 50 pcr-confirmed sars-cov-2 patient immune sera in a sars-cov-2 luciferase neutralization assay (fig. 5 ). as judged by the spearman test (ρ = 0.86, p < 0.0001), we observed that the magnitude of the total rbd-binding ig antibody strongly correlated with the levels of neutralizing antibodies in sars-cov-2 patients ( currently, patients who have had a documented sars-cov-2 infection identified by rt-pcr or a serologic test, and who are clear of symptoms for at least 14 days, are recruited for convalescent plasma donation. we evaluated the neutralizing potency in patient samples collected between 1 and 40 days with a titer of at least 1:160 (fig. 5d ). we observed that 32% of patients (7/22) developed weak to no neutralizing antibodies even 21 days after onset of symptoms, suggesting that days after the start of symptoms is a poor determinant of the levels of sars-cov-2 neutralizing antibodies in the patients included in our study, particularly within the early convalescent phase (<6 weeks). to evaluate whether a simple rbd elisa can be used as a surrogate for neutralizing potency in sars-cov-2 patients, we analyzed the relationship between the level of total ig antibody to rbd and a neutralizing antibody titer of at least 1:160. we observed that 22/24 people who had a substantial total ig binding antibody to rbd (>1.5 od) also developed a robust neutralizing antibody titer (fig. 5e) . notably, only 3/26 people who developed a relatively weak rbd-binding antibody had a neutralizing antibody titer higher than 1:160. one subject (p54) neither seroconverted for rbd antigen nor developed neutralizing antibodies to sars-cov-2 ( fig. 4d and e, and fig. s2 ). serology is critical to understanding the transmission, pathogenesis, mortality rate and epidemiology of emerging viruses. in the few months after the discovery of sars-cov-2 as a human pathogen, scientists have developed a large number of antibody assays and many commercial tests are now available. although none of the assays have been fully validated yet, the fda has granted emergency use authorization (eua) for multiple tests, while stressing the need for further validation. investigators have already encountered problems with the specificity and sensitivity of commercial assays rushed to market (4, 22) . widespread use of inaccurate antibody assays could lead to policies that exacerbate the current sars-cov-2 pandemic instead of containing it. to address the need for reliable antibody-based diagnostic assays, we focused on the rbd domain of the spike protein because this region is poorly conserved between different covs and is also known to be a major target of human antibodies (19) . a major concern with using a protein domain instead of a full-length protein or whole virion for antibody detection is possible reduction in assay sensitivity. however, we observed that over 95% of sars-cov-2 patients developed antibodies to the rbd 9 days after onset of symptoms. although our study included only a few recent convalescent sera and relatively large numbers of presumably positive samples from past common human cov infections, the high specificity of the rbd antigen was also evident with the serum specimens from animals that were hyperimmunized with other zoonotic covs. some patients infected with sars-cov-2 had antibodies that cross-reacted with the rbd of sars-cov-1. we have not tested the more distantly related rbd ag from mers cov or the serum samples from individuals with confirmed mers infection. since sars-cov-1 and mers cov seroprevalence are very low in humans, the sars-cov-2 antibody cross-reactivity with sars-cov-1 is unlikely to pose diagnostic challenges. other recent studies that have been published or under peer review also support the high specificity and sensitivity of the sars-cov-2 rbd for antibody detection (4, 17, 18) . amanat and colleagues tested samples from sars-cov-2 patients collected at the beginning of the epidemic in the usa and reported that the full length s protein and the rbd performed well for specific antibody detection (17) . okba and colleagues compared the performance of different sars-cov-2 antigens for antibody detection using samples from 10 sars-cov-2 patients in europe (4) . for the sars-cov-2 spike rbd, they observed levels of specificity and sensitivity that were comparable to our results reported here. the s2 subunit, which comprises conserved regions between covs, was less specific than the rbd (4). perera and colleagues evaluated the performance of the rbd for antibody detection using samples from 24 sars-cov-2 patients in hong kong (18) . they also observed high specificity and sensitivity when patients were tested 10 days or more after onset of illness. our study with 77 specimens from 63 documented sars-cov-2 patients, which includes patients presenting to hospitals in north carolina and georgia with varying levels of severity, together with these recent studies conducted in new york, europe and hong kong, strongly support the use of sars-cov-2 rbd as an antigen for antibody detection. we designed the assay for separate detection of rbdspecific total ig and igm. as the pandemic is ongoing and most infections are likely to have occurred within the past few months, infected individuals have variable levels of antigen-specific igg, igm and iga (fig. 4c ). to maximize assay sensitivity and to prevent different antibody isotypes competing for binding sites and reducing assay signal, we measured total ig. we did not observe any decrease in assay specificity by designing the assay to monitor levels of total ig instead of igg binding to the rbd even at high serum concentration or with hyperimmune sera. our study showed that igm and iga antibodies can also be detected using rbd-based serological assays. both iga and igm antibodies are relatively short lived and indicative of a recent exposure. when conducting large scale population level surveillance for sars-cov-2 antibodies, it will be possible to distinguish recent from remote infections by measuring both total ig and igm (or iga) binding to the rbd. antibody assays that correlate with protective immune responses in individuals who have recovered from sars-cov-2 infection and also reflect herd immunity at a population level are urgently needed to define each individual's risk of disease and to identify communities at high risk for new waves of infection. in animal studies with sars-cov-1, virus-neutralizing antibodies were strongly correlated with protective immune responses (19) . we observed a striking correlation between the levels of rbd antibodies in patients and the ability of patient sera to neutralize sars-cov-2 virus. other groups have recently reported finding a strong correlation between spike/rbd antibodies and sars-cov-2 neutralization in patients infected with sars-cov-2 (4, 17, 18) . our results point out that roughly one-third of patients develop very low or no neutralizing antibodies to sars-cov-2 and that ig and igm antibodies are useful predictors of neutralizing antibody levels in patients in the early convalescent phase (<6 weeks). as people developing a high level of rbd-binding antibodies (>1.5 od) also have a robust neutralizing response, a simple rbd-based elisa can be a useful tool to identify blood plasma donors. while further studies are needed to fully evaluate rbd antibodies as correlate of protective immunity, the results to date indicate that rbd antibodies are a promising correlate of protection in the early convalescent phase. a simple antibody detection assay that also predicts individuallevel risk of disease will be a major advance for vaccine development and immunogenicity of vaccines because sars-cov-2 neutralization assays are time-consuming and require bsl-3 containment. one sars-cov-2 patient (p54) who tested positive for viral rna and required hospitalization did not develop rbdspecific ig, igm or neutralizing antibodies, even at 16 days after the onset of symptoms. this was the only person among the 68 pcr positive subjects who did not seroconvert by 9 days after onset of symptoms in the rbd-based assay. while we cannot rule out the possibility of a false positive pcr test result, others have also reported rare instances where people infected with sars-covs have atypical, dampened immune responses (23) . further studies are needed to establish the frequency and significance of atypical antibody responses in sars-cov-2 patients and characterize the serological repertoire and epitopes targeted by the antibodies in convalescent sera. as sars-cov-2 infections in the southeastern u.s. have started to increase relatively recently, all convalescent samples used in this study were collected within 90 days following onset of symptoms. in most patients, the convalescent sera had high end-point titers (>1:1000) in the rbd ig elisa supporting the utility of this assay even as antibody levels start to wane over time. we need to prioritize studies to prospectively monitor sars-cov-2 patients to determine the long-term kinetics of antibody levels and the performance of antibody detection assays over time. all the sars-cov-2 human immune sera used for this study were collected from symptomatic patients that included many with serious illness requiring hospitalization. the research community currently does not know if individuals experiencing mild/inapparent symptoms after sars-cov-2 infection have similar kinetics and levels of rbdbinding antibodies as those experiencing symptomatic infections. studies must be done with individuals experiencing mild/inapparent sars-cov-2 infections to define the kinetics and levels of rbd antibodies before implementing large population-level antibody testing. the goal of the study was to evaluate the performance of rbd-based spike antigen for reliable detection of sars-cov-2-specific antibodies. we produced properly folded rbd from the spike proteins of sars and common-cold hcovs in mammalian cells and used this antigen to evaluate a large panel of human sera from documented sars-cov-2 patients and control subjects, and hyperimmune sera from animals exposed to zoonotic covs. we also used a sars-cov-2 luciferase neutralization assay to assess the dynamics of the neutralizing antibody response and its association with the rbd-binding activity. the structure coordinate sets of the spike proteins, spike protein complexes with their cognate receptor ace2 and monoclonal antibodies were obtained from the protein data bank (pdb). the structures were aligned to the reference spike protein using the pymol molecular graphics system (version 1.2r3pre, schrödinger, llc). molecular figures were drawn using pymol. the pdb coordinates used for the containing human serum albumin secretion signal sequence, three purification tags (6xhistidine tag, halo tag, and twin-strep tag) and two tev protease cleavage sites was cloned into the mammalian expression vector pαh. s1 rbds were expressed in expi293 cells (thermofisher) and purified from the culture supernatant by nickel-nitrilotriacetic acid agarose (qiagen). to generate virus replicon particles (vrps), the sars-cov-2 s gene was inserted into pvr21 3526 as previously described (24) . in summary, the sars-cov-2 s gene was ligated into pvr21 following digestion by restriction endonuclease sites, paci and apai. t7 rna transcripts were generated using the sars-cov-2-s-pvr21 construct in conjunction with plasmids containing the venezuelan equine encephalitis virus envelope glycoproteins and capsid protein. the rna transcripts were then electroporated into baby hamster kidney fibroblasts and monitored for cytopathic effect. vrp were harvested 48 hours after electroporation and purified via high-speed ultra-centrifugation. to generate serum samples against sars-cov-2, 10-week-old balb/c mice (jackson labs) were inoculated via footpad injection with the vrp and boosted with the same dose one time three weeks later. serum samples were then collected from individual animals at 2 weeks post-boost and pooled for use in assays. all human specimens used in these studies were obtained after informed consent under good clinical research practices (gcp) and compliant with oversight by the relevant institutional review boards (irbs). a list of the sars-cov-2 patient samples included in the study with basic demographic and clinical information can be found in table s1 . emory university school of medicine specimens: specimens were obtained from patients with symptomatic illness and clinical testing confirming sars-cov-2 by pcr (cdc sars-cov-2 test). de-identified specimens were shared with researchers at unc consistent with local irb protocols (emory irb# 00110683 and 00022371). blood plasma donor study: convalescent sera was obtained from donors who volunteered for plasma collections at the unc donation center. fresh sera collected as part of the standard plasmapheresis procedure were saved for research from donors who signed informed consent. unc irb 20-1141 is conducted under good clinical research practices (gcp) and is compliant with institutional irb oversight. all donors had confirmed sars-cov-2 infection by nasopharyngeal swab indicating the presence of sars-cov-2 rna as performed by eua approved qrt-pcr in a us laboratory with a clinical laboratory improvement amendments (clia) certification. all donors had recovered from their sars-cov-2 illness and were at least 14 days post last symptoms. donors who presented for plasma collection prior to 28 days from their last symptoms had a confirmed negative nasopharyngeal rt-pcr test done within 72 hours prior to donation. healthy unexposed donors: samples from healthy u.s. adult donors were obtained by the la jolla institute for immunology (lji) clinical core or provided by a commercial vendor (carter blood care) for prior, unrelated studies between early 2015 and early 2018, at least one year before the emergence of sars-cov-2. the lji institutional review board approved the collection of these samples (lji; vd-112). samples from the caribbean, central america and south asia were obtained from archived samples at unc collected before december 2019 for other studies. human and animal specimens from bei resources: the following reagents were obtained through bei resources, niaid, nih as part of the human microbiome project: pooled sera obtained from rabbits dosed with a recombinant sars-cov spike protein (nrc-772), monoclonal anti-sars-cov s protein (similar to 240c) (nr-616), anti-porcine respiratory coronavirus (prcov; isu-1) serum obtained from pig (nr-460), anti-porcine transmissible gastroenteritis virus obtained from pig (nr-458), anti-porcine respiratory coronavirus (prcov; isu-1) serum obtained from guinea pig (nr-459), anti-sars coronavirus obtained from guinea pig (nr-10361), anti-bovine coronavirus (mebus) obtained from guinea pig (nr-455), anti-feline infectious peritonitis virus, 79-1146 obtained from guinea pig (nr-2518), anti-avian table s1 ). in-house rbd ig and igm elisa all serum specimens tested by elisa assay were heat-inactivated at 56°c for 30 min to reduce risk from any possible residual virus in serum. briefly, 50 μl of spike rbd antigen at 4 μg/ml in tris buffered saline (tbs) ph 7.4 was coated in the 96-well high-binding microtiter plate (greiner bio-one cat # 655061) for 1 hour at 37°c. then the plate was washed three times with 200 μl of wash buffer (tbs containing 0.2% tween 20) and blocked with 100 μl of blocking solution (3% milk in tbs containing 0.05% tween 20) for 1 hour at 37°c. the blocking solution was removed, and 50 μl of serum sample at 1:20 or indicated dilutions in blocking buffer was added for 1 hour at 37°c. the plate was washed in the wash buffer, 50 μl of alkaline phosphatase-conjugated secondary goat anti-human secondary antibody at 1:2500 dilution was added for 1 hour at 37°c. for measuring total ig, a mixture of anti-igg (sigma cat # a9544), anti-iga (abcam cat # ab97212), and anti-igm (sigma cat # a3437] were added together. for measuring specific antibody isotype, only secondary goat anti-human igg or iga or igm was used. the plate was washed, and 50 μl p-nitrophenyl phosphate substrate (sigma fast, cat no n2770) was added to the plate and absorbance measured at 405nm using a plate reader (biotek epoh, model # 3296573). for testing animal sera, the secondary antibody was matched to the species as follows: goat antimouse igg (sigma, a3688), goat anti-rabbit igg (abcam, ab6722), goat anti-pig igg (abcam, ab6916), and goat antiguinea pig igg (abcam, ab7140). full-length viruses expressing luciferase were designed and recovered via reverse genetics and described previously (25, 26) . viruses were tittered in vero e6 usamrid cells to obtain a relative light units (rlu) signal of at least 20x the cell only control background. vero e6 usamrid cells were plated at 20,000 cells per well the day prior in clear bottom black-walled 96-well plates (corning 3904). neutralizing antibody serum samples were tested at a starting dilution of 1:20, and were serially diluted 4-fold up to eight dilution spots. antibody-virus complexes were incubated at 37°c with 5% co2 for 1 hour. following incubation, growth media was removed and virus-antibody dilution complexes were added to the cells in duplicate. virus-only controls and cell-only controls were included in each neutralization assay plate. following infection, plates were incubated at 37°c with 5% co2 for 48 hours. after the 48 hour incubation, cells were lysed and luciferase activity was measured via nano-glo luciferase assay system (promega) according to the manufacturer's specifications. sars-cov-2 neutralization titers were defined as the sample dilution at which a 50% reduction in rlu was observed relative to the average of the virus control wells. each data points in fig. 1e, fig. 2 , fig. 3b and 3c, fig. 4 and 5 are presented as means of technical duplicates. the correlation of rbd binding and neutralization titers shown in fig. 5a and fig. 5b was evaluated using a spearman correlation coefficient (rs) and the associated two-tailed p-value (graphpad prism, version 8). receiver operating characteristic (roc) analyses were performed to establish cutoff values for sars-cov-2 seropositivity using spss software. statistical analyses were performed using spss software ver. 26.0 (ibm, armonk, ny, usa). immunology.sciencemag.org/cgi/content/full/5/48/eabc8413/dc1 fig. s1 . titration curves of sera from sars-cov-2 positive patients. fig. s2 . seroconversion of sars-cov-2 neutralizing antibodies. fig. s3 . estimation of rbd elisa assay cutoff. table s1 . summary of samples tested and associated characteristics (excel spreadsheet). table s2 . raw data file (excel spreadsheet). covs. (e) binding characterization of the spike rbd antigens with immune sera and a monoclonal antibody. sars-cov-1 monoclonal antibody (240c), serum from a mouse immunized with vrp expressing sars-cov-2 or sars-cov-1 spike protein, serum from a rabbit immunized with sars-cov-1 spike protein and an archived human sample collected before sars-cov-2 were tested for binding against rbd spike antigens from sars-cov-2, sars-co-v-1, hcovα (nl63) and hcovβ (hku-1). the cutoff values determined by the receiver operating (roc) curve analysis (fig s3) for the elisa assay are indicated by the broken line. scatter plots were generated using individual serum binding to rbd antigen (y-axis) versus sars-cov-2 neutralizing antibody titers (x-axis). the nonparametric spearman correlation coefficient (rs) and the associated two-tailed p-value were calculated (graphpad prism, version 5.0). (c) relationship between sars-cov-2 neutralizing antibody titer and days after onset of symptoms. (d) total ig antibody binding to rbd as a surrogate for identifying people with high sars-cov-2 neutralizing antibodies. a total of 50 serum samples collected between 1 and 39 days after onset of symptoms from pcr-confirmed sars-cov-2 subjects were measured for ig and igm binding to spike rbd antigen and sars-cov-2 neutralization assay. the fdarecommended neutralizing antibody titer for plasma therapy (1:160) is indicated by the broken green line. virology, epidemiology, pathogenesis, and control of covid-19 the important role of serology for covid-19 control profiling early humoral response to diagnose novel coronavirus disease (covid-19) severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019 chronological evolution of igm, iga, igg and neutralisation antibodies after infection with sarsassociated coronavirus mers-cov antibody responses 1 year after symptom onset two-year prospective study of the humoral immune response of patients with severe acute respiratory syndrome origin and evolution of pathogenic coronaviruses development and clinical application of a rapid igm-igg combined antibody test for sars-cov-2 infection diagnosis cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests an outbreak of human coronavirus oc43 infection and serological cross-reactivity with sars coronavirus. can falsepositive results in a recombinant severe acute respiratory syndrome-associated coronavirus (sars-cov) nucleocapsid-based western blot assay were rectified by the use of two subunits (s1 and s2) of spike for detection of antibody to sars-cov antigenic cross-reactivity between severe acute respiratory syndrome-associated coronavirus and human coronaviruses 229e and oc43 a serological assay to detect sars-cov-2 seroconversion in humans serological assays for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) the spike protein of sars-cov-a target for vaccine and therapeutic development cross-reactive antibody response between sars-cov-2 and sars-cov infections potent binding of 2019 novel coronavirus spike protein by a sars coronavirusspecific human monoclonal antibody rapid point-of-care testing for sars-cov-2 in a community screening setting shows low sensitivity neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient cohort and their implications. medrxiv development of a broadly accessible venezuelan equine encephalitis virus replicon particle vaccine platform reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus competing interests: the authors declare that they have no competing interests. data and materials availability: the recombinant rbd antigens from the spike proteins used in this study are available under a standard mta with the university of north carolina. please contact lakshmanane premkumar (prem@med.unc.edu) or aravinda m acknowledgments: we gratefully acknowledge bei resources (https://www.beiresources.org) for the prompt processing and shipping of the reagents. we are grateful for the expert procedural care provided by the unc hospital and blood donor center and to the patients and blood donors providing samples for the study. key: cord-340472-9ijlj4so authors: li, wenhui; zhang, chengsheng; sui, jianhua; kuhn, jens h; moore, michael j; luo, shiwen; wong, swee-kee; huang, i-chueh; xu, keming; vasilieva, natalya; murakami, akikazu; he, yaqing; marasco, wayne a; guan, yi; choe, hyeryun; farzan, michael title: receptor and viral determinants of sars-coronavirus adaptation to human ace2 date: 2005-03-24 journal: the embo journal doi: 10.1038/sj.emboj.7600640 sha: doc_id: 340472 cord_uid: 9ijlj4so human angiotensin-converting enzyme 2 (ace2) is a functional receptor for sars coronavirus (sars-cov). here we identify the sars-cov spike (s)-protein-binding site on ace2. we also compare s proteins of sars-cov isolated during the 2002–2003 sars outbreak and during the much less severe 2003–2004 outbreak, and from palm civets, a possible source of sars-cov found in humans. all three s proteins bound to and utilized palm-civet ace2 efficiently, but the latter two s proteins utilized human ace2 markedly less efficiently than did the s protein obtained during the earlier human outbreak. the lower affinity of these s proteins could be complemented by altering specific residues within the s-protein-binding site of human ace2 to those of civet ace2, or by altering s-protein residues 479 and 487 to residues conserved during the 2002–2003 outbreak. collectively, these data describe molecular interactions important to the adaptation of sars-cov to human cells, and provide insight into the severity of the 2002–2003 sars epidemic. sars coronavirus (sars-cov) is the etiological agent of severe acute respiratory syndrome (sars), an acute pulmonary syndrome that, when it emerged in the winter of 2002-2003, resulted in the death of approximately 800 individuals, close to 10% of those infected fouchier et al, 2003; ksiazek et al, 2003; kuiken et al, 2003) . despite concerns that sars-cov would re-emerge, last winter (2003) (2004) , only a handful of individuals were found infected by the virus. these individuals appeared to have much less severe symptoms, and no secondary transmission was observed peiris et al, 2004; song et al, 2005) . severe cases of sars were also reported in 2004, but these resulted from laboratory infections (normile, 2004) . the coronavirus spike (s) protein mediates infection of receptor-bearing cells (gallagher and buchmeier, 2001; holmes, 2003; . angiotensin-converting enzyme 2 (ace2) is a functional receptor for sars-cov, and binds the sars-cov s protein with high affinity . several lines of evidence suggest that ace2 is a physiologically relevant receptor during infection. tissue expression of the receptor corresponds to the localization of virus in infected individuals and animals (harmer et al, 2002; chan et al, 2004; ding et al, 2004; hamming et al, 2004) . also, the efficiency of infection in humans, mice, and rats correlates with the ability of the ace2 of each species to support viral replication subbarao et al, 2004; wentworth et al, 2004) . antibodies that block ace2 association (sui et al, 2004) protect mice against infection (sui et al, 2005) . finally, although many cell lines do not express ace2, most cell lines shown to support sars-cov infection or replication detectably express this receptor nie et al, 2004) . although dc-signr (l-sign, cd209l) and dc-sign (cd209) have recently been shown to enhance infection of ace2-expressing cells (jeffers et al, 2004; marzi et al, 2004; yang et al, 2004) , these proteins do not appear to mediate efficient infection in the absence of ace2 (jeffers et al, 2004; marzi et al, 2004) . unlike many type i fusion proteins, including those of other coronaviruses, the s protein of sars-cov is not cleaved in the virus-producing cell (xiao et al, 2003; moore et al, 2004) . however, two domains corresponding to the s1 and s2 proteins of processed coronaviruses can be defined (gallagher and buchmeier, 2001) . the s1 domain mediates receptor association, whereas the s2 domain is membraneassociated and likely undergoes structural rearrangements that mediate membrane fusion. a discrete receptor-binding domain (rbd) of the s protein has been defined at residues 318-510 of the s1 domain (xiao et al, 2003; babcock et al, 2004; wong et al, 2004) . this rbd binds ace2 with higher affinity than does the full s1 domain . early cases of sars in 2002 were reported to have occurred in animal traders and restaurant workers handling wild mammals, and sars-cov has been isolated from two such mammals, palm civets (paguma larvata) and raccoon dogs (nyctereutes procyonoides) (guan et al, 2003) . palm civets have also been implicated in the minor 2003-2004 sars outbreak (zhong, 2004) . the sequences of the s-protein genes of 13 viruses isolated from palm civets, and one from raccoon dogs, have been determined (guan et al, 2003; song et al, 2005) . interestingly, the rbds of these animal s proteins are highly conserved except residue 479, which varies between asparagine and basic amino acids, but differ at several positions from the rbds of viruses isolated during the 2002-2003 outbreak. although different from palm-civet-derived rbds, the latter rbds were themselves highly conserved in the more than 100 s-protein genes obtained during the severe human outbreak zhang et al, 2004) . full sequence has also been published for two s-protein genes obtained during the mild 2003-2004 outbreak song et al, 2005) . these genes are nearly identical and contain elements common to s protein isolated during the earlier human outbreak and to that isolated from palm civets. here we describe the s-protein-binding domain of human ace2 by characterizing chimeras of human and rat ace2. by introducing four residues of human ace2 into rat ace2, we convert this receptor, which supports little or no s-proteinmediated infection, into a receptor that binds the s1 domain and supports infection, with efficiency close to that of human ace2. by characterizing additional ace2 variants, we localize the s-protein-binding domain primarily to a-helix 1 of ace2 and to a loop leading to b-sheet 5. we then show that a representative s protein from the mild 2003-2004 outbreak, and one from palm civets, mediates more efficient infection of cells expressing palm-civet ace2 compared to cells expressing human receptor. in contrast, s protein from the severe 2002-2003 outbreak efficiently binds and utilizes both receptors. two regions of the s-protein-binding site on ace2, and two residues in the rbd of these s proteins, largely determine these differences. our data describe s-protein adaptations, and their receptor counterparts, that permit efficient infection of human cells. these adaptations, absolutely conserved during 2002-2003 outbreak, may in part account for the unusual severity of sars. we have previously shown that s-protein-mediated infection of cells expressing human ace2 is substantially more efficient than that of cells expressing the same levels of rat ace2 . we first investigated whether this difference localized to the ace2 enzymatic or collectrin domain. the latter domain is defined by its close homology with a small, kidney-expressed protein of the same name (zhang et al, 2001) . figure 1a shows that a fusion protein comprising the sars-cov s1 domain and the fc domain of human igg1 (s1-ig) efficiently precipitated human ace2, as well as an ace2 chimera with the human catalytic domain and the rat collectrin domain. in contrast, s1-ig could not precipitate rat ace2 or an ace2 chimera with the rat catalytic domain and the human collectrin domain. the ability of these chimeric receptors to bind s1-ig was also reflected in their ability to support s-protein-mediated infection ( figure 2c ). these data indicate that differences in the ability of rat and human ace2 to support infection are localized to the catalytic domain of the receptor. guided by the structure of the ace2 catalytic domain (towler et al, 2004) , we made a series of human ace2 variants in which one or a few solvent-exposed residues were altered to their rat ace2 counterparts. two variants consistently bound s1-ig less efficiently than did wild-type human ace2. introduction of rat residues 82-84 (nfs), which include a glycosylation site at asparagine 82 not present in human ace2, partially inhibited s1-ig association ( figures 1b and 2a ) and s-protein-mediated entry ( figure 2c ). alteration of lysine 353 to a histidine residue present on the rat receptor interfered more dramatically with s1-ig association, and also partially inhibited s-proteinmediated entry. introduction of human residues 82-84 (myp) and lysine 353 into rat ace2 resulted in substantial figure 1 introduction of rat ace2 residues into the human ace2 catalytic domain interferes with s-protein association. (a) hek293t cells were transfected with plasmids encoding human or rat ace2, or chimeras of these receptors in which residues 1-600, corresponding to the ace2 catalytic domain, were exchanged. transfected cells were metabolically labeled with [ 35 s]cysteine and [ 35 s]methionine, and lysed. lysates were immunoprecipitated with protein a-sepharose together with either the s1 domain of the sars-cov (tor2) s protein fused to the fc domain of human igg1 (s1-ig), or with an antibody (1d4) recognizing a tag present at the carboxyterminus of each of the ace2 variants, and analyzed by sds-page. (b) hek293t cells were transfected with plasmids encoding human ace2, rat ace2, or human ace2 variants in which residues corresponding to those of rat ace2 were introduced at the indicated position. transfected cells were analyzed as in (a) and precipitated ace2 was quantified by phosphorimaging. values indicate the ratio of protein precipitated by s1-ig to that precipitated by 1d4. error bars indicate the range of two or more experiments. increases in s1-ig binding, as assayed both by immunoprecipitation and by flow cytometry (figure 2a and b). combination of both sets of residues resulted in a rat ace2 variant that bound s1-ig and supported s-protein-mediated infection comparably to human ace2 (figure 2a -c). these data suggest that residues 82-84 and, more so, lysine 353 participate in s-protein association with human ace2. we subsequently altered a number of additional residues of human ace2 in the vicinity of residues 82-84 and 353 to alanine or, in some cases, aspartic acid. alteration of two residues on the first helix of ace2, at lysine 31 and tyrosine 41, substantially interfered with the s1-ig association, as did residues adjacent to lysine 353, at aspartic acid 355 and at arginine 357, both within ace2 b-sheet 5 ( figure 3a) . figure 3b -d shows three views of the crystal structure of human ace2, in which residues that convert rat ace2 to an efficient sars-cov receptor are shown in red, and additional residues whose alteration interferes with s1-ig association are shown in yellow. green indicates residues whose alteration did not affect s1-ig binding. the c-terminal collectrin domain is not well ordered in the structure, but is used here to position the molecule with respect to the cell membrane. as shown in the figures, the s-protein-binding site on ace2 is localized above the deep cleft that harbors the catalytic site and on the upper left of the structure when viewed facing that cleft. the crystal structure of ace2 has been solved in two distinct conformations, an open one ( figure 3b -d) and a conformation in which the cleft is closed around the ace2 inhibitor mln-4760 (dales et al, 2002; towler et al, 2004) . these two conformations likely reflect free and substrate-bound states of the enzyme, respectively. we investigated whether the large conformational change associated with inhibitor binding interfered with s1-ig binding or s-protein-mediated infection. as previously reported, mln-4760 inhibits ace2 activity in the low nanomolar range (dales et al, 2002) , and no ace2 enzymatic activity was detected in the presence of 100 nm inhibitor ( figure 4a ). however, 100 nm mln-4760 did not interfere with immunoprecipitation of ace2 by s1-ig, nor did this inhibitor interfere with s-protein-mediated infection ( figure 4b and c). consistent with these observations, the distances among a and b carbons of residues 31, 41, 353, 355, and 357 in the s-protein-binding site of ace2 varied by less than 0.4 å between the inhibitor-bound and -unbound structures (towler et al, 2004) . in contrast, distances between these residues and residues across the cleft typically varied by greater than 6 å in the two structures. these data suggest that the s-protein-binding region of ace2 is not perturbed by inhibitors or substrates that induce large conformational changes in the receptor. consistent with these studies, we have also observed, using the assay shown in figure 4a , that s1-ig does not interfere with the enzymatic activity of ace2 (data not shown). the s protein used in the studies above was obtained from a patient infected during the severe 2002-2003 sars-cov outbreak. another outbreak, during the winter of 2003-2004, caused much less severe symptoms in the few individuals figure 2 introduction of human ace2 residues into rat ace2 converts rat ace2 to an efficient sars-cov receptor. (a) hek293t cells transfected with plasmids encoding the indicated human and rat ace2 variants or with vector alone were analyzed as in figure 1 (light gray), or by flow cytometry (dark gray). flow cytometry values indicate the ratio of mean fluorescence intensity (m.f.i.) observed using s1-ig to that using the 9e10 antibody, which recognizes a tag present at the amino-terminus of each ace2 variant. error bars indicate the range of two or more experiments. (b) representative example of an immunoprecipitation experiment used in (a). (c) murine leukemia viruses (mlv) expressing green fluorescent protein (gfp), lacking its endogenous envelope glycoprotein (mlv-gfp), and pseudotyped with the s protein of sars-cov (tor2 isolate) were incubated with hek293t cells transfected with plasmids encoding the indicated human or rat ace2 variants. amount of ace2-expressing plasmid was adjusted to maintain comparable receptor expression levels, as indicated, by flow cytometry using the antibody 9e10 that recognizes an amino-terminal myc tag on these receptors. gfp expression in cells was quantified by flow cytometry to measure infection of cells by pseudotyped viruses. error bars indicate range of two experiments. infected and resulted in no documented human-to-human transmissions liang et al, 2004; zhong, 2004; song et al, 2005) . we compared an s protein of virus isolated during this latter outbreak (gd03t0013; accession number ay525636; denoted gd herein) with that of virus obtained during the 2002-2003 outbreak (tor2; ay274119) and with that isolated from palm civets (sz3; ay304486) (guan et al, 2003; marra et al, 2003; rota et al, 2003; he et al, 2004; song et al, 2005) . figure 5a shows that the s1 domains of all three s proteins efficiently bound palm-civet ace2 (accession number ay881174), whereas only the s1 domain of tor2 efficiently bound human ace2. of note, the s1 domain of virus isolated during the 2003-2004 outbreak bound palmcivet ace2 much more efficiently than it bound human ace2. we then investigated the rbd of each of these s-protein variants. the ability of these s1 domains to bind palm-civet and human ace2 was reflected in the ability of their respective rbds to bind these receptors ( figure 5b ). we also assayed the ability of the entire s protein to mediate infection of cells expressing human or palm-civet ace2. the efficiency of entry was consistent with the ability of the s1 domain of each variant to bind each ace2 ( figure 6c ). these data are consistent with the hypothesis that the palm civet is a source of sars-cov, and suggest that the apparent lack of severity of disease during the 2003-2004 outbreak may be due in part to incomplete adaptation of gd virus to human ace2. the rbds of tor2 and sz3 differ by four residues ( figure 6a ). we investigated which of these residues contribute to the ability of tor2 rbd to bind efficiently human ace2. each residue in the tor2 rbd was altered to its sz3 counterpart. alteration of two residues, at positions 479 and 487, interfered with the association of the tor2 rbd with human ace2 ( figure 6b ). surface plasmon resonance studies further demonstrated a greater than 20-fold decrease of affinity for human ace2 when either residue 479 or 487, but not when residue 344 or 360, is altered to its palm-civet counterpart (table i and supplementary figure 2) . notably, alteration of threonine 487 to serine also affected the ability of the tor2 rbd to associate with palm-civet ace2 ( figure 6b ). introduction of tor2 residues at 479 or 487 substantially increased the ability of the full sz3 s protein to infect cells expressing human ace2. introduction of sz3 residues at these positions into the tor2 s protein resulted in a 2-to 3-fold decrease in infection of these cells figure 1 except that the indicated solvent-accessible residues common to human and rat ace2 were modified in human ace2 to either alanine or aspartic acid. (b) representation of the crystal structure of human ace2, with the collectrin domain oriented downward and viewed from the side of the cleft bearing the enzymatic active site. residues of rat ace2 whose alteration to the corresponding human residues converted rat ace2 to an efficient sars-cov receptor are shown in red. human ace2 residues whose alteration substantially decreased s1-ig association are shown in orange. residues whose alteration did not affect s1-ig association are shown in green. low-resolution electron density associated with the collectrin domain is represented by a small b-sheet and a-helix at the base of the figure. (c) a view identical to that in (b) except that the molecule has been rotated 901 about the vertical axis. (d) a view identical to that in (c) except that the molecule has been rotated 901 about the horizontal axis. ( figure 6c ). these data suggest that adaptation of s protein to human ace2 is facilitated by alteration of residue 479 to asparagine and of 487 to threonine. we also investigated determinants on civet ace2 that participate in its ability to facilitate efficient infection by gd and sz3. two regions within the s-protein-binding site differed significantly between human and palm-civet ace2 (see figure 7d and e). a region of a-helix 1 (residues 30-40) varied at six residues between human and civet ace2. likewise, a loop initiating ace2 a-helix 3 differed by four residues (90-93). a glycosylation site at asparagine 90 of human ace2 is part of this latter region and is not present in palm-civet ace2. figure 7a shows that the s1 domains of gd and sz3 bound substantially more efficiently when either of these regions was introduced into human ace2, and that introduction of both regions resulted in receptor binding comparable to that with wild-type palm-civet ace2. these observations were also reproduced in infection assays ( figure 7b ). these data demonstrate that residues within the s-protein-binding domain of ace2 largely determine the efficiency with which gd and sz3 s proteins bind and utilize human and palm-civet ace2. figure 7c compares the ability of eight rbd variants to bind to human and civet ace2, as well as to the chimeric molecules assayed in figure 7a and to a point-mutation variant of palm-civet ace2, in which aspartic acid 354 was altered to a glycine present in human ace2. rbd variants were generated from that of tor2 (left panels) or sz3 (right panels), and altered at positions 479, 487, or both, as indicated. the panels of figure 7c permit several conclusions. first, the rough equivalence between the left and right panels indicates that, as implied by figure 6b and c, s-protein residues 479 and 487 account for most of the differences between tor2 and sz3 rbd. second, no consistent differences were observed between palm-civet ace2 and its variant with glycine at residue 354, indicating little or no contribution of this residue to s-protein association. third, consistent with infection data in figure 6c , the presence of threonine at residue 487 enhanced the affinity of most rbds for civet and human ace2, and for variants of these receptors. (compare, for example, k479/s487 rbd variants with k479/t487 variants for their ability to precipitate each ace2 variant.) fourth, and in contrast, substitution of lysine 479 for asparagine in most contexts increased the ability of each rbd variant to associate with human, but not with palm-civet, ace2. (compare the binding of n479/t487 rbd with that of k479/t487 variants for binding to human ace2 (lane 1) and palm-civet ace2 (lane 6); likewise for n479/s487 and k479/s487 variants.) this enhancement was also observed for ace2 chimeras containing human a-helix 1 residues (lanes 1 and 2), but not those of palm civet (lanes 3, 5, and 6), whereas residues 90-93 did not determine sensitivity to rbd residue 479. fifth, consistent with infection data in figure 7b and supplementary figure 3 , all rbds bound ace2 variants bearing residues 90-93 of palm-civet ace2 substantially more efficiently than they bound equivalent variants with human ace2 residues at these positions (compare lanes 1 and 2 and lanes 3 and 4 in each panel). thus, the data of figure 7c indicate that a lysine at s-protein residue 479 interferes with rbd association with human, but not palmcivet, ace2. supplementary figure 4 shows data consistent with a steric interaction between lysine 31 of human ace2 and lysine 479 of the sz3 s protein. our data also show that alteration of s-protein serine 487 to threonine increases rbd affinity for both human and civet ace2. finally, they suggest that no s protein studied has fully adapted to human ace2 residues 90-93, consistent with a recent zoonotic transmission of the virus. figure 7d -f summarizes our findings. figure 7d shows amino-acid sequences of regions critical to s-protein association for palm-civet, rat, and human ace2. figure 7e shows ace2 oriented with the c-terminal membrane-associated collectrin domain facing away from the viewer. red indicates residues whose alteration transformed rat ace2 to an efficient sars-cov receptor. orange indicates additional residues common to rat and human ace2 whose alteration also interferes with s-protein association. yellow indicates residues along the a-helix 1 ridge that are unique to palm-civet ace2, and which permit efficient association with rbd isolates from palm civet and likely interact with lysine 479 of the palm-civet rbd. k31 of human ace2, which interferes with palm-civet rbd lysine 479, is labeled with white text in figure 7e . four residues at the beginning of a-helix 4 that permit more efficient binding and infection by all s proteins assayed are shown in cyan, and the glycosylation site in this region, present in human but not palm-civet ace2, is shown in green. ace2 is a functional receptor for sars-cov, and is likely to play a critical role in viral replication in an infected host hamming et al, 2004; nie et al, 2004) . here we describe the s-protein-binding domain of ace2. in particular, residues along the first a-helix, and lysine 353 and proximal residues at the n-terminus of b-sheet 5, participate in sprotein binding and in infection. by altering histidine 353 in rat ace2 and modifying a glyosylation site that may alter the shape of a-helix 1, we converted rat ace2 to an efficient receptor for sars-cov. this s-protein-binding region of ace2 remains intact in the presence of an inhibitor that dramatically alters the overall conformation of ace2 (dales et al, 2002; towler et al, 2004) , consistent with the inability of this inhibitor to block infection, and with the inability of the s protein to modulate ace2 activity. although there can be multiple constraints on interspecies transmission of viruses (webby et al, 2004) , s-protein alterations are sufficient to extend or alter the host range of a number of coronaviruses (kuo et al, 2000; casais et al, 2003; haijema et al, 2003; schickli et al, 2004) . we have shown that entry is the primary barrier to sars-cov infection of murine surface plasmon resonance experiments in which the indicated rbd-ig tor2 variants shown in figure 6b bound to immobilized anti-human antibody were assayed for association with soluble human ace2. the experiment is representative of two performed with similar results. table listing amino-acid differences among the rbds of the s proteins of the indicated isolates. residues critical to the differential association of these rbds with palm-civet and human ace2 are shown in gray. (b) experiment similar to that shown in figure 5b except that individual residues within the tor2 rbd have been altered to the corresponding residues in the sz3 rbd. (c) hiv-1-luciferase pseudotyped with s protein of the tor2, gd, or sz3 viruses, or with the indicated sz3 or tor2 variant, was incubated with hek293t cells transfected with plasmid encoding human ace2 or with palm-civet ace2. infection, measured as luciferase activity of cell lysates, was assayed 2 days postinfection. the figure shows the mean and range of two experiments. species-specific ace2 determinants of differential s-protein association. (a) hek293t cells transfected with plasmid encoding human or palm-civet ace2, with human ace2 bearing the indicated palm-civet residues, or with vector alone were analyzed by flow cytometry using s1-ig variants of the tor2, gd, and sz3 isolates. error bars indicate the range of two or more experiments. (b) hek293t cells transfected with plasmid encoding the ace2 variants used in (a) were incubated with hiv-1-luciferase virus pseudotyped with the s proteins of tor2, gd, or sz3 viruses. infection was assayed as in figure 6c . (c) hek293t cells transfected with plasmid encoding human ace2, human ace2 variants bearing the indicated palm-civet residues, palm-civet ace2, or the palm-civet ace2 variant d354g were metabolically labeled and lysed. cell lysates were immunoprecipitated with an anti-tag antibody recognizing an amino-terminal tag on these ace2 variants (a-myc), or with rbd-ig of tor2 or sz3, or with their variants with the indicated alterations of residues 479 and 487. the experiment is representative of at least two with similar results. (d) amino-acid content of critical regions of ace2 from human, palm civet, and rat. orange indicates human-ace2 residues whose alteration interferes with tor2 s-protein association. red indicates rat-ace2 residues whose alteration to their human counterparts converts rat ace2 to an efficient sars-cov receptor. yellow indicates residues of palm-civet ace2 that accommodate s-protein lysine 479 of sars-cov isolated from palm civets. cyan indicates additional residues of palm-civet ace2 that, when introduced into human ace2, result in more efficient association with all s proteins assayed. this effect may be due to the loss of glycosylation at asparagine 90 of human ace2, shown in green. cells . these observations suggest that s-protein changes may be critical to or sufficient for the adaptation of sars-cov to human cells. accordingly, we compared the s proteins derived from the 2002-2003 outbreak (tor2), from the less severe 2003-2004 outbreak (gd), and from apparently healthy palm civets (sz3) (guan et al, 2003; he et al, 2004) . strikingly, the receptor-binding regions of each of these s proteins bound palm-civet ace2 efficiently, but only that from the 2002-2003 outbreak bound human ace2 with comparable efficiency. these data are consistent with the absence of human-to-human transmission during the 2003-2004 outbreak, and with recent transmission of sars-cov from palm civets to humans (guan et al, 2003; zhong, 2004; song et al, 2005) . differences among these s proteins permitted identification of key changes necessary for adaptation to the human receptor. in particular, changes at s-protein residues 479 and 487 appear to be critical for high-affinity association with human ace2. the alteration at 479 to a small, uncharged residue is a consistent property of all described sars-cov obtained from humans, whereas most civet-derived viruses retain a basic residue at this position (guan et al, 2003; marra et al, 2003; rota et al, 2003; he et al, 2004; zhang et al, 2004; song et al, 2005) . our data indicate that residue 479 interacts with residues along a ridge formed by ace2 a-helix 1, and in particular with lysine 31, which is present in human but not palm-civet ace2. alteration of s-protein residue 479 to the asparagine found in virus isolated from humans appears to accommodate this human ace2 lysine. differences at s-protein residue 487 are also of interest. a threonine at position 487 is absolutely conserved in all of the more than 100 s proteins isolated during the severe 2002-2003 outbreak. in contrast, the s proteins of viruses isolated during the 2003-2004 outbreak, and all 14 animal sars-cov isolated, had a serine at this position (guan et al, 2003; he et al, 2004; zhang et al, 2004; song et al, 2005) . a threonine at position 487 increased affinity of most rbds assayed for both human and palm-civet ace2, and all chimeras thereof, and substantially enhanced the efficiency with which palmcivet-derived s protein infected cells expressing human ace2. these observations indicate that the additional methyl group of threonine 487 participates in the efficiency of infection of human and non-human cells. s-protein alterations at residues 479 and 487 are important for high-affinity association with human ace2, and for efficient infection of cells expressing this receptor. knowledge of these residues may be useful in assessing the risk posed by any new sars-cov outbreak. our data also show that, even with these and other changes outside the rbd, sars-cov is imperfectly adapted to its human receptor. in particular, introduction of residues 90-93 of civet ace2 into the human receptor increased binding of, and infection mediated by, all s proteins assayed. this effect may be due to removal of a glycosylation site at position 90 to which no sars-cov has fully adapted. this observation raises the possibility that soluble human ace2 lacking this glycosylation would more effectively inhibit sars-cov replication than wild-type human ace2. we have previously shown that replication of sars-cov in a murine cell line is limited by the low affinity of the s protein for murine ace2 (li et al, 2004) . moreover, the affinity of s protein for the receptors of rats, mice, and humans correlates with the ability of virus to replicate in these animals. the lower affinity of palm-civet-derived s protein for the palmcivet receptor is consistent with this pattern in that no overt disease was manifest in animals from which this virus was isolated (guan et al, 2003) , but disease was observed in palm civets challenged with isolates obtained during the 2002-2003 outbreak . together, these observations suggest that the affinity of s protein for ace2 is an important determinant in the overall rate of viral replication and in the severity of disease. if so, adaptations within the s protein that are critical for high-affinity association with human ace2 may have contributed to the unusual severity of sars. plasmid encoding a codon-optimized form of the sars-cov s protein of the tor2 isolate (accession number ay274119) has been previously described moore et al, 2004) . plasmids encoding the corresponding s proteins of the gd03t0013 isolate, isolated during the mild 2003-2004 outbreak (accession number ay525636; denoted gd herein), and the sz3 isolate, isolated from palm civets (accession number ay304486), were generated de novo by recursive pcr. plasmids encoding the s1 domain (residues 12-672) and the rbd (residues 318-510) of the tor2 s protein, fused to the fc domain of human igg1 (s1-ig and rbd-ig, respectively), have been previously described wong et al, 2004) . corresponding s1-ig and rbd-ig variants of the gd and sz3 isolates and variant ace2 molecules were generated by mutagenesis using the quikchange method (invitrogen). human, rat, and palm-civet ace2 molecules were amplified from cdna of corresponding tissue by pcr, and cloned into a vector encoding previously described amino-and carboxy-terminal tags . association of s1-ig or rbd-ig with ace2 variants was determined by flow cytometry and by immunoprecipitation. flow cytometry using ace2-expressing cells has been previously described wong et al, 2004) . briefly, hek293t cells were transfected with a plasmid encoding ace2 variants, or with vector alone. at 2 days post-transfection, cells were detached in pbs/5 mm edta and washed with pbs/0.5% bsa. s1-ig or rbd-ig, or variants thereof, or the anti-tag antibody 9e10, were added to 10 6 cells, and the mixture was incubated on ice for 1 h. cells were washed three times with pbs/0.5% bsa, and then incubated for 30 min on ice with antihuman igg fitc conjugate (sigma). cells were again washed with pbs/0.5% bsa, and analyzed. immunoprecipitations were performed as previously described wong et al, 2004) . briefly, hek293t cells were transfected with plasmid encoding ace2 variants and radiolabeled with [ 35 s]cysteine and [ 35 s]methionine. after 2 days, transfected cells were harvested and lysed in pbs buffer containing 1% chapso. cell lysates were incubated with protein a-sepharose beads together with 2 mg s1-ig or rbd-ig variants, or with the antibodies 1d4, recognizing a carboxy-terminal c9 tag on ace2, or 9e10, recognizing an amino-terminal myc tag. protein a-sepharose beads were washed three times in pbs/0.5% chapso, and analyzed by sds-page. immunoprecipitated ace2 variants were quantified by phosphorimaging. tor2 rbd-ig variants were also assayed by surface plasmon resonance using a biacore 3000. a 200 nm portion of purified rbd-ig of tor2 variants was bound to an anti-human antibody (sigma i-2136) immobilized on a cm5 sensor chip. soluble human ace2 in hbs-ep buffer (biacore) was introduced at a flow rate of 20 ml/min at concentrations of 700, 200, 40, 8, 1.6, and 0 nm. kinetic parameters were determined with bia-evaluation software (biacore). infection with s-protein-pseudotyped retrovirus mlv expressing gfp and pseudotyped with sars-cov s-protein variants has been previously described . briefly, mlv virions were generated by cotransfecting plasmid encoding mlv gag and pol genes, the pqcxix vector (bd sciences) expressing gfp, and plasmid encoding s-protein variants. at 48 h post-transfection, cell supernatants were normalized for reverse transcriptase activity and incubated with hek293t cells transfected with ace2 variants. at 48 h postincubation, gfp fluorescence of infected cells was measured by flow cytometry. in some cases, cells were preincubated for 1 h with the ace2 inhibitor mln-4760 or with nh 4 cl before infection, and equivalent concentrations were maintained during infection. infection was also assayed with a lentivirus expressing a luciferase reporter gene and pseudotyped with s-protein variants, as previously described (sui et al, 2005) . briefly, 293t cells were cotransfected with plasmid encoding s-protein variants, a plasmid (pcmvdr8.2) encoding hiv-1 gag-pol, and a plasmid (phiv-luc) encoding the firefly luciferase reporter gene under control of the hiv-1 long terminal repeat. at 2 days post-transfection, viral supernatants were harvested and 3 ìl of s-protein-pseudotyped virus was used for infection of 6000 ace2-expressing 293t cells in a 96-well plate. infection efficiency was quantitated by measuring the luciferase activity in the target cells with an eg&g berthold microplate luminometer lb 96v. the enzymatic activity of ace2 was assayed using a fluorogenic substrate, 7-methoxycoumarin-yvadapk(2,4-dinitrophenyl)-oh (r&d systems). cleavage of this peptide by ace2 removes the 2,4-dinitrophenyl moiety that quenches the fluorescence of the 7-methoxycoumarin moiety. a 1 mg portion of a soluble form of ace2 was incubated in 100 mm tris buffer with varying concentrations of the ace2 inhibitor mln-4760 (dales et al, 2002) . fluorescence was monitored at 5 min intervals using an excitation wavelength of 330 nm and emission wavelength of 450 nm. supplementary data are available at the embo journal 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associated with the extended host range of viruses from persistently infected murine cells cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association evaluation of human mab 80r in immunoprophylaxis of sars by an animal study, epitope mapping and analysis of spike variants ace2 x-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis molecular constraints to interspecies transmission of viral pathogens mice susceptible to sars coronavirus a 193-amino acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme 2 civets are equally susceptible to experimental infection by two different severe acute respiratory syndrome coronavirus isolates the sars-cov s glycoprotein: expression and functional characterization ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign collectrin, a collecting duct-specific transmembrane glycoprotein, is a novel homolog of ace2 and is developmentally regulated in embryonic kidneys reconstruction of the most recent common ancestor sequences of sars-cov s gene and detection of adaptive evolution in the spike protein management and prevention of sars in china key: cord-333264-jdvb8px4 authors: hanke, leo; vidakovics perez, laura; sheward, daniel j.; das, hrishikesh; schulte, tim; moliner-morro, ainhoa; corcoran, martin; achour, adnane; karlsson hedestam, gunilla b.; hällberg, b. martin; murrell, ben; mcinerney, gerald m. title: an alpaca nanobody neutralizes sars-cov-2 by blocking receptor interaction date: 2020-09-04 journal: nat commun doi: 10.1038/s41467-020-18174-5 sha: doc_id: 333264 cord_uid: jdvb8px4 sars-cov-2 enters host cells through an interaction between the spike glycoprotein and the angiotensin converting enzyme 2 (ace2) receptor. directly preventing this interaction presents an attractive possibility for suppressing sars-cov-2 replication. here, we report the isolation and characterization of an alpaca-derived single domain antibody fragment, ty1, that specifically targets the receptor binding domain (rbd) of the sars-cov-2 spike, directly preventing ace2 engagement. ty1 binds the rbd with high affinity, occluding ace2. a cryo-electron microscopy structure of the bound complex at 2.9 å resolution reveals that ty1 binds to an epitope on the rbd accessible in both the ‘up’ and ‘down’ conformations, sterically hindering rbd-ace2 binding. while fusion to an fc domain renders ty1 extremely potent, ty1 neutralizes sars-cov-2 spike pseudovirus as a 12.8 kda nanobody, which can be expressed in high quantities in bacteria, presenting opportunities for manufacturing at scale. ty1 is therefore an excellent candidate as an intervention against covid-19. s ars-cov-2 was first identified as the etiologic agent of the novel pneumonia covid-19, in late 2019. in the comparatively short time since then, it has achieved pandemic status, causing more than 13.3 million cases, leading to more than 600,000 deaths. accordingly, the who declared the pandemic to be a public health emergency of international concern. a safe and effective vaccine is urgently needed, but requires time to develop. in the meantime, and indeed also in the post-vaccine era, highly specific and potent antiviral interventions are needed. many generic or repurposed candidates are in trials, but so far results have been unremarkable. since the virus is newly emerged, specifically designed drugs have not yet reached late phase trials. when available, specific antiviral drugs or antibody therapies will be used to protect individuals at risk and their widespread use will allow immunologically naïve populations to exit lockdowns more safely. the virus is closely related to sars-cov-1, both being members of the lineage 2 betacoronaviruses. cell entry of both viruses is achieved by first binding to the cell surface expressed receptor angiotensin-converting enzyme 2 (ace2), followed by conformational changes in the viral spike glycoprotein trimer and subsequent membrane fusion. the affinity of sars-cov-2 receptor-binding domain (rbd) for ace2 is considerably higher than that for sars-cov-1 1,2 , supporting efficient cell entry and likely contributing to pathogenesis. the rbd is a globular domain situated on the distal surface of the spike protein. two conformations have been observed in the stabilized trimer. specifically, one conformation where one rbd is ace2accessible while the other two are not, and one conformation where all three rbds are down, i.e. receptor inaccessible 2, 3 . as the receptor-engaging part of the spike, the rbd is an attractive target for coronavirus neutralization, and a number of conventional neutralizing monoclonal antibodies that target the rbd and block receptor binding have already been isolated from convalescent patients [4] [5] [6] . camelid-derived single domain antibody fragments, also called vhhs or nanobodies, offer several advantages over conventional antibodies as candidates for specific therapies. despite being approximately one-tenth of the size of a conventional antibody, they retain specificity and affinity similar to conventional antibodies, while being far easier to clone, express, and manipulate. they are readily expressed in bacteria in large quantities and show high thermal stability and solubility, making them easily scalable and cost effective. their modularity means that they can be oligomerized to increase avidity or to increase serum half-life 7 . critical to their use as antivirals in humans, they can easily be humanized with existing protocols 8 . importantly, they have proven to be highly potent inhibitors of viral infections in vivo; particularly respiratory infections 9, 10 . here, we describe the isolation, evaluation, and molecular determination of an alpaca-derived nanobody, ty1, directed to the rbd of the sars-cov-2 spike glycoprotein. we demonstrate that the monomeric 12.8 kda ty1 molecule potently neutralizes sars-cov-2 spike pseudovirus. the nanobody binds with high affinity to the rbd in a manner that occludes ace2 interaction. we have also determined the mechanism of neutralization to be due to direct interference with rbd binding to ace2. altogether, these results highlight the great potential of ty1 as a sars-cov-2 antiviral agent. isolation of a sars-cov-2 neutralizing nanobody. we immunized one alpaca with sars-cov-2 s1-fc and rbd on a 60day immunization schedule. we generated a phage display library and performed two consecutive rounds of phage display, followed by an elisa-based binding screen (fig. 1a) . we isolated one nanobody, ty1, that binds specifically to the rbd of the sars-cov-2 spike glycoprotein. in parallel we performed next generation sequencing (ngs) on the baseline and post-enrichment libraries, and quantified variant frequency before and after each enrichment step. ty1 exhibited the greatest fold-change in frequency among all nanobody variants, increasing over 10,000-fold from baseline to after the second enrichment round (fig.1b) . we report the amino acid sequence of ty1 in fig. 1c . to determine whether ty1 neutralized sars-cov-2 we employed an in vitro neutralization assay using lentiviral particles pseudotyped with the sars-cov-2 spike protein. ty1 neutralized sars-cov-2 pseudotyped viruses at an ic 50 of 0.77 µg/ml (54 nm) (fig. 2a) . no neutralization of a lentivirus pseudotyped with vsv-g by ty1 was evident, and a control nanobody produced and purified in the same way, but specific for influenza a virus nucleoprotein 11 , showed no evidence of neutralization of sars-cov-2 pseudotyped viruses. when ty1 was expressed in mammalian cells as an fc-fusion protein the potent neutralization could be further increased to~12 ng/ml (fig. 2a to confirm that ty1 is directed specifically against the sars-cov-2 spike protein, we characterized the specificity of ty1 by flow cytometry. we site-specifically conjugated a fluorophore to the c-terminus of ty1 by means of a sortase a reaction and copper-free click chemistry (ty1-as635p) and stained untransfected cells and cells transiently transfected with sars-cov-2 spike under permeabilizing conditions (fig. 2b) . while untransfected and unstained cells displayed similar signals, cells expressing the viral spike protein showed a strong shift in fluorescence intensity when stained with ty1-as635p. the apparent double peak likely reflected the varying efficiency of this transient transfection. to determine if the same probe can be exploited to recognize the viral spike protein in immunofluorescence, we infected vero e6 cells with infectious sars-cov-2 at moi 1 for 24 h, and stained the fixed and permeabilized cells with ty1-as635p and anti-dsrna antibody (fig. 2c) . while uninfected cells showed no signal, infected cells were strongly labeled with both dsrna antibody and ty1-as635p. thus, ty1 recognized the viral spike glycoprotein with high specificity in its native conformation in sars-cov-2-infected cells. importantly, the low background in both experiments also suggested that ty1 is a highly specific and suitable tool for research, diagnostics, and therapy. to understand the mechanism of neutralization, we evaluated the effect of ty1 on rbd binding to ace2. we site-specifically conjugated a fluorophore to the c-terminus of the rbd (rbd-as635p) and used this probe to stain ace2 expressing hek293t cells (fig. 2d) . preincubation of rbd-as635p with unlabeled ty1 resulted in a strong reduction of ace2 staining, while preincubation with the control nanobody np-vhh1 11 , specific for influenza a virus nucleoprotein np, had no such effect. this result indicated that ty1 directly prevents binding of sars-cov-2 rbd to its host cell receptor ace2. ty1 binds to rbd with high affinity. specific and high-affinity binding of ty1 to the rbd was also demonstrated in kinetic bio layer interferometry (bli) experiments. dipping of surfaceimmobilized nanobodies into monomeric rbd solutions at 550 nm yielded binding responses with fast association kinetics and amplitudes reaching 1.5 nm only for ty1 but not for np-vhh1 (red and blue curves, respectively, in fig. 3a) . titration experiments performed under normal (280 mm) and high salt (680 mm) conditions revealed concentration-dependent kinetic response curves for binding of rbd to ty1 ( fig. 3b and supplementary fig. 1a , respectively). the derived semi-log concentration-response . infectivity relative to cells infected with pseudotyped virus in the absence of nanobody is shown. neutralization by ty1 was repeated in duplicate across six assays, neutralization by ty1-fc was repeated in duplicate across two assays, and the error bars represent the standard deviation. b cells were transfected with a plasmid harboring the sars-cov-2 spike for 24 h. cells were fixed, permeabilized, and stained with ty1-as635p (black and red) or left unstained (gray). cells were analyzed by flow cytometry. cell counts are presented as % of max (representative histogram). c vero e6 cells were infected with sars-cov-2 at a moi of 1 for 24 h. cells were fixed, permeabilized, and stained for dna (blue), dsrna (green), and with ty1-as635p (red). pictures were taken by fluorescence microscopy and representative examples are shown. scale bar, 20 µm. d ace2 expressing hek293t cells were trypsinized, fixed, and stained with rbd-as635p alone (blue), or preincubated with np-vhh1 (green) or ty1 (red). cells were analyzed by flow cytometry. nature communications | https://doi.org/10.1038/s41467-020-18174-5 article curves revealed sigmoidal line-shapes with fitted apparent k dvalues of 8 ± 1.5 and 13 ± 1.5 nm (mean value ± standard deviation) for binding at normal and high salt conditions, respectively. local fits to individual sensorgrams applying the standard 1:1 binding model appeared reasonable for the association phases at lower to intermediate rbd concentrations, as well as for all dissociation curves when fits were allowed to stay above zero (gray lines fig. 3b and s1a 1st panel). however, the model deviated from the observed data at higher rbd concentrations. instead almost perfect fits were obtained when the same data were analyzed in terms of a bayesian two-dimensional distribution of k d and k off -rate constants to address heterogeneous ligand site populations on the sensor surface [12] [13] [14] . for the two titrations at normal and high salt conditions, distinct peaks at k d and k off -rate values of 5-9 nm and 4-5 × 10 −3 s −1 were obtained ( fig. 3b and supplementary fig. 1a 4th panel) . in both conditions, a second elevated plateau with k d and k off -rate values of about 7 nm and 1-4 × 10 −7 contributed significantly to the observed sensorgrams. since most high-affinity protein-protein interactions in the nmrange have dissociation rates in the 1 × 10 −3 s −1 range 15 , we attribute the first defined peak as the relevant ty1:rbd interaction. the second broad plateau is likely caused by rbd competition and rebinding effects on the sensor surface, as well as heterogeneous ligand populations 13, 16, 17 . the orthogonal biophysical method isothermal titration calorimetry (itc) 18 confirmed the high affinity binding of ty1 to rbd with a k d of 9 nm (with estimated bounds of 1 and 70 nm) characterized by an exothermic enthalpy of about −10 ± 0.5 kcal/mole (fig. 3c , left panel). exothermic binding was already evident from the three initial relatively constant negative spikes that were caused by the injection of ty1 to rbd (fig. 3c , right panel). the amplitude of the following three to four spikes returned to baseline demonstrating saturation of the available rbd sites by ty1 binding. notably, return to baseline was accompanied by the appearance of preceding positive spikes (fig. 3c , left panel and supplementary fig. 1b) . these spikes were also detected when ty1 was injected into the buffer (hbs) and thus treated as ty1 dilution effects during data analysis. injection of np-vhh1 into rbd did not cause any binding or dilution heat changes above background noise. it should be noted that the itc measurements were performed at the lowest possible protein concentrations to derive k d -values in the low nm range, while still being able to detect interaction heat above background noise signals that were at about −0.17 μcal/s (maximum spike amplitude) and ±0.05 μcal/s, respectively. altogether, we concluded from these results that rbd bound to ty1 with high affinity of about 5-10 nm. ty1 binds to the rbd in 'up' and 'down' conformation. to understand the structural basis underlying the potent neutralization of sars-cov-2 we performed a cryo-em structure determination of the prefusion-stabilized spike ectodomain in complex with ty1. the cryo-em reconstruction reaches an overall resolution of 2.9 å (0.143 fsc; supplementary table 1) with strong variation of estimated local resolution from high resolution in the core of the spike trimer to relatively low resolution in the top of the spike ( fig. 4a and supplementary fig. 2 ). nevertheless, this reconstruction clearly shows that the spike retains only one main conformation with one rbd 'up' and two rbds 'down'. importantly, all three rbds are decorated in their upper parts with a ty1 nanobody. the nanobodies retain a similar binding orientation to the rbd whether the rbd is found in the 'up' or 'down' conformation ( fig. 4a, b) and each has a solvent-excluded surface area of~860 å 2 , which is in line with the strong affinity observed in the biophysical-interaction studies. primary interactions with the rbd are through the cdrs. specifically, cdr1 interacts primarily with rbd t470 and v483-e484, and cdr3 interacts primarily with rbd y449, f490, and q493. interestingly, cdr2 does not form any major interactions with the rbd, instead it stabilizes the conformation of cdr1 in the rbd bound mode and thereby acts indirectly to potentiate the ty1-rbd binding. since ace2 can only be bound by an rbd in the 'up' conformation, the cryo-em reconstruction clearly shows that ace2 binding is sterically hindered from two sides (fig. 4c) . specifically, ace2 binding is blocked both by the ty1 nanobody bound to the rbd in the 'up' conformation and the neighboring rbd in the 'down' conformation. hence, ace2 binding is sufficiently hindered with any two of the available three binding rbd sites in the spike trimer. the current coronavirus pandemic has drastic consequences for the world's population, and vaccines, antibodies, or antivirals are urgently needed. neutralizing antibodies can block virus entry at an early step of infection and potentially protect individuals that are at high risk of developing severe disease. we report the identification and characterization of a sars-cov-2 rbdspecific single domain antibody fragment (nanobody) termed ty1 that potently neutralizes the virus. we identified ty1 by binding assay after two consecutive rounds of phage display, simultaneously monitoring sequence enrichment by ngs. although ty1 exhibited the greatest fold-enrichment in the ngs analysis, multiple additional nanobodies exhibited enrichment of varying extent across both rounds. as the correlation between phage display enrichment and neutralization is likely imperfect, further analyses of our libraries may yield other potent sars-cov-2 neutralizing nanobodies. in addition to neutralization activity, we also show that ty1 can be used as a detection reagent in flow cytometry and immunofluorescence demonstrating its suitability as a research tool and for diagnostics. glycans on spike glycosylation sites n165, n234, and n343 shield the rbd from antibodies, especially when the rbd is in down conformation 19, 20 . indeed, in the rbd-down conformation, the glycan on n165 points towards the ty1-binding epitope, likely not leaving sufficient space to accommodate a conventional antibody. in agreement with that, fab fragments from convalescent patients bound the rbd only in the up conformation and to an epitope that only minimally overlaps with the ty1 epitope 21 . it should be noted that the nanobody ty1 can be readily produced in bacteria at very high yield (in excess of 30 mg/l culture), making it an excellent candidate for a low-cost, scalable antiviral agent against sars-cov-2, and we provide the amino acid sequence, encouraging direct exploitation as such. interestingly, while ty1 contains the hallmark (hydrophobic) amino acids of variable-heavy chains in framework 2, only one arginine (instead of tryptophan) in framework 4 demonstrates that this antibody fragment derives from a heavy-chain only antibody 22 . nevertheless, ty1 expresses extremely well, but exchanging the hydrophobic residues in framework 2 may further improve this nanobody. while nanobodies capable of binding sars-cov-2 spike have recently been isolated, these were generated after sars-cov-1 spike immunization 23 , or pcr maturation 24 . also, in both cases a fusion to human fc domain is required for neutralization of sars-cov-2, precluding expression in bacterial culture. naive libraries of human single-domain antibodies (sdabs) have also been screened to identify sars-cov-2 spikespecific nanobodies 25, 26 , but they lack detailed structural information. other synthetic rbd-specific nanobodies have been published, but they lack information on their neutralization potential 27 . ty1 represents the first single-domain antibody isolated from an animal specifically immunized with a sars-cov-2 protein. future work will aim to improve the potency and potential efficacy of ty1 through various strategies. for example, mutational scanning may yield potency improvements to ty1. also, since ty1 already neutralizes as a monomeric protein, the generation of homodimeric or trimeric fusion constructs is expected to further increase its neutralization activity. indeed, fusion of ty1 to a human igg1-fc dramatically improved the ic 50 of this molecule, to~12 ng/ml. additional strategies will explore linkerbased constructs that chain multiple copies of ty1 together, which may provide similar improvements in potency while retaining the possibility of being expressed in bacteria. ty1 may additionally be a useful component of a bi-specific or tri-specific antibody, which could combine epitope specificities to increase the mutational barrier to viral escape. based on our work, we hope that ty1 will be investigated as a candidate for antiviral therapy. cells and virus. vero e6 cells (atcc-crl-1586) and hek293t cells (atcc-crl-3216) were maintained in dulbecco's modified eagle medium (gibco) supplemented with 10% fetal calf serum and 1% penicillin-streptomycin and cultured at 37°c in a humidified incubator with 5% co 2 . a hek293t cell line engineered to overexpress human ace2 (hek293t-ace2) was generated by the lentiviral transduction of hek293t cells. briefly, lentiviruses were produced by cotransfecting hek293t cells with a plasmid encoding vsv-g (addgene cat#12259), a lentiviral gag-pol packaging plasmid (addgene cat#8455), and a human ace2 transfer plasmid. virions were harvested from the supernatant, filtered through 0.45 µm filters, and used to transduce hek293t cells. all cell lines used for experiments were negative for mycoplasma as determined by pcr. infectious sars-cov-2 28 was propagated in vero e6 cells and titrated by plaque assay. proteins and probes. the plasmid for expression of the sars-cov-2 prefusionstabilized spike ectodomain with a c-terminal t4 fibritin trimerization motif was obtained from ref. 2 . the plasmid was used to transiently transfect freestyle 293f cells using freestyle max reagent (thermo fisher scientific). the s ectodomain was purified from filtered supernatant on streptactin xt resin (iba lifesciences), followed by size-exclusion chromatography on a superdex 200 in 5 mm tris ph 8, 200 mm nacl. the rbd domain (rvq-vnf) of sars-cov-2 was cloned upstream of an enterokinase cleavage site and a human igg1 fc. this plasmid was used to transiently transfect freestyle 293f cells using the freestyle max reagent. the rbd-fc fusion was purified from filtered supernatant on protein g sepharose (ge healthcare). the protein was cleaved using bovine enterokinase (genscript) leaving a flag-tag at the c-terminus of the rbd. enzyme and fc-portion were removed on his-pur ni-nta resin (thermo fisher scientific) and protein g sepharose (ge healthcare), respectively, and the rbd was purified by sizeexclusion chromatography on a superdex 200 in 50 mm tris ph 8, 200 mm nacl. in addition, the rbd domain (rvq-vnf) was cloned upstream of a sortase a recognition site (lpetg) and a 6xhis tag and expressed in freestyle 293f cells as described above. rbd-his was purified from filtered supernatant on his-pur ni-nta resin, followed by size-exclusion chromatography on a superdex 200. the nanobodies were cloned for expression in the phen plasmid with a cterminal sortase recognition site (lpetg) and a 6xhis tag. this plasmid was used to transform bl21 cells for periplasmic expression. expression was induced with 1 mm iptg at od600 = 0.6; cells were grown overnight at 30°c. nanobodies were retrieved from the periplasm by osmotic shock and purified by ni-nta affinity purification and size-exclusion chromatography. biotinylated and fluorescent probes were generated using sortase a as described in refs. 29, 30 . in brief, nanobodies were site-specifically biotinylated on the cterminus using sortase a 5 m. nanobody at a concentration of 50 μm was incubated with sortase a 5 m (5 μm), gggk-biotin (200 μm) in 50 mm tris, ph 7.5, 150 mm nacl, 10 mm cacl 2 , for 2 h at 25°c. unreacted nanobody and sortase was removed with ni-nta resin and excess gggk-biotin was removed using zeba spin desalting columns (0.5 ml, 7k mwco, thermo fisher scientific). to generate the fluorescently labeled probes, first a dibenzocyclooctyne-amine (dbco-amine, sigma aldrich) was attached via sortase a to the nanobody or the rbd (reaction conditions: 50 μm rbd or nanobody, 50 μm sortase a 5 m, 8 mm dbco-amine in 50 mm tris ph 7.5, 150 mm nacl, 10 mm cacl 2 , 2 h, 25°c). unreacted probe, sortase and excess dbco-amine were removed using ni-nta resin and pd-10 columns (ge healthcare), respectively. abberior star 635p-azide (abberior gmbh) was attached to the dbco-labeled proteins in a copper-free click chemistry reaction. unreacted fluorophore was removed on pd-10 column (rbd) or size-exclusion chromatography (nanobody). for mammalian expression, the sequence encoding the nanobody ty1 was cloned upstream of a human igg1. this plasmid was used to transiently transfect freestyle 293f cells using the freestyle max reagent. the ty1-fc fusion was purified from filtered supernatant on protein g sepharose followed by sizeexclusion chromatography. alpaca immunization. alpaca immunization and phage display was performed similarly as described in refs. 31, 32 . in brief, the adult male alpaca tyson at pre-clinics, germany, was immunized four times in a 60-day immunization schedule. sars-cov-2 s1-sheep-fc (native antigen company, sku: rec31806) was used for the first two immunizations, and sars-cov-2 rbd produced in freestyle 293f cells was used for the last two immunizations. the animal study protocol was approved by the preclinics animal welfare officer commissioner and registered under the registration no. 33.19-42502-05-17a210 at the lower saxony state office for consumer protection and food safety-laves and is compliant with the directive 2010/63/eu on animal welfare. library generation and nanobody isolation. four days after the final boost, rna was isolated from pbmcs (rna plus mini kit, qiagen). for cdna synthesis, superscript iii rt (thermo fisher scientific) was used with a combination of oligo (dt), random hexamers, or gene-specific primers (al.ch2, atggagaggac gtccttgggt, and al.ch2.2 ttcggggggaagayraagac) 32 . all primer sequences are listed in supplementary table 2 . nanobody sequences were pcr amplified and cloned into a phagemid vector for expression as piii fusion. tg1 cells (lucigen) were transformed with this library by electroporation. cells were inoculated with vcsm13 helper phage, and the resulting phage was enriched in two consecutive rounds of phage display on rbd immobilized on magnetic beads. after the second round of phage display, individual bacterial colonies were picked in a 96-well format, grown until od = 0.6 and nanobody expression was induced by addition of 1 mm iptg. after 16 h incubation at 30°c, bacterial supernatant was used as primary detection reagent in an elisa coated with rbd or s ectodomain. bound nanobodies were detected with anti-e tag (bethyl laboratories, 1:10,000) secondary antibody. positive clones were sequenced and cloned into the phen expression vector for further characterization. amino acid sequence of ty1. qvqlvetggglvqpggslrlscaasgftfss vymnwvrqapgkgpewvsrispnsgnigytdsvkgrftisrdnakn tlylqmnnlkpedtalyycaiglnlssssvrgqgtqvtvss ngs and analysis of nanobody libraries. plasmids from nanobody libraries before enrichment, and after each enrichment step, were amplified for 13 cycles using q5 high-fidelity 2x master mix (neb) according to manufacturer's instructions, using primers: nb-ngs-fw: cactctttccctacacgacgctc ttccgatctctcgcggcccagccggccatgg and nb-ngs-rv: ggagttc agacgtgtgctcttccgatctaccggcgcaccactagtgca, annealing at 72°c. illumina indexing primers were added using an additional 9 cycles, with kapa hifi. amplicons were size selected using agencourt ampure xp beads (bead ratio: 1:1), and were pooled at ratios of 6:2:1 for pre:post-1:post-2 libraries, to account for the reduction in diversity expected during enrichment, and sequenced on an illumina miseq using the miseq reagent kit v3 (2 × 300) ms-102-3003. paired-end reads were merged using usearch11 33 , and then processed in the julia language, primarily using the nextgensequtils.jl package 34 (analysis code is available here: https://github.com/murrellgroup/ty1). briefly, reads are trimmed of primer sequences, and deduplicated, maintaining read frequencies. variant frequencies are calculated as combined frequency of any reads matching a variant within 3% nucleotide divergence, using a kmer-based distance approximation for rapid database search. any reads with counts >3 from the second enrichment library are searched for their variant frequencies across all databases. when calculating enrichment, to avoid zeros due to sampling and to regularize against over-sensitivity to low-frequency baseline variants, all frequencies are increased by the reciprocal of the size of the pre-enrichment database. neutralization assay. pseudotyped viruses were generated by the co-transfection of hek293t cells with plasmids encoding the sars-cov-2 spike protein harboring an 18 amino acid truncation of the cytoplasmic tail 35 , a plasmid encoding firefly luciferase, and a lentiviral packaging plasmid (addgene cat#8455) using lipofectamine 3000 (invitrogen). media was changed 12-16 h after transfection, and pseudotyped viruses were harvested at 48 and 72 h post transfection, filtered through a 0.45 µm filter, and stored at −80°c until use. pseudotyped virus neutralization assays were adapted from protocols previously validated to characterize the neutralization of hiv 36 , but with the use of hek293t-ace2 cells. briefly, pseudotyped viruses sufficient to generate~100,000 rlus were incubated with serial dilutions of nanobodies for 60 min at 37°c. approximately 15,000 hek293t-ace2 cells were then added to each well and the plates were incubated at 37°c for 48 h. luminescence was then measured using bright-glo (promega) per the manufacturer's instructions on a gm-2000 luminometer (promega) with an integration time of 0.3 s. flow cytometry. cells were trypsinized and fixed in 4% formaldehyde/pbs and stained with rbd-as635p under non-permeabilizing conditions or with ty1-as635p under permeabilizing conditions. fluorescence was quantified using a bd facscelesta and the flowjo software package. immunofluorescence. vero e6 cells were seeded onto coverslips in a 24-well plate and incubated overnight at 37°c/5% co 2 . cells were infected with sars-cov-2 at a moi of 1 for 24 h. cells were fixed with 4% (v/v) formaldehyde, permeabilized in 0.5% triton x-100 and blocked in 5% horse serum. cells were incubated with anti-dsrna antibody (1:2000, j2 scicons, rnt-sci-10010200) for 1 h at room temperature followed by 1 h staining with the secondary antibody anti-mouse-alexa fluor 488 (1:2000, thermo fisher scientific, a-21202), hoechst (1:1000, invitrogen) and ty1-as635p (0.5 µg/ml). coverslips were mounted in mounting media and images were obtained using zeiss axiovert microscope and processed using adobe photoshop. biophysical bli and itc. bli was performed using single-use high-precision streptavidin biosensors (sax) on an eight-channel octet red instrument according to manufacturer's protocols (fortebio) 16 . assays were performed in 2xpbs comprising 0.05% tween-20 (pbst). biotinylated nanobodies ty1 and np-vhh1 were loaded at concentrations between 30 and 250 nm followed by quenching using biocytin to reach final sensor loads of between 0.15 and 0.7 nm. for the comparative binding test, the eight sensors were divided into two sets, each comprising double sample as well as single reference and single control sensors. sample and reference sensors were loaded with respective nanobodies. the sax control was only quenched. loading of the two sets was performed consecutively to reach similar immobilization levels, while subsequent association and dissociation phases were performed simultaneously. for association, the sample and control sensors were dipped into rbd, while the reference sensor was dipped into pbst. for titration experiments, all sensors were loaded simultaneously. during association one of the sensors was used as reference and only dipped into pbst. raw data were preprocessed, analyzed, and fitted by applying the 1:1 binding model as implemented in the manufacturer's software. bayesian analysis to obtain the two-dimensional distribution of k d and k off -rate values were performed using evilfit [12] [13] [14] . the shown titration data were processed applying reference sensor subtraction and savitzky-golay filter operations. for itc, proteins were exchanged to 2xhbs-buffer (50 mm hepes, 300 mm nacl, ph 7.5) and isolated as single peak populations by superdex-200 hr10/300 size-exclusion chromatography. itc measurements were performed using an itc200 calorimeter (ge healthcare). the cell temperature was set to 37°c and the syringe stirring speed to 750 rpm. before each experiment, the rbd and nanobodies were loaded into the cell and syringe at concentrations of 4 and 75 μm, respectively. data and binding parameters were analyzed using the microcal peakitc software (malvern). the integrated heat versus molar ratio plots of the ty1:rbd interactions were obtained by subtracting the ty1 dilution heat uptake from the binding data. the np-vhh1:rbd data were only baseline-corrected, since dilution effects were not evident. raw and processed bli/itc data were imported into rstudio for visualization and further analysis [37] [38] [39] . data along with analysis r scripts will be made publicly available via github and/or datadryad. cryo-em sample preparation and imaging. spike trimer (0.7 mg/ml) and ty1 (1.3 mg/ml) were mixed in a 1:8 molar ratio and incubated on ice for 5 min. a 3-μl aliquot of the sample solution was applied to glow-discharged cryomatrix holey grids with amorphous alloy film (zhenjiang lehua technology) in a vitrobot mk iv (thermo fisher scientific) at 4°c and 100% humidity (blot 10 s, blot force 3). cryo-em data collection was performed with epu 2.7 (thermo fisher scientific) using a krios g3i transmission-electron microscope (thermo fisher scientific) operated at 300 kev in the karolinska institutet 3d-em facility. images were acquired in nanoprobe eftem mode with a slit width of 10 ev using a gif 967 energy filter (ametek) and a k3 detector (ametek) during 2.4 s with a dose rate of 4.1 e − /px/s resulting in a total dose of 38 e − /å2 fractionated into 40 movie frames. motion correction, ctf-estimation, fourier binning (to 1.02 å/px), picking and extraction in 428 pixel boxes were performed on the fly using warp 40 . a total of 13,589 micrographs were selected based on an estimated resolution cutoff of 4 å and defocus below 2 microns and 573,036 particles were picked by warp. extracted particles were imported into cryosparc v2.15.0 41 for 2d classification, 3d classification, and non-uniform 3d refinement. the particles were processed with c1 symmetry throughout. after 2d classification (300 classes) 354,678 particles were retained and used to build three ab-initio 3d reconstructions. these were further processed for heterogeneous refinement that resulted in one reconstruction showing high-resolution structural features in the core of the spike. one round of homogenous refinement followed by non-uniform refinement resulted in a final reconstruction to an overall resolution of 2.9 å (0.143 fsc) using 210,832 particles. localized reconstruction 42 were performed using particles where all parts of the spike except the n-terminal domains, the rbds, and the nanobodies had been subtracted 43 . the combined effects of these two approaches significantly increased the level of density detail in the upper part of the spike. model building and structure refinement. a structure of the 2019-ncov spike protein trimer 2 (pdb: 6vsb) was used as a starting model for model building. the model was extended and manually adjusted in coot 44 . the nanobody structure was homology modeled using swiss-model 45 taking pdb:5jmr 46 as a template. the missing regions of the rbd domains were built based on the rbd-ace2 crystal structure (pdb: 6lzg) 47 . for model building and refinements, a composite map was made using phenix 48 utilizing the particle center-of-mass focused reconstruction and the map from the localized reconstruction described above. structure refinement and manual model building were performed using coot and phenix in interspersed cycles with secondary structure and geometry restrained. all structure figures and all em density-map figures were generated with ucsf chimerax 49 . reporting summary. further information on research design is available in the nature research reporting summary linked to this article. the sequence of ty1 is deposited in the ncbi genbank sequence data base and is available under the accession code mt784731. bli and itc data are available in https:// github.com/derpaule/ty1_octet_itc and https://doi.org/10.5061/dryad.gb5mkkwmz, respectively. next generation sequencing data is deposited at the sra, under bioproject id prjna638614. jupyter notebooks to reproduce the ngs data processing are available at: https://github.com/murrellgroup/ty1. the cryo-em density map of sars-cov-2 spike glycoprotein with ty1 nanobodies bound was deposited in the electron microscopy data bank (emdb) with accession code emd-11526. the corresponding model was deposited in the protein data bank (pdb) with accession code 6zxn. source data are provided with this paper. received: 19 june 2020; accepted: 8 august 2020; cell entry mechanisms of sars-cov-2 cryo-em structure of the 2019-ncov spike in the prefusion conformation structure, function, and antigenicity of the sars-cov-2 spike glycoprotein potent neutralizing antibodies against sars-cov-2 identified by high-throughput single-cell sequencing of convalescent patients' b cells human neutralizing antibodies elicited by sars-cov-2 infection a human neutralizing antibody targets the receptor-binding site of sars-cov-2 nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics general strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold single-domain antibodies targeting neuraminidase protect against an h5n1 influenza virus challenge generation and characterization of alx-0171, a potent novel therapeutic nanobody for the treatment of respiratory syncytial virus infection the antiviral mechanism of an influenza a virus nucleoprotein-specific single-domain antibody fragment bayesian analysis of heterogeneity in the distribution of binding properties of immobilized surface sites the role of mass transport limitation and surface heterogeneity in the biophysical characterization of macromolecular binding processes by spr biosensing combined affinity and rate constant distributions of ligand populations from experimental surface binding kinetics and equilibria a guide to simple and informative binding assays determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the octet designing binding kinetic assay on the bio-layer interferometry (bli) biosensor to characterize antibody-antigen interactions characterization of protein-protein interactions by isothermal titration calorimetry sitespecific glycan analysis of the sars-cov-2 spike developing a fully glycosylated full-length sars-cov-2 spike protein model in a viral membrane structures of human antibodies bound to sars-cov-2 spike reveal common epitopes and recurrent features of antibodies nanobodies: natural single-domain antibodies structural basis for potent neutralization of betacoronaviruses by single-domain camelid antibodies neutralizing nanobodies bind sars-cov-2 spike rbd and block interaction with ace2 humanized single domain antibodies neutralize sars-cov-2 by targeting spike receptor binding domain identification of human single-domain antibodies against sars-cov-2 sybodies targeting the sars-cov-2 receptor-binding domain inhibition of sars-cov-2 infections in engineered human tissues using clinical-grade soluble human ace2 site-specific protein labeling via sortase-mediated transpeptidation production of unnaturally linked chimeric proteins using a combination of sortase-catalyzed transpeptidation and click chemistry how lamina-associated polypeptide 1 (lap1) activates torsin lama pacos) as a convenient source of recombinant camelid heavy chain antibodies (vhhs) error filtering, pair assembly and error correction for next-generation sequencing reads long-read amplicon denoising isolation of potent sars-cov-2 neutralizing antibodies and protection from disease in a small animal model optimization and validation of the tzm-bl assay for standardized assessments of neutralizing antibodies against hiv-1 r: a language and environment for statistical computing (team rc, other elegant graphics for data analysis welcome to the tidyverse real-time cryo-electron microscopy data preprocessing with warp cryosparc: algorithms for rapid unsupervised cryo-em structure determination localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes bacteriophage φ29 scaffolding protein gp7 before and after prohead assembly features and development of coot swiss-model: homology modelling of protein structures and complexes the structure of a furin-antibody complex explains noncompetitive inhibition by steric exclusion of substrate conformers structural and functional basis of sars-cov-2 entry by using human ace2 phenix: a comprehensive python-based system for macromolecular structure solution ucsf chimerax: meeting modern challenges in visualization and analysis equilibrium analysis of high affinity interactions using biacore the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/10.1038/s41467-020-18174-5. peer review information nature communications thanks the anonymous reviewers for their contribution to the peer review of this work. peer reviewer reports are available.reprints and permission information is available at http://www.nature.com/reprintspublisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. key: cord-295482-qffg6r91 authors: wong, alan h. m.; tomlinson, aidan c. a.; zhou, dongxia; satkunarajah, malathy; chen, kevin; sharon, chetna; desforges, marc; talbot, pierre j.; rini, james m. title: receptor-binding loops in alphacoronavirus adaptation and evolution date: 2017-11-23 journal: nat commun doi: 10.1038/s41467-017-01706-x sha: doc_id: 295482 cord_uid: qffg6r91 rna viruses are characterized by a high mutation rate, a buffer against environmental change. nevertheless, the means by which random mutation improves viral fitness is not well characterized. here we report the x-ray crystal structure of the receptor-binding domain (rbd) of the human coronavirus, hcov-229e, in complex with the ectodomain of its receptor, aminopeptidase n (apn). three extended loops are solely responsible for receptor binding and the evolution of hcov-229e and its close relatives is accompanied by changing loop–receptor interactions. phylogenetic analysis shows that the natural hcov-229e receptor-binding loop variation observed defines six rbd classes whose viruses have successively replaced each other in the human population over the past 50 years. these rbd classes differ in their affinity for apn and their ability to bind an hcov-229e neutralizing antibody. together, our results provide a model for alphacoronavirus adaptation and evolution based on the use of extended loops for receptor binding. c oronaviruses are enveloped, positive-stranded rna viruses that cause a number of respiratory, gastrointestinal, and neurological diseases in birds and mammals 1, 2 . the coronaviruses all possess a common ancestor and four different genera (alpha, beta, gamma, and delta) that collectively use at least four different glycoproteins and acetylated sialic acids as host receptors or attachment factors have evolved [3] [4] [5] . four coronaviruses, hcov-229e, hcov-nl63, hcov-oc43, and hcov-hku1 circulate in the human population and collectively they are responsible for a significant percentage of the common cold as well as more severe respiratory disease in vulnerable populations 6, 7 . hcov-229e and hcov-nl63 are both alphacoronaviruses and although closely related, they have evolved to use two different receptors, aminopeptidase n (apn) and angiotensin converting enzyme 2 (ace2), respectively 8, 9 . the more distantly related betacoronaviruses, hcov-oc43 and hcov-hku1, are less well characterized and although hcov-oc43 uses 9-o-acetylsialic acid as its receptor 10 , the receptor for hcov-hku1 has not yet been determined [11] [12] [13] . recent zoonotic transmission of betacoronaviruses from bats is responsible for sars and mers, and in these cases infection is associated with much more serious disease and high rates of mortality [14] [15] [16] . like hcov-nl63, sars-cov uses ace2 17 as its receptor and the observation that mers-cov uses dipeptidyl peptidase 4 18 highlights the fact that coronaviruses with new receptor specificities continue to arise. the coronavirus spike protein (s-protein) is a trimeric singlepass membrane protein that mediates receptor binding and fusion of the viral and host cell membranes 19 . it is a type-1 viral fusion protein possessing two regions, the s1 region that contains the receptor-binding domain (rbd) and the s2 region that contains the fusion peptide and heptad repeats involved in membrane fusion [20] [21] [22] [23] [24] [25] . the coronavirus s-protein is also a major target of neutralizing antibodies and one outcome of hostinduced neutralizing antibodies is the selection of viral variants capable of evading them, a process known to drive variation [26] [27] [28] . as shown by both in vivo and in vitro studies, changes in host, host cell type, cross-species transmission, receptor expression levels, serial passage, and tissue culture conditions can also drive viral variation [29] [30] [31] [32] [33] . rna viruses are characterized by a high mutation rate, a property serving as a buffer against environmental change 34 . a host-elicited immune response, the introduction of antiviral drugs, and the transmission to a new species provide important examples of environmental change 35 . nevertheless, the means by which random mutations lead to viral variants with increased fitness and enhanced survival in the new environment are not well characterized. given their wide host range, diverse receptor usage and ongoing zoonotic transmission to humans, the coronaviruses provide an important system for studying rna virus adaptation and evolution. the alphacoronavirus, hcov-229e, is particularly valuable as it circulates in the human population and a sequence database of natural variants isolated over the past fifty years is available. moreover, changes in sequence and serology have suggested that hcov-229e is changing over time in the human population [36] [37] [38] . reported here is the x-ray structure of the hcov-229e rbd in complex with human apn (hapn). the structure shows that receptor binding is mediated solely by three extended loops, a feature shared by hcov-nl63 and the closely related porcine respiratory coronavirus, prcov. it also shows that the hcov-229e rbd binds at a site on hapn that differs from the site where the prcov rbd binds on porcine apn (papn), evidence of an ability of the rbd to acquire novel receptor interactions. remarkably, we find that the natural hcov-229e sequence variation observed over the past fifty years is highly skewed to the receptor-binding loops. moreover, we find that the loop variation defines six rbd classes (classes i-vi) whose viruses have successively replaced each other in the human population. these rbd classes differ in their affinity for hapn and their ability to be bound by a neutralizing antibody elicited by the hcov-229e reference strain (class i). taken together, our results provide a model for alphacoronavirus adaptation and evolution stemming from the use of extended loops for receptor binding. characterization of the hcov-229e rbd interaction with hapn. to define the limits of the hcov-229e rbd, we expressed a series of soluble s-protein fragments and measured their affinity to a soluble fragment (residues 66-967) 39 of hapn, the hcov-229e receptor. the smallest s-protein fragment made (residues 293-435) bound hapn with an affinity (k d of 0.43 ± 0.1 µm) similar to that of the entire s1 region (residues 17-560) ( table 1, supplementary fig. 1a , b) and this fragment was used in the structure determination. to confirm the importance of the table 1 analysis of the hapn ectodomain (residues 66-967, wt and mutants) interaction with fragments of the hcov-229e sprotein (wt and mutants) using surface plasmon resonance hcov-229e rbd-hapn interaction for viral infection, we showed that both the rbd and the hapn ectodomain inhibited viral infection in a cell-based assay (fig. 1a, b, c) . crystals of the hcov-229e rbd-hapn complex were obtained by co-crystallization of the complex after size exclusion chromatography. the crystallographic data collection and refinement statistics are shown in table 2 . the asymmetric unit contains one hapn dimer (and associated rbds) and one hapn monomer (and associated rbd) that is related to its dimeric mate by a crystallographic two-fold rotation axis. both dimers (noncrystallographic and crystallographic) are found in the closed conformation and are essentially identical to that which we previously reported 39 for hapn in its apo form (rmsd over all cα atoms of 0.34 å). each apn monomer is bound to one rbd as shown in fig. 2a . the hcov-229e rbd-hapn interaction buries 510 å 2 of surface area on the rbd and 490 å 2 on hapn. the hcov-229e rbd is an elongated six-stranded β-structural domain with three extended loops (loop 1: residues 308-325, loop 2: residues 352-359, loop 3: residues 404-408) at one end that exclusively mediate the interaction with hapn (fig. 2b ). loop 1 is the longest and it contributes~70% of the rbd surface buried on complex formation (figs. 2c and 3g). within loop 1, residues cys 317 and cys 320 form a disulfide bond that makes a stacking interaction with the side chains of hapn residues tyr 289 and glu 291 (fig. 2c) . the c317s/c320s rbd double mutant showed no binding to hapn at concentrations up to 15 μm (table 1 , supplementary fig. 1d , and supplementary table 1), evidence of the importance of the stacking interaction and a likely role for the disulfide bond in defining the conformation of loop 1. notably, loop 1 contains three tandemly repeated glycine residues (residues 313-315) whose nh groups donate hydrogen bonds to the side chain of asp 288 and the carbonyl oxygen of phe 287 of hapn (fig. 2c) ; mutation of hapn residue asp 288 to alanine leads to a~10-fold reduction in affinity ( (fig. 2c) ; the importance of trp 404 of loop 3 is evidenced by the fact that mutating it also ablates binding (table 1 , supplementary fig. 1f , and supplementary table 1 ). hcov-229e and prcov bind at different sites on apn. as with hcov-229e, the porcine respiratory alphacoronavirus, prcov, also uses apn as its receptor 40 . as our complex shows, hcov-229e binds at a site on hapn (h-site) that differs from the site on papn (p-site) used by prcov (fig. 3a, b) . glu 291 in hapn, a residue in the hapn-rbd interface, is an n-glycosylated asparagine (asn 286 ) in papn and attempts to dock the hcov-229e rbd at the h-site on papn leads to a steric clash with the n-glycan ( supplementary fig. 3a ). consistent with this observation, the hcov-229e rbd cannot bind to a mutant form of hapn (e291n/k292e/q293t) that possesses an n-glycan at position 291, as we have shown ( 41 . across species, the sequence identity at the h-and p-sites is only~60% ( fig. 3c and supplementary fig. 3c ) and the receptor-binding loops of these viruses must be accommodating the remaining apn structural differences on receptors from species that they do not infect. together these results provide evidence that the extended receptor-binding loops of these alphacoronaviruses possess conformational plasticity. the observation that hcov-229e and prcov bind to different sites on apn has important consequences. among species, apn is found in open/intermediate and closed conformations and conversion between them is thought to be important for the catalysis of its substrates 39, 42 . the hcov-229e rbd binds to hapn in its closed conformation and structural comparison shows that the h-site does not differ between the open and closed conformations. this is to be contrasted with the p-site of papn that differs in the open and closed conformations. indeed, the prcov rbd has recently been shown to bind to papn in the open conformation as a result of p-site interactions made possible in the open form 42 . these differences in binding and receptor conformation are reflected in the fact that enzyme inhibitors that promote the closed conformation of apn block tgev infection 42 , but not hcov-229e infection 8 , and the fact that the prcov s-protein 42 , but not hcov-229e 43 , inhibits apn catalytic activity. the receptor-binding loops of hcov-229e vary extensively. sequence data from viruses isolated over the past 50 years provides a wealth of data on the natural variation shown by hcov-229e ( supplementary fig. 5 ). with reference to the hcov-229e rbd-hapn complex reported here, we now show that 73% of the amino acids in the receptor-binding loops and supporting residues vary among the sequences analyzed (52 sequences in total), while only 11% of the rbd surface residues outside of the receptor-binding loops show variation (fig. 4a, b) . moreover, for the eight variants where full genome sequences were reported, the receptor-binding loops represent the location at which the greatest variation in the entire genome is observed (fig. 4c) . analysis of the hcov-229e rbd-hapn interface further shows that of the 16 rbd surface residues that are fully or partially buried on complex formation, 10 of them vary in at least one of the 52 sequences analyzed and a pairwise comparison of the sequences suggests that many of these positions can vary simultaneously ( supplementary fig. 5 ). finally, we show that the six invariant interface residues on the rbd (gly 313 , gly 315 , cys 317 , cys 320 , asn 319 , and arg 359 ) constitute only 45% of the viral surface area buried, the very region expected to be the most highly conserved from a receptor-binding standpoint. the fig. 4 naturally occurring hcov-229e sequence variation. a color-coded amino-acid sequence conservation index (chimera) mapped onto a ribbon representation of the hcov-229e rbd. blue represents a high percentage sequence identity and red represents a low percentage sequence identity among the 52 viral isolates analyzed. b surface representation in the same orientation as in (a, left), and rotated 180°(right). the asn-glcnac moiety of the nglycans are shown in stick representation. color coding as in a. c amino-acid sequence variation shown by the eight viral isolates whose entire genome sequences have been reported. the entire protein coding region of the viral genome was treated as a continuous amino acid string (8850 residues in total). amino acid differences among the eight sequences were analyzed in 100 residue bins and for each bin the sum was plotted. green-colored bins correspond to residues in the s-protein and purple-colored bins correspond to residues in the rbd. the horizontal dotted line denotes the average number of aminoacid differences per bin across the protein-coding region of the whole viral genome. d alignment of the sequences selected for each of the six classes. the "|" symbol demarcates every 10 residues in the alignment. e representative images showing hcov-229e infection of l-132 cells in the presence of: pbs, monoclonal antibody 9.8.e12 at two different concentrations, and monoclonal antibody 2.8h5 at two different concentrations (anti-hcov-oc43 antibody). the nucleus is stained blue and green staining indicates viral infection. magnification (×200) and scale bar = 10 µm. f statistical quantification of the monoclonal antibody inhibition experiment. error bars correspond to standard deviations obtained from three independent experiments remaining 55% (i.e., 279 å 2 ) of the viral surface area buried is made up of 10 residues that differ in their variability and the role they play in complex formation (supplementary table 2 ). loop variation leads to phylogenetic classes. phylogenetic analysis of the hcov-229e rbd sequences found in the database showed that they segregate into six classes ( supplementary fig. 6 ). class i contains the atcc-740 reference strain (originally isolated in 1967 and deposited in 1973) and related lab strains, while classes ii-vi, represent clinical isolates that have successively replaced each other in the human population over time since the 1970s. to characterize these classes, a representative sequence from each was selected; for class i, the rbd of the reference strain, also used in our structural analysis, was selected. to simplify characterization, the rbds of the other five classes were synthesized with the class i sequence in all but the loop regions (fig. 4d) . as observed for class i, the other rbds do not bind to the hapn mutant that introduces an n-glycan at glu 291 (supplementary fig. 4d) , an observation suggesting that they all bind at the same site on hapn. the rbds bound hapn with añ 16-fold range in affinity (k d from~30 to~440 nm). these differences in affinity are largely a result of differences in k off with little difference in k on (table 3 and supplementary fig. 7) . table 2 shows the identity of the loop residues that have shown variation. of those buried in the rbd-hapn interface, residues 314, 404, and 407 are particularly noteworthy as they undergo considerable variation in amino-acid character. residue 314, for example, accounts for 9% of the total buried surface area on complex formation and changes from gly to val to pro in the transition from classes i to vi. variation of this sort provides insight into how changes in receptor-binding affinity might be mediated during the process of viral adaptation. each of the six rbd classes were also characterized using a neutralizing mouse monoclonal antibody (9.8e12) that we generated against the hcov-229e reference strain (class i). as shown in fig. 4e, f, 9 .8e12 inhibits hcov-229e infection of the l132 cell-line. this antibody binds to the class i rbd with a k d of 66 nm (k on = 6.3 × 10 5 m −1 s −1 , k off = 0.041 s −1 ) and as shown by a competition binding experiment, it blocks the rbd-hapn interaction ( supplementary fig. 8a, b) . in contrast, 9.8e12 shows no binding to the other five rbd classes at a concentration of 1 μm (supplementary fig. 8c ), strong evidence that the receptorbinding loops of the class i rbd are important for antibody binding and that loop variation can abrogate antibody binding. consistent with this observation, non-conserved amino-acid changes both within and outside of the rbd-hapn interface are observed across all classes (supplementary table 2 ). correlating structure and function with natural sequence data is a powerful means of studying viral adaptation and evolution. to this end, we have delimited the hcov-229e rbd and determined its x-ray structure in complex with the ectodomain of its receptor, hapn. we found that three extended loops on the rbd are solely responsible for receptor binding, and that these loops are highly variable among viruses isolated over the past 50 years. a phylogenetic analysis also showed that the rbds of these viruses define six rbd classes whose viruses have successively replaced each other in the human population. the six rbds differ in their receptor-binding affinity and their ability to be bound by a neutralizing antibody (9.8e12) and taken together, our findings suggest that the hcov-229e sequence variation observed arose through adaptation and selection. antibodies that block receptor binding are a common route to viral neutralization and exposed loops are known to be particularly immunogenic 44 . loop-binding neutralizing antibodies are elicited by the alphacoronavirus tgev 40 , and the receptorbinding loops of hcov-229e mediate the binding of the neutralizing antibody, 9.8e12. as shown by the sequences of the viral isolates analyzed, the rbds differ almost exclusively in their receptor-binding loops. 9.8e12 blocks the hapn-rbd interaction and it can only bind to the rbd (class i) found in the virus that elicited it. this observation shows that loop variability can abrogate neutralizing antibody binding. indeed, the successive replacement or ladder-like phylogeny observed, when the sequence of the hcov-229e rbd is analyzed, is characteristic of immune escape as shown by the influenza virus 45, 46 . taken together, our results suggest that immune evasion contributes to if not explains the extensive receptor-binding loop variation shown by hcov-229e over the past 50 years. hcov-229e infection in humans does not provide protection against different isolates 37 , and viruses that contain a new rbd class that cannot be bound by the existing repertoire of loop-binding neutralizing antibodies provide an explanation for this observation. neutralizing antibodies that block receptor binding can also be thwarted by an increase in the affinity/avidity between the virus and its host receptor. increased receptor-binding affinity/avidity allows the virus to more effectively compete with receptor blocking neutralizing antibodies, a mechanism thought to be important for evading a polyclonal antibody response 47 . in addition, an optimal receptor binding affinity is thought to exist in a given environment. as such, adaptation in a new species, changes in tissue tropism, and differences in receptor expression levels can all lead to changes in receptor binding affinity 29, 31, 48 . recent cryoem analysis has shown that the receptor-binding sites of hcov-nl63, sars-cov, mers-cov, and by inference hcov-229e, are inaccessible in some conformations of the prefusion s-protein trimer [21] [22] [23] [24] [25] . although the ramifications of this structural arrangement are not yet clear, restricting access to the binding site has been proposed to provide a means of limiting bcell receptor interactions against the receptor-binding site 23 . how this might work in mechanistic terms is also not clear given the need to bind receptor. however, in a simple model, the inaccessible s-protein conformation(s) would be in equilibrium with a less stable (higher energy) but accessible s-protein conformation (s). the energy difference between these conformations is a barrier to binding that decreases equally the intrinsic free energy of binding of both the viral receptor and the b-cell receptor and relative binding energies may be the key. both soluble hapn and values after ± correspond to the residual standard deviation reported by scrubber 2. two experiments were performed nature communications | doi: 10.1038/s41467-017-01706-x article antibody 9.8e12 can inhibit hcov-229e infection in a cell-based assay, an indication that their binding energies (k d of 430 and 66 nm, respectively) are sufficient to efficiently overcome the barrier to binding. however, b-cell receptors bind their antigens relatively weakly prior to affinity maturation 49 and they would be much less able to do so. the dynamics of the interconversion between accessible and inaccessible conformations may also be a factor in the recognition of inaccessible antibody epitopes 50, 51 , and further work will be required to establish if and how restricting access to the receptor binding site enhances coronavirus fitness. the cryoem structures also show that the receptor-binding loops make intra-and inter-subunit contacts in the inaccessible prefusion trimer. this suggests the intriguing possibility that the magnitude of the energy barrier, or the dynamics of the interconversion between accessible and inaccessible conformations, might be modulated by loop variation during viral adaption. immune evasion and cross-species transmission involve viral adaptation and we posit that the use of extended loops for receptor binding represents a strategy employed by hcov-229e and the alphacoronaviruses to mediate the process. such loops can tolerate insertions, deletions, and amino acid substitutions relatively free of the energetic penalties associated with the mutation of other protein structural elements. indeed, our analysis of the six rbd classes shows that the receptor-binding loops possess a remarkable ability to both accommodate and accumulate mutational change while maintaining receptor binding. among the six classes, 73% of the loop residues show change and only 45% of the receptor interface buried on receptor binding has been conserved. as we have shown, variation in the receptorbinding loops can abrogate neutralizing antibody binding and it will also increase the likelihood of acquiring new receptor interactions by chance. in this way, the selection of viral variants capable of immune evasion and/or cross-species transmission will be facilitated 27, 28, [52] [53] [54] . cross-species transmission involves the acquisition of either a conserved (i.e., a similar interaction with a homologous receptor) or a non-conserved receptor interaction (i.e., an interaction with a non-homologous receptor, or an interaction at a new site on a homologous receptor) in the new host. hcov-229e binds to a site on hapn that differs from the site where prcov 40 binds to papn (fig. 3a, b) , and hcov-nl63 is known to bind the nonhomologous receptor, ace2 55 . clearly, conserved receptor interactions have not accompanied the evolution of these alphacoronaviruses ( fig. 3d-g) . in mechanistic terms, receptor-binding loop variability and plasticity would facilitate the acquisition of both conserved and non-conserved receptor interactions. however, compared to conserved receptor interactions, the successful acquisition of non-conserved interactions would be expected to be relatively infrequent and more likely to require viral replication and mutation in the new host to optimize receptor-binding affinity. many coronaviruses have originated in bats 3, 4 and it is tempting to speculate that viral transmission between bats has facilitated the emergence of non-conserved receptor interactions. bats account for~20% of all mammalian species and they possess a unique ecology/biology that facilitates viral spread between them 56, 57 . moreover, the barriers to viral replication in a new host are lower among closely related species 58, 59 . it follows that the viral replication required to optimize non-conserved receptor interactions in the new host would be facilitated by transmission between closely related bat species. by a similar reasoning, the use of conserved receptor interactions requiring little optimization would facilitate large species jumps. several bat coronaviruses showing a high degree of sequence similarity with hcov-229e have recently been identified 60, 61 and an analysis of how they interact with bat apn will inform this discussion. predicting the emergence of new viral threats is an important aspect of public health planning 62 and our work suggests that rna viruses that use loops to bind their receptors should be viewed as a particular risk. rna viruses are best described as populations 34 , and extended loops-inherently capable of accommodating and accumulating mutational change-will enable populations with loop diversity. such populations will provide routes to escaping receptor loop-binding neutralizing antibodies, optimizing receptor-binding affinity, and acquiring new receptor interactions, interrelated processes that drive viral evolution and the emergence of new viral threats. protein expression and purification. the soluble ectodomain of hapn (residues 66-967) was expressed and purified from stably transfected hek293s gnt1-cells (atcc crl-3022) as described previously 39 . the various soluble forms of the hcov-229e s-protein were expressed and purified from stably transfected hek293s gnt1-cells for x-ray crystallography, and from hek293t (atcc crl-3216) and/or hek293f (invitrogen 51-0029) cells for cell-based and biochemical characterization, as described previously 63 . point mutations were generated using the infusion hd site-directed mutagenesis protocol (clontech). in all cases, the target proteins were secreted as n-terminal protein-a fusion proteins with a tobacco etch virus (tev) protease cleavage site following the protein-a tag. harvested media was concentrated 10-fold and purified by igg affinity chromatography (igg sepharose, ge). the bound proteins were liberated by on-column tev protease cleavage and further purified by anion exchange chromatography (hitrap q hp, ge). protein crystallization. the rbd of the s-protein of hcov-229e (residues 293-435) and the soluble ectodomain of hapn (residues 66-967) were mixed in a ratio of 1.2:1 (rbd:hapn) and the complex was purified by superdex 200 (ge) gel filtration chromatography in 10 mm hepes, 50 mm nacl, ph 7.4. the complex was concentrated in gel filtration buffer to 10 mg/ml for crystallization trials. crystals were obtained by the hanging drop method using a 1:1 mixture of stock protein and well solution containing 8% peg 8000, 1 mm gssg, 1 mm gsh, 5% glycerol, 1 µg/ml endo-β-n-acetylglucosaminidase a 64 and 100 mm mes, ph 6.5 at 298 k. crystals were typically harvested after 3 days and flash-frozen with well solution supplemented with 22.5% glycerol as cryoprotectant. data collection and structure determination. diffraction data were collected at the canadian light source, saskatoon, saskatchewan (beamline cmcf-08id-1) at a wavelength of 0.9795 å. data were merged, processed, and scaled using hkl2000 65 ; 5% of the data set was used for the calculation of r free . phases were obtained by molecular replacement using the human apn structure as a search model (pdb id: 4fyq) using phaser in phenix 66 . manual building of the hcov-229e rbd was performed using coot 67 . alternate rounds of manual rebuilding and automated refinement using phenix were performed. secondary structural restraints and torsion-angle non-crystallographic symmetry restraints between the three monomers in the asymmetric unit were employed. ramachandran analysis showed that 96% of the residues are in the most favored region, with 4% in the additionally allowed region. data collection and refinement statistics are found in table 2 . a stereo image of a portion of the electron density map in the hcov-229e-hapn interface is showed in supplementary fig. 9 . figures were generated using the program chimera 68 . buried surface calculations were performed using the pisa server. surface plasmon resonance binding assays. surface plasmon resonance (biacore) assays were performed on cm-5 dextran chips (ge) covalently coupled to the ligand via amine coupling. the running and injection buffers were matched and consisted of 150 mm nacl, 0.01% tween-20, 0.1 mg/ml bsa, and 10 mm hepes at ph 7.5. response unit (ru) values were measured as a function of analyte concentration at 298 k. kinetic analysis was performed using the global fitting feature of scrubber 2 (biologic software) assuming a 1:1 binding model. for experiments using hapn as a ligand, between 300 and 400 ru were coupled to the cm-5 dextran chips. for experiments using 9.8e12, 1900 ru was immobilized. viral inhibition assay. hcov-229e was originally obtained from the american type culture collection (atcc vr-740) and was produced in the human l132 cell line (atcc ccl5) which was grown in minimum essential medium alpha (mem-α) supplemented with 10% (v/v) fbs (paa). the l132 (1 × 10 5 ) cells were seeded on coverslips and grown overnight in mem-α supplemented with 10% (v/v) fbs. for inhibition assays in the presence of soluble hapn, wild-type hcov-229e (10 5.5 tcid 50 ) was pre-incubated with the fragment (residues 66-967) diluted in pbs for one hour at 37°c before being added to cells for 2 h at 33°c. for inhibition assays in the presence of the soluble sprotein fragments, the different fragments, diluted in pbs, were added to cells and kept at 4°c on ice for 1 h. medium was then removed and cells were inoculated with wild-type hcov-229e (10 5 tcid 50 ) for 2 h at 33°c. for both inhibition assays, after the 2-h incubation period, medium was replaced and cells were incubated at 33°c with fresh mem-α supplemented with 1% (v/v) fbs for 24 h before being analyzed by an immunofluorescence assay (ifa). cells on the coverslips were directly fixed with 4% paraformaldehyde (pfa 4%) in pbs for 30 min at room temperature and then transferred to pbs. cells were permeabilized in cold methanol (−20°c) for 5 min and then washed with pbs for viral antigen detection. the s-protein-specific monoclonal antibody, 5-11h.6, raised against hcov-229e (igg1, produced in our laboratory by standard hybridoma technology), was used in conjunction with an alexafluor-488-labeled mouse-specific goat antibody (life technologies a-21202), for viral antigen detection 69 . after three washes with pbs, cells were incubated for 5 min with dapi (sigma-aldrich) at 1 µg/ml to stain the nuclear dna. to determine the percentage of l-132 cells positive for the viral s-protein, 15 fields containing a total of 150-200 cells were counted, at a magnification of ×200 using a nikon eclipse e800 microscope, for each condition tested in three independent experiments. green fluorescent cells were counted as s-protein positive and expressed as a percentage of the total number of cells. statistical significance was estimated by the analysis of variance (anova) test and tukey's test post hoc. monoclonal antibodies (igg1, produced in our laboratory by standard hybridoma technology) raised against hcov-229e (9.8e12) or hcov-oc43 (2.8h5, negative control) that were found to be s-protein specific were tested in an infectivity neutralization assay. wild-type hcov-229e (10 5.5 tcid 50 ) was preincubated with the antibodies (1/100 of hybridoma supernatant) for 1 h at 37°c before being added to l-132 cells for 2 h at 33°c. cells were washed with pbs and incubated at 33°c with fresh mem-α supplemented with 1% fbs (v/v) for 18 h before being analyzed by an immunofluorescence assay (ifa). statistical significance was estimated by an anova test, followed by post hoc dunnett (twosided) analysis. comparative sequence analysis of hcov-229e viral isolates. the protein sequence of the hcov-229e p100e isolate rbd (residues 293-435) was used to perform a search of the non-redundant protein sequence database using blastp. 70 and the residue-specific sequence conservation index was mapped onto the surface of the rbd using the "render by conservation" tool in chimera 68 . percentage identity is mapped using a color scale with blue indicating 100% identity and red indicating 30% identity. the protein-coding regions of the eight sequences for which the entire genome were reported (genbank identifier numbers: nc_002645.1, jx503060.1, jx503061.1, kf514433.1, kf514430.1, kf514432.1, af304460.1, and ku291448.1) were aligned using muscle. the entire protein-coding region of the viral genome was treated as a continuous amino-acid string (8850 residues in total). protein residues that were not identical among the eight sequences were counted as a difference and plotted in 100 residue bins. the sequence aak32191.1 was chosen as the representative of class i and the loop sequences of abb90507.1, abb90514.1, abb90519.1, abb90523.1, and afr45554.1 were combined with the non-loop sequences of aak32191.1 to generate the rbds of classes (ii-vi), respectively. data availability. coordinates and structure factors for the hcov-229e rbd in complex with human apn were deposited in the protein data bank with pdb id: 6atk. the authors declare that all other data supporting the findings of this study are available within the article and its supplementary information files, or are available from the authors upon request. received: 29 may 2017 accepted: 6 october 2017 a decade after sars: strategies for controlling emerging coronaviruses epidemiology, genetic recombination, and pathogenesis of coronaviruses discovery of seven novel mammalian and avian coronaviruses in the genus 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stability, and pathogenesis conformational dynamics of single hiv-1 envelope trimers on the surface of native virions hiv-1 fitness cost associated with escape from the vrc01 class of cd4 binding site neutralizing antibodies spread of mutant middle east respiratory syndrome coronavirus with reduced affinity to human cd26 during the south korean outbreak escape from human monoclonal antibody neutralization affects in vitro and in vivo fitness of severe acute respiratory syndrome coronavirus crystal structure of nl63 respiratory coronavirus receptorbinding domain complexed with its human receptor bats as "special" reservoirs for emerging zoonotic pathogens bats as viral reservoirs host phylogeny constrains cross-species emergence and establishment of rabies virus in bats jumping species-a mechanism for coronavirus persistence and survival evidence for an ancestral association of human coronavirus 229e with bats surveillance of bat coronaviruses in kenya identifies relatives of human coronaviruses nl63 and 229e and their recombination history what can we predict about viral evolution and emergence? simple piggybac transposon-based mammalian cell expression system for inducible protein production synthesis of neoglycoenzymes with homogeneous n-linked oligosaccharides using immobilized endo-beta-n-acetylglucosaminidase a processing of x-ray diffraction data collected in oscillation mode phenix: a comprehensive python-based system for macromolecular structure solution coot: model-building tools for molecular graphics ucsf chimera--a visualization system for exploratory research and analysis persistent infection of human oligodendrocytic and neuroglial cell lines by human coronavirus 229e muscle: multiple sequence alignment with high accuracy and high throughput the work was supported by cihr operating grants to j.m.r. and p.j.t. and a canada research chair to p.j.t. the canadian light source is acknowledged for synchrotron data collection. supplementary information accompanies this paper at doi:10.1038/s41467-017-01706-x.competing interests: the authors declare no competing financial interests.reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. key: cord-314676-ndke9agh authors: gollapalli, pavan; b. s, sharath; rimac, hrvoje; patil, prakash; nalilu, suchetha kumari; kandagalla, shivanandha; shetty, praveenkumar title: pathway enrichment analysis of virus-host interactome and prioritization of novel compounds targeting the spike glycoprotein receptor binding domain–human angiotensin-converting enzyme 2 interface to combat sars-cov-2 date: 2020-11-04 journal: journal of biomolecular structure & dynamics doi: 10.1080/07391102.2020.1841681 sha: doc_id: 314676 cord_uid: ndke9agh sars-cov-2 has become a pandemic causing a serious global health concern. the absence of effective drugs for treatment of the disease has caused its rapid spread on a global scale. similarly to the sars-cov, the sars-cov-2 is also involved in a complex interplay with the host cells. this infection is characterized by a diffused alveolar damage consistent with the acute respiratory disease syndrome (ards). to explore the complex mechanisms of the disease at the system level, we used a network medicine tools approach. the protein-protein interactions (ppis) between the sars-cov and the associated human cell proteins are crucial for the viral pathogenesis. since the cellular entry of sars-cov-2 is accomplished by binding of the spike glycoprotein binding domain (rbd) to the human angiotensin-converting enzyme 2 (hace2), a molecule that can bind to the spike rdb-hace2 interface could block the virus entry. here, we performed a virtual screening of 55 compounds to identify potential molecules that can bind to the spike glycoprotein and spike-ace2 complex interface. it was found that the compound ethyl 1-{3-[(2,4-dichlorobenzyl) carbamoyl]-1-ethyl-6-fluoro-4-oxo-1,4-dihydro-7-quinolinyl}-4-piperidine carboxylate (the s54 ligand) and ethyl 1-{3-[(2,4-dichlorobenzyl) carbamoyl]-1-ethyl-6-fluoro-4-oxo-1,4-dihydro-7-quinolinyl}-4 piperazine carboxylate (the s55 ligand) forms hydrophobic interactions with tyr41a, tyr505b and tyr553b, leu29a, phe495b, respectively of the spike glycoprotein, the hotspot residues in the spike glycoprotein rbd-hace2 binding interface. furthermore, molecular dynamics simulations and free energy calculations using the mm-gbsa method showed that the s54 ligand is a stronger binder than a known sars-cov spike inhibitor ssaa09e3 (n-(9,10-dioxo-9, 10-dihydroanthracen-2-yl) benzamide). communicated by ramaswamy h. sarma covid-19 is a coronavirus disease caused by the severe acute respiratory syndrome coronavirus-2 (sars-cov-2). the antiviral therapeutics acting on the virus exhibit various modes of action, like disabling the viral rna synthesis and virus replication or blocking virus attachment to the host cell receptors (angiotensin-converting enzyme 2, ace2) or to the viral structural proteins in order to inhibit the viral selfassembly process (canrong et al., 2020) . the host-sars-cov interaction is established through various strategies and various host cellular mechanisms are utilized by the virus for its successful multiplication during the infection (zumla et al., 2020) . the mechanism of the viral infection can be elucidated systematically by identifying the protein-protein interactions (ppis) during the virus-host interplay (yang et al., 2019; chuang et al., 2019) . viruses induce malfunction of the host cell by mimicking interacting domains of the host proteins, thus manipulating the signalling networks and cellular responses for their benefit (pawson & warner, 2007) . in such a way, by interacting with the host proteins, viruses (e.g. sars-cov-2) alter host responses at a systems level. one of the most effective ways to develop a potential drug for sars-cov-2 within a short period of time is by repurposing the existing compounds. recently, several compounds with anti-covid 19 properties were identified by this approach. combination therapy was found to be more efficient in combating certain viruses like hiv and coronavirus and hence the synergistic effect of lopinavir, oseltamivir, and ritonavir was used against the sars-cov-2 protease (mpro). a better insight into the interaction between these three drugs and mpro was obtained by performing molecular docking and molecular dynamic simulations (nisha muralidharan et al., 2020) . a comparative analysis of sars-cov-2 mpro with proteases of other viruses from the coronoviridea family and further virtual screening of phytochemicals and active ingredients of ayurvedic anti-tussive medicines in india, and the synthetic anti-viral drugs revealed several potential sars-cov-2 mpro inhibitors, such as delta d-viniferin, myricitrin, chrysanthemin, myristicin, taiwanhomoflavone a, lactucopicrin 15-oxalate, nympholide a, afzelin, biorobin, hesperidin and phyllaemblicin b. these molecules showed an equally strong binding to other sars-cov-2 targets, e.g. rdrp and hace-2 (joshi et al., 2020) . recent studies using molecular dynamic (md) simulations of isothymol-ace2 docked complex revealed that isothymol is a functional inhibitor of ace2 activity and the components of ammoides verticillata essential oils can be used as potential inhibitors of the ace2 receptor-sars-cov-2 interaction (abdelli et al., 2020) . in silico studies on the binding affinity of a truncated ace2 (tace2) for spike glycoprotein rbd by protein-protein docking and md simulations demonstrated that the tace2 has a high binding affinity for the rbd when compared to the intact ace2 and thus forms a more stable complex (basit et al., 2020) . drugs that can interfere with the sars-cov-2 rbd binding to human ace2 (hace2) can potentially prevent sars-cov-2 from entering human cells. nine short peptides that have this potential were designed by liu et al. (2020) and md simulations of the free peptides and their sars-cov-2 rbd-bound forms showed a high binding affinity of peptides to sars-cov-2 spike glycoprotein (lupala et al., 2020) . in the present work, we employed computational approaches to model protein-protein interactions of the host-virus complex and functional enrichment and pathway analysis of the gene/protein set was performed. as was already said, the virus entry into the host cell is initiated by its binding to human ace2 via the receptor-binding domain (rbd) of the spike glycoprotein and hence serves as a potential drug target (lupala et al., 2020) . therefore, the genes/ proteins which are the first neighbours of the spike glycoprotein in the interaction network were used to gain mechanistic insights into the virus-host interplay. this information was then used for the virtual screening of a small library of compounds against the spike glycoprotein rbd. the top hit molecules from this screening were then docked to the sars-cov-2 spike glycoprotein rbd-ace2 interface, after which molecular dynamic simulations of the top scored compound and a reference ligand were performed to compare their binding affinities. the search tool for the retrieval of interacting genes/ proteins database specific for viral-host interactions (stringvirus v11.0) was used to construct the network of the human-sars coronavirus protein-protein interactions (cook et al., 2018) . given the set of viral proteins, the stringvirus database generates a ppi network between the query proteins and their associated human proteins, with emphasis on primary interactions. the sars-cov-2 shares a high nucleotide sequence identity of 79.7% with the human sars-cov . hence, human protein data associated with the sars-cov were used here to construct the protein-protein interaction network. first, based on the virus seed proteins, an interaction network was constructed associated with the human proteins. these interactions were derived based on different sources: text mining, experiments, databases, co-expression, neighbourhood, gene fusion, and cooccurrence with a mean confidence level of 0.4. later, the number of interactions was increased to 200. cystoscope 3.3.0 (su et al., 2014) with default settings was used for the network visualization to analyse and calculate the properties of the nodes. several topological measures, i.e. degree (k), betweenness centrality (bc), eccentricity, closeness centrality (cc), network density, diameter, average number of clusters, average shortest path length, and clustering coefficient were adopted to evaluate nodes of the ppi network (albert & barab asi, 2002; barabasi and oltvai, 2004) . these topological parameters were calculated using the networkanalyzer (fienner et al., 2013) . the input and output values of the node are received as mathematical functions (jeanquartier et al., 2015) . a comprehensive analysis and visualization of a functionally enriched set of genes was performed using cluego (bindea et al., 2009 ), a cytoscape plug-in that significantly improves the biological interpretation of large lists of genes. a functionally organized go/pathway term network was created by integrating gene ontology (go) terms as well as kegg pathways. we considered the first neighbours of the hub spike glycoprotein for the functional enrichment analysis. a total of 76 neighbours were found to interact with the spike glycoprotein. parameters specified for protein/gene list enrichment analysis were set as follows: statistical testenrichment/depletion (two-side hypergeometric test), correlation test-bonferroni step down, min go level-3, max go level-8, kappa score threshold-0.4, go fusion-false, go group-true and p 0.05. the drug-like compounds were collected from the zinc15 database (http://zinc15.docking.org) (sterling & irwin, 2015) , by using the search term 'spike glycoprotein'. the structures of all the compounds were obtained in smiles format. the three-dimensional (3 d) conformation of compounds was protonated at the physiological ph and biologically relevant tautomers were generated for each molecule. a known inhibitor of the sars-cov spike-glycoprotein, ssaa09e3 (n-(9,10-dioxo-9,10-dihydroanthracen-2-yl) benzamide), which prevents the fusion of the viral membrane with the host cellular membrane and blocks the interaction of the sars-spike glycoprotein with the ace2 receptor (adedeji et al., 2013) was taken as the reference molecule. the 3d structure of the coronavirus spike glycoprotein receptor-binding domain (rbd) complexed with the ace2 receptor (pdb entry: 6lzg; resolution: 2.50 å) was obtained from the rcsb protein data bank (wrapp et al., 2020; berman et al., 2000) . both the receptor and the ligand molecules were prepared for docking using the ucsf chimera 1.14 program (pettersen et al., 2004) . initial docking calculations were performed using autodock vina (adt) (trott & olson, 2009) with a modified python script on ubuntu 16.04 lts platform. to detect the probable binding sites for all ligands with the spike glycoprotein rbd (chain b) and hace2 (chain a), we employed a blind docking procedure for both chains separately. thereafter, we opted for the spike glycoprotein rbd active pocket sites rather than the hace2 receptor because hace2 is expressed in various types of human cells and targeting hace2 might cause more side effects. before docking, the hace2 domain (chain a) was deleted from the original pdb complex (6lzg). additionally, ligand and water molecules were removed from the structure, polar hydrogen atoms and gasteiger charges were added. all 55 ligand structures collected from the zinc15 database (sterling & irwin, 2015) and the reference ssaa09e3 ligand were imported into ucsf chimera 1.14 as smiles strings, after which their structure was optimized using openbabel 2.3.2 (o'boyle et al., 2011). as a final step before docking, receptor and ligand molecules were saved in the pdbqt format using mgl 1.5.6 of autodocktools (adt) (morris et al., 2009) . initially, the spike glycoprotein rbd-hace2 complex was used to obtain the interface residues by using pdbsum (laskowski et al., 2018) . a grid map of size 16 â 16 â 16 å was generated with a 1.0 å spacing to cover the interface area (centred at à34.655, 30.216, 0.971). for initial compound screening, both exhaustiveness and the number of binding modes were set to 10. docking calculations were first performed for the spike glycoprotein rbd (chain b). the top five molecules underwent a second round of docking, with the exhaustiveness parameter set to 100 for a better conformation search. docking was conducted for both the spike glycoprotein rbd alone (chain b) and the spike glycoprotein rbd-ace2 complex (at the interface). top hit molecules were then analysed and visualized using maestro 12.3 (schr€ odinger release 2020) and chimerax (goddard et al., 2018) . md simulations for all ligands (the reference ssaa09e3 ligand and the s54, s5, s21, s43, and s55 ligands) were run in complex with the sars-cov-2 spike glycoprotein (6lzg). docked positions with the highest affinities to the protein were used as starting points for the md simulations. amber ff14sb force field (maier et al., 2015) was used to model the enzyme and gaff2 force field (as implemented in antechamber (wang et al., 2006) , was used in the case of ligands. such protein-ligand complexes were solvated in a truncated octahedral box of tip3p water molecules spanning a 12 å thick buffer, and na þ and clions were added according to machado and pantano (2020) to achieve a neutral environment with a salt concentration of 0.15 m (with the number of water molecules 41647, na þ ions 124, and clions 99 in the case of the standard ssaa09e3 ligand, number of water molecules 41633, na þ ions 124, and clions 99 in the case of the s54 ligand, number of water molecules 41650, na þ ions 124, and clions 100 in the case of the s5 and s43 ligands, number of water molecules 41659, na þ ions 124, and clions 100 in the case of the s21 ligand, and number of water molecules 41634, na þ ions 124, and clions 99 in the case of the s55 ligand). such structures were then submitted for geometry optimization in the amber16 program (case et al., 2014) , employing periodic boundary conditions in all directions. for the first 1500 cycles, the complex was restrained and only water molecules were optimized, after which another 2500 cycles of optimization followed where both water molecules and the complex were unrestrained. optimized systems were gradually heated from 0 to 300 k and equilibrated during 30 ps using nvt conditions, followed by productive and unconstrained md simulations of 300 ns employing a time step of 2 fs at constant pressure (1 atm) and temperature (300 k), the latter held constant using langevin thermostat with a collision frequency of 1 ps à1 . bonds involving hydrogen atoms were constrained using the shake algorithm (ryckaert et al., 1977) , while the long-range electrostatic interactions were calculated employing the particle mesh ewald method (darden et al., 1993) . the non-bonded interactions were truncated at 11.0 å. analysis of the trajectories was performed using the cpptraj module of ambertools16 (roe & cheatham, 2013 ). the binding energy, dg bind , of simulated complexes was calculated using the mm-gbsa (molecular mechanics -generalized born surface area) protocol (genheden & ryde, 2015; hou et al., 2011) , available as a part of ambertools16 (case, 2016) . mm-gbsa is a method for the calculation of dg bind from snapshots of md trajectory (ferenczy, 2015) with an estimated standard error of 1-3 kcal/mol (genheden & ryde, 2015) . dg bind is calculated in the following manner: where the symbol < > represents the average value over 100 snapshots collected from a 30 ns part of the corresponding md trajectories. the whole trajectory was divided into 10 parts of 30 ns length and dg bind was calculated for all 10 parts of the simulation and reported as mean ± standard deviation. the calculated mm-gbsa binding free energies were decomposed into specific residue contribution on a per-residue basis according to established procedures. this protocol calculates the contributions to dg bind arising from each amino acid side chains and identifies the nature of the energy change in terms of interaction and solvation energies, or entropic contributions (gohlke et al., 2003; rastelli et al., 2010) . in this case, the entropy term was not calculated. identification of potential metabolic sites of a drug can give key information of its pharmacokinetic and pharmacodynamic characteristics. drugs are commonly metabolized by a special class of enzymes which are known as cytochrome p450 (cyp) enzymes. in this concern, by using the smartcyp 3.0 tool (rydberg et al., 2019) , metabolic sites for cyp mediated metabolism were predicted for the top hit molecule. the protein-protein interaction was constructed by assembling the sars-cov associated human proteins using the stringvirus database. based on various experimentally collected data, we obtained nearly 360 human proteins associated with sars-cov (supplementary material, table 1 ). it was also found that these proteins are involved in crucial pathways of the viral infection. the core part (the core network) of the human sars-cov-host ppi network (the giant network) generated by using the string database consisted of 374 nodes and 5827 edges ( figure 1 ). the number of edges connected to a designated node is termed a degree, implying the significance of the protein in the biological interactions. the highest degree in the core network was found to be 43, while the average degree was 15.6. the ppi network is characterized by a small number of highly connected nodes, while most of the nodes have only a few connections. the nodes which degrees or bc are in the top 5% were considered as the key nodes, i.e. the critical points. out of 374 nodes in the network, the top 10 nodes with the highest bc values were: the spike glycoprotein, acvr1b, cd44, alb, myc, b2m, creb1, phb2, stat3, and il6. these include both the viral and the human proteins, with the spike glycoprotein identified as the hub node that was further validated as an important target protein. to distinguish these nodes in the network and their roles, they are highlighted in a different colour (figure 1, supplementary material) . the spike glycoprotein was identified as the hub protein with the highest degree and the second highest bc value, while acvr1b is the second hub protein with the highest bc value and the second highest degree. the proteins which are directly interacting (first neighbours) with the spike glycoprotein are shown in figure 2 (supplementary material). the sars-cov spike glycoprotein, identified as the key protein, is involved in binding to the ace2 receptor, a human cell receptor, through its receptor-binding domain (rbd). rbd-up conformation of the spike glycoprotein is a prerequisite for the formation of the rbd-ace2 complex (walls et al., 2020) . a drastic conformational change is found to be triggered in the s2 domain of spike glycoprotein due to the specific interaction between the receptor-binding domain and ace2 receptor, which leads to the viral fusion with the cellular membrane and the nucleocapsid release into the cytoplasm of the host cell (lan et al., 2020) . the second identified hub protein (acvr1b) in the interactome is a transmembrane serine/threonine kinase activin type-1 receptor. network analyzer v.3.3.1 was employed to evaluate the confidence of the core interactome, using the power law fit of the form y ¼ ax b : power law uses the least square method to determine the topological parameters and considers the points with positive coordinate values for the fit. the betweenness centrality (bc), closeness centrality (cc), and topology correlation coefficient scores of 0.770, 0.321, and 0.250, respectively, were considered as network topology parameters. additionally, the neighbourhood connectivity (0.472) and the shortest path length distribution were also considered in the analysis. the hub proteins in the interactome were determined by using network topology parameters like bc and topological correlation coefficient with a cut-off value of 0.7 and 0.6, respectively. these topological parameters considered for the network generation by using the above cut-off values were graphically plotted ( figure 3a -d, supplementary material). the extended global network topological measures of the two protein-protein interaction networks, i.e. the giant or the core network and the backbone or the subnetwork, are presented in table 1 . therefore, the biological process is essentially regulated by the bottleneck node in the interactome ( functional enrichment analysis was carried out using cluego, a plug-in for cytoscape. a total of 41 go terms were collected, out of which 29, 6, and 6 go terms corresponded to biological processes (figure 2(a) ), molecular function (figure 2(b) ) and pathways (figure 2(c) ), respectively (listed in the supplementary material, tables 3-5). genes related to the specific go terms are presented in figure 3 . the protein interaction network from stringvirus showed a dense network of the spike glycoprotein with 76 first neighbour nodes. we carried out a functional enrichment analysis of these protein interactions, which showed that the proteins in the network play a major role in biological processes related to the viral entry into the host cell (go:0046718), the heterophilic cell-cell adhesion via plasma membrane cell adhesion molecules (go: 0007157), the transition metal ion homeostasis (go: 0055076), the natural killer cell-mediated immunity (go:0042267), the glycogen catabolic process (go: 0005980) and the regulation of humoral immune response mediated by circulating immunoglobulins (go: 0002923), as well as molecular functions, such as the viral receptor activity (go: 0001618), the mhc protein binding (go:0042287), the phosphorylase kinase activity (go:0004689) and the mannose binding (go:0048029). enriched pathways (reactome) included immunoregulatory interactions between a lymphoid and a non-lymphoid cell, dap12 interactions, glycogenolysis, and the complement cascade. the go term related to the reactions is reported as the genes involved in exocytosis of tertiary granule membrane proteins (r-has-6798747, clec15a, clec5a, and ola1). the interaction of sars-cov spike glycoprotein with cellular receptors is indispensable for the viral entry into the host cells. from these enriched go terms, we have identified the genes associated with the viral entry into the host cell and the viral receptor activity: cd209, cd55, clec4m, clec5a, and clecag. the clec4m (c-type lectin domain family 4 member m) acts as an attachment receptor for ars-cov (marzi et al., 2004) . it was demonstrated that both the ace2 and clec4m (cd2009) are highly expressed in human lung microvascular endothelial cells and lymphatic endothelial cells, respectively (jing et al., 2007) . the ace2 and other proteins are associated with clec4m through a primary interaction and act as a receptor for binding of the viral spike glycoprotein (figures 2 and 3) . several studies have demonstrated that the interaction between the spike glycoprotein and the ace2 receptor is found to be crucial for the viral entry into the host and thus targeting this mechanism and identifying the inhibitors to interrupt this interaction could result with promising lead compounds (zumla et al., 2016) . recent reports showed that the sars-cov-2 spike glycoprotein uses the ace2 receptor to enter the host cell and that the rbds of sars-cov-2 and sars-cov spike glycoproteins bind with similar affinity to the human ace2 receptor (walls et al., 2020) . the ligand-binding specificity of the spike glycoprotein rbd was detected by running blind docking and setting both the exhaustiveness and the number of modes to 10. using blind docking, we analysed the cavity at the spike glycoprotein rbd-hace2 interface. the results suggest that the majority of ligands' best scored poses were found in the a and c pockets. additionally, most of the ligands bind at the interface (the c pocket) and have higher binding affinities compared to the a pocket ( figure 4) . therefore, targeting this position may contribute to the interruption of the interaction between the spike glycoprotein rbd and the hace2. due to its high ranking and position advantages, we used this cavity (the c pocket) for further studies under the assumption that targeting this region may induce conformational changes that could inhibit virus-host interactions and prevent viral entry. among the 55 compounds identified from the zinc15 database, binding energies for most of the ligands were found to be between à6 and à7 kcal/mol (46 ligands). one ligand (s28) showed a lower affinity with the binding energy of à5.8 kcal/mol and eight ligands showed binding energies of à7 kcal/mol or lower (supplementary material, table 6 ). among them, the top 5 ligands were selected based on the interaction pattern with the spike glycoprotein rbd (chain b) for the second round of the docking by setting the exhaustiveness parameter to 100. analysis of the top 5 molecules interacting with the spike glycoprotein alone showed a good inhibition potential. the s05 molecule forms two h-bonds, with asn501b and gly496b of spike glycoprotein and has a binding energy of à7.9 kcal/mol. similarly, calculated binding energies for the s54 and s55 ligands were à8.4 and à7.9 kcal/mol, respectively. however, they form only one favourable h-bond interaction, with gly498b and asn501b, respectively. on the other hand, the other three molecules form no h-bonds with the spike glycoprotein rbd (table 2) . after this, we merged the hace2 domain with the spike glycoprotein rbd-s54 complex, and found a hydrophobic interaction between spike glycoprotein rbd and tyr41a of figure 3 . functional assessment analysis of the spike glycoprotein first neighbour proteins. the genes recognized as close neighbours of the spike glycoprotein are highlighted in different colours based on their functional enrichment: genes for the viral entry into the host cell and the viral receptor activity are shown in green, genes for the heterophilic cell-cell adhesion via plasma membrane cell adhesion molecules in grey, genes for the transition metal ion homeostasis in light blue, genes for the natural killer cell-mediated immunity in dark blue, genes for the glycogen catabolic process in red, genes for the regulation of humoral immune response mediated by circulating immunoglobulin in pink, genes for the mhc protein binding in sky blue, and genes for the immunoregulatory interactions between a lymphoid and a non-lymphoid cell in yellow. the genes shown in the subnetwork (orange) show the interaction of clec4m protein with the human ace2 protein (receptor for the spike glycoprotein), and the genes which are involved in the viral entry into the host. the hace2 domain (yan et al., 2020) . the binding analysis of the s54 ligand compared to the reference ssaa093 ligand is shown in figure 5 . the results suggest that the h-bond interaction with the asn501b residue of the spike glycoprotein may play a key role in destabilizing hace2 (chain a) by forming hydrophobic interactions with the tyr41a residue since tyr41a was found to play a crucial role in the interaction of the spike glycoprotein rbd-hace2 complex formation (lan et al., 2020; yan et al., 2020) . these results encouraged us to investigate the complicated role of the spike glycoprotein rbd-hace2 complex in virus entry into the cells. to focus on the role of amino acid residues asn501b and tyr41a, we performed a docking simulation of the top 5 ligands at the interface of the spike glycoprotein rbd-hace2 complex (amino acid residues were taken from pdbsum) by setting exhaustiveness to 100 (supplementary material, table 7 and figure 4) . these calculations showed significant h-bond interactions of all 5 ligands and the reference ssaa093 ligand with amino acids his34a, arg403b, asp30a, asn33a, and gly496b at the interface. equally, all 5 ligands form hydrophobic interactions with amino acids tyr505b, tyr495b, tyr453b, pro389a, ala387a, val93a, and leu29a. the binding modes of s54 and the reference ssaa093 ligand at the interface of spike-ace2 are shown in figure 6 . s55, which has a similar fingerprint as s54, forms two hbond interactions with asp30a and gly496b and forms favourable hydrophobic interactions with val93a, leu29a, tyr495b, phe497b, and tyr505b. tyr505b, tyr495b, and tyr453b were also found to play a key role in forming interaction with the spike glycoprotein alone. interestingly, robetta alanine scanning (kortemme et al., 2004) results also replicated the docking results by confirming the mutagenic amino acid residue tyr41a (hace2) with a ddg complex score of 4.88 kcal/mol and tyr505b (spike glycoprotein) with ddg complex score of 1.54 kcal/mol. the docking calculations at the spike glycoprotein rbd-hace2 interface showed an even better binding energy and formation of h-bond interactions, and the detailed interaction analysis for the top 5 ligands is reported in table 2 . among the top 5 ligands, s54 and s55 were found to show good binding energies, hbond interactions, hydrophobic interactions, as well as alanine scanning by forming favourable interaction with tyr41a and tyr505b. therefore, apart from h-bond interacting amino acids asn501b, asp30a, asn33a, gly496b, the hydrophobic residues tyr41a and tyr505b were also found to be important hotspots responsible for the binding of the spike glycoprotein to the hace2 domain. interestingly, s54 forms interactions with hotspot residues tyr41a and tyr505b along with the h-bond interactions at the interface of the spike glycoprotein rbd (gly496b) and the hace2 domain (asp30a, asn33a). these observations clearly explain the main reason behind the tighter affinity of the sars-cov-2 spike glycoprotein to hace2 when compared to that of sars-cov (masters, 2006) . our results suggest that s54 is a potent inhibitor of both spike glycoprotein alone, as well as the spike glycoprotein rbd-hace2 complex. structure-based drug design showed that the interaction of s54 at the interface of the spike-hace2 involves forming 3 hydrogen bonds and favourable hydrophobic interactions, which play a major role in destabilizing the spike-hace2 interaction, thus inhibiting viral entry into human cells. the top 5 molecules were subjected to molecular fingerprinting analysis (fp) with maccs and morgan circular fingerprint method to check their similarity against all the 55 molecules collected form zinc15 database. the importance of the fp method selection for virtual screening was highlighted and the difference in results obtained by different fp approaches was analysed (cereto-massagu e et al., 2015; matsuyama & ishida, 2018) . the extended connectivity fingerprint diameter (ecfp) offers the highest precision on average, according to database search by compound similarity based on fp (riniker & landrum, 2013) . in our analysis, the compound s54 and s55 are structurally similar, with both having piperazine, benzene, and 1h-quinolin-4-one in the structure. the only difference between these two compounds is the presence of the nitrogen atom in the piperazine ring of the s55 compound, which makes the compound more rotatable. further, molecular fingerprinting analysis showed a similarity score of 0.79 between s54 and s55 in morgan circular fingerprint. other lead compounds such as s5, s21 and s43 also share common traits, such as the chloro-fluorobenzene functional group. the s5 compound along with chloro-fluorobenzene, contains also the 5-azaspiro [3.5] nonane functional group and the s21 compound contains pyrrolidine and cyclohexane rings along with the chloro-fluorobenzene group. finally, s43 compound contains a piperidine ring along with the chloro-fluorobenzene group. among these, the s43 compound shows the similarity of score 0.60 and 0.63 with s5 and s21, respectively (supplementary material, table 8 ). md simulations were carried out for all the top five compounds (s54, s5, s21, s43, and s55) and the reference ssaa093 ligand complexed with the spike glycoprotein. all complexes were found to be stable throughout the entire duration of the simulation (300 ns). however, since only the ligands s54 and s55 had dg bind significantly lower than the reference ssaa093 ligand, they will be discussed more thoroughly (the complete mm-gbsa results are shown in table 9 supplementary material). figure 7 shows the backbone mass-weighted root-meansquare deviation (rmsd) for the s54, s55 and the reference ssaa093 ligand complexes through time. it can be seen that all three complexes achieve the equilibrium state very early and remain stable for the entire duration of the simulation. for the ssaa093 and the s54 ligand, this can also be seen in the intermolecular h-bond graph (figure 8) , where the number of h-bonds for all three ligands remains constant through time (with the reference ssaa093 ligand forming on average 1.39 ± 0.62, ligand s54 1.33 ± 0.78), while the ligand s55 (1.42 ± 1.25 intermolecular h-bonds) undergoes a slight conformational change around the 150 ns mark, but without any significant influence on the protein structure. this indicates that the docking procedure was successful in finding the correct binding poses for the ssaa093 and the s54 ligands since these ligands did not need to optimize their conformation inside the binding pocket, while the s55 ligand had to go through some conformational changes to obtain the optimal pose. this is in accordance with the docking results, where the binding affinity of the s54 and s55 ligands are almost the same, while the mm-gbsa results differ. binding energies for all the complexes were calculated using the mm-gbsa protocol. since all the complexes are stable throughout the entire simulation, their entire trajectories (300 ns) were divided into ten segments of 30 ns. dg bind was calculated for all segments individually and the final dg bind was calculated as mean ± standard deviation. for the reference ssaa093 ligand dg bind ¼ à23.09 ± 6.01 kcal/mol, for the s54 ligand dg bind ¼ à32.13 ± 4.16 kcal/mol, and for the s55 ligand dg bind ¼ à44.55 ± 6.65 kcal/mol (table 3) . dg bind for ligands s5, s21, s43 are à20.85 ± 2.74 kcal/mol, à26.89 ± 3.31 kcal/mol, and à26.06 ± 1.33 kcal/mol, respectively (supplementary material, table 9 ). it has to be emphasized that since the entropy term was not calculated, these results are overestimated in their absolute terms (genheden & ryde,2015) . however, since all ligands bind to the same binding site of the same protein, the entropic contribution in both cases would also be approximately the same. therefore, this method can be used in predicting relative binding energies in biomolecular complexes and their comparison (homeyer & gohlke, 2012) . for this reason, the obtained dg bind of the tested compounds should only be analysed relative to each other. that being said, the mm-gbsa results are in accordance with the h-bonds analysis: the s55 ligand forms a slightly higher number of intermolecular h-bonds than the s54 ligand and has a slightly higher binding affinity, similar to the docking results. additional decomposition of dg bind (supplementary material, table 9 (a), 9 (b), and 9 (f)) show that throughout the simulation time, s54 and s55 ligands are more stable in their binding sites, with a lower standard deviation of all types of interactions, excluding the non-polar solvation interaction for the s54 ligand, and with significantly more favourable van der waals and electrostatic interactions. from table 3 it is also visible which amino acid residues contribute the most in the binding of the tested ligands. in the case of the reference ligand and the s54 ligand, the most contributing residue is pro389a, but for the s54 ligand, its contribution is significantly higher. additionally, the s54 ligand forms strong bonds with residues lys26a and thr92a, which are not present in the list of top 10 contributing residues in the case of the standard ssaa093 ligand. on the other hand, the standard ssaa093 ligand forms a much stronger bond with the his34a residue than the s54 ligand. however, even with these differences, the most important amino acid residues for these two complexes are more similar than compared to the complex with the s55 ligand. as opposed to them, in the case of the s55 ligand, eight of the ten most important amino acid residues come from the b chain, which is a result of its position deeper inside the interaction pocket (figures 9 and 10) . given that the structural difference between ligands s54 and s55 is only in one nitrogen atom (piperidine and piperazine rings, respectively), it goes to show how small structural changes can have a significant influence on ligand binding. an interesting observation can also be made when comparing the per residue root-mean-square fluctuation (rmsf) for all three complexes ( figure 11 ). while the a chain has practically the same rmsf in all three cases, rmsf for the chain b shows significant differences in residues 355-395. for all amino acid residues except ser375, rmsf is the lowest for the s55 ligand, followed by s54 ligand and the reference ligand, which corresponds to their decreasing binding affinities. it also seems figure 9 . overlay of the spike glycoprotein complexes with the reference ssaa093 ligand (tan), the s54 ligand (light blue), and the s55 ligand (pink) after 300 ns md simulation with top contributing amino acid residues (yellow) shown. figure 10 . binding interactions of the s54 and the s55 ligand at the interface of the spike glycoprotein rbd-hace2 complex after 300 ns md simulations. that the s55 ligand stabilizes the chain b the most, which is in accordance with the fact that, among the three ligands, it has the strongest interactions with it. however, this part of the chain b is not in vicinity of the binding pocket, so the exact stabilization mechanism remains unknown. all pharmacokinetic properties of s54 and s55 were performed using preadmet and molinspiration tool/online server (jan et al.,2020). the results are in the acceptable range and follow lipinski's rule of five (supplementary material, tables 10 and 11). additionally, the metabolic site analysis of s54 (zinc000043069833) and s55 (zinc000095551034) has predicted that it is not an inhibitor of cytochrome enzymes, but is a cyp 3a4 substrate with 4 putative metabolic sites: c17, c15, c12, c27 and c7, c8, c11, respectively which is depicted in supplementary material, figure 5a and 5b. the database smiles arbitrary target specification-based fragment was used by smartcyp in combination with an accessibility descriptor to obtain the ranking for the site of metabolism (soms) (hashem & mahrouse, 2018) . here, the primary site of cyp-mediated metabolism for the s54 ligand was predicted to be c17 and c15 of the amine group and c12 of the aryl fluoride group. similarly, the s55 ligand belongs to quinoline-3-carboximide class of organic compounds, where the quinoline ring is substituted by one carboxiamide group at the 3-position. these compounds were found to inhibit the nipah virus glycoprotein g/f-mediated cell-cell fusion expressed in african green monkey vero cells after 24 h relative to untreated control (niedermeier et al., 2009) . in this study, we adopted a systems biology method to construct an extended ppi network of sars-cov and associated human proteins. our findings suggest that the spike glycoprotein has the highest degree and the second-highest bc, and acvr1b has the second highest degree and the highest bc. the spike glycoprotein is mainly involved in binding to the human ace2 receptor, while human acvr1b is involved in the transmembrane receptor protein serine/threonine kinase signalling pathway. both proteins are essential in the viral entry and causing infection in humans. furthermore, studies on the sars-cov spike glycoprotein rbd inhibition with the top five ligands were successfully carried out using molecular dynamics approach. ligands s54 and s55 were found to be selectively interacting with the tyr41a and tyr505b hotspots inside the binding pocket via formation of an inclined tape over the binding site with the oh group. these results demonstrate the likelihood of the ethyl 1-f3[(2,4-dichlorobenzyl) carbamoyl]-1-ethyl-6-fluoro-4-oxo-1,4dihydro-7-quinolinylg-4 piperidine carboxylate (the s54 ligand) and ethyl 1-f3-[(2,4-dichlorobenzyl) carbamoyl]-1-ethyl-6-fluoro-4-oxo-1,4-dihydro-7-quinolinylg-4 piperazine carboxylate (the s55 ligand) activity to block the virus spike glycoprotein rbd from docking to hace2. the trajectory analysis of the spike rbd-hace2-s54/s55 complexes also displayed structural stability and lower binding free energy when compared to the complex with the reference ssaa093 ligand. however, these computationally validated results need to be investigated on in vivo models before classifying molecules as potential covid-19 inhibitors. authors provided critical feedback and helped shape the research, analysis and manuscript. no potential conflict of interest was reported by the author(s). in silico study the inhibition of angiotensin converting enzyme 2 receptor of covid-19 by ammoides verticillata components harvested from western algeria novel inhibitors of severe acute respiratory syndrome coronavirus entry that act by three distinct mechanisms statistical mechanics of complex networks network biology: understanding the cell's functional organization truncated human angiotensin converting enzyme 2; a potential inhibitor of sars-cov-2 spike glycoprotein and potent covid-19 therapeutic agent the protein data bank cluego: a cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks molecular fingerprint similarity search in virtual screening viruses.string: a virus-host protein-protein interaction database particle mesh ewald: an nálog(n) method for ewald sums in large systems computation of drug-binding thermodynamics the mm/pbsa and mm/gbsa methods to estimate ligand-binding affinities ucsf chimerax: meeting modern challenges in visualization and analysis insights into protein-protein binding by binding free energy calculation and free energy decomposition for the ras-raf and ras-ralgds complexes in vitro metabolism study of a novel p38 kinase inhibitor: in silico predictions, structure elucidation using ms/ms-i free energy calculations by the molecular mechanics poisson-boltzmann surface area method assessing the performance of the mm/pbsa and mm/gbsa methods. 1. the accuracy of binding free energy calculations based on molecular dynamics simulations integrated web visualizations for protein-protein interaction databases discovery of potential multi-target-directed ligands by targeting host-specific sars-cov-2 structurally conserved main protease computational alanine scanning of protein-protein interfaces structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor pdbsum: structural summaries of pdb entries computational network biology: data, models, and applications computational analysis on the ace2-derived peptides for neutralizing the ace2 binding to the spike protein of sars-cov-2 split the charge difference in two! a rule of thumb for adding proper amounts of ions in md simulations ff14sb: improving the accuracy of protein side chain and backbone parameters from ff99sb dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus the molecular biology of coronaviruses stacking multiple molecular fingerprints for improving ligand-based virtual screening autodock4 and autodocktools4: automated docking with selective receptor flexibility computational studies of drug repurposing and synergism of lopinavir, oseltamivir and ritonavir binding with sars-cov-2 protease against covid-19 open babel: an open chemical toolbox oncogenic re-wiring of cellular signaling pathways ucsf chimera-a visualization system for exploratory research and analysis fast and accurate predictions of binding free energies using mm-pbsa and mm-gbsa open-source platform to benchmark fingerprints for ligand-based virtual screening ptraj and cpptraj: software for processing and analysis of molecular dynamics trajectory data numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes smartcyp: a 2d method for prediction of cytochrome p450-mediated drug metabolism a small-molecule inhibitor of nipah virus envelope protein-mediated membrane fusion schr€ odinger release 2020-1: maestro (2020). schr€ odinger, llc zinc 15-ligand discovery for everyone biological network exploration with cytoscape 3 sars-cov-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading structure, function, and antigenicity of the sars-cov-2 spike glycoprotein automatic atom type and bond type perception in molecular mechanical calculations cryo-em structure of the 2019-ncov spike in the prefusion conformation structural basis for the recognition of sars-cov-2 by full-length human ace2 understanding human-virus protein-protein interactions using a human protein complex-based analysis framework a pneumonia outbreak associated with a new coronavirus of probable bat origin coronaviruses -drug discovery and therapeutic options the authors are thankful to registrar, nitte (deemed to be university), mangalore, india for providing all the facilities to complete this work. the authors also acknowledge the university of zagreb, university computing centre (srce) for granting computational time on the isabella cluster. . per residue root-mean-square fluctuation (rmsf) for complexes with the reference ssaa093, the s54 ligand, and the s55 ligand. key: cord-336150-l8w7xk0b authors: rathore, jitendra singh; ghosh, chaitali title: severe acute respiratory syndrome coronavirus-2 (sars-cov-2), a newly emerged pathogen: an overview date: 2020-08-25 journal: pathog dis doi: 10.1093/femspd/ftaa042 sha: doc_id: 336150 cord_uid: l8w7xk0b coronavirus disease 2019 (covid-19) is a viral pneumonia, responsible for the recent pandemic, and originated from wuhan, china, in december 2019. the causative agent of the outbreak was identified as coronavirus and designated as severe acute respiratory syndrome coronavirus 2 (sarscov-2). few years back, the severe acute respiratory syndrome coronavirus (sarscov) and the middle east respiratory syndrome coronavirus (mers-cov) were reported to be highly pathogenic and caused severe infections in humans. in the current situation sars-cov-2 has become the third highly pathogenic coronavirus that is responsible for the present outbreak in human population. at the time of this review, there were more than 14 007 791 confirmed covid-19 patients which associated with over 597 105 deaths in more then 216 countries across the globe (as reported by world health organization). in this review we have discussed about sars-cov, mers-cov and sarc-cov-2, their reservoirs, role of spike proteins and immunogenicity. we have also covered the diagnosis, therapeutics and vaccine status of sars-cov-2. on december 31, 2019, several cases of severe pneumonia were reported from wuhan, china. the causative agent of the outbreak was identified as betacoronavirus. genome sequencing revealed that it is closely related to the sars-cov (severe acute respiratory syndrome coronavirus) which had emerged in 2003, and is designated as sars-cov-2 (gorbalenya et al. 2020; zhou et al. 2020) . in a very short duration, more than 80 000 infectious cases including more than 3000 deaths were reported in china as on march 15, 2020. at the time of this review (18 may, 2020), the disease, termed as covid-19 (corona virus disease 2019), had already become pandemic and spread to more than 216 countries and territories, including community transmissions in countries like the united states, germany, france, spain, japan, singapore, south korea, iran, italy and india. as on july 19, more than 14 007 791 cases and 597 105 deaths had been reported globally, with the rapid growth of numbers in many countries. for the up-to-date information about covid-19, visit the world health organization (who) website (https://www.who.int/emer gencies/diseases/novel-coronavirus-2019). the bats are likely to be the origin of sars-cov-2, but the role of an intermediate host cannot be ruled out at this stage. initial studies showed that sars-cov-2, can use angiotensinconverting enzyme 2 (ace2) from bats, cats, civet cats, swine, ferrets, non-human primates (nhps) and humans as a receptor (letko, marzi and munster 2020; wan et al. 2020a; zhou et al. 2020) . a pet dog in hong kong and a tiger in bronx zoo in the united state of america tested positive with sars-cov-2 infection, indicating that canine ace2 can also be recognized by sars-cov-2. pangolins, which are endangered animals and are illegally imported into southern china (guangdong and guangxi provinces), have been considered as a potential intermediate host (lam et al. 2020; zhang et al. 2020b) . the initial reports showed that in most of the covid-19 cases there was mild to moderate infection. however, approximately 20% of the cases were reported severe (chen et al. 2020; wang et al. 2020a) . in this review, we will discuss about sars-cov, mers-cov and sarc-cov-2. we have also discussed about various reservoirs, associated with them. in the end, we have covered the role of spike proteins and their immunogenicity along with the diagnosis, therapeutics and vaccine status of sars-cov-2. zoonotic coronaviruses are becoming a global concern as there was emergence of earlier two coronaviruses, sars-cov and mers-cov (middle east respiratory syndrome coronavirus) which created a havoc and recently emergence of the third highly pathogenic sarc-cov-2. it has been observed that members of the family coronaviridae are known to infect a wide range of vertebrates and humans. before the outbreak of sars (severe acute respiratory syndrome), only two coronaviruses including hcov-229e and hcov-oc43 were known to infect humans. however, post-sars outbreak, the sars coronavirus (sars-cov), human coronavirus hcov-nl63, human coronavirus hcov-hku1 and mers-cov have been isolated from humans. similar to sars-cov and mers-cov, the newly isolated sarc-cov-2 is highly pathogenic in humans and causes severe acute respiratory distress (shi, guo and rottier 2016) . the genomes of coronaviruses consist of a positive and single-stranded rna genome of about 30 kb. the 5 terminus encodes the enzyme viral replicase/transcriptase, which is involved in virus replication, whereas the 3 terminus encodes viral structural proteins and virus group specific accessory proteins. functional studies of these viral proteins in detail are essential for antiviral drug screening and vaccine development. the earliest available genome sequencing data of sars-cov-2 made it possible to compare it with the genomes of sars-cov and other coronaviruses. results showed that sars-cov-2 belongs to the genus betacoronavirus and subgenus sarbecovirus, which also includes sars-cov. however, the mers-cov belongs to another subgenus, merbecovirus (lu et al. 2020; zhou et al. 2020) . the comparison study also showed that there is 79% nucleotide similarity between sars-cov-2 and sars-cov. the essential surface glycoprotein of sars-cov-2 known as spike (s) protein, essential for host cell receptor binding, showed only 72% similarity with sars-cov at the nucleotide level. the genomic organization of sars-cov-2 resembles those of other betacoronaviruses, including 5'-orf1ab-s (surface glycoprotein)-orf3a-e (envelope)-m (membrane glycoprotein)-n-3' as shown in fig. 1 . comparative genome analysis of ratg13, a virus from a rhinolophusaffinis (i.e. horseshoe) bat sampled from yunnan province in china in 2013, with sars-cov-2, showed that sars-cov-2 has 96% similarity at the nucleotide sequence level . although the sars-cov-2 and ratg13 have high similarity, yet they differ in some genomic features, such as sars-cov-2 contains a polybasic (furin) cleavage site insertion (residues prra) between the s1 and s2 subunits of the surface glycoprotein s protein (coutard et al. 2020) . polybasic insertion may increase the infectivity of the virus, as it is absent in other related betacoronaviruses. however, similar polybasic insertions are observed in different human coronaviruses, such as hcov-hku1 and highly virulent strains of avian influenza viruses. therefore, whether polybasic insertion between s1 and s2 subunits of s protein occurs due to the natural evolution in sars-cov-2 or by other means is going to be the topic of debate in the future. however, an independent insertion(s) of the amino acids paa at the s1/s2 cleavage site was also observed in armyn02 virus (having 72% similarity in spike protein and 97% similarity in replicase nucleotide) isolated from rhinolophus bat in yunnan province in mid-2019, indicating that these insertion events may be a natural part of ongoing coronavirus evolution . the receptor-binding domain (rbd) of s protein is essential to interact with the ace2 receptor present on the surface of the target cells of the host. therefore, comparative sequence analysis was performed and results showed that there is 85% similarity in rbd between sars-cov-2 and ratg13, but they share only one amino acid among the six key amino acid residues. further, due to the proteinaceous nature of spike, structural comparisons were also performed, suggesting that the rbd domain of the sars-cov-2 is well suited to interact with the human ace2 receptor. interestingly the same receptor was also utilized by sars-cov to cause infection (wrapp et al. 2020) . the sars-cov-2 is closely related to sars-cov and mers-cov having bat as reservoirs, but there are huge biological differences in the former as compared to the other two. the sars-cov-2 is markedly more infectious and has very different epidemiological dynamics. moreover, the mers-cov has never been able to fully adapt to human transmission (sabir et al. 2016) , whereas there is the remarkable local and global spread of sars-cov-2. as in the case of sars and mers, the intermediate host including civets and camels, respectively, played an important role and may be considered as a true reservoir host (sabir et al. 2016) . therefore, due to the ecological separation between a bat (reservoir) and humans, 'intermediate' or 'amplifying' mammalian host is a must to acquire mutations in sars-cov-2, and is essential for the efficient human transmission. to determine the intermediate host, it is essential to perform a wider sampling of animals that live close to human populations or available in wet markets for human consumption. surprisingly, there was discovery of viruses, closely related to sars-cov-2 from the malayan pangolins (manisjavanica) that are illegally imported into southern china (guangdong and guangxi provinces). it has been observed that rbd domain of guangdong pangolin viruses are particularly closely related to sars-cov-2. there is a 97% amino acid sequence similarity and contain all six critical key mutations that are essential for binding to the ace2 receptor in these viruses. however, the rest of the genome is highly divergent from sars-cov-2. hence, the evolution of coronaviruses in animal reservoirs as well as in intermediate hosts is required to explain the emergence of sars-cov-2 in humans. it might be possible that due to its asymptomatic infection, the virus could have acquired some of its essential mutations during a period the ''cryptic'' spread in humans before it was first detected in december 2019. recombination is another possibility, which cannot be ruled out as sarbeviruses, and coronaviruses experience widespread recombination. the genome of sarbeviruses experience recombination at multiple locations, including spike protein. there are studies, which showed that recombination does occur among sars-cov-2, ratg13 and the guangdong pangolin covs (lam et al. 2020) . the genome of rmyn02 too has been impacted by recombination . because of the small recombinant region, which may likely change as we increase the sample size of viruses related to sars-cov-2, it would be difficult to determine the pattern and genomic ancestry of recombination. a total of 8422 cases with 916 deaths in 29 countries including china were reported due to the human respiratory disease during 2002-2003, caused by the sars-cov. the studies showed that bats acted as a natural reservoir of sars-cov that caused the outbreaks (chan-yeung and xu 2003) . later, the sars-cov like similar antibody and genomic sequences were also discovered in rhinolophus bat, such as in r. ferrumequinum, r. pearsoni, r. sinicus, r. pusillus and r. macrotis (lau et al. 2005) . the comparative study of the genomes revealed that bat sars-like covs (sl-cov) have 78-92% nucleotide sequence identities with sars-cov and also among themselves, and hence display great genetic diversity. further, the phylogenetic analysis pointed out that rhinolophus bat might be the direct progenitor of human sars-cov (hon et al. 2008) . various sars-cov groups were isolated in different epidemic periods and hosts. several methods have been adopted to investigate the selective pressure. results have shown that the most functional proteins of sars-cov adopted the stepwise adaptive evolutionary pathway. for example, the spike protein showed strong positive selection in the early as well as middle phases, and not in the late phase. however, the replicase enzyme experienced positive selection only in humans, and assembly proteins experienced the same in the middle and late phases. interestingly, no such positive selection was observed in any proteins of bat sars-like-cov. however, specific amino acid sites that may be the targets of positive selection in each group were identified (tang et al. 2009 ). later in 2010, a study suggested the presence of two distinct genotypes of bt-slcov in r. sinicus (i.e. rp3/rs672 and hku3/rs806). the results also showed the evidence for the recombinant origin of rp3 and rs672. the phylogenetic study showed that their major parent has a relatively closer relationship with hu-scovs. therefore, there may be a possibility for the presence of a bt-scov lineage in r. sinicus, that may have hu-scovs as their direct ancestor, as reported earlier (hon et al. 2008) . however, these speculations are based on studies done on limited strains only, therefore an extensive analysis for the prevalence of such genotype is required for its credibility (yuan et al. 2010) . in 2012, globally, 64 human cases were confirmed resulting in 38 deaths by june 17, 2013, by a disease having symptoms similar to sars, that emerged in saudi arabia (who 2013). later, it was found that the disease was caused by a virus designated as a novel human coronavirus, mers-cov, phylogenetic data showed that it belonged to lineage c of the betacoronavirusgenus and was highly similar to bat coronaviruses hku4 (tylonycterispachypus) and hku5 (pipistrelluspipistrellus; lau et al. 2013) . comparative genomic results showed that mers-cov has a 50% nucleotide identity in the entire genome with hku4 and hku5. moreover, the rna dependent rna polymerase (rdrp) gene has 82% nucleotide identity. later, while studying the mode of entry into the cell it was confirmed that mers-cov uses dipeptidyl peptidase 4 (dppiv), also known as cd26. as dppiv is evolutionarily conserved among mammals, therefore mers-cov can infect a broad range of mammalian cells (humans, pigs, monkeys and bats) and may be efficient in cross-host transmission (raj et al. 2013) . similar to the case for sars-cov and mers-cov , the bat is still a probable species for origin of sars-cov-2, as it shares 96% whole-genome identity with a bat cov, batcov ratg13, from rhinolophusaffinis from yunnan province ). however, sars-cov and mers-cov before entering humans pass through intermediate hosts, such as civets or camels (cui, li and shi 2019) . this fact indicates that sars-cov-2 was probably transmitted to humans by other animals. by comparing the overall genome identity, it was concluded that pangolin-cov genome sequence is 90.55% identical to ratg13 and 91.02% identical to sars-cov-2. however, there was 96.2% identity between sars-cov-2 and ratg13 . other sar like covs (sl-covs) are also showed similarity with pangolin-cov, as its was 85.65% similar to zxc21 and 85.01% with zc45. in a comparative genome analysis between pangolin-cov and sars-cov-2 (genbank: mn908947), result showed 45.8-100% coverage range (average coverage 76.9%). moreover, pangolin-cov genes shared high average nucleotide (93.2%) and amino acid identity (94.1%) with sars-cov-2 (genbank mn908947). similar results were obtained when pangolin-cov genes were compared with ratg13 where 92.8% nucleotide and 93.5% amino acid identity was observed (zhang, wu and zhang 2020) . interestingly, some of the pangolin-cov genes showed higher amino acid sequence identity to sars-cov-2 genes than to ratg13genes. for example orf1b of pangolin-cov 73.4%, the spike (s) protein 97.5%, orf7a 96.9% and orf10 is 97.3% identical to sars-cov-2. similarly, orf1b 72.8%, the spike (s) protein 95.4%, orf7a 93.6% and orf10 is 94.6% identical to ratg13. the high s protein amino acid identity governs the functional similarity between sars-cov-2 and pangolin-cov. a comprehensive phylogenetic analysis was performed based on the nucleotide sequences of whole-genome sequence, rna-dependent rna polymerase gene (rdrp), non-structural protein genes orf1a and orf1b, and main structural proteins encoded by the s and m genes. results showed that in all phylogenetic trees, pangolin-cov, ratg13 and sars-cov-2 were clustered into a well-supported group designated as ''sars-cov-2 group'' which represents a novel betacoronavirus group. however, within this group, ratg13 and sars-cov-2 were grouped together, and pangolin-cov was their closest common ancestor (zhang, wu and zhang 2020) . recently, an extensive study including localized genomic analysis and the pattern of evolutionary recombination was done. the results showed that the strong purifying selection among coronaviruses from distinct host species as well as cross-species infections is responsible for the origin of sars cov-2 ). therefore, we may summarize the origin and intermediate hosts of sars-cov, mers-cov and sars-cov-2 as shown in fig. 2 . in the s1 domain of s protein, followed by fusion with cell membrane. sars-cov is responsible to cause severe acute respiratory syndrome. sars-cov utilizes angiotensin-converting enzyme 2 (ace2) receptor present on the surface of host cells, as shown in fig. 3 . sarc like-covs and sars-covs have identical genetic organizations with high sequence identities. the schematic representation of spike protein (s) from sars-cov-2 is shown in fig. 4 . however, there is some important exception at the n' terminus of spike protein (s), essential for receptor binding in covs. there is a study to investigate the receptor usage by full-length s of sl-cov, sars-cov and a series of s chimeras. different ace2 receptors from human, civet, or horseshoe bat were expressed in cell lines by using human immunodeficiency virus-based pseudovirus system. several important observations were made in the study. first, the sl-cov s was unable to use any of the three receptors. second, the sars-cov s was unable to enter the cells expressing bat ace2. third, the chimeric s enters the cells with different efficiencies for different constructs via human ace2. fourth, a minimal insert region (amino acids 310-518) was sufficient to convert the sl-cov s from non-ace2 binding to human ace2 binding, indicating that the sl-cov s is largely compatible with sars-cov s protein, both in structure and function (ren et al. 2008) . detailed structural study of human sars-cov rbd complexed with human ace2 receptors was performed. results revealed that it is the truncations in the receptor-binding motif (rbm) region of sl-cov spike protein, which abolished its human ace2-binding ability (li 2008) ). therefore, we may hypothesize that the sl-cov found in horseshoe bats is not the direct ancestor of human sars-cov. moreover, it has been observed that the human sars-cov, as well as its closely related civet sars-cov spike proteins, were not able to use a horseshoe bat (r. pearsoni) ace2 as a receptor for cell entry (ren et al. 2008) . these findings highlight a critical missing link (an intermediate host) in the bat-to-civet/human transmission chain of sars-cov (hou et al. 2010 ). an earlier study showed that ace2 from horseshoe bat could not function as a receptor for sars-cov. however, changing 3 amino acids (40, 41 and 42 amino acids) from she to fyq was found adequate to convert the nonfunctional bat ace2 into a fully active receptor for sars-cov. further, an ace2 molecule from a fruit bat, which naturally has the fyq motif, supports sars-cov entry into the cells thus causing infection. this result indicates that there must be a wide host range for sars-cov-related viruses among different bat populations (yuan et al. 2010) . in the case of sars-cov-2, the structural bioinformatics approaches accurately predicted that sars-cov-2 spikes bind human ace2 (wan et al. 2020b ). when cell lines over-expressed the transmembrane protein 'angiotensin-converting enzyme 2' (ace2) from humans, bats, pig or civet cats and were infected with sars-cov-2, results showed that they became hypersensitized to infection, thus indicating that ace2 is a sars-cov-2 receptor . the binding studies also revealed that receptor-binding domains on the sars-cov-2 s proteins have a high affinity to human ace2 (wrapp et al. 2020 ) which makes it more virulent. however, apart from ace2 interaction, the n-terminal domain (ntd) of the sars-cov-2 s proteins may show binding to alternative host-cell receptors . sars-cov-2 s proteins have also acquired a furin protease cleavage site, by acquiring several basic residues (rrar/s). the sars-cov-2 furin substrate site facilitates the prime cleavage step, which further sensitizes s proteins for subsequent activation of cleavages occurring on susceptible target cells, and finally facilitates virus to enter the cells and cause infection (qing and gallagher 2020) . human sars-cov and sars-like coronavirus (sl-cov) in bats have a similar genomic organization; therefore their corresponding gene products are highly conserved. as far as s protein is concerned, it has only a 63-64% sequence identity at the n-terminal region. it is the n-terminal region of coronavirus s protein that is responsible for receptor interaction. when the immunogenicity of the sl-cov s protein was analyzed and compared with that of sars-cov, results revealed that they shared only a limited number of immunogenic epitopes in their s proteins. moreover, major neutralization epitopes were also different ). in another study, a pseudovirus expressing full-length sl-cov s protein was used to raise mouse sera and monoclonal antibody. series of constructs expressing truncated s protein were prepared and analyzed with elisa, as well as western blot. results showed that amino acids 280-455 and amino acids 561-666 are two immunogenic determinants in mice. further, it was also shown that 280-455 amino acids are more immunogenic, as it was recognized by polyclonal as well as monoclonal antibodies. earlier studies also showed that amino acids 528-635 from sars-cov are immunodominant determinants (he et al. 2004 ). due to the high sequence similarity with sl-cov s protein in the same region, the amino acids 561-666 of s protein also demonstrated immune response in mouse (zhou et al. 2013 ). in a cross-reactivity test with antibodies against rbd domain sars-cov, some of the sl-cov strains (wivi) have shown positive results whereas some strains (shc014) failed too. this difference in reactivity is due to the low sequence identity in the rbd domain of shc014 and high sequence identity in the rbd domain of wivi with rbd domain of sars-cov (zeng et al. 2017 ). detection of novel coronavirus is done by different molecular biology techniques including real-time reverse transcription pcr (rrt-pcr), reverse transcription pcr (rt-pcr), reverse transcription loop-mediated isothermal amplification (rt-lamp), multiplex nucleic acid amplification, real-time rt-lamp and microarray-based assays . who also recommended a pan-coronavirus assay for characterization and confirmation.). viral culture and rt-pcr are among the most efficient and reliable methods for the diagnosis of sars-cov-2 infection. these methods are time consuming and generally takes hours to detect the nucleic acid and many days to isolate the virus from the samples. apart from that, specialized equipments and expertise are also required. to overcome these limitations, rapid diagnosis of sars-cov-2 infection can be done with rapid antigen detection (rad) tests. in rad tests, the immobilized sars-cov-2 antibody on the device can detect viral antigen in the sample. the results of rad tests are prompt and interpreted without specialized instrument. hence, rad tests could be beneficial reduce the workload in diagnostic laboratories and hospitals (mak et al. 2020) . however, as per the who, rad tests for sars â�� cov-2 antigen detection, further needs evaluation and is not recommended for clinical diagnosis (laboratory testing strategy recommendations for covid-19: interim guidance 2020). the immune response to sars-cov2 in the early weeks of the infection can be detected using enzyme-linked immunosorbant assay (elisa), automated chemiluminescence immunoassay (clia), and lateral flow immunoassay (lfia), plaque reduction neutralization tests (prnt), or a combination of these methods (espejo et al. 2020) . the most commonly antigens used in these assays were the spike glycoprotein s1 including the receptor binding domain (jin et al. 2020 ), the nucleocapsid protein or both (pang et al. 2020) . application of inhibitor to halt virus interactions with the host may be one of the prophylactic methods. in this direction, an engineered pan-cov fusion inhibitor has been designed and designated as ek1 peptide. it has shown promising results in mice by inhibiting the infection in five human coronaviruses, including sars-cov, mers-cov and three bat-sl-covs (xia et al. 2019) . it has also been reported that intranasal application of engineered ek1 peptide before or post viral infection showed protection in human dpp4-transgenic mice against mers-cov infection, indicating its potential prophylactic and therapeutic effect. another approach is designing of neutralizing antibody, which may block the interaction with the host cell. the s proteins of sars-cov and mers-cov are immunogenic. the rbd domains of sars-cov and mers-cov are known to have nonsequential epitopes that induce a more potent neutralizing antibody and give protection against sars-cov and mers-cov (du et al. 2009; zhou et al. 2019) . the modification on the structural basis for mers-cov s-rbd amino acid has improved the efficacy against mers-cov infection (zhou et al. 2019) . therefore, we may suggest that sars-cov-2 s-rbd or modified s-rbd of another related coronavirus could be used as target to develop a vaccine against sars-cov-2. recently, neutralizing monoclonal antibodies and nanobodies against the rbd domain of s protein showed protection against sars-cov and mers-cov (du et al. 2009; zhou et al. 2019) . although the ntd and s2 unit of s protein from sars-cov and/or mers-cov was also studied to develop neutralizing antibodies, but the efficacy was found to be very low (du et al. 2009 ). therefore, rbd of s protein sars-cov-2 would be a key target for developing neutralizing antibodies as shown in fig. 5 . cross protection by the antibodies developed against sars-cov, has been observed against bat-sl-cov-w1v1 and bat-sl-cov-shc014 (zeng et al. 2017) . therefore, the development of cross-neutralizing antibodies can be another possible way for urgent prevention and treatment of sars-cov-2 infection. currently, plasma therapy in which polyclonal antibodies from recovered sars-cov-2-infected patients have been used to treat sars-cov-2 infection is also being considered. researchers are working hard to develop monoclonal abs (mabs) and once such antibodies are produced, the next step will involve in vitro testing for neutralizing and/or crossneutralizing activity as well as in vivo evaluation for protective efficacy in available covid-19 animal models. preclinical and clinical trials testing the safety and and efficacy before they are approved for clinical applications are also necessary. recently, 1000 memory b cells specific to sars-cov-2 s1 or rbd (receptor binding domain) have been purified. among these, 178 antibodies showed positive results in antigen binding assays with the top 17 binders having ec 50 below 1 nm specific for rbd. further, among 11 neutralizing antibodies, eight of them have shown an ic 50 value within 10 nm, whereas 414-1 best among all have ic 50 of 1.75 nm. in epitope mapping, three main epitopes recognized by monoclonal antibodies have been identified in rbd domain. interestingly, 515-5 monoclonal antibody from same study, also showed cross-neutralizing property in the sars-cov pseudovirus assay (wan et al. 2020a) . in another study, 61 sars-cov-2-neutralizing monoclonal antibodies were isolated from five infected patients. 19 among them have shown positive result in in vitro neutralization assay and nine among them shown 50% virus-inhibition at the concentrations of 1-9 ng/ml. epitope mapping showed that receptor-binding domain (rbd) and the n-terminal domain (ntd), both are immunogenic in nature. further, structural studies of these monoloclonal antibodies have proven that one is targeting rbd, second one is targeting ntd and a third bridging rbd and ntd. therefore, several of these monoclonal antibodies are promising candidates for clinical development as potential therapeutic and/or prophylactic agents against sars-cov-2 (l et al. 2020). due to the high sequence identity of s protein between sars-cov-2 and its closely related sars-cov , sars-cov nabs have been tested for its cross-reactivity and/or cross-neutralizing activity against sars-cov-2 infection. interestingly, a sars-cov rbd-specific human neutralizing mab, cr3022, have shown the binding of sars-cov-2 rbd with high affinity and may recognize an epitope on the rbd that does not overlap with the ace2-binding site (tian et al. 2020) . further, sars-cov-2 entry and infection may be blocked by crossreacting the sera isolated by convalescent sars patients or from animals specific for sars-covs1 (hoffmann et al. 2020) . moreover, it has been observed that polyclonal antibodies against the rbd domain of sars-cov have been cross-reacted with the rbd protein of sars-cov-2. they cross-neutralized sars-cov-2 infection in hek293t cells expressing the human ace2 receptor. such findings may open new avenues for the potential development of sars-cov rbd-based vaccines that might eventually prevent sars-cov-2 and sars-cov infection (tai et al. 2020) . it may be possible that sars-cov rbd-targeting neutralizing antibodies could be applied for treatment/prophylaxis of sars-cov-2 infection in the current absence of a specific vaccine against sars-cov-2. remdesivir has been recently recognized as a promising antiviral drug in cultured cells, mice and nonhuman primate (nhp) models, against rna viruses including sars-cov and mers-cov (sheahan et al. 2017) . it is currently under clinical trials for the treatment of ebola virus infection (mulangu et al. 2019) . recently studies have shown that ec 90 value of remdesivir against 2019-ncov in vero e6 cells was 1.76 î¼m. these data suggest that its working concentration is likely to be achieved in nhps remdesivir have shown the efficient in vitro antiviral activity against sars-cov-2. however, the controversial evidence of clinical improvement in severe covid-19 patients has been reported recently in france. the five covid-19 patients admitted in icu and treated with remdesivir. treatment showed significant reduction of sars-cov-2 viral load from upper respiratory in most of the cases, however but two patients died with active sars-cov-2 replication in their lower respiratory tract. remdesivir treatment was interrupted for its side effects among four patients due the complexity in such critically ill patients (dubert et al. 2020 ). the first covid-19 case in the united states was intravenously treated with remdesivir (iv) (holshue et al. 2020) . within 24 h of remdesivir treatment, the patient showed recovery sign. as the viral loads was decreasing before remdesivir treatment, therefore it cannot be determined if further viral load reduction and clinical improvement were as the direct result of remdesivir treatment. in another study, the compassionate use of remdesivir (n = 53) reported 68% improved oxygenation, 47% discharge and 13% death. this study was not most significantly as it lacks of a paired control group (grein et al. 2020) . recent study at the national institutes of health (nih), showed preliminary results of the adaptive covid-19 treatment trial (actt, n = 1063). in this randomized controlled trial (rct), remdesivir treatment showed 31% faster time to recovery as comparative to the placebo group (p < 0.001). the mortality rate was also showed reduction in remdesivir group, however it was not statistically significant (8% vs. 11.6%, p = 0.059). so far, remdesivir has not shown any significant benefit in the reduction of mortality rate. currently, remdesivir is recommended by the nih for hospitalized severe covid-19 cases as defined by oxygenation needs (clinical management of covid-19). another drug, like chloroquine (c), has recently been reported as a potential broad-spectrum antiviral drug (savarino et al. 2006) . it inhibits the virus infection by increasing endosomal ph, which is essential for virus/cell fusion, as well as by interfering with the glycosylation of cellular receptors of sars-cov (vincent et al. 2005) . recent studies demonstrated that chloroquine is effective at both entry, as well as at postentry stages of the sars-cov-2 infection in vero e6 cells . apart from antiviral activity, chloroquine also has immune-modulating activity. therefore, it may synergistically enhance antiviral effect in vivo. chloroquine gets widely distributed in the whole body after oral administration, including lungs. the ec 90 value of chloroquinein vero e6 cells against the sars-cov-2 was 6.90î¼m, therefore it could be clinically achievable . the effect of hydroxychloroquine (hcq) and chloroquine (cq) in vitro was also tested by yao et al. in a systematic way, they had divided the experiment into two phases: treatment study and prophylaxis. in the treatment study, they determined the ec 50 values for chloroquine. results showed that it was 23.90 î¼m and 5.47 î¼m at 24 and 48 h, respectively. however, in the case ofhydroxychloroquine,the ec 50 values were 6.14 î¼m and 0.72 î¼m at 24 and 48 h, respectively. on the other hand in the prophylaxis study for chloroquine, the ec 50 values were more than100 î¼m and 18.01 î¼m at 24 and 48 h, respectively. similarly, for hydroxychloroquine, the ec 50 values were 6.25 î¼m and 5.85 î¼m at 24 and 48 h, respectively (yao et al. 2020) . hence, they found that hydroxychloroquine is more effective in vitro than chloroquine for both prophylaxis and treatment. a study in united states, where covid-19 patients hospitalized within 24 h of diagnosis was treated with hydroxychloroquine alone (hcq) or with hydroxychloroquine and azithromycin (hcq + azm) or no hcq as treatments. among patients, there was no significant reduction in mortality rate or in the need of ventilation with hydroxychloroquine alone or with hydroxychloroquine and azithromycin (magagnoli et al. 2020) . a new york hospital stated the qtc prolongation associated with hcq + azm (n = 84; chorin et al. 2020) . it was amplified from a baseline of 435 â± 24 ms to a maximal value of 463 â± 32 ms (p < 0.001) on day 3.6 â± 1.6 of the treatment. till date, researchers present conflicting data's related to the treatment with cq and hcq. therefore, significant randomized control tests (rcts) with improved study designs are required to examine the efficacy and the clinical benefits of hcq/cq treatment over its risks. currently, the nih recommendation are against cq/hcq and hcq + azm treatment for covid-19, except for clinical trials. due to the potential toxicity, nih recommendations are also against the high-dose of cq (600 mg) twice daily for 10 days in all settings (coronavirus disease 2019 (covid-19) treatment guidelines). nitazoxanide has demonstrated potent in vitro activity against sars cov-2, with an ec 50 at 48 h of 2.12 î¼m in vero e6 cells . this potent activity is consistent with ec 50 values for nitazoxanide and its active metabolite, tizoxanide, against mers-cov in llc-mk2 cells where ec 50 values of 0.92 î¼m and 0.83 î¼m respectively, have been demonstrated (rossignol 2016) . dexamethasone, a synthetic glucocorticoid, has antiinflammatory and immunosuppressive properties. there is a hyper inflammatory response involved in the clinical course of patients with pneumonia due to sars-cov-2. the elevated level of c-reactive protein (crp) in sars-cov-2 patients has significantly decreased from 129.52 to 40.73 mg/l at time of discharge. 71% of the patients were discharged home with a mean length of stay of 7.8 days. none of the patients had escalation of care, leading to mechanical ventilation (selvaraj et al. 2020) recently, a randomized, controlled clinical trial in the united kingdom save the lives of people seriously ill with covid-19 when treated with dexamethasone. results showed the reduction of number of death by one-third (ledford 2020) . dexamethasone may be useful for the short-term in severe sars-cov-2 patients as it inhibit the protective function of t cells and block b cells from making antibodies (theoharides and conti 2020). the development of successful vaccine for humans can take several years. as no coronavirus vaccines are available in the market as of now, therefore, the development of a vaccine for the first time can be difficult and time-consuming. however, a mrna-based vaccine has been co-developed by moderna and the vaccine research center at the national institutes of health. in this vaccine, the target antigen's mrna, encapsulated in lipid nanoparticles are injected into vaccinee and antigen expresses in vivo. the phase i clinical trial has been recently started (clini-caltrials.gov: nct04283461). curevac is also using the same platform but they are still in the pre-clinical phase. apart from this, there are various other approaches including dna vaccine, recombinant protein-based vaccine, recombinant vector vaccine, inactivated vaccine and attenuated vaccine. different research companies, universities and institutes are targeting s protein of sars-cov-2 to develop recombinant proteinbased vaccines including novavaxexpress2ion, ibio, sichuan clover biopharmaceutical, baylor college of medicine and the university of queensland. similarly, cansino biologics, geovax vaxart and the university of oxford are using viral-vector-based vaccine platform especially focused on the s protein. applied dna sciences and inovio are using dna vaccines platform again focused on the s protein (amanat fatima 2020). apart from the above-mentioned platforms, the whole microorganism based vaccine platform like inactivated and attenuated virus vaccine is also in consideration. codagenix with the serum institute of india is using live attenuated vaccine platforms. the recombinant vector-based platform (adenovirus vector) is adopted by johnson and johnson, and on the other hand, sanofi is also using the same platform (recombinant influenza vector) to develop a vaccine against sars-cov-2. at this stage, it is difficult to predict the best platform for a vaccine against sars-cov-2 as all the above mentioned platforms have some advantages as well as disadvantages (amanat fatima 2020). coronaviruses have shown the capability to jump species boundaries and adapt to new hosts. therefore, we may face more such kind of outbreaks in the future. role of the intermediate host is also of major importance, as they provide direct pathway for virus transmission in humans. the enormous diversity of viruses in animals and their ongoing evolution makes it important to limit our exposure to animal pathogens as much as possible. based on the metagenomic data it is predicted that the pangolin-cov is most closely related to sars-cov-2. pangolin-cov genome showed 91.02% nucleotide identity with the sars-cov-2 genome. due to a very limited knowledge of this novel virus, it is difficult to explain the significant number of amino acid substitutions that occurred between the sars-cov-2 and sars or sl-covs. for example, in sars-s-cov, six mutations occurred in the regions other than that of the rbd domain, but interestingly no amino acid substitutions were present in the receptor-binding motifs that directly interact with human receptor ace2 protein. therefore, such differences that could affect sars-cov-2 transmission property as compared to sars-cov are of importance for future investigation. sars-cov-2 continues to infect people globally; therefore it is imperative to develop new, safe, accurate, fast and simple new technologies for detecting sars-cov-2. apart from diagnosis, effective prophylactic and therapeutic agents are also required to control and prevent infection. various therapeutic agents including dexamethasone have shown promising results in the in vitro studies to control infection. however, there is an urgency to develop vaccine against coronaviruses. in this direction, studies on neutralizing antibodies from sars-cov and mers-cov against s protein and its many fragments including s1-ntd, rbd and s2 may provide important guidelines for development of vaccine against sars-cov-2. apart from neutralizing antibody against s protein, other approaches including dna vaccine, recombinant vector vaccine, inactivated vaccine and attenuated vaccine are also in pipeline to develop vaccines against sars-cov-2. so far, the traditional public health measures including detection of active cases, isolation of such cases, tracing of all contacts and their quarantine, maintaining social distancing, as well as community quarantine were found to be successful. only after this pandemic ends, we will be in a position to assess the social, health and economic impact of such a massive outbreak. therefore, we must learn lessons for our future from such outbreaks, as new viruses will keep coming. none declared. sars-cov-2 vaccines: status report epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study the qt interval in patients with covid-19 treated with hydroxychloroquine and azithromycin clinical management of covid-19 covid-19) 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respiratory syndrome (sars)-like coronavirus and its implications on the direct ancestor of sars coronavirus angiotensin-converting enzyme 2 (ace2) proteins of different bat species confer variable susceptibility to sars-cov entry diagnostic value and dynamic variance of serum antibody in coronavirus disease 2019 laboratory testing strategy recommendations for covid-19: interim guidance identification of 2019-ncov related coronaviruses in malayan pangolins in southern china genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats coronavirus breakthrough: dexamethasone is first drug shown to save lives functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses structural analysis of major species barriers between humans and palm civets for severe acute respiratory syndrome coronavirus infections potent neutralizing monoclonal antibodies directed to multiple epitopes on the sars-cov-2 spike bats are natural reservoirs of sars-like coronaviruses emergence of sars-cov-2 through recombination and strong purifying selection genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding outcomes of hydroxychloroquine usage in united states veterans hospitalized with covid-19 evaluation of rapid antigen test for detection of sars-cov-2 virus a randomized, controlled trial of ebola virus disease therapeutics potential rapid diagnostics, vaccine and therapeutics for 2019 novel coronavirus (2019-ncov): a systematic review dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc difference in receptor usage between severe acute respiratory syndrome (sars) coronavirus and sars-like cronavirus of bat origin nitazoxanide, a new drug candidate for the treatment of middle east respiratory syndrome coronavirus co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia new insights into the antiviral effects of chloroquine short-term corticosteroids in sars-cov2 patients: hospitalists' perspective broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses coronavirus: epidemiology, genome replication and the interactions with their hosts characterization of the receptorbinding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine differential stepwise evolution of sars coronavirus functional proteins in different host species dexamethasone for covid-19? not so fast potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody chloroquine is a potent inhibitor of sars coronavirus infection and spread remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro human igg neutralizing monoclonal antibodies block sars-cov-2 infection receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus who| novel coronavirus summary and literature update -as of 17 cryo-em structure of the 2019-ncov spike in the prefusion conformation a pan-coronavirus fusion inhibitor targeting the hr1 domain of human coronavirus spike in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) intraspecies diversity of sars-like coronaviruses in rhinolophus sinicus and its implications for the origin of sars coronaviruses in humans cross-neutralization of sars coronavirus-specific antibodies against bat sars-like coronaviruses recent advances in the detection of respiratory virus infection in humans probable pangolin origin of sars-cov-2 associated with the covid-19 outbreak identification of immunogenic determinants of the spike protein of sars-like coronavirus immunogenicity difference between the sars coronavirus and the bat sars-like coronavirus spike (s) proteins a pneumonia outbreak associated with a new coronavirus of probable bat origin advances in mers-cov vaccines and therapeutics based on the receptor-binding domain key: cord-306438-db2rqz4d authors: kalathiya, umesh; padariya, monikaben; mayordomo, marcos; lisowska, małgorzata; nicholson, judith; singh, ashita; baginski, maciej; fahraeus, robin; carragher, neil; ball, kathryn; haas, juergen; daniels, alison; hupp, ted r.; alfaro, javier antonio title: highly conserved homotrimer cavity formed by the sars-cov-2 spike glycoprotein: a novel binding site date: 2020-05-14 journal: j clin med doi: 10.3390/jcm9051473 sha: doc_id: 306438 cord_uid: db2rqz4d an important stage in severe acute respiratory syndrome coronavirus 2 (sars-cov-2) life cycle is the binding of the spike (s) protein to the angiotensin converting enzyme-2 (ace2) host cell receptor. therefore, to explore conserved features in spike protein dynamics and to identify potentially novel regions for drugging, we measured spike protein variability derived from 791 viral genomes and studied its properties by molecular dynamics (md) simulation. the findings indicated that s2 subunit (heptad-repeat 1 (hr1), central helix (ch), and connector domain (cd) domains) showed low variability, low fluctuations in md, and displayed a trimer cavity. by contrast, the receptor binding domain (rbd) domain, which is typically targeted in drug discovery programs, exhibits more sequence variability and flexibility. interpretations from md simulations suggest that the monomer form of spike protein is in constant motion showing transitions between an “up” and “down” state. in addition, the trimer cavity may function as a “bouncing spring” that may facilitate the homotrimer spike protein interactions with the ace2 receptor. the feasibility of the trimer cavity as a potential drug target was examined by structure based virtual screening. several hits were identified that have already been validated or suggested to inhibit the sars-cov-2 virus in published cell models. in particular, the data suggest an action mechanism for molecules including chitosan and macrolides such as the mtor (mammalian target of rapamycin) pathway inhibitor rapamycin. these findings identify a novel small molecule binding-site formed by the spike protein oligomer, that might assist in future drug discovery programs aimed at targeting the coronavirus (cov) family of viruses. the global pandemic developing from december 2019 by a strain of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) can cause coronavirus disease 2019 disease. this emergent variant adds to the additional coronavirus strains that can infect humans including human coronavirus oc43 (hcov-oc43), human coronavirus hku1 (hcov-hku1), sars-cov, human coronavirus 229e (hcov-229e), human coronavirus nl63 (hcov-nl63), and human coronavirus nl63 (hcov-nl63) [1] [2] [3] [4] [5] [6] . coronaviruses (covs) are positive-sense, enveloped, single-stranded rna viruses that are classified taxonomically as a family coronavirdiae and order nidovirales [4] . there are four genera of covs, including αcov, βcov, δcov, and γcov; most δcovs and γcovs target avians, whilst αcovs and βcovs infect rodents and bats [1, 7, 8] . severe acute respiratory syndrome cov (sars-cov) outbreaks have also emerged previously creating an epidemic [2, 4, [9] [10] [11] [12] [13] . although the mortality of mers-cov, sars-cov, and sars-cov-2 is substantial, there are no preventative vaccines or drugs available to treat patients infected with the virus [9, 11, 12] . the current public health emergency of international concern (pheic) by the world health organization (who) has declared sars-cov-2 (covid-19; a novel βcov) as a pandemic threat. the data obtained from who (08/may/2020) suggest that the virus has caused 3,759,967 infections, 259,474 deaths, and it has affected over 200 countries. the open reading frame 1ab (orf1ab) of sars-cov-2 encodes for three proteins that are broadly recognized as drug targets, since they are key components for infections and disease progression: the sars-cov-2 protease [14, 15] , the rna-dependent rna polymerase (rdrp) [14, 16, 17] , and the sars-cov-2 spike (s) glycoprotein [15, [18] [19] [20] . the sars-cov-2 protease processes the polyproteins that are translated from the viral rna, and it has been heavily studied using small molecules inhibitors [15] . to penetrate the host, the sars-cov-2 makes use of homotrimeric class i glycosylated fusion spike protein [18, 21, 22] . fusion of the viral and host cell membranes is facilitated by the spike glycoprotein, which undergoes a significant conformational change upon fusion [18, 21, 22] . sars-cov-2 studies suggest [18, 23, 24] that the spike glycoprotein functions as a homotrimer. the recognition and subsequent fusion of the viral and cellular membranes are triggered by the s1 subunit of the spike protein, which binds the host cell receptor; angiotensin converting enzyme-2 (ace2) [16, [25] [26] [27] [28] [29] [30] [31] . several insights from structural biology are consistent with the role for this domain in affecting the infection rate of the virus. this host-virus interaction is mediated by the receptor binding domain (rbd) domain from s1 subunit of sars-cov-2 spike glycoprotein that forms a hinge-like conformation [18, 32] , i.e., "down" and "up" states that represents the host cell receptor-inaccessible and receptor-accessible [18] . this receptor-accessible "up" conformation exists in a highly fluctuating state [33] [34] [35] [36] . binding to the host target destabilizes the pre-fusion homotrimer, which sheds off the s1 subunit, and allows for the transition of the s2 subunit to a highly stable postfusion conformation [18] . interestingly, protein-mediated cell-cell fusion assays suggest that sars-cov-2 spike protein displays an elevated plasma membrane fusion capacity when compared to that of sars-cov [32, 37] . several studies have aimed to define the mechanism of binding of sars-cov-2 to the host cell receptor [38] . molecular dynamics simulations of the spike (rbd)-ace2 complex, over 10 ns indicated that spike(rbd)-ace2 binding free energy for sars-cov-2 is better than for the sars-cov [39] . similarly, other studies have shown that the sars-cov-2 spike protein has a better binding affinity to ace2 at two different "up" angles of the rbd domain than the sars-cov [40] . structural features at the spike-ace2 interface suggest that residues q493 and p499 from the spike rbd domain are responsible for maintaining protein-protein stability [41] . using a virtual high-throughput screening approach, small-molecules have been identified that can interact with the rbd domain of sars-cov-2 spike protein [42] . natural compounds present in curcuma sp., citrus sp., alpiniagalanga, and caesalpiniasappan could also target the rbd domain of the sars-cov-2 spike glycoprotein, the protease domain (pd) from ace2, and the sars-cov-2 protease [20] . a set of b cell and t cell epitopes derived from the spike and nucleocapsid proteins that map identically to sars-cov-2 proteins, were identified as potential vaccine candidates [23] . applying an integrative, antiviral drug repurposing methodology, the interplay between the cov-host interactome and drug targets in the human protein-protein interaction network have been defined [43] . bioinformatics methodologies were used to identify neutralizing antibodies that might interact with interfaces formed by the spike glycoprotein and the ace2 host cell receptor [24] . by targeting the rbd domain of the spike protein using docking experiments, kanishka et al. identified small molecule inhibitors [44] . in the majority of studies, the most common strategy is focused on targeting the interface formed by sars-cov-2 spike glycoprotein and the ace2 host cell receptor (i.e., spike(rbd)-ace2). currently, there are no robust drugs for wide-spread dissemination available against coronaviruses including; the sars-cov-2 virus. due to the relatively rapid spread in the current outbreak and the relatively high mortality rate (3.5%), more rapid development of new or repurposed antiviral drugs is of high value. although the majority of drug discovery programs target classically druggable enzymes encoded by the virus, such as the viral rna polymerase inhibited by remdesivir [13, 17, 45] , there is a paucity of information concerning the other regions of spike glycoprotein outwith the ace2-binding domains, especially the domains interacting with the viral membrane. the sars-cov-2 spike protein is a homotrimer composed of three monomers (chains a, b, and c; figure 1a ). each monomeric protein contains an n-terminal ace2 binding domain (receptor binding domain; rbd), a central helix/heptad repeat, and a c-terminal region that interacts with the plasma membrane [18] . homotrimer spike protein assembly from monomeric forms can be rate limiting in cells, suggesting a possible space for intervention on the viral life cycle [46] . our current study focuses on understanding the variability of the trimer spike glycoprotein in sars-cov-2 with respect to the genomes from other coronavirus strains, and identifying the changes in the molecular properties due to conformational flexibility in the spike protein. the analysis suggests that residues in the s2 subunit are less variable compared to the other regions. in addition, the molecular dynamics simulations (mds) identified that residues from the rbd domain obtained substantial flexibility which may be an obstacle in finding active hits. by contrast, residues in the s2 subunit (trimer cavity) showed the least flexibility representing a novel binding region for ligands. this information was used to identify potentially novel drug pockets or the active site regions specifically in the oligomeric sars-cov-2 spike glycoprotein. we performed md simulations on the monomeric and trimeric form of the sars-cov-2 spike glycoprotein, and developed a virtual screening using a food and drug administration (fda) approved chemical library. we identified and focused on an apparent cavity formed by three subunits (the homotrimer), that our simulations suggest can mediate dynamic movements that mimic a "bouncing spring" or a "sarrus linkage (converting a circular motion to a linear motion or vice versa)" when interacting with the ace2 host cell receptor. this motion might be important in the fusion of the virion and the host cell membrane. we hypothesized that such a cavity formed by three monomers or subunits of the spike protein (i.e., chains a, b, and c) might form an acceptor for small molecules, and we asked whether small molecules could be identified with a relatively high binding energy. we identified several known compounds with predicted binding energy of gbvi/wsa dg (generalized-born volume integral/weighted surface area) from −35 to −71 kcal/mol, some of which are already proposed for clinical trials including an mtor (mammalian target of rapamycin) pathway inhibitor, sirolimus (rapamycin; a macrolide type; nct03901001 not yet recruiting) [47] [48] [49] and ritonavir (open-label trial in hospitalized adults with severe covid-19) [48, [50] [51] [52] . a recent study that screened hundreds of approved molecules in a sars-cov-2 assay using artificial intelligence-enabled phenomic assays [53] , also identified sirolimus (rapamycin) as a promising candidate. in addition to the macrolides, one of the top hits we have identified, chitosan, has a recently reported derivative inhibiting sars-cov-2 coronavirus replication in cell lines [54, 55] . a previous study has also shown that the chitosan derivatives can interact with the spike protein and block its interaction with the host receptor [56] . our data suggest a mechanism whereby chitosan (and possibly its derivatives), as well as macrolide type molecules, might bind to a pocket formed by the spike protein trimer and provide a novel domain to focus on for future drug discovery projects. [18, 19] . (c) receptor binding domain (rbd) illustrating the "up" or "open" (pdb id. 6vsb [18] ) and "down" or "closed" (pdb id. 6vxx [31] ) conformation. a total of 791 viral genome sequences were downloaded from the global initiative on sharing all influenza data platform (gisaid) [57] , in order to define the evolutionary variability in different domains of the spike glycoprotein. only genomes with high coverage and complete sequences were selected. further filtering was applied to obtain complete sequences on the targeted domains which reduced the total number of strains to 768. total protein sequences were acquired from 3 frame translation using the transeq tool from emboss (european molecular biology open software suite) package (version 6.5.7) (european bioinformatics institute (embl-ebi), hinxton, cambridge, uk) [58] . the amino acid chains from the spike glycoprotein were aligned to the reference protein (pdb id. 6vsb [18, 59] ) using muscle [60] . variations in the amino acid or the residue changes were scanned on the entire spike protein sequence, along with two areas of interest in the multiple alignment file, focusing on a subset of the s2 subunit (hr1, ch, and cd domains) and the rbd domain ( figure 1 and tables s1-s3). the cryo-em (cryogenic electron microscopy) homotrimer structure of sars-cov-2 spike glycoprotein was retrieved from the protein data bank database (http://www.rcsb.org/pdb; pdb id. 6vsb; figure 1 ) [18, 59] . in addition, the missing amino acid (residues range: 67-78, 96-98, 143-155, 177-186, 247-260, 329-334, 444-448, 455-490, 501-502, 621-639, 673-686, 812-814, and 829-852) coordinates in the structure of sars-cov-2 spike glycoprotein were built using the swissmodel ( figure 1 ) [61] . molecular dynamics simulations were carried on the model systems as per the standardized pipelines [62] [63] [64] (detailed method explained in the supplementary materials; file s1). the gromacs 4.6.5 [65] program (gromacs; groningen machine for chemical simulations, university of groningen, groningen, the netherlands) was used to perform md calculations assigning the charmm27 forcefield [66] . we performed 100 ns molecular dynamics simulations on two systems: (i) the monomeric form and (ii) the homotrimer form of the spike protein. in our analysis of the md simulations, the dynamics of the monomeric form of the spike protein serves as control to the homotrimer, which is the functional unit. initially, the model systems were energy minimized, which provides a base-line model structure and resolves poorly-resolved conformations often found in crystal structures [13, 18, 62, [67] [68] [69] [70] . a simulation box of solvent atoms is then added to enhance simulation realism. following that, using the npt (number of particles (n), system pressure (p), and temperature (t); isobaric-isothermal) thermodynamic ensemble, equilibration of the systems was performed to adjust solvent molecules with counter ions in the simulation box [71] . these equilibrated systems were subsequently used to perform the final md production runs for 100 ns, and results were analyzed using gromacs [56] , biovia discovery studio (dassaultsystèmes, biovia corp., san diego, ca, usa), chimera, and visual molecular dynamics (vmd) tools [71] [72] [73] . structure-based virtual screening (sbvs) is an application of in silico methods that identify promising lead molecules from chemical libraries or databases. these methods are computational counterparts of experimental biological evaluation methods, such as high-throughput screening (hts). fda approved drug libraries were retrieved from target molecule corp. (targetmol; www.targetmol.com) and selleck chemicals (selleckchem; www.selleckchem.com) vendors. the sbvs against the sars-cov-2 spike glycoprotein was performed using the molecular operating environment (moe; chemical computing group inc., montreal, qc, canada) package [74, 75] . receptor-ligand binding or docking using the charmm27 forcefield [66] was evaluated using the gbvi/wsa ∆g scoring function [76] . the compounds showing best energies with the spike protein were selected for further analysis. gbvi/wsa ∆g is a forcefield based scoring function which determines the free energy of binding of the ligand from a given position [75] . in addition, we have also selected the compounds that showed comparatively stable interactions with the homotrimer spike protein. applying the "triangle matcher"placement method, receptor-ligand docking was performed defining the receptor as rigid and ligands as flexible [74, 75] . we were interested to define the evolutionary variance in the sars-cov-2 spike protein. understanding regions of high and low variance can identify domains that may be functionally conserved and potentially important to the virus life cycle, or those under positive evolutionary pressure whose selection might avoid the immune system. examining the variability of the spike protein in sars-cov-2 and its different domains, a total of 791 genome viral sequences were retrieved from gisaid [57] . a global view of the mutation space of the virus is presented in figure 2a , which represents the amino acid substitutions in bins of 10 aminoacids across the spike glycoprotein. these hotspots of variation are mostly confined to the ntd and the rbd domains (figures 1b and 2b) . we investigated the variability in the entire sequence of the spike protein, focusing on the regions that showed low-variability in the structure (figure 2 ). by investigating the variations in the residue changes across the entire spike protein sequence or all the regions of lower variability (figure 2a and table s1 ), the s2 subunit exhibited the lowest sequence variability (residue range: 816-1141; figures 1b and 2c) . moreover, previous studies have identified that the active site region for this spike protein is located in the rbd domain which interacts with the ace2 host cell receptor [18, 20, 26, [38] [39] [40] 42, 44] . comparing the variability of the rbd domain and s2 subunit domains, the rbd domain was shown to contain more mutations in its region compared to the s2 subunit (hr1, ch, and cd) domains (figure 2b ,c, and tables s1-s4). these data suggest that during mutation by natural selection, the viral-host "arms race" might operate more frequently on the rbd domain. by contrast, the s2 subunit conservation is suggestive of an important core function where mutations cannot be tolerated. these findings prompted our focus on the s2 subunit as an important region to investigate for identifying potentially novel druggable pockets. we next traced the dynamics of different domains in the spike protein using md simulations (figure 1b ). the simulated model systems of the spike protein in the monomer and the homotrimer forms were first processed to check the stability of the protein. stability of the simulated spike protein in both forms in the solvent environment was traced by rmsds (root mean square deviation), a time dependent change in the non-hydrogen atoms (figure 3a ). the rmsd plots (figure 3a) suggest that the trimer form of the spike protein is more stable compared to the monomer form. in addition, chain a in trimer has a higher rmsd (~2 å) compared to the other two chains which is a consequence of the fact that the "up" (or ace2-active) conformation [18] induces flexibility. since the monomer form has a higher rmsds compared to the trimer (figure 3a) , we performed independent triplicates (mds was repeated three times) of md simulation for the monomer form ( figure s1 ). the findings from these replicates indicate that the monomer form has a higher rmsd compared to the trimer spike protein (figure 3a and figure s1 ). the root mean square fluctuations (rmsf) were computed on the cα atoms of each residue from the spike protein, in order to trace their flexibility and thereby define the motions of different domains (figure 3b ). the rmsf findings in both forms (monomer and trimer) indicated that the amino acids in the rbd domain (residue range: 329-521) were highly fluctuating (figures 1b and 3b ). in addition, the triplicate md simulations of the monomer form, also suggests that the rbd domain has a higher rmsf in all three simulation replicates ( figure s1 ). these analyses correlate with previous studies [33] [34] [35] [36] 39] . particularly, amino acids ranging from 470-490, responsible for interacting with the ace2, that were highly fluctuating. furthermore, examining other regions of the spike protein suggests that the s2 subunit domains (residue range: 850-1141; hr1, ch, and cd) showed the least fluctuations within the entire protein sequence (figure 3b ). this correlates with the cryo-em studies performed on the spike protein; that the s2 subunit is more stable [18] compared to the rbd domain, and that this subunit is responsible for a highly stable postfusion conformation of the spike protein [18, 32] . from the perspective of designing drugs, the more stable or less flexible a region is within a protein, the more accurately we can trace a better hit molecule. in the case of the spike protein, the rmsf findings guided us towards focusing on the s2 subunit ( figure 3b) . moreover, by tracing the residues involved in the h-bond interactions between two monomers (i.e., chains a-b, a-c, or b-c) of the homotrimer, we observed that the rbd domain residues were also involved in intermolecular interactions with each other and with high occupancy (%). this suggests that intermolecular interactions between chains in the homotrimer might equilibrate the spike protein, and might stimulate conversion from a "down" to "up" conformation of the rbd domain that interacts with the ace2 receptor ( figure s2 and table s5 ). the structural dynamics over the time course for spike protein in the monomer and the homotrimer form was monitored during md simulations (figure 3c,d) . the monomeric spike protein in the solvent environment exhibited a movement from the "up" active state towards the "down" inactive state for the rbd domain (figure 1c, figure 3c , and movie s1). these correlate with the previous findings that the rbd domain can form two different conformations, i.e., "down" and "up" states, which represents the host cell receptor-inaccessible and receptor-accessible, respectively [33] [34] [35] [36] . the sars-cov-2 spike protein has a better binding affinity to the ace2 receptor at two different "up" angles of the rbd domain compared to the sars-cov [40] . figure 3c (and movie s1) describes the conformational change in other regions of the spike protein, when the rbd domain moves towards an "up" to "down" state in the monomeric form. 3d (and movie s2) represents the dynamics of the homotrimer spike protein, suggesting that the rbd domain of chain a opens more widely in its "up" state. domains hr1, ch, and cd close to the viral transmembrane exhibited the least movement (figure 3d ) during md simulations. in addition, exploration of the structural orientation of these s2 subunit domains (figure 3d and movie s2) suggests that they form a large pocket or cavity using three chains (or monomers) from a homotrimer spike protein. the slight movement observed in homotrimer during md simulation of this cavity (movies s1 and s2), and the structural orientation suggest that it could work as "bouncing spring" or "sarrus linkage". one may postulate that, when the spike protein interacts with the ace2 receptor, this "bouncing spring" or "sarrus linkage" movement may be important in the fusion of the virion with the host membrane. additionally, this cavity from the spike protein could work as a platform for the design or development of new drug leads against this protein (figure 3d ). such molecules might alter the trimer stability upon viral entry or upon viral coat assembly. there have been several studies performed to design drugs specific for the sars-cov-2 spike protein [20, [38] [39] [40] 42, 44] ; however, most of them are focused on the rbd domain. in addition, from our md simulation and variability analysis (figures 2 and 3) , the rbd domain is highly flexible and variable, therefore, drugging this variable site may be an obstacle in finding active hit molecules. by targeting the less variable s2 region, such as the cavity formed by the homotrimer (figures 2 and 3d) we suggest that this might be a novel approach to develop small molecule drug leads. we next investigated the targetability of the trimer cavity formed by the s2 subunit (hr1, ch, and cd domains) in the spike protein (figures 2 and 3 ) using the moe (chemical computing group inc.) package [74, 75] , before using it for high-throughput virtual screening (or sbvs) using a library of fda approved drugs. the "alpha shapes" construction [75, 77] geometric method was used to compute the possible residues that can be considered for ligand docking from this trimer cavity in the spike protein (figure 4a ). high-throughput virtual screening is a powerful computational approach that is increasingly being used in the drug discovery process, through the in silico identification of novel hits from large compound databases [78] . we applied the sbvs approach to dock the molecules to the trimer cavity and to check its feasibility as a target. ligand binding to this cavity might reduce or increase the "bouncing spring" movement in the spike protein, as observed in md simulations (figure 3 , movies s1 and s2). this perturbation might affect its interactions with the host cell receptor or the hinge movement of the rbd domain. the compounds that exhibit a relatively high binding affinity towards the sars-cov-2 spike glycoprotein trimer cavity with a binding affinity −35 to −71 kcal/mol (gbvi/wsa dg) were recorded. from the list of ligands showing best binding, the compounds that were already validated or suggested to be/can be active against the sars-cov-2 virus includes: chitosan [54] [55] [56] , rapamycin [47] [48] [49] , everolimus (rad001) [49] , paclitaxel [79] , ritonavir [48, [50] [51] [52] , selameerin (selamectin) [80] , and danoprevir [52] (table 1) . among these molecules rapamycin and everolimus drugs were previously identified as mtor pathway inhibitors [47, 50, [81] [82] [83] . the antibacterial or antiparasitic drugs from the list are chitosan [84] or selameerin (selamectin) [85] , respectively. paclitaxel, has been found to be previously target bcl-2 and microtubule associated functions [86, 87] . in addition, the fda approved drugs that target the protease are: ritonavir [88] and danoprevir (itmn-191) [89] . by docking known drugs within the trimer cavity of spike protein, the relative selectivity of the cavity suggests that the majority of higher-affinity drugs will have a molecular weight (mw) ≥~700 g/mol (table 1 and figure s3 ). however, this is with the certainty that compounds with high mw can form more interactions with the spike protein, in addition, our finding highlights the possibility that the trimer cavity can occupy large ligands deep inside the binding pocket (figure 4a ). particularly, a specific class of ligands (mostly macrolide type) were found to exhibit a better fit to the trimer cavity ( figure 4a) ; for example, rapamycin [47] [48] [49] , everolimus (rad001) [49] , paclitaxel [79] , and selameerin (selamectin) [80] (figure 4a ). the intermolecular interactions between the spike protein and the compounds suggest that residues from all three monomers (chains a, b, and c) are actively involved in binding to the drugs. in addition, placement of the compounds inside the trimer cavity suggests that they make use of the pocket space (forming different conformation) to form stable interactions with the spike protein (figure 4a ). the ligands that were found interacting with the homotrimer cavity with high binding affinity were also docked with an interface formed by the spike proteins (rbd domain; pdb id. 6lzg [90] ) that interact with the ace2 receptor. sbvs, structure-based virtual screening. in order to check the selectivity of these ligands to the trimer cavity, we docked this same subset (table 1) with the rbd domain of the viral spike protein (figure 4b ; pdb id. 6lzg [90] ). the rbd domain is involved in interacting with the ace2 host cell receptor [16, 26, 30, [38] [39] [40] [41] [42] 90, 91] . the docking suggests that all compounds from table 1 have better binding affinity to the trimer cavity compared to that of the rbd domain. in addition, chitosan [54] [55] [56] 84 ] (a linear polysaccharide; −37.29 kcal/mol) could form a linear conformation in its structure when binding tothe rbd domain (figure 4b and table 1 ), whilst the same ligand (due to its molecular structural nature) can form a slightly folded shape (as shown in 2d-diagram; figure 4a ) within the trimer cavity. by contrast, everolimus [49] (a macrolide type) exhibits high affinity for the trimer pocket, and very little selectivity for the rbd domain (table 1) . moreover, ligands (figure 4b ) that interact with the rbd domain overlap with the region bound by cr3022 (a neutralizing antibody isolated from a convalescent sars patient that, interacts with the receptor binding domain of the sars-cov-2 spike protein [92] ). the sars-cov-2 virus causing covid-19 disease uses the fusion spike glycoprotein to penetrate into the host cell, and therefore a detailed understanding of this protein forms a critical intervention point in the viral life-cycle. we interrogated the spike protein with a diversity of computational approaches. first, the variability in spike protein from 791 different viral genome sequences was evaluated. residues in the s2 subunit (residue range: 816-1141; hr1, ch, and cd domains) were found to be less evolutionarily variable compared to other regions or domains. by contrast, residues h49y, q239k, v367f, v483a, s943p, k986p, and v987p were found to be the most common amino acid substitutions in the spike protein from related viruses. secondly, md simulations revealed that residues in rbd domain (residue range: 329-521) were more flexible compared to residues in the s2 subunit, making it more complicated for drug design strategies. an examination of less variable regions revealed that the hr1, ch, and cd domains (s2 subunit) located close to the viral transmembrane formed a large cavity or pocket that is formed from three spike monomers. the md simulation traced an "up" active state and a "down" inactive state of the spike protein in its monomer form. slight movement of the trimer cavity within this structural orientation suggests that it could work as "bouncing spring" or "sarrus linkage" when interacting with the host cell receptor. the conversion between "up" and "down" states in the monomer form of spike protein using the in silico methods is defined by the md field to be relatively fast. nevertheless, there are different structural isoforms that have been identified on the spike protein using different experimental methods or virus strains. this indicates that, although conversion may be quick, there are structural endpoints which are "stable". using the recent cryo-em structure of the sars-cov-2 spike protein [18] ; an asymmetric hinge-like movement was observed in only one of the three rbd domains in the s1 subunit, which was also observed in mers-cov and sars-cov [33, 34] . however, there are also other structures where all three rbd domains are in the "up" or "down" conformation [18, 31, 33, 34, 40] . these data suggest a physiological relevance due to heterogeneous protein conformational dynamics. for example, asymmetric conformational flexibility might have a functional role, perhaps in evading the exposure of b-cell epitopes (only one rbd domain is in the "up" conformation) and/or optimized interaction with the ace2 receptor depending on virus strain. in addition, because of the "bouncing spring" mechanism (communication between the trimer pocket and the rbd domain conformation), it is possible that these different spike protein conformational isoforms provide another avenue to develop drug discovery programs that exploit and/or circumvent these dynamics. our investigation into the genomic variation within virus strains, as well as our findings from the md simulations, identified a conserved trimer cavity or pocket formed by the s2 subunit in the spike protein. these findings suggest that a novel target, "the trimer cavity formed by spike protein oligomerization", may be suitable to manipulate viruses of this class. targeting the trimer pocket might identify a new functional class of drugs against this protein. applying the sbvs approach, we docked drug libraries against the trimer cavity with the hypothesis that such a ligand might perturb the predicted "bouncing spring" movement and the homotrimer formation. protein-ligand docking identified severalhits that have already been published or proposed to inhibit the sars-cov-2 virus in cell systems. for example, our studies suggest an action mechanism for molecules such as chitosan and macrolide types (e.g., rapamycin). based on the sequence variability of the coronavirus, including our findings from md simulations of the spike protein, a conserved trimer cavity (hr1, ch, and cd domains) is a feature of the spike protein in most coronaviruses. consistent with this, previous work has shown that the molecule ek1 exhibited potent inhibitory activity against all human coronaviruses (hcovs) tested through binding to the c-terminal hr1 domain [37] . additionally, the "up" and "down" conformations of rbd domain observed during md simulations, supports that concept that the spike protein can also be a target of a possible igg therapeutic [92] . from the list of the top compounds identified that dock into the trimer cavity, some of them have already been validated or suggested as sars-cov-2 virus inhibitors in cells, including; a chitosan derivative [54] [55] [56] , rapamycin [47] [48] [49] , everolimus (rad001) [49] , paclitaxel [79] , ritonavir [48, [50] [51] [52] , selameerin (selamectin) [80] , and danoprevir [52] . among these, a modified polymeric version of the chitosan drug (a top hit in our analysis) was recently shown to inhibit cov replication with evidence that the molecule inhibits the binding of the viral spike protein to the host ace2 receptor [54] [55] [56] . the protein-protein interaction map or the network-based methodologies [14, 43] suggest that sirolimus (rapamycin) emerges as a common potential drug lead for repurposing against covid-19. this rapamycin (mtor inhibitor) drug was found previously to disrupt larp1 (la-related protein 1) and mtorc1 (mammalian target of rapamycin complex 1) binding, and has been shown to reduce mers infection by~60% in vitro [47] . the postulated geroprotectors, such as sirolimus (rapamycin) and its close derivative, the rapalog everolimus (rad001), decreased infection rates in a small sample of elderly patients [49] . moreover, the drugs sirolimus (rapamycin) and ritonavir are currently in clinical trials for repurposing against covid-19 [48, 50, 51] . sirolimus (rapamycin) is registered in a clinical trial (nct03901001 not yet recruiting) designed to evaluate adjunctive use of sirolimus (rapamycin) and oseltamivir in patients hospitalized with influenza [47, 48] . ritonavir, a hiv protease inhibitor is in an open-label trial in hospitalized adults with severe covid-19 [48, 51] . the data from this small-sample clinical study showed that danoprevir boosted by ritonavir is safe and well tolerated in all patients [52] . selamectin is a potential drug for treating covid-2019 found active against the pangolin coronavirus gx_p2v, a workable model for sars-cov-2 research [80] . the antitumor drug paclitaxel increases cellular methylglyoxal to virucidal levels, providing a rationale for repurposing doxorubicin and paclitaxel for covid-19 treatment [79] . nevertheless, whether the hit molecules we have identified that dock into the trimer cavity and impact on the virus life cycle requires orthogonal validation. we hope the findings of our study can help to understand the function of the highly conserved spike interspecies transmission and emergence of novel viruses: lessons from bats and birds severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting 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inhibitor for the potential oral treatment of hcv infection the first-in-class peptide binder to the sars-cov-2 spike protein structural basis of receptor recognition by sars-cov-2 a highly conserved cryptic epitope in the receptor-binding domains of sars-cov-2 and sars-cov this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the international centre for cancer vaccine science project is carried out within the international research agendas programme of the foundation for polish science co-financed by the european union under the european regional development fund. authors would also like to thank the pl-grid infrastructure, poland for providing their hardware and software resources. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord-343107-oj1re34k authors: zhou, haixia; chen, yingzhu; zhang, shuyuan; niu, peihua; qin, kun; jia, wenxu; huang, baoying; zhang, senyan; lan, jun; zhang, linqi; tan, wenjie; wang, xinquan title: structural definition of a neutralization epitope on the n-terminal domain of mers-cov spike glycoprotein date: 2019-07-11 journal: nat commun doi: 10.1038/s41467-019-10897-4 sha: doc_id: 343107 cord_uid: oj1re34k most neutralizing antibodies against middle east respiratory syndrome coronavirus (mers-cov) target the receptor-binding domain (rbd) of the spike glycoprotein and block its binding to the cellular receptor dipeptidyl peptidase 4 (dpp4). the epitopes and mechanisms of mabs targeting non-rbd regions have not been well characterized yet. here we report the monoclonal antibody 7d10 that binds to the n-terminal domain (ntd) of the spike glycoprotein and inhibits the cell entry of mers-cov with high potency. structure determination and mutagenesis experiments reveal the epitope and critical residues on the ntd for 7d10 binding and neutralization. further experiments indicate that the neutralization by 7d10 is not solely dependent on the inhibition of dpp4 binding, but also acts after viral cell attachment, inhibiting the pre-fusion to post-fusion conformational change of the spike. these properties give 7d10 a wide neutralization breadth and help explain its synergistic effects with several rbd-targeting antibodies. m iddle east respiratory syndrome coronavirus (mers-cov), a novel lethal human virus in the family of coronaviridae, was first identified in saudi arabia in june 2012 1 . infection by this pathogen causes an acute respiratory disease designated as mers, with symptoms that are very similar to those of sars 2 . globally, mers-cov infections have been confirmed in 27 countries causing 803 deaths (http://www.who. int/emergencies/mers-cov/en/). interspecies transmission from dromedary camels to humans is considered to be one major route of transmission in the middle east region 3, 4 . however, many infected patients without camel exposure and a recent mers outbreak in korea demonstrated that large-scale human-tohuman transmissions can occur through close contacts 5 . due to its potential for mutating toward efficient human-to-human transmission and causing a pandemic, mers-cov was listed as a category c priority pathogen by the us national institute of allergy and infectious diseases. monoclonal antibodies (mabs) with potent neutralizing activity have become promising candidates for both prophylactic and therapeutic interventions against viral infections 6 . on coronaviruses, the component primarily targeted by mabs is the homotrimeric spike (s) glycoprotein of the virion. as a typical class i fusion glycoprotein, the s trimer of highly pathogenic coronaviruses such as mers-cov and sars-cov, which mediates receptor recognition and membrane fusion during viral entry [7] [8] [9] [10] [11] [12] , undergoes protease cleavage into the s1 and s2 subunits, positional change of the receptor-binding domain (rbd) in the s1 subunit for receptor binding, dissociation of the s1-receptor complex, and finally formation of a six-helix bundle by the s2 subunits. a series of rbd-targeting antibodies against mers-cov, which block the binding of the s trimer to the cellular receptor dpp4, have been reported and characterized [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] . these antibodies exhibited high potency in inhibiting the infectivity of pseudotyped and live mers-cov in cells and animal models. the neutralizing epitopes and mechanisms of antibodies including 4c2, d12, m336, mers-27, jc57-14, cdc-c2, mers-4, and mers-gd27 were further elucidated at the atomic level by structural and functional studies [19] [20] [21] [22] [23] [24] [25] . sequence comparisons of different mers-cov strains have shown that most naturally occurring mutations of the s glycoprotein are located on the rbd of the s1 subunit and the s2 subunit. considering the rapid evolution and high genome variation of rna viruses, more mutations on the rbd may enable the new strains to escape neutralization by currently known rbd-targeting antibodies. therefore, new mabs targeting other functional regions of the mers-cov s glycoprotein and/or neutralizing by different mechanisms are important for developing effective prophylactic and therapeutic interventions against mers-cov infection. although several mabs targeting non-rbd regions have recently been reported, their neutralizing epitopes and mechanisms remain unclear 20, 21, 26 . in this study, we isolated and characterized the mouse mab 7d10 by combining structural, biochemical, and functional studies. the 7d10 antibody recognizes the ntd of mers-cov s glycoprotein and neutralizes the infectivity of pseudotyped and live virus with a potency comparable to those of the most active rbd-targeting antibodies. we also found that the epitope and mechanism of 7d10, which are different from those of rbdtargeting antibodies, enable it to have a better neutralizing breadth and to work synergistically with other antibodies against different mers-cov strains. all these results indicate that 7d10 is a very promising candidate for the future combined use of different antibodies in our battle against mers-cov. characterization of neutralizing mab 7d10 targeting the ntd. to generate mers-cov neutralizing mabs with epitopes outside the rbd, mice were immunized with recombinant mers-cov s protein (residues 1-1297). subsequently, the spleenocytes were harvested and fused with sp2/0 myeloma cells, and the hybridoma cell lines were screened for positive clones by elisa with the s protein 27 . the positive clones were further tested for their reactivity to different s fragments, including the s1 subunit ntd (residues 18-353), rbd (residues 367-606), and the s2 subunit (residues 726-1297). one ntd-specific mab, named as 7d10, was finally isolated with an ec 50 of approximately 0.31 μg ml −1 in elisa (fig. 1a) . it exhibited no crossreactivity with the rbd at a concentration of 4 μg ml −1 (fig. 1b) . we further assessed the potential of 7d10, in the form of crude extracts from mouse ascites, for inhibiting mers-cov entry into susceptible huh7 cells and vero e6 cells with either pseudotyped or infectious viruses. as expected, 7d10 was able to neutralize the infectivity of pseudotyped and live mers-cov (fig. 1c, d) . the neutralizing activity of 7d10 was dose-dependent, with an ic 50 of approximately 0.18 μg ml −1 against pseudotyped virus and practically the same ic 50 of approximately 0.2 μg ml −1 against live virus (emc strain) (fig. 1c, d) . images illustrating the reduced pfu formation, corresponding to the rate of neutralization of live mers-cov, are shown in fig. 1e . antibody isotyping showed that 7d10 belongs to the igg1 subtype. sequencing further determined that the heavy chain germline v and j segments are ighv1-12*01 and ighj2*03, while those of light chain are igkv03-12*01, igkj1*01, and igkj1*02, respectively (supplementary table 1) . we also generated a chimeric version of 7d10 (7d10-h) by combining the v segments of 7d10 with the human igg1 backbone, which was efficiently expressed and purified in freestyle 293-f cells ( supplementary fig. 1a ). the bio-layer interferometry (bli) experiment showed that the affinity constant of the binding between 7d10-h and ntd was approximately 25 nm (table 1 and supplementary fig. 1b) . the ic 50 of the purified 7d10-h against cell entry by pseudotyped mers-cov was approximately 0.06 μg ml −1 (supplementary fig. 1c ). we also investigated the protective efficacy of 7d10-h against infection of pseudotyped mers-cov using r26-hdpp4 mice model with a human dpp4 inserted into the rosa26 locus by crispr/cas9, which could also been productively infected by high-titer mers-cov pseudovirus, with effects comparable to the authentic infection 28 . bioluminescence of the fluc reporter showed that the pseudovirus infection in the mice was clearly prevented by 7d10-h and rbd-specific mab mers-4 when both antibodies were administered by the intraperitoneal injection with a dose of 200 μg per mouse ( supplementary fig. 1d ). the recombinant chimeric 7d10-h, which retained the activities as the mouse 7d10 and protected r26-hdpp4 mice against challenge of pseudotyped mers-cov, was utilized in subsequent binding and neutralization experiments. overall structure of the 7d10 scfv bound to the ntd. to structurally characterize the 7d10 and its binding to the spike protein, we determined the crystal structure of the antibody scfv (7d10-scfv) in complex with the ntd at a resolution of 3.0 å with a final r work of 0.188 and r free of 0.224. statistics of diffraction data collection, processing, and structure refinement are listed in table 2 . there were three complexes of 7d10-scfv bound to ntd per asymmetric unit. the refined model contains residues tyr18 to ser353 of mers-cov ntd, glu1 to ser120 of the v h and asp26 to lys136 of the v l . n-linked glycans attached to asn66, asn104, asn125, asn155, asn166, asn222, asn236, and asn244 of the ntd are also included in the model. it has been previously shown that the mers-cov ntd folds into a galectin-like structure, which can be separated into top, core and bottom subdomains (fig. 2a) . upon binding, the 7d10-scfv contacts the top subdomain of the ntd and the asn222-linked glycans with its heavy and light chains ( fig. 2a and supplementary fig. 2 ). all three cdrs of the heavy chain and the cdr1 and cdr3 of the light chain participate in the binding (fig. 2a) . the buried surface between the 7d10-scfv and the ntd encompasses approximately 551 å 2 for the heavy chain and 320 å 2 for the light chain. structural features of the interface between 7d10 and ntd. the binding interface between 7d10-scfv and ntd consists of 12 residues and asn222-linked glycans from the ntd, as well as 15 residues from all 6 cdrs except for lcdr2 (fig. 2b, c) . the interacting residues from the ntd are tyr18, asp20, pro23, asp24, val26, ser28, glu188, ser191, asn226, lue234, arg235, and asn236. together with the asn22-linked nag508, nag509 and man519, they form the conformational epitope recognized by 7d10 (fig. 2b) . the residues recognizing 7d10 are ser31, tyr32, asn33 from the hcdr1, tyr52, asn55, and ser59 from the hcdr2, arg98, tyr99, asn101, tyr102, and tyr105 from the hcdr3, tyr59, and tyr61 from the lcdr1, and arg121 and asp122 from the lcdr3 (fig. 2c) . specifically, 7d10 hcdr1 residues ser31, tyr32, and asn33 interact with pro23 and asp24 from the ntd, and a formed hydrogen bond is from 7d10 asn33 to ntd asp24 ( fig. 2d and supplementary fig. 2 crystal structure of 7d10-scfv bound to ntd and the binding interface. a an overall structure of the ntd/7d10-scfv complex in which the ntd, n222-linked glycans on the ntd, 7d10 v l , and 7d10 v h are colored in blue, gray, magenta, and cyan, respectively. b epitope on the ntd recognized by 7d10. the ntd is represented as blue surface, on which the protein region bound by 7d10 is displayed in orange and the n222-linked glycans are displayed as gray sticks. c 7d10 residues that are involved in the binding. the v l and v h are colored in magenta and cyan, respectively, and the residues interacting with 7d10 are displayed in orange. d interactions between the 7d10 v h residues and the corresponding residues of ntd. e interactions between the 7d10 v l residues and the corresponding residues of ntd. f zoom-in view of interactions between n222-linked glycans and 7d10 interacting with tyr18, asp20, pro23, asp24, and arg235 of the ntd (fig. 2d ). tyr99 and asn101 of 7d10 form two hydrogenbonding interactions with asp24 of the ntd (supplementary table 2 ). for the light chain, the lcdr1 and lcdr3 residues tyr59, tyr61, arg121, and asp122 interact with glu188, ser191, arg235, and asn236 of the ntd, and a salt bridge is formed between arg121 of lcdr3 and glu188 of the ntd (fig. 2e and supplementary table 2) . a prominent feature at the interface is the extensive recognition of asn222-linked glycans by all three heavy chain cdrs ( fig. 2f and supplementary table 3 ). specific hydrogen-bonding interactions occur between tyr58 and arg98 of 7d10 and the nag508 and man519 glycans, respectively ( fig. 2f and supplementary table 2 ). confirmation of the neutralizing epitope. to confirm the epitope and its critical residues, we performed a mutagenesis study by introducing single mutations to all 13 ntd recognized residues including trp18, asp20, pro23, asp24, val26, ser28, glu188, ser191, asn222, asn226, lue234, arg235, and asn236. we first examined the effects of these ntd mutations on the binding by 7d10-h. the 7d10-h bound the wild-type ntd with an affinity of approximately 25 nm (table 1 and supplementary fig. 3 ). by contrast, the d24a and r235a mutations dramatically reduced the binding, to a level that was undetectable by bli experiment (table 1 and supplementary fig. 2 ). the e188a and n222q mutations reduced by the binding affinity by 148-fold to 3.7 μm and 112-fold to 2.8 μm, respectively (table 1 and supplementary fig. 3 ). all the other nine mutations had variant unequal effects on the binding by reducing the affinity in the range of 2-to 15-fold (table 1 and supplementary fig. 2 ). the effects of these mutations on the neutralizing activity of 7d10-h were in consistent with the changes of binding affinity. pseudotyped mers-cov bearing d24a, e188a, or r235a mutation in the spike glycoprotein escaped the neutralization by 7d10-h (table 1 and supplementary fig. 4 ). the ic 50 values of 7d10 against pseudotyped mers-cov bearing d20a, v26a, or n222q mutation were increased approximately by 60-, 181-, and 50-fold (table 1 and supplementary fig. 4 ). the binding and neutralization assays collectively revealed that asp24, val26, glu188, arg235, and asn222-linked glycans are critical for recognition and neutralization of mers-cov by 7d10. sequencing of multiple clinical isolates had revealed that the mers-cov s glycoprotein is evolving at an average rate of 1.12 × 10 −3 substitutions per site per year 29 . alignments of the deposited sequences in the ncbi identified 22 naturally changing residues from the prototype emc sequence including v26f, v26i, v26a, d158y, l411f, t424i, a482y, l506f, d509g, v530l, v534a, e536k, d537e, v810i, q833r, q914h, r1020h, r1020q, a1193s, t1202i, g1224s, and v1314a, which are located in the ntd (residues 18-353) and rbd (residues 367-606) of the s1 subunit, and the s2 subunit (residues 752-1297). several residue changes on the rbd, such as those occurring on d506, d509, and e536, indeed enabled the mers-cov to escape the neutralization of antibodies targeting the rbd 20, 21 . considering that most of the mutations are outside the ntd, we speculated that 7d10-h would have a better tolerance for these naturally occurring mutations. we generated pseudotyped mers-cov bearing the emc strain s glycoproteins and its mutants harboring all the 22 listed residue changes. the neutralization assays showed that 7d10-h showed effective neutralizing activity against almost all pseudotyped mers-cov variants. only the two mutations v26f and v26a on the ntd increased the ic 50 value of 7d10-h by more than 150-fold and significantly reduced its neutralization activity (fig. 3a, b) , which confirmed the results of the structural and biochemical studies of the binding interface. all other naturally occurring mutations, most of them on the rbd and the s2 subunit did not affect the neutralization capability of 7d10-h (fig. 3a, b) , indicating that 7d10 would have a wide neutralization breadth against different variants of mers-cov. combination of 7d10 with other rbd-targeting antibodies. the current available mers-cov antibody epitopes with solved structures are all on the rbd, which can be grouped into three categories: (1) epitope of mers-4; (2) epitopes of mers-27, d12, 4c2, and jc57-14; and (3) epitopes of m336, mca1, cdc-c2, and the newly reported mers-gd27 ( supplementary fig. 5 ) 25 . in our study of the rbd-specific mab mers-4, we also found synergism with the ntd-targeting mab 5f9 25 . thus, the elucidation of the epitope targeted by 7d10, which added a category outside the rbd ( supplementary fig. 5 ), prompted us to study the combined effect of 7d10 together with the three representative antibodies mers-4, mers-27, and mers-gd27 in the neutralization of pseudotyped mers-cov by titrating the neutralizing potency of an equimolar mixture of the two antibodies and comparing the dose response with that observed in neutralization assays performed with the individual antibody alone. as shown in the fig. 4 , the combination index (ci) values of mers-gd27 combined with 7d10 at fa values of effective dose 50%, 75%, 90%, and 95% (ed 50 , ed 75 , ed 90 , and ed 95 , respectively) were 0.26, 0.25, 0.24, and 0.24, respectively. as a ci value of 1 indicates an additive effect, <1 indicates synergism, and >1 indicates antagonism, the combination of 7d10 and mers-gd27 worked in a clearly synergistic manner. meanwhile, the combination index (ci) values of combined mers-4 with 7d10 at fa values of effective dose 50%, 75%, 90%, and 95% (ed 50 , ed 75 , ed 90 , and ed 95 ) were 0.25, 0.27, 0.30, and 0.33, respectively. thus, the combination of mers-4 and 7d10 also demonstrated synergism, in particular at relatively lower concentrations. however, the percent neutralization obtained using combined mers-27 and 7d10 showed no obvious difference of half maximal inhibitory concentration (ic 50 ) compared with that of 7d10 alone. the combination index (ci) values of combined mers-27 and 7d10 at fa values of effective dose 50%, 75%, 90%, and 95% (ed 50 , ed 75 , ed 90 , and ed 95 ) were 0.82, 0.87, 0.94, and 1.00, respectively. it indicated that the combination of 7d10 with mers-27 exhibited neither synergy nor antagonism. mechanism of 7d10 neutralization. a major reported mers-cov neutralization mechanism relies on inhibiting the binding of the s trimer with the cellular receptor dpp4. the epitopes of these reported antibodies all reside in the rbd responsible for receptor binding. the fact that the 7d10 epitope is outside the rbd indicated that it may have a different neutralizing mechanism. we first examined if 7d10 is still able to inhibit the receptor binding by the s trimer. the facs analysis of cellsurface staining showed that the scfv and fab fragments of 7d10-h did not inhibit the staining of huh7 cells by the s trimer, while the 7d10-h slightly reduced the staining (fig. 5a , supplementary table 4 and supplementary fig. 6 ). by contrast, the rbdtargeting mab mers-4 was much more potent than 7d10-h in inhibiting the binding of the s trimer to huh7 cells. moreover, the fab and scfv fragments of mers-4 retained nearly the same potency in the inhibition ( fig. 5a and supplementary table 4 ). surface plasmon resonance (spr) analysis confirmed these conclusions by showing that 7d10-h, and not its fab or scfv fragments, could interfere with the binding of the s trimer to chipcoupled dpp4 in a dose-dependent manner ( supplementary fig. 7) , while the igg, fab, and scfv of mers-4 all inhibited the binding ( supplementary fig. 7) . to investigate why the igg, fab, and scfv of 7d10 inhibit receptor binding differently, we constructed models of their binding to the s trimer. the mers-cov s trimer structure was determined by cryo-em with the rbd in standing or lying positions, and only the standing rbd could bind to the dpp4 receptor. after superimposing the ntd/7d10-scfv crystal structure onto the s trimer, we observed no steric clashes between three ntd-bound scfv fragments and one or two rbdbound dpp4 receptors ( fig. 5b and supplementary fig. 8 ). the s trimer with three rbd-bound receptors was not considered because the cryo-em study of the mers-cov s trimer only revealed conformations with one or two standing rbds. when the scfv was replaced with the fab, there were also no steric clashes between the fab and dpp4 receptor (fig. 5c ). it is more complicated to model the binding of 7d10-h to the s trimer, considering that the igg form has two binding sites and the intrinsic flexibility. we found that binding of the 7d10-h igg to the ntd in certain orientations could inhibit the binding of dpp4 due to steric clashes, while there were still no steric clashes with the 7d10-h bound in some other orientations (fig. 5d, e) . these results provided a structural explanation for the inability of 7d10-h scfv and fab to inhibit the binding of the s trimer to the dpp4 receptor. they may also explain why the 7d10-h igg form is not as potent as the mers-4 igg, fab, and scfv which all directly bind to the rbd. in parallel with biochemical studies, we also examined the neutralizing activities of 7d10-h igg, fab, and scfv. the 7d10-h fab and scfv did not interfere with the binding of the s trimer to the dpp4 receptor. however, they were still able to inhibit the cell entry of pseudotyped mers-cov with ic 50 value of 0.26 μg ml −1 and 0.28 μg ml −1 , respectively (fig. 6a) . although the 7d10-h fab and scfv are less active than the igg in infection inhibition, they were still comparable to the fab or scfv fragments of several reported rbd-targeting antibodies such as mers-4 fab (ic 50 : 1.49 μg ml −1 ) and mers-4 scfv (ic 50 : 0.55 μg ml −1 ) ( supplementary fig. 9a ). these results collectively indicated that neutralization by 7d10-h involves other mechanism besides interfering with the initial receptor binding. we tested and compared the neutralizing activity of 7d10 in pre-attachment and post-attachment settings. after the cell attachment, 7d10 was still able to inhibit infection by pseudotyped mers-cov with an ic 50 of 0.55 μg ml −1 (fig. 6b) . in comparison, mers-4, which is more potent than 7d10 in inhibiting receptor binding, exhibited very weak neutralization after receptor binding (supplementary fig. 9b) . the above results, especially the retaining activity of 7d10 after viral attachment indicated that 7d10 would also interfere with the prefusion to postfusion conformational transition of the s glycoprotein required for membrane fusion. this transition 30, 31 . we showed that the mers-cov s glycoprotein in the prefusion state is sensitive to the digestion of proteinase k (fig. 6c) . previous studies have demonstrated that cleavage at the s1/s2 site by trypsin and the binding with cellular receptor greatly enhanced the prefusion to postfusion transition of the spike glycoprotein 31 . consistently, the amount of a 50 kda and proteinase-k-resistant band of the s glycoprotein representing the postfusion six-helix bundle was at the maximum level in the presence of trypsin and dpp4 (fig. 6c) . and the addition of 7d10-h fab obviously reduced the intensity of the band (fig. 6c) . meanwhile, we analyzed the full-length mers-cov s trimer embedded in the membrane of pseudotyped virus and the trigger we used to induce the conformational transition was the incubation with huh 7 cells that endogenously expressing dpp4 receptor. after incubating the pseudotyped virus with huh 7 cells for 1 h at 37°c, a proteinase-k resistant band on the sds-page gel appeared and the addition of 7d10-h, 7d10-h fab, or 7d10 scfv all clearly decreased the intensity of this band ( supplementary fig. 10) . thus, these biochemical results strongly suggest that 7d10 could also exert its neutralizing activity in the postattachment stage after receptor-binding by inhibiting the conformational transition of the s glycoprotein required for membrane fusion (fig. 6d) . since dpp4, which is a critical step for viral cell attachment. in this study, we first isolated the neutralizing mouse antibody 7d10 targeting the ntd of the s glycoprotein. neutralization assays showed that 7d10 is highly potent and its activity is comparable to that of the most potent rbd-targeting antibodies. structural determination of 7d10 scfv bound to the ntd and mutagenesis studies revealed the epitope and key residues on the ntd for binding and neutralization at atomic level. comparisons of 7d10 scfv, fab, and igg forms in dpp4-binding competition and neutralization assays indicated that its activity is not solely dependent on the inhibition of dpp4 binding. further experiments indicated that the neutralizing activity of 7d10 after cell attachment is through the inhibition of prefusion to postfusion conformational transition of the s glycoprotein trimer, which mediates the fusion of viral and cell membranes. we also showed that 7d10 has a wide neutralization breadth against mers-cov variants bearing naturally occurring mutations and exhibited synergistic effects with several rbd-targeting antibodies. these results collectively revealed an antibody epitope and neutralization mechanism on the s glycoprotein, which would contribute to the global efforts to control mers-cov infection and transmission by providing alternatives for mers-cov immunotherapy. similar the ntds of the s protein of other betacoronaviruses such as mhv, bcov and hku1, that of mers-cov also folds into a galectin-like structure. although the galectin domain is a typical carbohydrate-recognition domain, the betacoronavirus ntds can include structural variations that enable more diverse functions in viral infection. the examples, include the ntd of bcov that retains the glycan-binding activity recognizing 5-n-acetyl-9-oacetylneuraminic acid (neu5,9ac2) and the ntd of mhv that evolved specific protein-protein interactions with its cellular receptor ceacam1, and both interactions are important for the viral cell attachment 36, 37 . however, there is still no report on the glycan or protein-binding activities of the mers-cov ntd. in fact, crystallographic structure determination showed that the glycanbinding site on the mers-cov ntd is occupied by a short helix (residues 222-231) and the asn222-linked glycan, indicating that it is not able to bind glycans in the same way as the ntd of bcov 11 . notably, the asn222-linked glycan is involved in the recognition by 7d10, whereby nag508 and man519 undergoes specific hydrogen-bonding interactions with tyr58 and arg98 of 7d10, respectively. the ntd n222q mutation also dramatically reduced the binding and neutralization by 7d10, but did not dramatically affect the cell infection of pseudotyped mers-cov ( supplementary fig. 11) . therefore, the asn222-linked glycan serves as an important anchor point for the binding of 7d10 to the mers-cov ntd. as the largest class i viral fusion protein, the coronavirus s glycoprotein is expected to undergo a prefusion to postfusion conformational transition to mediate the interaction between viral and cellular membrane proteins, although structural studies just began to shed light on this recently. the s glycoprotein of betacoronaviruses mhv and hku1, whose structures have been determined by the cryo-em method, all adopt a similar prefusion homotrimeric architecture 38, 39 . interestingly, in the prefusion architecture of the s trimer of highly pathogenic mers-cov and sars-cov, two major conformational states were observed. a 7d10-h igg was tested for neutralizing activity against pseudotyped mers-cov before or after receptor binding. vrc01 mab was used as unrelated control. c the effect of 7d10-h fab on the conformational change of the mers-cov s trimer was probed by western blotting using an anti-mers-cov s2 polyclonal antibody. refolding to the postfusion conformation was detected by the appearance of a proteinase-k resistant band. trypsin was used at 5 μg ml −1 and proteinase k at 10 μg ml −1 . digestion experiments and western blots were performed in triplicates, and a representative result is shown for each of them. d a cartoon representation designed by us showing the neutralization mechanism by which 7d10 blocks mers-cov entry. on the one hand, some virus particles can not bind to dpp4 due to steric hindrance caused by 7d10 binding. on the other hand, 7d10 still recognizes the particles when the up receptor-binding domain (rbd) binds to dpp4, and may inhibit the prefusion to postfusion transition of the s2 subunit and the initiation of membrane fusion. source data are provided as a source data file major difference between them is the change of the rbd in the s1 subunit from a down to an up position, which was proposed to be a prerequisite for the binding of the s trimer to their respective cellular receptor dpp4 and ace2 9 . this proposal was recently confirmed by our cryo-em study of the sars-cov s trimer in complex with ace2, and we also showed that ace2-binding could induce the dissociation of the s1 subunit, which results in the falling apart of the prefusion s trimer and the transition to the prefusion state of the s2 subunit 12 . a major neutralization mechanism of antibodies against mers-cov is to directly or indirectly compete with the cellular receptor dpp4 for binding to the rbd. in theory, antibodies that interfere with the coronavirus membrane fusion process other than receptor binding would also have a neutralizing activity, and the 7d10 mab targeting the ntd we studied is one such example. here, we showed that 7d10 neutralization is not solely dependent on dpp4-binding competition, and its inhibition of the s trimer conformational transition after cell attachment also plays a significant role in the neutralization. we suggested that the binding of 7d10 may stabilize the prefusion architecture of the s trimer, even after the binding of dpp4 receptor. the stabilization of viral fusion protein at one conformational state for neutralization has also been observed and studied in other viruses such as hiv. a recent study revealed that the hiv env trimer is intrinsically dynamic with three major and distinct prefusion conformations 40 . among them, the closed, ground-state conformation is dominant and could be remodeled to another two conformations by cd4 receptor binding, which is essential for the subsequent prefusion to postfusion transition 40 . the binding of neutralizing antibodies, whether inhibiting the binding of the cd4 receptor (such as vrc01) or not (such as 2g12 and pgt145) all resulted in the stabilization of the ground-state conformation of the env, which finally disfavors its prefusion to postfusion state transition required for viral entry 40, 41 . to the best of our knowledge, our study offers the first structural definition of the neutralizing epitope of an antibody targeting the s ntd of mers-cov. as we summarized in supplementary table 5 , a total of six anti-ntd mabs have been reported 20, 21, 26, 42 . all of them neutralize the infection of pseudotyped mers-cov emc strain with high potency except for mab 1.10f3. the mab 5f9 and our 7d10 showed the same neutralizing activity against live mers-cov in plaque reduction neutralization testing. notably, the mouse mab g2 can greatly relieve the symptom of dpp4-transgenic mice infected following mers-cov infection and our 7d10-h can inhibit the infection of pseudotyped mers-cov in r26-hdpp4 mice. however, the specific neutralizing epitopes and mechanisms of 5f9, g2, jc57-13, and fib-h1 are largely unknown. in addition, the combination of different antibodies is supposed to be an effective strategy to combat mers-cov infection as it continues to spread among multiple animal species and to probe and adapt to the human population [43] [44] [45] . an effective combination would require the candidate antibodies to bind to disparate epitopes or with distinct mechanisms and hence display additive or synergistic effects, as the mabs mers-4 and 5f9 we mentioned before 25 . although the exact mechanism that leads to the synergy or additive is uncertain, our 7d10-h with mers-gd27 or mers-4 antibodies demonstrated a synergy in inhibiting the infectivity of pseudotyped mers-cov, while 7d10-h and mers-27 antibodies together had an additive effect. consequently, 7d10 is currently the most comprehensively studied ntd-targeting mab with a different epitope and working mechanism, which makes it an excellent candidate, in combination with other rbd-targeting neutralizing antibodies or alone, in our battle against mers-cov infection. three weeks after the initial immunization, these mice were boosted twice at 2-week intervals. cells collected from the spleens of sacrificed animals were fused with cultured sp2/0 cells at a 10:1 ratio in the presence of peg1450 (sigma). hat selection medium was used for the fused hybridoma cultures. after 2-weeks of incubation, the positive hybridomas were selected via s-coated elisa, and the positive clones were subjected to limited dilutions and downstream validation. for large-scale mab production, ascites fluid from mice inoculated with the hybridomas was collected and purified by the caprylic acid-ammonium sulfate precipitation method. protein expression and purification. the coding sequence of the mers-cov spike glycoprotein ectodomain (emc strain, spike residues 1-1290) was ligated into the pfastbac-dual vector (invitrogen) with a c-terminal t4 fibritin trimerization domain and a hexa-his-strep tap tag to facilitate further purification processes. briefly, the protein was prepared using the bac-to-bac baculovirus expression system, purified by sequentially applying strep-tactin and superose 6 column (ge healthcare) with hbs buffer (10 mm hepes, ph 7.2, 150 mm nacl). fractions containing mers-cov s glycoprotein were pooled and concentrated for subsequent biochemical analyses. the sequence encoding mers-cov s1 ntd (residues 18-353) with a c-terminal hexa-his tag was inserted into the eukaryotic expression vector pvax. freestyle 293-f cells were transfected with the plasmid using polyethylenimine (pei) (sigma). after 72 h, the supernatant was collected and the ntd was purified using nta sepharose (ge heathcare) and superdex 200 high performance column (ge healthcare) with hbs buffer (10 mm hepes, ph 7.2, 150 mm nacl). the sequence encoding the 7d10 v l and v h were separately cloned into the backbone of antibody expression vectors containing the constant regions of human igg1. the chimeric antibody 7d10-h was expressed in freestyle 293-f cells by transient transfection and purified by affinity chromatography using protein a sepharose and gel-filtration chromatography. the purified 7d10-h was exchanged into phosphate-buffered saline (pbs), and was digested with papain protease (sigma) over night at 37°c. the digested antibody was then passed back over protein a sepharose to remove the fc fragment, and the unbound fab in the flow through was additionally purified using a superdex 200 high performance column (ge healthcare). the gene encoding the 7d10 v l followed by v h with a connecting triple gggs linker and a c-terminal hexa-his tag was synthesized and cloned into the eukaryotic expression vector pvrc8400. freestyle 293-f cells were transfected the plasmid in the presence of pei (sigma). the cell-culture supernatant was collected 72 h after the transfection, and the 7d10 scfv was collected and captured on nta sepharose (ge healthcare). the bound 7d10 scfv was eluted with hbs buffer containing 500 mm imidazole and was then further purified by gel-filtration chromatography using a superdex 200 high performance column (ge healthcare). complex preparation and crystallization. the mers-cov ntd and the scfv fragment of 7d10 were mixed at a molar ratio of 1:1.2, incubated for 2 h at 4°c and further purified by gel-filtration chromatography. the purified complex concentrated to approximately 10 mg ml −1 in hbs buffer (10 mm hepes, ph 7.2, data collection and structure determination. to collect the diffraction data, all crystals were flash-cooled in liquid nitrogen after being incubated in reservoir solution containing 20% (v/v) glycerol. the diffraction images were collected on the bl17u beamline at the shanghai synchrotron research facility (ssrf) 46 with the wavelength of 0.9796 å. all images were processed with hkl2000 47 . the structure was solved by molecular replacement using phaser from the ccp4 suite 48 . the search models were the mers-cov ntd structure (pdb id: 5vyh) and the structures of the variable domain of the heavy and light chains available in the pdb with the highest sequence identities. subsequent model building and refinement were performed using coot and phenix, respectively 49, 50 . there are 94% of most favored, 5.3% of allowed and 0.3% of disallowed ramachandran plot in the final refinement model. all structural figures were generated using pymol 51 . neutralizing assay of pseudotyped mers-cov. 293t cells cultured in 100 mm dish were co-transfected with 6 μg of pcdna3.1-mers-spike or its mutants and 24 μg of pnl4-3.luc.re. the supernatants containing sufficient pseudotyped mers-cov were harvested 48-72 h post-transfection. subsequently, the 50% tissue culture infectious dose (tcid 50 ) was determined by infection of huh7 cells. for the neutralization assay, 100 tcid 50 per well of pseudoytped virus were incubated with 16 or 8 serial 1:3 dilutions of purified antibodies, fabs or scfvs for 1 h at 37°c, after which huh7 cells (about 1.5 × 10 4 per well) were added. after incubation for 72 h at 37°c, the neutralizing activities of antibodies were determined by the luciferase activity and presented as ic 50 , calculated using the dose-response inhibition function in graphpad prism 5 (graphpad software inc.) cell entry of pseudotyped virus. the concentration of the harvested pseudotyped virions was normalized by p24 elisa kit (beijing quantobio biotechnology co., ltd., china) before infecting the target huh7 cells. the infected huh7 cells were lysed at 48 h after infection and viral entry efficiency was quantified by comparing the luciferase activity between pseudotyped viruses bearing the mutant-and wildtype mers-cov spike glycoproteins. postattachment neutralization assay. for the postattachment pseudotyped virus neutralization assay, huh7 cells, upon reaching a density of 1.5 × 10 4 per well in a 96-well plate, were incubated with 100 tcid 50 per well of pseudotyped virus at 4°c for 1 h. after removing the supernatant, 200 μl of pbs was added twice to each well to wash the un-bond pseudotyped viruses. a total of 16 serial 1:3 dilutions of purified antibodies in dmem (10% fbs) were then added to the huh 7 cells with attached pseudotyped viruses, as well as dmem (10% fbs) alone as control. neutralization activities were determined based on the luciferase activity after incubation for 72 h at 37°c and also presented as ic 50 , calculated using the dose-response inhibition function in graphpad prism 5 (graphpad software inc.) cooperativity of mabs for neutralization. synergistic, additive, and antagonistic interaction between 7d10 and mers-gd27, 7d10, and mers-27, as well as 7d10 and mers-4 for virus neutralization were evaluated by the median effect analysis method using compusyn software as previously reported 52, 53 . the measured neutralization values were input to the program as fractional effects (fa) ranging between 0.01 and 0.99 for each of the two antibodies and for both in combination. ci values were calculated in relation to fa values. a logarithmic ci value of 0 indicates an additive effect, <0 indicates synergism, and >0 indicates antagonism. live mers-cov neutralization assay. the neutralizing activity of the mabs against live mers-cov was also determined in dpp4-expressing vero e6 cells. upon reaching a density of 5 × 10 4 per well in a 12-well plate, cell monolayers were infected with 30-35 plaque-forming units (pfu) of live virus in the presence or absence of the mab. after three days of incubation at 37°c, the inhibitory capacity of the mabs was assessed by determining the numbers of plaques compared with the potent mers-cov anti-rbd and anti-n9 mabs. murine model of mers-cov pseudovirus infection. the mers-cov susceptible animal model hdpp4-knockin mouse, which was established by inserting human dipeptidyl peptidase 4 (hdpp4) into the rosa26 locus using crispr/cas9, resulting in global expression of the transgene in a genetically stable mouse line 28 , was used in this experiment. mice (n = 5) were challenged by intraperitoneal injection (i.p.) with doses of 1.27 × 10 7.5 tcid 50 of pseudotyped mers-cov. 7d10-h and mers-4 were administered i.p. to r26-hdpp4 mice at a dose of 200 μg per mouse prior to challenge with pseudovirus. mice (n = 4 for the pbs group and n = 3 for the 3c11 group) were also administered pbs or control mab 3c11 (mab of anti-na of h5n1, at a dose of 400 μg per mouse) and challenged using the same i.p. dose of pseudovirus. the ivis-lumina ii imaging system (xenogen, baltimore, md, usa) was used to detect bioluminescence. prior to measuring luminescence, the mice were anesthetized using an i.p. injection of sodium pentobarbital (240 mg kg −1 ). the exposure time was 60 s, and fluorescence intensity in regions of interest was analyzed using living image software (caliper life sciences, baltimore, md, usa). different wavelengths were used for detecting pseudovirus and tdtomato fluorescence. the substrate, d-luciferin (50 mg kg −1 , xenogen-caliper corp., alameda, ca, usa), was injected i.p. and imaging was conducted 10 min later. the relative intensities of emitted light were represented as colors ranging from red (intense) to blue (weak) and quantitatively presented as photon flux in photons s −1 cm −2 sr −1 . binding studies using bli. binding kinetics of mers-cov ntd and its mutants with 7d10 were studied using a fortébio octet htx instrument. assays with agitation set to 1000 rpm in hbs buffer (10 mm hepes, ph 7.2, 150 mm nacl) supplemented with 0.01% (v/v) tween 20 were performed at 25°c in solid black tilted-bottom 96-well plates (greiner bio-one). 7d10 (20 μg ml −1 ) was used to load anti-human igg fc capture probes for 300 s to capture levels of 0.5-1 nm. biosensor tips were then equilibrated for 300 s in hbs buffer supplemented with 0.01% (v/v) tween 20 prior to binding assessment with different concentrations of wild-type or mutant mers-cov ntd for 120 s, followed by dissociation for 240 s. data analysis and curve fitting were performed using octet software, version 9.0. binding competition assays by spr. real-time binding and analysis by spr were conducted on a biacore t200 instrument with cm5 chips (ge healthcare) at room temperature. for all the analyses, hbs buffer consisting of 10 mm hepes, ph 7.2, 150 mm nacl and 0.005% (v/v) tween 20 was used, and all proteins were exchanged to the same buffer. the blank channel of the chip was used as the negative control. dpp4 (20 μg ml −1 ) was immobilized on the chip at about 100 response units. soluble mers-cov spike trimer (s) at the same gradient in the present or absence of the concentration gradient of iggs, fabs, or scfvs was flowed over the chip surface. after each cycle, the sensor surface was regenerated with 7.5 mm naoh. data were analyzed using the biacore t200 evaluation software by fitting to a 1:1 langmuir binding model. facs analysis of cell-surface staining. the binding between recombinant soluble mers-cov spike trimer (s) and human dpp4 expressed on the surface of huh7 cells was measured using fluorescence-activated cell sorting (facs). all cellsurface staining experiments were performed at room temperature. soluble mers-cov spike trimer (s) with strep-tag (1 μg) was incubated with monoclonal antibodies (mabs) in advance at molar ratios of 1:1, 1:3, 1:9, and 1:27 for 1 h. huh7 cells were trypsinized and then incubated with s or s and mabs mixtures for 1 h. after washing the un-bound s with pbs 3 times, the huh7 cells were then stained with streptavidin apc (bd ebioscience) for another 45 min. cells were subsequently washed with pbs 5 times and analyzed by flow cytometry on a facs aria iii machine (bd ebiosciences). western blots. totally, 10 μl pseudotyped mers-cov was thawed and mixed with 2 μg of antibodies (igg, fab or scfv) for 1 h. the virus alone or the mixture was incubated with 20 μl of huh7 cell suspension for another 1 h at 37°c. an equal volume of buffer and proteinase-k (final concentration of 10 μg ml −1 ; thermo_fisher) was then added and incubated 1 h at 4°c. for the soluble s, 1 μg of the s trimer was incubated with 3 μg of the dpp4 ectodomain or 3 μg of 7d10 fab for 1 h on ice. trypsin (final concentration of 5 μg ml −1 ; thermo_-fisher) was then added to these samples and incubated 30 min at 37°c. subsequently, the samples were supplemented with 10 μg ml −1 proteinase-k and incubated 30 min at 4°c. 6× sds-page loading buffer was then added to all samples prior to boiling at 100°c. samples were run on a 4-12% gradient tris-mops-gel (genscript) and transferred to polyvinylidene fluoride membranes. an anti-s2 mers-cov s polyclonal antibody (1:2000 dilution; thermo_fisher; cat#pa5-81788) and an hrp-conjugated goat anti-rabbit secondary antibody (1:500 dilution; huaxingbio; cat#hx2027) were used for western blotting. ai600 was used to develop images. reporting summary. further information on research design is available in the nature research reporting summary linked to this article. the source data underlying figs. 1a-d, 3, 4, and 6a-c and supplementary figs. 1a-c, 3, 4, 7, 9-11 are provided as a source data file. crystal structures presented in this work has been deposited in the protein data bank (pdb) and are available with accession code 6j11. isolation of a novel coronavirus from a man with pneumonia in saudi arabia the emerging novel middle east respiratory syndrome coronavirus: the "knowns" and "unknowns middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation human infection with mers coronavirus after exposure to infected camels, saudi arabia environmental contamination and 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and allele-specific residues are critical for mers-cov neutralization by an exceptionally potent germline-like antibody structural basis for the neutralization of mers-cov by a human monoclonal antibody mers-27 structural definition of a unique neutralization epitope on the receptor-binding domain of mers-cov spike glycoprotein a novel neutralizing monoclonal antibody targeting the nterminal domain of the mers-cov spike protein receptor-binding domain of severe acute respiratory syndrome coronavirus spike protein contains multiple conformation-dependent epitopes that induce highly potent neutralizing antibodies a human dpp4-knockin mouse's susceptibility to infection by authentic and pseudotyped mers-cov spread, circulation, and evolution of the middle east respiratory syndrome coronavirus two-step conformational changes in a coronavirus envelope glycoprotein mediated by receptor binding and proteolysis unexpected receptor functional mimicry elucidates activation of coronavirus fusion 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native virions broadly neutralizing antibodies and the search for an hiv-1 vaccine: the end of the beginning towards a solution to mers: protective human monoclonal antibodies targeting different domains and functions of the merscoronavirus spike glycoprotein hiv therapy by a combination of broadly neutralizing antibodies in humanized mice human monoclonal antibodies against highly conserved hr1 and hr2 domains of the sars-cov spike protein are more broadly neutralizing improving neutralization potency and breadth by combining broadly reactive hiv-1 antibodies targeting major neutralization epitopes automatic crystal centring procedure at the ssrf macromolecular crystallography beamline processing of x-ray diffraction data collected in oscillation mode phaser crystallographic software coot: model-building tools for molecular graphics phenix: building new software for automated crystallographic structure determination pymod 2.0: improvements in protein sequence-structure analysis and homology modeling within pymol quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors drug combination studies and their synergy quantification using the chou-talalay method we would like thank dr. changfa fan (division of animal model research, institute for laboratory animal resources, national institutes for food and drug control) for help in providing the r26-hdpp4 mouse model and experimental method. we thank dr. jianhua he and the staff scientists at the ssrf bl17u beam line, as well as dr. shilong fan at the x-ray crystallography platform of the tsinghua university technology center for assistance in diffraction data collection. this work was supported by the national key plan for scientific research and development of china (grants 2016yfd0500307 and 2016yfd0500301), the national natural science foundation of china (grants 31470751 and u1405228), and the national major project for control and prevention of infectious disease in china (2016zx10004001-003). h.z., w.t., l.z. and x.w. designed the experiments. y.c., k.q. and w.t. isolated the antibody 7d10 and sequenced the corresponding v l and v h genes. b.h. carried out the neutralizing assay with live mers-cov. h.z. and s.z. expressed, purified, and crystallized the protein, and h.z. carried out the bli and spr analysis. h.z. conducted all the neutralizing assays based on pseudotyped mers-cov with the help of w.j. h.z. conducted dpp4-competition assays and the western blots analysis. p.n. performed the protection assay in mice. h.z. and j.l. collected the diffraction data. h.z. and x.w. processed the diffraction data, determined, and analyzed the structure. h.z. and x.w. wrote the paper with contributions from l.z. and w.t. supplementary information accompanies this paper at https://doi.org/10.1038/s41467-019-10897-4.competing interests: the authors declare no competing interests.reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ peer review information: nature communications thanks the anonymous reviewers for their contribution to the peer review of this work. peer reviewer reports are available.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. key: cord-333703-1ku3jc9s authors: kraus, aurora; casadei, elisa; huertas, mar; ye, chunyan; bradfute, steven; boudinot, pierre; levraud, jean-pierre; salinas, irene title: a zebrafish model for covid-19 recapitulates olfactory and cardiovascular pathophysiologies caused by sars-cov-2 date: 2020-11-08 journal: biorxiv doi: 10.1101/2020.11.06.368191 sha: doc_id: 333703 cord_uid: 1ku3jc9s the covid-19 pandemic has prompted the search for animal models that recapitulate the pathophysiology observed in humans infected with sars-cov-2 and allow rapid and high throughput testing of drugs and vaccines. exposure of larvae to sars-cov-2 spike (s) receptor binding domain (rbd) recombinant protein was sufficient to elevate larval heart rate and treatment with captopril, an ace inhibitor, reverted this effect. intranasal administration of sars-cov-2 s rbd in adult zebrafish recombinant protein caused severe olfactory and mild renal histopathology. zebrafish intranasally treated with sars-cov-2 s rbd became hyposmic within minutes and completely anosmic by 1 day to a broad-spectrum of odorants including bile acids and food. single cell rna-seq of the adult zebrafish olfactory organ indicated widespread loss of expression of olfactory receptors as well as inflammatory responses in sustentacular, endothelial, and myeloid cell clusters. exposure of wildtype zebrafish larvae to sars-cov-2 in water did not support active viral replication but caused a sustained inhibition of ace2 expression, triggered type 1 cytokine responses and inhibited type 2 cytokine responses. combined, our results establish adult and larval zebrafish as useful models to investigate pathophysiological effects of sars-cov-2 and perform pre-clinical drug testing and validation in an inexpensive, high throughput vertebrate model. 2 species that have been reported as naturally susceptible to sars-cov-2 include rhesus and 47 cynomolgus macaques (munster et al., 2020; rockx et al., 2020) , ferret (kim et al., 2020) , cat 48 (shi et al., 2020) , and syrian hamster (chan et al., 2020) . mice, by contrast, are not 49 spontaneously permissive to the virus, but mice expressing the human ace2 receptor provide a 50 useful animal model (bao et al., 2020; jia et al., 2020; lutz et al., 2020) . all these mammalian 51 models have unique advantages and disadvantages for the study of immune responses to sars-52 cov-2 and other host-pathogen interactions but do not allow rapid, whole organismal, high 53 throughput, and low-cost preclinical testing of drugs and immunotherapies. 54 55 as model vertebrates, zebrafish are permissive to human viral pathogens including influenza a 56 (gabor et al., 2014) , herpes simplex virus type 1 (burgos et al., 2008) , chikungunya virus 57 (palha et al., 2013) and human noroviruses gi and gii (van dycke et al., 2019). zebrafish offer 58 many advantages over other animal models due to their high reproductive ability, rapid 59 development, low maintenance costs, and small transparent bodies. importantly, zebrafish 60 olfactory, immune, and cardiovascular physiology share a significant degree of conservation 61 with humans (postlethwait et al., 2020; saraiva et al.) . genetically, more than 80% of disease 62 related genes have a zebrafish orthologue (howe et al., 2013) . the zebrafish innate immune 63 system is already developed in the transparent larval stages and members of all major groups of 64 mammalian cytokines have been identified in the zebrafish genome (gomes and mostowy, 2020; 65 zou and secombes, 2016) . academic laboratories and the pharmaceutical industry use zebrafish 66 larvae in preclinical studies for assessing efficacy and toxicity of candidate drugs for several 67 diseases (taylor et al., 2010) . zebrafish is a proposed model for covid-19 and has recently 68 been used in one vaccination study (galindo-villegas, 2020; ventura-fernandes et al., 2020). 69 this study aims to elucidate the physiopathology of wildtype zebrafish in response to sars-70 cov-2. 71 72 sars-cov-2 infection causes a wide litany of symptoms, ranging from asymptomatic to mild or 73 severe disease (menni et al., 2020) . apart from respiratory symptoms, multi-organ pathologies 74 are often reported with heterogeneous symptoms such as olfactory and taste loss, cardiac 75 dysfunction, renal pathologies, neurological damage, muscle and joint pain, gastrointestinal 76 symptoms, clotting disorders and others (tabata et mudd et al., 2020), and elevated type 2 cytokine levels (lucas et al., 2020) . importantly, this 84 cytokine pattern is in sharp contrast to that found in patients experiencing mild or moderate 85 symptoms, who are able to control exacerbated type 1 and type 3 cytokine responses (lucas et 86 al., 2020). 87 88 sars-cov-2 enters the human host cells when sars-cov-2 spike (s) protein receptor binding 89 domain (rbd) binds to angiotensin-converting enzyme 2 (ace2) on a permissive host cell, then 90 a serine protease, such as tmprss2, cleaves the spike protein s1/s2 site to facilitate fusion of 91 the virion with the host cell membrane (hoffmann et al., 2020a (hoffmann et al., , 2020b . ace2 is expressed in 92 many different cell types across many organs in the human body including lung, olfactory 93 sustentacular cells, enterocytes, and endothelial cells (albini et ace2, the use of ace inhibitors is being considered as a therapeutic intervention in covid-19 112 patients (lopes et al., 2020) . importantly, drugs currently used to treat the covid-19 can be 113 pro-arrhythmic and therefore there is a need to incorporate cardiovascular damage into the list of 114 targets of therapeutic interventions in covid-19 and for models that replicate human cardia 115 physiology (kochi et al., 2020) . 116 117 a hallmark of sars-cov-2 infection is acute loss of smell (cooper et al., 2020) . viral-induced 118 anosmia is not unique to sars-cov-2 infections since viruses such as rhinoviruses, influenza, 119 parainfluenza and coronaviruses are known to be the main cause of olfactory deficits in humans 120 (suzuki et al., 2007; imam et al., 2020) . in mice and humans, ace2 expression is detected in 121 sustentacular cells, olfactory stem cells known as horizontal and globose basal cells in the 122 olfactory epithelium, and vascular cells (pericytes) in the olfactory bulb (brann et al., 2020 the present study reports for the first time that zebrafish larvae exposed to sars-cov-2 appear 134 to mount innate immune responses that resemble cytokine responses of mild covid-19 patients. 135 recombinant sars-cov-2 s rbd is sufficient to cause olfactory, renal and cardiovascular 136 pathologies in larvae and adult zebrafish. we also identify potential mechanisms of sars-cov-137 2 induced anosmia by scrna-seq. our findings support the use of zebrafish as a novel 138 4 vertebrate model to elucidate sarscov-2 pathophysiology and to screen drugs and other 139 therapies targeting covid-19. 140 141 142 143 results 144 145 phylogenetic analyses of ace2 molecules in vertebrates 146 comparative analysis of ace2 molecules in vertebrates indicated that ace2 molecules are well 147 conserved in vertebrates with a 72%-73% similarity and 57.5%-58% identity between zebrafish 148 ace2 and human ace2, respectively (table s1 ). examination of ace2 amino acid motifs in 149 the region involved in binding sars-cov-2 s protein revealed zebrafish ace2 has 50%/64% 150 sequence similarity with the corresponding human ace2 region compared to 71%/78% in 151 macaques ace2 or 57%/71% in ferret ace2 ( systemic injection of recombinant sars-cov-2 protein into adult zebrafish has been shown to 159 induce some toxicity (ventura-fernandes et al., 2020). in order to determine whether 160 recombinant sars-cov-2 s rbd protein causes inflammatory responses in zebrafish larvae, we 161 exposed 5 dpf larvae to sars-cov-2 s rbd recombinant protein for 3 hours (h) and measured 162 cytokine responses by qpcr. as shown in figure 1a , 3 h immersion with sars-cov-2 s rbd 163 protein induced a significant downregulation in ifnphi1 expression and significant increase in 164 expression of ccl20a.3, a pro-inflammatory chemokine. no changes in ace2, tnfα, il1b and 165 il17a/f3 expression were observed ( figure 1a ). these results indicate that rapid immune 166 responses occur in zebrafish larvae exposed to sars-cov-2 s rbd. we next evaluated the 167 effects of sars-cov-2 rbd s on zebrafish larva heart function to validate zebrafish larvae as a 168 model for covid-19 cardiac manifestations. we immersed 7-and 5-days post fertilization (dpf) 169 zebrafish larvae with sars-cov-2 s rbd, or with vehicle, and measured heart rate after 3 h. 170 as shown in figure 1b , 7 dpf and 5 dpf zebrafish treated with sars-cov-2 s rbd had 171 significantly higher heart rates compared to vehicle treated controls. as a positive control for the 172 recombinant protein, we used animals treated with the same dose of recombinant infectious 173 hematopoietic necrosis virus (ihnv) glycoprotein (r-ihnvg), a rhabdovirus known to cause 174 severe endothelial damage in zebrafish (ludwig et al., 2011) . r-ihnvg caused a severe decrease 175 in larval zebrafish heart rate compared to control treated animals ( figure 1b -c). to determine if 176 increased heart rate induced by sars-cov-2 s rbd was dependent on ace2 binding, we co-177 incubated 5 dpf larvae with captopril and reverted sars-cov-2 s rbd induced heart 178 dysfunction. captopril had no effect on r-ihnvg induced bradycardia ( figure 1b ). ventricular 179 trace analyses showed marked differences in rhythm patterns in each treatment group ( figure 180 1d). importantly, the captopril and sars-cov-2 s rbd treated animals, despite having similar 181 heart rates to those of the vehicle treated controls, displayed a unique ventricular trace pattern, 182 warranting future studies regarding the potential cardioprotective role of captopril in combined, these results indicate that zebrafish exposed to sars-cov-2 s rbd protein 184 5 experience tachycardia and suggest that zebrafish larvae constitute a valuable pre-clinical model 185 to test the effects of drugs for covid-19 on cardiac activity in vivo. 186 187 zebrafish 189 anosmia is one of the earliest manifestations of sars-cov-2 infection in humans (cooper et 190 al., 2020) . we have previously shown that ihnv glycoprotein protein is sufficient to induce 191 rapid nasal immune responses as well as neuronal activation in teleost fish (sepahi et ( figure 2d ). loss of the epithelial mosaic structure characteristic of the teleost olfactory 202 epithelium was observed on days 1, 3 and 5 post-treatment ( figure 2e ). by day 5, loss of entire 203 apical lamellar areas due to severe necrosis was observed in the olfactory lamellae of all treated 204 animals compared to controls ( figure 2f ). significant loss of olfactory cilia was recorded in all 205 animals treated with sars-cov-2 s rbd at all time points ( figure 2h ). these results indicate 206 the sars-cov-2 s rbd is sufficient to cause inflammation, edema, hemorrhages, ciliary loss, 207 and necrosis in the olfactory organ of zebrafish. hence, olfactory damage can be caused by 208 indirect mechanisms and in the absence of active sars-cov-2 replication in this tissue. 209 210 toxicity effects of intranasal sars-cov-2 s rbd delivery were also evaluated in distant tissues 211 such as the kidney, a target organ of sars-cov-2. acute kidney injury (aki) incidence varies 212 from 0.9% to 29% in covid-19 patients (su et al., 2020) . renal damage, especially aki, is also 213 common in patients with ras dysfunction such as diabetic patients who suffer from 214 hypertension (ribeiro-oliveira et al., 2008; advani, 2020). a recent study in zebrafish injected 215 with the n-terminal part of sars-cov-2 s protein reported inflammation and damage in several 216 tissues of adult zebrafish including kidney 7 and 14 d post-injection (ventura-fernandes et al., 217 2020). in the present study, histological examination of the head-kidney of zebrafish who 218 received sars-cov-2 s rbd intranasally revealed renal tubule pathology characteristic of aki 219 3 h post-treatment ( figure s1 ). pathology was not as severe at later time points, but vacuolation 220 of the renal tubule epithelium was still visible 5 days post-treatment ( figure s1 ). we did not 221 observe signs of glomerulopathology in treated animals compared to controls. together, these 222 results indicate that intranasal delivery of sars-cov-2 s rbd is sufficient to cause 223 nephropathy in adult zebrafish but that pathology is not as severe as when the protein is delivered 224 by injection. 225 226 intranasal delivery of sars-cov-2 s rbd causes anosmia in adult zebrafish 227 228 adult zebrafish exposed to sars-cov-2 s rbd had a significant reduction of olfactory 229 responses to food extracts of ~50% of preexposure olfaction within minutes as measured by 230 6 electro-olfactogram (eog), indicating an instant effect of the protein on olfactory function (fig. 231 3a). reduction of olfaction was sustained for least one hour of recording, but the olfactory organ 232 remained still semi functional. zebrafish treated with pbs never lost olfaction at any time point. 233 to further quantify the degree of olfactory reduction due to sars-cov-2 s rbd, we took 234 advantage of the two easily accessible and isolated olfactory chambers present in zebrafish. we 235 exposed one naris to the sars-cov-2 s rbd protein and the other naris, from the same animal, 236 to pbs and waited 3 h or 1d before measuring olfaction by eog. at 3 h we observed a 37-70% 237 reduction in food and bile olfactory responses between the treated and untreated naris and a 238 complete loss of olfactory function to both odorants 1 d post-treatment (fig. 3b) . the reduction 239 of olfactory sensitivity for food extract was smaller than that found for bile, probably due to the 240 lower number of osns involved in bile acid detection compared to amino acids found in food 241 (hansen et al., 2003) . our results indicate that sars-cov-2 s rbd-induced-anosmia is not 242 specific for a subset of osns, since both food extracts and bile olfactory signals were suppressed 243 in sars-cov-2 s rbd treated zebrafish. this fact, together with the 1d time to develop 244 complete anosmia and disrupt olfactory epithelial structure, support the hypothesis that sars-245 cov-2 s rbd damage may occur first on sustentacular cells, with subsequent impacts on osn 246 viability and function. 247 single-cell analysis of the zebrafish olfactory organ 249 250 to understand the impact of sars-cov-2 s rbd on zebrafish oo, we performed single cell clusters, 3 endothelial cell (ec) clusters and 7 leucocyte (lymphoid and myeloid) clusters ( figure 258 4a-b). 259 of the 8 ncs, neuron1 and neuron2 corresponded to mature osns. neuron1 expressed markers 260 of ciliated osns (ompa and ompb) in addition to several olfactory receptor (or) genes 261 (buiakova et al., 1996) . neuron2, on the other hand, expressed markers of microvillus osns 262 (trpc2b, s100z, and gnao) and many vomeronasal receptors (vr) such as v2rh32 as well as or 263 gene ( figure s2 ) (kraemer et al., 2008 neuron7 and neuron8 expressed cell cycle and early neuronal progenitor markers as well as 278 tmprss13b and tmprss4a, however the majority of cluster identifying genes in these two clusters 279 are undescribed ( figure s2 ). 280 scs are supporting cells that exist in the neuroepithelium around osns and in humans, they 281 express ace2 (bryche et al., 2020) . scs in the olfactory epithelium can directly arise from 282 horizontal basal cells (hbcs) (yu and wu, 2017). we found 5 clusters of scs in our datasets. subpopulation closely related to the sustentacular4 cluster ( figure s2 ). 292 we identified three clusters of ecs that all express tmprss13 and tmprss4. while we did not 293 detect ace2 expression in any cell clusters, we detected ace2 mrna in adult zebrafish olfactory 294 organ, and at low levels in the olfactory bulb ( figure s3 ). ace2 expression levels have previously 295 shown to be low in neuronal tissues and therefore may be hard to detect by scrna-seq (song et 296 al., 2020). endothelial1 and 2 clusters expressed the endothelial markers sox7 and tmp4a (yao et 297 al., 2019). all three clusters broadly expressed genes associated with tight junctions (tjp3, jupa, 298 ppl, cldne, and cgnl1) as well as many keratin genes ( figure s2 ). interestingly, endothelial3 299 cluster also expressed the calcium channel trpv6 and a slew of non-annotated genes. 300 there are copious amounts of immune cells in the teleost olfactory organ ( intranasal delivery of sars-cov-2 s rbd induces inflammatory responses and 318 widespread loss of olfactory receptor expression in adult zebrafish olfactory organ 319 320 the cellular landscape of the zebrafish olfactory epithelium was affected by sars-cov-2 s 321 rbd treatment and time ( figure 4a -d). this was especially evident in the proportions of 322 8 neuronal cell types 3 d post-treatment when the proportion of mature, omp + ciliated osn was 323 much lower compared to controls and the 3 h treated group. in contrast, neuronal progenitors 324 expressing cell cycle markers (aubk, ecrg4, and mki67) and neuronal differentiation and 325 plasticity markers (neurod1, neurod4, gap43, sox11, and sox4) were expanded 3 d post-treatment 326 ( figure 4b -c). further, we detected a noticeable decrease in the proportion of cells belonging to 327 the lymphocyte2 cluster, a cluster that expressed markers of treg cells (foxp3b) 3 h post 328 intranasal delivery of recombinant sars-cov-2 s rbd but this change was not noticeable at 329 day 3 ( figure 4c ). at 3 d, we observed a third lymphocyte cluster, not detected at 3 h, highly 330 expressing the tcr subunit zap70 as well as plac8 onzin related protein (ponzr1), a molecule 331 that has immunoregulatory roles during th1 type immune responses in mammals ( figure s2 in olfactory neuronal clusters were enriched in processes such as neuron differentiation, sensory 367 system development, and sensory organ morphogenesis, whereas downregulated genes belonged 368 9 to sensory perception of smell, detection of chemical stimulus and gpcr signaling pathway 369 ( figure 5f ). functional enrichment analyses in metascape showed that the top non-redundant 370 enriched clusters in both upregulated and downregulated genes in zebrafish osns 3 h post-371 treatment was sensory perception of smell ( figure 5g ). the same was true 3 days post-treatment, 372 but processes such as regeneration, neuron development, neuron fate commitment and the p53 373 signaling pathway were also enriched within the upregulated genes ( figure 5h ). combined, 374 these results suggest that presence of sars-cov-2 s rbd in the olfactory organ instigates 375 harmful effects on osns within hours and that the magnitude of the osn damage increases by 3 376 days post-treatment. further, these analyses indicate that neuronal regeneration and 377 differentiation processes were initiated by day 3 in order to begin repair of olfactory damage. our study allowed us to dissect how each cell type in the zebrafish olfactory organ responds to 387 sars-cov-2 s rbd. our results indicated unique responses by sc clusters and ec clusters to 388 treatment (figures 4 and 6) . at 3 h, we detected increased expression of apoeb, of transcription 389 factors foxq1a and id2b, the transcriptional regulator nfil3-5, two tumor necrosis factor receptor 390 superfamily members (tnfrsf11b and tnfrsf9a) as well as tcima (transcriptional and immune 391 response regulator), whose mammalian ortholog pcim regulates immune responses as well as 392 endothelial cell activation and expression of inflammatory genes ( figure 6a ) (kim et al., 2009 ). 393 further, at 3 h we observed downregulation of the pro-inflammatory cytokine il17af/3 as well as 394 glutathione peroxidase gpx1b, the transcription factor notch1b, basal cell adhesion molecule 395 bcam, guanine nucleotide-binding protein subunit gamma gng8, and the calcium binding s100z 396 in sc and ec from sars-cov-2 s rbd treated olfactory organs relative to vehicle treated. at 3 397 d post-treatment, we observed significant increased expression of the gene that encodes brain 398 natriuretic peptide (nppc), a vasodilating hormone, the pro-inflammatory chemokine ccl19a.2, 399 the m2 macrophage marker arg2, the transcription factors foxq1a and sox11a, tubulin beta 5 400 tubb5, and the epithelial mitogen epgn, among others ( figure 6b ). downregulated genes at 3 d 401 post-treatment included hsp70.3, apoeb, the osteoblast specific factor b postb, the desmosomal 402 component periplakin (ppl), the vasoconstricting endothelin 2 (edn2), the heparin binding 403 molecule latexin (ltx) involved in pain and inflammation, and cd74b, a part of the mhc-ii 404 complex ( figure 6b ). combined, these data indicated immune regulatory responses in sc and 405 ec clusters early after sars-cov-2 s rbd treatment, followed by transcriptional changes with 406 potential vasoactive effects by day 3. 407 408 go and enrichment set analyses indicated that sc and ec clusters initially undergo 409 transcriptional changes enriched in metabolic responses, response to stress, and cell 410 differentiation ( figure 6c ). later on, at day 3, sc and ec responses were enriched in genes 411 involved not only in the stress response but also in immune responses and responses to wounding 412 ( figure 6d ). similar results were identified using metascape, which showed that the 413 inflammatory response to wounding was moderately enriched in the downregulated genes at 3 h, 414 whereas by day 3, response to wounding became the top enriched set among the upregulated 415 genes ( figure 6e -f). 416 417 exposure of wildtype zebrafish larvae with sars-cov-2 does not support viral replication 418 419 zebrafish larvae have been used as models to investigate several human viruses. infecting 420 zebrafish larvae in a bsl-3 laboratory by immersion in contaminated water is comparable to 421 infecting a cell line. we first checked the stability of sars-cov-2 in zebrafish water overtime, 422 in the absence of any animals. we found that sars-cov-2 viral loads in the water remained 423 stable throughout the experiment ( figure 7a -b). we exposed wildtype ab zebrafish larvae to 424 live sars-cov2 and examined viral mrna abundance over time to determine if zebrafish 425 larvae can support viral replication. we detected no increases in the viral n copy numbers over 426 time and a steady decline in e gene copy numbers in both water from wells containing larvae and 427 virus as well as in the larval tissue ( figure 7c -f). these results indicate that wild-type zebrafish 428 larvae cannot support efficient sars-cov-2 replication as suggested by the in silico 429 comparative sequence analyses of the zebrafish ace2 molecule. 430 431 exposure of zebrafish larvae to sars-cov-2 decreases ace2 expression and triggers pro-432 inflammatory cytokine responses 433 434 in order to determine whether exposure of zebrafish larvae with live sars-cov-2 causes 435 changes in ai, we measured ace2 mrna levels in control and sars-cov-2 exposed larvae over 436 time. ace2 expression was significantly downregulated as early as 6 h post-infection. ace2 437 expression inhibition was sustained over the time course of the experiment with the greatest 438 decrease occurring 2 days post-infection (dpi) ( figure 8a ). we next evaluated changes in 439 expression of cytokine and chemokine genes to establish whether zebrafish mount inflammatory 440 responses that resemble the patterns of mild or severe sars-cov-2 infection. il1β expression 441 was significantly upregulated at 6h, 1 dpi (2-3 fold) and 2 dpi (10 fold) and significantly 442 downregulated at 4 dpi ( figure 8b ). we detected a significant increase in tnfa expression in 443 sars-cov-2 exposed larvae 2 dpi ( figure 8c ). ifnphi1 and ifnphi3 are the two main type i ifn 444 genes involved in larval zebrafish antiviral responses (levraud et al., 2019) . we detected a 445 significant up-regulation of ifnphi1 at 1 and 2 dpi, whereas expression was inhibited at 4 dpi. 446 interestingly, ifnphi3 expression followed a very different pattern compared to ifnphi1, which 447 was significantly downregulated 2 dpi but significantly upregulated at 4 dpi ( figure 8d -e). mxa 448 expression was significantly downregulated at all time points ( figure 8f ). il17af/3 expression 449 was significantly elevated 1, 2 and 4 dpi ( figure 8g ). expression levels of il22, a member of the 450 il10 family, were downregulated 6 hpi and 1 dpi ( figure 8h ) whereas the type ii cytokine 451 il4/il13b was downregulated at 6 hpi, 1 dpi and 2 dpi ( figure 8i ). further, a significant increase 452 in the expression of the chemokine ccl20a.3 was detected in infected larvae at 1 and 2 dpi 453 compared to controls ( figure 8j) . a moderate increase in ccl19a.1 expression was observed at 2 454 dpi followed by a strong down-regulation (15 fold) at 4 dpi ( figure 8k ). taken together, these 455 data indicate that exposure to sars-cov-2 induces a significant antiviral and pro-inflammatory 456 immune response in wildtype zebrafish larvae. this response involved type i ifn, tnfa, il1b, il17 457 and ccl20, reminiscent of covid-19 patients with mild disease. 458 459 the current covid-19 pandemic has propelled the investigation of sars-cov-2 and the 461 development of animal models that help identify therapeutic interventions and vaccines for 462 covid-19. thus far, all animal models reported are mammals, and therefore breeding, genetic 463 manipulation, and animal housing in bsl-3 laboratories make these models costly and not 464 readily available in large numbers. zebrafish can overcome many of the limitations of 465 mammalian models thanks to their transparent bodies, short life-span, low maintenance costs and 466 production of large numbers of embryos. we therefore performed the simplest infection 467 procedure, where sars-cov-2 was added to the water of zebrafish larvae. in this manner, bsl-468 3 trained personnel with no experience in zebrafish microinjection can readily expose larvae to 469 sars-cov-2 without the need of animal protocols in a similar fashion to in vitro cell culture 470 infections. exposure of wildtype zebrafish larvae to sars-cov-2 in the water did not however 471 result in any detectable viral replication. downregulated in response to sars-cov-2 exposure. combined, these data suggest the sars-505 cov-2 induces some type i ifn responses in zebrafish larvae while inhibits others. future 506 12 studies are clearly needed to ascertain the role of teleost type i ifn in the anti sars-cov-2 507 immune response. 508 509 s protein is a structural protein of sars-cov-2 and therefore the target of several vaccine trials. 510 therefore, we exposed zebrafish larvae to sars-cov-2 s rbd protein and investigated 511 transcriptional and physiological responses. rapid changes in gene expression were detected in 512 treated larvae, including up-regulation of the chemokine ccl20a.3 and the down-regulation of 513 ifnphi3. the ccl19/ccl20 axis appears to be critical in teleost antiviral innate responses, as 514 previous studies have shown very rapid responses in larvae exposed to the rhabdovirus svcv 515 (sepahi et al., 2019) . this change was also detected in the larvae that were exposed to the live 516 sars-cov-2 virus in the present study. we further detected a significant down-regulation of 517 type i ifn ifnphi1 gene in larvae exposed to sars-cov-2 s rbd protein. 518 519 examination of ace2 transcriptional changes in zebrafish larvae exposed to sars-cov-2 520 revealed a consistent down-regulation in expression throughout the course of infection. 521 interestingly, we did not observe any changes in ace2 expression after 3h immersion with sars-522 cov-2 s rbd protein. recently, enterocytes were found to be the main cell type expressing 523 ace2 in 5 dpf-old zebrafish larvae (postlethwait et al., 2020); and therefore it is possible that the 524 down-regulation in ace2 expression observed in our experiments was the result of enterocyte 525 responses to sars-cov-2. however, an olfactory epithelial cell cluster was not identified in this 526 dataset, probably because these cells constitute too small a fraction of the cells of an entire larva. 527 importantly, we exposed larvae to the virus at 3 dpf, when the olfactory pit is already sampling 528 the surrounding water, while the gut fully opens only at 4 dpf. thus, changes in ace2 expression 529 levels in the olfactory pit of the zebrafish larvae cannot be ruled out at this point. 530 531 previous work has shown that ace2 knockdown in mice protects from sars-cov infection 532 (kuba et al., 2005) . thus, down-regulation of zebrafish ace2 expression may have protected 533 larvae from sars-cov-2 infection in our experiments. our data agree with studies in mouse 534 lungs, where suppression of ace2 gene expression was consistently observed following sars-535 cov-2 infection (chen et al., 2020). interestingly, changes in ace2 levels can occur in response 536 viruses that do not require ace2 for host entry (chen et al., 2020). thus, although further 537 studies are warranted, our data suggest that ace2 is involved in antiviral sars-cov-2 responses 538 in zebrafish. 539 540 we took advantage of the zebrafish fish model which allows for easy live imaging of heart beats 541 in transparent larvae. we detected in vivo cardiac/heart responses in larval zebrafish exposed to 542 sars-cov-2 s rbd protein characterized by tachycardia. cardiac arrhythmia is a common 543 symptom among covid-19 patients and current research efforts aim to understand how sars-544 cov-2 infection impacts cardiovascular function (libby, 2020) . our findings underscore that 545 sars-cov-2 s rbd is able to cause tachycardia in the zebrafish larval model and that this 546 model can be used for rapid evaluation of drug treatments for covid-19. as a proof of concept, 547 we used captopril, an ace inhibitor currently being evaluated in human clinical trials 548 (nct04355429). captopril treatment ameliorated tachycardia in zebrafish larvae exposed to 549 sars-cov-2 s rbd recombinant protein. our studies therefore suggest the beneficial use of 550 captopril in covid-19 patients undergoing cardiac arrhythmia, but clearly further studies are 551 13 required to fully translate these findings to the clinic and to determine the duration and timing of 552 captopril treatment in covid-19 patients. 553 554 a recent report in zebrafish adults indicated that injection of recombinant sars-cov-2 s n 555 terminal protein caused histopathology of several tissues including the liver, kidney, brain and 556 ovary (ventura-fernandes et al., 2020). additionally, some animals succumbed to injection with 557 the recombinant protein. we did not detect any mortalities neither in larvae nor in adults in any 558 of our experiments, perhaps suggesting that mortalities were due to the injection procedure rather 559 than the protein treatment itself. of note, the dose used in the present study was considerably 560 lower than the dose delivered in the ventura-fernadez study, perhaps explaining the differences 561 in toxicity between both studies. we observed histological damage following a single intranasal 562 delivery of sars-cov-2 protein, specifically at the site of delivery, the olfactory organ, whereas 563 more transient and moderate damage was detected in the renal tubules. renal damage may have 564 occurred by direct uptake of sars-cov-2 s rbd by the kidney once the protein reached the 565 bloodstream following intranasal administration or, alternatively, by activation of ras or 566 inflammatory cascades at the olfactory organ. thus, toxicity of sars-cov-2 s protein in adult 567 zebrafish may be less severe when delivered intranasally than by injection, and future studies 568 should evaluate whether current vaccine candidates also exert similar effects and whether 569 different administration routes cause the same side-effects or not. this is particularly important 570 as the intranasal route appears promising for some vaccine candidates study did not determine when zebrafish recover olfactory function following sars-cov-2 s 597 14 rbd intranasal treatment, but based on our histopathological observations, the olfactory organ 598 was still severely damaged 5 days after treatment, suggesting that recovery of olfactory function 599 may take several weeks in our model. our findings therefore indicate that similar to humans, 600 zebrafish suffer from olfactory pathology and loss of smell in response to sars-cov-2 s rbd 601 protein. thus, olfactory pathophysiology appears to occur even in the absence of viral replication 602 raising the possibility that nasal vaccines for covid-19 may also cause transient anosmia in 603 humans. 604 605 in conclusion, the present study reports that both adult and larval wild-type zebrafish can be 606 useful models to advance our understanding of covid zebrafish larvae in responses to sars-cov-2 s rbd. animals were exposed to sars-cov-2 s 639 rbd protein (r-spike, 2 ng/ml) for 3 h at 28.5°c or vehicle. changes in gene expression were 640 measured by rt-qpcr using rps11 as the house-keeping gene. each data point represents a pool 641 of 4 larvae/well. data are expressed fold-change compared to vehicle controls using the pffafl 642 method. 643 (b) average heart beat per minute of 7 dpf (n=6) zebrafish larvae after 3 h of incubation with 644 vehicle, 2 ng/ml r-spike, or 2 ng/ml r-ihnvg. 645 (c) average zebrafish heart beats per minute in 5 dpf zebrafish larvae (n=8) after 3 h of 646 incubation with vehicle, 2 ng/ml r-spike, or 2 ng/ml r-ihnvg with and without treatment with 647 12mm of captopril. heart beats were recorded for 3 min at 20 under a nikon ti microscope 648 and (c-d) mean viral loads quantified as log of sars-cov-2 n gene and sars-cov-2 e gene copy 726 numbers in control supernatants from well with larvae not exposed to virus, and supernatants 727 from wells with larvae that were exposed to 10 4 pfu of sars-cov-2 for 6 h, 1 d, 2 d and 4 d. 728 each sample represents the supernatant of one well containing 12 larvae. 729 (e-f) mean viral loads quantified as log of sars-cov-2 n gene and sars-cov-2 e gene copy 730 numbers in control larvae and larvae exposed to 10 4 pfu of sars-cov-2 for 6 h, 1 d, 2 d and 4 731 d. each sample point represents one well containing 12 larvae. 732 larval infections began at 2 dpf after mechanical dechorionation at 1 dpf. * p-value<0.05; ** p-733 value<0.01 *** p-value<0.001. results are representative of two independent experiments. 734 735 for all experiments, wild type ab zebrafish were obtained from zirc (oregon, usa). for the 764 intranasal delivery of sars-cov-2 s rbd protein into adult zebrafish, female and male adult 765 zebrafish were obtained from dr. wong's laboratory at the university of nebraska due to 766 lockdown of zirc during the pandemic. all fish were maintained in a filtered aquarium system 767 at 28℃ with a 14 h light and 10 h dark cycle at the university of new mexico aquatics animal 768 facility. all experiments with adults utilized a mix of male and female animals, and the larvae 769 sex is indeterminable. animals were fed ad libitum gemma complete nutrition (skretting). ab 770 larvae were obtained by batch-crossing ab adults allowing for natural fertilization. the morning 771 of fertilization, larvae were collected at n=50 per petri dish and kept in e3 medium containing 772 0.002% methylene blue. in the afternoon, larvae were placed in fresh e3 medium without 773 methylene blue and non-surviving embryos were removed. larvae were maintained at 28.5℃ in 774 e3 medium until 4dpf when they are slowly changed to system water. 775 776 sars-cov-2 777 the sars-cov-2 isolate, a cdc isolate from a us patient (usa-wa1/2020), was obtained 778 from bei resources. the strain was grown at a low moi to minimize generation of 779 noninfectious particles and low passaged virus was used in all experiments described. the virus 780 was propagated on vero e6 cells and viral loads quantitated by rt-qpcr and by plaque forming 781 assays as we have described previously (bradfute et al, 2020). 782 783 intranasal delivery of sars-cov-2 s rbd recombinant protein to adult zebrafish 785 adult zebrafish were anesthetized for 1 min in 0.04 mg/ml tricaine-s (syndel) solution and then 786 moved to an absorbent boat where their gills were still covered with anesthetic solution for 787 administration of solutions to nares. using a microloader tip (eppendorf, 930001007), 5 μl of 20 788 ng/μl sars-cov-2 s rbd (kindly provided by dr. f. krammer) was directly pipetted into each 789 naris, while 5 μl of sterile pbs was applied in control fish. after inoculation, animals were 790 recovered in a separate tank supplemented with o2 before returning to their rearing tank until the 791 end of the experiment. euthanasia was performed on ice to ensure rapid death without perturbing 792 the combined supernatants were centrifuged for 10 min at 400g in supplemented neurobasal medium 820 and cells were counted with a hemocytometer. viability was estimated by trypan blue staining. 821 cells were then strained twice through flowmi 10 μm strainers and loaded onto the chromium 822 controller with a viability of > 85%. cell libraries were generated according to 10x genomics 823 protocols at the university of new mexico cancer center genomics core facility and sequenced 824 on an illumina novaseq 6000 at the university of colorado genomics and microarray core 825 facility. sequencing depth and statistics of the scrna-seq run are shown in figure s2 . sras for 826 this project can be found on ncbi under bioproject #prjna668529. 827 fastqs were run through the cell ranger v3.0 pipeline with default settings using the grcz11 828 zebrafish genome. output matrices were loaded to r (v1.2.5001) as a seurat object (package 829 seurat v3.1.1). first, cells with less than 200 or greater than 2500 features, and greater than 5% 830 mitochondrial features were removed. after counts were normalized using the "lognormalize" 831 method and a scale factor of 10000, 2000 variable genes were selected using the 'vst" method. 832 data was scaled, and pca dimensional reduction was run. jackstraw analysis determined the 833 vehicle control to have 38 significant principal components (pcs) and the treated samples to 834 have 40 significant pcs which were used for clustering analysis. sars-cov-2 s rbd treated 835 samples were integrated with the vehicle treated sample and clustered together using 30 836 significant pcs and a resolution of 0.5. cluster markers were identified with "findallmarkers" 837 in seurat and exported for cluster identification. differential expression analysis was done with 838 seurat "findmarkers" in default settings for each cluster and exported for gene ontology 839 analysis. 840 gene ontology (go) analysis was done with web-based guis metascape and shinygo v0.61 841 which draw multiple currently maintained databases (ensembl, entrz, kegg among others). 842 biological process webs were created using biological process output from shinygo v0.61 in 843 prism graphpad. biological processes bar graphs were produced by metascape. 844 845 electro-olfactogram recordings 846 adult ab zebrafish were anesthetized and received 2 µl of recombinant sars-cov-2 s rbd 847 protein (50 ng/µl) in pbs or pbs alone. after 3 h or 1 day, zebrafish were anaesthetized (0.1 g 848 ms222/l), placed in a v-shape stand and supplied with aerated water containing ms222 849 anesthetic (0.05 g/l). the nasal flap was removed with sterile fine forceps to expose the 850 olfactory rosettes to a continuous tank water source. olfactory responses to zebrafish food 851 extract or goldfish bile were measured by electrical recordings as detailed in sepahi et al., 2019. 852 the food extract was prepared as a filtered solution of 1l tap water and 0.1 g of dry food pellets. 853 water food extracts were separated in 200 ml aliquots and kept frozen until the recording day. a 854 0.5 ml mix of bile fluid from 50 adult goldfish was aliquoted in 10 µl and kept frozen until the 855 day of the recording. before each recording, bile aliquots were diluted 1:1000 in water from the 856 eog system. there were no significant differences in olfactory responses between males and 857 females, hence responses of both sexes were averaged together. the percentage reduction in 858 olfactory activity was calculated by dividing the amplitude of the olfactory signal at time x by 859 amplitude of the olfactory signal at time 0 *100. percentage of olfactory signal reduction 860 between control and treated naris was calculated as follows (amplitude response to odorant in 861 control naris (mv) -amplitude response to odorant in treated naris (mv))/amplitude response to 862 odorant in control naris (mv)) * 100. 863 (sigma) at 42℃, stabilized for 5 min at rt and then imaged to record heart-beat activity. 872 873 zebrafish larvae heart-beat recordings and analysis 874 as the agarose solidified, animals were adjusted to the microscope stage (approx. 5 min) then 875 hearts were recorded using brightfield avi for 3 min at 16.667 frames/s at rt. avi images were 876 then opened in imagej with the time series analyzer v3 plugin. circular rois were drawn in 877 either the atrium or ventricle and average intensity was extracted. the maximum average 878 intensity peaks were identified and counted per 60s as bpm. data were analyzed by one-way 879 anova with tukey's post hoc. 880 881 infection of zebrafish fish larvae with sars-cov-2 882 ten animals were placed in each well in 12-well plates containing 2 ml of tank water and 883 transferred to bsl3 facility the day before infection. gene expression analyses by rt-qpcr 903 whole larvae rna was extracted using trizol. for tissue homogenization bead beater tubes are 904 preloaded with 1.5 g 1.0 mm dia zirconia beads, 1.5 g 2.0 mm zirconia beads and 500 µl trizol. 905 samples were loaded into the tubes and bead beat at 4350 rpm for 45 s. tubes were then 906 centrifuged at 7,000 rpm for 7 min. the homogenate/lysates were transferred to clean 1.5 ml 907 microfuge tubes and spun at 7,000 rpm for 7 min to pellet debris. supernatants were then 908 processed to extract the total rna using a standard chloroform/phenol extraction protocol. rna 909 was quantified by nanodrop and samples were normalized and 1 µg of rna was used to 910 synthesize cdna using the superscript iii first strand system (thermofisher, 18080051). qpcr 911 was performed using ssoadvanced supermix (biorad, 1725270) and primers listed in table s3 912 (supplemental methods). gene expression changes were quantified using the pfaffl method 913 (pfaffl, 2001 glycerophospholipid biosynthesis apoptosis microtubule polymerization or depolymerization gpcr signaling, coupled to cyclic nucleotide 2nd messenger negative regulation of cell differentiation iron uptake and transport circadian regulation of gene expression sensory perception of smell developmental process anatomical structure development response to wounding response to stress immune system response cell chemotaxis multicellular organism development acute kidney injury: a bona fide complication of diabetes the sars-cov-2 990 receptor, ace-2, is expressed on many different cell types: implications for ace-inhibitor-and 991 angiotensin ii receptor blocker-based cardiovascular therapies myocardial injury and covid-19: possible mechanisms savalan the pathogenicity of sars-cov-2 in hace2 transgenic mice the establishment 1000 of neuronal properties is controlled by sox4 and sox11 imbalanced host response to sars-cov-2 1007 drives development of covid-19 macrophage-mediated neuroprotection and neurogenesis in the olfactory 1010 epithelium cov-2 entry genes in the olfactory system suggests mechanisms underlying covid-19-1015 associated anosmia massive transient damage of the olfactory 1019 epithelium associated with infection of sustentacular cells by sars-cov-2 in golden syrian 1020 hamsters olfactory marker protein (omp) gene 1024 deletion causes altered physiological activity of olfactory sensory neurons zebrafish as a new model for herpes simplex virus type 1 infection integrating single-cell 1031 transcriptomic data across different conditions, technologies, and species simulation of the clinical and pathological 1036 manifestations of coronavirus disease 2019 (covid-19) in golden syrian hamster model: 1037 implications for disease pathogenesis and transmissibility individual variation of the sars-cov-2 receptor ace2 gene expression and regulation acute inflammation regulates neuroregeneration 1044 through the nf-κb pathway in olfactory epithelium chronic inflammation directs an olfactory stem 1047 cell functional switch from neuroregeneration to immune defense covid-19 and the chemical 1052 senses: supporting players take center stage keiland broad host range of sars-cov-2 1056 predicted by comparative and structural analysis of ace2 in vertebrates smell 1060 and taste disorders during covid-19 outbreak: cross-sectional study on 355 patients an inflammatory cytokine 1065 signature predicts covid-19 severity and survival promoter transgenes direct macrophage-lineage expression in zebrafish expressions and significances of the angiotensin-converting enzyme 2 gene, the receptor of 1072 sars-cov-2 for covid-19 influenza a virus infection in zebrafish recapitulates mammalian 1076 infection and sensitivity to anti-influenza drug treatment covid-19: real-time dissemination of scientific information to 1079 fight a public health emergency of international concern a mechanistic model and therapeutic 1083 interventions for covid-19 involving a ras-mediated bradykinin storm shinygo: a graphical gene-set enrichment tool for 1086 animals and plants epigenetic contribution of high-1089 mobility group a proteins to stem cell properties differential downregulation of ace2 by the 1093 spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus 1094 nl63 the case for modeling human infection in zebrafish covid-19 and the 1101 cardiovascular system: implications for risk assessment, diagnosis, and treatment options impaired type i interferon activity and 1106 exacerbated inflammatory responses in severe covid-19 patients crystal structure of 1109 zebrafish interferons i and ii reveals conservation of type i interferon structure in vertebrates correlation between olfactory receptor cell type and function in the channel catfish a multibasic cleavage site in the 1117 spike protein of sars-cov-2 is essential for infection of human lung cells sars-cov-2 cell entry 1122 depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor the zebrafish reference genome 1127 sequence and its relationship to the human genome angiotensin-converting enzyme 2 protects from severe acute lung failure is sars-cov-2 1134 (covid-19) postviral olfactory dysfunction (pvod) different from other pvod? ace2 mouse models: a toolbox for cardiovascular 1139 and pulmonary research tc1(c8orf4) is a novel endothelial inflammatory regulator enhancing nf-κb activity infection and rapid transmission of sars-cov-2 in ferrets cardiac and 1150 arrhythmic complications in patients with covid-19 structural and functional 1154 diversification in the teleost s100 family of calcium-binding proteins intranasal vaccination with a lentiviral vector strongly 1158 protects against sars-cov-2 in mouse and golden hamster preclinical models a crucial role of angiotensin converting enzyme 2 (ace2) in sars 1163 coronavirus-induced lung injury arrhythmias and sudden cardiac death in the covid-19 pandemic sars-cov-2 1170 productively infects human gut enterocytes genes in zebrafish and humans define an ancient arsenal of antiviral immunity angiopoietin-like 4 increases pulmonary tissue leakiness and 1178 damage during influenza pneumonia the heart in covid-19: primary target or secondary bystander? jacc basic 1181 to composition and divergence of coronavirus spike proteins and host ace2 receptors 1185 predict potential intermediate hosts of sars-cov-2 continuing versus suspending angiotensin-converting enzyme inhibitors and angiotensin 1190 receptor blockers : impact on adverse outcomes in hospitalized patients with severe acute 1191 respiratory syndrome coronavirus 2 ( sars-cov-2 ) -the brace corona trial longitudinal analyses reveal immunological misfiring in 1196 severe covid-19 whole-body analysis of a viral infection: vascular 1200 endothelium is a primary target of infectious hematopoietic necrosis virus in zebrafish 1201 larvae covid-19 preclinical models: human 1204 angiotensin-converting enzyme 2 transgenic mice characterization of the 1207 immune barrier in human olfactory mucosa real-time tracking of self-1211 reported symptoms to predict potential covid-19 targeted immunosuppression 1215 distinguishes covid-19 from influenza in moderate and severe disease respiratory disease 1220 in rhesus macaques inoculated with sars-cov-2 real-time whole-body visualization 1224 of chikungunya virus infection and host interferon response in zebrafish cc chemokine receptor 9 expression defines a 1229 subset of peripheral blood lymphocytes with mucosal t cell phenotype and th1 or t-1230 regulatory 1 cytokine profile type i and type iii interferons -induction evasion, and application to combat covid-19 angiotensin ii induced proteolytic cleavage of myocardial ace2 is 1237 mediated by tace/adam-17: a positive feedback mechanism in the activating a reserve neural stem cell population in vitro 1242 a new mathematical model for relative quantification in real-time rt-1245 pcr an intestinal cell type in zebrafish 1248 is the nexus for the sars-cov-2 receptor and the renin angiotensin-aldosterone system that 1249 contributes to covid-19 comorbidities the renin-angiotensin system and diabetes: an update. vasc. 1253 health risk manag comparative 1257 pathogenesis of covid-19, mers, and sars in a nonhuman primate model innate immune signaling in the 1262 olfactory epithelium reduces odorant receptor levels: modeling transient smell loss in covid-19 1263 patients interplay between sars-cov-2 1266 and the type i interferon response molecular and neuronal homology between the olfactory systems of zebrafish and mouse. sci. 1270 rep tissue 1273 microenvironments in the nasal epithelium of rainbow trout ( oncorhynchus mykiss two distinct cd8α + cell populations and establish regional immunity olfactory sensory neurons 1279 mediate ultrarapid antiviral immune responses in a trka-dependent manner coronavirus 2 placenta-specific 8 limits ifnγ production by cd4 t cells in vitro and promotes establishment of 1288 influenza-specific cd8 t cells in vivo neuroinvasion of sars-cov-2 in human and mouse brain alterations in smell or taste in mildly symptomatic outpatients with sars-1296 cov-2 infection renal histopathological analysis of 26 postmortem findings of patients with 1300 covid-19 in china identification of viruses in patients with postviral olfactory dysfunction clinical characteristics of covid-19 in 104 people with 1309 sars-cov-2 infection on the diamond princess cruise ship: a retrospective analysis small molecule screening in 1313 zebrafish: an in vivo approach to identifying new chemical tools and drug leads proinflammatory cytokines in the olfactory mucosa result in covid-19 induced anosmia a robust human norovirus 1323 replication model in zebrafish larvae zebrafish 1327 studies on the vaccine candidate to covid-19, the spike protein: production of antibody and 1328 adverse reaction a rampage through the body neuronal wiskott-aldrich syndrome protein regulates 1335 tgf-β1-mediated lung vascular permeability receptor recognition by the 1338 novel coronavirus from wuhan: an analysis based on decade-long structural studies of 1339 sars coronavirus clinical characteristics of 138 hospitalized patients with coronavirus-infected pneumonia in wuhan, china remdesivir and chloroquine effectively inhibit the recently emerged novel 1348 coronavirus (2019-ncov) in vitro the zebrafish activating immune receptor nitr9 signals via 1352 dap12 sars and mers: 1355 recent insights into emerging coronaviruses sox transcription factors in endothelial 1358 differentiation and endothelial-mesenchymal transitions spatiotemporal photolabeling of neutrophil trafficking 1361 during inflammation in live zebrafish regeneration and rewiring of rodent olfactory sensory neurons severe acute respiratory syndrome coronavirus 2 infects and 1368 damages the mature and immature olfactory sensory neurons of hamsters angiomotin-like protein 1 controls endothelial 1374 polarity and junction stability during sprouting angiogenesis metascape provides a biologist-oriented resource for the analysis of 1378 systems-level datasets clinical 1381 characteristics of 3062 covid-19 patients: a meta-analysis sars-cov-2 receptor ace2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell 1386 subsets across tissues the function of fish cytokines single-cell rna-seq data 1391 analysis on the receptor ace2 expression reveals the potential risk of different human organs 1392 vulnerable to 2019-ncov infection key: cord-355807-q3bngari authors: yepes-pérez, andres f.; herrera-calderon, oscar; quintero-saumeth, jorge title: uncaria tomentosa (cat’s claw): a promising herbal medicine against sars-cov-2/ace-2 junction and sars-cov-2 spike protein based on molecular modeling date: 2020-10-29 journal: journal of biomolecular structure & dynamics doi: 10.1080/07391102.2020.1837676 sha: doc_id: 355807 cord_uid: q3bngari covid-19 is a novel severe acute respiratory syndrome coronavirus. currently, there is no effective treatment and vaccines seem to be the solution in the future. virtual screening of potential drugs against the s protein of severe acute respiratory syndrome corona virus 2 (sars-cov-2) has provided small molecular compounds with a high binding affinity. unfortunately, most of these drugs do not attach with the binding interface of the receptor-binding domain (rbd)–angiotensin-converting enzyme-2 (ace-2) complex in host cells. molecular modeling was carried out to evaluate the potential antiviral properties of the components of the medicinal herb uncaria tomentosa (cat’s claw) focusing on the binding interface of the rbd–ace-2 and the viral spike protein. the in silico approach starts with protein–ligand docking of 26 cat’s claw key components followed by molecular dynamics simulations and re-docked calculations. finally, we carried out drug-likeness calculations for the most qualified cat’s claw components. the structural bioinformatics approaches led to the identification of several bioactive compounds of u. tomentosa with potential therapeutic effect by dual strong interaction with interface of the rbd–ace-2 and the ace-2 binding site on sars-cov-2 rbd viral spike. in addition, in silico drug-likeness indices for these components were calculated and showed good predicted therapeutic profiles of these phytochemicals found in u. tomentosa (cat’s claw). our findings suggest the potential effectiveness of cat’s claw as complementary and/or alternative medicine for covid-19 treatment. communicated by ramaswamy h. sarma the severe acute respiratory syndrome corona virus 2 (sars-cov-2) is a part of coronavirus family (cov) and was initially identified in wuhan, china. covid-19 (coronavirus disease 2019) is highly contagious in humans, which has rapidly spread and caused an unprecedented pandemic, with a large number of deaths and economic crisis in the world (prajapat et al., 2020) . according to the latest report of the world health organization (who), over 32.7 million cases and 991 000 deaths of covid-19 were confirmed as of september 27, 2020 (world health organization, 2020 . in developing countries of latin america and the carribean, the public health has been the most affected because people do not have the opportunities to access a modern health system and medicines . phytotherapy based on natural products might be a proper alternative for treating viral diseases (akram et al., 2018) . according to who estimates, about 80% of the population in developing countries uses traditional medicine in primary health care, mainly medicinal plants (world health organization, 2018) . the selection of natural products for the study of their biological properties has been addressed through three fundamental methodologies: the selection of random natural sources, the selection based on chemotaxonomy (screening of similar compounds in organisms belonging to the same family or genus) and selection based on ethnomedicine (heinrich, 2002) . ethnomedicine has been considered the most effective therapy and consists of the study of natural products that have a long history of use in some communities for the treatment of certain diseases and are part of the phytotherapeutic arsenal of popular knowledge (wu & tan, 2019) . on the other hand, uncaria tomentosa (willd. ex schult.) dc. named cat's claw ('uña de gato' in spanish) is a woody vine indigenous to the peruvian amazon and other tropical areas of south and central america that belongs to rubiaceae family (sandoval et al., 2000) . currently, the raw material of u. tomentosa is dispensed in public hospitals of the social health insurance (essalud-peru) as complementary medicine service (cms) (gonzales et al., 2010) . traditionally, extracts prepared by roots and barks decoction are used against several diseases, such as allergies, arthritis, inflammations, rheumatism infections and cancer (araujo et al., 2018) . bioactive constituents of u. tomentosa extracts include proanthocyanidins [proanthocyanidin b2 (the main component), proanthocyanidin b4, proanthocyanidin c1, an epicatechin trimer, epiafzelechin-4b!8-epicatechin and an epicatechin tetramer] (batiha et al., 2020; navarro-hoyos et al., 2017) , oxindole alkaloids (isopteropodine, pteropodine, rhynchophylline, mytraphylline, speciophylline, uncarine f and uncarine e), indole alkaloidal glucosides (cadambine, 3dihydrocadambine and 3-isodihydrocadambine) (batiha et al., 2020; kura s et al., 2009; laus et al., 1997; lima-junior et al., 2013; lock et al., 2016; navarro et al., 2019 snow et al., 2019) , quinovic acid glycosides (pavei et al., 2012) , tannins (ostrakhovich et al., 1997) , polyphenols, catechins, beta sitosterol (aquino et al., 1997; navarro et al., 2019) and proteins (lenzi et al., 2013) , which individually or synergistically contribute to their therapeutic properties. in regards to the antiviral properties of u. tomentosa, the alkaloid fraction has been demonstrated to be the most effective on human monocytes infected with dengue virus-2 (denv) in vitro (reis et al., 2008) . another study revealed that only the alkaloidal fraction has inhibitory activity on dengue virus, and the negative effect was observed with the nonalkaloidal fraction (lima-junior et al., 2013) . in another study, the antiherpetic activity of u. tomentosa seems to be associated with polyphenols or with their synergistic effect with pentacyclic oxindole alkaloids or quinovic acid glycosides (caon et al., 2014) . u. tomentosa hydroethanolic extracts have demonstrated a significant in vitro inhibitory effect on the replication of herpes simplex virus type 1, and the inhibition of viral attachment in the host cells was characterized as the main mechanism of its antiviral activity (terlizzi et al., 2016) . sars-cov-2 contains four structural proteins, namely the spike (s), membrane (m), envelope (e) and nucleocapsid (n) proteins. the s protein is responsible for the host attachment and fusion of the viral and host-cell membranes (wu et al., 2020) . otherwise, the angiotensin-converting enzyme 2 receptor (ace-2r) is the host cellular receptor with a higher affinity to sars-cov-2 (jamwal et al., 2020) . this process is triggered when the s1 subunit of s protein binds to a host-cell receptor (han & kr al, 2020) . to engage a host-cell receptor, the receptor-binding domain (rbd) of s1 undergoes transient hinge-like conformational motions (receptoraccessible or receptor-inaccessible states). u. tomentosa's constituents could block the virus from binding to human cell receptors and disrupt the virus cycle helping to prevent the protein maturation of sars-cov-2 and limit its infection spread . several molecular targets have been identified as the main druggable key of sars-cov-2 for new antiviral discovery. moreover, its x-ray structure has been recently released, hence allowing possible computational analysis. in fact, several computational studies have already been undertaken on this system including a long 20 ls molecular dynamics (md) study and virtual screening of several databases (huang et al., 2020) . with neither drugs nor vaccines approved against covid-19 yet, finding strategies to diminish the impact of the pandemic is fundamental. medicinal herbs and, more particularly, those with demonstrated antiviral activities as u. tomentosa could slow down the spreading of the disease. particularly in developing countries, in which the accessibility to these plants is easier and more economically viable, adding these medicinal herbs to the general medical kit may be beneficial. here, our study stands on an in silico strategy reminiscent to those applied at the early stage of current state-of-the-art drug discovery pipelines and includes (1) protein-ligand docking of all bioactive compounds of u. tomentosa against focusing both on the binding interface of the rbd-ace-2 and inside sars-cov-2 rbd spike protein, (2) simulations of ligand pathway of the best predicted compounds from step 1 to evaluate convenient entrance mechanism of the compounds to the binding site, (3) md simulation to assess the stability of the best protein-ligand complexes from 1, (4) calculation of pharmacokinetics parameters for the most qualified compounds resulting from the previous parts of the docking protocol. this study demonstrates the antiviral potential of u. tomentosa-based products to be applied as a rapid phytotherapeutic option for covid-19. calculated binding affinity of the main constituents of the u. tomentosa (table 1) was explored for its ability to disrupt the sars-cov-2/ace-2 complex and inhibit sars-cov-2 spike protein of novel coronavirus findings, a facile therapeutic option for anti-coronavirus therapy. to this purpose, the crystal structures of sars-cov-2/ace-2 complex and sars-cov-2 spike protein were downloaded from the protein data bank (pdb entry code 6m17 and 6vyb, respectively) (yan et al., 2020) and all bounded ligands, ions and solvent molecules were manually removed using the ds visualizer 2.5 program. for docking studies, the structures of the selected proteins were parameterized using autodock tools (trott & olson, 2009) . to facilitate the formation of hydrogen bonds, polar hydrogens were added. ligands used in this study are major components of the u. tomentosa extracts and a sulfated heparin octasaccharide (taken from pdb 5ue2), a potent sars-cov-2 inhibitor in vitro reported in the literature (kwon et al., 2020) . the 2d structures of the 26 cat's claw constituents were obtained as mol.2 files from the zinc database (chemaxon, 2016) . the resultant compounds were submitted to marvinsketch 8.3 (morris et al., 1998) to correct the protonation states of the ligands at physiological ph 7.4. in addition, the geometry optimization of all ligands was carried out using the hf/6-31g ã level of theory. then, the structures were parameterized using autodocktools to add full hydrogens to the ligands, to assign rotatable bonds and saving the resulting structure in the required format for use with autodock. all possible flexible torsions of the ligand molecules were defined using autotors in pdb autodocktools (morris et al., 2009; walls et al., 2020) to promote the calculated binding with the target structure. our docking protocol was performed using autodock vina and default procedures to dock a flexible ligand to a rigid protein. docking simulation of ligands was carried out on the interface between the sars-cov-2 and ace-2 (pdb code: 6m17) (yan et al., 2020) , where both proteins residues are in proximity. next, we used the cryo-em structure of sars-cov-2 spike protein (pdb code: 6vyb) in their open state (lipinski et al., 2012) to explore the potential inhibition of components of the cat's claw, selecting ace-2-binding pocket to this study. once a potential binding site was identified, 26 compounds which are the major components of the cat's claw extracts were docked to this enzymes-site to determine the most probable and the most energetically favorable binding conformations. to accomplish rigorous docking simulations involving a grid box to the identified catalytic site, autodock vina 1.1.2 (trott & olson, 2009 ) was used. the exhaustiveness was 20 for each protein-ligand pair (number of internal independent runs). the active site was surrounded by a docking box of 40 â 40 â 40 å with a grid spacing of 1 å. affinity scores (in kcal mol -1 ) given by autodock vina for all compounds were obtained and ranked based on the free energy binding theory (more negative value means greater binding affinity). the resulting structures and the binding docking poses were graphically inspected to check the interactions using the ds visualizer 2.5 (http://3dsbiovia.com/ products/) or the pymol molecular graphics system 2.0 programs. molecular interaction stability of protein-ligand complexes obtained by docking simulations were verified through md simulations by using the gromacs program (abraham et al., 2015) considering the sars-cov-2/ace-2 interface, as well as the sars-cov-2 spike protein active site and the best docking pose for proanthocyanidin c1, qag-2, proanthocyanidin b2 and 3-dihydrocadambine, respectively. force field parameters for protein and ligands were derived independently. for the selected protein, the amber03 force field was selected and assigned using the pdb2gmx tool of the gromacs program packages, meanwhile ligand force field parameters were prepared with the generalized amber force field (gaff) using the molecular geometries previously optimized with the hf/6-31g ã level of theory in gas phase, (foresman et al., 1992; glendening et al., 2012; roothaan, 1951) with the gamess-us program (schmidt et al., 1993) . in addition, each ligand was verified as a minimum through a harmonic vibrational normal mode analysis. atomic charges were obtained with the merz-kollman scheme (singh & kollman, 1984) by fitting a restricted electrostatic potential (resp) model by the gamess-us program (bayly et al., 1993) , and the output file was used into the resp sub-program of the ambertools program package (cornell et al., 1995) . assignment of gaff force field parameters was carried out by the antechamber program (wang et al., 2006) and the required input files for molecular dynamics simulations were prepared using the acpype python interface. protein and protein-ligand complexes were solvated in a rectangular box of tip3p waters. the obtained system was neutralized adding seven-sodium counter ions to neutralize the net negative charge of the protein, and then physiological conditions (298 k, ph 7.4, 0.9% nacl solution) were established (hammad et al., 2020) . to remove spurious contact, molecular geometries were optimized with the steepest descent algorithm with 100,000 steps, protein backbones atoms were constrained with a force constant of 1000 kj mol à1 . then, the md simulations were allowed to run for 1000 ps in the npt ensemble. in addition, 50 ns in the npt ensemble were calculated for the production stage. all simulations were carried out under periodic boundary conditions. a cubic box with the size of 25 â 25 â 25 nm was used. a 12 å cutoff distance was used to calculate nonbonded interactions. electrostatic interactions were treated with the ewald particle mesh (pme) method (nishizawa & nishizawa, 2010) ; while van der waals interactions were introduced by using the cut-off scheme. finally, table 1 . best binding energy (kcal mol -1 ) based on autodock scoring of the main constituents of the u. tomentosa into the rbd/ace-2 interface and sars-cov-2 spike protein binding domain (rbd) (pdb id: 6vyb). best binding energy rbd/ace-2 interface (kcal mol -1 ) best binding energy sars-cov-2 rbd (kcal mol -1 ) spiroxindole alkaloids uncarine f -7.1 -6.1 speciophylline -6.8 -6.8 mitraphylline -6.8 -6.2 pteropodine -6.7 -6.2 isopteropodine -6.7 -6.3 isomitraphylline -6.6 -6.1 rynchophylline -6.0 -5.5 isorynchophyllin -6.0 -5.7 indole glycosides alkaloids 3-isodihydrocadambine -7.6 -6.4 cadambine -7.6 -6.1 3-dihydrocadambine -7.0 -7.1 polyhydroxylated triterpenes uncaric acid -7.0 -5.5 floridic acid -7.0 -5.5 pht-1 -6.9 -5.6 quinovic acid glycosides qag-2 -8.2 -6.8 qag-5 -8.0 -6.1 qag-4 -7.8 -6.8 qag-6 -7.8 -6.8 3) b a hepos: heparin octasaccharide taken from pdb 5ue2 was used as positive control. b estimated by kwon et al. (2020) . the v-rescale thermostat at 300 k with a coupling constant of 1.0 ps was used and the pressure was kept constant at 1 atm using the parinello-rahman barostat (parrinello & rahman, 1981) with a coupling constant of 2.0 ps and a compressibility factor of 4.5 â 10 à5 bar à1 . all covalent bonds were constrained using the lincs algorithm and the contact list was updated every 40 fs. drug-likeness prediction along with further adme properties presents a wide of opportunities for a rapid new antiviral drug discovery. the drug-like and adme properties for the most active components of the u. tomentosa extract (constituents having the highest binding affinity) were screened using openaccess cheminformatics platforms such as molinspiration (for molecular weight -mw, rotatable bonds and polar surface area -psa descriptors), alogps 2.1 (for log p o/w descriptor) and the pre-admet 2.0 to predict four pharmaceutical relevant properties such as intestinal permeability (app. caco-2), albumin-binding proteins (k hsa ), madin-darby canine kidney (mdck line) cells permeation and intestinal absorption (%hia). these parameters establish movement, permeability, absorption and action of potential drugs (ertl et al., 2000) . the interpretation of both mdck and caco-2 permeability using preadmet is as follows: (1) permeability lower than 25: low permeability; (2) permeability between 25 and 500: medium permeability; (3) permeability higher 500: high permeability. this study was performed to identify whether certain components of u. tomentosa extracts have potential therapeutic effects against covid-19. to this purpose, a database of 26 compounds that have shown prevalence on the herbal therapeutic activity has been generated ( figure 1 ) (aquino et al., 1990 (aquino et al., , 1997 batiha et al., 2020; keplinger et al., 1998; kitajima et al., 2000; lima-junior et al., 2013; lock et al., 2016; montoro et al., 2004; navarro et al., 2019 pavei et al., 2012; peñaloza et al., 2015; snow et al., 2019; vera-reyes et al., 2015) . our initial hypothesis is that cat's claw should contain molecules with highest therapeutic profiles against sars-cov-2, by disrupting sars-cov-2/ace-2 association or by inhibiting sars-cov-2 spike protein. during covid-19 host infection, sars-cov-2 enters human epithelial cells through a first molecular recognition of rbd to the ace-2 protein. when coronaviruses bind directly to the peptidase domain (pd) of ace-2, it results in the loss of their primary physiological role, which includes vasoconstriction and blood pressure regulation. in consequence, binding of sars-cov-2 rbd to the human ace-2 receptor is associated strongly with cardiovascular diseases, such as hypertension, heart attack and chronic nephropathies. blocking the binding of sars-cov-2 to the human ace-2 receptor may result in the most promising approach to prevent virus entry into human cells. recently, the cryo-em cocrystal structures of the rbd of sars-cov-2 with human ace-2 have been solved (yan et al., 2020) , which open the possibility to design better and more specific inhibitors for suppression of viral infection. thus, to study the effectiveness of cat's claw against sars-cov-2/ace-2 complex, docking approaches were carried out in the ace-2-rbd binding interface as the druggable site, to establish the interaction between the selected site and the main constituents of cat's claw. we also performed molecular docking studies to find a potential association of constituents of cat's claw to the sars-cov-2 spike protein. this approach also could conduce to block the sars-cov-2 spike protein interaction with human receptor ace-2. hence, in this article, the structure of spike glycoprotein (pdb id: 6vyb) is to be considered as an additional druggable target. in addition, we have also performed the docking of a sulfated heparin octasaccharide (hepos) as positive reference, which have been recently reported as an effective in vitro inhibitor of sars-cov-2 by its interaction against the spike protein rbd (kwon et al., 2020) . overall, docking approach revealed that the most components founded in cat's claw could block sars-cov-2/ace-2 association because they displayed significant binding affinity at interface of ace-2-rbd complex in the range between -6.0 to -8.6 kcal mol -1 (table 1) . on the other hand, when structures were docked against the sars-cov-2 spike protein, good dock scores were obtained (specially, proanthocyanidins series) ranging from -5.5 to -7.2 kcal mol -1 . thus, we performed a rigorous exploration of the docking solutions obtained from these compounds when docking occurred against sars-cov-2-related enzymes. hence, based on the analysis of these different results and visual inspection, a clear behavior appears along the molecular docking that could be summarized as follows in the following sections. the recognition of sars-cov-2 by human ace-2 can be divided into several contacts. from the ace-2 structure: a 1 helix domain comprises 12 critical aminoacids, such as gln24, thr27, asp30, lys31, his34, glu35, asp38, tyr41, gln42, glu37, phe390 and gln388; a 2 helix domain located around met82 and four residues (lys353, gly354, asp355 and arg357) between b 3 and b 4 sheets are needed to recognize sars-cov-2. from sars-cov-2 structure, ten residues are essential for ace-2 binding, such as lys417, gln493, tyr453, tyr505, tyr449, asn501, gln498, thr500, gln474 and phe486. therefore, these key binding domains were considered in this article to explore the ability of u. tomentosa to disrupt sars-cov-2 rbd interaction to human ace-2 (yan et al., 2020) . in general, all compounds as part of u. tomentosa show docked structures that fit well into the rbd/ace-2 interface, particularly along a 1 , a 2 , b 3 and b 4 domains of human ace-2 ( figure 2 ) and with good predicted docking scores that range from -6.0 to -8.6 kcal mol -1 (table 1) . calculations revealed that most of ligands were located between b 3 and b 4 sheets, and a reduced group very close to a 1 and a 2 helix of ace-2. since during viral infection a 1 , a 2 , b 3 and b 4 domains are responsible for the recognition of sars-cov-2 by human ace-2, our findings open the possibility to use u. tomentosa against sars-cov-2/ace-2 association. notably, most predicted complexes have the interaction fingerprint with those of critical residues as part of ace-2-rbd binding interface. protein-ligand interaction analysis of the 26 constituents to the rbd/ace-2 complex revealed that the most of constituents strongly bonded to ace-2 through his34, asp30, glu37, phe390, gln388, lys353, thr27, glu35 residues, meanwhile with sars-cov-2 rbd showed critical contacts with tyr505, tyr453, lys417, tyr449 residues. due to these compounds as part of cat's claw and interaction with key aminoacids inside ace-2-rbd binding interface, we are encouraged to believe that u. tomentosa could affect the interaction of sars-cov-2 spike protein with ace-2. note that this approach may be useful as a rapid therapeutic option to prevent or treat covid-19. a simple view showed that all ligands were able to interact with those critical aminoacids involved in the molecular recognition of rbd to the ace-2 protein, where at least one compound from each chemical series showed great ability to bind to the sars-cov-2-rbd complex along the interface. hence, we focused on five compounds (those with highest negative energy value obtained after docking on each series) such as proanthocyanidin c1 (-8.6 kcal mol à1 ), qag-2 (-8.2 kcal mol à1 ), 3-isodihydrocadambine (-7.6 kcal mol à1 ), uncarine f (-7.1 kcal mol -1 ) and uncaric acid (-7.0 kcal mol à1 ), which had higher or comparable affinity than positive reference hepos (-7.3 kcal mol à1 ) to the sars-cov-2-rbd complex (table 1) . at this point it is worth mentioning that the docking calculations involving positive reference give a value in very good agreement with experimental one (-7.3 kcal mol à1 ), hence providing with a certain amount of confidence regarding the autodock scoring function of this project. a rigorous view of 2d plots ligand interactions into ace-2-rbd interface generated from ds visualizer program revealed which interactions are involved by the most docking active ligands and how their structures affect them. thus, the most active docking ligand proanthocyanidin c1 (which had affinity of à8.6 kcal mol à1 ) was able to interact with those critical aminoacids for binding sars-cov-2 spike to the human ace-2 receptor. we found that proanthocyanidin c1 establishes three strong hydrogen bond interactions between the hydroxyl groups in flavone moiety with tyr505 (sars-cov-2) and asp30 (ace-2 receptor) residues, one p-alkyl interaction with lys417 residue and several hydrophobic interactions between the molecule and the glu37, gln388, lys353, phe390, thr37 and his34 residues. in addition to these critical aminoacids, proanthocyanidin c1 revealed notable binding interactions with human ace-2 receptor very close to interface through four hydrogen contact, such as ala386, thr92, lys26 and ala337 that could may also affect the interaction of rbd with ace-2 (figure 3(a) ). furthermore, a closer look at the best possible binding pose of qag-2 (which display a high docking score of à8.2 kcal mol à1 ), reveals strong interactions at the contact interface between the two proteins. thus, it was found that qag-2 forms two hydrophobic interactions with critical aminoacids lys353 and asp355 located between b 3 and b 4 sheets in the ace-2 protein. we also observed that qag-2 displays one interaction at interface with val407 (from spike protein) through strong hydrogen bond. in addition, various hydrophobic contacts were observed between molecule and ser375, phe377, val510, cys379, ile410, gly404, asp405, phe356, ala386, arg408 and gly502 at the sars-cov-2 rbd-ace-2 interface (figure 3(b) ). note that the deprotonated carboxylate moiety does not form any important interaction, therefore does not play any specific role in the qag-2 binding at rbd-ace-2 interface. this fact can be explained primarily because positively charged residues (lysine, arginine or histidine) do not surround this moiety at the interface. on the other side, 3-isodihydrocadambine (-7.6 kcal mol -1 ) has key contacts with residues in the rbd/ace-2 binding interface (figure 3(a) ). thus, molecule showed several interactions with critical residues of human ace-2, which are essential for sars-cov-2 spike binding, among them, one hydrogen bond contact and one p-alkyl interaction with glu37 residue. importantly, the protonated nitrogen atom in the b-carboline moiety forms one salt bridge interaction with the carboxyl in asp30 (from ace-2 protein) and three hydrophobic interactions with phe390, thr27 and gln388. furthermore, 3-isodihydrocadambine also had a crucial h-bond contact with tyr505 amino acid of sars-cov-2 spike protein, which is well-reported as an initial contact point during ace-2 recognition. 3-isodihydrocadambine also showed one p-cation interaction between fused aromatic ring in the b-carboline moiety and the key residue lys417 and eight van der waals contacts with spike protein residues, including glu406, asp405, gln409, phe456 leu455, asp405, tyr453 and tyr421. further interactions were also observed that might promote the disruption of the interactions between sars-cov-2-rbd and ace-2, among them two h-bonds formed per arginine residues 393 (from ace-2 protein) and 403 (from spike protein) interacting with the sugar moiety. interestingly, uncarine f binds at rbd/ace-2 interface with binding affinity of à7.1 kcal mol à1 through several interactions with those base residues for the recognition of sars-cov-2 to the human ace-2 (figure 3(b) ). besides two strong hydrogen contacts with his34 of human ace-2 and tyr505 from the sars-cov-2 spike protein, uncarine f showed several hydrophobic interactions with key aminoacids at the interface, such as lys353, asp30, phe390, glu37, tyr453 and gln388. finally, analysis of the 2d-interaction map for uncaric acid (which had docking score of à7.0 kcal mol à1 ) revealed crucial contacts with essential aminoacids residues for ace-2-rbd binding. thus, uncaric acid binds at asp30 residue of ace-2 through a hydrogen bonding with one of the hydroxyl groups. furthermore, hydrophobic interactions between molecule and five of those key residues at interface were displayed as follows: his34, tyr453, phe390, gln388 and tyr505. notably, uncaric acid had five p-alkyl interactions with other interface residues that probably could promote the ace-2-rbd binding cleavage, such as leu29 (d), val93 (d), lys26 (d), pro389 (d) and lys417 (f). in accordance to abovementioned, our docking studies shows that several components of u. tomentosa may have the ability to disrupt the association of sars-cov-2 spike protein with the human ace-2 receptor. among these components, proanthocyanidin c1 (-8.6 kcal mol à1 ), qag-2 (-8.2 kcal mol à1 ), 3-isodihydrocadambine (-7.6 kcal mol à1 ), uncarine f (-7.1 kcal mol à1 ) and uncaric acid (-7.0 kcal mol à1 ) had good predicted binding affinity for binding interface and they can naturally access it without noticeable energetic cost. these findings suggest that u. tomentosa may be a viable treatment option during initial stage of the covid-19 infection. recently, cryo-em structure of the sars-cov-2 spike glycoprotein was resolved in their closed and open states (pdb id: 6vxx and 6vyb, respectively) (walls et al., 2020) . sars-cov-2 spike protomer consists of five functional domains, namely as ntd, rbd, fp, hr1 and cd (figure 4 ). because rbd domain is responsible for the binding to the host cell receptor (ace-2), we focused our docking investigations inside the several residues in the sars-cov-2 rbd have been identified as essential in the association to the human ace-2 during coronavirus (covid-19) infection, including tyr449, tyr453, gly496, thr500, tyr505, lys417, gln493, asn501 and gln498 (lan et al., 2020) . therefore, to demonstrate the ability of constituents of u. tomentosa to block binding of the sars-cov-2 spike protein to human ace-2 receptor, we performed molecular docking studies around aforementioned critical amino acids, meaning this docking runs were carried out inside ace-2 binding surface of rbd. from a general perspective, promising docking scores were obtained when the major constituents from u. tomentosa bind to the rbd of sars-cov-2 (ranging from -5.5 to -7.2 kcal mol à1 ). thus, to compare the best binding pose of the most docking-active components of u. tomentosa and hepos (positive reference) inside sars-cov-2 rbd, figure 5 illustrates the most stable binding poses based on autodock scoring listed in table 1 for all constituents on rbd binding domain. the superposition of the positive reference (hepos) and the best conformation obtained theoretically for selected docked compounds showed that the major constituents in the ethanolic extract of u. tomentosa can accommodated themselves into stable conformations occupying this binding site during docking process. notably, all main constituents of u. tomentosa had at least two interactions with those key aminoacids for sars-cov-2 rbd binding to human ace-2, through h-bonds, p-contacts or hydrophobic contacts, making this herb a promising treatment that may be used in the early stages of the covid-19 infection. among them, four exhibited high potential to bind rbd: 3-dihydrocadambine (in brown), proanthocyanidin b4 (in purple blue), proanthocyanidin b2 (in light blue) and proanthocyanidin c1 (in hot pink) ( figure 5) , which had the highest docking score of -7.1, -7.2, -7.2 and -7.0 kcal mol à1 , respectively, that would be comparable to that reported for the potent inhibitor hepos of -7.3 kcal mol à1 . as such, in searching those critical contacts that could blocks rbd-ace-2 interaction, an exhaustive analysis has been undertaken to the docking results for those components as mentioned in figure 6 . as shown in figure 7 , our findings revealed that the most docking active molecules complexed with the sars-cov-2 rbd had an interaction fingerprint involving seven critical residues implied in the sars-cov-2 spike attachment to the human receptor ace-2: tyr453, gly496, lys417, asn501, tyr505, gln498 and asn501. thus, 3-dihydrocadambine displays two strong h-bond through binding between sugar and acetyl moiety and key tyr453 and gly446 residues, respectively. notably, tyr453 also established one p-p stacking interaction with the fused aromatic ring in the b-carboline moiety. in addition, critical residues lys417 and arg403 from rbd was involved in the binding event by forming three p-cation contacts with 3-dihydrocadambine, while hydrophobic interactions formed with asn501, tyr449, gln493, tyr505 residues clearly favored its affinity for sars-cov-2 spike protein. however, a visual inspection to the 2d-protein-ligand interaction plot of 3-dihydrocadambine shows that the protonated nitrogen formed two unfavorable positive-positive interactions (represented in red dotted lines) that could have great significance in the stability of protein-ligand complex. proanthocyanidin b2 was well-fitted into the functional domain rbd and its hydroxyl groups formed four hydrogen bonding and four hydrophobic contacts with critical residues for binding to ace-2, including gly496, asn501, gln493 and tyr505, tyr453, lys417, gln498, respectively. one further hbond with ser494 and one hydroxyl group were predicted by docking when proanthocyanidin b2 binds to sars-cov-2 rbd. finally, proanthocyanidin c1 was able to bind sars-cov-2 rbd through three strong h-bonds with those critical residues (gly496, lys417, asn501) crucial to association with human ace-2. also, further van der waals interactions were predicted to form between proanthocyanidin c1 and crucial gln498, tyr453, gln493, tyr505, gln498 residues that may be stabilizing the binding event to the rbd domain. with higher confidence on the viability of our docking predictions of the best compound with highest binding affinity, such as proanthocyanidin c1 (-8.6 kcal mol à1 ) and qag-2 (-8.2 kcal mol à1 ) into the rbd/ace-2 interface and 3-dihydrocadambine (-7.1 kcal mol à1 ) and proanthocyanidin b2 (-7.2 kcal mol à1 ) within sars-cov-2 rbd binding domain, we further evaluated the stability of the docked complexes throughout md simulations for 50.0 ns. to accomplish this aim, we first calculated the root mean square deviation (rmsd) for ligands for 50 ns of md simulation at real natural conditions into the selected binding pocket. the md simulation results (figure 8(a-d) ) showed that the rmsd of the systems reached equilibrium after around %5 ns of simulation time. in general, after equilibration, small fluctuations in the rmsd were observed, suggesting substantial stability for all complexes during the simulations, which fall within the ideal range around 2 å (smaller rmsd values indicate higher stability of the simulation) (gohlke et al., 2000; kramer et al., 1999) . a rigorous exploration of the rmsd values for ligand-sars-cov-2 rbd complexes shows that structures of 3-dihydrocadambine and proanthocyanidin b2 display good signals of stability during 50 ns of md simulation with rmsds values of 1.866 ± 0.300 and 2.233 ± 0.304 å, respectively (figure 8(a,b) ). importantly, similar behavior had the docked complex composed of proanthocyanidin c1 and qag-2 into the rbd/ace-2 interface, which showed remarkable stability throughout the simulation time period at rmsd values of 2.151 ± 0.237 and 1.775 ± 0.361, respectively (figure 8(c,d) ). a closer look at rmsd plot for qag-2 into the rbd/ace-2 interface revealed that ligand gradually stabilized after 5 ns, which is an indication of its higher conformational flexibility within the interface between rbd and ace-2 proteins. taken together, these findings suggest binding stability of ligands toward the active domains of the sars-cov-2 rbd and rbd/ ace-2 viral targets. the radius of gyration (r g ) represents the compactness of the protein structure and conformational stability of the whole systems (i.e. protein-ligand complexes). if the radius of gyration remained relatively constant, the complex was considered to be stably folded; otherwise, it was considered to be unfolded. in this scenario, radius of gyration values was calculated in order to observe the conformational alterations and dynamic stability of each viral protein within the 50 ns simulation time. figure 9 (a-d) illustrates r g values for the protein and ligand in the complexes, respectively. as shown in figure 9 (a-d), calculated r g values for ligands into the sars-cov-2 rbd protein for 3-dihydrocadambine (5.065 ± 0.144 å), figure 8 . backbone rmsd values of (a) 3-dihydrocadambine within sars-cov-2 rbd active site (red) and protein without ligand (blue). b: proanthocyanidin b2 within sars-cov-2 rbd binding site (red) and protein without ligand (blue). c: proanthocyanidin c1 at rbd/ace-2 interface and rbd/ace-2 complex without ligand (blue). d: qag-2 into rbd/ace-2 interface (red) and rbd/ace-2 complex without ligand (blue). proanthocyanidin b2 (4.356 ± 0.087 å) and inside rbd/ace-2 binding interface for proanthocyanidin c1 (5.542 ± 0.095 å) and qag-2 (6.818 ± 0.083 å) remained relatively constant during the simulation, therefore each protein-ligand complex could be considered to be stably folded. in addition, although rmsd values would show that the ligands undergo a significant shift within the active domain, estimated r g values suggest that overall shape of the protein was stable upon binding of the ligand during the 50-ns md simulation. finally, these observations are also supported by 2d-binding interactions plots and 3d representation of the selected ligands into the binding pocket, respectively (see supporting information figures s1-s4 ). in addition, to show the conformational changes of the ligands into the viral proteins active site along the first 50 ns window md simulation (figure 9 ), these plots revealed that after md simulations the key protein-ligand interactions initially shown by the docking results were maintained and the 3-dihydrocadambine and proanthocyanidin b2 within sars-cov-2 rbd, as well as proanthocyanidin c1 at rbd/ace-2 remained stable in the binding pocket compared to the initial docking pose. thus, crucial binding ligand interactions with lys417, arg403, tyr453, gln493, asn501, tyr505, ser494, tyr449, tyr495 phe497 and gly496 were maintained after 50-ns md simulation into the binding pocket of sars-cov-2 rbd. similarly, in comparison with the docking results, those key ligand interactions with amino acid residues identified as essential for maintaining rbd-ace-2 stability are also present after md simulations. besides, 3d representations of the selected ligands into each binding pocket were used with the aim to compare the best conformation poses from md simulation and docking, hence we plotted the superposition of the docked complex 3d-structures before and after 50-ns md simulation into the active cavity (see supporting information figures s5-s8 ). in general, there is no significant difference between the structures extracted after 50-ns md simulation and the docking pose of ligands, only slight translational and rotational motions were observed. the obtained md simulation results suggest (1) the conformation of the binding pocket and ligands were stable during the md simulations, (2) ligands do not leave the binding pocket while running md simulation and (3) active pocket in both selected viral targets favored ligands binding, suggesting not only the rationality and validity of our docking studies, but also proposes that many of these constituents of u. tomentosa could act as a dual inhibitors of the sars-cov-2 spike protein and rbd/ace-2 complex, which are mostly responsible for the attachment and internalization of the novel coronavirus in the human host. in the final stage, selected ligands were redocked into the sars-cov-2 rbd (3-dihydrocadambine and proanthocyanidin b2) or inside the rbd/ace-2 interface (proanthocyanidin c1 and qag-2) starting from the mean geometries of the last 3 ns md simulations trajectories, aiming to obtain the correct binding energies and poses. the vina re-docked results are summarized in table 2 and the best ligand bound receptor figure 9 . radius of gyration (r g ) graphs: (a) for 3-dihydrocadambine into the binding cavity (red) and sars-cov-2 spike protein without ligand (blue). b: for proanthocyanidin b2 onto active site (red) and sars-cov-2 spike protein without ligand (blue). c: for proanthocyanidin c1 within binding cleft and rbd/ace-2 complex without ligand (blue and violet). d: for qag-2 into the binding site (red) and rbd/ace-2 complex without ligand (blue and violet). conformations are shown in the supporting information figures s9-s12 . notably, after re-docked process 3-dihydrocadambine displays higher binding energy (-7.5 kcal.mol à1 ) than the initial predicted docking score (-7.1 kcal.mol à1 ). 2d and 3d diagrams of protein-ligand interactions from 3d coordinates showed that 3-dihydrocadambine had a significant conformational change in their binding modes in comparison with observed binding poses initially predicted by docking. as can be seen from table 2 , interactions profile into the sars-cov-2 rbd binding site was substantially altered, showing contacts with those critical residues, including asn501 (h-bond), gly496 (three h-bond), tyr453 (h-bond), tyr505 (p-cation with cationic nitrogen). on the other hand, despite proanthocyanidin b2 showed a re-docking score significantly lower than the initial docking. however, readers can observe that proanthocyanidin b2 is well-accommodated inside the rbd binding pocket highlighting key contacts with tyr453, ser494, lys417, gln493, arg403, tyr495, tyr505, glu406 and phe497 that are essential for sars-cov2 binding to human ace-2 receptors. in addition, predicted binding poses were compared in supporting information figure s10 , thus the best binding pose obtained from re-docking studies is considerably different to that retrieved from the initial docking, this finding could be strongly associated with a lower binding energy after re-docking. interestingly, when proanthocyanidin c1 was re-docked using the mean geometry of the last 3 ns md simulation trajectory, a higher binding score (-9.0 kcal mol à1 ) than the initial docking was obtained (-8.6 kcal mol à1 ). as shown in supporting information figure s11 , proanthocyanidin c1 fitwell into the rbd/ace-2 interface interacting with those critical residues at the junction of sars-cov-2 to human ace-2, such us glu37, his34, asp30, gln388, tyr505, phe390, lys353, glu354, lys417. a 3d-comparison between the best initial docking pose and the best re-docking pose into the active site clearly revealed that this binding pose significantly favors proanthocyanidin c1 binding. finally, re-docking calculations into rbd/ace-2 interface for qag-2 showed a very similar binding affinity (-8.1 kcal mol à1 ) to the initial docking prediction (-8.2 kcal mol à1 ). a close view to 2d and 3d ligand-protein diagrams plot revealed that qag-2 fit-well inside the binding pocket, and is located closed to b3 and b4 sheets in the ace-2 protein. re-docked process confirms that qag-2 is capable of binding to aminoacids residues that are critical for the recognition of the sars-cov-2 by full-length human ace-2. after the re-dock analysis, we identified that qag-2 formed two hydrogen bond interactions with key gly354 and tyr505 residues, respectively. similarly, ligand was able to bind through van der waals contacts with three critical residues asp355, gln388 and glu37, which could have an effect on stabilizing the binding event. as can be seen in supporting information figure s12 , 3d-plots comparison between starting pose docking and docking pose after re-docking for qag-2 revealed similar binding modes at rbd/ace-2 interface, only a slight shift was observed. taken together, these computational results clearly evidenced that constituents of u. tomentosa are capable of binding to sars-cov-2 spike protein through strong interactions with those key aminoacids crucial for the viral attachment to human ace-2 in stable complexes. our investigations could be a new strategy for inhibiting the recognition of the sars-cov-2 rbd by ace-2, and therefore might interfere with the entry of coronavirus to its host cells. computational modeling demonstrated that components of this herb may cause ace-2 and spike protein cleavage, because they would interact with key residues within rbd/ace-2 interface forming very stable complexes. hence, we firmly believe that ethanolic extract of cat's claw may be a novel herbal-based therapeutic option to treat covid-19 infection because its components may cause disruption of sars-cov-2 rbd/ace-2 complex or could block attachment of sars-cov-2 to the entry receptor ace-2. calculated drug-likeness profiles play a critical role in assessing the quality of novel antiviral candidates. early predictions of pharmacokinetic behavior of the promising antiviral compounds based on its structure could help find safer and effective leads for preclinical testing. here, we calculated and analyzed various drug-likeness indices for the most qualified components of u. tomentosa predicted by docking studies (table 3) . to this purpose, ten pharmacokinetics parameters were calculated as drug-likeness filter for speciophylline, uncarine f, uncaric acid, cadambine, 3-isodihydrocadambine, 3-dihydrocadambine, proanthocyanidin b2, proanthocyanidin c1, proanthocyanidin b4, epiafzelechin-4b-8, qag-1, qag-2, qag-4, qag-5 and qag-6, respectively. results obtained revealed the druggability of the selected components from ethanolic extract of cat's claw, demonstrating their potential as likely orally active antiviral. despite major components had more than two violations to the rule of 5 and druggability, predicted values for speciophylline, uncarine f, uncaric acid and cadambine displayed favorable physiochemical profiles compared to positive reference hepos, which clearly displayed important violations in rof-5. the discussion focused on these four active compounds showed that according to lipinski rule of five (rof-5) (no more than one violation is acceptable) (lipinski et al., 2012) , compounds could be used as orally dosed drugs in humans. the predicted human intestinal absorption (% hia) for speciophylline, uncarine f and uncaric acid revealed greater hia value in the range between 81% and 90%, while cadambine having relatively low values of 27% may be acceptable within 95% of marketed drugs. note that, greater hia values denote that these compounds could be absorbed throughout the intestinal segments upon oral administration. on the other hand, we calculated the most important physicochemical property to correlated passive molecular transport through membranes and drug-membrane interactions, such as polar surface area (psa) (ertl et al., 2000) . predicted psa showed favorable values for compounds ranging from 82 to 158 å 2 , indicating they would penetrate more efficiently through the infected host cells. in addition, the partition coefficient between n-octanol and water (log p o/w ) was calculated in order to explore the ability of constituents to pass through lipid bilayers (veber et al., 2002) . notably, speciophylline, uncarine f and uncaric acid presented values within ideal range for approved-drugs (ranging from 1.7 to 4.20). binding to serum albumin (expressed as log k hsa ) is the most important parameter for distribution and transport of antiviral drugs in the systemic circulation (colmenarejo, 2003; zhivkova, 2015) . early prediction of this parameter reduces the amount of wasted time and resources for drug development candidates in the antiviral therapy and management. we found that ligands fit well within the recommended values range (ranging from à1.5 to 1.5), showing log k hsa numbers between -0.592 and 0.58. finally, we also predicted the passive transmembrane permeation using caco-2 cell monolayers or mdck cells as models. currently, both models are used as a simplified in vitro model for intestinal absorption in drug development (broccatelli et al., 2016; pham-the et al., 2018; press & di grandi, 2008) . our results showed that in comparison with reference drugs, speciophylline, uncarine f and uncaric acid have 72-342 nm/s. from such observations, these compounds displayed great values of human intestinal absorption (% hia) above 81%, very similar to the reference drugs values (above 96%). given the aforementioned results, we believe at least four components of cat's claw here reported may provide favorable characteristics as the drug like, hence u. tomentosa may constitute itself a promissory option to fight against covid-19 infection. by integrating in silico approaches, this article evidenced that several components of u. tomentosa could act disrupting the association of sars-cov-2 spike protein with the human ace-2 receptor or by blocking the rbd-ace-2 interaction during covid-19 virulence. further, we identified that various constituents of cat's claw bears optimal pharmacokinetics properties to be used orally as a potential antiviral response. therefore, we believe that ethanolic extract of u. tomentosa should be taken into consideration as a rapid response to the covid-19 during the early stages of infection. covid-19 outbreak that emerged from wuhan, china has acquired pandemic status and severe acute respiratory <0.0 ----5 a molecular weight of the hybrid (150-500). b polar surface area (psa) (7.0-200 å 2 ). c n-on number of hydrogen bond acceptors <10. d n-ohnh number of hydrogens bonds donors 5. e octanol water partition coefficient (log p o/w ) (-2.0 to 5.0). f binding-serum albumin (k hsa ) (-1.5 to 1.5). g human intestinal permeation (<25 poor, >500 great). h madin-darby canine kidney (mdck) cells permeation. i human intestinal absorption (% hia) (>80% is high, <25% is poor). j heparin octasaccharide used as positive reference in this work. syndrome requires the attention of academics to discover the possible safe and effective drug to ameliorate its effects worldwide. in the present study, 26 constituents of u. tomentosa were docked on the binding interface of the rbd-ace-2 and inside sars-cov-2 rbd spike protein of novel corona virus. it was observed that the components of u. tomentosa such as proanthocyanidin c1, qag-2, 3-isodihydrocadambine, uncarine f and uncaric acid had a good predicted binding affinity for interface of the rbd-ace-2 as compared to the sulfated heparin octasaccharide (hepos). likewise, 3-dihydrocadambine, proanthocyanidin b4, proanthocyanidin b2 and proanthocyanidin c1 had the highest docking score on sars-cov-2 spike glycoprotein in their open state, whereas md simulations at 50 ns demonstrated both the feasibility of the binding free energy predicted by docking protocols and the stability of the docked protein-ligand complexes. virtual prediction adme revealed that speciophylline, uncarine f and uncaric acid presented values of druggability according to lipinski rule, demonstrating their potential bioavailability as likely orally active antiviral. based on our findings and its ancestral use in the traditional medicine from south american countries, u. tomentosa can be performed as an herbal supplement with the safety and efficacy parameters at both preclinical and clinical stages to evaluate its effectiveness in the treatment of novel coronavirus disease . furthermore, all components found in u. tomentosa could work in synergism by different mechanisms to combat the spread of sars-cov-2. no potential conflict of interest was reported by the author(s). andres f. yepes-p erez http://orcid.org/0000-0001-6975-5119 oscar herrera-calderon http://orcid.org/0000-0001-7264-0961 jorge quintero-saumeth http://orcid.org/0000-0002-6544-9429 gromacs: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. softwarex, 1-2, 19-25 antiviral potential of medicinal plants against hiv, hsv, influenza, hepatitis, and coxsackievirus: a systematic review new polyhydroxylated triterpenes from uncaria tomentosa triterpenes and quinovic acid glycosides from uncaria tomentosa uncaria tomentosa improves insulin sensitivity and inflammation in experimental nafld uncaria tomentosa a wellbehaved electrostatic potential based method using charge restraints for deriving atomic charges: the resp model a second generation force field for the simulation of proteins, nucleic acids, and organic molecules predicting passive permeability of drug-like molecules from chemical structure: where are we? antimutagenic and antiherpetic activities of different preparations from uncaria tomentosa (cat's claw) chemaxon -software solutions and services for chemistry and biology. marvinsketch, version in silico prediction of drug-binding strengths to human serum albumin fast calculation of molecular polar surface area as a sum of fragment-based contributions and its application to the prediction of drug transport properties toward a systematic molecular orbital theory for excited states natural bond orbital methods. wires computational molecular science knowledge-based scoring function to predict protein-ligand interactions the world summit of harmonization on traditional, alternative and complementary medicine pharmacophore development, drug-likeness analysis, molecular docking, and molecular dynamics simulations for identification of new ck2 inhibitors computational design of ace2-based peptide inhibitors of sars-cov-2 ethnomedicine and drug discovery de novo design of protein peptides to block association of the sars-cov-2 spike protein with human ace2 an updated insight into the molecular pathogenesis, secondary complications and potential therapeutics of covid-19 pandemic uncaria tomentosa (willd.) dc. -ethnomedicinal use and new pharmacological, toxicological and botanical results two new 19-hydroxyursolic acid-type triterpenes from peruvian "una de gato" (uncaria tomentosa) evaluation of the flexx incremental construction algorithm for protein-ligand docking effect of alkaloid-free and alkaloid-rich preparations from uncaria tomentosa bark on mitotic activity and chromosome morphology evaluated by allium test sulfated polysaccharides effectively inhibit sars-cov-2 in vitro structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor alkaloids of peruvian uncaria tomentosa effects of aqueous fractions of uncaria tomentosa (willd.) d.c. on macrophage modulatory activities searching therapeutic strategy of new coronavirus pneumonia from angiotensin-converting enzyme 2: the target of covid-19 and sars-cov uncaria tomentosa alkaloidal fraction reduces aracellular permeability, il-8 and ns1 production on human microvascular endothelial cells infected with dengue virus experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings bioactive compounds from plants used in peruvian traditional medicine identification and quantification of components in extracts of uncaria tomentosa by hplc-es/ms autodock4 and autodocktools4: automated docking with selective receptor flexibility automated docking using a lamarckian genetic algorithm and an empirical binding free energy function fractioning of proanthocyanidins of uncaria tomentosa. composition and structure-bioactivity relationship proanthocyanidin characterization and bioactivity of extracts from different parts of uncaria tomentosa l. (cat's claw molecular dynamics simulation analyses of viral fusion peptides in membranes prone to phase transition: effects on membrane curvature, phase behavior and lipid-water interface destabilization antioxidant activity of the extract from uncaria tomentosa polymorphic transitions in single crystals: a new molecular dynamics method hplc-pda method for quinovic acid glycosides assay in cat's claw (uncaria tomentosa) associated with uplc/q-tof-ms analysis chemical composition variability in the uncaria tomentosa (cat's claw) wild population in silico assessment of adme properties: advances in caco-2 cell monolayer permeability modeling drug targets for corona virus: a systematic review permeability for intestinal absorption: caco-2 assay and related issues immunomodulating and antiviral activities of uncaria tomentosa on human monocytes infected with dengue virus-2 new developments in molecular orbital theory cat's claw inhibits tnfa production and scavenges free radicals: role in cytoprotection general atomic and molecular electronic structure system an approach to computing electrostatic charges for molecules the amazon rain forest plant uncaria tomentosa (cat's claw) and its specific proanthocyanidin constituents are potent inhibitors and reducers of both brain plaques and tangles inhibition of herpes simplex type 1 and type 2 infections by oximacrov r , a cranberry extract with a high content of a-type proanthocyanidins (pacs-a) autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading molecular properties that influence the oral bioavailability of drug candidates monoterpenoid indole alkaloids and phenols are required antioxidants in glutathione depleted uncaria tomentosa root cultures structure, function, and antigenicity of the sars-cov-2 spike glycoprotein a novel coronavirus outbreak of global health concern automatic atom type and bond type perception in molecular mechanical calculations annex 1 who guidelines on good herbal processing practices for herbal medicines coronavirus disease (covid-19): weekly epidemiological update. world health organization severe acute respiratory syndrome coronavirus 2: from gene structure to pathogenic mechanisms and potential therapy interaction between gut microbiota and ethnomedicine constituents structural basis for the recognition of sars-cov-2 by full-length human ace2 studies on drug-human serum albumin binding: the current state of the matter key: cord-341396-0tn06al2 authors: ni, ling; ye, fang; cheng, meng-li; feng, yu; deng, yong-qiang; zhao, hui; wei, peng; ge, jiwan; gou, mengting; li, xiaoli; sun, lin; cao, tianshu; wang, pengzhi; zhou, chao; zhang, rongrong; liang, peng; guo, han; wang, xinquan; qin, cheng-feng; chen, fang; dong, chen title: detection of sars-cov-2-specific humoral and cellular immunity in covid-19 convalescent individuals date: 2020-05-03 journal: immunity doi: 10.1016/j.immuni.2020.04.023 sha: doc_id: 341396 cord_uid: 0tn06al2 summary the world health organization has declared sars-cov-2 virus outbreak a world-wide pandemic. however, there is very limited understanding on the immune responses, especially adaptive immune responses to sars-cov-2 infection. here, we collected blood from covid-19 patients who have recently become virus-free and therefore were discharged, and detected sars-cov-2-specific humoral and cellular immunity in 8 newly discharged patients. follow-up analysis on another cohort of 6 patients 2 weeks post discharge also revealed high titers of igg antibodies. in all 14 patients tested, 13 displayed serum neutralizing activities in a pseudotype entry assay. notably, there was a strong correlation between neutralization antibody titers and the numbers of virus-specific t cells. our work provides a basis for further analysis of protective immunity to sars-cov-2, and understanding the pathogenesis of covid-19, especially in the severe cases. it has also implications in developing an effective vaccine to sars-cov-2 infection. *these authors contributed equally to this work. 1 2 #to whom correspondence should be addressed: chen dong, chendong@tsinghua.edu.cn; 3 or fang chen, anzhenchenfang@163.com; cheng-feng qin, qincf@bmi.ac.cn. 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 1 introduction 1 at the end of 2019, patients with coronavirus disease 2019 were identified 2 in wuhan, china (wang et al., 2020) , infected by a novel coronavirus, now named as severe 3 acute respiratory syndrome coronavirus 2 (sars-cov-2). the world health organization 4 (who) first declared this outbreak a public health emergency of international concern 5 (phelan et al., 2020) and subsequently a world-wide pandemic (di pierro et al., 2020) . the genome sequence of sars-cov-2 bears 96% (zhou et al., 2020) and 79.5% 7 identity to that of a bat coronavirus and sars-cov, respectively (zhu et al., 2020) . like 8 sars-cov and mers-cov, sars-cov-2 belongs to the beta genus coronavirus in the 9 corornaviridae family (lu et al., 2020) . clinically, several papers showed that most covid-10 19 patients developed lymphopenia as well as pneumonia with higher plasma levels of pro-11 inflammatory cytokines in severe cases (chan et al., 2020; huang et al., 2020; wu et al., 12 2020), suggesting that the host immune system is involved in the pathogenesis (mahallawi 13 et al., 2018; nicholls et al., 2003) . patients infected by sars-cov or mers-cov were 14 previously reported to have antibody responses (ko et al., 2017; shi et al., 2004; wang et al., (thevarajan et al., 2020) . one covid-19 patient in 1 finland was shown to possess a low level of neutralizing antibody titer (haveri et al., 2020) . however, virus-specific t lymphocytes and their relationships with neutralizing antibody 3 titers in covid-19 patients remains uncharacterized. in this study, we collected blood from covid-19 patients who have recently become 5 virus-free and therefore were discharged, and analyzed their sars-cov-2-specific antibody 6 and t cell responses. whereas the remaining 6 were 2 weeks post discharge (follow-up patients, patients #9-14). only three travelled to wuhan city within the past 3 months. in line with the previous reports 16 (wang et al., 2016) , 2 patients (#5, 10) showed lymphopenia (normal range is 1.1-3.2x10e9 17 cells per l). sera from three healthy donors (wang et al., 2016) were obtained before the 18 sars-cov-2 outbreak (healthy donor #1-3). 3 additional healthy donors (#4-6) who had 19 been in close contacts with the patients were recruited in this study. human ab serum 20 collected from healthy male ab donors in the us (gemcell, ca) was used as a negative 21 control. in order to detect anti-viral immune responses, we first constructed recombinant pet28-23 n-6xhis by linking 6 copies of his tag to the c-terminus of np in the pet28-n vector 24 (biomed, cat. number: bm2640). escherichia coli transformed with pet28-n-6xhis was 25 lysed and tested by coomassie blue staining to confirm np expression at 45.51 kda. np 26 was further purified by ni-nta affinity chromatography and gel filtration. the purity of np 27 was approximately 90% ( figure s1a ). the presence of np was subsequently confirmed by 28 anti-flag antibody ( figure s1b ). the receptor-binding domain (rbd) of s protein (s-rbd) 1 and main protease (lan et al., 2020) were produced by a baculovirus insect expression 2 system and purified to a purity of 90% ( figure s1a ). using sera from patients and healthy donors, igg and igm against sars-cov-2 np, 4 main protease and s-rbd antigens were analyzed. there was no significant antibody 5 response to main protease in sera from several patients (data not shown), suggesting that it 6 may not serve as an antigen for humoral immunity. we thus focused on np and s-rbd. the 7 individual serum samples were then performed by serial dilutions to get optimal dilutions 8 ( figure 1a ). dilution of 1:50 was used for igm and 1:450 for igg. np-and s-rbd-specific 9 igm and igg antibodies were both detected in the sera of newly discharged patients, 10 compared with healthy donor groups. anti-sars-cov-2 igg antibodies were also more 11 obviously observed than igm in the follow-up patients (#9-14), when compared with healthy 12 donors ( figure 1b ). in addition, values from the serum dilution curves were calculated to 13 determine the area under the curve (auc) values. compared to control sera, covid-19 14 patient sera showed significantly higher auc for np-and s-rbd-specific igg antibodies 15 ( figure 1c ). taken together, these findings indicate that covid-19 patients mounted igg 16 and igm responses to sars-cov-2 proteins, especially np and s-rbd, and also suggest 17 that infected patients could maintain their igg amounts, at least for two weeks after 18 discharge. in addition, igg isotypes was further tested in 14 patients and 6 controls. as shown in 20 figure 1d , anti-np and s-rbd igg was mainly igg1 isotype, and the newly discharged and 21 follow-up patients showed similarly amounts of anti-np igg1. of interest, one patient (pt#5) showed higher amounts of anti-np igg3, whereas anti-s-rbd igg3 was detected in two 23 patients (pt#4-5). however, we did not detect igg2 to either np or s-rbd proteins (data not 24 shown). since the rbd of the s protein has been shown to bind to human angiotensin 1 converting enzyme 2 (ace2) (zhou et al., 2020) , the existence of antibodies against it may 2 suggest neutralization of sars-cov-2 infection. to assess this, we performed pseudovirus 3 particle-based neutralization assay, since there was a significantly positive correlation in the 4 neutralizing antibody titers between pseudovirus and sars-cov-2 ( figure 2a ). as shown in 5 figure 2b and 2c, patients #1, 2, 4, 5 and 8, all within the newly discharged group, had high 6 neutralizing antibody titers. these results demonstrate that most recently discharged 7 patients had strong humoral immunity to sars-cov-2. among the follow-up patients, all had 8 neutralizing antibody titers with the exception of patient #9 being negative. as expected, 9 there was a significant correlation between neutralizing antibody titers and auc of anti-s to explore cellular immune responses to sars-cov-2, we isolated peripheral blood 16 monocytic cells (pbmcs) from the whole blood and phenotypically analyzed them by flow 17 cytometry ( figure 3a ). we found that compared to newly discharged patients, there was a 18 trend towards an increased frequency of nk cells in the follow-up patients ( figure 3b ). however, there was no significant difference in terms of the percentages of t cells among 20 those two groups and the healthy donors. to assess virus-specific cellular immunity, we then treated pbmcs with recombinant np, 22 main protease and s-rbd, followed by ifn-γ elispot analysis. the results were considered 23 positive if there were at least 2-fold increase in the numbers of ifn-γ-secreting t cells in the 24 subject than in the healthy donors. as shown in figure 3c , compared with healthy donors, 25 the numbers of ifn-γ-secreting np-specific t cells in patients #1, 2, 4, 5 and 8 were much 26 higher than other patients, suggesting that they had developed sars-cov-2-specific t cell responses. of note, patients #1, 2, 4, 5 and 8 developed both strong humoral and cellular 1 immune responses. main protease-specific t cells were detected in patient #1, 2 and 5, 2 while patients # 1, 2, 4, 5, 6, 7 and 8 showed s-rbd-specific t cells. although the numbers 3 of ifn-γ-secreting s-rbd specific t cells were much lower than those of np-specific t cells, 4 they could be detected in more patients than those for other viral proteins. in the follow-up 5 patients, only patient #10 who showed lymphopenia before treatment still had a high number 6 of ifn-γ-secreting t cells in response to np, main protease and s-rbd ( figure 3c ), which 7 suggests that anti-viral t cells may not be maintained at high numbers in the pbmcs in the 8 recovered patients. more interestingly, when combining all 14 patients in our analysis, there 9 was a significant correlation between the neutralizing antibody titers and the numbers of npin this study, we characterized sars-cov-2-specific humoral and cellular immunity in 2 recovered patients. both were detected in newly discharged patients. in addition, the 3 neutralizing antibody titers significantly correlated with the numbers of np-specific t cells. these findings suggest both b and t cells participate in immune-mediated protection to viral 5 infection. our work has thus provided a basis for further analysis of protective immunity to 6 sars-cov-2, and understanding the pathogenesis of covid-19, especially in the severe 7 cases. it has also implications in designing an effective vaccine to protect and treat sarsin our study, production of s-rbd-specific antibodies were readily detected in recovered 10 patients. moreover, we observed virus-neutralization activities in these recovered patients. not surprisingly, a significant correlation between neutralizing antibody titers and auc of 12 anti-s-rbd igg, but not anti-np igg, was observed. anti-s-rbd igg might be useful in nonetheless, in our study and the one mentioned above, most patients developed 27 measurable neutralization antibodies after infection, suggesting that the viral infection does 1 not curtail adaptive immunity. however, unlike the above-mentioned study, we did not find 2 any correlation between neutralizing antibody titers and patient's age, which could be due to 3 our small sample size. our results thus need further confirmation in a large cohort of covid-4 19 patients. in addition, our analysis could not differentiate cd4 + and cd8 + t cell responses, 5 due to the limitation in the amounts of pbmcs obtained and availability of instrumentation. the plasmid (pet28-n-6xhis) generated in this study will be made available on request 7 from the lead contact without restriction. the study did not generate any unique dataset or code. committee at tsinghua university. informed consent was obtained from all subjects for 19 being included in the study. all patient data were anonymized before study inclusion. see 20 table 1 for full patient information, including age, sex, and health status. cell lines 23 huh-7 cells originally taken from a liver tumor in a japanese male were cultured in dmem 1 supplemented with 10% fbs. cells were grown at 37 °c in a 5% co2 setting. the od value at 450 nm was calculated. neutralizing antibody assay pseudovirus expressing the sars-cov-2 s protein was produced as described previously 19 (deng et al., 1997) . pnl43luci and gp-pcaggs were co-transfected into 293t cells. 48 highlights: 1. sars-cov-2-specific antibodies are detected in covid-19 convalescent subjects. 2. most covid-19 convalescent individuals have detectable neutralizing antibodies. 3. cellular immune responses to sars-cov-2 are found in covid-19 convalescent human convalescence sera notes: pt, patient; f, female; m, male; p, positive; n, negative; bt, before treatment; na key: cord-321918-9jwma2y6 authors: xiu, siyu; dick, alexej; ju, han; mirzaie, sako; abdi, fatemeh; cocklin, simon; zhan, peng; liu, xinyong title: inhibitors of sars-cov-2 entry: current and future opportunities date: 2020-06-15 journal: j med chem doi: 10.1021/acs.jmedchem.0c00502 sha: doc_id: 321918 cord_uid: 9jwma2y6 [image: see text] recently, a novel coronavirus initially designated 2019-ncov but now termed sars-cov-2 has emerged and raised global concerns due to its virulence. sars-cov-2 is the etiological agent of “coronavirus disease 2019”, abbreviated to covid-19, which despite only being identified at the very end of 2019, has now been classified as a pandemic by the world health organization (who). at this time, no specific prophylactic or postexposure therapy for covid-19 are currently available. viral entry is the first step in the sars-cov-2 lifecycle and is mediated by the trimeric spike protein. being the first stage in infection, entry of sars-cov-2 into host cells is an extremely attractive therapeutic intervention point. within this review, we highlight therapeutic intervention strategies for anti-sars-cov, mers-cov, and other coronaviruses and speculate upon future directions for sars-cov-2 entry inhibitor designs. coronaviruses (covs) are enveloped positive-stranded rna viruses. they belong to the order of nidovirales and are classified into four genera: α, β, γ, and δ. 1 coronaviruses are animal viruses with circulating reservoirs in mammals and birds. for most coronaviruses, the lifecycle can be dissected into four steps, including viral entry, replication, assembly, and release. 2 until last year, six strains of coronaviruses have been identified that are pathogenic to humans. among them are cov-nl63, cov-oc43, cov-hku1, and cov-229e that could cause mild respiratory tract diseases. 3 however, two of the β-covs, the severe acute respiratory syndrome coronavirus (sars-cov), and the middle east respiratory syndrome coronavirus (mers-cov) have caused severe epidemics in the past. 4, 5 in april 2003, sars-cov was responsible for 8098 infections, with a fatality rate of ∼10% by the end of september 2003. 6 mers-cov emerged from its zoonotic reservoir in 2012 and infected 2494 people with a fatality rate of ∼34% by the end of 2019. 7 both outbreaks having such high fatality rates, highlight the need for surveillance of coronavirus emergence. while efforts for the development of antivirals against sars-cov or mers-cov are still in process, a new coronavirus (sars-cov-2) has emerged from an epicenter located in wuhan, china, in december 2019. 8 sars-cov-2 is highly contagious and has quickly spread in and beyond china. as of may 28, 2020, there have been more than 5 596 550 diagnosed cases around the world, with 353 373 confirmed deaths (figure 1 ). 9 the united states of america and brazil reporting the majority of the confirmed cases in the americas, with 1 658 896 and 391 222 cases, respectively. recently the genome of sars-cov-2 was determined, which revealed 80% identity with that of some sars-cov strains (gz02, bj01, tor2, sz3, pc4-227) and interestingly 96% identity to the bat coronavirus batcov ratg13. 11 the receptor-binding spike (s) protein is highly divergent from other covs and displays nucleotide sequence identities of 75% or less to all other previously described sars-covs. however, again, the new sars-cov-2 s protein shares 93.1% identity to the ratg13 s protein. 11 the glycoprotein or s protein is responsible for receptor recognition and viral entry into host cells. the spike protein can be divided into two domains; s1 is responsible for angiotensin-converting enzyme ii(ace2) recognition, the recently identified host cell receptor, and s2 mediates membrane fusion (figure 2 ). 12 structural alignment of sars-cov-2 s protein with sars-cov s protein shows that both s proteins are similarly with a root-mean-square deviation (rmsd) of 3.8 å over 959 cα atoms, while the s2 domain, responsible for membrane fusion, display the most substantial similarities with an rmsd of 2.0 å ( figure 2c ). engagement of the host cell receptor ace2 is important for viral entry; however, subsequent entry steps can vary and are cell-type specific. sars-cov can enter the host cell via both clathrin (endosomal) and nonclathrin pathways (nonendosomal); however, both pathways are dependent upon ace2 binding. 13, 14 the clathrin-mediated pathway includes the s protein binding to ace2 and subsequent dynamin/clathrinmediated internalization of endosomal vesicles that maturate to late endosomes. within the late endosomes and lysosomes, acidification of the internalized endosomes and h + -dependent activation of the cellular cathepsin l proteinase takes place that cleaves and activates the s protein, therefore initiating viral fusion with the endosomal/lysosomal membrane (figure 3 ). in the case of sars-cov, cell culture studies revealed that the entry process is delayed with a lag phase of around 30 min, suggesting substantial maturation requirements. 15 in accordance with findings that mouse hepatitis coronavirus (mhv) and feline coronavirus (fcv) infections of hela cells are also heavily dependent on endosomal maturation, the clathrindependent entry and endosomal maturation are key to entry across coronaviridae. 16 for sars-cov-2, a recent study also confirms that virus can use host cell receptor cd147 to gain entry into the host cells besides ace2. 17 in addition to the endosome-mediated entry pathway, host proteases also play critical roles in the nonendosomal entry of coronaviruses. 5 host proteases such as the transmembrane protease serine 2 (tmprss2) and tmprss11d can cleave the s protein at the s1/s2 cleavage site (figure 2 ) to prime and activate the s protein for membrane fusion during the nonendosomal pathway. 18 a recent study also confirms that tmprss2 expressing veroe6 cells are highly susceptible to sars-cov-2 infection, highlighting the importance of tmprss2 in the replication cycle. 19 mers-cov can also be activated by furin (serine endoprotease) to initiate the nonclathrin mediated membrane fusion event. 20 interestingly, in the new sars-cov-2 s figure 3 . entry model of sars-cov-2 into the host cell. binding of the s1 domain within the spike (s) protein to the cellular ace2 receptor triggers conformational changes in the s2 domain that results in internalization and subsequent membrane fusion ((a) endosomal/clathrindependent pathway). the endosomal pathway is facilitated by a low ph and the ph-dependent cysteine protease cathepsin l. alternatively, sars-cov-2 can enter the cell via the nonendosomal/clathrin-independent pathway (b). during this route, ace2 recognition by the sars-cov-2 s protein (comparable to route a) is followed by additional activation/cleavage of the s protein into s1 and s2 domains by cell membrane-associated serine proteases such as tmprss2 and tmprss11d. the figure was prepared with https://biorender.com/. . genome organization of sars-cov-2. genome organization of the sars-cov-2 and location the central genes within the genome (numbers in brackets). 29 the figure was prepared with https://biorender.com. protein, additional amino acid insertions at the s1/s2 cleavage site results in an "rrar" furin recognition site absent in sars-cov s protein. 21 this polybasic insertion sequence has possible implications for the sars-cov-2 replication cycle and its increased pathogenicity. indeed, polybasic furin sites have been observed in hemagglutinin (ha) proteins of highly virulent avian and human influenza viruses, and similar furinlike processing events are also observed for other rna viruses such as ebola virus and marburg virus, human immune deficiency virus (hiv), and flaviviruses. 22 to activate the s protein for membrane fusion with the cellular membrane, structural rearrangements within the s2 domain are required. two heptad repeats, hr1 (dark blue in figure 2 ) and hr2 can interact to form a six-helix bundle (6-hb), a common postfusion structure shared by all type i viral glycoproteins, to bring viral and cellular membranes in close proximity. additionally, the s2 domain contains a membrane interacting domain or fusion peptide that is exposed upon specific triggers such as receptor binding or low endosomal ph. to date, three membrane interacting regions with hostmembrane destabilizing effects have been identified in the sars-cov s protein: two conserved sequences across coronaviridae, with residues 798−815 23 and residues 864− 886, 24 both c-terminal positioned at the second cleavage site in the s protein termed s2′ at arg 797 and a less conserved third region with membrane disordering properties residues 770−788. 25 once in the host cell, the viral particle uncoats and is ready for transcription and translation. 26 the first orf codes for approximately 67% of the genome and is separated into open reading frames (orf) 1a and 1b (figure 4 ). orf1a and orf1b are translated into polyproteins pp1a (4382 amino acids) and pp1ab (7073 amino acids) that are processed by 3-c-like protease (3clpro) and papain-like protease (plpro). the processing of these polyproteins produces a variety of nonstructural proteins (nsps), including rna-dependent rna polymerase (rdrp) and helicase, to catalyze viral genome replication and protein synthesis. 27 the remaining orfs in the sars-cov-2 genome code for accessory and structural proteins. following further assembly, the mature virions are transported to the cell surface in vesicles and released by exocytosis. 28 any protein involved in the replication process could be a potential target for the development of antiviral agents. as mentioned previously, zhang et al. determined the fulllength genome sequence of sars-cov-2 and revealed that the virus was very similar (89.1% nucleotide similarity) to a group of sars-like coronaviruses. 30 simultaneously, shi et al. found that sars-cov-2 shares 96% sequence identity at a wholegenome level to a bat coronavirus, and importantly, they confirmed that sars-cov-2 utilizes the same cell entry receptor, ace2, as sars-cov. 11 recently, the cryo-em structure of full-length human ace2 bound to the rbd of the sars-cov-2 was solved, providing an important structural foundation for intervention strategies. 31 conservation analysis also revealed that the rdrp and the 3clpro are highly conserved between sars-cov-2 and sars-cov. 32 therefore, it is widely accepted that sars-cov-2 would behave similarly to sars-cov with regards to viral entry and replication. being the first step in the infection process, the entry of pathogenic viruses into susceptible cells is an extremely attractive intervention point. as with other well-known viruses, such as hiv-1 and ebola, viral entry of coronaviruses is a complex multiple-step process with numerous interactions and processing points that, in theory, could be targeted. 33 in this review, we summarize case studies and highlight efforts in designing entry inhibitors against sars-cov, mers-cov, and other coronaviruses that can provide important information to combat the current sars-cov-2 outbreak. ■ host cell ace2 receptor recognition by the sars-cov-2 spike (s) as a promising antiviral target binding of the sars-cov-2 spike (s) protein to the cellular ace2 receptor represents the first encounter (in both the endosomal and nonendosomal pathway) in the viral replication cycle and provides prophylactic intervention opportunities. 34 sars-cov-2 spike (s) recognizes with its rbd the cellular ace2 receptor with high affinity (k d = 14.7 nm) 12 as judged by surface plasmon resonance (spr) interaction analysis, and intervention at the rbd-ace2 interface can potentially disrupt infection efficiency. recently the cryo-em and crystal structures of sars-cov-2's rbd in complex with ace2 were solved and provide important structural guidance for inhibitor design ( figure 5 ). 31 the interface can be divided into three contact sides, mainly polar in nature, and is similar to the sars-cov-ace2 complex. 35, 36 in this structure, an extended loop of the rbd contacts an arch-like helix α1 of the proteolytic domain (pd) of ace2 via an n-(cluster 1), central (cluster 2), and cterminal (cluster 3) portion ( figure 5 purple box). additionally, helix α2 and loop 3−4 (connecting β3 and β4) of ace2 provide limited contacts. at the n terminus of α1 (cluster 1), gln498, thr500, and asn501 of the rbd interact via hydrogen bonds with tyr41, gln42, lys353, and arg357 from ace2. the middle portion (cluster 2) of the rbd loop contacts via tyr453, the ace2 pd at residue his34. at the c terminus of α1 (cluster 3), gln474 of rbd contacts gln24 of ace2, and phe486 of rbd interacts with met82 of ace2 through van der waals interactions ( figure 5 ). the structures of the rbds from the sars-cov-2-ace2 complex and the sars-cov-ace2 complex are quite similar, with an rmsd of 0.68 å over 139 cα atoms ( figure 6 ). 31 a comparison of both structures, however, also highlights some deviations at all three clusters summarized in table 1 . these deviations need to be considered carefully during the inhibitor design process. in addition, helix α2 and the loop 3−4 connecting β3 and β4 are also contributing to the interface. sars-cov-2 s protein monomer was obtained from pdb 6vsb and rbd-ace2 complex from pdb 6vw1. boxes 1, 2, and 3 highlight polar clusters 1, 2, and 3, respectively. ■ targeting the rbd peptide analogues, monoclonal antibodies, and protein chimeras as rbd inhibitors. both sars-cov and sars-cov-2 use ace2 to gain entry into the host cells. as such, this critical interaction can be blocked to stop viral entry. 19 this strategy was first demonstrated by hsiang et al. using a biotinylated enzyme-linked immunosorbent assay (elisa), hsiang et al. reported the disruption of the sars-cov s protein-ace2 interaction by small peptides. from a total of 14 designed peptides, peptides sp-4, sp-8, and sp-10 ( figure 7 and table 2 ) significantly blocked the interaction of the sars-cov s protein with ace2 with ic 50 values of 4.30, 6.99, and 1.88 nm, respectively. additional immunofluorescence assay (ifa) studies with s-protein-pseudotyped retroviruses, revealed a novel mechanism of infection inhibition of vero e6 cells by sp-10. 37 38 in light of the successful inhibition of sars-cov with this linked peptide, a similar strategy could potentially be effective against the new sars-cov-2. the recently solved cryoem structure of sars-cov-2 in complex with the human ace2 receptor can provide a structural rationale for the peptide design. 31 monoclonal antibodies (mab) have potential applications for diagnosis, prophylaxis, and treatment of established and evolving viral infections. 39−41 prabhakar et al. isolated specific antibodies from b cells in xenomouse immunized with sars-cov. further investigation revealed that several abs directly react with the rdb domain, and a combination of two abs (4d4 and 3c7) displayed near-complete neutralization efficiency as compared to a single ab application. 42 two additional potent monoclonal antibodies, mab201 and mab68, could be isolated from transgenic mice immunized with the soluble ectodomain of sars-cov s protein. 43 this mab could bind sars-cov s protein directly with affinities of 34 nm (mab 201) and 83 nm (mab 68) as judged by spr analysis. mice that received 40 mg/kg of mab 201 or mab 68 before sars-cov infection showed complete protection from reinfection of lung tissues. 43, 44 cross-reactivity of mabs is highly desirable, and dimitrov et al. identified the human mab m396 that binds sars-cov with high affinity (k d = 20 nm). 45 mice that received 200 μg of m396 were nearly completely protected from infection by urbani and gd03 virus strains. 46 m396 did compete with the sars-cov receptor, ace2, for binding to the rbd, suggesting that m396 inhibits sars-cov-ace2 binding as the predominant mechanism of action. 45 however, sars-cov-2 showed some complexities for rbd directed antibodies. for instance, wrapp et al. tested crossreactivity of three antibodies, including s230, m396, and 80r, against sars-cov-2 rbd. despite the partly high degree of structural homology between the sars-cov-2 and sars-cov, no binding to the sars-cov-2 rbd was detected for any of the three antibodies at the concentration of 1 μm. it can be concluded that sars-cov antibodies will not necessarily be cross-reactive for sars-cov-2. 12 in a different approach, hu et al. generated a novel chimeric recombinant protein recently by connecting the extracellular domain of human ace2 to the fc region of human immunoglobulin igg1. these chimeric constructs displayed high-affinity for the sars-cov-2 and sars-cov rbd binding and potently neutralized sars-cov and sars-cov-2 in vitro, with ic 50 values between 0.8 and 0.1 μm, respectively. these journal of medicinal chemistry pubs.acs.org/jmc perspective recombinant chimeras also showed cross-reactivity and could have, therefore, useful applications for diagnosis, prophylaxis, and treatment of sars-cov-2. 47 using the velocimmune platform, pascal et al. generated several human, noncompeting monoclonal antibodies that target mers-cov s protein and block viral entry into host cells. among them, two antibodies, regn3051 and regn3048, can significantly inhibit mers-cov pseudoparticles, with ic 50 values of 460 and 180 pm, respectively. 48 in addition, regn3051 and regn3048 showed a good performance in a novel transgenic mouse model, which was developed by replacing the mouse dpp4 coding sequence with that encoding human dpp4. results suggested that both regn3051 and regn3048 were able to potently reduce mers-cov specific rna levels in the lungs at a 200 μg per mouse dose compared with the isotype control antibody. at the 20 μg dose, regn3051 was more effective at decreasing mers-cov rna levels compared with regn3048 at the same dose. 48 recently, in the common marmoset model of mers-cov infection, de wit et al. tested the prophylactic and therapeutic efficacy of regn3051 and regn3048. data demonstrated that their protection might be more effective in a prophylactic treatment process rather than treatment of mers-cov. 49 in the latest attempt, chen et al. identified sars-cov-2 rbd specific antibodies from samples of 26 recovered covid-19 patients using an rbd-specific elisa binding study. among them, 311mab-31b5 and 311mab-32d4 effectively neutralized pseudovirus entry, with ic 50 values of 0.0338 and 0.0698 μm, respectively. 50 recently, in an elisa based (cross)reactivity assay, assessing antibody-containing supernatants of a collection of 51 sars-s hybridoma's derived from immunized transgenic h2l2 mice that encode chimeric immunoglobulins, wang et al. identified a chimeric mab 47d11 that targets rbd. 47d11 exhibited cross-neutralizing activity of sars-cov-s protein and sars-cov-2-s protein pseudotyped vsv infection with ic 50 values of 0.19 and 0.57 μm, respectively. 51 brouwer et al. used cross-sectional blood samples from three pcr-confirmed sars-cov-2-infected individuals to screen for binders to a soluble prefusion-stabilized s protein of sars-cov-2 using an elisa-based approach. all three blood samples did bind to the prefusion-stabilized s protein and prompted subsequent sorting of sars-cov-2 s proteinspecific b cells for mab isolation. nineteen nabs could be identified that target a diverse range of antigenic sites on the s protein and showed remarkable picomolar inhibiting activities with the two most potent ic 50 values of 0.010 and 0.007 μg/ ml (cova1-18 and cova2-15, respectively) against live sars-cov-2 virus. 52 large antibody libraries are crucial in response to rapidly emerging pathogens. using eight large phage-displayed vh, scfv, and fab libraries and panning against the rbd of the sars-cov-2, li et al. identified an exceptional potent (k d to rbd of 160 pm as judged by biolayer interferometry) mab igg1 ab1 that competes with ace2 in vitro and protected transgenic mice expressing hace2 from high-titer intranasal sars-cov-2 challenge. 53 in two different assays using replication-competent sars-cov-2 in a microneutralizationbased assay, 100% neutralization at <400 nm, and in a luciferase reporter gene assay, an ic 50 of 200 nm was reported. moreover, transgenic mice expressing human ace2 administrated with 0.3 mg of ig1 ab1 prior intranasal infection with sars-cov-2 did not show any detectable replicationcompetent virus, demonstrating the preventive effect of igg1 ab1. 53 small molecules targeting the rbd. besides peptides, mab, and protein chimeras, small molecules are still the preferred modality for a drug. this is due to improved pharmacokinetics, stability, and dosage logistics compared to proteins or peptides. 54, 55 in addition, small molecules have advantages compared to peptides/proteins regarding dissemination logistics in remote areas and the high expenses of peptide/protein production. 54, 55 to identify small molecule entry inhibitors against the sars-cov s protein, sarafianos et al. screened a chemical library composed of 3000 compounds according to lipinski's rule of five 56 and identified an oxazole-carboxamide derivative, ssaa09e2 (1, table 3), that blocks the binding of the rbd of 57 lundin et al. screened a library of 16 671 diverse compounds and found a small molecule inhibitor, k22 (2), which was able to inhibit hcov-229e with an ic 50 value of 0.7 μm and cc 50 value of 110 μm. studies for mechanism showed that k22 targeted a very early step in the hcov-229e life cycle and may interact with viral particles, thus inactivating their binding. 58 ■ targeting the cellular receptor peptide analogues as ace2 inhibitors. human angiotensin-converting enzyme (ace) is a highly glycosylated type i integral membrane protein and has been identified as a fundamental regulator of the renin−angiotensin system (ras) in humans and is an important target in regulation of blood pressure homeostasis. ace2 is a human homologue of ace. 59 it contains a single zinc-binding catalytic domain, which is 42% similar to the human ace active region. 60 ace2 can catalyze the cleavage of angiotensin i into angiotensin 1-9, and angiotensin ii into the vasodilator angiotensin 1-7 and its organ-and cell-specific expression also suggests a role in the regulation of cardiovascular and renal function and fertility. 60 ace2 is a functional receptor to the sars-cov during viral entry, and recent research demonstrated that sars-cov-2 also utilizes ace2 for infection. 61 however, ace2 cannot be inhibited by ace inhibitors, so there is an urgent need to develop specific ace2 inhibitors that would prevent infection by both sars-cov and sars-cov-2. one of the first efforts to target the ace2 receptors was documented by liu et al. using a novel epitope assembling assay, liu et al. identified linear b-cell immuno-cross-reactive epitopes of sars-cov s protein by synthesizing 22 longer peptides. five of these peptides showed serologically highly cross-reactivity in all tested sars patients sera. among them, peptide s 471-503 could significantly block the binding of rbd to ace2. s 471-503 , derived from the s1 fragment ( figure 7 and table 2 ) could target ace2, and showed antiviral activity against sars-cov infection in vitro, with an ec 50 value of 41.6 μm, providing an important basis to explore the antiviral potential of s 471-503 against sars-cov-2. 62 another peptide derived from the rbd, rbd-11b, located in s1 of the sars-cov s protein, is crucial for binding to the host cells ace2 receptor 62 (figure 7 and table 2 ). given the vital role of this motif, meyer et al. confirmed the binding to ace2 of a synthesized peptide mimicking this region ( 438 ykyryl 443 ) with a k d of around 46 μm. moreover, rbd-11b displays no toxicity, as judged by an mtt (3-(4,5)dimethylthiahiazo-(-z-y1)-3,5-di-phenytetrazoliumbromide) cell proliferation assay, on veroe6 cells. in addition, rbd-11b showed antiviral activity to hcov-nl63 at a peptide concentration of 7 mm in caco2 cells, which also used ace2 as a functional receptor. 63 constrained peptides are receiving more attention in the drug development field, combining the best attributes of antibodies and small molecules. linear peptides are often highly flexible and unstructured in solution, only forming structures upon target binding. this can sometimes reduce the affinity of such peptides for their target by an entropic penalty mechanism. however, stabilization methods such as cyclization or hydrocarbon stapling can increase the physicochemical characteristics and drug-like properties while negating the entropic penalty of binding and having a positive impact on affinity. 64 using a constrained peptide library displayed on filamentous phages, ladner et al. identified several peptides inhibiting ace2 function with the most potent being dx600 (table 2) . dx600, an n-terminal acetylated and c terminal amidated peptide, was a potent ace2 peptide inhibitor with an ic 50 value of 10 nm and a k i value of 2.8 nm. dx600 did not inhibit ace activity and thus is specific to ace2. in addition, dx600 was chemically stable and not hydrolyzable by ace2. 65 although it is not clear whether dx600 can inhibit coronavirus, as an effective ace2 inhibitor, anticoronavirus tests should be conducted in the future. small molecule as ace2 inhibitors. as discussed previously, peptide and constrained peptide inhibitors have inherent caveats concerning their use as drugs. 64 therefore, screening for small molecule inhibitors, guided by information gleaned from the previous studies is the next logical step. a virtual screen targeting the ace2 catalytic site with around 140 000 compounds combined with a molecular docking approach led to the identification of naae (n-(2-aminoethyl)-1 aziridine-ethanamine) (3, table 3 ). 3 showed a dosedependent inhibition of ace2 catalytic activity with an ic 50 value of 57 μm and a k i of 459 μm. despite its micromolar potency in inhibiting a sars-cov pseudotyped virus, cytotoxicity data is not available to date. 66 chloroquine (4) currently has applications for malaria and amoebiasis treatment. interestingly, nichol et al. showed that chloroquine could also block the interaction of rbd of sars-cov to ace2 under cell culture conditions with an ed 50 value of 4.4 μm. 67 recently, wang et al. found that 4 blocked sars-cov-2 virus infection, with an ic 50 value of 1.13 μm and a cc 50 > 100 μm in vero e6 cells. 68 chloroquine possibly increases endosomal ph required for virus/cell fusion as well as impairs with the terminal glycosylation of the cellular ace2 receptor, thereby reducing the affinity of sars-cov/sars-cov-2 to ace2. besides its antiviral activity, chloroquine may synergistically enhance its antiviral effect with immunemodulating activity in vivo. 68 at present, chloroquine is carried out in clinical research in china for the treatment of sars-cov-2 (chictr2000029609). 69 hydroxychloroquine (5) is an analogue of chloroquine, which shares the same mechanism of action as chloroquine but displays a more tolerable safety profile. 70 71 recent studies suggest that 4 and 5 could cause ventricular arrhythmias, 72 qt prolongation, 72,73 retinopathy, 74 and other cardiac-related toxicity, which may pose a particular risk to critically ill patients. although both show antiviral activity, safety, and effectiveness, they require further clinical research. turner et al. identified that the sars-cov receptor, ace2, undergoes proteolytic shedding, releasing an enzymatically active ectodomain during viral entry. 75 further research identified that a disintegrin and metalloproteinase (adam17) is responsible for shedding regulation of ace2. inhibiting adam activity with the adam-specific inhibitor gw280264x (6) reduced shedding of ace2 at 1 nm against sars-cov. 76 another enzyme involved in ace2 shedding is tace (tnf-α converting enzyme, a member of the adam family). two tace inhibitors, tapi-0 (7) and tapi-2 (8), reduced ace2 shedding against sars-cov, with ic 50 values of 100 and 200 nm, respectively. 75 perhaps the most promising small molecule described to date is the very potent ace2 inhibitor mln-4760 (9). 9 can inhibit the catalytic activity of ace2 with an ic 50 of around 440 pm. 77 the crystal structure of the apo and 9 bound ace2 complex revealed a significant subdomain movement of the nterminal and c-terminal subdomains of ace2 upon 9 binding. this movement is important to position critical residues to stabilize the bound inhibitor. its high potency makes 9 a very attractive candidate for sars-cov-2 interference; however, no antiviral coronavirus data is available at this time. milewska et al. synthesized several polymer-based compounds showing prominent anticoronaviral activity. among them, a cationically modified chitosan derivative, n-(2hydroxypropyl)-3-trimethylammonium chitosan chloride (htcc, 10), and hydrophobically modified htcc (hm-htcc, 11) were found that could inhibit hcov-nl63 replication. for both tested polymers, their ic 50 values were relatively low in llc-mk2 cells, amounting to ∼50 nm for 10 and ∼230 nm for 11. cc 50 values were ∼0.8 and ∼1 μm for 10 and 11, respectively. 78 recent research showed that 10 and 11 blocked the interaction of hcov-nl63 with its ace2 receptor and thus interfered with the process of viral entry. 79 despite the availability of many compounds with inhibitory effects on ace2, the corresponding admet data in a preclinical model is not available. regardless, direct inhibition of ace2 is probably not a viable therapeutic modality, however. this is due to its important normal physiological roles, in addition to its lung injury protective role in acute respiratory distress syndrome from a variety of causes, including sars-cov infection. 80, 81 as such, directly inhibiting ace2 as an antiviral strategy appears to be physiologically unsound, and virally targetted blockers of its interaction with the sars-cov/sars-cov-2 s protein hold greater promise. membrane fusion is a crucial step in the mers/sars infection cycle in both described pathways (see section 1). within the endosomal/clathrin-dependent route, internalized viral particles need to fuse with the endosomal membrane to escape the endosomal/lysosomal environment. this is achieved via a conformational change of the s protein (s2 domain) within the acidic milieu followed by membrane fusion activation by the host protease cathepsin l. membrane fusion is also essential during the nonendosomal/clathrin-independent route to fuse with cellular membranes facilitated by host protease cleavage of the s protein by cell membrane-associated proteases such as tmprss2. 19 in conclusion, the s2 domain of the sars-cov s protein and host proteases such as 84, 85 hr1 and hr2 can interact with each other to form a 6-hb to bring viral and cellular membranes close (for exact location, see figure 2 ). on the basis of this requirement, bosch et al. obtained peptides corresponding to region hr2 within the hr. hr2-8 displayed in an infection inhibition assay with pseudotyped sars-cov s protein in vero cells an ec 50 value of 17 μm (figure 7 and table 2 ). 84 moreover, hr2-8 demonstrated concentration-dependent inhibition of hcov-nl63 infection with an ic 50 value of 0.5 μm and a cc 50 value of 20 μm. 86 on the basis of these initial results, further development of the hr2-8 peptide is necessary to develop a more potent human coronaviruse (hcov) peptide inhibitor. similarly, ngai et al. obtained three hr derived peptides, including hr1-a, gst-removed-hr2, and hr2 peptide, with remarkable inhibitory activity against sars-cov ( figure 7 and table 2 ). virus entry inhibition studies suggested that hr1-a, derived from the hr1 region, had an ec 50 value of 1.61 μm. gst-removed-hr2 peptide and hr2 peptide, derived from the hr2 region, had ec 50 values of 2.15 and 0.34 μm, respectively. 87 hr2p, spanning residues 1251−1286 in hr2 domains, could effectively inhibit mers-cov infection and s protein-mediated membrane fusion (figure 7 and table 2 ). this study indicates that hr2p could specifically inhibit mers-cov in vero cells, with an ic 50 value of ∼0.6 μm and a cc 50 value of >1000 μm. hr2p also demonstrated high selectivity, as indicated by its high selectivity index (si > 1667). importantly, the introduction of arg, lys, or glu residues into the hr2p peptide increased stability, solubility, and anti-mers-cov activity. 88 to improve the stability, solubility, and antiviral activity of hr2p, channappanavar et al. designed and synthesized an hr2p analogue named hr2p-m2. hr2p-m2 strongly blocked s protein-mediated cell−cell fusion in a dose-dependent manner at ic 50 values of 0.55 μm in vitro. in vivo, hr2p-m2 intranasal administration to ad5/ hdpp4 transgenic mice protected them from mers-cov infection and reduced the lung viral titers by more than 1000fold. moreover, combination treatment with ifn-β was demonstrated to enhance the protective effect. 89 the development of a drug with broad-spectrum hcov inhibitory activity is increasingly becoming an attractive approach. xia et al. found that the ek1 peptide showed pan-cov fusion inhibitory activity against multiple hcovs ( figure 7 and table 2 ). 90 further investigation revealed that ek1 directly reacts with the hr1 region and can competitively inhibit viral 6-hb formation. the pseudovirus assay suggested that the antiviral activity of ek1 against hcov-oc43, hcov-nl63, and hcov-229e infection with ic 50 values of 1.81, 6.02, and 3.35 μm, respectively. in vitro cytotoxicity assay determined that ek1 is not cytotoxic at concentrations up to 1 mm. mice that received 5 mg/kg of ek1 were nearly completely protected from infection by hcov-oc43 and 200 μg of ek1 against mers-cov infection. recently, this team found that ek1 could also potentially inhibit sars-cov-2 with an ic 50 value of 2.38 μm in pseudovirus assay and an ic 50 value of 0.19 μm in fusion inhibitory assay. 91 to improve the inhibitory activity of ek1 against sars-cov-2, they conjugate the cholesterol molecule to the ek1 peptide and found that a new peptide, ek1c4, exhibited highly potent inhibitory activity inhibit sars-cov-2 s-mediated membrane fusion and pseudovirus infection with ic 50 values of 1.3 and 15.8 nm, the cc 50 of ek1c4 was 5 μm, and the selectivity index was >136. in the oc43-infected mouse model, mice that received 0.5 mg/kg of ek1c4 were nearly completely protected from infection by hcov-oc43. these data suggested that ek1c4 could be used for inhibition and treatment of infection by currently circulating sars-cov-2. 92 mers-5hb, a polypeptide derived from the hr1 and hr2 region, was synthesized by gong et al., and affinity analysis demonstrated a low k d value of 0.24 nm, and an ic 50 value of 1 μm against mers-cov and cc 50 > 50 μm. hr derived peptides is a highly promising strategy for viral fusion inhibition. successful hr peptides have been used in the past to block entry of other virus families such as the hiv with the gp41 derived peptide fuzeon (t20), the only approved fusion inhibitor for hiv-1 treatment to date. 93 therefore, hr derived peptides highlight a promising strategy for inhibitor development combating the new sars-cov-2. xia et al. reported that two peptide-based membrane fusion inhibitors, 229e-hr1p and 229e-hr2p (figure 7 and table 2 ), targeting the hcov-229e s protein hr1 and hr2 domains, could competitively inhibit the viral autologous 6-hb formation and inhibit hcov-229e s protein-mediated viruscell membrane fusion with ic 50 values of 5.7 and 0.3 μm, respectively. moreover, neither 229e-hr1p nor 229e-hr2p had significant cytotoxicity to huh-7 and a549 cells at concentrations up to 1000 μm. in addition, 229e-hr2p potentially inhibited pseudotyped and live hcov-229e infection with ic 50 values of 0.5 and 1.7 μm, respectively. 94 the s2 domain is the most conserved motif between the sars-cov and the new sars-cov-2 s protein. 92 it represents an ideal immunogen for the generation of a novel or repurposing sars-cov s2 domain targeting mabs with cross-reactive potential. 95 sasazuki et al., for example, could successfully isolate the human mab 5h10 from immunized kunming (km) mice. 5h10 displayed an anti-sars-cov neutralizing activity of around 5 μg/ml. cell fusion assays indicate that 5h10 can inhibit viral fusion and entry rather than viral attachment to the surface of host cells or cleavage of the s protein. consequently, the s protein of sars-cov might be the direct target of 5h10; however, further studies are required to confirm this hypothesis. 96 tan et al. identified mab 1a9 (ic 50 value between 25 and 50 μg/ml), an anti-sars-cov s2 domain mab, that binds to a conserved loop region between the hr1 and hr2 domains of the s2 domain. 97 tsunetsugu-yokota et al. found that antibody skot20 can inhibit sars-cov with an ec 50 value of 5 μg/ml in vero e6 cells sars-cov. mutational studies indicate that skot20 restrict conformational changes within the s2 domain, essential for viral entry. 98 99 on the basis of this approach, they identified two small molecules, tgg (12, table 4 ) and luteolin (13) , that can bind avidly to the sars-cov s2 protein and inhibit viral entry of sars-cov into vero e6 cells with ic 50 values of 4.5 and 10.6 μm, respectively. cytotoxicity assay showed that the cc 50 of 12 and 13 were 1.08 and 0.155 mm, respectively. therefore, the selectivity index of 12 and 13 were 240.0 and 14.62, respectively. further acute toxicity suggested that the 50% lethal doses of 12 and 13 were ∼456 and 232.2 mg/kg, respectively. these indicated that these small molecules could be used at relatively high concentrations in mice. 98 quercetin (14), an analogue of 13, also showed antiviral activity against sars-cov, with an ic 50 arbidol (16), a broad-spectrum drug, has been licensed for decades in russia and china against influenza by binding to the ha protein to block the viruses−cell fusion. 103 recently, wang et al. identified that 16 efficiently inhibited sars-cov-2 virus infection in vitro with an ic 50 value of 4.11 μm, a cc 50 value of 31.79 μm, and an si of 7.73. 104 vankadari compared protein sequence analysis and found that a small region of the s2 domain (aa947−aa1027) of the sars-cov-2 spike glycoprotein resembles that of the influenza virus h3n2 ha. so the mechanism of 16 was to target the sars-cov-2 spike glycoprotein and blocked its trimerization, which may inhibit host cell adhesion and hijacking. 105 in january 2020, in wuhan, china, a clinical pilot trial conducted with 36 patients with sars-cov-2 virus infection received 400 mg 16 three times a day for 9 days; 31 untreated sars-cov-2 patients served as a control group. in this trial, patients with 16 showed a tendency to decrease viral load as determined by rt-pcr and reduced mortality (0% vs 16%), as compared to the control group. 106 the hr regions of sars-cov and sars-cov-2 s protein share a high degree of conservation, and the described small molecules as fusion inhibitors can have potential applications in inhibiting sars-cov-2 fusion. indeed, targeting virus surface protein is a promising antiviral strategy, whether inhibiting rbd or s2 domain. during clathrin-dependent viral entry, the host cellular cathepsin l protease plays a key role in infection efficiency by activation of the s protein into a fusogenic state to escape the late endosomes, and cathepsin l (lysosomal endopeptidase) cleavage is believed to expose a hydrophobic fusion peptide essential to initiate membrane fusion. 107 in light of its vital role in the sars cov infection cycle, cathepsin l is a desirable target to interfere with virus−cell entry. 83 cathepsin l consists of a pro-and a mature-domain. in a low ph milieu, the pro-domain is autocatalytically cleaved to obtain the papain-like folded mature-domain consisting of an n-terminal helical domain and a c-terminal β-sheet domain (figure 8 ). 108 a well conserved cys-his-asn triad in the active site is crucial for substrate binding and catalysis. in light of its importance in the sars-cov-2 replication cycle, cathepsin l is a highly desirable target that will be described in the following section. 109 teicoplanin is a glycopeptide antibiotic, with applications in the treatment of serious infections caused by gram-positive bacteria such as streptococcus and staphylococcus aureus. 110 interestingly, teicoplanin was shown to block the entry of sars, mers, and ebola virus by specifically inhibiting the cathepsin l activity. 111 more recently, zhang et al. showed that teicoplanin could also block the entry of the new sars-cov-2 pseudoviruses with an ic 50 value of 1.66 μm. as a routinely used clinical antibiotic, teicoplanin could be potentially used immediately to combat the current sars-cov-2 outbreak. 112 small molecules as cathepsin l inhibitors. human cathepsin l plays numerous critical roles in diverse cellular settings associated with human diseases. 113 previous studies also highlighted the feasibility of targeting this cysteine endopeptidase with small molecules with implications for possible intervention strategies of sars-cov-2 infection. 113 a high-throughput screen (hts) of a 1000-compound library that resulted in the identification of mdl28170 (17 , table 4 ) by bates et al., and in an antiviral activity assay, 17 specifically inhibited cathepsin l-mediated substrate cleavage and blocked sars-cov viral entry, with an ic 50 value of 2.5 nm and ec 50 value in the range of 100 nm. however, despite its potent inhibitory activity, no cytotoxicity data for 17 is currently available. 83 two small molecules, cid 16725315 (18) and cid 23631927 (19) , were reported by diamond et al. as viral entry inhibitors of the sars-cov. in a cathepsin l inhibition assay, 19 could block cathepsin l with an ic 50 value of 6.9 nm, while 18 showed slightly weaker potency with an ic 50 value of 56 nm. interestingly, besides inhibiting sars-cov, compound 19 (ec 50 value of 273 nm) showed some inhibition activity for ebola virus infection (ec 50 value of 193 nm) of human embryonic kidney 293t cells. importantly, 19 did not show any sign of toxicity to human aortic endothelial cells at 100 μm. this data offers a new promising point for the treatment of sars and ebola virus infections. 114 recently, in a cell-based assay screen of ∼14 000 compounds, ssaa09e1 (20) was identified that could specifically bind to the cathepsin l proteinase and interference sars-s protein during viral entry, with an ic 50 value of 5.33 μm. in a pseudotype-based assay in 293t cells, the ec 50 value of 20 was around 6.4 μm, and no cytotoxicity was detected below 100 μm. 57 using sars-cov entry assays, zhou et al. screened 2100 cysteine protease inhibitors with confirmed activity to inhibit human cathepsins. among them, k11777 (21) demonstrated the most robust activity. results demonstrated that 21 blocked sars-cov pseudovirus entry at an ic 50 value of 0.68 nm while no toxicity was observed, cc 50 value >10 μm. interestingly, for other coronaviruses, 21 showed broadspectrum antiviral activity with ic 50 values of 1.48, 6.78, and 46.12 nm against hcov-229e, hcov-nl63, and mers-cov, respectively. 115 inhibitors of cell membrane-associated tmprss2. either the endosomal cysteine proteases cathepsin l or the cell membrane-associated serine protease tmprss2 can facilitate sars-cov virus entry into host cells by cleavage of the viral s protein. 19 this cleavage exposes fusion-competent motifs known as fusion peptides, and importantly, for sars-cov, the interference of both proteases is required for efficient inhibition of virus replication. 19 matsuyama et al. identified camostat (22 , table 4 ), a commercially available serine protease inhibitor that can efficiently prevent sars-cov infections at 10 μm by inhibiting tmprss2 activity. however, even at high concentrations (100 μm) of 22, the inhibition of viral entry via sars s protein-mediated cell fusion never exceeded 65% (inhibition efficiency), indicating that despite the inhibition of tmprss2, 35% of virus entry takes place via the endosomal cathepsin pathway. therefore, they examined the activity of pseudotyped viruses when treated with a combination of (23,25)trans-epoxysuccinyl-l-leucylamindo-3methylbutane ethyl ester (est, a cathepsin inhibitor) and 22. the results suggested that simultaneous treatment with est and 22 remarkably blocked infection (>95%). 116 similarly, poḧlmann et al. reported that 22 could prevent the viral entry of sars-cov-2. importantly, full inhibition efficiency was attained when treated with both 22 and e-64d (a cathepsin inhibitor). both studies indicate that sars-cov and sars-cov-2 enter cells via a similar mechanism, showing the potential of 22 as a promising candidate for further development as a sars-cov-2 treatment. 19 inhibitors of the furin cleavage site in the coronavirus spike proteins. elevated levels of furin expression were able to facilitate mers-cov pseudovirion infection, and viral entry could be reduced by furin sirna silencing. 20 decanoyl-rvkr-chloromethylketone (23, dec-rvkr-cmk), a furin inhibitor, was shown to block mers-cov s protein-mediated entry as well as virus infection, with journal of medicinal chemistry pubs.acs.org/jmc perspective an ic 50 value of 75 μm in hek-293t cells. furthermore, when cathepsin inhibitor camostat was used in combination with 23, a significant inhibition in infectivity was characterized compared to camostat alone. 20 recently, bestle et al., showed that the potent peptidomimetic inhibitor mi-1851 (24) could prevent proteolytic processing of the s protein from sars-cov-2 by endogenous furin in hek293 cells. however, no antiviral data is available for 24 yet. 117 the peculiar furin-like cleavage site (s1/s2-site in figure 2 ) in sars-cov-2 that is absent in the sars-cov and other sars-like covs indicates that furin inhibitors could play a significant role in blocking the viral entry process. 117, 118 ■ host factor inhibitors sars-cov-2 cell entry also relies on host cell factors. therefore, these host cell factors can play an essential role as targets for sars-cov-2 inhibition. 119 chlorpromazine (25 table 5 ) is an antipsychotic drug developed for the treatment of schizophrenia. it has also been reported to inhibit the infection of hepatic c virus (hcv), 120 mouse hepatitis virus (mhv-2), 27 and alphavirus. 121 (28), an abelson kinase signaling pathway inhibitor that could inhibit abelson tyrosine−protein kinase 2 (abl2) to block mers-cov virion fusion with endosomal membranes with an ic 50 value of 10 μm. 28 showed no cytotoxic effects in vero cells at 100 μm. 123, 124 another abl inhibitor, dasatinib (29) , was active against both mers-cov and sars-cov, with ic 50 values of 5.4 and 2.1 μm, respectively. 125 on the basis of an hts assay using cytopathic-effect ( phenotypic screening methods are usually used to identify firstin-class drugs without knowing the actual target and mechanism of action of the drug, while target-based screening identifies best-in-class drugs. 127−129 although the phenotypic screening approach often is limited in terms of capacity compared to in silico target-based screening, it can have advantages in identifying cell-active compounds providing information on drug solubility or cell uptake. 127−129 many drugs, especially natural products, have an unknown mechanism of action but were shown to inhibit coronavirus entry. 130 hsiang et al. screened a library of 121 chinese herbs using a biotinylated enzyme-linked immunosorbent assay to search for active compounds that could potentially inhibit sars-cov s protein binding to ace2. further studies identified emodin (31 , table 5 ), the active component from polygonum multiflorum and rheum officinale, could block the interaction of sars-cov s protein to ace2, with an ic 50 value of 200 μm in an s protein-pseudotyped retrovirus assay using vero e6 cells. however, the mechanism of action of 31 still needs to be determined. 131 sarafianos et al. found that ssaa09e3 (32), a benzamide derivative, could prevent sars-cov virus−cell membrane fusion in pseudotyped-based and antiviral-based assays, with an ic 50 value of 9.7 μm, but a cc 50 value of 20 μm indicates additional unknown cellular targets. 57 out of an hts, ve607 (33) was identified using a phenotype-based screen from a 50 240 structurally diverse small-molecule compound library. pseudotype virus entry assay suggested ve607 can specifically inhibit sars-cov virus entry into cells with an ec 50 value of 3 μm and inhibited sars-cov plaque formation with an ic 50 of 1.6 μm. 132 a similar hts approach was employed by zhang et al. for screening a compound library consisting of 727 structurally diverse small molecules. eighty-four compounds were identified with significant anticoronavirus potential. further studies revealed that 51 compounds inhibited virus entry, while 19 others interfered with viral replication. 133 natural products should, however, be considered with caution due to their unknown mechanism of action and possible toxic side effects. the recent sars-cov-2 outbreak, with its high fatality rate, has raised global concerns and was declared as a global pandemic by the who. the number of infections continues to rise, and numerous research groups around the globe have prioritized the identification and development of new covid-19 treatments. still, there are no effective treatments to date. viral entry is the first step in the viral life cycle and represents an attractive intervention point by blocking the coreceptor interaction or the virus−cell membrane fusion event. sars-cov-2 and other coronaviruses have similar infection mechanisms. this is especially true for sars-cov and cov-nl63, which share the same human ace2 receptor crucial for viral entry. therefore, already developed inhibitors against known hcovs could potentially be used to combat sars-cov-2. these efforts identified a large number of inhibitors, including peptides, antibodies, small-molecule compounds, and natural products with anticoronavirus activity. although many inhibitors demonstrated efficacy in inhibiting coronavirus virus infection, no specific prophylactic or postexposure therapy is currently available for hcovs. one of the main reasons causing this is that most of the potenial agents were not adequately evaluated for in vitro and in vivo studies. most drugs are in the preclinical stage and stopped in animal models due to poor bioavailability, safety, and pharmacokinetics so that few entered human trials. in light of the urgency of the current outbreak, repositioning of already approved drugs is increasingly becoming a promising approach, especially with toxicity and safety data in hand. the most effective measure to prevent viral diseases is vaccination. coronavirus vaccine development mainly focused on s protein, and some of them reported can inhibit sars, 134−136 and mers. 137 although vaccination strategies were developed in the context of previous epidemics, no vaccine for sars-cov-2 infections is yet available. since the recent sars-cov-2 outbreak, research groups around the world are now stepping up to develop vaccines targeting sars-cov-2, and vaccine research routes include nucleic acid vaccines, viral vector vaccines, inactivated vaccines, and recombinant protein vaccines. typical vaccine development is time, resource, and financially consuming, although this pandemic has created initiatives that hope to speed the development of a sars-cov-2 vaccine. even the most optimiztic views regarding an effective sars-cov-2 vaccine being created are at least one year away. even after creation, other hurdles for the sars-cov-2 include global implementation and distribution, and different strategies for containing this contagion should be explored simultaneously as the vaccine efforts. in addition to small-molecule inhibitors, monoclonal antibodies, and vaccine development, convalescent sera from sars-cov-2 survivors (convalescent-phase sera) is an additional option for covid-19 treatment. passive immunization was well established for viral infection prophylaxis. 138 by metaanalysis of studies about the 1918 influenza, h1n1 influenza epidemic demonstrated that early treatment of convalescent blood products decreased the risk ratio caused by pneumonia from 37% to 16%. 139 nevertheless, the appropriate titer of the convalescent-phase sera antibody remains to be determined, which was required for therapeutic efficacy to inhibit sars-cov-2. research carried out with mers-cov suggested that sera from patients recovering from infections 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infection: a study protocol key: cord-260406-qvc2fb0c authors: chen, long; liu, bo; sun, peng; wang, wenjun; luo, shiqiang; zhang, wenyuan; yang, yuanfan; wang, zihao; lin, jian; chen, peng r. title: severe acute respiratory syndrome coronavirus‐2 spike protein nanogel as a pro‐antigen strategy with enhanced protective immune responses date: 2020-10-26 journal: small doi: 10.1002/smll.202004237 sha: doc_id: 260406 cord_uid: qvc2fb0c prevention and intervention methods are urgently needed to curb the global pandemic of coronavirus disease‐19 caused by severe acute respiratory syndrome coronavirus‐2 (sars‐cov‐2). herein, a general pro‐antigen strategy for subunit vaccine development based on the reversibly formulated receptor binding domain of sars‐cov‐2 spike protein (s‐rbd) is reported. since the poor lymph node targeting and uptake of s‐rbd by antigen‐presenting cells prevent effective immune responses, s‐rbd protein is formulated into a reversible nanogel (s‐rbd‐ng), which serves as a pro‐antigen with enhanced lymph node targeting and dendritic cell and macrophage accumulation. synchronized release of s‐rbd monomers from the internalized s‐rbd‐ng pro‐antigen triggers more potent immune responses in vivo. in addition, by optimizing the adjuvant used, the potency of s‐rbd‐ng is further improved, which may provide a generally applicable, safer, and more effective strategy for subunit vaccine development against sars‐cov‐2 as well as other viruses. the outbreak of severe acute respiratory syndrome coronavirus-2 (sars-cov-2) infection has caused a pandemic of coronavirus disease-19 (covid19) , posing a great threat to human life globally. [1] till mid-june of 2020, more than 9 million individuals were tested positive for covid-19, with a death toll over 470 000 worldwide. [2] early efforts have focused on finding small-molecule drugs such as favipiravir, chloroquine, enzyme 2 (hace2). [1] the s-rbd of sars-cov-1 has been used as a candidate subunit vaccine to prevent virus entry into cells. [9] when formulated with adjuvants, sars-cov-1 rbd can elicit protective immune responses. [10] since sars-cov-2 s-rbd possesses 80% sequence similarity to sars-cov-1 rbd with an even higher binding affinity to human ace2, [11] it should also be a suitable antigen for subunit vaccine development. nevertheless, the poor pharmacokinetics and low immunogenicity greatly hindered the use of s-rbd for subunit vaccine development. [12] a critical reason for the low immunogenicity of s-rbd lies in its poor targeting ability to lymph nodes, which is crucial for antigen uptake and processing by dendritic cells (dcs) and macrophages. [13] as nano particle formulations have been shown to enhance cell permeability and potency of anti-cancer drugs, [14] we envisioned that formulating s-rbd into redox-responsive nanogels may serve as a pro-antigen with improved lymph node targeting and dc and macrophage accumulation, which can lead to synchronized release of internalized s-rbd monomers with enhanced protective immune responses. herein, we report a pro-antigen strategy based on the reversibly formulated spike protein nanogel (s-rbd-ng) for subunit vaccine development against sars-cov-2 (scheme 1). we started by overexpressing and purifying sars-cov-2 s-rbd from yeast cells and then verified its integrity via sds-page and western blotting analysis ( figure s1 , supporting information). since sars-cov-2 s-rbd contains two n-linked glycosylation sites, the observed mass from lc-ms analysis was heterogeneous but higher than the calculated molecular weight without n-glycosylation ( figure s2 , supporting information). we further used enzyme-linked immunosorbent assay (elisa) to prove that recombinant human ace2 can bind to s-rbd expressed in yeast ( figure s3 , supporting information). the affinity was measured to be ≈7.65 nm ( figure s4 , supporting information), which was consistent with the reported value. [15] furthermore, the interaction can be competed with an s-rbd targeting sars-cov-2 neutralizing nanobody, which indicated a similar binding pattern of the yeast-expressed s-rbd ( figure s5 , supporting information). [16] we then used amine reactive, redoxresponsive reversible chemical crosslinkers to generate protein nanogels ( figure 1a) . two crosslinkers with different spacer groups were synthesized ( figure 1b) , which contain an internal disulfide bond that is reduced upon uptake by antigen-presenting cells (apcs) to disassemble the ngs back to protein monomers. notably, whereas reduction of crosslinker 1 (cl1) would generate a thiol group on the protein, crosslinker 2 (cl2) would undergo a rearrangement to regenerate the native amine on the protein upon reduction ( figure 1c ). s-rbd protein was treated with both crosslinkers at different equivalents and sds-page analysis showed that ngs formed by cl2 exhibited a slightly higher efficiency than formed by cl1 and all ngs could be reduced to monomers ( figure 1d ). the crosslinked bands were further collected and subjected to dynamic light scattering (dls) and transmission electron microscopy ( figure 1e ,f). in contrast to the native s-rbd that had a diameter of ≈2 nm, as measured by dls ( figure s6 , supporting information), its average diameter was increased to ≈25 nm upon crosslinking, which confirmed the formation of s-rbd ngs. as the uptake of antigens by apcs (dcs and macrophages) is critical for antigen processing and cross-presentation, we first examined the internalization of s-rbd-ngs by these cells. confocal microscopy was used to quantify the uptake by dc2.4 or raw 264.7 cells after incubation with ngs formulated using different s-rbd/crosslinker ratios (figure 2 , figures s7 and s8 , supporting information). compared to the s-rbd monomer, enhanced cellular uptake of s-rbd-ngs prepared with both cl1 and cl2 was observed, and quantitative analysis of the imaging data showed an approximately fourfold enhancement of uptake with 50 equivalents of crosslinkers ( figure 2b ,d). since reduction of cl2 can regenerate native proteins, we used cl2 at a 50:1 ratio with s-rbd to produce ngs for the following study. scheme 1. the design of reversibly formulated sars-cov-2 s-rbd protein nanogel (s-rbd-ng) as a pro-antigen strategy for subunit vaccine development for covid-19. s-rbd was formulated with redox-responsive crosslinkers as a pro-antigen with enhanced lymph node targeting and antigen presenting cell (apc) accumulation. synchronized regeneration of s-rbd monomers from the internalized s-rbd-ng pro-antigen triggered more potent immune responses to neutralize sars-cov-2. next, we tested whether s-rbd-ng could improve lymph node targeting ability in vivo. cy5-labeled s-rbd or s-rbd-ng were administered to c57bl/6n mice via intramuscular injection, and inguinal lymph nodes were collected after 24 h ( figure 3a) . ex vivo imaging showed significantly higher accumulation of s-rbd-ng in lymph nodes compared to s-rbd monomers ( figure 3b ). further quantitative analysis showed an ≈3.9-fold enhanced accumulation of s-rbd-ng compared to s-rbd alone ( figure 3c ). dcs and macrophages in the inguinal lymph nodes were further analyzed by flow cytometry, and enhanced uptake of s-rbd-ng was observed in these cells ( figure 3d and figure s9 , supporting information). the mechanism underlying the enhanced uptake of s-rbd-ng by lymph nodes and apcs remains unclear. since the immune system has evolved the ability to capture and process nanosized viruslike particles, [17] the nanoparticles could be filtered and accumulate in the lymphoid organs (e.g., liver, spleen, and lymph nodes), followed by rapid uptake and phagocytosis by apcs, the major cell types responsible for capturing these nanoparticles in a size-dependent manner. [18] in addition, the change in surface charge may play an important role in lymph node uptake. [19] encouraged by the lymph node targeting and dcs/macrophage uptake results, we next examined the immunogenicity of s-rbd-ng in vivo. c57bl/6n mice were immunized intramuscularly with pbs, s-rbd (50 µg per mouse), or s-rbd-ng (50 µg per mouse) in the presence or the absence of an aluminum hydroxide adjuvant (100 µg per mouse), one of the most commonly used adjuvants for vaccine development. mice were further boosted with the same dosage on days 14 and 28, and sera were collected one week after each immunization ( figure 4a ). s-rbd-specific serum igg was detected using elisa, and the titers were calculated. one week after the first immunization, the igg titers were still below our detection limit (lower than the lowest dilution factor 50, data not shown) for all groups. after the second round of immunization, s-rbd-specific serum igg titers were increased to ≈10 4 for s-rbd-ng treated groups, both in the presence and absence of aluminum hydroxide adjuvant ( figure 4b,c) . notably, after the third round of immunization, the titers for the s-rbd-ng-treated group reached ≈10 5 , while the s-rbd monomer-treated group had a titer less than 10 4 . quantitative comparison showed that s-rbd-ng induced 27.6-and 8.3-fold higher titers than the s-rbd monomer in the absence or presence of aluminum hydroxide adjuvant. taken together, these results showed that s-rbd-ng possessed higher immunogenicity than s-rbd, and disassembly of this internalized proantigen elicited more potent and rapid immune responses. next, we examined whether the toll-like receptor 1/2 agonist pam3csk4, another frequently used adjuvant in vaccine development, could further boost the immunogenicity of s-rbd-ng. [20] indeed, coadministration of s-rbd-ng with pam3csk4 stimulated similar but more potent immune responses ( figure 4f,g) . the s-rbd-specific igg titer reached ≈10 6 after the third round of immunization. this suggested that the vaccination titer of s-rbd ngs could be further improved by optimizing the adjuvant used. since blocking the interaction between spike protein and ace2 is crucial for preventing sars-cov-2's entry into host cells, we investigated whether the sera from the immunized mice were able to inhibit this interaction. a competitive elisa strategy was employed in which the sera were used to compete with hace2 for binding to the immobilized s-rbd. indeed, the sera from s-rbd-ng-immunized groups (either in the absence or presence of adjuvant) efficiently blocked the s-rbd-hace2 interaction ( figure s10 , supporting information), which was consistent with the aforementioned titer measurement. therefore, s-rbd-ng induced the development of specific antibodies that could target and block the interaction between s-rbd and hace2. finally, we used the pseudovirus to further test the utility of s-rbd-ng as a pro-antigen for subunit vaccine development for sars-cov-2 neutralization. the sars-cov-2 pseudovirus contains the spike protein shell and harbors a luciferase gene as a reporter (termed spike-pv-luc). the cos7 cell line stably expressing hace2 (cos7-hace2) was generated to mimic human cells. expression of hace2 was first validated by immunofluorescence ( figure s11 , supporting information). the neutralization activity of the sera from different immunized mice was assessed by monitoring the transduction efficiency of spike-pv-luc. indeed, the inhibition of spike-pv-luc transduction by immunized sera was consistent with the titer measured ( figure 5) . in particular, sera from mice immunized with s-rbd-ng and pam3csk4 almost completely inhibited pseudovirus entry under both dilution factors. sera from s-rbd-ng immunized mice inhibited pseudovirus transduction in a dilution-dependent manner, both in the presence or absence of the aluminum hydroxide adjuvant. in contrast, no inhibition was observed using sera from pbs-or s-rbd immunized mice. interestingly, when the sera were used at a higher concentration (20-fold dilution), the pseudovirus transduction was enhanced. this may be due to the antibodydependent enhancement, [21] which indicated that antibodies from sars-cov-2 infected blood may facilitate virus entry. to further confirm the results, another spike pseudovirus harboring the gfp gene as the reporter (termed spike-pv-gfp) was prepared. inhibition of spike-pv-gfp transduction into cos7-hace2 cells by immunized sera was observed by confocal microscopy imaging ( figure 5b ). the sera from mice immunized with s-rbd-ng were found to neutralize the sars-cov-2 pseudovirus. to show that our pro-antigen strategy can be generally applicable to other viruses, we formulated the recombinant s1 subunit of sars-cov-1 as ngs and the uptake by raw 264.7 cells was validated. indeed, the intracellular uptake of the resulting sars-cov-s1-ng was greatly enhanced ( figure s12 , supporting information). since many other viruses such as ebola virus also depend on the envelopeattached glycoproteins for entering host cells, we envision that our pro-antigen strategy may be extended to these viruses for subunit vaccine development. in conclusion, we developed a generally applicable pro-antigen strategy by employing the reversibly formulated s-rbd-ng to enhance the immunogenicity of sars-cov-2 spike proteins. s-rbd-ng showed improved lymph node targeting and accumulation in apcs, which can be rapidly converted into s-rbd monomers after internalization, leading to more potent immune responses during in vivo immunization. these results demonstrated the advantages of s-rbd-ng over s-rbd monomer for future subunit vaccine development. notably, s-rbd-ng alone was able to induce rapid and potent immune responses, which offers the possibility of developing subunit vaccine without the use of adjuvants. the immunized sera were further shown to block the interaction between the spike protein and hace2, which is crucial for virus entry into host cells. finally, in the pseudovirus neutralization assay, sera from s-rbd-ng-immunized groups effectively neutralized the pseudovirus in a concentration-dependent manner. the s-rbd-ngbased pro-antigen strategy within the lymph node niche can elicit more rapid and potent immune responses and may serve as a potential subunit vaccine candidate against sars-cov-2. supporting information is available from the wiley online library or from the author. figure 5 . neutralization of sars-cov-2 spike pseudovirus using immunized mouse sera. a) transduction inhibition of the spike-pv-luc by different sera. spike-pv-luc was pre-incubated with sera from different groups at 1:40 or 1:20 dilution and then added to cos7-hace2. transduction efficiency was assessed by luciferase reporter. data are presented as mean ± sem. n = 3 or 4. b) transduction inhibition of spike-pv-gfp by different sera. spike-pv-gfp was pre-incubated with sera from different groups at 1:20 dilution and then added to cos7-hace2. transduction efficiency was assessed by confocal microscopy imaging. scale bar: 50 µm. 13, 592; c) key: cord-321854-cy8vyb6j authors: ripperger, tyler j.; uhrlaub, jennifer l.; watanabe, makiko; wong, rachel; castaneda, yvonne; pizzato, hannah a.; thompson, mallory r.; bradshaw, christine; weinkauf, craig c.; bime, christian; erickson, heidi l.; knox, kenneth; bixby, billie; parthasarathy, sairam; chaudhary, sachin; natt, bhupinder; cristan, elaine; el aini, tammer; rischard, franz; campion, janet; chopra, madhav; insel, michael; sam, afshin; knepler, james l.; capaldi, andrew p.; spier, catherine m.; dake, michael d.; edwards, taylor; kaplan, matthew e.; scott, serena jain; hypes, cameron; mosier, jarrod; harris, david t.; lafleur, bonnie j.; sprissler, ryan; nikolich-žugich, janko; bhattacharya, deepta title: orthogonal sars-cov-2 serological assays enable surveillance of low prevalence communities and reveal durable humoral immunity. date: 2020-10-14 journal: immunity doi: 10.1016/j.immuni.2020.10.004 sha: doc_id: 321854 cord_uid: cy8vyb6j we conducted a serological study to define correlates of immunity against sars-cov-2. relative to mild covid-19 cases, individuals with severe disease exhibited elevated virus-neutralizing titers and antibodies against nucleocapsid (n) and the receptor binding domain (rbd) of spike protein. age and sex played lesser roles. all cases, including asymptomatic individuals, seroconverted by 2 weeks post-pcr confirmation. spike rbd and s2 and neutralizing antibodies remained detectable through 5-7 months post-onset, whereas α-n titers diminished. testing of 5882 members of the local community revealed only 1 sample with seroreactivity to both rbd and s2 that lacked neutralizing antibodies. this fidelity could not be achieved with either rbd or s2 alone. thus, inclusion of multiple independent assays improved the accuracy of antibody tests in low seroprevalence communities and revealed differences in antibody kinetics depending on the antigen. we conclude that neutralizing antibodies are stably produced for at least 5-7 months after sars-cov-2 infection. reduction neutralization test (prnt) titers, which we quantified as the final dilution at which 90% 134 viral neutralization occurred (prnt 90 ) ( figure 1a) . rbd to determine if rbd was capable of distinguishing between sars-cov-2 exposed and 141 uninfected individuals and to set preliminary thresholds for positive calls, we initially tested 1:40 142 serum dilutions of samples from 30 pcr+ sars-cov-2 infected individuals and 32 samples 143 collected prior to september, 2019, well before the onset of the current pandemic ( figure s1d) . 144 using this test data set, we established a preliminary positive cutoff od 450 value of 0.12, equal 145 to 3 standard deviations above the mean values of the negative controls. we next used this 146 preliminary threshold to test an expanded cohort of 320 negative control samples collected prior 147 to 2020. ( figure 1b ). reactivity to rbd was clearly distinguishable for the majority of positive 148 samples from negative controls ( figure 1b) . however, 6.5% of the expanded negative control 149 group displayed rbd reactivity that overlapped with pcr+ individuals (figure 1b, blue shade) , 150 some of whom may have been early into disease and had not yet generated high levels of 151 antibodies. to quantify the sensitivity of the assay relative to time of diagnosis, we measured 152 antibody levels to rbd and plotted these values against time following sars-cov-2 pcr+ 153 confirmation. whereas the sensitivity was modest within the first two weeks, after 2 weeks, 42 of 154 43 samples showed high elisa signal ( figure 1c ). based on these data, samples were 155 considered seropositive at od 450 numbers above 0.39, a value slightly above the highest od 156 obtained from the 352 subjects in the negative control group (figure 1b) . sera were considered 157 negative at od 450 values below 0.12. finally, we created an indeterminate call at od 450 values between 0.12-0.39, as we observed some overlap between negative controls and pcr-159 confirmed samples in this range (figure 1b, blue shade) . 160 we next applied this assay to community testing and obtained serum samples from 5882 to improve the positive predictive value, we considered the use of an orthogonal 174 antigenically distinct test. previous studies have used full length s protein as a secondary 175 screen following rbd elisas (amanat et al., 2020). while this improves the sensitivity of the 176 assay and is perfectly reasonable in high seroprevalence communities such as new york city, 177 rbd is part of s and is not antigenically distinct. thus, a false positive for rbd would 178 presumably also be apparent in s elisas. we therefore first tested nucleocapsid (n) protein, as 179 several other commercial serological tests quantify antibodies to this antigen (bryan et al., 2020; 180 burbelo et al., 2020) . igg antibody titers to n protein in our collected sample cohort showed a 181 strong correlation to prnt 90 titers (figure 2a) . a weaker correlation was observed between n-182 reactive igm levels and prnt 90 titers ( figure s2a) . we next assayed reactivity to n antigen overlapped substantially between negative and positive controls ( figure 2b) . moreover, 5 185 confirmed covid-19 samples showed very weak reactivity to n ( figure 2b) . because of the 186 relatively poor performance of n protein as an antigen in our hands, we next tested the s2 187 domain of s protein as another candidate to determine seropositivity. rbd is located on the s1 188 domain, rendering s2 antigenically distinct (bosch et al., 2003; li, 2016; wrapp et al., 2020) . 189 igg antibody titers to s2 correlated well with prnt 90 titers ( figure 2c) , consistent with reports 190 of s2-specific neutralizing antibodies to sars-cov-1 and sars-cov-2 (duan et al., 2005; 191 song et al., 2020). assessment of s2 serum reactivity in the pre-2019 cohort revealed that 192 approximately 3.3% of these samples overlapped with signals in pcr-confirmed covid-19 193 samples ( figure 2d) . we thereafter employed a threshold of od 450 >0.35, as our cutoff for s2 194 positivity, which was 5 standard deviations above the average seroreactivity from the original 195 32-samples from the negative control cohort. specificity control testing using 272 negative 196 control sera showed that reactivities of negative samples against rbd and s2 were largely 197 independent of one another, as samples with high signal for one antigen rarely showed similar 198 background for the other (figure 2e ). based on these data, we chose to rely on combined rbd 199 and s2-reactivities as accurate indicators of prior sars-cov-2 exposure. 200 with this improved combinatorial rbd and s2 assay to exclude false positives, we re-201 examined the original samples from the cohort of 5882 subjects that displayed rbd od 450 202 values greater than 0.12 ( figure 1d-e) . of the 13 non-neutralizing samples that displayed high 203 (od 450 >0.39) rbd reactivity, 12 lacked s2 reactivity ( figure 2f ). in contrast, the remaining 60 204 rbd+ neutralizing samples all displayed substantial reactivity to s2 ( figure 2f) . five of the 9 205 samples that fell below the rbd cutoff, yet still neutralized virus, displayed strong reactivity to 206 s2 ( figure 2f ). based on these data, we established a scoring criterion of rbd od 450 >0.39, s2 207 and all other samples as seronegative. applying these criteria to 320 samples obtained prior to 209 2020 would lead to 317 negative, 3 indeterminate, and 0 positive calls. using these same (atyeo et al., 2020). we therefore examined our data for these trends. first, in our pcr-223 confirmed cohort, we plotted igg titers relative to the time of disease onset, stratified by disease 224 severity. severe disease (hospital admission) correlated with significantly higher antibody titers 225 against rbd and n than those with mild disease, who were symptomatic but did not require 226 hospital admission, whereas s2 titers were not statistically significantly different (figure 3a-c) . 227 neutralizing titers were also higher in those with severe disease relative to mild cases ( figure 228 3d). through campus screening efforts, we also identified 6 pcr+ individuals who either never 229 developed symptoms or had only a brief and mild headache or anosmia. although previous 230 reports suggested that such individuals may infrequently seroconvert or frequently serorevert 231 ko et al., 2020) . given that older adults, as well as those of male sex, exhibit disproportional 234 morbidity and mortality from covid-19, we also sought to test whether humoral immunity in 235 these subjects may be quantitatively reduced . contrary to this expectation, we 236 did not observe any adverse impact of advanced age on humoral immunity (figure 3e-h) . before settling to a more stable nadir at later timepoints, as would be expected for all acute viral 248 infections. we considered the possibility that we may have missed subjects that had 249 seroreverted prior to their antibody test, thereby incorrectly raising our estimates of the durability 250 of antibody production. therefore, to examine the duration of igg production in more depth, a 251 subset of seropositive individuals with relatively low titers was tested longitudinally up to 226 252 days post-onset. these data again revealed stable rbd and s2 igg levels at later stages of 253 convalescence (figures 4a-b) . however, n-reactive igg levels were quite variable and most 254 samples approached the lower limit of detection at later timepoints ( figure 4c) . a direct 255 comparison in matched subjects of the changes in rbd, s2, and n igg titers over time 256 confirmed the variability in n responses and rapid decline in a subset of individuals ( figure 4d ). 257 of time in all but one subject ( figure 4e) , which showed evidence of neutralizing antibodies that 259 did not quite reach a prnt 90 titer of 20 ( figure s4) . these data suggest persistent neutralizing, 260 rbd, and s2-specific antibodies, but variable and often declining n-reactive titers during 261 convalescence. together, these data are consistent with the maintenance of functionally 262 important antibody production for at least several months after infection, and caution against the 263 use of α-n antibodies to estimate immunity or seroprevalence. here, we demonstrated that using two antigenically distinct serological tests can greatly 267 remedy specificity problems that are exacerbated in low sars-cov-2 seroprevalence 268 communities. rbd and s2 seroreactivity behaved independently for sars-cov-2-unexposed 269 individuals, thereby suggesting that the theoretical false positive rate of the overall assay is the 270 product of the two tests. using neutralization assays to confirm these results, we found our 271 empirically determined false positive rate to be <0.02% (1/5882), consistent with the 272 independence of the rbd and s2 tests. the tight co-incidence between rbd/s2 positivity and 273 the presence of neutralizing antibodies, even in low seroprevalence populations, is especially 274 valuable for identifying individuals who likely have some degree of immunity. surprisingly, 275 nucleocapsid (n), which is used by several commercial serological tests as an antigen, did not 276 perform as well in our assays, with high false positive and negative rates. the reasons for the differences in antibody responses across antigens are difficult to 306 explain, given the identical inflammatory environment in which these responses arose. one 307 possibility is that the avidities of germline precursors differ for n-and s-protein specificities. for 308 both memory and plasma cells, there appears to be a 'sweet spot' of antigen avidity that taken together, we have reported a highly specific serological assay for sars-cov-2 323 exposure that is usable in very low seroprevalence communities, and that returns positive 324 results that are highly co-incident with virus neutralization. using this assay, we characterize the 325 responses in different subject populations by age, sex and disease severity, we demonstrate 326 that antibody production persists for at least 3 months, and we suggest explanations for some 327 reports that concluded otherwise. 328 329 j o u r n a l p r e -p r o o f limitations of current study: one caveat to our study is that in our community testing cohort 330 we may have missed individuals who were seropositive initially but then seroreverted by the 331 time of the antibody test. second, the latest timepoint post-disease onset in our study is 226 332 days. it remains possible that antibody titers will wane substantially at later times. additional 333 serial sampling of pcr-confirmed mild cases will be required to test these possibilities. another the graphical abstract for this study was created on biorender.com. further information and requests for resources and reagents should be directed to and will be 442 fulfilled by the lead contact, deepta bhattacharya (deeptab@arizona.edu). 443 this study did not generate new unique reagents. 445 the data generated in this study and corresponding analyses have been described in main and 447 table 1 , as well as below in the text. subjects were recruited in three ways. first, targeted 456 recruitment was used to recruit confirmed positive covid-19 pcr test subjects with severe 457 covid-19, defined as one that needed hospitalization into the banner-university medical 458 center. second, targeted recruitment was used to recruit subjects with confirmed positive 459 covid-19 pcr test who did not require hospitalization (mild/moderate covid-19 cases). aliquoting, serum was used for the elisa assay with or without freezing and thawing as 469 described below. finally, sera from 352 subjects recruited into the above two irb protocols prior 470 to september, 2019, served as negative controls for assay development. based on local and 471 general prevalence, it would be expected that 96-98% of these subjects have previously 472 encountered seasonal coronaviruses (gorse et al., 2010) . freezing and thawing had no effect 473 on levels of antibodies detected by elisa or prnt. 474 lenti-x tm 293t cells (takaro bio usa) were grown at 37ºc, 5% co 2 in high glucose 476 dmem supplemented with 10% fetal bovine serum, non-essential amino acids, 477 penicillin/streptomycin, glutamine, and sodium pyruvate. the serum/plasma dilution that contained 10 or less plaques was designated as the nt90 titer. 544 statistical analyses were performed in graphpad prism (v8) and microsoft excel (v16.40). the 548 threshold for indeterminate seropositivity to rbd was calculated as 3 standard deviations above 549 the average od value of the pre-pandemic negative control group. rbd seropositivity was 550 established with an indeterminant range from an od value 3 standard deviations above the 551 mean od value of the negative control cohort (od 450 =.12) to an od slightly above the highest 552 od value observed in the negative control cohort (od 450 =0.39). readings above od 450 =0.39 553 were considered seropositive. the seropositive threshold to s2 was determined by calculating 554 j o u r n a l p r e -p r o o f the od value 5 standard deviations above the average od (od 450 =0.35) of the pre-pandemic 555 negative control cohort. 556 correlation r values between antibody titers and neutralizing titers were determined using a 558 pearson correlation. p values to compare non-linear regression fits of antibody and 559 neutralization titers over time grouped by disease severity, patient age, and patient sex were 560 calculated in graphpad prism. null hypothesis was set for a single curve to fit all subject 561 groups, which was rejected with less than 95% confidence. loess soothing splines were 562 generated in graphpad prism. pseudo-r 2 values were calculated using the squared correlation 563 between the predicted outcomes and the actual outcomes from the fitted model (efron, 1978) . the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the 592 performance characteristics of the abbott architect detection of nucleocapsid antibody to sars-cov-2 is more sensitive than antibody to spike protein in covid-19 patients the time course of the 602 immune response to experimental coronavirus infection of man disappearance of 605 antibodies to sars-associated coronavirus after recovery information for laboratories about coronavirus (covid-19) early release -antibody responses to sars-cov-2 at 8 weeks 610 a human sars-cov neutralizing antibody against epitope on s2 protein regression and anova with zero-one data: measures of residual variation retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus 618 s protein prevalence of antibodies to four human coronaviruses is lower in nasal secretions than in serum complete mapping of mutations to the sars-cov-2 spike receptor-binding domain that escape antibody recognition immune response to sars-cov-2 in iceland persistence of igg antibodies in sars-cov infected healthcare workers serology detection 638 of igm and igg antibodies in patients with coronavirus disease 2019 rapid decay of anti antibodies in persons with mild covid-19 evidence for sustained mucosal and systemic antibody responses to sars-cov-2 antigens in covid-19 patients dynamics and significance of the 649 antibody response to sars-cov-2 infection zbtb32 restricts the duration of memory b cell recall responses murine cytomegalovirus infections, but not other repetitive challenges furin cleavage site is key to sars neutralizing antibody production in asymptomatic and mild comparison with pneumonic covid-19 patients findings from investigation and analysis of re-665 positive cases identification and characterization of the 668 constituent human serum antibodies elicited by vaccination structure, function, and evolution of coronavirus spike proteins clinical features of covid-19 in elderly patients: 672 a comparison with young and middle-aged patients clinical and immunological assessment of asymptomatic sars-cov-2 675 infections antibody responses to sars-cov-2 in patients with covid-19 lifetime of plasma cells in the bone marrow ene-covid): a nationwide, population-based seroepidemiological 684 study. the lancet the receptor-binding domain of the 687 viral spike protein is an immunodominant and highly specific target of antibodies in sars-cov-688 2 patients memory 690 b cells, but not long-lived plasma cells, possess antigen specificities for viral escape mutants igm antibodies against severe acute respiratory syndrome 694 clinical infectious diseases the behaviour of recent isolates of human respiratory coronavirus in vitro 696 and in volunteers: evidence of heterogeneity among 229e-related strains cumulative incidence 700 and diagnosis of sars-cov-2 infection in new york measuring sars-cov-2 704 neutralizing antibody activity using pseudotyped and chimeric viruses robust t cell immunity in 707 convalescent individuals with asymptomatic or mild covid-19 longitudinal evaluation and decline of 710 antibody responses in sars-cov-2 infection humoral immunity due to long-712 the extent of affinity 714 maturation differs between the memory and antibody-forming cell compartments in the primary 715 immune response cross-reactive serum and memory b cell responses to spike 718 protein in sars-cov-2 and endemic coronavirus infection structural genomics of sars-cov-2 indicates evolutionary conserved 721 functional regions of viral proteins seroconversion of a city: longitudinal monitoring of sars-cov-2 seroprevalence in new york city. medrxiv 2020.06.28 hospital-wide sars-cov-2 antibody screening in 3056 staff in a tertiary center iga dominates the early neutralizing antibody response to 730 sars-cov-2 serocov-pop): a population-based study. the lancet intrinsic constraint on plasmablast growth and extrinsic limits of plasma cell survival a sars-cov-2 surrogate virus neutralization test based on 740 antibody-mediated blockage of ace2-spike protein-protein interaction covid-19 re-infection by a phylogenetically distinct sars-coronavirus-2 strain confirmed by whole genome sequencing repeated in vivo stimulation of t and b cell responses in old mice generates protective 748 immunity against lethal west nile virus encephalitis sars-cov-2 infection induces robust, neutralizing 752 antibody responses that are stable for at least three months divergent transcriptional programming of class-specific b cell memory by escape from neutralizing 758 antibodies by sars-cov-2 spike protein variants the emergence of sars-cov-2 in europe cryo-em structure of the 2019-ncov spike in the prefusion 764 conformation viral rna level, serum antibody responses, and transmission risk in 767 discharged covid-19 patients with recurrent positive sars-cov-2 rna test results: a 768 population-based observational cohort study protective 'immunity' by pre-existent neutralizing 770 antibody titers and preactivated t cells but not by so-called 'immunological memory cd80 and pd-l2 define functionally 774 distinct memory b cell subsets that are independent of antibody isotype key: cord-353748-y1a52z8e authors: bhattacharya, rajarshi; gupta, aayatti mallick; mitra, suranjita; mandal, sukhendu; biswas, swadesh r. title: a natural food preservative peptide nisin can interact with the sars-cov-2 spike protein receptor human ace2 date: 2021-01-02 journal: virology doi: 10.1016/j.virol.2020.10.002 sha: doc_id: 353748 cord_uid: y1a52z8e nisin, a food-grade antimicrobial peptide produced by lactic acid bacteria has been examined for its probable interaction with the human ace2 (hace2) receptor, the site where spike protein of sars-cov-2 binds. among the eight nisin variants examined, nisin h, nisin z, nisin u and nisin a showed a significant binding affinity towards hace2, higher than that of the rbd (receptor binding domain) of the sars-cov-2 spike protein. the molecular interaction of nisin with hace2 was investigated by homology modeling and docking studies. further, binding efficiency of the most potent nisin h was evaluated through the interaction of hace2:nisin h complex with rbd (receptor-binding domain) of sars-cov-2 and that of hace2:rbd complex with nisin h. here, nisin h acted as a potential competitor of rbd to access the hace2 receptor. the study unravels for the first time that a globally used food preservative, nisin has the potential to bind to hace2. the ongoing global outbreak of covid-19, a severe life-threatening infectious respiratory disease caused by a recently discovered severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has drastically affected human life with over eighteen millions of cases of infection globally (https://coronavirus.jhu.edu/map.html). until now, no specific antiviral medication is available for covid-19, but extensive efforts are underway worldwide. although vaccines are thought to be the most powerful weapon to fight against virus invasion, it may take quite a long time to go from the lab to successful applications in humans. considering the acute crisis of covid-19 pandemic, there is an urgent need for developing effective antiviral therapeutics for the prevention and treatment of covid-19. it is well accepted that the spike protein on the outer surface of sars-cov-2 is a crucial recognition factor for its attachment and entry to the host cells (shang et al., 2020) . the viral infection in humans is initiated by binding of rbd (receptor binding domain) of spike protein to human angiotensin-converting enzyme 2 (hace2) receptor (wang et al., 2020) . therefore, a therapeutic agent that blocks hace2 might prevent the interaction of spike protein of sars-cov-2 and thereby could reduce the establishment of infection. although small non-proteinaceous molecules are commonly preferred as therapeutics, they are not effective in blocking protein-protein interactions (ppis) particularly, where a deep binding pocket may be missing at the interface (arkin et al., 2014) . on the contrary, peptides are more suitable for disrupting ppis by specifically interacting with the interfaces. more importantly, small peptides have reduced immunogenicity (sorolla et al., 2020) . hence, peptides are potentially the ideal candidates for application as novel therapeutics. the recently described peptides are all small, synthetic and costly, and have not produced promising results against sars-cov-2 (du et al., 2005) . the peptides recently designed computationally (han and král, 2020) against the sars-cov-2 has to be synthesized prior to practical application, hence such peptides are not natural and food-grade. the present study attempts to investigate the ability of food-grade nisin a and its natural variants to block the interaction between hace2 and the spike protein of sars-cov-2, a key step of covid-19 disease initiation. nisin, a pentacyclic antibacterial peptide with 34 residues, is produced by certain strains of food-grade lactococcus lactis, widely used for cheese manufacturing (fox and wood, 1971; lubelski et al., 2008; juncioni et al., 2009) . nisin belongs to a group of cationic peptide antimicrobials collectively called type a (i) lantibiotics (smith and hillman, 1016) . it was first identified in fermented milk cultures and is now globally used as a natural and safe food preservative in a variety of food products around the world, such as processed cheese, dairy desserts, milk, fermented beverages, meat and canned foods (hurst, 1981; fons et al., 2009; mitra et al., 2011) . it has been approved by the european union (e234), world health organization (who) as well as by the us food and drug administration (fda). currently, nisin is licensed in over 50 countries (shin et al., 2015) . because of the high safety profile over the past 40 years of usage and its strong antimicrobial action against a wide range of food spoilage and pathogenic bacteria, nisin has been extensively studied. it also has multiple applications in biomedicine including bacterial infections, cancer, oral diseases and other veterinary and research field (shin et al., 2015) . since the discovery of nisin a, eight natural variants of nisin have been discovered which include nisin a, z, f, q, h, u, u2 and p (garcia-gutierrez et al., 2020) . nisin z producing organisms are very common in nature (mitra et al., 2011; vos et al., 1993) . the structures of eight variants of nisin were analyzed in the present study. all nisin peptides were aligned to show their identity and modeled on swiss-model web server. hace2 and rbd domain of 2019-cov-2 were also modeled on the same platform to increase the acceptability of the structures. all the peptides and rbd were docked with hace2 using haddock server. the binding affinity of the peptides was examined by docking analysis based on z-score, binding affinity and buried surface area. structurally, nisin is a unique molecule containing unusual amino acids including dehydroalanine and dehydrobutyrine, formed by dehydration of serine and threonine residues, respectively. these two residues are stereo-and regio-specifically coupled to the thiol group of the cysteines to form lanthionine and β-methyl lanthionine introduced enzymatically at post-translational level (cotter et al., 2012) . nisin is thus a thioether-bridged pentacyclic peptide. the crystal structure of nisin has not been developed. the peptide molecule adopts different conformations depending on the environment. the structure of nisin cannot be described in terms of regular secondary-structure elements, due to the presence of the ring systems in which 65% of the residues are incorporated. however, the nmr structure is available in pdb database, which was used in this study as template to generate the model structures of the nisin variants. the nmr structure of nisin has determined two structured domains: an n-terminal domain (residues 3-19) containing three lanthionine rings, a, b and c; and a c-terminal domain (residues 22-28) containing two intertwined lanthionine rings numbered d and e (hilbers, 1996) . these domains are flanked by regions showing structural flexibility. the four-residue rings b, d and e of nisin all show a β-turn structure, which is closed by the thioether linkage. the backbones of the rings b and d form type i1 β-turns. the c-terminal domain consists of three consecutive β-turns. the nmr data will help us to locate residues in nisin interacting with hace2. the present study attempts to evaluate the potential of nisin variants to interact with hace2 by predicting nisin binding site using nisin-hace2 docking computation with the nmr structure of nisin in the pdb database. this is the first report on the potential of widely used food-grade antibacterial peptide nisin to bind with hace2 and predicting the possibility of nisin as therapeutic against covid-19. the work is significant in finding a solution to prevent the infection by novel coronavirus sars-cov-2. amino acids sequences of eight nisin variants: nisin z ( (park et al., 2019) . esprit 3 software (robert and gouet, 2014) was used to represent the msa using blosum 62 algorithm. homology models of all nisin variants were done using the swissmodel web server (waterhouse et al., 2018) using nisin z (smtl id:1wco.1) as template. the steriochemical property of each of the models was evaluated by ramachandran plot using volume, area, dihedral angle reporter (vadar) server (willard et al., 2003) (fig. s1) . similarly, the rbd (receptorbinding domain) of spike protein of sars-cov-2 and hace2 receptor was modeled using smtl id: 6lzg.1 and smtl id: 6m18.1, respectively. all the models of nisin variants were superimposed together to determine their structural differences using read scoring matrix in pymol software. (the pyolecular graph). molecular docking was performed to test the binding affinity of all nisin variants towards hace2. in order to understand the comparative binding strength, multi-body docking were done between hace2:nisin h complex with the rbd of sars-cov-2 and hace2:rbd complex with nisin h. the solvated docking software, haddock (melquiond et al., 2016) was used without defying any restraints for such study. most reliable model was selected by lowest haddock score value. the score is calculated as where evdw is the intermolecular van der waals energy, eelec the intermolecular electrostatic energy, edesol represents an empirical desolvation energy. active site residues of hace2 (k31, e35, d38, m82) responsible of rbd spike binding were selected for docking. the residues surrounding the active loci were considered as passive. the interacting residues were visualized using discovery studio. (systèmes, 2017) prodigy@bonvin lab web server (xue et al., 2016) was used to calculate δg to predict the affinity of nisin h for hace2 at 25 • c with other parameters remained under default condition. grand average of hydropathy score of hace2 was calculated with exapassyprotparam webserver. (gasteiger et al.walker, 2005) in multiple sequence alignment (fig. 1) of amino acid residues of eight nisin variants (nisin a, z, q, h, p, u, u2 and f), nisin z shared 82.35% amino acid sequence similarity with nisin h, whereas nisin p, u, u2, q and f shared only 70.97%, 67.74%,67.74%, 76.47% and 79.41%, respectively with nisin h (table s2) . nisin a was found to be closely related to nisin z (97.06% identity) with only a single amino acid difference (his27asn). in contrast, nisin h differs from nisin a by five different amino acids at positions 1, 6, 18, 21 and 31 with 85.29% identity. nisin p is shorter than nisin h (34 residues) by three residues from the c-terminus. nisin h differs from nisin f by 7 residues, f1i, m6l, t18g, y21 m, h27 n, i30v and k31h. nisin q is different from nisin h due to the presence of isoleucine, leucine, valine, glycine, leucine, asparagine, valine and histidine at positions 1, 6, 15, 18, 21, 27, 30 and 31, respectively. nisin u and u2 differed from nisin h by ten amino acids. the residual surface accessibility is present at the bottom of the alignment (fig. 1) . the model structures of all nisin variants, hace2, rbd of spike protein built on using swiss-model web server were validated for steriochemical properties using ramachandran plot (fig. s2) . we considered the number of amino acids in the disallowed regions except for glycine and proline because of their chirality and imino group, respectively. homology model of nisin p and u2 had no disallowed amino acids. nisin h and u had only one residue in disallowed region, whereas two residues were found in the disallowed region for nisina, f, q and z. the rmsd (c-alpha) from all the superimposed variants of nisin was found 0.191. these signify that all the nisin models were structurally similar to one another. the binding efficiency of nisins with hace2 was further evaluated from docking studies. best haddock model of nisin variants in complex with hace2 was analyzed for three parameters viz. z-score, buried surface area, and binding affinity. the z-score indicates how many standard deviations from the average of the cluster is located in terms of score (the more negative the better). z-score of hace2-sars-cov-2 rbd, hace2-nisin a, hace2-nisin z, hace2-nisin h, hace2-nisin q, hace2-nisin u, hace2nisin u2, hace2-nisin f, and hace2-nisin p was predicted as − 1.5,-1.6,-1.9,-2.1,-1.4,-1.7,-0.8,-1.4, and − 1.5. hence, both nisin h and nisin z were lowest than rest of the nisin variants as well as rbd of spike protein. burried surface area of nisin z and nisin h with hace2 were found higher, 2332.4 å 2 and 2395.1 å 2 , respectively in contrast to 2092 å 2 for the rbd. this suggests that nisin h and nisin z had better binding efficiency for hace2. the binding affinity of docked structures of all eight variants of nisin in complex with hace2 was calculated as δg derived from analysis with prodigy for each complex in comparison with the rbd of spike protein of sars-cov-2. δg of hace2-sars-cov-2, hace2-nisin a, hace2-nisin z, hace2-nisin h, hace2-nisin q, hace2-nisin u, hace2-nisin u2, hace2nisin f, and hace2-nisin p was − 11 kcal/mol, − 10.6 kcal/mol, − 10.8 kcal/mol, − 11.3 kcal/mol, − 10.5 kcal/mol, − 10.5 kcal/mol, − 12.3 kcal/mol, − 12.5 kcal/mol, and − 11.4 kcal/mol, respectively. thus δg of hace2-nisin z and hace2-nisin h are much higher conferring strong binding affinity than that of hace2-rbd.gravy score of nisin a, z, h, q, u, u2, f, p and rbd-sars-cov-2 was calculated as 0. 415, 0.406, 0.185, 0.524, 0.542, 0.439, 0.171, 0.185, − 0.258, respectively (table 1) . from the gravy score of all nisin variants, nisin h turned out to be more hydrophilic than nisin a and nisin z and will thus more potent to interact with the hydrophobic groove of hace2 than others variants of nisin. from the docking analysis it is evident that nisin z and nisin h interacts to hace2 more efficiently. the interacting residues and atoms are given in (table s1 ). the hydrogen bonds (k31:c19, k31:t13, k31: k12, e35:k12, e35:c19, e35:n20, d38:k22, d38:c27, m82:c7, k353: n27) and hydrophobic bonds (m82:i4, m82:c7, k31:c19, y83:c7, k353: c28) are the major binding force for hace2-nisinz interaction. interacting residues of nisin z was predicted as i4, c7, k12, t13, c19, n20, k22, and c27. all interacting residues of nisin z were hydrophilic in nature. the residues in nisin h interacting with the hace2 include hydrogen bond of t13:k31, c19:k31, k12:k31, t8:k31, p9:k31, k12: e35, k22:d38, n20:e35, c26:d38, h27:d38, c28:k353, t23:k353 (fig. 2) and hydrophobic bond of c19:k31 and y21:k31, c7:m82, a24: k353, c26:k353, c28:k353. among all these interacting residues, t8, p9, c11, k12, t13, c19, k22, c26 were highly conserved among all the nisin variants. like rbd, surface accessible hydrophilic residues, t8, p9, c11, k12, t13, k22, and c26 were found to be involved in binding to hydrophobic groove of hace2. it was found that nisin z and nisin h recognized five common residues (k31, e35, d38, m82, k353) in hace2 that were also recognized by rbd of spike. the binding efficiency of preformed hace2:nisin h complex was performed by competitive tertiary docking with rbd of sars-cov-2 (fig. 3) . as, nisin h had already occupied the active site residues of hace2 with strong hydrogen bond and hydrophobic interactions (table s3) , rbd of sars-cov-2 could not get access the active residues of hace2 with reasonable efficiency by overcoming the binding strength of hace2:nisin h interaction with δg of − 11.3 kcal/mol (binding affinity of rbd:hace2 complex is − 11 kcal/mol). on the contrary when nisin h was allowed to interact with the hace2:rbd complex, it was found that nisin h could be able to interact with active residues (k31 and m82) of hace2 from the hace2:rbd complex (table s4) . nisin h, being more potent candidate could able to interfere in the interaction between rbd-hace2. there is high possibility that nisin would be able to competitively displace bound sars-cov-2 because of its higher binding affinity towards the ace2 receptor compared to that of the virus. furthermore nisin being a non-synthetic molecule and smaller in size, will ensure high bioavailability. based on such study, we hypothesize that nisin h, z, a and u could be an eligible competitor of rbd of sars-cov-2 for having the same binding patch in hace2. recently, several peptides computationally designed to target the spike protein of sars-cov-2 have been reported (han and král, 2020; baig et al., 2020) as a strategy to prevent their interaction with ace 2 receptor for tackling covid-19 infection. from an application perspective, it would be advantageous of using nisin as an effective treatment option over the reported designed peptides for several reasons, including its natural occurrence, food-grade status, extreme stability and ease of manufacturing through microbial fermentation, cost effectiveness, delivery at high concentration, etc. however, further experimental validation is required to confirm nisin binding to hace2. among all analyzed nisin variants, nisin z, nisin a, nisin u and nisin h were most effective in interacting with human endothelial cell surface-receptor hace2, the site where rbd of spike of sars-cov-2 binds to initiate infection. compared to the rbd of viral spike protein, nisin binds with the hace2 receptor with higher affinity. nisin being a low molecular weight peptide and readily bioavailable in the system, its binding to hace2 is expected to over-rule the interaction possibility of the rbd of spike of sars-cov-2 and could essentially exclude the virus entry to the host cell. since nisin is a heat stable natural food grade peptide, can be produced cost effectively, even in large quantity through microbial fermentation, the present work will create greater interest among researchers to develop a new nisin-based treatment strategy for covid-19, either through oral or nasal applications. however, further experimental validation is necessary to determine its doses and mechanistic application to check the competition of nisin and spike protein of sars-cov-2 for accessing the human. the authors declare that they have no conflict of interest. small-molecule inhibitors of protein-protein interactions: progressing toward the reality identification of a potential peptide inhibitor of sars-cov-2 targeting its entry into the host cells bacteriocins -a viable alternative to antibiotics? molecular modeling and chemical modification for finding peptide inhibitor against severe acute respiratory syndrome coronavirus main proteinase microbial ecology in health and disease mechanisms of colonisation and colonisation resistance of the digestive tract part 2: bacteria/bacteria interactions mechanisms of colonisation and colonisation resistance of the digestive tract first evidence of production of the lantibiotic nisin protein identification and analysis tools on the expasy server computational design of ace2-based peptide inhibitors of sars-cov-2 surface location and orientation of the lantibiotic nisin bound to membrane-mimicking micelles of dodecylphosphocholine and of sodium dodecylsulphate introduction, i. a. hurst nisin biotechnological production and application: a review biosynthesis, immunity, regulation, mode of action and engineering of the model lantibiotic nisin the haddock2.2 web server: user-friendly integrative modeling of biomolecular complexes potential application of the nisin z preparation of lactococcus lactis w8 in preservation of milk the embl-ebi search and sequence analysis tools apis deciphering key features in protein structures with the new endscript server structural basis of receptor recognition by sars-cov-2 biomedical applications of nisin therapeutic potential of type a ( i ) lantibiotics , a group of cationic peptide antibiotics precision medicine by designer interference peptides: applications in oncology and molecular therapeutics dassault syst mes biovia properties of nisin z and distribution of its gene , nisz , in lactococcus lactis structural and functional basis of sars-cov-2 entry by using human ace2 swiss-model: homology modelling of protein structures and complexes vadar: a web server for quantitative evaluation of protein structure quality structural bioinformatics prodigy: a web server for predicting the binding affinity of protein-protein complexes we most sincerely acknowledge the research grant (bt/pr16246/ get/119/73/2016) received from dbt, department of biotechnology, govt. of india, new delhi. supplementary data to this article can be found online at https://doi. org/10.1016/j.virol.2020.10.002. rb curated, analyzed and interpreted the data. amg helped in docking studies. s mitra, s mandal and srb supervised the work. all the authors write, review and edited the manuscript. key: cord-323967-2mo915u1 authors: miersch, shane; li, zhijie; saberianfar, reza; ustav, mart; blazer, levi; chen, chao; ye, wei; pavlenco, alia; subramania, suryasree; singh, serena; ploder, lynda; ganaie, safder; leung, daisy; chen, rita e.; case, james brett; novelli, guiseppe; matusali, giulia; colavita, francesca; copabianchi, maria r.; jain, suresh; gupta, j.b.; amarasinghe, gaya; diamond, michael; rini, james; sidhu, sachdev s. title: tetravalent sars-cov-2 neutralizing antibodies show enhanced potency and resistance to escape mutations date: 2020-11-01 journal: biorxiv doi: 10.1101/2020.10.31.362848 sha: doc_id: 323967 cord_uid: 2mo915u1 recombinant neutralizing antibodies (nabs) derived from recovered patients have proven to be effective therapeutics for covid-19. here, we describe the use of advanced protein engineering and modular design principles to develop tetravalent synthetic nabs that mimic the multi-valency exhibited by iga molecules, which are especially effective natural inhibitors of viral disease. at the same time, these nabs display high affinity and modularity typical of igg molecules, which are the preferred format for drugs. we show that highly specific tetravalent nabs can be produced at large scale and possess stability and specificity comparable to approved antibody drugs. moreover, structural studies reveal that the best nab targets the host receptor binding site of the virus spike protein, and thus, its tetravalent version can block virus infection with a potency that exceeds that of the bivalent igg by an order of magnitude. design principles defined here can be readily applied to any antibody drug, including iggs that are showing efficacy in clinical trials. thus, our results present a general framework to develop potent antiviral therapies against covid-19, and the strategy can be readily deployed in response to future pathogenic threats. to date, all clinically advanced candidate nabs against sars-cov-2 infection have been 72 derived by cloning from b-cells of recovered covid-19 patients or from other natural 73 sources 9,17,19-23 . here, we applied an alternative strategy using in vitro selections with phage-74 displayed libraries of synthetic abs built on a single human framework derived from the highly 75 validated drug trastuzumab. this approach enabled the rapid production of high affinity nabs 76 with properties optimized for drug development. moreover, the use of a highly stable framework 77 enabled facile and modular design of ultra-high affinity nabs in tetravalent formats that retained 78 favorable drug-like properties and exhibited neutralization potencies that greatly exceeded those 79 of the bivalent igg format. these methods provide a general means to rapidly improve the 80 potency of virtually any nab targeting sars-cov-2 and its relatives, and thus, our strategy can be 81 applied to improve covid-19 therapies and can be adapted in response to future pathogenic 82 threats. 83 84 85 results 86 using a phage-displayed human antigen-binding fragment (fab) library similar to the 88 highly validated library f 24 , we performed four rounds of selection for binding to the biotinylated 89 rbd of sars-cov-2 immobilized on streptavidin-coated plates. screening of 384 clones for 90 binding to cov-2 rbd, revealed 348 fab-phage clones that bound to the rbd but not to 91 streptavidin. fab-phage were screened by elisa and those that exhibited >50% loss in binding 92 to rbd in the presence of 200 nm ace2 were sequenced, revealing 34 unique clones (fig. 1a) , 93 deemed to be potential nabs and converted into the full-length human igg1 format for 94 purification and functional characterization. 95 to estimate affinities, elisas were performed with serial dilutions of igg protein binding 96 to biotinylated s protein trimer captured with immobilized streptavidin, and these assays showed 97 that three iggs bound with ec50 values in the sub-nanomolar range (fig. 1b,c and table 1 ). elisas 98 also confirmed that each igg could partially block the binding of biotinylated ace2 to immobilized 99 s protein (fig. 1d) . moreover, similar to the highly specific igg trastuzumab, elisas showed that 100 the three iggs did not bind to seven immobilized proteins that are known to exhibit high non-101 specific binding to some iggs, and lack of binding to these proteins has been shown to be 102 predictive of good pharmacokinetics in vivo (fig. 1e) 25, 26 . we also used biolayer interferometry 103 (bli) to measure binding kinetics and determine avidities more accurately, and all three 104 antibodies exhibited sub-nanomolar dissociation constants (table 1, fig. s1 ), in close accord with 105 the estimates determined by elisa. igg 15033 exhibited the highest avidity, which was mainly 106 due to a two-or seven-fold faster on-rate than igg 15031 or 15032, respectively, and thus, we 107 focused further efforts on this ab. 108 we took advantage of the precision design of our synthetic ab library to rapidly improve 109 the affinity of ab 15033. the synthetic library was designed with tailored diversification of key 110 positions in all three heavy chain complementarity-determining regions (cdrs) and the third cdr 111 of the light chain (cdr-l3). consequently, we reasoned that the already high affinity of ab 15033 112 could be further improved by recombining the heavy chain with a library of light chains with naïve 113 diversity in cdr-l3. following selection for binding to the rbd, the light chain library yielded 114 numerous variants, 17 of which were purified in the igg format and analyzed by bli (fig. s2) . 115 several of the variant light chains resulted in iggs with improved binding compared with igg 116 15033, and in particular, igg 15033-7 (fig. 1b) exhibited significantly improved avidity (kd = 300 117 or 39 pm, respectively) due to an off-rate that was an order of magnitude slower (table 1, fig. 118 s2) . 119 120 to understand the molecular basis for antagonism of ace2 binding, we solved the x-ray 122 crystal structures of the sars-cov-2 rbd in complex with fab 15033 or 15033-7 at 3.2 or 3.0 å 123 resolution, respectively ( fig. 2a) . as expected, backbone superposition showed that the two 124 complexes were essentially identical (rmsd = 0.17 å). however, there were differences in side 125 chain interactions due to sequence differences in the cdr-l3 loop, which explained the enhanced 126 affinity of fab 15033-7 compared with fab 15033 (fig. 2b) . although the side chains of tyr 108l in 127 fab 15033 and his 108l in fab 15033-7 both make hydrogen bonds with the side chain of tyr 473 in 128 the rbd, the bond mediated by his 108l is shorter, and thus, likely to be stronger. moreover, in 129 fab 15033-7, the side chain of his 108l also makes an intramolecular hydrogen bond with the side 130 chain of thr 109l , which tyr 108l and arg 109l are incapable of making in fab 15033, and this 131 interaction may stabilize the cdr-l3 loop of fab 15033-7 in a conformation that is favorable for 132 antigen recognition. thus, the crystal structures show that the two substitutions in the cdr-l3 133 loop of fab 15033-7 relative to fab 15033 act in a cooperative manner to mediate favorable 134 intermolecular contacts with the rbd, and also, intramolecular interactions that stabilize the loop 135 in a conformation that may be better positioned to interact with the rbd. 136 we next analyzed the structures to understand how the abs could function as antagonists 137 of rbd binding to ace2. binding of fab 15033-7 to the rbd involves an extensive interface, with 138 1130 and 1112 å 2 of surface area buried on the epitope or paratope, respectively, and 59% or 139 41% of the structural paratope is formed by the light or heavy chain, respectively (fig. 2c) . 140 comparison of the fab and ace2 epitopes on the rbd revealed extensive overlap, with 79% or 141 69% of the fab or ace2 epitope occluded by the other ligand (fig. 2c) . thus, direct steric 142 hinderance explains the blockade of ace2 binding by fabs 15033 and 15033-7 (fig. 1d) . 143 we also used cryogenic electron microscopy to visualize fab 15033 in complex with the s 144 protein trimer (fig. s3a) . this analysis revealed that all three rbds in a single trimer were 145 positioned in an "up" conformation, which was similar to the conformation bound to ace2, and 146 the three rbds were bound to three fab molecules. notably, the c-termini of the three fabs were 147 positioned close to each other and pointed away from the s protein, suggesting that a single igg 148 may be able to present two fabs in a manner that would enable simultaneous engagement of 149 two rbds on a single s protein. indeed, this was confirmed in single particle negative stain 150 electron micrographs of igg 15033 and the s protein, which revealed that the two fabs of a single 151 igg bound two rbds on a single s protein trimer with a pincer-like grip (fig. s3b) . taken together, 152 the x-ray crystallography and electron microscopy showed that fabs 15033 and 15033-7 block 153 ace2 binding to rbd by direct steric hinderance, and simultaneous binding of fabs to multiple 154 rbds on the s protein trimer enables the iggs to inhibit ace2 binding with enhanced potency due 155 to avidity. 156 157 next, we explored whether we could further enhance the avidity of nabs by taking 159 advantage of modular design strategies to engineer tetravalent formats. each sars-cov-2 160 particle displays multiple s protein trimers, suggesting that multivalent fab binding could 161 enhance avidity, especially since a single igg 15033 molecule can utilize both fab arms to bind a 162 single s protein trimer. we reasoned that additional fab arms added to an igg may further 163 enhance avidity by interacting with rbds on s protein trimers close to the trimer engaged by the 164 core igg. thus, we designed tetravalent versions of 15033 and 15033-7 by fusing additional fabs 165 to either the n-or c-terminus of the igg heavy chain to construct molecules termed fab-igg or 166 igg-fab, respectively (fig. 3a) . consistent with our hypothesis, the tetravalent molecules 167 exhibited higher avidity, and consequently, greatly reduced off rates compared with their 168 bivalent counterparts, and dissociation constants were in the low single-digit picomolar range 169 ( fig. 3b, table 1 ). 170 our ultimate aim was to produce therapeutic abs that could be used to treat covid-19 in 171 patients. aside from high affinity and specificity, effective ab drugs must also possess favorable 172 biophysical properties including high yields from recombinant expression in mammalian cells, 173 high thermodynamic stability, and lack of aggregation and excessive hydrophobic surface area. 174 all iggs and tetravalent molecules were produced in high yields by transient expression in 175 expi293f cells (160-200 mg/l, table 1 ). all proteins were highly thermostable with melting 176 temperatures of the ch3/fab domain ranging from 81-87 o c, which exceeded the melting 177 temperature of the trastuzumab fab (79.5 o c, table 1 ). size exclusion chromatography revealed 178 that all iggs eluted as a predominant monodisperse single peak with elution volumes nearly 179 identical to that of trastuzumab ( fig. 3c and table 1) , and the monomeric fraction was calculated 180 to be 91 to >95% ( table 1) to explore neutralization of potential escape mutants, we generated hiv-gag-based 205 lentivirus-like particle (vlps) pseudotyped with the sars-cov-2 s protein. we confirmed ace2-206 dependent uptake of the pseudotyped vlps by hek-293 cells stably over-expressing exogenous 207 ace2, and we showed that uptake was inhibited by either fc-tagged rbd (rbd-fc) or igg 15033. 208 within this system, we generated a panel of 44 pseudotyped vlp variants, each containing a 209 single alanine substitution at an rbd position within or close to the ace2-binding site. twenty of 210 these vlp variants exhibited a >4-fold reduction in internalization compared with the wild-type 211 (wt) vlp, suggesting that these wt side chains contributed favorably to the interaction between 212 the rbd and ace2. the remaining 24 vlp variants were internalized with high efficiency, and 213 these represent good mimics of escape mutants, which maintain strong ace2-mediated 214 infectivity but may potentially reduce binding to nabs that compete directly with ace2. 215 with the panel of 24 vlp variants that mimicked potential escape mutants, we surveyed 216 the effects on cellular uptake after treatment with various nabs (fig. 4b) . we defined as escape 217 mutants those vlp variants for which cellular uptake in the presence of 50 nm nab was >5% of 218 the uptake in the absence of the nab. based on this definition, we found that 6 of the mutations 219 enabled escape from igg 15033, whereas only three mutations enabled escape from igg 15033-220 7. presenting the 15033 paratope in tetravalent formats resulted in nabs that could neutralize 221 more variants than igg 15033, and most importantly, tetravalent nabs containing the 15033-7 222 paratope strongly neutralized all variants except one. as expected, these results showed that 223 enhancing the avidity of the igg paratope for the s protein enhanced both potency and resistance 224 to escape mutations. moreover, similar enhancements were also achieved by the presentation 225 of paratopes in tetravalent rather than bivalent formats, and the most effective nabs were those 226 that presented the optimized paratope in the tetravalent format. 227 228 229 discussion 230 sars-cov-2 has wreaked havoc on global health and economics, and along with its relatives 231 sars-cov and mers, has shown that viral outbreaks and pandemics will continue to plague the 232 world in the future. consequently, it is essential for the scientific community to adapt the most 233 advanced drug development technologies to combat not only covid-19, but also, pathogenic 234 disease in general. in this context, we have deployed advanced synthetic antibody engineering 235 to rapidly develop human nabs, which are potent therapeutic candidates in the natural igg 236 format, and are even better neutralizing agents in the synthetic tetravalent formats that our 237 modular design strategies enable. most importantly, the enhanced affinities and potencies 238 afforded by tetravalent nabs are achieved without compromising any of the favorable 239 characteristics that make igg molecules ideal drugs. moreover, tetravalent nabs resist potential 240 escape mutants, which further augments the power of these molecules as drugs to combat not 241 only sars-cov-2, but also, its relatives that may emerge in the future. 242 covid-19 has also exposed the need for drug development to respond to viral outbreaks 243 15031 15032 15033 15034 15035 15036 15037 15038 15039 15040 15041 15042 15043 15044 15045 15046 15047 15048 15049 15050 15051 15052 15053 15054 15055 15056 15057 15058 15059 15060 15061 15062 15063 15064 15033 ab 28 29 36 37 38 56 57 65 107 108 109 114 115 116 30 35 36 37 38 39 55 56 57 58 59 62 63 64 65 66 107 108 109 110 111 for the rbd, residues in the ace2-binding site are also shown as colored surfaces, and the following color scheme was used: red, contacts with both fab 15033-7 and ace2; blue, contacts with fab 15033-7 only; yellow, contacts with ace2 only. fab 15033-7 residues that contact the rbd are colored magenta or cyan if they reside in the light or heavy chain, respectively. the cdr-l3 residues that differ between 15033-7 and 15033 are shown as red spheres. the vlps were treated with 50 nm of the indicated nab (x-axis) and uptake by ace2-expressing hek-293 cells was measured in triplicate and results are representative of n=2 independent experiments. the heat map shows uptake normalized to uptake in the absence of nab. boxed cells indicate vlps that represented escape mutants for a given nab, as defined by >5% uptake with nab treatment compared with untreated control (the percent uptake is shown in each cell). remdesivir for the treatment of covid-19 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prophylaxis for sars coronavirus infection in ferrets prophylactic and postexposure efficacy of a 642 potent human monoclonal antibody against mers coronavirus sars-cov-2 structure and replication 646 characterized by in situ cryo-electron tomography structures and distributions of sars-cov-2 spike proteins on intact 649 virions entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease 652 studies in humanized mice and convalescent humans yield a sars-cov-2 655 antibody cocktail cross-neutralization of sars-cov-2 by a human monoclonal 658 sars-cov antibody neutralizing antibodies against sars-cov-2 identified by high-throughput single-cell 661 sequencing of convalescent patients' b cells isolation of potent sars-cov-2 neutralizing antibodies and protection from 664 disease in a small animal model igg-neutralizing monoclonal antibodies block the sars-cov-2 infection a panel of human neutralizing mabs targeting 670 sars-cov-2 spike at multiple epitopes broad 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spike antigen diversity synthetic antibody libraries yield novel anti-egfr antagonists high-throughput 709 screening of formulations to optimize the thermal stability of a therapeutic monoclonal 710 antibody neutralizing antibody and soluble ace2 inhibition of a replication-competent vsv-713 sars-cov-2 and a clinical isolate of sars-cov-2 a highly conserved 715 cryptic epitope in the receptor binding domains of sars-cov-2 and sars-cov molecular characterization of sars-cov-2 from the first case of 719 covid-19 in italy a simple method of estimating fifty percent endpoints -scienceopen crystal violet assay for determining viability of 724 cultured cells primary structure of the streptomyces enzyme endo-beta-n-acetylglucosaminidase h -726 phaser 730 crystallographic software features and development of coot towards 735 automated crystallographic structure refinement with phenix.refine imgt unique numbering for immunoglobulin and t cell receptor variable 739 domains and ig superfamily v-like domains key: cord-329392-fufattj8 authors: den hartog, gerco; schepp, rutger m; kuijer, marjan; geurtsvankessel, corine; van beek, josine; rots, nynke; koopmans, marion p g; van der klis, fiona r m; van binnendijk, robert s title: sars-cov-2–specific antibody detection for seroepidemiology: a multiplex analysis approach accounting for accurate seroprevalence date: 2020-08-08 journal: j infect dis doi: 10.1093/infdis/jiaa479 sha: doc_id: 329392 cord_uid: fufattj8 background: the covid-19 pandemic necessitates better understanding of the kinetics of antibody production induced by infection with sars-cov-2. we aimed to develop a high-throughput multiplex assay to detect antibodies to sars-cov-2 to assess immunity to the virus in the general population. methods: spike protein subunits s1 and receptor binding domain, and nucleoprotein were coupled to microspheres. sera collected before emergence of sars-cov-2 (n = 224) and of non-sars-cov-2 influenza-like illness (n = 184), and laboratory-confirmed cases of sars-cov-2 infection (n = 115) with various severities of covid-19 were tested for sars-cov-2–specific igg concentrations. results: our assay discriminated sars-cov-2–induced antibodies and those induced by other viruses. the assay specificity was 95.1%–99.0% with sensitivity 83.6%–95.7%. by merging the test results for all 3 antigens a specificity of 100% was achieved with a sensitivity of at least 90%. hospitalized covid-19 patients developed higher igg concentrations and the rate of igg production increased faster compared to nonhospitalized cases. conclusions: the bead-based serological assay for quantitation of sars-cov-2–specific antibodies proved to be robust and can be conducted in many laboratories. we demonstrated that testing of antibodies against multiple antigens increases sensitivity and specificity compared to single-antigen–specific igg determination. coronavirus disease 2019 (covid-19) caused by the newly emerged severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has resulted in a pandemic in a largely immune-naive population. the presence of specific antibodies is currently being investigated to assess the induction of an immune response in patients and to assess the degree of exposure and immunity in the general population [1] [2] [3] . as it is a recently emerged coronavirus variant, the kinetics and degree of immunity induced following contact with the virus and covid-19 disease are largely unknown. sars-cov-2 expresses a spike protein, highly similar to spike of sars-cov, which binds to angiotensin converting enzyme 2 (ace2) [4, 5] . binding of antibodies to the receptor binding domain (rbd) of spike neutralizes the ability of the virus to infect cells [6] . in addition, antibodies are detected against other viral proteins, including nucleoprotein (n) [7] . n is shielded within the virion and therefore n-specific antibodies are probably unable to neutralize the virus. although n may not be involved in neutralization of the virus, antibodies to n could provide an indicator of exposure to the virus. antibodies to n induced by sars-cov reportedly recognize n of sars-cov-2 but not of seasonal coronaviruses [8] . estimates of the prevalence of seroconversion as proxy for protection of the general population may support health decision making, including the decision to lift lockdown measures. to appropriately apply an assay for serosurveys we need to know the precision of the assay, that is the sensitivity and specificity, which are variable between currently available tests [9, 10] . performing and sustaining large studies to assess changing population immunity requires high-throughput screening assays that are robust and accurate [11] . many countries now aim to assess the protective status of the general population for covid-19 using antibody assays. to guarantee high specificity, the assay should be validated with a representative number of sera from patients infected with other coronaviruses and other pathogens causing influenza-like illness (ili), but this is often lacking [11] [12] [13] . to date, covid-19 prevalence of seroconverted individuals is relatively low and there is a risk of significant overestimation if an assay has insufficient specificity (supplementary table 1 ). thus, high specificity is important at this stage [11, 12] . our laboratory has extensive experience in developing multiplex assays to quantify antibodies to many bacterial and viral pathogens in the general population, of which most are part of the national immunization program [1, [14] [15] [16] [17] . we developed a high-throughput and highly quantitative bead-based multiplex immunoassay to assess the prevalence of seropositivity in the general population, and also anticipating the introduction of future sars-cov-2 vaccines. by multiplexing a broader range of sars-cov-2 antigens in a single assay we may generate a better understanding of the proportion of persons that have seroconverted. moreover, in a multiplex assay positivity can be compared among antigens to provide a more detailed evaluation of the antibody levels and to enhance assay performance [17] . the developed assay was tested on samples from covid-19 patients with various severities of disease collected at multiple timepoints to determine the kinetics of seroconversion. serum samples were obtained from the following cohorts: (1) a random selection of individuals (n = 224) from a national (dutch) cohort representing all age groups and obtained 3 years prior to sars-cov-2 emergence (pienter3 study, netherlands trial register number nl5467); (2) individuals (supplementary table 2 ) with proven non-sars-cov-2 ili caused by human coronaviruses (n = 110, hcov ili) or other viruses (n = 74, non-hcov ili) obtained from the national institute for public health and the environment (rivm), bilthoven, the netherlands (trial register number nl4666) [18] , and from erasmus medical center, rotterdam, collected prior to the sars-cov-2 outbreak and at least 2 weeks after polymerase chain reaction (pcr) detection of the virus; and (3) the steps in assay validation were similar to recently developed bead-based multiplex immunoassays for cmv, ebv, and rsv, with minor modifications as described below [16, 17] . for the multiplex bead-based immune assay the following antigens obtained from sino biological were used: sars-cov-2 monomeric spike s1 (40591-v08h), rbd (40592-v08b), and nucleoprotein (n) (40588-v08b). microplex fluorescent beads were activated in 50 mm 2-(n-morpholino)ethanesulfonic acid (mes) ph 5.5. the proteins were diluted to a concentration of 0.2 mg/ml in phosphate-buffered saline (pbs) ph 7.4 and added at 5 µg per 75 µl of activated beads. an internal reference sample was created by pooling 13 sera of covid-19 patients with varying immunoglobulin g (igg) concentrations. an arbitrary antibody concentration unit of 100 was assigned on the basis of the mean fluorescence intensity (mfi) signal in the upper limit of linearity of a 3-fold serial dilution of the reference sample. sera (25 µl) diluted 1:400 and 1:8000 in sm01 buffer (surmodics) plus 2% fetal calf serum were incubated with antigen-coated beads for 45 minutes at room temperature at 750 rpm in the dark. following incubation, samples were washed 3 times with pbs, incubated with phycoerythrin-conjugated goat anti-human igg for 30 minutes and washed. samples were acquired on an lx200 or fm3d (luminex). mfi was converted to arbitrary units (au/ml) by interpolation from a 5-parameter logistic standard curve, using bioplex manager 6.2 (bio-rad laboratories) software and exported to microsoft excel. different batches of antigen-conjugated beads were incubated with serially diluted sera to test linearity and parallelism between bead conjugations, reference, and serum samples. assay robustness was tested by analyzing a serum panel by 3 different operators on independent days using 2 different bead and 2 reference batches. the ability to discriminate igg concentrations between covid-19 patients and controls was evaluated by receiver operator characteristic (roc) analyses. to select the optimal assay defaults, both the youden j statistic, which balances between sensitivity and specificity, and a specificity-optimized cutoff (specificity of at least 98.5% for low-prevalence settings of 5%-10%) were selected. data were entered into graphpad prism 8.4.1 to generate graphs and perform statistical analyses. reproducibility of the assay was evaluated using r 2 and coefficient of variation (% cv) calculated by standard deviation divided by average × 100. for the roc analyses antibody concentrations of cross-sectional pienter3 participants (n = 224), ili patients with coronavirus (n = 74), or other viral infection (n = 110) were used as the negative control group and pcr-confirmed covid-19 samples (n = 115) with various clinical severities were used in the positive group. we selected for serum samples that were obtained more than 10 days post onset of disease symptoms to meet a reasonable degree of seroconversion, as shown in recent reports [8, 19] . both the youden j statistic-determined cutoff and the specificity-optimized cutoff (specificity of at least 98.5%) were determined. to compare differences in concentrations, data were logtransformed and tested with either a t test between 2 groups, or 1-way anova and tukey's multiple comparison test to compare multiple groups and adjusted p values reported. antibody kinetics was fitted using a nonlinear 4-parameter least square fit in graphpad prism 8.4.1. we prepared a reference serum by pooling 13 pcr-confirmed covid-19 sera and tested serial dilutions in the multiplex assay consisting of distinct fluorescent beads coupled to sars-cov-2 nucleoprotein (n), s1, and the s1 subunit rbd ( figure 1a ). this was repeated for varying batches of beads to assess consistency of performance. the assay was able to quantify concentrations in a 1000 to 10 000-fold concentration range, using a single dilution of the serum. to reliably quantify antibody concentrations between the reference serum and test samples, we confirmed that the reference and a selection of samples display the same rate of decline of fluorescence signal with increasing dilutions, which is referred to as parallelism ( figure 1b) . these data show that the triplex assay is a highly quantitative assay to detect antibodies to sars-cov-2. applying an assay in large population and longitudinal studies requires reproducibility of assay results. therefore, antibody concentrations were determined in independent experiments performed on 6 different days, using a selection of 214 samples for rbd and 268 samples for n and s1 with different concentrations of sars-cov-2 antibodies ( figure 1c ). in addition, the reproducibility test was performed by 3 different technicians using different bead batches and references to reflect the expected maximum variability of the assay over time. comparison of sample data determined on 2 independent assays runs resulted in an r 2 of 0.982, 0.985, and 0.988 for n, s1, and rbd, respectively ( figure 1c ). the obtained % cvs were 19.1, 25.5, and 14.6 for n, s1, and rbd, respectively, showing that the assay results were reproducible. sera of 115 pcr-confirmed covid-19 patients after 10 days of symptoms were tested in the assay and the results compared to a control panel of 408 sera collected prior to the outbreak of sars-cov-2. in covid-19 patients, high concentrations of igg were observed to all 3 antigens (figure 2a ). despite clear discrimination of igg concentrations between groups of control and covid-19 patients, some samples overlapped between the 2 groups. therefore, the specificity and sensitivity of the assay to discriminate between covid-19 patients and controls using igg concentrations was evaluated by an established statistical standard to analyze assay performance, the roc analyses. for the roc analyses, concentration data of hospitalized and nonhospitalized covid-19 disease cases were included to provide a realistic evaluation of the performance of the assay (figure 2a ). the area under the curves ranged from 0.9839 to 0.9859 ( figure 2b ). the roc generated cutoff concentrations of 14.8, 0.85, and 8.21 au/ml using the roc youden j statistic. to gain a higher specificity of the assay optimized for a low population seroprevalence, the cutoff concentrations were 19.7, 2.37, and 19.1 for n, s1, and rbd, respectively ( figure 2c ). the latter cutoffs resulted in a specificity of 98.5%, 99.0%, and 98.5% at a sensitivity of 89.4%, 84.4%, and 83.6% for n, s1, and rbd, respectively. to study how our assay discriminates between antibodies of individuals with different laboratory-confirmed viral infections, antibodies were measured in a cross-sectional population panel (n = 224), a panel of noncorona ili patients (non-hcov ili, n = 74), and non-sars-cov-2 corona ili patients (hcov ili, n = 110) and compared to pcr-confirmed covid-19 patients' samples. some of the covid-19 patients were admitted to hospital (n = 50) because of severe covid-19 and these were compared to nonhospitalized covid-19 cases (n = 65). for each of the 3 negative control groups the majority of the samples had concentrations below the cutoff for all 3 antigens ( figure 3a ). the number of falsepositive samples ranged from 5 to 6 out of 404 or 408 samples tested for the different antigens. the non-hcov and hcov ili panels were from persons infected with multiple different non-sars viruses including 4 different endemic coronavirus (supplementary table 2 ). the proportion of false positives did not increase by testing the convalescent sera from patients with a laboratory (pcr)-confirmed infection with either of the 4 seasonal coronaviruses ( figure 3b , and data not shown), indicating that the antigens used in the assay are selective for sars-cov-2-induced antibodies. comparison of pcrconfirmed sars-cov-2 patients samples shows that all hospitalized patients induced antibodies to n and the majority of hospitalized patients induced antibodies to s1 and rbd. the majority of the nonhospitalized cases showed antibody concentrations above the cutoff for n, whereas around 10% of the nonhospitalized patients did not produce antibodies above the cutoffs for s1 and rbd. overall, the concentrations of antibodies in serum samples from patients that were hospitalized were significantly higher compared to patients that were not hospitalized. following infection, an immune response is initiated, resulting in the production of serum antibodies. to study the time between onset of disease symptoms and the development of antibodies, paired serum samples were collected from the majority of patients. data were separated for patients that were either admitted to the hospital or not ( figure 4a and 4b) . apart from the paired samples from 2 patients that were obtained before 7 days after onset of disease, all other hospitalized cases showed seroconversion for all 3 antigens tested ( figure 4a ). in line with other reports, hospitalized covid-19 patients seroconverted around day 10 of disease onset. of 53 nonhospitalized cases, 48 seroconverted, whereas 5 showed slight increases in concentrations but failed to formally cross the cutoff value for any of the 3 analytes to be regarded a specific seroconversion. hospitalized patients reached a plateau of antibody production shortly after 2 weeks from onset of symptoms, which took at least 25 days for the nonhospitalized cases (4-10 fold lower slope; figure 4c and 4d). as a consequence of the slower increase of antibody concentrations the time to detectable antibodies was delayed, especially with respect to antibodies reacting to s1 and rbd. the variance in the nonhospitalized cases was high compared to the hospitalized cases, which is illustrated by the lower r 2 of the nonlinear least square fit of the 2 patient groups. cutoff (au/ml) figure 2 . ability of the assay to identify covid-19 patients. a, control sera (n = 408) and covid-19 sera (n = 115) collected after day 10 of symptoms were tested and compared for concentrations of igg. median concentration and 95% confidence intervals are shown. b, the sera tested in (a) were analyzed by roc. c, the roc data were used to determine youden j statistic cutoff (lower cutoff) and a specificity-optimized cutoff of at least 98.5% specificity (higher cutoff). abbreviations: au, arbitrary unit; igg, immunoglobulin g; n, nucleoprotein; rbd, receptor binding domain; roc, receiver operator characteristic; s1, spike protein subunit 1. table 2 ) and concentration data of ili patients are shown to confirm that the assay discriminates sars-cov-2-specific antibodies from antibodies induced by various laboratory-confirmed viral infections. abbreviations: au, arbitrary unit; covid-19, coronavirus disease 2019; hcov, human coronavirus; mers-cov, middle east respiratory syndrome coronavirus; n, nucleoprotein; non-hcov, noncoronavirus; rbd, receptor binding domain; rsv, respiratory syncytial virus; s1, spike protein subunit 1. the engagement of different structural sars-cov-2 proteins in 1 serological determination (multiplex testing) instead of 1 protein could improve the sensitivity and the specificity. if only 1 analyte is analyzed, the sensitivities for hospitalized cases were 94.1%, 94.3%, and 100% for rbd, s1, and n, respectively, using the specificity-optimized cutoff (table 1) . using the roc youden j statistic cutoff the sensitivities were 97.1% for both s1 and rbd and 100% for n. nonhospitalized cases typically had lower concentrations of igg, which reduced the sensitivity: 76.3% for s1 and up to 82.7 % for n using the specificity-optimized cutoff. using the youden j statistic cutoff, the sensitivity increased to 91.3% for s1. in this multiplex approach an increased sensitivity can be obtained by evaluating a sample as positive when either 1 of the antibody concentrations determined is higher than the set cutoff (logical or analysis in table 1 ). any combination of antigen reached a sensitivity of 100% when n was used in hospitalized cases and ranged from 90.4% (s1 or rbd) up to 95.1% (n or s1 or rbd) using the specificity-optimized cutoff. applying the youden j statistic cutoff resulted in a sensitivity for nonhospitalized cases of at least 92.8% (n or s1) up to 98.8% (n or s1 or rbd). the specificity of the youden j analyses using n or s1 or rbd dropped to 90.9%. this specificity is far too low for serosurveillance purposes in areas of low prevalence. the specificity-optimized cutoff (95.8%-97.8%) is clearly better, which may be considered adequate if the true prevalence in the population is above 20%. because in most countries the overall covid-19 seroprevalence is currently under 20%, high specificity is required to provide reliable seroprevalence estimates (illustrated in supplementary table 1 ). this could be achieved by defining a sample positive when at least 2 antibody test results in multiplex are above the cutoff. this resulted in a specificity of 100% for any of the combinations and both the specificityoptimized and the youden j statistic-determined cutoffs (logical and; table 1 ). as expected, this increased specificity comes at the expense of the sensitivity. here, if only s1 and rbd are taken into consideration, this combination resulted in the highest possible sensitivity of 87.3% and 97.1% for nonhospitalized and hospitalized patients, respectively. we aimed to develop a high-throughput quantitative assay to measure true concentrations of antibodies to spike s1, spike rbd, and n of sars-cov-2. the assay presented here uses a very small sample volume, which can be obtained from, for example, fingerstick blood, while retaining highly quantitative output. this bead-based multiplex immunoassay generates robust results and is able to discriminate covid-19 with different degrees of disease severity, especially from day 10 of disease onward. the results of the assay presented here provide detailed insight into the performance of the assay in terms of parallelism between the references and sera containing different concentrations of antibodies. in addition, we show consistency of assay results when the same samples are measured on independent days, by different investigators using different batches of reagents, basically incorporating all potential variability. large population studies are in high demand to provide insight into the spread of the virus and the protective status of the population, which can be used for policy makers to manage the pandemic or lift the lockdown measures [2, 3, 11, 12] . assays results have to be accurate to generate reliable seroprevalence data of the general population. in addition to knowing the performance of an assay, we need to understand how the majority of infections in the general population relate to the induction of detectable antibodies. our data comparing hospitalized and nonhospitalized cases revealed that milder disease results in both lower levels of antibodies and later seroconversion, which is in line with previous reports [19, 20] . also, comorbidities may play a role in the production of specific serum antibodies following infection, which warrants further study. approximately 10% of the nonhospitalized cases in our selection did not show any seroconversion at all, indicating that such mild infections may not be detected by serological assays. however, assay performance could be improved by adding other sars-cov-2 proteins or subunits of these to further improve the sensitivity of the assay to detect low seroconversion in some cases. essential performance characteristics of assays aiming to identify seroprevalence in the population are the specificity and sensitivity. the specificity and sensitivity determine the positive and negative predictive value (ppv and npv) of the assay given the prevalence of seropositivity in the population [21] . in current low-prevalence settings insufficient specificity will generate a low ppv, resulting in a significant overestimation of the proportion of seropositive individuals (illustrated in supplementary table 1 ). however, the accuracy of the reported sensitivity and specificity of an assay also highly depends on the patient selection used for this evaluation, for example, using sera of severe covid-19 patients will result in beneficial statistics of an assay because of the acknowledged higher antibody concentration and seroconversion rate [22] . these statistics will not apply in a population serosurvey where the majority of persons will not develop severe covid-19. for this reason, we included a heterogeneous group of covid-19 patients' samples, consequently reducing sensitivity. scoring samples positive if at least 2 of the analytes generated positive results improved the specificity of the assay to 100% at a sensitivity > 90%. at a true seroprevalence of 5%, this would provide a seroprevalence estimate of 4.5% and therefore would be much more accurate than using a single analyte. we recommend transparent reporting of underlying assay performance using heterogeneous panels of controls and covid-19 patients. furthermore, implementation of international reference materials as being distributed by, for example, the national institute for biological standards and control, to facilitate comparison of seroepidemiological data between studies and countries is greatly recommended [1, 23] . from an immunological point of view, it needs to be established which sars-cov-2-specific antibodies correlate with protection. antibodies to rbd of s1 have been shown to associate with neutralization of the virus in vitro, and preliminary data indicate that the antibodies reported in our assay correlate quantitatively with virus neutralization in vitro as well [6] . the data presented here show detection of total igg. another study has shown that igg subclasses are not equally induced by sars-cov-2 infection, with a bias towards the production of igg3, at least in the first weeks after infection [24] . infection with sars-cov-2 also induces the production of iga and igm, which can contribute to protection and in vitro neutralization of the virus, but these isotypes are currently not captured by our assay [7, 8, 25] . follow-up studies are needed to establish the longevity of the production of antibodies, the degree of protection antibodies confer through various fc receptor-mediated and other mechanisms, and how b-cell memory is induced. such studies should also consider different viral loads detected in a patient and degree of severity of covid-19. in conclusion, we developed a robust multiplex assay to detect antibodies to sars-cov-2 in small blood volumes. our study is unique in validating the assay against hcov and non-hcov ili panels. because of the differences in seroconversion rates and quantitative antibody concentrations among nonhospitalized covid-19 cases, which represents the majority of patients in the general population, further investigation is required to improve assay performance for serosurveys in general. we show the advantages of multiplexed analysis in determining seroconversion and provide a framework for reliable seroprevalence estimates in different settings. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. immune surveillance for vaccine-preventable diseases developing antibody tests for sars-cov-2 towards the next phase: evaluation of serological assays for diagnostics and exposure assessment ace2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of 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cellular immunity in covid-19 convalescent individuals longitudinal change of severe acute respiratory syndrome coronavirus 2 antibodies in patients with coronavirus disease 2019 acknowledgments. the authors acknowledge jorgen de jonge and puck van kasteren for critically reviewing our manuscript and gert-jan godeke for providing technical assistance.financial support. this work was supported by the national institute for public health and the environment, the netherlands.potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-351760-698voi9y authors: han, hui-ju; liu, jian-wei; yu, hao; yu, xue-jie title: neutralizing monoclonal antibodies as promising therapeutics against middle east respiratory syndrome coronavirus infection date: 2018-11-30 journal: viruses doi: 10.3390/v10120680 sha: doc_id: 351760 cord_uid: 698voi9y since emerging in 2012, middle east respiratory syndrome coronavirus (mers-cov) has been a global public health threat with a high fatality rate and worldwide distribution. there are no approved vaccines or therapies for mers until now. passive immunotherapy with neutralizing monoclonal antibodies (mabs) is an effective prophylactic and therapeutic reagent against emerging viruses. in this article, we review current advances in neutralizing mabs against mers-cov. the receptor-binding domain (rbd) in the spike protein of mers-cov is a major target, and mouse, camel, or human-derived neutralizing mabs targeting rbd have been developed. a major problem with neutralizing mab therapy is mutant escape under selective pressure, which can be solved by combination of neutralizing mabs targeting different epitopes. neutralizing mabs are currently under preclinical evaluation, and they are promising candidate therapeutic agents against mers-cov infection. middle east respiratory syndrome (mers) emerged in 2012 in saudi arabia with the death of a man with pneumonia; the causative agent was subsequently identified as mers-cov, which belonged to lineage c betacoronaviruses [1] . with dromedary camels (camelus dromedarius, also known as arabian camel) as direct sources and bats as potential reservoirs [2] , mers-cov has been frequently introduced into human populations. once mers-cov is introduced into a person, person-to-person transmission might occur, and is responsible for approximately 40% of mers cases globally [3] . mers-cov has been a consistent threat to humans. as of october 2018, mers-cov has caused 2254 laboratory-confirmed human cases, including 800 deaths in 27 countries, with the fatality rate as high as 35% (http://www.who.int/emergencies/mers-cov/en/). although mers cases are primarily reported in the middle east, facilitated by international travelling, mers-cov can also be a worldwide threat, which is well illustrated by the mers outbreak in south korea in 2015 [4] . given the potential risk of causing worldwide public health emergencies and the absence of licensed vaccines and antiviral therapeutics, the world health organization has listed mers-cov in the "list of blueprint priority diseases" (http://www.who.int/blueprint/priority-diseases/en/). vaccines are the most important approach against viral infections, but usually take a long time to develop. they are also unable to provide either immediate prophylactic protection or treat ongoing viral infections. neutralizing monoclonal antibodies (mabs) have recently emerged as a powerful tool to provide prophylactic and therapeutic protection against emerging viruses [5] . potent neutralizing mabs can be achieved by various technologies, such as hybridoma technology, humanized mouse, phage or yeast display, and single b cell isolation [5] . mers-cov is a single, positive-stranded rna virus of about 30 kb, which encodes four major viral structural proteins-including spike (s), envelope (e), membrane (m) and nucleocapsid (n)-as well as several accessory proteins [6] . the s protein (1353 aa) plays an important role in virus infection and consists of a receptor-binding subunit s1 (aa 18-751) and a membrane-fusion subunit s2 (aa 752-1353). s1 mediates viral attachment to host cells and s2 mediates virus-cell membrane fusion [7] . the s1 subunit contains a receptor-binding domain (rbd) (aa 367-606) [8] that can bind to cell receptor dipeptidyl peptidase 4 (dpp4, also known as cd26), and mediates viral attachment target cells [9] . the rbd consists of a core subdomain and a receptor-binding motif (rbm) (aa 484-567). the schematic representation of mers-cov s protein is shown in figure 1a . neutralizing mabs binding to the s protein of mers-cov can prevent viral attachment to the cell receptor and inhibit viral entry [7] . the s protein of mers-cov is a key target for antivirals, and rbd is the most popular focus. in this study, we review the current knowledge on neutralizing mabs targeting the rbd of mers-cov. stable hybridoma cell lines were generated by fusing myeloma cells with splenocytes of mice that were immunized with mers-rbd protein. two neutralizing mabs, 4c2 and 2e6, had high affinity for the rbd of mers-cov and blocked both pseudovirus and live mers-cov entry into cells with high efficacy [10] . humanized 4c2 showed similar neutralizing activity in cell entry tests. in vivo tests indicated that 4c2 could significantly reduce the virus titers in the lungs of ad5-hcd26-transduced mice which were infected with mers-cov, highlighting its potential application in humans not only for preventing but also treating mers-cov infection. crystallization of the 4c2 fab/mers-rbd complex showed that the 4c2 recognized conformational epitopes (y397-n398, k400, l495-k496, p525, v527-s532, w535-e536, and d539-q544), which were partially overlapped the receptor-binding footprint in the rbd of mers-cov. the 4c2 complex interfered with mers-cov binding to dpp4 by both steric hindrance and interface-residue competition. 2e6 competed with 4c2 to bind to mers-rbd, indicating that they recognized proximate or overlapping epitopes [10] . neutralizing mab mersmab1 was obtained by fusing myeloma cells with splenocytes of a mouse that was immunized with recombinant mers-cov s1 [11] . mersmab1 effectively blocked the entry of pseudovirus and live mers-cov into cells. structural analysis showed that mersmab1 bound to the rbd of mers-cov through recognizing conformational epitopes, and all of the residues critical for mersmab1 binding were located on the left ridge of rbm. mersmab1 neutralized mers-cov by competitively blocking the binding of mers-cov rbd to dpp4. based on escape mutant analysis of the key residues on the rbd, it was found that residue l506, d510, r511, e513, and w553 were critical for mersmab1 binding to the rbd, while mutation of e536, d539, or e565 did not affect the interaction of mersmab1 and the rbd at all [11] . an ultra-large nonimmune human antibody-phage display library was constructed with b cells of unimmunized donors. with a unique spanning strategy, seven human neutralizing mabs with varying neutralization efficacy to mers-cov were identified [12] . binding detection demonstrated that the epitopes of these mabs lay within aa 349-590 of the s protein, which overlapped a large part of the rbd of mers-cov. binding competition assays showed that these mabs recognized at least three distinct epitope groups, which was further confirmed by escape studies. with no cross-epitope resistance, these mabs neutralized mers-cov by competitively blocking the binding of the rbd of mers-cov to dpp4. escape mutant assays showed that five residues were critical for neutralization of these mabs, namely l506, t512, y540, r542, and p547. of the seven mabs, 3b11 exhibited the best neutralization activity against both pseudovirus and live mers-cov infectivity in cells. moreover, under the selective pressure of these mabs, the igg form of 3b11 was superior, since it did not induce neutralization escape [12] . in vivo tests demonstrated that 3b11 reduced lung pathology in rhesus monkeys infected with mers-cov [13] . with its high neutralizing activity and suppression of mutant escape, 3b11 in the igg form is a promising therapeutic mab against mers-cov. three human mabs-m336, m337, and m338-were identified from a large naïve human phage display antibody library, which was constructed with peripheral blood mononuclear cells from healthy volunteers [14] . the binding sties of the three mabs were within the rbd of mers-cov (aa 377-588), therefore they neutralized mers-cov by competing with dpp4 binding to the rbd. the three mabs also competed with each other to bind to the rbd of mers-cov, and mutant analysis showed that the three mabs possessed overlapping but distinct epitopes. of the three mabs, m336 neutralized both pseudovirus and live mers-cov infectivity in cells with exceptional potency (m336 inhibited 90% mers-cov pseudovirus infection at a concentration of 0.039 g/ml, and neutralized live mers-cov with ic 95 of 1 g/ml and ic 50 of 0.07 g/ml). residues in the rbd crucial for m336 binding were l506, d510, e536, d539, w553, and v555 [14] . in vivo study demonstrated that prophylaxis with m336 reduced virus titers in the lung of rabbits infected with mers-cov [15] , and m336 also provided transgenic mice expressing human dpp4 with full prophylactic and therapeutic protection from mers-cov [16] . however, another study with a non-human primate, the common marmoset showed that m336 could only alleviate the severity of the disease, and did not provide complete protection against mers-cov [17] . igg+ memory b cells were isolated from a mers patient, and were subsequently immortalized with epstein-barr virus. a neutralizing mabs, lca60, was identified, and was the first fully human neutralizing mab with naïve heavy and light chain pairs [18] . lca60 efficiently neutralize mers-cov infectivity in cells. in vivo study showed that lca60 provided balb/c mice transduced with adenoviral vectors expressing human dpp4 (hdpp4) with both prophylactic and postexposure protection against mers-cov. furthermore, the neutralizing efficacy of lca60 was evaluated in ifn-α/β receptor-knockout mice that were more stringent models of mers-cov infection. after transducing with hdpp4, these mice showed more profound clinical symptoms when challenged with mers-cov [19] ; administration of lca60 reduced mers-cov titer in the lungs of these mice more effectively (lung viral titer reduced by three logs in one day for ifn-α/β receptor-knockout mice vs. three days for balb/c mice) [18] . with naïve heavy and light chain pairs, lca60 was more potent than 3b11 and comparable to m336. cross-competition experiment demonstrated that lca60 competed with 3b11 to bind to the rbd. lca60 interacted with rbd residues around k493, and the lca60 footprint on the rbd was partially overlapped with that of dpp4. four residues in the rbd affected the binding of lca60-namely t489, k493, e536, and e565-which were conserved in all mers-cov isolates. moreover, compared with dpp4, the binding affinity of lca60 to rbd was significantly higher (~500-fold). therefore, one major neutralization mechanism of lca60 was to competitively inhibit the interaction of the rbd with dpp4. interestingly, virus escape studies demonstrated that under the selective pressure of lca60, a mutant variant (v33a) in the n-terminal domain (ntd) of mers-cov s1 subunit was also generated [18] . a gmp-approved cell line (lca60.273.1) that expresses lca60 in high concentrations has been established, highlighting its application as promising therapeutics against mers-cov infection [20] . hybridoma b cells producing neutralizing mabs against the s protein of mers-cov were generated by immunizing humanized transgenic mice (velocimmune mice) with dna encoding the mers-cov s protein. two fully human neutralizing mabs, regn3051 and regn3048, were obtained [21] . the two mabs bound with high affinity to distinct epitopes on the rbd of mers-cov, which were conserved during the natural evolution of mers-cov. mutation as a result of selective pressure by one mab should not affect the binding of the other mab. regn3051 neutralized a broad range of mers-cov isolates, the prototype emc/2012 strain and all clinical mutants including a431p, s457g, s460f, a482v, l506f, d509g, and v534a. with the exception of v534a variant, regn3048 achieved similar neutralizing activity. in vivo study demonstrated that regn3051 and regn3048 reduced mers-cov replication in humanized dpp4 mice in both prophylactic and therapeutic settings [21] . when evaluated in the common marmoset, both mabs seemed to be more effective for prophylaxis rather than for treatment of mers-cov infection [22] . an anti-mers-cov phage display antibody library was constructed with the peripheral b cells of a mers survivor, and a human neutralizing mab against mers-cov, mca1, was identified [23] . mca1 showed potent neutralizing activity against mers-cov in cell entry tests. in vivo, mca1 completely inhibited the replication of mers-cov in common marmosets when administrated prophylactically or therapeutically. structure analysis of the mca1 fab-rbd complex showed that mca1 formed direct contacts with the receptor-binding site (rbs) subdomain on the rbd. epitopes on the rbs critical for mca1 binding were d510, w535, e536, d539, y540, r542, and q544. superimposed structure analysis of mca1-rbd and hdpp4-rbd complexes showed that the binding interface of mca1 was largely overlapped with that of hdpp4. therefore, the neutralizing mechanism of mca1 was achieved by competing with dpp4 for binding to the rbd [23] . two potent human neutralizing mabs, mers-4 and mers-27, were derived from a nonimmune human yeast display antibody library, which was constructed with spleen and lymph node polyadenylated rna from normal humans [24] . [24] . further structural analysis showed that mers-4 bound to unique epitopes and caused conformational changes in the rbd interface critical for accommodating dpp4, therefore indirectly disrupting the interaction between the two. moreover, mers-4 also demonstrated synergistic effects with m336 and 5f9 (a ntd-specific mab). the special neutralizing mechanism made mers-4 a valuable addition for the combined use of mabs against mers-cov infection [25] . thirteen ultrapotent neutralizing mabs, which all targeted the rbd of mers-cov were generated following a protocol for the rapid production of antigen-specific human mabs [26] . briefly, antibody-secreting b cells were isolated from the whole blood of a mers patient, and the antibody genes were amplified and cloned into vectors to transfect human cell lines for mab production. of the 13 mabs, mers-gd27 and mers-gd33 exhibited the strongest neutralizing activity against both pseudovirus and live mers-cov in cell infection tests. mers-gd27 directly competed with dpp4 to bind to the rbd to dpp4, and the crystal structure of mers-gd27 showed that its epitopes were almost completely overlapped with dpp4-binding sites. mers-gd27 and mers-gd33 recognized distinct epitopes on the rbd, and had a low level of competing activity. the combined use of the two mabs demonstrated synergistic effects in neutralization against pseudotyped mers-cov. mutant analysis demonstrated that residues l506, d509, v534, e536, and a556 on rbd were important for the neutralizing activity of mers-gd27, and residue r511was critical for mers-gd33 [26] . moreover, in vivo study found that mers-gd27 could provide both prophylactic and therapeutic protection for hdpp4-trangenic mice against mers-cov infection [27] . dromedary camels exposed to mers-cov showed mild clinical signs but developed exceptionally potent neutralizing antibodies. camelid species naturally produced heavy chain-only antibodies (hcabs) [28] , which are dimeric and devoid of light chains, and their antigen recognition region is solely formed by the variable heavy chains (vhhs) (also called nanobodies, nbs). vhhs or nbs have long complementarity-determining region 3 (cdr3) loops and are capable of binding to unique epitopes not accessible to conventional antibodies [29] . notably, camelid vhhs are relatively stable and can be produced with high yields in prokaryotic systems [30] . because of their small size; good tissue permeability; and cost-effective production, storage, and transportation [31] [32] [33] , vhhs or nbs have been gaining acceptance as antiviral agents. a vhh complementary dna library was constructed with the bone marrow of dromedary camels infected with mers-cov. four vhhs (vhh-1, vhh-4, vhh-83, and vhh-101) with high neutralizing activity were identified by direct cloning and screening of the phage display antibody library [34] . the four vhhs competed for a single epitope that partially overlapped with the rbd-dpp4 interface. mutant analysis showed that the four vhhs did not bind to the d539n variant, which was a critical residue on the rbd for dpp4 binding [8, 35] . therefore, these vhhs most likely neutralized mers-cov by blocking its binding to dpp4. of the 4 vhhs, vhh-83 showed the best neutralizing activity and epitope recognition. vhh-83 efficiently blocked the entry of mers-cov into cells, and it also prophylactically protected k18 transgenic mouse expressing hdpp4 from mers-cov infection. to extend the half-life of vhh-83 in serum, it was linked to a human fc domain lacking the ch1 exon to construct the chimeric camel/human hcab-83, which showed similar neutralizing activity as vhh-83. the chimeric camel/human hcab-83 was highly stable in mice and provided k18 mice with fully prophylactic protection against mers-cov infection [34] . alpacas were immunized with recombinant mers-cov rbd-containing a c-terminal human igg1 fc tag, and vhhs were amplified from their peripheral blood mononuclear cells to construct a vhh phage display library. a neutralizing nb, nbms10, which bound with high affinity to the rbd of mers-cov and blocked the binding of rbd to dpp4, was identified [36] . to extend its in vivo half-life, the human-fc-fused version, nbms10-fc, was constructed. nbms10 competed with dpp4 to bind to rbd, indicating that the binding site of nbms10 on rbd overlapped with that of dpp4. the binding site of the nbms10 on the rbd was mapped to be around residue d539, which is part of a highly conserved conformational epitope at the receptor-binding interface in almost all the natural mers-cov published to date. nbms10 did not neutralize psuedotyped mers-cov bearing a mutation in d539, confirming that residue d539 was critical for nbms10 binding. nbms10 efficiently neutralized the cell entry of live mers-cov. moreover, nbms10 showed potent prophylactic and therapeutic efficacy in protecting hdpp4-transgenic mice against mers-cov infection [36] . for their exceptionally high neutralization activity in vitro and in vivo, these newly identified neutralizing mabs are promising candidate therapeutics against the infection of mers-cov. however, the use of a single neutralizing antibody bears the risk of selecting escape mutants, a fact that has been observed for lca60 and other described antibodies [12, 18, 37] . notably, the majority of these escape mutations had little impact on viral fitness and the interaction of dpp4 with the rbd [12] . moreover, mutants of mers-cov during natural infection have also been reported [38] . escape from neutralization is a major concern with therapeutic neutralizing mabs, however, this potential problem can be solved by combining mabs that target distinct epitopes and show different neutralizing mechanisms [37] . this strategy can take advantage of the synergistic effects while decreasing the possibility of viral escape. currently, most of the mers-cov neutralizing mabs compete with dpp4 binding to the rbd, and residues on the rbd critical for mab neutralization are identified by mutant analysis. almost all of the residues identified critical for mab neutralization are located in rbm, and overlap with those critical for dpp4 binding ( figure 1b) . with the availability of crystal structure of mab fab-rbd complex, the neutralization mechanism of these mabs will be better illustrated. based on the crystal structure of rbd-dpp4, it was found that several conserved residues in the rbd are critical for the interaction of the rbd with dpp4 (y499, l506, d510, e513, e536, d537, d539, y540, r542, w553, and v555) [35, 39] . development of therapeutic neutralizing mabs targeting those critically conserved residues might be important for combating mers-cov. moreover, a study found a mouse-derived neutralizing mab, 5f9, which bound to a possible linear epitope in the ntd of the mers-cov s1 subunit, exhibited efficient neutralizing activity against pseudovirus and live mers-cov in cell entry tests. this study highlighted the important role of ntd during the infection process of mers-cov. ntd might have significant implications for the development of prophylactic and therapeutic mabs against mers-cov infection [40] . although the in vitro neutralizing potency of 5f9 was approximately 10-fold lower than that of the rbd-targeting neutralizing mabs [40] , it may provide an alternative for the immunotherapy against mers-cov, once the virus mutates and is no longer susceptible to rbd-specific mabs. so far, there is a lack of appropriate animal models to mimic the pathology of merd-cov in humans. commonly-used laboratory animals-such as wild-type mouse, ferret, hamster, and guinea pig-are not susceptible to mers-cov infection due to differences in critical amino acids in the s-binding domain of their dpp4 [41] [42] [43] . new zealand rabbits, hdpp4-transduced/transgenic mice, camelids and non-human primates (rhesus macaque and common marmoset) are susceptible to mers-cov infection, however, rabbits showed asymptomatic infection [44] ; dromedary camels displayed different clinical manifestations to that of humans [45] ; rhesus macaque only showed transient lower respiratory infection [46] , while common marmoset developed progressive pneumonia [47] ; hdpp4-trangenic mouse expressed hdpp4 extensively, and resulted in multiple organ damage [48] ; hdpp4-transduced mouse only exhibited mild transient clinical diseases [19] . with robust animal models, the protective effects of these neutralizing mabs will be better evaluated. furthermore, ongoing efforts on developing therapeutic neutralizing mabs against mers-cov should also consider the different target populations (dromedary camels and humans) and their protective efficacy. isolation of a novel coronavirus from a man with pneumonia in saudi arabia evidence for zoonotic origins of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission probable 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spike protein in a novel humanized mouse model of mers-cov infection prophylactic and therapeutic efficacy of mab treatment against mers-cov in common marmosets human neutralizing monoclonal antibody inhibition of middle east respiratory syndrome coronavirus replication in the common marmoset potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein structural definition of a unique neutralization epitope on the receptor-binding domain of mers-cov spike glycoprotein ultrapotent human neutralizing antibody repertoires against middle east respiratory syndrome coronavirus from a recovered patient a novel human mab (mers-gd27) provides prophylactic and postexposure efficacy in mers-cov susceptible mice naturally-occurring antibodies devoid of light-chains molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies nanobodies: natural single-domain antibodies application of camelid heavy-chain variable 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spike protein wild-type and innate immune-deficient mice are not susceptible to the middle east respiratory syndrome coronavirus the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase 4-mediated entry of the middle east respiratory syndrome coronavirus asymptomatic middle east respiratory syndrome coronavirus infection in rabbits experimental infection of dromedaries with middle east respiratory syndrome-coronavirus is accompanied by massive ciliary loss and depletion of the cell surface receptor dipeptidyl peptidase 4 an animal model of mers produced by infection of rhesus macaques with mers coronavirus infection with mers-cov causes lethal pneumonia in the common marmoset multi-organ damage in human dipeptidyl peptidase 4 transgenic mice infected with middle east respiratory syndrome-coronavirus funding: this study was supported by a grant from national natural science funds of china (nos. 31570167). the authors declare that they have no conflict of interest. key: cord-327711-welf0eb1 authors: zhou, daming; duyvesteyn, helen me; chen, cheng-pin; huang, chung-guei; chen, ting-hua; shih, shin-ru; lin, yi-chun; cheng, chien-yu; cheng, shu-hsing; huang, yhu-chering; lin, tzou-yien; ma, che; huo, jiandong; carrique, loic; malinauskas, tomas; ruza, reinis r; shah, pranav nm; tan, tiong kit; rijal, pramila; donat, robert f.; godwin, kerry; buttigieg, karen; tree, julia; radecke, julika; paterson, neil g; supasa, piyasa; mongkolsapaya, juthathip; screaton, gavin r; carroll, miles w.; jaramillo, javier g.; knight, michael; james, william; owens, raymond j; naismith, james h.; townsend, alain; fry, elizabeth e; zhao, yuguang; ren, jingshan; stuart, david i; huang, kuan-ying a. title: structural basis for the neutralization of sars-cov-2 by an antibody from a convalescent patient date: 2020-06-13 journal: biorxiv doi: 10.1101/2020.06.12.148387 sha: doc_id: 327711 cord_uid: welf0eb1 the covid-19 pandemic has had unprecedented health and economic impact, but currently there are no approved therapies. we have isolated an antibody, ey6a, from a late-stage covid-19 patient and show it neutralises sars-cov-2 and cross-reacts with sars-cov-1. ey6a fab binds tightly (kd of 2 nm) the receptor binding domain (rbd) of the viral spike glycoprotein and a 2.6å crystal structure of an rbd/ey6a fab complex identifies the highly conserved epitope, away from the ace2 receptor binding site. residues of this epitope are key to stabilising the pre-fusion spike. cryo-em analyses of the pre-fusion spike incubated with ey6a fab reveal a complex of the intact trimer with three fabs bound and two further multimeric forms comprising destabilized spike attached to fab. ey6a binds what is probably a major neutralising epitope, making it a candidate therapeutic for covid-19. conversion to the post-fusion form where the s2 subunit engages the host membrane whilst dispensing with s1 2,3 . neutralising human monoclonal antibodies that recognise the ace2 receptor binding site for sars-cov-1 and sars-cov-2 are generally not cross-reactive between the two viruses and are susceptible to escape mutation 6-10 (indeed a natural mutation y495n has already been identified at this site (gisaid 11 : accession id: epi_isl_429783 wienecke-baldacchino et al.)). in contrast cr3022 (derived from a sars-cov-1 patient) cross-reacts strongly with sars-cov-2 (methods, fig. 1 ) and has been shown to recognise a cryptic, conserved epitope on the rbd distinct from the binding epitope of ace2 7, [12] [13] [14] . that this is not uncommon for sars-cov-1 antibodies is suggested by similar observations for 47d11 15 . to isolate sars-cov-2 spike-reactive monoclonal antibodies, we cloned antibody genes from blood-derived plasmablasts of a covid-19 patient in the convalescent phase. one of these, ey6a was shown by elisa to bind s1 of sars-cov-2 and cross react with sars-cov-1 (fig. 1) . binding of ey6a to sars-cov-2-infected cells was detected by immunofluorescence ( fig. 1 ). surface plasmon resonance (spr) measurements for ey6a fab showed high affinity binding to immobilised sars-cov-2 rbd (kd = 2 nm, although the value for immobilised ey6a igg was somewhat higher) as derived from the kinetic data (methods, extended data fig. 1 , extended data table 1 ). spr studies showed that there was some interdependence of ey6a and cr3022 binding, which varied depending on which component was immobilised on to the sensor chip; ace2 blocking assays confirmed a somewhat asymmetric blocking effect (extended data fig. 2 ). with rbd stably expressed on 5 mdck-siat1 cells (mdck-rbd), ey6a did not block binding of ace2 to the rbd, whereas with ace2 stably expressed on mdck-siat1 cells (mdck-ace2) ey6a blocked the interaction of rbd with ace2. in this assay, ey6a exhibits around 7 times stronger ace2 blocking than cr3022 13 (ey6a, ic50 = 54 nm; cr3022, ic50 = 347 nm) and has equivalent ace2 inhibition compared to ace2-fc (ic50 = 54 nm) and vhh72-fc (ic50 = 69 nm) 2 . these observations are suggestive of an indirect effect by ey6a once bound to the rbd, consistent with an allosteric or weak direct interaction. this was supported by an spr competition assay with immobilised cr3022, which binds distant from the ace2 binding site (extended data fig. 2 ) 12 . this showed complete competition with ey6a for rbd binding suggesting they recognise the same or overlapping epitopes, and indicated that ey6a binds the sars-cov-2 rbd more tightly. two independent neutralisation tests, both using live wild type sars-cov-2 showed strong neutralisation. a neutralisation test for ey6a based on quantitative pcr detection of virus in the supernatant bathing infected vero e6 cells after 5 days of culture, showed a ~1000-fold reduction in virus signal (methods, extended data fig. 3 ) indicating that it is highly neutralising. this was further corroborated by a plaque reduction neutralisation test (prnt) at phe porton down (methods and extended data table 2 ) using sars-cov-2 virus and ey6a which gave an nd50 of ~10.8 µg/ml (70 nm) (calculated according to grist 16 ) . a separate prnt implementation at oxford gave a slightly higher nd50 of ~30 µg/ml, consistent with a shorter incubation time of antibody with virus at lower temperature (extended data fig. 4 ). to elucidate the epitope of ey6a, we determined the crystal structures of the deglycosylated sars-cov-2 rbd in complex with ey6a fab alone and in a ternary complex incorporating a nanobody (nb) which has been shown to compete with ace2 (for data on a closely related nb see huo 2020, submitted), as a crystallisation chaperone. the crystals of the binary complex diffracted to 3.8 å resolution (methods, extended data table 3 ) and those of the ternary complex to 2.65 å. the interaction between ey6a and the rbd was identical in both complexes (extended data fig. 5 ). the higher resolution ternary complex, which showed that there was no interaction between ey6a and the nb, permitted a full interpretation of the detailed interactions (figs. 2 and 3) and has been refined to give an rwork/r-free of 0.216/0.262 and good stereochemistry (methods, extended data table 3 ). residues 333-527 of the rbd, 1-136 and 141-224 of the heavy chain and 1-215 of the light chain of ey6a and 2-126 of the nb are well defined fig. 2a ,b. the nb recognises an epitope adjacent to and slightly overlapping the ace2 receptor binding site and binds the rbd orthogonally to ey6a (fig. 2b,c) . ey6a binds essentially the same epitope as cr3022 12, 13 but with a different pose corresponding to a rotation of 73° about an axis perpendicular to the rbd α3 helix (central to both epitopes) (fig. 2d,e) . the fab complex interface buries 564 and 361 å 2 of surface area for the cdrs of the heavy and light chains respectively. the ey6a interaction is mediated by the cdr loops h1, h2, h3, l1 and l3 contacting predominantly α3 but also α2 and the β2-α3, α4-α5 and α5-β4 loops of the rbd (fig. 3 and extended data fig. 6 ). a total of 16 residues from the heavy chain and 11 from the light chain participate in the interface together with 31 residues from the rbd. for the heavy chain these form potentially 6 hydrogen bonds and a single salt bridge between d99 (of h3) and k386 of the rbd and the light chain interface residues contribute an additional 6 hydrogen bonds. hydrophobic interactions further increase the binding affinity (fig. 3) . of the 31 residues involved in the interaction 21 are conserved between the cr3022 and ey6a epitopes ( fig. 3 and extended data fig. 6 ). conformational changes are introduced into the rbd by binding to ey6a at the α2 (residues 365-371) and α3 (residues 384-388) helices (extended data fig. 6 ), similar to those seen for the cr3022 complex 12 . comparison of the epitope residues for ey6a, cr3022 12 and vhh-72 17 shows that there is a very substantial overlap (extended data fig. 6 ), although the bulk of the molecules extend in different directions, such that vhh-72 directly blocks ace-2 binding 17 . in the first pre-fusion spike structures (pdb ids: 6vsb 2 , 6vxx, 6vyb 4 ), where residues 986 and 987 in the linker between two helices in s2 were mutated to a pro-pro sequence to prevent the conversion to the post-fusion helical conformation, the rbds were found in either one 'up' two 'down' or all three 'down' configuration, and in both cases the epitope is inaccessible. in the 'down' position it is packed against another rbd of the trimer and the nterminal domain (ntd) of the neighbouring protomer. a recent publication for the wild type spike identifies a more closed form 18 where the s1 portion of the spike is tightened up. the structure is not yet deposited however, and so we have looked at the role of the epitope in the down rather than fully closed form, which will be broadly similar. here the ey6a epitope packs down tightly against the s2 'knuckle' bearing the pro-pro mutations, forming a buried protein-protein interface and making the epitope completely inaccessible. we assume that in the closed form this interaction will be even tighter and is probably responsible for maintaining the spike in the pre-fusion state. even when the rbd is in the 'up' configuration, the epitope remains largely inaccessible and substantial further movement of the rbd would be required to permit interaction unless more than one rbd was in the up conformation 12 . to investigate how the fab insinuates itself into the spike, we performed cryo-em analysis. spike ectodomain was mixed with a 6-fold molar excess of ey6a fab and incubated at room temperature (21 °c) with an aliquot taken at 5 hours, applied to cryo-em grids and frozen (methods). unbiased 2d class averages revealed three major particle classes with over onethird comprising a trimeric spike/ey6a complex (some of which are self-associated) (methods, extended data table 4 and 121°. in addition, the orientations of the vh domains relative to their associated rbds differ slightly from that of the crystal structure (by 5°, 2° and 7°, respectively). the quality of the density suggests that these likely samples selected from a continuous distribution (extended data fig. 10 ). the majority of the remaining particles form either a roughly 2-fold symmetric structure or a triangular association (methods, extended data table 4 and figs. 7-11). reconstructions of these particles were anisotropic due to a preferential orientation of the particles on the grid which was somewhat mitigated by collecting data with 30° tilt to yield reconstructions at 4.4 å and 4.7 å, respectively, in the plane of the grid but significantly worse resolution perpendicular to the grid (extended data fig. 10 ). the reconstructions were sufficiently clear to allow the unambiguous fitting of ey6a-rbd complexes (extended data fig. 11 ), however the density for what we assume are the n-terminal domains is poor in both reconstructions and we did not attempt to fit a model. these structures likely represent a residual well-structured fragment from the unfolding of the pre-fusion state of the spike (sds page analysis shows that the spike polypeptide remains largely uncleaved, extended data fig. 12 ). the 'dimeric' and 'trimeric' structures are formed by different lateral associations and these also differ from that seen for similarly structurally degraded spike-cr3022 complexes 12 (extended data fig. 11 ). convalescent serum has shown promise in patients severely ill with covid-19 19, 20 , thus immunotherapeutics have potential for treating covid-19 even at a relatively late stage in the disease. to this end, it is desirable to find a combination of antibodies that neutralise the virus by different mechanisms to mitigate against immune evasion and antibody dependent enhancement. one neutralisation mechanism is blocking receptor attachment. we propose that attachment at the ey6a epitope is a further major neutralisation mechanism. in support of this, the epitope recognised by ey6a has been reported for several antibodies 12, 13, 21, 22 and nanobodies 17,23 raised against sars-cov-2, sars-cov-1 and mers. for sars-cov-1, cr3022 has also been shown to neutralise synergistically with ace2 blocking antibodies 7 . despite the spatial separation of the ey6a and ace2 epitopes we find some cross-talk between the two binding events. the ey6a epitope is extremely unusual, since it is completely inaccessible in the pre-fusion spike trimer. this raises the question of what the mechanism of neutralisation might be. in the pre-fusion state the ey6a/cr3022 epitope rests down upon the upper end of the helixturn-helix between heptad repeat 1 (hr1) and the central helix (ch) of s2, essentially putting a lid on the spring-loaded extension of the helix which occurs on conversion to the postfusion state in the vicinity of the mutations designed to prevent conversion between the preand post-fusion conformation 24 (fig. 5 ). the residues of the epitope are crucial to these protein-protein interactions, and therefore highly conserved, explaining why it has, to date, proved impossible to generate mutations that escape binding of the antibody 7,12 . ey6a binding to the isolated rbd is tight (at ~2 nm it is roughly an order of magnitude tighter than cr3022) and, remarkably, the binding pose on top of the spike allows three fabs to bind simultaneously around the central axis (whereas cr3022 fab cannot be accommodated). in line with this, a major portion of spike molecules incubated for 5 h with ey6a are still in the intact pre-fusion state, with only about 1/3 being converted. simple modelling suggests that a similar packing could occur for intact antibodies (extended data fig. 13 ). in general, we would expect binders at this epitope to neutralise by displacing the 'lid' on the hr1/ch turn, reducing the stability of the pre-fusion state and therefore reducing the barrier to conversion to the more stable post-fusion trimer. this conversion is hindered in the construct we have used by the presence of the proline mutations at the turn between the helices. premature conversion would prevent later attachment to the cell and block infectivity. the kinetics of this process will determine the effectiveness of the antibody in neutralisation and ultimately protection. since the rbd is a relatively small domain there might also be an interplay between separate epitopes, thus we saw allosteric effects between ey6a and ace2 binding and similarly vhh-72, which binds an overlapping epitope to ey6a, strongly inhibits ace-2 binding by virtue of its different angle of attack 17 . the reason for the cross-talk between this study was designed to isolate sars-cov-2 antigen-specific human mabs from peripheral plasmablasts in humans with natural sars-cov-2 infection, to characterize the antigenic specificity and phenotypic activity of sars-cov-2 spike-reactive mab, and to determine the structure of antibody in complex with viral antigen. fresh peripheral blood mononuclear cells (pbmcs) were separated from whole blood by density gradient centrifugation and cryopreserved pbmcs were thawed. pbmcs were stained with a mix of fluorescent-labelled antibodies to cellular surface markers, including anti-cd3 (bd biosciences, usa), anti-cd19 (bd biosciences, usa), anti-cd27 (bd biosciences, usa), anti-cd20 (bd biosciences, usa), anti-cd38 (bd biosciences, usa), anti-igg (bd biosciences, usa) and anti-igm (bd biosciences, usa). plasmablasts were selected by gating on cd3-cd20-cd19+cd27hicd38hiigg+igm-events and were isolated in chamber as single cell as previously described 26 . sorted single cells were used to produce human igg monoclonal antibodies as previously described 26 . expression vectors that carry variable domains of heavy and light chains were transfected into the 293t cell line for expression of recombinant full-length human igg monoclonal antibodies in serum-free transfection medium. to determine the individual gene segments employed by vdj and vj rearrangements and the number of nucleotide mutations and amino acid replacements, the variable domain sequences were aligned with germline gene segments using the international immunogenetics (imgt) alignment tool (http://www.imgt.org/imgt_vquest/input). ey6a igg used for neutralisation and making fab: antibody was expressed using nanobody: this was derived from a naïve library followed by affinity maturation as described deglycosylation of rbd:10 µl of endoglycosidase f1 (~1mg/ml) was added to rbd (~2 mg/ml, 3 ml) and incubated at room temperature for two hours. rbd was then loaded to a superdex 75 hiload 16/60 gel filtration column (ge healthcare) for further purification using buffer 10 mm hepes ph 7.4, 150 mm nacl. purified rbd was concentrated using a 10 kda ultrafiltration tube (amicon) to 12 mg/ml. the neutralization activity of monoclonal antibody-containing supernatant was measured using a sars-cov-2 (strain cdc-4) infection of vero e6 cells 27 . briefly, vero e6 cells were preseeded in a 96 well plate at a concentration of 2 x 10 4 cells per well. on the following day, monoclonal antibody-containing supernatant were mixed with an equal volume of 100 tcid50 virus preparation and incubated at 37 °c for 1 hour. the mixture was added into seeded vero e6 cells and incubated at 37 °c for 5 days. the cell control, virus control, and virus back-titration were setup for each experiment. at day 5, the culture supernatant was harvested from each well and the viral rna was extracted by the automatic labturbo system (taigen, taiwan) following the manufacturer's instructions. for the most part, except that the specimen was pretreated with proteinase k prior to rna extraction. real-time reverse transcription polymerase chain reaction was performed in a 25-µl reaction containing 5 µl of rna 28 sars-cov-2 (australia/vic01/2020) 29 plaque reduction neutralization tests were performed using passage 4 of sars-cov-2 victoria/01/2020 29 . virus suspension at appropriate concentrations in dulbecco's modification of eagle's medium containing 1 % fbs (d1; 100 µl) was mixed antibody (100 µl) diluted in d1 at a final concentration of 50 µg/ml, 25 µg/ml, 12.5 ug/ml or 6.125 µg/ml, in triplicate, in wells of a 24 well tissue culture plate, and incubated at room temperature for 30 minutes. thereafter, 0.5 ml of a single cell suspension of vero e6 cells in d1 at 5 x 105/ml was added, and incubated for 2 h at 37 o c before being overlain with 0.5 ml of d1 supplemented with carboxymethyl cellulose (1.5 %). cultures were incubated for a further 4 days at 37 o c before plaques were revealed by staining the cell monolayers with amido black in acetic acid/methanol. purified and deglycosylated rbd and ey6a fab were combined in an approximate molar ratio of 1:1 at a concentration of 6.5 mg/ml. nb was also combined with ey6a-6his fab and rbd in a 1:1:1 molar ratio with a final concentration of 5.7 mg/ml. these two complexes were separately incubated at room temperature for one hour. initial screening of crystals was performed in crystalquick 96-well x plates (greiner bio-one) with a cartesian robot using the nanolitre sitting-drop vapour diffusion method as previously described 31, 32 . crystals were soaked in a solution containing 25% glycerol and 75% reservoir solution for a few seconds and then mounted in loops and frozen in liquid nitrogen prior to data collection. diffraction data were collected at 100 k at beamline i03 of diamond light source, uk. diffraction images of 0.1° rotation were recorded on an eiger2 xe 16m detector with an exposure time of 0.01 s per frame, beam size 80×20 µm and 100% beam transmission. data were indexed, integrated and scaled with the automated data processing program xia2-dials 33, 34 . the data set for the binary complex of 360° was collected from a single frozen crystal to 3.8 å resolution with 20-fold redundancy. the crystal belongs to space group p3121 with unit cell dimensions a = b = 166.6 å and c = 270.8 å. the structure was determined by molecular replacement with phaser 35 using search models of antibody cr3022 fab and the rbd of the rbd/cr3022 fab complex (pdb id 6yla; 12 ). there are three rbd/ey6a complexes in the crystal asymmetric unit, resulting in a crystal solvent content of ~75%. for the ternary complex, a data set of 360° rotation with data extending to 2.6 å was collected on beamline i03 of diamond with exposure time 0.008 s per 0.1° frame, beam size 80×20 µm and 100% beam transmission). the crystal also belongs to space group r3 but with unit cell dimensions (a = b = 178.1 å and c = 87.8 å). there is one rbd/ey6a/nb complex in the asymmetric unit and a solvent content of ~61%. one cycle of refmac5 36 was used to refine atomic coordinates after manual correction in coot 37 to the protein sequence from the search model. for both the binary and ternary complexes the final refinement used phenix 38 resulting in rwork = 0.219 and rfree = 0.259 for all data to 3.8 å resolution for the binary complex and to rwork = 0.216 and rfree = 0.262 for all data to 2.64 å resolution for the ternary complex. there is well ordered density for a single glycan at the glycosylation site n343 in the rbd. data collection and structure refinement statistics are given in extended data table 3 . structural comparisons used shp 39 , residues forming the rbd/fab interface were identified with pisa 40 , figures were prepared with pymol (the pymol molecular graphics system, version 1.2r3pre, schrödinger, llc). spike protein, following sec purification, was buffer exchanged into 2 mm tris ph 8.0, 200 mm nacl, 0.02 % nan3 buffer using a desalting column (zeba, thermo fisher). a final concentration of 0.18 mg/ml was incubated with ey6a fab (in the same buffer) in a 6:1 molar ratio (fab to trimeric spike) at room temperature for 5 hrs. control grids of spike alone after incubation at room temperature for 5 hrs were also prepared. each grid was prepared using 3 µl sample applied to a freshly glow-discharged on high for 20 grids were screened on a titan krios microscope using serialem operating at 300 kv (thermo fisher). movies were collected on a k3 detector on a titan krios operating at 300 kv in super resolution mode, with a calibrated super resolution pixel size of 0.415 a/pix at both 0° and 30° tilt. to compensate for the poorer contrast with tilted data, it was necessary to use a higher dose rate for the latter dataset. alignment and motion correction was performed using relion3.1's implementation of motion correction 41 , with a 5-by-5 patch-based alignment. all frames were binned by two, resulting in a final calibrated pixel size of 0.83 å/pixel. contrast-transfer-function (ctf) of full-dose and non-weighted micrographs was estimated within a cryosparc wrapper for gctf-v1.06 42 . images were then manually inspected and those with poor ctf-fits were discarded. particles were then picked by unbiased blob picking in cryosparc v.2.14.1 43 and subjected to rounds of 2d classification. for the spike-ey6a dataset (structure a), 2,096,246 spike-like particles were used to make a template to pick particles from the untilted dataset, which were then filtered by 2d classification to 110,096 particles and then further refined by 3d classification with an ab initio model set. for the 30 ° dataset, 124,194 particles were used as a template, and filtered by 2d classification to a set of 84,230 particles and then, as before, further refined by unbiased 3d classification. the two particle sets were then refined together, with a final set of 144,680 particles. for b and c (triangular ring and 'dimeric' form), particles from both the zero and 30° datasets were combined in a similar manner to the spike-ey6a dataset using the 'exposure group utilities' module in cryosparc. both particle sets (b, 41372 particles and c, 119,343 particles) were then reclassified and the best class refined with non-uniform refinement. for b, c3 symmetry was imposed at this final refinement stage, resulting in an appreciable improvement in resolution, as indicated by inspection and gold-standard fsc = 0.143 (4.7 versus 5.9 å, see extended data table 4 ). the em density of spike/ey6a was fitted with the structure of a closed form of spike (pdb id 6vxx) apart from the rbds and ey6a fab which were fitted with rbd/ey6a of the ternary crystal structure using coot 37 . due to the lower resolution, rbd and ey6a are only fitted to the 'dimeric' and 'trimeric' em density. the spike/ey6a structure was refined with phenix 38 real space refinement, first as a rigid body and then by positional and bfactor refinements. only rigid body refinement was applied to the 'dimeric' and the 'trimeric' complexes. the statistics of em data collection and structure refinement are shown in extended data table 4 these authors contributed equally: d.z., hmed, c.-p.c. ey6a binds the s1 subunit of sars-cov-2 and cross react with s1 of sars-cov-1. b, antibody cr3022 similarly binds the s1 subunit of sars-cov-2 and cross react with sars-cov-1 s1, but with lower affinity. c, convalescent serum from a covid-19 patient was also included as a control and in this case binding to mers and oc43 spike proteins also investigated. d, binding of ey6a on the sars-cov-2 infected cells in immunofluorescence assay. anti-influenza h3 mab bs 1a was included as a control. sars-cov-2 spike sars-cov-2 s1 sars-cov-1 s1 mers spike oc43 spike performed cryo-em sample preparation, screening and processing and j.raedecke performed cryo-em data collection, and j.ren refined the cryo-em structures helped prepare materials, perform experiments and analysed data. all authors read and approved the manuscript real estimates of mortality following covid-19 infection cryo-em structure of the 2019-ncov spike in the prefusion conformation. science (80-. ) cryo-em structure of the sars coronavirus spike glycoprotein in complex with its host cell receptor ace2 structure, function, and antigenicity of the sars-cov-2 spike glycoprotein dynamical asymmetry exposes 2019-ncov prefusion spike human monoclonal antibodies block the binding of sars-cov-2 spike protein to angiotensin converting enzyme 2 receptor human monoclonal antibody combination against sars coronavirus: synergy and coverage of escape mutants potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association potent cross-reactive neutralization of sars coronavirus isolates by human monoclonal antibodies human neutralizing antibodies elicited by sars-cov-2 infection global initiative on sharing all influenza data -from vision to reality neutralization of sars-cov-2 by destruction of the prefusion spike a highly conserved cryptic epitope in the receptor-binding domains of sars-cov-2 and sars-cov. science (80-. ) potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody a human monoclonal antibody blocking sars-cov-2 infection diagnostic methods in clinical virology. x article structural basis for potent neutralization of betacoronaviruses by single-domain camelid antibodies distinct conformational states of sars-cov-2 spike protein deployment of convalescent plasma for the prevention and treatment of covid-19 treatment of 5 critically ill patients with covid-19 with convalescent plasma structural basis for neutralization of sars-cov-2 and sars-cov by a potent therapeutic antibody early release-severe acute respiratory syndrome coronavirus 2−specific antibody responses in coronavirus disease identification of human single-domain antibodies against sars-cov-2 immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen the production of glycoproteins by transient expression in mammalian cells epitope-associated and specificity-focused features of ev71-neutralizing antibody repertoires from plasmablasts of infected children sequence variation among sars-cov-2 isolates in taiwan detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr isolation and rapid sharing of the 2019 novel coronavirus (sar-cov-2) from the first patient diagnosed with covid-19 in australia beitrag zur kollektiven behandlung pharmakologischer reihenversuche a procedure for setting up high-throughput nanolitre crystallization experiments. i. protocol design and validation a procedure for setting up high-throughput nanolitre crystallization experiments. crystallization workflow for initial screening, automated storage, imaging and optimization xia2: an expert system for macromolecular crystallography data reduction dials: implementation and evaluation of a new integration package phaser crystallographic software refmac5 for the refinement of macromolecular crystal structures coot: model-building tools for molecular graphics macromolecular structure determination using x-rays, neutrons and electrons: recent developments in phenix crystal structure of cat muscle pyruvate kinase at a resolution of 2.6 å inference of macromolecular assemblies from crystalline state new tools for automated high-resolution cryo-em structure determination in relion-3 real-time ctf determination and correction algorithms for rapid unsupervised cryo-em structure determination multiple ligand-protein interaction diagrams for drug discovery we acknowledge the bd facsaria™ cell sorter service provided by the core instrument the authors declare no competing interests. correspondence to david i. stuart or kuan-ying a. huang. the coordinates and structure factors of the sars-cov-2 rbd/ey6a crystallographic complexes are available from the pdb with accession codes xxx and vvv respectively. em maps and structure models are deposited in emdb and pdb with accession codes xxx and yyy for the pre-fusion spike, and xxxxx and yyyy for the dimeric complex respectively. the data that support the findings of this study are available from the corresponding authors on request. key: cord-335316-x2t5h5gu authors: madariaga, m. l. l.; guthmiller, j.; schrantz, s.; jansen, m.; christenson, c.; kumar, m.; prochaska, m.; wool, g.; durkin, a.; oh, w. h.; trockman, l.; vigneswaran, j.; keskey, r.; shaw, d. g.; dugan, h.; zheng, n.; cobb, m.; utset, h.; wang, j.; stovicek, o.; bethel, c.; matushek, s.; giurcanu, m.; beavis, k.; disabato, d.; meltzer, d.; ferguson, m.; kress, j. p.; shanmugarajah, k.; matthews, j.; fung, j.; wilson, p.; alverdy, j. c.; donington, j. title: clinical predictors of donor antibody titer and correlation with recipient antibody response in a covid-19 convalescent plasma clinical trial date: 2020-06-23 journal: nan doi: 10.1101/2020.06.21.20132944 sha: doc_id: 335316 cord_uid: x2t5h5gu background: convalescent plasma therapy for covid-19 relies on the transfer of anti-viral antibody from donors to recipients via plasma transfusion. the relationship between clinical characteristics and antibody response to covid-19 is not well defined. we investigated predictors of convalescent antibody production and quantified recipient antibody response in a convalescent plasma therapy clinical trial. methods: multivariable analysis of clinical and serological parameters in 103 confirmed covid-19 convalescent plasma donors 28 days or more following symptom resolution was performed. mixed effects regression models with piecewise linear trends were used to characterize serial antibody responses in 10 convalescent plasma recipients with severe covid-19. results: mean symptom duration of plasma donors was 11.9 and 7.8% (8/103) had been hospitalized. antibody titers ranged from 0 to 1:3,892 (anti-receptor binding domain (rbd)) and 0 to 1:3,289 (anti-spike). multivariable analysis demonstrated that higher anti-rbd and anti-spike titer were associated with increased age, hospitalization for covid-19, fever, and absence of myalgia (all p<0.05). fatigue was significantly associated with anti-rbd (p=0.03) but not anti-spike antibody titer (p=0.11). in pairwise comparison among abo blood types, ab donors had higher anti-rbd titer than o negative donors (p=0.048) and higher anti-spike titer than o negative (p=0.015) or o positive (p=0.037) donors. eight of the ten recipients were discharged, one remains on ecmo and one died on ecmo. no toxicity was associated with plasma transfusion. after excluding two ecmo patients and adjusting for donor antibody titer, recipient anti-rbd antibody titer increased on average 31% per day during the first three days post-transfusion (p=0.01) and anti-spike antibody titer by 40.3% (p=0.02). conclusion: advanced age, fever, absence of myalgia, fatigue, blood type and hospitalization were associated with higher convalescent antibody titer to covid-19. despite variability in donor titer, 80% of convalescent plasma recipients showed significant increase in antibody levels post-transfusion. a more complete understanding of the dose-response effect of plasma transfusion among covid-19 patients is needed to determine the clinical efficacy of this therapy. convalescent plasma therapy has historically been used as a treatment during epidemics (1) . in this therapy, neutralizing anti-viral antibodies, as well as non-neutralizing antibodies and other immunomodulators, are transferred via plasma transfusion from those who have recovered from disease to those currently infected (2) (3) (4) . for patients with severe covid-19, convalescent plasma therapy has safely led to improvement in clinical and radiographic parameters (5) (6) (7) (8) (9) (10) . once adequate numbers of people convalesced and supply chain logistics were established, providing plasma therapy to a large number of patients has proven feasible (11) . efficacy of convalescent plasma therapy relies on a robust antibody response in convalescent plasma donors. measurements of antibody response among patients with covid-19 demonstrate that the majority develop igm and igg within 2 weeks of symptom onset, with specificity towards receptor binding domain (rbd) and spike protein viral epitopes correlating with virus neutralization (12) (13) (14) . strikingly, a small proportion of recovered covid-19 patients show no detectable antibodies to these epitopes (12, 15) . the relationship between host characteristics, disease course and variability in antibody response to covid-19 is poorly understood. the aim of this study was to establish a translational convalescent plasma program to investigate the relationship between clinical and serological parameters in convalescent plasma donors and define the antibody response of convalescent plasma recipients. this was a prospective open label clinical study to assess the feasibility, safety and immunological impact of delivering anti-sars-cov-2 convalescent plasma to hospitalized patients aged 18 years or older with severe or life-threatening covid-19 disease within 21 days from the onset of their illness. this study was conducted at university of chicago medicine (ucm) from april 10, 2020 to may 17, 2020 . the final date of follow-up was may 25, 2020. we used existing hospital infrastructure and personnel to build the convalescent plasma program at a time when state-wide shelter-in-place orders were active, elective procedures were not being performed, and non-covid-19-related research activities were halted. the donor enrollment team consisted of two surgeons, two surgical residents, and three physician assistants. a dedicated study coordinator was present at the ucm blood donation center to facilitate whole blood donation and collect research samples. recipients were selected during daily videoconference with infectious disease. one surgeon visited the hospital covid-19 unit daily to obtain consent and research samples. plasma donors were age 18 or older, able to donate blood per standard ucm blood donation center guidelines, had a documented covid-19 polymerase chain reaction (pcr) positive test, and complete resolution of symptoms at least 28 days prior to donation. recruitment occurred via social media, news outlets, word-of-mouth and announcements in university and community bulletins. the ucm infectious disease team provided an institutional list of patients with a positive pcr test for covid-19, and their physicians were emailed to request permission to contact the patient for donor participation. interested plasma donors were directed to fill out a short screening survey online. potential donors meeting study criteria were screened for eligibility, reported symptoms and comorbidities, consented, and were scheduled for donation at the ucm blood donation center in a single telephone encounter. after meeting the ucm blood donation center eligibility, whole blood was collected and processed according to standard ucm blood donation center procedures. standard whole blood donation was used for plasma collection because it fit into preexisting ucm blood bank infrastructure and workflow therefore facilitating rapid deployment of a collection process, and allowed red blood cell and unused plasma units to be used in the regular blood bank inventory. during blood donation, a single research sample was collected at the same time as blood samples for standard immunohematology testing and infectious disease screening. leukocyte filters used in separation of constituent blood parts were also collected for research. eligibility for convalescent plasma recipients included: age 18 or older, laboratoryconfirmed covid-19, within 21 days from the start of illness and severe or life-threatening covid-19 as defined by the united states food and drug administration (fda) (16). severe covid-19 was defined as dyspnea, respiratory frequency ≥ 30/min, blood oxygen saturation ≤ 93%, partial pressure of arterial oxygen to fraction of inspired oxygen ratio < 300, and/or lung infiltrates > 50% within 24 to 48 hours. life-threatening covid-19 was defined as respiratory failure, septic shock, and/or multiple organ dysfunction or failure. patients who were pregnant, received pooled immunoglobulin in the past 30 days or had a history of transfusion reaction were excluded from this study. recipients had routine pre-transfusion testing, in keeping with institution policies. on the day of enrollment, an emergency investigational new drug (eind) application was filed and approved for each recipient by the fda (16). subsequently, one abo-compatible unit of convalescent plasma (~300 ml) was transfused over 4 hours. repeat administration of convalescent plasma occurred in one recipient (r7). blood samples and nasopharyngeal swabs were obtained at day 0, 1, 3, 7, 14 post transfusion. the primary outcome was feasibility as defined by the collection of convalescent plasma and its administration into hospitalized patients. secondary outcomes included type and duration of respiratory support, cardiac arrest, transfer to intensive care unit (icu), length of stay, mortality, complications of plasma administration, process outcomes, and antibody titer of plasma donors and recipients. levels of anti-rbd and anti-spike antibodies were measured by enzyme-linked immunosorbent assay (elisa) in blood samples at time of donation and plasma recipients, as previously described (17) . nasopharyngeal specimens were obtained by flocked swabs in plasma recipients and analyzed by rt-pcr to detect sars-cov-2 rna. study data were collected and managed using redcap electronic data capture tools hosted at ucm (18, 19) . donor patient characteristics were compared using the chi-squared test for categorical variables and the two-sample t test for continuous variables. univariate regression analysis for antibody titer (anti-rbd and anti-spike) was conducted against age, sex, body mass index (bmi), previous pregnancy, previous blood donation, blood type, symptoms (fever, cough, sore throat, dyspnea, abdominal pain, aguesia, anosmia, fatigue, myalgia, headache), co-morbidities (respiratory, cardiovascular, renal, diabetes, autoimmune disease, cancer, liver disease), smoking history, travel in the past 3 months to the united states, asia or europe, symptom duration, interval from symptoms resolution to plasma donation, and hospitalization. pairwise comparison using t tests without adjusting for multiple comparisons was used to compare antibody titers among different abo blood groups. we conducted multivariable analyses to identify prediction models for anti-rbd and anti-spike antibody titers among convalescent plasma donors. best subset variable selection method was chosen to identify the subset of predictors that maximizes the adjusted r-squared among all possible models. to compare daily change in recipient antibody response, we fit mixed effects regression models with piecewise linear trend with a change point at 3 days after intervention for log-transformed antibody titers. we considered recipients on extra-corporeal membrane oxygenation (ecmo) (r3 and r6) separately from recipients not on ecmo (r1, 2, 4, 5, 7, 8, 9, 10), because ecmo recipients had different baseline characteristics. data analysis was performed using software r, version 3.6.3. mixed effects regression models were fit using the lmer function of the lme4 package (20) . data analysis was conducted within rstudio environment, and r markdown files with fully reproducible data analysis can be obtained from the authors upon request. this study was approved by the institutional review board (irb20-0523). all participants (plasma donors and plasma recipients) gave written informed consent prior to inclusion in the study. analysis was performed by mlm and mg. this clinical trial was registered at clinicaltrials.gov with identifier nct04340050. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. . https://doi.org/10.1101/2020.06.21.20132944 doi: medrxiv preprint 697 potential plasma donors were recruited to our study over 35 days (table 1 ). the average age was 43.5 years (range 18 to 87), the majority were female (63.1%), and 37% had never donated blood before. potential donors with confirmed positive covid-19 pcr (n=384, 55%) were more likely to be male, have ageusia and anosmia, and lack cough, sore throat and dyspnea compared to the 313 symptomatic patients who had clinical signs of covid-19 but were never tested (table 1) . among plasma donors (n=103) who donated as of publication, average symptom duration was 11.9±5.91 days, 9 (8.7%) had respiratory comorbidities such as asthma, chronic obstructive pulmonary disease or obstructive sleep apnea, and 8 (7.8%) had been previously hospitalized for covid-19 ( table 1 ). the average interval between symptom start and plasma donation was 45.1±8.02 days. donor antibody titers measured on day of plasma donation ranged from 0 to 1:3,892 (anti-rbd) and from 0 to 1:3,288.7 (anti-spike) ( table 1 ). in univariable regression analysis, higher average anti-rbd and anti-spike antibody titers were associated with plasma donors who were older, male, had higher bmi, had fever and had been hospitalized (p<0.05, supplemental table 1 ). in a pairwise comparison among abo groups without adjusting for multiple comparisons, ab donors had higher anti-rbd titer than o negative donors (p=0.048) and higher anti-spike titer than o negative (p=0.015) or o positive (p=0.037) donors. to determine predictors of anti-rbd and anti-spike antibody titer, we performed best subset multivariable analysis including age, sex, blood type, history of previous blood donation, fever, cough, fatigue, myalgia, symptom duration, hospitalization and travel in the united states within the past 3 months. significant predictors of anti-rbd antibody titer were age (p=0.02), fever (p<0.01), previous hospitalization (p<0.01), lack of myalgia (p=0.01), and fatigue (p=0.03) (r-squared=0.40, adjusted r-squared=0.32, table 2 ). significant predictors of anti-spike antibody titer were age (p=0.02), fever (p=0.01), previous hospitalization (p=0.01), and absence of myalgia (p<0.01) (r-squared=0.35, adjusted r-squared=0.26, table 2 ). o positive blood type was associated with lower anti-rbd (p=0.05) but did not meet significance threshold for antispike (p=0.07). ten hospitalized patients with severe or life-threatening covid-19 received plasma on day 0 ( figure 1 , table 3 ). plasma recipients were on average 61.9 years old (range 30 to 86) and 40% female. the average time from start of symptoms to plasma transfusion was 12 days (range 2 to 21) and the average time from hospital admission to plasma transfusion was 6 days (range 2 to 17). at the time of plasma transfusion, two patients were on ecmo, one patient was mechanically ventilated, two patients were on high-flow nasal cannula (hfnc), four patients were on nasal cannula and one patient was on room air. five patients had received other therapies for covid-19 before transfusion, including remdesivir, tocilizumab, anakinra and hydroxychloroquine. two plasma recipients were on chronic immunosuppression after transplantation. figure 2 shows selected clinical and laboratory parameters of convalescent plasma recipients. only one recipient (r8) had fever prior to transfusion and this resolved by day 3 post-transfusion. r3 and r6 remained on ecmo throughout the study period. in the remaining 8 recipients, oxygen requirements improved to room air or nasal cannula. the sequential organ failure assessment (sofa) score (21) was calculated for recipients on mechanical ventilation or ecmo and showed a general trend towards improvement; notably both ecmo patients were weaned off vasopressor and intra-aortic balloon pump support by 7 days post-transfusion. levels of inflammatory marker c-reactive protein (crp) were variable. crp decreased in six recipients (r1, r2, r5, r6, r9, r10). sars-cov2 np swab pcr remained positive in 5 patients and turned negative in 4 patients; 1 patient (r6) had been positive for sars-cov2 17 days prior to plasma transfusion but was negative for sars-cov2 on day of transfusion ( figure 1 ). at last follow-up, 1 patient on ecmo remained in the hospital (r6), 1 patient on ecmo was transitioned to comfort care and died on day 30 after plasma transfusion (r3), 4 patients were discharged to rehabilitation facilities and 4 patients were discharged to their place of residence ( figure 1 ). on day of transfusion, anti-rbd antibody titers were undetectable in 3 recipients (r1, r2, r10) and anti-spike antibody titers were undetectable in 3 recipients (r1, r8, r10) ( table 3 and figure 3 ). both patients on ecmo had very high antibody titer at day 0 which decreased in the days after transfusion (figure 3 ). the remaining plasma recipients showed increase in antibody titer within the first three days after transfusion (r1, 2, 4, 5, 7, 8, 9) with the exception of r10 who did not show any antibody titer until day 7 (anti-spike) and day 14 (anti-rbd) after transfusion ( figure 3 ). we performed a mixed effects model for log-transformed reciprocal antibody titer adjusting for donor antibody titer level looking at the first 3 days post-transfusion among the non-ecmo patients. after plasma transfusion, recipient anti-rbd antibody titer increased on average by 31% per day (p=0.01) and recipient anti-spike antibody titer increased on average by 40.3% per day (p=0.01) (figure 4 ). among the two ecmo recipients, recipient antibody response was not significantly changed until three days after plasma transfusion (decreasing by 9.2% per day for anti-rbd titer and 8.2% per day for anti-spike titer, p<0.01) (figure 4) . we monitored the clinical status of the recipients before, during and immediately after transfusion. no recipients experienced toxicity associated with plasma transfusion. there was no clinical deterioration or worsening of disease status immediately related to plasma transfusion. convalescent plasma transfusion was safe in high-risk individuals in our study: immunosuppressed patients after stem cell and lung transplants and a patient with end-stage renal disease on dialysis. we developed a translational convalescent plasma treatment program within the existing hospital infrastructure during the covid-19 pandemic that provided a new therapeutic option for patients while assessing the antibody profile of both convalescent and hospitalized patient populations. our multivariable analysis demonstrated that clinical characteristics can predict serological response of antibodies associated with virus neutralization (12) . higher anti-rbd and anti-spike antibody were more likely found in convalescents who were older, hospitalized, had fever, and lacked myalgia. fatigue also significantly predicted higher anti-rbd but not antispike antibody titer. variability in convalescent populations and immune response to viral infection may explain why recovery is not always marked by seroconversion (12, 15) . indeed, in our study four plasma donors (as well as four plasma recipients) had undetectable antibody titers. disparate plasma donor populations and geography may explain why symptom duration and elapsed time from symptom onset was associated with antibody response in new york city (13) but not among our patients in chicago. disparate plasma donor populations and geography may also explain antibody variability. these data highlights that the impact of variability in antibody type and titer on virus-neutralizing activity and long-term immunity is unknown. interestingly, we found that antibody titers significantly differed across abo blood type groups, (24) . further studies on the relationship between abo polymorphism and antibody titer may uncover genetic determinants of the host response to covid-19. recipients received plasma with a range of antibody titer from 1:73 to 1:3,892 (anti-rbd) and 1:69 to 1:2,921 (anti-spike). despite this, 80% of recipients demonstrated a significant increase in anti-spike and anti-rbd antibody titer in the 3 days post transfusion that was independent of donor antibody titer and were discharged after clinical improvement. interestingly, recipient antibody titer continued to increase up to 14 days in four recipients (r1, 2, 8, 10); in contrast, the two most severely ill patients on ecmo who had the highest antibody titers (up to 1:13,833 anti-spike antibody in r6) showed a decrease in antibody titer after receiving plasma on day 20-21 of illness. importantly we demonstrate the safety of transfusing convalescent plasma in immunosuppressed patients after lung transplantation and stem cell transplantation. none of the plasma recipients in this study deteriorated after convalescent plasma transfusion, consistent with the safety profile of other trials (5) (6) (7) (8) (9) 11) . repeat plasma dose in recipient r7 was also welltolerated. pre-clinical models of sars-cov and clinical experience of other viral illness had raised concern about the potential for non-neutralizing antibody to cause antibody dependent enhancement of disease, which was not seen here despite variable titers of donor antibodies (25) (26) (27) . the variability in post-transfusion recipient antibody titer and clinical response seen here and in other studies (5, 6, 28, 29) indicates that the therapeutic activity of convalescent plasma depends on the timing of treatment and composition of convalescent plasma. indeed, plasma contains more than 1,000 proteins, including albumin, immunoglobulins, complement, and coagulation factors as well as organic compounds such as cytokines (4) . convalescent plasma drawn shortly after natural infection (1, (5) (6) (7) (8) may be enriched for populations of protective antibodies not present in plasma derived from long-recovered or rarely-hospitalized donors studied here. furthermore, immunomodulatory and non-virus neutralizing antibody effects such as stimulation of the host humoral immune response and facilitating viral uptake into cells via fc-receptors to increase viral antigen presentation to other effector cells may contribute to disease recovery. taken together, while randomized controlled efficacy trials for convalescent plasma therapy in covid-19 are currently underway, establishing effective anti-covid-19 plasma-based therapy will require both an understanding of the precise dose and type of virusneutralizing antibody and in-depth characterization of plasma donor-recipient pairs. the availability of a pre-existing hospital-based blood collection facility within our medical center significantly eased the procurement of convalescent plasma and will allow us to assess immunological characteristics of donor-recipient pairs in future studies. such hospitalbased blood collection facilities have been declining in number across the united states for several decades (30) . cultivating region-specific convalescent plasma inventory may potentially facilitate the identification and isolation of antibodies with specific activity against local virus strains and be a useful model for future outbreaks. in addition, convalescent plasma derived from whole blood collection is a rapidly scalable technique that requires basic phlebotomy and blood separation rather than a dedicated apheresis personnel and equipment. furthermore, a significant proportion (36.3%) of our plasma donors had never donated blood before, indicating that a convalescent plasma donation program can serve as important community outreach during a time when patients avoid hospitals that are perceived as unsafe (31) . in summary, development of a convalescent plasma program is feasible, rapidly deployable and economical when existing resources of equipment, space and personnel are used. establishing the clinical predictors of high antibody titer and understanding the serological posttransfusion response may guide patient selection and shed light on antibody response to covid-19. further work characterizing convalescent plasma donor and recipient pairs is needed to elucidate mechanisms of convalescent plasma therapy and demonstrate optimal viral epitope therapeutic targets. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. . , interval of symptoms to plasma donation, blood type were not significantly associated with anti-rbd or anti-spike antibody titer. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. . https://doi.org/10.1101/2020.06.21.20132944 doi: medrxiv preprint treatment of influenza pneumonia by the use of convalescent human serum: preliminary report the convalescent sera option for containing covid-19 convalescent plasma as a potential therapy for covid-19 convalescent plasma in covid-19: possible mechanisms of action effectiveness of convalescent plasma therapy in severe covid-19 patients treatment of 5 critically ill patients with covid-19 with convalescent plasma treatment with convalescent plasma for critically ill patients with sars-cov-2 infection use of convalescent plasma therapy in two covid-19 patients with acute respiratory distress syndrome in korea patients with convalescent plasma in convalescent plasma treatment of severe covid-19: a matched control study early safety indicators of covid-19 convalescent plasma in 5,000 patients humoral immune response and prolonged pcr positivity in a cohort of 1343 sars-cov 2 patients in the new york city region temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019 seroconversion in humans: a detailed protocol for a serological assay, antigen production, and test setup research electronic data capture (redcap)--a metadata-driven methodology and workflow process for providing translational research informatics support the redcap consortium: building an international community of software platform partners fitting linear mixed-effects models using lme4 the sofa (sepsisrelated organ failure assessment) score to describe organ dysfunction/failure. on behalf of the working group on sepsis-related problems of the european society of intensive care medicine abo blood group and susceptibility to severe acute respiratory syndrome relationship between the abo blood group and the covid-19 susceptibility inhibition of the interaction between the sars-cov spike protein and its cellular receptor by anti-histo-blood group antibodies anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection current studies of convalescent plasma therapy for covid-19 may underestimate risk of antibody-dependent enhancement treatment with convalescent plasma for influenza a (h5n1) infection use of convalescent plasma therapy in sars patients in hong kong continued decline in blood collection and transfusion in the united states-2015 delayed access or provision of care in italy resulting from fear of covid-19 we thank all the plasma donors for their willingness to help in a time of need and the blood bank staff for their excellent care. we thank samantha guerrero, alyssa anneken, bruce boehrnsen and rohit allada for helping us establish the infrastructure for this study. we thank the university of chicago and department of surgery, university of chicago for providing support for this study. this study was funded by the department of surgery, university of chicago and the national institute of allergy and infectious diseases (niaid) collaborative influenza aka, above the knee amputation; chf, congestive heart failure; dm, diabetes mellitus; dvt, deep venous thrombosis; esrd, endstage renal disease; htn, hypertension; nafld, non-alcoholic fatty liver disease; pe, pulmonary embolism; pvd, peripheral vascular disease.all rights reserved. no reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity.the copyright holder for this prep this version posted june 23, 2020. . https://doi.org/10.1101/2020.06.21.20132944 doi: medrxiv preprint key: cord-319571-fspmgg4s authors: sehailia, moussa; chemat, smain title: antimalarial-agent artemisinin and derivatives portray more potent binding to lys353 and lys31-binding hotspots of sars-cov-2 spike protein than hydroxychloroquine: potential repurposing of artenimol for covid-19 date: 2020-07-22 journal: journal of biomolecular structure & dynamics doi: 10.1080/07391102.2020.1796809 sha: doc_id: 319571 cord_uid: fspmgg4s medicinal herbs have proved along history to be a source of multiple cures. in this paper, we demonstrate how hydroxychloroquine can act as a good inhibitor of sars-cov-2 spike protein receptor-binding-domain using molecular docking studies. we also unveil how hydroxychloroquine can interfere in the prevention of lys353 in hace2 from interacting with the corresponding binding hotspot present on the spike protein. further screening of artemisinin & derived compounds produced better vina docking score than hydroxychloroquine (-7.1 kcal mol(−1) for artelinic acid vs. −5.5 kcal mol(−1) for hydroxychloroquine). artesunate, artemisinin and artenimol, showed two mode of interactions with lys353 and lys31 binding hotspots of the spike protein. molecular dynamics analysis confirmed that the formed complexes are able to interact and remain stable in the active site of their respective targets. given that these molecules are effective antivirals with excellent safety track records in humans against various ailment, we recommend their potential repurposing for the treatment of sars-cov-2 patients after successful clinical studies. in addition, an extraction protocol for artemisinin from artemisia annua l. is proposed in order to cope with the potential urgent global demand. communicated by ramaswamy h. sarma the spread of covid-19 pandemic has triggered a race to unveil the secrets of severe sars-cov-2 and its underlying acute respiratory syndrome. although, functional importance of different targets has been linked to the viral replication and maturation of coronaviruses' family such as chymotrypsin-like protease(3clpro) or known as mpro (khan et al., 2020; muralidharan et al., 2020) or envelope protein (e) (gupta et al., 2020; boopathi et al., 2020) but it has been confirmed that the binding of the viral trimeric surface spike glycoprotein (sprotein) of sars-cov-2 to the human receptor angiotensin-converting enzyme 2 (hace2) is the first step in host infection . in fact, vankadari and wilce (2020) revealed that s1 domains of the spike protein has an open conformation, enabling it to interact with target host proteins. yan et al. (2020) identified the structure of hace2 as a dimer in complex with membrane protein, they also showed that the two trimeric sproteins of the receptor binding domain (rbd) of sars-cov-2 bind very tightly to the hace2 dimer. the latter is activated by a specific cellular enzyme called furin (hasan et al., 2020) . medicinal plants reap an important source of complex active molecules that have been proposed, and sometimes traditionally used for the treatment of several pathologies, such as cancer, autoimmune diseases, and infectious diseases. the scientific community are relying on different modes and various mechanisms of action these molecules hold to halt sars-cov-2 severity. recently, remdesivir and chloroquine have been proved to inhibit in vitro vero e6 cells of emerged novel coronavirus (2019-ncov) . chloroquine has been envisaged for sars-cov-1 (de clercq, 2006) , and has long been used for malaria therapy but has been replaced with hydroxychloroquine (hcq) due to the increased plasmodium falciparium parasite resistance, whereas an overdose of cq can cause acute poisoning and death (weniger, 1979) . they have shown similar activities in in-vivo essays against malaria parasite but accompanied by an increased risk of retinopathy (schrezenmeier & d€ orner, 2020) . nonetheless, hcq is considered safer than cq as the later has mediated cardiotoxic effects including rhythm disorders, and propels the development of cardiomyopathy in patients with rheumatic diseases (schrezenmeier & d€ orner, 2020) . for its turn, hcq has been found to be effective in inhibiting sars-cov-2 infection in vitro and attenuate inflammatory response . organic extracts of artemisia annua l. have been found to be more effective, faster, and less toxic than cq and hcq in treating malaria. a. annua contains a vital compound known as artemisinin, a sesquiterpene lactone with a peroxide linkage exhibiting low toxicity (table 1) , also the parent compound for semisynthetic derivatives chemically modified at the c-10 position to produce artesunate, artemether, arteether, artenimol (dihydroartemisinin), and artelinic acid (table 1) . these compounds, and in some cases their sodium salts, have been formulated as antimalarials for oral, rectal, and parenteral administration (woodrow et al., 2005) . several reports proved the efficiency of artemisinin derivatives as potent antivirals for hpv bovine viral diarrhea virus (bvdv) for the treatment of anal and cervical intraepithelial high-grade neoplasia, human herpes virus-6 (hhv-6), human immunodeficiency virus (hiv) and more particularly, artesunate, against human cytomegalovirus (hcmv) (d'alessandro et al., 2020) . this antiviral potency put artemisinin class of compounds as promising candidates for the treatment of patients suffering from sars-cov-2 virus. the encouraging results generated from the utilization of hcq to treat patients suffering from covid-19 pandemic further raises many questions surrounding its mode of action (million et al., 2020) . at the cellular level, direct and indirect mechanisms of cq and hcq are believed to inhibit immune activation by reducing toll-like receptor signaling and cytokine production and, in t cells, reducing cd154 expression (schrezenmeier & d€ orner, 2020) ; however, the absence of binding assay studies between the sprotein and hace2 protein in the presence of hcq opens the door to two main possibilities (vincent et al., 2005) : the first possibility revolves around hcq prevention of terminal glycosylation of hace2 protein which consequently impacts the final attachment between the sprotein and hace2 protein, whereas the second possibility revolves around hcq interaction with the receptor binding domain (rbd) of sprotein, thus preventing its docking on hace2 receptor. to further expand on the second possibility, we elected to perform computational studies based on molecular docking to help us understand more about the mode of interaction between hcq and the rbd of sars-cov-2 sprotein, and eventually, how such interaction prevents the sprotein from docking on the hace2. another class of antimalarial and antiviral molecules comprised of artemisinin and artemisinin derived compounds are investigated to reveal how effective these molecules act with binding sites of sprotein, then ultimately how their mode of interaction occur. this study aims to propose also an extraction protocol for artemisinin from artemesia annua l. in order to cope with the potential urgent global demand. the pdb file of sars-cov-2 sprotein rbd-hace2 complex (pdb ref. 6lzg, version 1.0) was obtained from the research collaboratory for structural bioinformatics (rcsb) protein data bank (pdb) (http://www.rcsb.org/structure/6lzg). ucsf chimera1.14 was used to visualise the structure of the ligand and/or protein-complex structure, to perform the various functions associated with ligand and protein preparations, and acting as an interface to enable molecular docking calculations using locally hosted autodock vina software (pettersen et al., 2004; trott & olson, 2010) . prior to molecular docking, the hace2 protein (part a, protein section coloured in greenfigure 2 ) was deleted from the pdb file of the complex. in addition, all non-standard residues including that of water were also removed. the structure of each ligand was incorporated into ucsf chimera using smiles string followed by structure minimisation. the pdbqt files of the sprotein rbd and each ligand was generated after adding all hydrogens and charges to each structure. the number of binding modes was set to 10 with exhaustiveness of search set to 8. the maximum energy difference was set to 3 kcal mol à1 . the best scoring pose for each molecule was analysed in terms of its interaction with the receptor binding motif (rbm). the x, y, z coordinates of the grid box covered the full area of the receptor binding motif of the sprotein, in our case, for center (x ¼ à40.34, y ¼ 27.28, z ¼ à7.72) whereas for size (x ¼ 24.05, y ¼ 51.38, z ¼ 21.79). in the case of the receptor, all hydrogen atoms were added to the structure, charges were merged and nonpolar hydrogen were removed; water molecules and side chains of non-standard residues were also ignored. for the studied ligands, charges were merged and non-polar hydrogens were also removed. the obtained molecular docking results were then aligned with the pdbqt file of the sprotein rbd-hace2 complex in order to visualise the type of interactions of each docked molecule in the sprotein-hace2 binding interface. computations were performed at the al-farabi cluster computer of the ecole nationale polytechnique oran -maurice audin (algeria). all molecular dynamics (md) studies are performed using groningen machine for chemical simulations (gromacs) which is a software package designed to perform md simulations of proteins, lipids and nucleic acids. amber force field was utilised during the parameterisation of each protein complex system followed by solvation with tip3p water molecules; water.tip3p force field in a cubic periodic box with 10 å spacing protein-box edge was applied. na þ ions were introduced to neutralise the overall charge. further energy minimisation was performed using steepest descent and conjugate gradient algorithms. the system was subjected to 100 ns md at 300 k and pressure of 1 bar, the latter value was maintained using berendsen barostat. the generated trajectory files were analysed using visual molecular dynamics (vmd) software. most explanations associated with hcq mode of action are based on findings revolving around the mode of action of cq on sars-cov infection (simmons et al., 2004; yang et al., 2004) . amongst the cited reasons are: (i) cq can increase the value of endosomal ph which can reduce the transduction of sars-cov pseudo-type viruses (simmons et al., 2004; yang et al., 2004) , (ii) cq can raise the possibility of affecting the endosome-mediated fusion if added at the initial stage of the infection (vincent et al., 2005) , and (iii) once the virus is inside the cell, cq can inhibit the production of glycoproteins in vesicular stomatitis, thus preventing virus replication (dille & johnson, 1982) . previously, vincent et al. (2005) showed that introduction of cq prevents terminal glycosylation of ace2 receptor protein of the host cell, thus destabilizing the recognition mode of the sprotein on the surface of the virus, albeit such action did not impact the level of expression of surface hace2 proteins of the host cell. therefore, we elected to perform computational docking studies of hcq as safe cq substitute against sprotein rbd of sars-cov-2 to further study the nature of such interaction and explore its inhibition potential against sprotein. our molecular docking studies of hcq on the rbd of sars-cov-2 sprotein produced a vina score of à5.5 kcal mol à1 (table 1 ). the obtained scoring is relatively moderate given that vina score of the best molecule, i.e. physalin b, previously docked on a homology model of sars-cov-2 sprotein rbd was à7.2 kcal mol à1 (micholas & jeremy, 2020) . analysis of our docking results showed that the hydroxyl group (oh) of hcq molecule formed strong hydrogen bonding with asn501 side chain residing on one part of the receptor binding motif (rbm) of the sprotein (figure 1 ). in the sprtoein-hace2 complex, asn501 of sars-cov-2 sprotein forms favourable hydrogen bonding with tyr41 of hace2 while at the same time helps neutralising the charge of lys353 (lan et al., 2020) . therefore, the resulting hydrogen bonding between the oh group of hcq and asn501 can play a role in destabilising other interactions with hace2 residues, e.g. tyr41, lys353, gly354 and asp355, which were already found to play key roles in the interaction with the sprotein furthermore, when our initial docking results were aligned with the structure of sprotein-hace2 complex, we successfully observed significant clash between the aminoalkyl side-chain of hcq and the lys353 residue side-chain of hace2 ( figure 2 ). equally, lys353(o) was found to form one hydrogen bonding with gln502 of the sprotein in the complex as reported by lan et al. (2020) ; at the same time, lys353(n) forms important hydrogen bonding with gln496 while maintaining a salt bridge with asp38 (lan et al., 2020; yan et al., 2020) . similar to lan et al. (2020) findings, lys353 and lys31 were both found to be important hotpots in hace2 responsible for binding to the sprotein of sar-cov-2. it is believed that the latter specie developed key mutations to help stabilise and/or neutralise both lysine hotspots via introduction of asn501 (to neutralise lys353) and gln 493 & leu 455 (to neutralise lys31) of hace2 protein, thus ensuring tighter incorporation of both hotspots deep into the hydrophobic pockets of the sprotein, which would explain the main reason behind the higher affinity of sars-cov-2 sprotein to hace2 compared to that of sars-cov (lan et al., 2020) . therefore, it is very likely that selective interaction of hcq with the surface of sars-cov-2 sprotein through the formation of an inclined tape over the hydrophobic pocket responsible for hosting the lys353 hotspot (the oh group in this case is acting like a hook by forming a hydrogen bond with asn501), can be responsible for the prevention of tighter binding with hace2 protein via restricting penetration of lys353 into its finally assigned destination on the sprotein rbd (figure 2 ). similar to asn501 in sars-cov-2, thr487 in sars-cov sprotein was previously reported by shang et al. (2020) to interact with tyr41, lys353, gly354 and asp355 in hace2 protein. we advocate here that similarity in the hydrogen bond networking system between both types of sproteins (i.e. that of sars-cov and sars-cov-2) and that of hace2 may be used to explain the supposed efficacy of hcq in inhibiting sars-cov and sars-cov-2 interaction with hace2. on the other hand, our molecular docking results also showed good interaction between the quinoline aromatic ring in hcq and his34 in hace2; in normal circumstances, the latter residue interacts well with leu455 and gln493 of the sprotein. by doing that, hcq can also disturb interaction in the middle region of the binding interface between the sprotein and hace2 (figure 2) . previously, samarth and kirk (2020) studied the interaction of hydroxychloroquine/azithromycin with sars-cov-2 sprotein-hace2 complex using a virtualised quantum mechanical modelling approach. in agreement with our study, the authors found that hcq had low potency of interaction with the studied complex compared to azithromycin; they also recommended molecular docking studies to further strengthen their rationale. our approach to study the interaction of molecules with the receptor binding motif (rbm) of the sprotein prior to complexation with hace2 has helped properly analyse the type of interaction with the sprotein, and how such interaction could prevent hace2 from docking onto the rbm of sprotein. in addition, we successfully addressed some of the aspects surrounding the mechanism of action of hcq and artemisinin derivatives in preventing the interaction between the virus' sprotein and hace2 receptor via selectively interacting with the lys353 binding hotspot of sprotein. with this information on hand, we then elected to perform in-silico screening of other potent antimalarial compounds derived from the core structure of artemisinin; by doing so, we believe we can gain more insight about the potential use of such class of compounds as safer and more potent substitutes to hcq. in this regard, a total number of 11 compounds were successfully screened against sprotein rbd using the same molecular docking approach previously followed with hcq. the obtained results are shown in table 1 . analyses of the data show that artemisinin and its derivative compounds have scored better than hcq, with compounds in entry 10 & 11 of table 1 producing the least and closest vina score (-6.0 kcal mol à1 ) to hcq. on the other hand, artelinic acid (table 1 , entry 1) gave the best vina score of à7.1 kcal mol à1 ; however, this compound was discarded due to its high toxicity levels (li et al., 2007) . although artemisinin and its derivative compounds resulted in good vina scoring (-6.0 score 7.1 kcal mol à1 ), only those approved and prescribed as antimalarials were selected namely, artesunate (table 1 , entry 2), artemisinin (table 1 , entry 4) and artenimol (table 1, entry 6). these were found to possess good clinical records with very promising antiviral properties, thus enhancing their potential to be repurposed for the treatments of sars-cov-2. artesunate (table 1 , entry 2) with vina score of à6.8 kcal mol à1 . the calculated inhibition constant (ki) of each top scoring pose is also reported in table 1 . analysis of the data shows that artemisinin class of compounds possess much lower ki than hcq, thus enabling them to become good antiviral candidates against sars-cov-2. the elevated ki value of hcq also reflects the high cytotoxic concentration of hcq (cc 50 > 100 mm at multiplicity of infection (moi) ¼ 0.05) required to eradicate the virus during in-vitro assay studies as previously reported by liu et al. (2020) . besides its antiviral activity, artemisinin derivatives hold immunoregulatory properties and modulate neutrophils, t-cell and b-cell components of the immune system (yao et al., 2016) , thus enhancing system immunity and touting themselves as promising candidates to synergistically enhance their antiviral effect in vivo and treat inflammation-associated diseases (li et al., 2007) . the nature of interaction between the aforementioned three compounds and the rbd of sars-cov-2 sprotein was also analysed in order to see which molecule best influence the repulsion of hace2 lys353 and lys31 from binding to the inner hydrophobic pockets of the sprotein. by analysing the top scoring pose of artesunate (table 1 , entry 2), we observe that the molecule binds far away from the hydrophobic regions of lys353 and lys31 hotspot binding sites (figure 3) . furthermore, upon alignment of artesunate docking results with the structure of sprotein hace2 complex, no clashes were observed between artesunate structure and the other side-chains present in the hace2 protein. however, the carboxylic acid moiety of the artesunate side chain was observed to form hydrogen bonding with lys353(n), which can further neutralise the overall charge through the formation of a salt bridge, this can adversely lead to tighter interaction between hace2 and sprotein. therefore, in spite of the high vina scoring associated with artesunate, we predict that this molecule is unlikely to act as a good inhibitor, in its current form, to sars-cov-2 sprotein (figure 4) . in the case of artemisinin's (table 1 , entry 4) top pose, despite no hydrogen bonding is observed with the sprotein rbd, we notice a lateral incorporation of the six-membered ring cyclohexane group of artemisinin into the lys353 hotspot binding pocket, with the peroxy-bridge facing the peripheral hydrophilic surface of the binding region( figure 5 (a) , region coloured in blue next to the peroxy-bridge). such mode of interaction could well be used to prevent the penetration of lys353 side-chain into the hydrophobic pocket ( figure 5 ). artemisinin lateral penetration into these binding hotspots may reduce the random motion of a-helix and loops present in sprotein and its capability to attach with hac2 as hypothesized by gupta et al. (2020) where binding with phytochemicals reduces their motion present in the envelope (e) protein of sars-cov2, therefore inhibiting a modulation of ion channel activity and stop the pathogenesis caused via sars-cov2. artemisinin was also found to interact with lys31 hotspot binding pocket, although at slightly lower vina score (-5.6 kcal mol à1 ). the average score is perhaps attributed to the absence of hydrophilic surfaces close to the binding pocket ( figure 6) . therefore, the selective interaction of artemisinin with both lys353 and lys31 hotspot binding regions raises its possibility to be repurposed for the treatment of sars-cov-2 patients following successful clinical trials. artemisinin has better tolerance on human hepatoma cell line hepg2 with cc50 of 160 mm than artesunate cc50 of 20 mm (romero et al., 2005) . additionally, in vitro cytoxicity levels of artesunate against human epithelial cells (hela cells) and human foreskin fibroblasts (hff cells) report very low tolerance with cc50 of 71.7 mm and 7.1 mm respectively (he et al., 2013) . artenimol on the other hand showed a similar mode of interaction to that of artemisinin with both lys353 and lys31 hotspot binding sites, although at slightly lower vina scores of à6.4 and à5.4 kcal mol à1 , respectively (figure 7) . both artemisinin and artesunate are susceptible to cyp450 reduction to generate artenimol once incorporated into the human body, albeit at different conversion rates. we therefore recommend that artenimol can be prioritised for clinical trials to achieve the repurposing of such class of molecules for covid-19, however, careful considerations need to be taken into account given the water solubility characteristics of each compound (woodrow et al., 2005) . successful completion of md calculations permitted us to obtain two crucial graphs, i.e. that of root-mean square deviation (rmsd) vs. time (figure 8 ) and the root-mean square fluctuation (rmsf) of each residue in the three protein complexes, i.e. that of the sprotein rbm in complexation with hcq, artemisinin or artenimol (figure 9 ). rmsd represents a measure of the average change in the displacement of an atom for a particular frame compared to a reference frame. on the other hand, rmsf measures the local changes of each residue in the protein backbone. figure 8 indicates a good protein-ligand stability for all three complexes, with hcq protein complex showing the lowest rmsd value (0.22 å) followed by artenimol-protein complex (0.24 å) and artemisinin-protein complex (0.26 å). similarly, artemisinin-protein complex showed the highest deviation 0.4 å followed by artenimol-protein complex 0.37 å and hcq-protein complex 0.34 å. those values imply the currently, major production of artemisinin is based on solvent extraction from a. annua despite modest but not scalable enough trials to produce it chemically or semisynthetically via its precursor artemisinic acid in engineered bacteria (hale et al., 2007; dietrich et al., 2009 ). artemisinin is abundant in a. annua leaves (0.4-1.4%) and production includes several steps starting with screening and drying before biomass being processed generally via solvent leaching or percolation at 30-50 c using low polarity solvents like hexane, toluene or petroleum (chemat et al., 2019) . this operation is not selective for artemisinin, therefore terpenes, fatty acids and some pigments are inevitably co-extracted which calls for secondary refinery steps including adsorption, flash chromatography and sequential crystallization. herein, an indicative facile production setup is proposed to enhance worldwide production capacity ( figure 10 ). however, a fireproof equipment and facility is a pre-requisite to ensure safety and security measures are met. the plant comprises an extraction step (1) in which biomass is placed in hollow-fibre bags and processed at 40 c for 60 min using a solvent mixture of hexane/ethyl acetate (95:5 v/v) with solvent/biomass ratio of 6 to 1 (6 l for each kg) (chemat et al., 2017) . this step can be conducted by means of a stirring tank or a percolation type reactor to 1: extraction reactor; 2: frame and plate filter-press (or vibrating-screener/decanter); 3: adsorption column bed; 4: clarification column bed; 5: crystallization stirred reactor; 6: spray dryer; 7: distillation column; 8: solvent storage tank ensure up to 99% extraction yield is achieved. then, the mixture passes through a cloth or diaphragm plate and frame filter-press (2) in order to discard fine biomass particles and recover the extract. the latter is submitted to an adsorption bed column (3) filled with activated carbon aiming at the removal of pigments and tannins. another clarification stage is required to remove other impurities such as free fatty acids and pigments; for instance, an adsorption bed column (4) filled with celite 545 (merck) is recommended (chemat et al., 2017) . due to some affinity with activated carbon and celite 545, an artemisinin loss of 3-5% is expected. after that, the purified extract should be concentrated to at least 1/8 th its initial volume using an evaporator. the concentrated extract is submitted into a jacketed crystallization reactor (6) equipped with a stirring shaft set at tip-speed in the range of 100-150 rpm to control breakage effects and to generate a narrow particle size distribution (huter et al., 2018) . the cooling rate is set to 0.5 c/min to reach 4 c and is kept at this temperature for 60 min to let artemisinin crystals settle down. the crystals are sent into a spray drying system (7) to recover high purity crystals of 98-99% as a final product. the overall yield of artemisinin is expected to reach 60% from the initial content of artemisinin in a. annua leaves. the spent mother liquor is guided for another row of crystallization with longer residence time. the recovered crude crystals are washed with cold ethanol to recover purer artemisinin and increase the final yield. the inhibition of sars-cov-2 sprotein rbd with hcq was successfully studied using molecular docking techniques. hcq was found to selectively interact with the lys353 hotspot binding pocket via the formation of an inclined tape over the binding site with the oh group of hcq acting like a hook. artemisinin class of compounds were also found to interact the same binding pocket. in addition, artemisinin & derived molecules showed extra mode of interaction with the lys31 binding hotspot, although at slightly lower vina score. molecular dynamics studies confirmed that the formed complexes are able to interact and remain stable in the active site of their respective targets. these results demonstrate the likelihood of repurposing artemisinin as a less toxic substitute of hcq to block the sprotein rbd of the virus from docking onto hace2, while at the same time enhancing the immune system of the patient. more focus should be intended to study the in-vivo mode of action of artenimol as most artemisinin derivatives are converted to this compound once incorporated to the body. novel 2019 coronavirus structure, mechanism of action, antiviral drug promises and rule out against its treatment extraction mechanism of ultrasound assisted extraction and its effect on higher yielding and purity of artemisinin crystals from artemisia annua l. leaves biosynthesis of spathulenol and camphor stand as a competitive route to artemisinin production as revealed by a new chemometric convergence approach based on nine locations' field-grown artemisia annua l the use of antimalarial drugs against viral infection. microorganisms potential antivirals and antiviral strategies against sars coronavirus infections a novel semi-biosynthetic route for artemisinin production using engineered substrate-promiscuous p450(bm3) inhibition of vesicular stomatitis virus glycoprotein expression by chloroquine in-silico approaches to detect inhibitors of the human severe acute respiratory syndrome coronavirus envelope protein ion channel microbially derived artemisinin: a biotechnology solution to the global problem of access to affordable antimalarial drugs a review on the cleavage priming of the spike protein on coronavirus by angiotensin-converting enzyme-2 and furin unique and highly selective anticytomegalovirus activities of artemisinin-derived dimer diphenyl phosphate stem from combination of dimer unit and a diphenyl phosphate moiety systematic and model-assisted process design for the extraction and purification of artemisinin from artemisia annua l identification of chymotrypsin-like protease inhibitors of sars-cov-2 via integrated computational approach structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor toxicity evaluation of artesunate and artelinate in plasmodium berghei-infected and uninfected rats hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro repurposing therapeutics for covid-19: supercomputer-based docking to the sars-cov-2 viral spike protein and viral spike protein-human ace2 interface early treatment of covid-19 patients with hydroxychloroquine and azithromycin: a retrospective analysis of 1061 cases in marseille computational studies of drug repurposing and synergism of lopinavir, oseltamivir and ritonavir binding with sars-cov-2 protease against covid-19 ucsf chimera-a visualization system for exploratory research and analysis potential inhibitors of the enzyme acetylcholinesterase and juvenile hormone with insecticidal activity: study of the binding mode via docking and molecular dynamics simulations effect of artemisinin/artesunate as inhibitors of hepatitis b virus production in an "in vitro" replicative system energetics based modeling of hydroxychloroquine and azithromycin binding to the sars-cov-2 spike (s)protein -ace2 complex mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology structural basis of receptor recognition by sars-cov-2 characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading emerging wuhan (covid-19) coronavirus: glycan shield and structure prediction of spike glycoprotein and its interaction with human cd26. emerging microbes & infections chloroquine is a potent inhibitor of sars coronavirus infection and spread remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro review of side effects and toxicity of chloroquine. the bulletin of the world health organization structural basis for the recognition of sars-cov-2 by full-length human ace2 ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign immunomodulation of artemisinin and its derivatives a pneumonia outbreak associated with a new coronavirus of probable bat origin the authors would like to acknowledge the support of the directorate general of scientific research and technological development of the ministry of high education and scientific research in algeria. we are thankful to dr. djamila benrezkallah (djillali liabes university of sidi bel abbes, algeria) and al-farabi cluster computer of the ecole nationale polytechnique oran -maurice audin for running the md computations and insightful analysis of the molecular dynamics calculation. no potential conflict of interest was reported by the author(s). http://orcid.org/0000-0003-2123-9603 key: cord-338538-uea9kwge authors: shehata, mahmoud m.; mostafa, ahmed; teubner, lisa; mahmoud, sara h.; kandeil, ahmed; elshesheny, rabeh; boubak, thamer a.; frantz, renate; pietra, luigi la; pleschka, stephan; osman, ahmed; kayali, ghazi; chakraborty, trinad; ali, mohamed a.; mraheil, mobarak abu title: bacterial outer membrane vesicles (omvs)-based dual vaccine for influenza a h1n1 virus and mers-cov date: 2019-05-28 journal: vaccines (basel) doi: 10.3390/vaccines7020046 sha: doc_id: 338538 cord_uid: uea9kwge vaccination is the most functional medical intervention to prophylactically control severe diseases caused by human-to-human or animal-to-human transmissible viral pathogens. annually, seasonal influenza epidemics attack human populations leading to 290–650 thousand deaths/year worldwide. recently, a novel middle east respiratory syndrome coronavirus emerged. together, those two viruses present a significant public health burden in areas where they circulate. herein, we generated a bacterial outer membrane vesicles (omvs)-based vaccine presenting the antigenic stable chimeric fusion protein of the h1-type haemagglutinin (ha) of the pandemic influenza a virus (h1n1) strain from 2009 (h1n1pdm09) and the receptor binding domain (rbd) of the middle east respiratory syndrome coronavirus (mers-cov) (omvs-h1/rbd). our results showed that the chimeric antigen could induce specific neutralizing antibodies against both strains leading to protection of immunized mice against h1n1pdm09 and efficient neutralization of mers-cov. this study demonstrate that omvs-based vaccines presenting viral antigens provide a safe and reliable approach to protect against two different viral infections. acute respiratory infections are among the leading causes of disease and mortality in developing and developed countries [1, 2] . the severity of these acute infections is usually potentiated following the dissemination of the infection throughout the lower respiratory tract, leading to millions of human deaths worldwide each year [3] . annually, seasonal influenza epidemics attack 10-20% of the human population leading to 290-650 thousand deaths/year worldwide [4] . beside these epidemics, the world is confronted every 10-40 years with antigenically distinct pandemic influenza virus strains of wide geographical distribution and considerable human-to-human transmissibility resulting in high mortality rates [5] . in recent years, the world has been challenged with newly emerging influenza a virus (iav) infections, which have the potential to cause sporadic fatalities in the human population within limited epidemics. for instance, highly pathogenic avian influenza viruses (hpaiv) of the h5n1-subtype and low pathogenic avian influenza viruses (lpaiv) of the h7n9-, h10n8-and h6n1-subtypes [6] [7] [8] [9] have caused sporadic human infections. in addition, other iavs caused global pandemic outbreaks, such as the 2009 swine-origin h1n1 influenza virus (h1n1pdm09) [10, 11] . in 2012, a novel middle east respiratory syndrome coronavirus (mers-cov) emerged. by february 2019, a total of 2374 laboratory-confirmed human cases, including 823 associated deaths, were reported globally in 27 countries (case-fatality rate: 35.4%) [12] . the majority of these cases were reported from the arabian peninsula, specifically saudi arabia (cases = 1896; deaths = 732; case-fatality rate = 38.6%) [12] . to combat iav and mers-cov infections, vaccination represents an affordable and a facile way to protect against devastating epidemics and occasional pandemics. however, despite significant efforts to develop a safe and effective vaccine [13] , there are no approved vaccines for mers-cov till now. recent reports have also demonstrated that replication of recombinant iav vaccine strains in either embryonated eggs or in cell-culture systems allows viral adaptation, which may affect the antigenicity of the vaccine [14] [15] [16] . therefore, genetically and phenotypically stable vaccines represent a promising alternative to control iav and mers-cov infections [14] . outer membrane vesicles (omvs) are natural, spherical nanoparticles (50-250 nm) derived from gram-negative bacteria. omvs are released from both pathogenic and non-pathogenic bacteria and are highly immunogenic due to their components, including lipopolysaccharides (lps), bacterial outer membrane (om) proteins, lipids, immunogenic toxins, dna/rna and other periplasmatic and cytoplasmatic proteins [17, 18] . omvs from pathogenic bacteria have been commercially used to induce specific antibodies against different bacterial strains, including neisseria meningitidis serogroup b [19] . the composition of the omvs can be adapted and used as a vaccine platform via incorporation of heterologous antigens into the vesicles [20] . this engineering approach is advantageous because (i) it retains the antigens in their native conformation, (ii) it enables the omvs to target specific immune responses, and (iii) it provides multiple and commensurate protein antigens in a single production process [17] . however, bacteria-based vaccines are not well explored to deliver viral antigens. therefore, we engineered a stable omvs-based dual vaccine against h1n1pdm09 and mers-cov by producing omvs with a chimeric hemagglutinin (ha) comprising of both ha1 and ha2 from the h1n1pdm09 and the receptor binding domain (rbd) of mers-cov. mers-cov strain was isolated and grown in vero-e6 cells. the two viruses were used for preparation of the omvs-based dual vaccine and inactivated vaccines were used as positive control. influenza to construct the pmp-h1/rbd plasmid, three pcr fragments (f1, f2, and f3) encompassing (i) 5'-ncr and signal peptide of ha from cal-h1n1pdm2009, (ii) rbd of mers-cov and a 7-amino acid/peptide linker (gsagsag), (iii) the coding sequence and 3'-ncr of ha were amplified with sequence-specific primers (table 1) and phusion high-fidelity pcr master mix with hf buffer (invitrogen, carlsbad, ca) and then simultaneously ligated into linearized pmpccdb vector [21] . briefly, for the pcr amplification of each fragment, 25 µl of 2× phusion master mix, 2.5 µl of forward and reverse primers (10 µm/µl), and 50 ng of the according template dna were mixed and the reaction was then brought to a total volume of 50 µl using rnase-/dnase-free ddh 2 o. the plasmid pmp-ha-gi [21] encoding the ha of cal-h1n1pdm2009 was used as template dna for f1 and f3, while the plasmid pcdna3.1-spike-mers-cov encoding the spike protein from the isolate mers-cov/camel/egypt/hku-nrce-270/2013 was used as a template for f2. the pcr settings were: 95 • c for 1 min then 3 steps of 40 cycles (95 • c for 10 s, 56 • c for 30 s, and 72 • c for 2 min), with a final extension step at 72 • c for 10 min. the three amplified pcr fragments were then loaded onto a 1% agarose-gel for electrophoresis. separation and purification of the three specific fragments was done by using qiaquick gel purification kit according to manufacturer's (qiagen, germany) instructions. after purification the three fragments were digested by corresponding restriction enzymes, shown in table 1 . ligation of the three fragments and the linearized vector was performed using t4 dna ligase (promega, madison, wi, usa) by adding 5 µl of each purified fragment (20 ng/µl) to 2 µl 10× buffer, 2 µl t4 dna ligase, and 1 µl linearized vector (20 ng/µl). the mixture was then incubated overnight at 4 • c. transformation of escherichia coli dh5-α competent cells was performed by mixing 5 µl of the ligation reaction with 50 µl bacterial suspension (invitrogen, ca, usa) and subsequent incubation on ice for 30 min. the bacterial cells were then subjected to heat shock at 42 • c for 30 s in a water bath and were then chilled on ice for 2 min before adding 250 µl of soc media (invitrogen, ca, usa). the reaction tubes were then rotated at 250 rpm in a shaking incubator at 37 • c for 1 h. after an incubation time of 1 h, 100 µl of the transformed bacterial suspension was spread on ampicillin containing luria-bertani (lb) agar plate and incubated for 16 h at 37 • c. single colonies were then selected and incubated in 5 ml liquid lb for 16 h for subsequent plasmid isolation and the correct e. coli dh10ß competent bacteria (invitrogen, ca, usa) were transformed with pmp-h1/rbd according to manufacturer's instructions as described above with 20 ng of pmp-h1/rbd plasmid. individual colonies from ampicillin containing lb agar plate were picked and incubated in 5 ml liquid lb for 16 h. these cultures were then used to inoculate the large-volume cultures in the next step. omvs are typically purified from supernatants of transformed e. coli dh10ß cells. to this point, 2 liters (4 × 500 ml) of dh10ß cultures (inoculated with 5 ml of the starter culture) were grown in lb broth at 37 • c in an orbital shaking incubator at 180 rpm until reaching the exponential phase (od 600nm 1.0). the grown bacteria were pelleted at 6000× g for 15 min and the supernatant was sterile-filtered (millipore express plus membrane filter, pes, 0.22 µm) to remove residual bacteria. afterwards, the bacteria free supernatant was concentrated by ultrafiltration using krosflo research ii tff and a 100 kda hollow fiber membrane (spectrum labs, germany) to a final volume of 30 ml. the resulting filtrate (30 ml) was subjected to further ultracentrifugation at 150,000× g for 3 h and 4 • c in a sw41 ti rotor (beckman, ga, usa) to separate the omvs fraction. subsequently, the omvs containing pellet was resuspended in 300 µl pbs (dulbecco's phosphate buffered saline, biochrom gmbh), sterile filtered (millex-gv syringe filter unit, pvdf, 0.22 µm) and stored at −80 • c until use. the amount of isolated omvs was quantified by protein concentration measurement using bradford protein assay. enriched omvs (5 µg) from cultured dh10ß, either transformed with empty vector (control) or with pmp-h1/rbd, were mixed with 10 µl 4× sds sample buffer (40% glycerol, 240 mm tris/hcl (ph 6.8), 8% sds, 0.04% bromophenol blue, and 5% β-mercaptoethanol) and incubated for 5 min at 95 • c. the omvs samples were then separated on precast gradient nupage ® novex ® 4-12% bis-tris protein gels (invitrogen, usa) and subsequently transferred onto immobilon-fl polyvinylidene fluoride (pvdf) membranes (merck millipore). following protein transfer, the pvdf membrane was blocked using blocking buffer (1× tbs (20 mm tris-hcl, ph 7.6, 140 mm nacl) containing 5% non-fat dry milk) for 1 h at room temperature (rt). the membrane was washed once for 5 min using washing buffer (1× tbs-tween (20 mm tris-hcl, ph 7.6, 140 mm nacl, 0.05% tween20)). afterwards, detection of the viral ha1 protein was achieved using rabbit monoclonal antibodies recognizing influenza a virus hemagglutinin (abcam), diluted 1:1000 in blocking buffer. 1 h later, the membrane was washed three times for 5 min with washing buffer. next, the membranes were incubated in the dark for 1 h with the corresponding goat anti-rabbit irdye (li-cor, nebraska, usa), diluted 1:10,000 in blocking buffer containing a 1:1000 dilution of 10% sds. after three washing steps (5 min each), twice with washing buffer and once with 1× tbs, the proteins were visualized using an odyssey infrared imaging system and application software package (li-cor, nebraska, usa). the propagated virus was inactivated using 0.1% formaldehyde in 4 • c for 24 h. to ensure that there are no active viral particles following inactivation process of the inactivated control vaccines, mdck and vero e6 cells were inoculated with 100 µl of the inactivated strains. about 72 h post-inoculation, the cell-culture supernatant was then tested using either ha assay (for h1n1) or plaque assay (for mers-cov). a volume of 15 ml of the inactivated viral harvest was then carefully layered with 6 ml of 20% sucrose in an ultra-centrifugation tube and centrifuged in a sorvall mtx 150 ultracentrifuge (thermo scientific, ca, usa) at 28,000 rpm for 2 h at 4 • c. the pellets were further resuspended in 500 µl 1× pbs. the required amounts of viral antigen (µg) of each virus were mixed with imject alum adjuvant (invitrogen, ca, usa) in a ratio of 1:1 (v/v). the final antigen/adjuvant combination was continuously mixed for 30 min under cooling conditions to effectively adsorb the antigen into the surface of the adjuvant and generate optimal vaccine formulation. female balb/c mice (6-8 week-old) were reared and supplied from the animal house at the national research centre (nrc), egypt. mice were divided into 5 groups (7 mice/group). two groups of mice were intramuscularly injected with 5 µg of omvs-h1/rbd and omvs-empty. three other groups were used as controls including negative control group that was injected with sterile pbs and two positive control groups that were injected either with inactivated h1n1pdm09 or inactivated mers-cov. all animals received booster immunizations after 3 weeks. serum samples were collected at 0, 2, 4, 6, and 8 weeks after prime immunization. all mice sera were separated and stored at −20 • c until used. sera collected from immunized/control mice were treated with receptor-destroying enzyme (rde) from vibrio cholerae (denka seiken, tokyo, japan) and kept overnight at 37 • c. the rde was then inactivated by incubation at 56 • c for 1 h. diluted sera were incubated with four ha units of h1n1pdm and a 1.0% suspension of chicken red blood cells, incubated for 1 h at rt. hai titer is the reciprocal value of the dilution at which no agglutination was observed. titers <1:10 were considered as negative. plaque-reduction neutralization test (prnt) assay was performed to determine the efficacy of stimulated antibodies in sera from vaccinated/control balb/c mice to neutralize mers-cov. briefly, sera were inactivated by heating at 56 • c in a water bath for 30 min. sera were diluted two-fold serial dilution from 1:20 to 1:160 dilution in 40 µl of dmem/2% fbs. an equal amount of plaque forming unit in 40 µl dmem/2% fbs was added over sera dilutions. the serum/virus dilutions were then incubated at 37 • c for 1 h in a humidified incubator with 5% co 2 . afterwards, 50 µl of each dilution were inoculated into individual wells of 12-well tissue culture plates with confluent vero-e6 cell monolayers and incubated at 37 • c for 1 h. the plates were periodically undulated every 20 min to avoid cell drying. after 1 h of virus adsorption, inoculum was removed gently from the infected monolayer cells, washed with 1× pbs and covered with an overlay containing 1× mem media, 1% agar, 1% penicillin/streptomycin (pen/strep). the plates were left to solidify and incubated at 37 • c with 5% co 2 upside down until the formation of viral plaques were visible (3 days). the cell monolayers were then fixed with 3.4% formaldehyde solution for 1 h at rt, stained with 1% crystal violet solution (in 20% methanol) for 30 min at rt, and washed with water to visualize the plaques. the percent (%) of inhibition is calculated as following: % of plaque reduction = (virus control plaques count-sample plaques count)/(virus control plaques count) × 100 the prnt 50 is defined as the reciprocal of the antibody dilution required to reduce the number of mers-cov plaques in vero-e6 cells by 50% relative to the control wells. eight weeks after prime immunization, mice were anesthetized by intra-peritoneal injection with ketamine solution with doses adjusted to their individual body weight (2 µg/g). an infectious dose of 10 5.5 tcid 50 of influenza virus a/california/04/2009 (h1n1) wild type, was administered intra-nasally to all vaccinated and control groups. to ensure full separation between the groups and the absence of natural infection, an additional control group of five mice was added and not subjected to infection. body weight was monitored daily and mice showing a weight loss of more than 30% of their initial vaccines 2019, 7, 46 6 of 13 body weight were euthanized and recorded as dead. mice were kept under specific pathogen free (spf) conditions at the national research centre facility unit, egypt. all animal trials were conducted in accordance with the recommendations and guidelines of the egyptian animal welfare legislation. the ethics committee of the national research centre, egypt, approved the animal trial in mice (approval code: . all experiments with infectious virus were performed according to egyptian regulations for the propagation of influenza viruses. all experiments involving low pathogenic and highly pathogenic avian influenza a viruses were performed in biosafety level 2 and 3 (bsl2, 3) containment cabinets, respectively, approved for such use by the local authorities. as schematically represented in figure 1a , the pmp-h1/rbd construct comprises of the 3'-non-coding region (ncr) and the signal peptide (sp) of the ha gene from a/giessen/06/2009 (h1n1pdm2009), followed by the receptor-binding domain (rbd) of the mers-cov spike gene (rbd: 1099-1818 nt = 367-606 amino acids (aa)), a 7-aa flexible linker peptide (lp: gsagsag, [22] ) and the coding sequence plus the 5'-ncr of the ha gene. the pmp-h1/rbd is designed to link the rbd and ha0 fragments into a single polypeptide chain. the final fusion protein contains amino acid (aa) residues 1-240 of the rbd, aa residues 1-7 of the lp and aa residues 1-566 of the ha0. vaccines 2019, 7, x for peer review 6 of 13 their initial body weight were euthanized and recorded as dead. mice were kept under specific pathogen free (spf) conditions at the national research centre facility unit, egypt. all animal trials were conducted in accordance with the recommendations and guidelines of the egyptian animal welfare legislation. the ethics committee of the national research centre, egypt, approved the animal trial in mice (approval code: . all experiments with infectious virus were performed according to egyptian regulations for the propagation of influenza viruses. all experiments involving low pathogenic and highly pathogenic avian influenza a viruses were performed in biosafety level 2 and 3 (bsl2, 3) containment cabinets, respectively, approved for such use by the local authorities. as schematically represented in figure 1a , the pmp-h1/rbd construct comprises of the 3`-non-coding region (ncr) and the signal peptide (sp) of the ha gene from a/giessen/06/2009 (h1n1pdm2009), followed by the receptor-binding domain (rbd) of the mers-cov spike gene (rbd: 1099-1818 nt = 367-606 amino acids (aa)), a 7-aa flexible linker peptide (lp: gsagsag, [22] ) and the coding sequence plus the 5'-ncr of the ha gene. the pmp-h1/rbd is designed to link the rbd and ha0 fragments into a single polypeptide chain. the final fusion protein contains amino acid (aa) residues 1-240 of the rbd, aa residues 1-7 of the lp and aa residues 1-566 of the ha0. to produce outer-membrane vesicles (omvs), comprising an expressed mers-cov rbd and h1n1pdm2009 ha (omvs-h1/rbd) hybrid protein, the e. coli strain dh10ß was transformed with pmp-h1/rbd plasmid. the presence of the rbd from mers-cov and the h1-ha from h1n1pdm2009 in the omvs particles was examined by immunoblot analysis using 5 µg of purified omvs-h1/rbd. the blotting pattern confirmed the presence of rbd-linked ha viral protein; corresponding to ha0-rbd (77 kda + 25 kda equal 102 kda). in contrast, omvs isolated from untransformed dh10ß (omvs-empty) did not show any cross-reacting proteins (figure 1b) . to produce outer-membrane vesicles (omvs), comprising an expressed mers-cov rbd and h1n1pdm2009 ha (omvs-h1/rbd) hybrid protein, the e. coli strain dh10ß was transformed with pmp-h1/rbd plasmid. the presence of the rbd from mers-cov and the h1-ha from h1n1pdm2009 in the omvs particles was examined by immunoblot analysis using 5 µg of purified omvs-h1/rbd. the blotting pattern confirmed the presence of rbd-linked ha viral protein; corresponding to ha0-rbd (77 kda + 25 kda equal 102 kda). in contrast, omvs isolated from untransformed dh10ß (omvs-empty) did not show any cross-reacting proteins (figure 1b) . to assess the immunogenicity of the bivalent omvs-h1/rbd preparations, female balb/c mice were immunized with 5 µg/mouse of omvs-h1/rbd and omvs-empty in comparison with inactivated h1n1pdm2009 and inactivated mers-cov as a positive controls and pbs as a negative control. mice received a booster dose three weeks after prime immunization (figure 2a ). sera were collected every two weeks from week two to eight after prime immunization. ncr: non-coding region, sp: signal peptide, rbd: receptor binding domain (mers-cov), l: nucleotide sequence of peptide linker (ggtagcgccggtagcgccgga), ha1: hemagglutinin 1, and ha2: hemagglutinin 2. (b) immunoblotting pattern of omvs, extracted either from various control/non-transformed omvs (c1, c2, and c3) (empty omvs) or pmp-h1/rbd-transformed (omvs-h1/rbd) dh10ß (s1-s6), against antiserum of swine ha1 antibody. to assess the immunogenicity of the bivalent omvs-h1/rbd preparations, female balb/c mice were immunized with 5 µg/mouse of omvs-h1/rbd and omvs-empty in comparison with inactivated h1n1pdm2009 and inactivated mers-cov as a positive controls and pbs as a negative control. mice received a booster dose three weeks after prime immunization (figure 2a ). sera were collected every two weeks from week two to eight after prime immunization. at two weeks post-vaccination, mice vaccinated with inactivated h1n1pdm2009 virus or omvs-h1/rbd revealed a 2-3 × log2 increase in hai antibody titer as compared to the control and omvs-empty groups (figure 2b ). four weeks after vaccination we observed a drop in the hai titers in these mice due to the booster vaccination at week three. interestingly, the two groups vaccinated with omvs-h1/rbd or inactivated h1n1pdm2009 showed a significant increase in geometric mean hai antibody titers to 145.4 (7.2 log2) and 420 (8.7 log2) at week six, and at week eight, the geometric mean hai antibody titers decreased to 70.7 (6.1 log2) and 210 (7.7 log2), respectively. these results revealed that the vaccinated mice had developed a strong immunogenic response against the h1n1pdm2009 virus. in the negative pbs control group and in the omvs-empty group hai titers remained low, reflecting that all animals were indeed housed under influenza-free conditions (no natural infection). at two weeks post-vaccination, mice vaccinated with inactivated h1n1pdm2009 virus or omvs-h1/rbd revealed a 2-3 × log2 increase in hai antibody titer as compared to the control and omvs-empty groups (figure 2b ). four weeks after vaccination we observed a drop in the hai titers in these mice due to the booster vaccination at week three. interestingly, the two groups vaccinated with omvs-h1/rbd or inactivated h1n1pdm2009 showed a significant increase in geometric mean hai antibody titers to 145.4 (7.2 log2) and 420 (8.7 log2) at week six, and at week eight, the geometric mean hai antibody titers decreased to 70.7 (6.1 log2) and 210 (7.7 log2), respectively. these results revealed that the vaccinated mice had developed a strong immunogenic response against the h1n1pdm2009 virus. in the negative pbs control group and in the omvs-empty group hai titers remained low, reflecting that all animals were indeed housed under influenza-free conditions (no natural infection). in addition, the omvs-h1/rbd vaccinated mice showed a significant increase of neutralizing antibodies against the mers-cov strain hku-nrce-270 at week 2 and reached the highest neutralizing titer 160 (7.3 log2) at week eight compared to the control group (p < 0.001) (figures 2c and 3a) . control (1× pbs), inactivated h1n1pdm2009 and omvs-empty groups showed no neutralizing antibodies against mers-cov during the eight weeks of infection (figure 3b-d) . vaccines 2019, 7, x for peer review 8 of 13 in addition, the omvs-h1/rbd vaccinated mice showed a significant increase of neutralizing antibodies against the mers-cov strain hku-nrce-270 at week 2 and reached the highest neutralizing titer 160 (7.3 log2) at week eight compared to the control group (p < 0.001) (figures 2c and 3a) . control (1× pbs), inactivated h1n1pdm2009 and omvs-empty groups showed no neutralizing antibodies against mers-cov during the eight weeks of infection (figure 3b-d) . on the other hand, a plaque reduction neutralization test (prnt50) using sera from mice vaccinated with inactivated mers-cov showed complete neutralization (prnt50 titer, approximately >1:160) after eight weeks of first immunization of active mers-cov (figure 3e) . to investigate the protection level of vaccinated mice against h1n1pdm09 infection, vaccinatedand control groups of balb/c mice (8 weeks post-vaccination) (figure 2a) were infected with wild type cal-h1n1pdm2009 virus. to ensure full separation between the groups and the absence of natural infection, an additional control group of five mice was added and not subjected to infection. both groups, vaccinated either with inactivated h1n1pdm2009 or omvs-h1/rbd, showed no weight losses till 14 days p.i. (post infection) in comparison to the infected pbs control group. interestingly, these results showed that the vaccinated groups with omvs-h1/rbd and inactivated h1n1pdm2009 virus protected all mice from body weight loss (bwl) (figure 4a ) and mortality up to 14 days post challenge infection (figure 4b ). vaccines 2019, 7, x for peer review 9 of 13 interestingly, these results showed that the vaccinated groups with omvs-h1/rbd and inactivated h1n1pdm2009 virus protected all mice from body weight loss (bwl) (figure 4a ) and mortality up to 14 days post challenge infection (figure 4b ). in contrast, the control group of pbs-treated mice infected with cal-h1n1pdm2009 100% exhibited a bwl of more than 30% from day four to six p.i. resulting in euthanasia (figure 4a ). the mortality rate in this control group was 29% four days p.i. and increased gradually to 85% at day 5 p.i., and 100% at 6 days p.i. (figure 4b) . mortality in the omvs-empty group reached 57% at 13 days p.i. and resulted in euthanization of 4 mice (bwl ≥ 30%) (figure 4b ). the continuous evolution of h1n1pdm09 in swine and human populations, and the recent emergence of mers-cov infections with high mortality rate in humans has raised awareness of both viruses as serious emergent global health topics [23, 24] . since vaccination is the most important strategy to combat emerging human viral infections, an effective vaccine remains a necessity, particularly for the mers-cov. the mers-cov spike (s) protein plays an essential role during virus entry through the binding of its antigenic rbd region to the dpp4 host cell receptor [25] . the rbd is recognized as a major antigenic glycoprotein fragment for inducing a potent humoral and cellular neutralizing antibody (nab) immune responses [26] [27] [28] [29] [30] [31] . traditionally, influenza vaccines are produced by generating a natural or recombinant reassortant iav expressing the immunogenic ha antigen [14, 32] . the iav comprises of two subunits ha1 and ha2, hosting the antigenic sites to which specific and neutralizing antibodies are elicited to combat iavs strains during vaccination or natural infection [14, 33] . however, it was reported that the specificity of the vaccine produced in cell-culture and embryonated eggs is occasionally impaired by amino acid (aa) changes, due to seed strain adaptation, with a drastic impact on vaccine effectiveness [14] [15] [16] . the vaccine platform presented in this study depends on plasmid-based bacterial expression of recombinant viral antigen(s). these plasmids, encoding viral antigen(s), can be easily and quickly modified to insert non-synonymous changes in the encoding region of the antigen(s). additionally, the bacterial expression has lower mutation rates than eukaryotes [34] . this platform can be also a base for incorporating combinations of different viral antigens to address additional vaccines needed to combat seasonal h1n1, h3n2 and influenza b viruses. omvs had been introduced as a part of novel vaccine formulations carrying antigenic proteins eliciting protective responses in animal models from diverse microorganisms such as n. meningitis b, vibrio cholera, salmonella typhimurium, pseudomonas aeruginosa, gallibacterium anatis, acinetobacter baumannii, chlamydia trachomatis, shigella spp., and mycobacterium tuberculosis [35] [36] [37] [38] [39] [40] [41] . lps in the outer surface of omvs acts as a self-adjuvant that induces humoral and cellular immunity. therefore, omvs vaccines may be used without extra adjuvant to increase the immunogenicity and produce antiviral innate immune responses against various influenza virus infections via activation of macrophages [42] [43] [44] . despite that the exact role of lps in the context of omvs vaccines requires in contrast, the control group of pbs-treated mice infected with cal-h1n1pdm2009 100% exhibited a bwl of more than 30% from day four to six p.i. resulting in euthanasia (figure 4a ). the mortality rate in this control group was 29% four days p.i. and increased gradually to 85% at day 5 p.i., and 100% at 6 days p.i. (figure 4b) . mortality in the omvs-empty group reached 57% at 13 days p.i. and resulted in euthanization of 4 mice (bwl ≥ 30%) (figure 4b ). the continuous evolution of h1n1pdm09 in swine and human populations, and the recent emergence of mers-cov infections with high mortality rate in humans has raised awareness of both viruses as serious emergent global health topics [23, 24] . since vaccination is the most important strategy to combat emerging human viral infections, an effective vaccine remains a necessity, particularly for the mers-cov. the mers-cov spike (s) protein plays an essential role during virus entry through the binding of its antigenic rbd region to the dpp4 host cell receptor [25] . the rbd is recognized as a major antigenic glycoprotein fragment for inducing a potent humoral and cellular neutralizing antibody (nab) immune responses [26] [27] [28] [29] [30] [31] . traditionally, influenza vaccines are produced by generating a natural or recombinant reassortant iav expressing the immunogenic ha antigen [14, 32] . the iav comprises of two subunits ha1 and ha2, hosting the antigenic sites to which specific and neutralizing antibodies are elicited to combat iavs strains during vaccination or natural infection [14, 33] . however, it was reported that the specificity of the vaccine produced in cell-culture and embryonated eggs is occasionally impaired by amino acid (aa) changes, due to seed strain adaptation, with a drastic impact on vaccine effectiveness [14] [15] [16] . the vaccine platform presented in this study depends on plasmid-based bacterial expression of recombinant viral antigen(s). these plasmids, encoding viral antigen(s), can be easily and quickly modified to insert non-synonymous changes in the encoding region of the antigen(s). additionally, the bacterial expression has lower mutation rates than eukaryotes [34] . this platform can be also a base for incorporating combinations of different viral antigens to address additional vaccines needed to combat seasonal h1n1, h3n2 and influenza b viruses. omvs had been introduced as a part of novel vaccine formulations carrying antigenic proteins eliciting protective responses in animal models from diverse microorganisms such as n. meningitis b, vibrio cholera, salmonella typhimurium, pseudomonas aeruginosa, gallibacterium anatis, acinetobacter baumannii, chlamydia trachomatis, shigella spp., and mycobacterium tuberculosis [35] [36] [37] [38] [39] [40] [41] . lps in the outer surface of omvs acts as a self-adjuvant that induces humoral and cellular immunity. therefore, omvs vaccines may be used without extra adjuvant to increase the immunogenicity and produce antiviral innate immune responses against various influenza virus infections via activation of macrophages [42] [43] [44] . despite that the exact role of lps in the context of omvs vaccines requires further investigations, high amounts of lps could be a drawback due to its known endotoxicity and ability to induce excessive secretions of pro-inflammatory cytokines [45] . therefore, several ongoing investigations aim to produce genetically detoxified and less reactogenic lps to improve omv safety [42, 46, 47] . additionally, modified bacterial strains such as clearcoli™ bl21(de3), which do not trigger lps-related immune response, can be applied for omv production [48] . based on these observations we engineered the expression of antigenically-stable and immunogenic (omvs)-based bivalent vaccine that elicits protective antibodies (abs) following immunization to control infections with h1n1pdm09 and mers-cov. a recombinant construct comprising the ha of h1n1pdm09 fused to the rbd of the mers-cov s protein is expressed in an e. coli bacterial strain. the expressed bivalent antigens were incorporated within the released omvs (omvs-h1/rbd). this novel chimeric omvs-h1/rbd produced high levels of a neutralizing abs titer against influenza h1n1 virus at 8 weeks post immunization. stimulated neutralizing abs (humoral immunity) together with lps-induced cellular immunity could fully protect immunized mice after challenge with h1n1pdm09 without significant loss in body weight. surprisingly, the induced non-specific cellular immunity induced by omvs-empty could partially protect the mice. this emphasize the synergistic effect of humoral and cellular immunities secreted upon vaccination with the chimeric omvs-h1/rbd formulation. serum transfer experiments would be able to further elucidate the role of humoral immunity independent of cellular immunity [49] . additionally, omvs-h1/rbd-vaccinated mice demonstrated a significant increase in the neutralizing abs titer against mers-cov (1:160) at week 8 in comparison to the control group as in figure 2c . these findings ensured that omv vaccination platform can provide a protection by efficient neutralization of invading h1n1pdm09 and mers-cov. the data presented in this study are consistent with recent reports describing the potential of omvs as biologically active, stable and highly immunogenic vaccines to protect against iavs. a newly developed recombinant omvs bearing the conserved m2e protein (omvs-m2e) from iavs efficiently protected mice from an h1n1-and h3n2-type iav challenge [50, 51] . our study represents an extension of these studies and suggests the generation of omvs that incorporate combinations of different viral antigens to generate safe and efficient vaccines in animal husbandry and for humans. in summary, the results show that the generated (omvs-h1/rbd)-based vaccine presenting the antigenic stable chimeric fusion protein of h1-type ha of the pandemic influenza a virus (h1n1) strain and rbd of mers-cov induces specific neutralizing antibodies against h1n1pdm09 and mers-cov leading to protection of immunized mice against both viruses. these results demonstrate that omvs-based vaccines presenting viral antigens have the potential to be a vaccine platform that provides simultaneous protection against two 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recombinant outer membrane vesicles that display m2e elicit heterologous influenza protection key: cord-318444-sgm24q1i authors: walter, justin d.; hutter, cedric a.j.; zimmermann, iwan; wyss, marianne; earp, jennifer; egloff, pascal; sorgenfrei, michèle; hürlimann, lea m.; gonda, imre; meier, gianmarco; remm, sille; thavarasah, sujani; plattet, philippe; seeger, markus a. title: sybodies targeting the sars-cov-2 receptor-binding domain date: 2020-05-16 journal: biorxiv doi: 10.1101/2020.04.16.045419 sha: doc_id: 318444 cord_uid: sgm24q1i the covid-19 pandemic, caused by the novel coronavirus sars-cov-2, has resulted in a global health and economic crisis of unprecedented scale. the high transmissibility of sars-cov-2, combined with a lack of population immunity and prevalence of severe clinical outcomes, urges the rapid development of effective therapeutic countermeasures. here, we report the generation of synthetic nanobodies, known as sybodies, against the receptor-binding domain (rbd) of sars-cov-2. in an expeditious process taking only twelve working days, sybodies were selected entirely in vitro from three large combinatorial libraries, using ribosome and phage display. we obtained six strongly enriched sybody pools against the isolated rbd and identified 63 unique anti-rbd sybodies which also interact in the context of the full-length sars-cov-2 spike ectodomain. among the selected sybodies, six were found to bind to the viral spike with double-digit nanomolar affinity, and five of these also showed substantial inhibition of rbd interaction with human angiotensin-converting enzyme 2 (ace2). additionally, we identified a pair of anti-rbd sybodies that can simultaneously bind to the rbd. it is anticipated that compact binders such as these sybodies could feasibly be developed into an inhalable drug that can be used as a convenient prophylaxis against covid-19. moreover, generation of polyvalent antivirals, via fusion of anti-rbd sybodies to additional small binders recognizing secondary epitopes, could enhance the therapeutic potential and guard against escape mutants. we present full sequence information and detailed protocols for the identified sybodies, as a freely accessible resource. the ongoing pandemic arising from the emergence of the 2019 novel coronavirus, sars-cov-2, demands urgent development of effective antiviral therapeutics. several factors contribute to the adverse nature of sars-cov-2 from a global health perspective, including the absence of herd immunity [1] , high transmissibility [2, 3] , the prospect of asymptomatic carriers [4] , and a high rate of clinically severe outcomes [5] . moreover, a vaccine against sars-cov-2 is unlikely to be available for at least 12-18 months [6] , despite earnest development efforts [7, 8] , making alternative intervention strategies paramount. in addition to offering relief for patients suffering from the resulting covid-19 disease, therapeutics may also reduce the viral transmission rate by being administered to asymptomatic individuals subsequent to probable exposure [9] . finally, given that sars-cov-2 represents the third global coronavirus outbreak in the past 20 years [10, 11] , development of rapid therapeutic strategies during the current crises could offer greater preparedness for future pandemics. akin to all coronaviruses, the viral envelope of sars-cov-2 harbors protruding, club-like, multidomain spike proteins that provide the machinery enabling entry into human cells [12] [13] [14] . the spike ectodomain is segregated into two regions, termed s1 and s2. the outer s1 subunit of sars-cov-2 is responsible for host recognition via interaction between its c-terminal receptor-binding domain (rbd) and human angiotensin converting enzyme 2 (ace2), present on the exterior surface of airway cells [14, 15] . while there is no known host-recognition role for the s1 n-terminal domain (ntd) of sars-cov-2, it is notable that s1 ntds of other coronaviruses have been shown to bind host surface glycans [12, 16] . in contrast to spike region s1, the s2 subunit contains the membrane fusion apparatus, and also mediates trimerization of the ectodomain [12] [13] [14] . prior to host recognition, spike proteins exist in a metastable pre-fusion state wherein the s1 subunits lay atop the s2 region and the rbd oscillates between "up" and "down" conformations that are, respectively, accessible and inaccessible to receptor binding [12, 17, 18] . upon processing at the s1/s2 and s2' cleavage sites by host proteases as well as engagement to the receptor, the s2 subunit undergoes dramatic conformational changes from the pre-fusion to the post-fusion state. such structural rearrangements are associated with the merging of the viral envelope with host membranes, thereby allowing injection of the genetic information into the cytoplasm of the host cell [19, 20] . coronavirus spike proteins are highly immunogenic [21] , and several experimental approaches have sought to target this molecular feature for the purpose of viral neutralization [22] . the high specificity, potency, and modular nature of antibody-based antiviral therapeutics has shown exceptional promise [23] [24] [25] , and the isolated, purified rbd has been a popular target for the development of anti-spike antibodies against pathogenic coronaviruses [26] [27] [28] [29] . however, binders against the isolated rbd may not effectively engage the aforementioned pre-fusion conformation of the full spike, which could account for the poor neutralization ability of recently described single-domain antibodies that were raised against the rbd of sars-cov-2 [30] . therefore, to better identify molecules with qualities befitting a drug-like candidate, it would be advantageous to validate rbd-specific binders in the context of the full, stabilized, pre-fusion spike assembly [13, 31] . single domain antibodies based on the variable vhh domain of heavy-chain-only antibodies of camelids -generally known as nanobodies -have emerged as a broadly utilized and highly successful antibody fragment format [32] . nanobodies are small (12) (13) (14) (15) , stable, and inexpensive to produce in bacteria and yeast [33] , yet they bind targets in a similar affinity range as conventional antibodies. due to their minimal size, they are particularly suited to reach hidden epitopes such as crevices of target proteins [34] . we recently designed three libraries of synthetic nanobodies, termed sybodies, based on elucidated structures of nanobody-target complexes (fig. 1a) [35, 36] . sybodies can be selected against any target protein within twelve working days, which is considerably faster than natural nanobodies, which requires the repetitive immunization during a period of two months prior to binder selection by phage display [36] (fig. 1c) . a considerable advantage of our platform is that sybody selections are carried out under defined conditions -in case of coronavirus spike proteins, this offers the opportunity to generate binders recognizing the metastable pre-fusion conformation [13, 14] . finally, due to the feasibility of inhaled therapeutic nanobody formulations [37], virusneutralizing sybodies could offer a convenient and direct means of prophylaxis. here, we report of in vitro selection and characterization of sybodies against the rbd of sars-cov-2 spike protein. two independently prepared rbd constructs were used for in vitro sybody selections, and resulting single clones that could bind the full spike ectodomain were sequenced, expressed, and purified. six unique sybodies show favorable binding affinity to the sars-cov-2 spike, and five of these were also found to substantially attenuate the interaction between the viral rbd and human ace2. moreover, pairs of sybodies were identified that can simultaneously bind to the rbd. we present all sequences for these clones, along with detailed protocols to enable the community to freely produce and further characterize these sars-cov-2 binders. based on sequence alignments with isolated rbd variants from sars-cov-1 that were amenable to purification and crystallization [29, 38] , a sars-cov-2 rbd construct was designed, consisting of residues pro330-gly526 fused to venus yfp (rbd-vyfp). this construct was expressed and secreted from expi293 cells, and rbd-vyfp was extracted directly from culture medium supernatant using an immobilized anti-gfp nanobody [39], affording a highly purified product with negligible background contamination. initial efforts to cleave the c-terminal vyfp fusion partner with 3c protease resulted in unstable rbd, so experiments were continued with full rbd-vyfp fusion protein. to account for the presence of the vyfp fusion partner, a second rbd construct, consisting of a fusion to murine igg1 fc domain (rbd-fc), was commercially acquired. to remove any trace amines, buffers were exchanged to pbs via extensive dialysis. proteins were chemically biotinylated, and the degree of biotinylation was assessed by a streptavidin gel-shift assay and found to be greater than 90 % of the target proteins [40] . we note that while both rbd fusion proteins were well-behaved, a commercially acquired purified full-length sars-cov-2 spike ectodomain construct (ecd) was found to be aggregation-prone. very recently, we also produced an engineered spike protein ectodomain containing two point mutations known to stabilize the pre-fusion state, an inactivated furin cleavage site, and a c-terminal trimerization motif [13, 14, 31] . while this purified pre-fusion spike (pfs) had not yet been available for binder selections and characterization by grating-coupled interferometry, it was used to conduct elisas in order to identify selected sybodies which recognize the rbd in the pre-fusion context (see below). since both our rbd constructs bear additional fusion domains (fc of mouse igg1 and vyfp, respectively), sybody selections were carried out with a "target swap" approach (fig. 1b) . hence, selections with the three sybody libraries (concave, loop and convex) were started with the rbd-vyfp construct using ribosome display, and the rbd-fc construct was then used for the two phage display rounds (selection variant 1: rbd-vyfp/rbd-fc/rbd-fc) and vice versa (selection variant 2: rbd-fc/rbd-vyfp/rbd-vyfp). accordingly, there were a total of six selection reactions (table 1 , fig. 1b) . to increase the average affinity of the isolated sybodies, we included an off-rate selection step using the preenriched purified sybody pool after phage display round 1 as competitor. to this end, sybody pools of all three libraries of the same selection variant were sub-cloned from the phage display vectors into the sybody expression vector psb_init. subsequently, the two separate pools (all sybodies of selection variants 1 and 2, respectively) were expressed and purified. the purified pools were then added to the panning reactions of the respective selection variant in the second phage display round. thereby, rebinding of sybody-phage complexes with fast off-rates was suppressed. enrichment of sybodies against the rbd was monitored by qpcr. already in the first phage display round, the concave and loop sybodies of selection variant 2 showed enrichment factors of 7 and 3, respectively (table 1) . after the second phage display round (which included the off-rate selections step), strong enrichment factors in the range of 10-263 were determined. after sub-cloning the pools from the phage display vector pdx_init into the sybody expression vector psb_init, 47 clones of each of the 6 selections reactions ( table 1 , fig. 1b ) were picked at random and expressed in small scale. our standard elisa was initially performed using rbd-vyfp (rbd), spike ectodomain containing s1 and s2 (ecd), and maltose binding protein (mbp) as unrelated dummy protein. as outlined in the materials and methods section, elisa analysis revealed very high hit rates for the rbd and the ecd ranging from 81 % to 100 % and 66 % to 96 %, respectively (fig. 2 , table 1 ). the majority of the sybodies giving an elisa signal to the rbd also gave a clear signal the full-length spike protein (fig. 2 ). however, there was a total of 44 hits that only gave an elisa signal for rbd-vyfp, but not for the ecd. this could be due to the presence of cryptic rbd epitopes that are not accessible in the context of the full-length spike protein, or the respective sybodies may recognize the vyfp portion of the rbd-vyfp construct, though the selection procedure clearly disfavors the latter explanation. importantly, background binding to the dummy protein mbp was not observed for any of the analyzed sybodies, clearly showing that the binders are highly specific. we then sequenced 72 sybodies that were elisa-positive against rbd-vyfp as well as the full-length spike (12 for each of the 6 selection reactions numbered from sb#1-72, see also fig. 1b ). subsequent to sybody sequencing, we also performed the elisa using engineered pre-fusion-stabilized spike ectodomain (pfs) (fig. 2) , which was not available at the onset of the project. overall, the elisa signals for the ecd and pfs are highly similar. however, there are around 40 sybodies that bind to the ecd clearly stronger than to the pfs (yet the opposite scenario was never observed). this could be explained by the fact that the pfs forms a trimer, while the oligomeric state of the ecd is not clear. in addition, the ecd might adopt partially or completely a post-fusion state, whereas pfs is expected to predominantly adopt the pre-fusion state. trimer formation as well as pre-fusion stabilization might shield certain binding epitopes on the rbd in the context of the pfs, which might become accessible as the spike falls apart into monomers and/or transits to the post-fusion state. in light of our elisa data, the pfs construct will be a crucial element in any future sybody selection campaigns. sequencing results of 70 out of 72 sybody clones were unambiguous. out of these 70 clones, 63 were found to be unique and the respective clone names are indicated in the elisa figure (fig. 2 , table 2 ). of note, there were no duplicate binders identified in both selection variants, indicating that the two separate selection streams gave rise to completely different arrays of sybodies. as an additional note, one sybody identified from the supposed convex library turned out to belong to the concave library; spill-over of sybodies across libraries is occasionally observed. hence, there was a total of 23 concave, 22 loop and 18 convex sybodies, which were then aligned according to their library origin . as a final analysis, all sybody sequences were aligned to generate a phylogenetic tree, which shows a clear segregation across the three libraries and indicates a large sequence variability of the identified sybodies ( fig. 6 ). the 63 unique sybodies were individually expressed in e. coli and purified via ni-nta affinity chromatography and gel filtration. ultimately, 57 sybodies were sufficiently well-behaved, with respect to solubility, yield, and monodispersity, to proceed with further characterization. for a kinetic analysis of sybody interactions with the viral spike, we employed grating-coupled interferometry (gci) to probe sybody binding to immobilized rbd-vyfp or ecd. first, the 57 purified sybodies were subjected to an off-rate screen, which revealed six sybodies (sb#14, sb#15, sb#16, sb#42, sb#45, and sb#68) with strong binding signals and comparatively slow off-rates. binding constants were then determined by measuring on-and off-rates over a range of sybody concentrations, revealing affinities within a range of 20-180 nm to the sars-cov-2 spike (fig. 7 , table 3 ). of note, binding affinities were consistently equal or higher for the ecd as compared to the rbd-vyfp, in particular in case of sb#68 for which the off-rate differs by more than two-fold. this might indicate a binding avidity effect arising from binding epitopes clustering in the context of the spike trimer or differences with regards to the glycan structures (rbd-vyfp was produced in hek cells, whereas the ecd was produced in insect cells). to our surprise, the majority of purified and elisa-positive sybodies (51 out of 57) displayed binding affinities worse than 200 nm. this may be attributed to the presence of complex heterogeneous asnlinked glycans within the rbd, which could hinder the isolation of specific high-affinity binders. alternatively, given that the final elisa step of the selection process resulted in a substantial number of positive clones, insufficiently stringent conditions may have favored the high positive hit rate of lowaffinity binders. since virulence of sars-cov-2 is dependent on the ability of the viral rbd to bind to human ace2 (hace2), we sought to determine which of the 57 selected sybodies that were well-behaved upon purification could inhibit interaction between the isolated rbd and purified hace2. for this assessment, elisa plates were coated with purified hace2, and the binding of purified rbd to the 6 immobilized hace2 was measured in the presence or absence of an excess of each purified sybody (fig. 8 ). while the absence of any added sybody resulted in a strong elisa signal corresponding to rbd association with hace2, the pre-incubation of nearly all sybodies with the rbd resulted in an attenuated signal, implying that these binders inhibit rbd-hace2 association. this signal decrease relative to unchallenged rbd was modest for most sybodies, with an average signal reduction of about 50%, but five sybodies demonstrated exceptionally high apparent inhibition of rbd-hace2 interaction (sb#14, sb#15, sb#16, sb#42, and sb#45), showing ≥90% signal reduction. notably, the aforementioned kinetic analysis had shown that these sybodies were also among the strongest rbd binders. taken together, this data suggests that sb#14, sb#15, sb#16, sb#42, and sb#45 recognize a surface region on the rbd that overlaps with the hace2 binding site. while kinetic analysis had revealed sb#68 to be among the stronger binders to the sars-cov-2 ectodomain (kd ≈ 37 nm, fig. 7 , table 3 ), the hace2 competition elisa revealed that sb#68 does not inhibit hace2-rbd interaction to the same extent as other sybodies with comparable affinities (65% inhibition for sb#68, compared to >90% for sb#14, sb#15, sb#16, and sb#45). therefore, it was hypothesized that sb#68 may interact with a non-or partially-overlapping surface on the rbd, relative to the more strongly-inhibiting sybodies. using sb#15 as a representative of the hace2-inhibiting sybodies, we analyzed the ability of sb#15 and sb#68 to simultaneously associate with the rbd. first, elisa experiments demonstrate that incubation of sb#68 with the pre-fusion spike only slightly prevents the spike from binding to immobilized sb#15, whereas pre-incubation with sb#14, sb#15, sb#16, sb#42, or sb#45 completely prevents spike interaction with immobilized sb#15 (fig. 9 ). in agreement with the elisa data, gci experiments revealed that co-injection of sb#15 and sb#68 results in a clear (but not fully additive) increase of the response signal, relative to sb#15 or sb#68 injected alone, implying simultaneous binding of sb#15 and sb#68 (fig. 9 ). the control gci experiment involving the co-injection of sb#15 and sb#45 did not result in a similar signal increase ( fig. 9 ). in sum, this data plausibly suggests that sb#15 and sb#68 can simultaneously bind to the rbd. for the design of therapeutics against sars-cov-2, the fusion of such a pair of non-overlapping binders could provide benefits via increased overall avidity to the spike protein. we have demonstrated the ability of our rapid in vitro selection platform to generate sybodies against the sars-cov-2 rbd, within a two-week timeframe. characterization of these sybodies has identified a high-affinity subset of binders that also inhibit the rbd-ace2 interaction. we anticipate that the presented panel of anti-rbd sybodies could be of use in the design of urgently required therapeutics to mitigate the covid-19 pandemic, particularly in the development of inhalable prophylactic formulations [37] . furthermore, our identification of a pair of sybodies that can simultaneously associate with the rbd may offer an attractive foundation for the construction of a polyvalent sybodybased therapeutic. we have attempted to provide a complete account of the generation of these molecules, including full sequences and detailed methods, such that other researchers may contribute to their ongoing analysis. future work may include virus neutralization assays using the identified sybodies, as well as further selection campaigns targeting additional spike epitopes. finally, our recently described flycode technology could be utilized for deeper interrogation of selection pools, in order to facilitate discovery of exceptional sybodies that possess very slow off-rates or recognize rare epitopes [41] . a gene encoding sars-cov-2 residues pro330-gly526 (rbd, genbank accession qhd43416.1), downstream from a modified n-terminal human serum albumin secretion signal [42] , was chemically synthesized (geneuniversal). this gene was subcloned using fx technology [43] into a custom mammalian expression vector [44] , appending a c-terminal 3c protease cleavage site, myc tag, venus yfp [45] , and streptavidin-binding peptide [46] onto the open reading frame (rbd-vyfp). 100-250 ml of suspension-adapted expi293 cells (thermo) were transiently transfected using expifectamine according to the manufacturer protocol (thermo), and expression was continued for 4-5 days in a humidified environment at 37°c, 8% co2. cells were pelleted (500g, 10 min), and culture supernatant was filtered (0.2 µm mesh size) before being passed three times over a gravity column containing nhsagarose beads covalently coupled to the anti-gfp nanobody 3k1k [39], at a resin:culture ratio of 1ml resin per 100ml expression culture. resin was washed with 20 column-volumes of rbd buffer (phosphate-buffered saline, ph 7.4, supplemented with additional 0.2m nacl), and rbd-vyfp was eluted with 0.1 m glycine, ph 2.5, via sequential 0.5 ml fractions, without prolonged incubation of resin with the acidic elution buffer. fractionation tubes were pre-filled with 1/10 vol 1m tris, ph 9.0 (50 µl), such that elution fractions were immediately ph-neutralized. fractions containing rbd-vyfp were pooled, concentrated, and stored at 4°c. purity was estimated to be >95%, based on sds-page (not shown). yield of rbd-vyfp was approximately 200-300 μg per 100 ml expression culture. a second purified rbd construct, consisting of sars-cov-2 residues arg319-phe541 fused to a murine igg1 fc domain (rbd-fc) expressed in hek293 cells, was purchased from sino biological (catalogue number: 40592-v05h, 300 µg were ordered). purified full-length spike ectodomain (ecd) comprising s1 and s2 (residues val16-pro1213) with a c-terminal his-tag and expressed in baculovirus-insect cells was purchased from sino biological (catalogue number: 40589-v08b1, 700 µg were ordered). the prefusion ectodomain of the sars-cov2 spike protein (residues 1-1208) [13] , was transiently transfected into 50x10 8 suspension-adapted expicho cells (thermo fisher) using 3 mg plasmid dna and 15 mg of pei max (polysciences) per 1l procho5 medium (lonza) in a 3l erlenmeyer flask (corning) in an incubator shaker (kühner). one hour post-transfection, dimethyl sulfoxide (dmso; applichem) was added to 2% (v/v). incubation with agitation was continued at 31°c for 5 days. 1l of filtered (0.22 um) cell culture supernatant was clarified. then, a 1ml gravity flow strep-tactin®xt superflow® column (iba lifescience) was rinsed with 2 ml buffer w (100 mm tris, ph 8.0, 100 mm nacl, 1 mm edta) using gravity flow. the supernatant was added to the column, which was then rinsed with 5 ml of buffer w (all with gravity flow). finally, six elution steps were performed by adding each time 0.5 ml of buffer bxt (50mm biotin in buffer w) to the resin. all purification steps were performed at 4°c. to remove amines, all proteins were first extensively dialyzed against rbd buffer. proteins were concentrated to 25 µm using amicon ultra concentrator units with a molecular weight cutoff of 30 -8 50 kda. subsequently, the proteins were chemically biotinylated for 30 min at 25°c using nhs-biotin (thermo fisher, #20217) added at a 10-fold molar excess over target protein. immediately after, the three samples were dialyzed against tbs ph 7.5. during these processes (first dialysis/ concentrating/ biotinylation/ second dialysis), 20 %, 30 %, 65 % and 44% of the rbd-vyfp, rbd-fc, ecd and pfs respectively were lost due to sticking to the concentrator filter or due to aggregation. biotinylated rbd-vyfp, rbd-fc and ecd were diluted to 5 µm in tbs ph 7.5, 10 % glycerol and stored in small aliquots at -80°c. biotinylated pfs was stored at 4°c in tbs ph 7.5. sybody selections with the three sybody libraries concave, loop and convex were carried out as described in detail before [36] . in short, one round of ribosome display followed by two rounds of phage display were carried out. binders were selected against two different constructs of the sars-cov-2 rbd; an rbd-vyfp fusion and an rbd-fc fusion. mbp was used as background control to determine the enrichment score by qpcr [36] . in order to avoid enrichment of binders against the fusion proteins (yfp and fc), we switched the two targets after ribosome display (fig. 1b) . for the offrate selections we did not use non-biotinylated target proteins as described in the standard protocol, because we did not have enough purified protein at hand to do so. instead we sub-cloned all three libraries for both selections after the first round of phage display into the psb_init vector (10 8 clones) and expressed the six pools in e. coli mc1061 cells. then the pools corresponding to the same selection were pooled for purification. the two final pools were purified by ni-nta resin using gravity flow columns, followed by buffer exchange of the main peak fraction using a desalting pd10 column in tbs ph 7.5 to remove imidazole. the pools were eluted with 3.2 ml instead of 3.5 ml tbs ph 7.5 in order to ensure complete buffer exchange. these two purified pools were used for the off-rate selection in the second round of phage display at concentrations of approximately 390 µm for selection variant 1 (rbp-fc) and 450 µm for selection variant 2 (rbp-yfp). the volume used for off-rate selection was 500 µl. just before the pools were used for the off-rate selection, 0.5% bsa and 0.05% tween-20 was added to each sample. off-rate selections were performed for 3 minutes. elisas were performed as described in detail before [36] . 47 single clones were analyzed for each library of each selection. since the rbd-fc construct was incompatible with our elisa format due to the inclusion of protein a to capture an α-myc antibody, elisa was performed only for the rbd-vyfp (50 nm) and the ecd (25 nm) and later on with the pfs (25 nm). of note, the three targets were analyzed in three separate elisas. as negative control to assess background binding of sybodies, we used biotinylated mbp (50 nm). 72 positive elisa hits were sequenced (microsynth, switzerland). the 63 unique sybodies were expressed and purified as described [36] . in short, all 63 sybodies were expressed overnight in e.coli mc1061 cells in 50 ml cultures. the next day the sybodies were extracted from the periplasm and purified by ni-nta affinity chromatography (batch binding) followed by sizeexclusion chromatography using a sepax srt-10c sec100 size-exclusion chromatography (sec) column equilibrated in tbs, ph 7.5, containing 0.05% (v/v) tween-20 (detergent was added for subsequent kinetic measurements). six out of the 63 binders (sb#4, sb#7, sb#18, sb#34, sb#47, sb#61) were excluded from further analysis due to suboptimal behavior during sec analysis (i.e. aggregation or excessive column matrix interaction). kinetic characterization of sybodies binding onto sars-cov-2 spike proteins was performed using gci on the wavesystem (creoptix ag, switzerland), a label-free biosensor. biotinylated rbd-vyfp and ecd were captured onto a streptavidin pcp-sta wavechip (polycarboxylate quasi-planar surface; creoptix ag) to a density of 1300-1800 pg/mm 2 . sybodies were first analyzed by an off-rate screen performed at a concentration of 200 nm (data not shown) to identify binders with sufficiently high affinities. the six sybodies sb#14, sb#15, sb#16, sb#42, sb#45, and sb#68 were then injected at increasing concentrations ranging from 1.37 nm to 1 μm (three-fold serial dilution, 7 concentrations) in tbs buffer supplemented with 0.05 % tween-20. sybodies were injected for 120 s at a flow rate of 30 μl/min per channel and dissociation was set to 600 s to allow the return to baseline. sensorgrams were recorded at 25 °c and the data analyzed on the wavecontrol (creoptix ag). data were double-referenced by subtracting the signals from blank injections and from the reference channel. a langmuir 1:1 model was used for data fitting. purified recombinant hace2 protein (mybiosource, cat# mbs8248492) was diluted to 10 nm in phosphate-buffered saline (pbs), ph 7.4, and 100 μl aliquots were incubated overnight on nunc maxisorp 96-well elisa plates (thermofisher #44-2404-21) at 4°c. elisa plates were washed three times with 250 μl tbs containing 0.05% (v/v) tween-20 (tbst). plates were blocked with 250 μl of 0.5% (w/v) bsa in tbs for 2 h at room temperature. 100 μl samples of biotinylated rbd-vyfp (25 nm) mixed with individual purified sybodies (500 nm) were prepared in tbs containing 0.5% (w/v) bsa and 0.05% (v/v) tween-20 (tbs-bsa-t) and incubated for 1.5 h at room temperature. these 100 μl rbd-sybody mixtures were transferred to the plate and incubated for 30 minutes at room temperature. 100 μl of streptavidin-peroxidase (merck, cat#s2438) diluted 1:5000 in tbs-bsa-t was incubated on the plate for 1 h. finally, to detect bound biotinylated rbd-vyfp, 100 μl of development reagent containing 3,3′,5,5′-tetramethylbenzidine (tmb), prepared as previously described [36] , was added, color development was quenched after 3-5 min via addition of 100 μl 0.2 m sulfuric acid, and absorbance at 405 nm was measured. background-subtracted absorbance values were normalized to the signal corresponding to rbd-vyfp in the absence of added sybodies. purified sybodies carrying a c-terminal myc-his tag (sb_init expression vector) were diluted to 25 nm in 100 µl pbs ph 7.4 and directly coated on nunc maxisorp 96-well plates (thermofisher #44-2404-21) at 4°c overnight. the plates were washed once with 250 µl tbs ph 7.5 per well followed by blocking with 250 µl tbs ph 7.5 containing 0.5% (w/v) bsa per well. in parallel, chemically biotinylated prefusion spike protein (pfs) at a concentration of 10 nm was incubated with 500 nm sybodies for 1 h at room temperature in tbs-bsa-t. the plates were washed three times with 250 µl tbs-t per well. then, 100 µl of the pfs-sybody mixtures were added to the corresponding wells and incubated for 3 min, followed by washing three times with 250 µl tbs-t per well. 100 µl streptavidin-peroxidase polymer (merck, cat#s2438) diluted 1:5000 in tbs-bsa-t was added to each well and incubated for 10 min, followed by washing three times with 250 µl tbs-t per well. finally, to detect pfs bound to the immobilized sybodies, 100 µl elisa developing buffer (prepared as described previously [36] ) was added to each well, incubated for 1 h (due to low signal) and absorbance was measured at 650 nm. as a negative control, tbs-bsa-t devoid of protein was added to the corresponding wells instead of a pfssybody mixture. (47) 10 (12) 1) sb#51 belongs to the concave library (spill-over). 2) two sequencing reactions failed. sb#1 qvqlvesggglvqaggslrlscaasgfpvrkanmhwyrqapgkerewvaaimskgeqtvyadsve grftisrdnakntvylqmnslkpedtavyycrvfvgwhyfgqgtqvtvs sb#2 qvqlvesggglvqaggslrlscatsgfpvyqanmhwyrqapgkerewvaaiqsygdgthyadsvk grftisrdnakntvylqmnslkpedtavyycravyvgmhyfgqgtqvtvs sb#3 qvqlvesggglvqaggslrlscaasgfpvnyktmwwyrqapgkerewvaaiwsyghtthyadsvk grftisrdnakntvylqmnslkpedtavyycvvwvghnyegqgtqvtvs sb#4 qvqlvesggglvqaggslrlscaasgfpvyaqnmhwyrqapgkerewvaaiyshgywtlyadsvk grftisrdnakntvylqmnslkpedtavyycevqvgawytgqgtqvtvs sb#5 qvqlvesggglvqaggslrlscaasgfpvfsghmhwyrqapgkerewvaailsngdsthyadsvk grftisrdnakntvylqmnslkpedtavyycrvhvgahyfgqgtqvtvs sb#6 qvqlvesggglvqaggslrlscaasgfpveqgrmywyrqapgkerewvaaiishgtvtvyadsvk grftisrdnakntvylqmnslkpedtavyycyvyvgaqywgqgtqvtvs sb#7 qvqlvesggglvqaggslrlscaasgfpvlftymhwyrqapgkerewvaaiwssgnstwyadsvk grftisrdnakntvylqmnslkpedtavyycfvkvgnwyagqgtqvtvs sb#8 qvqlvesggglvqaggslrlscaasgfpvnagnmhwyrqapgkerewvaaiqsygrttyyadsvk grftisrdnakntvylqmnslkpedtavyycrvfvgmhyfgqgtqvtvs sb#9 qvqlvesggglvqaggslrlscaasgfpvssstmtwyrqapgkerewvaainsygwethyadsvk grftisrdnakntvylqmnslkpedtavyycyvyvggsyigqgtqvtvs sb#10 qvqlvesggglvqaggslrlscaasgfpvqshymrwyrqapgkerewvaaiestghhtayadsvk grftisrdnakntvylqmnslkpedtavyyctvyvgyeyhgqgtqvtvs sb#11 qvqlvesggglvqaggslrlscaasgfpvetenmhwyrqapgkerewvaaiyshgmwtayadsvk grftisrdntkntvylqmnslkpedtavyycevevgkwyfgqgtqvtvs sb#12 qvqlvesggglvqaggslrlscaasgfpvkasrmywyrqapgkerewvaaiqsfgevtwyadsvk grftisrdnakntvylqmnslkpedtavyycyvwvgqeywgqgtqvtvs sb#13 qvqlvesggglvqaggslrlscaasgfpvyasnmhwyrqapgkerewvaaiesqgymtayadsvk grftisrdnakntvylqmnslkpedtavyycwvivgeyyvgqgtqvtvs sb#14 qvqlvesggglvqaggslrlscaasgfpvqaremewyrqapgkerewvaaikstgtytayaysvk grftisrdnakntvylqmnslkpedtavyycyvyvgssyigqgtqvtvs sb#15 qvqlvesggglvqaggslrlscaasgfpvknfemewyrkapgkerewvaaiqsggvetyyadsvk grftisrdnakntvylqmnslkpedtavyycfvyvgrsyigqgtqvtvs sb#16 qvqlvesggglvqaggslrlscaasgfpvayktmwwyrqapgkerewvaaiesygikwtryadsv kgrftisrdnakntvylqmnslkpedtavyycivwvgaqyhgqgtqvtvs sb#17 qvqlvesggglvqaggslrlscaasgfpvagrnmwwyrqapgkerewvaaiyssgtyteyadsvk grftisrdnakntvylqmnslkpedtavyychvwvgslykgqgtqvtvs sb#18 qvqlvesggglvqaggslrlscaasgfpvkharmwwyrqapgkerewvaaidshgdttwyadsvk grftisrdnakntvylqmnslkpedtavyycyvyvgasywgqgtqvtvs sb#19 qvqlvesggglvqaggslrlscaasgfpvnshemtwyrqapgkerewvaaiqstgtvteyadsvk grftisrdnakntvylqmnslkpedtavyycyvyvgssylgqgtqvtvs sb#20 qvqlvesggglvqaggslrlscaasgfpveqremewyrqapgkerewvaaidsngnytfyadsvk grftisrdnakntvylqmnslkpedtavyycyvyvgksyigqgtqvtvs sb#21 qvqlvesggglvqaggslrlscaasgfpvkhhwmfwyrqapgkerewvaaiksygygteyadsvk grftisrdnakntvylqmnslkpedtavyycfvgvgthyagqgtqvtvs sb#23 qvqlvesggglvqaggslrlscaasgfpvyaaemewyrqapgkerewvaaissqgtityyadsvk grftisrdnakntvylqmnslkpedtavyycfvyvgksyigqgtqvsvs sb#25 qvqlvesggglvqaggslrlscaasgfpvhawemawyrqapgkerewvaairsfgssthyadsvk grftisrdnakntvylqmnslkpedtavyycnvkdfgthhyaydywgqgtqvtvs sb#26 qvqlvesggglvqaggslrlscaasgfpvntwwmhwyrqapgkerewvaaitswgfrtyyadsvk grftisrdnakntvylqmnslkpedtavyycnvkdkgmavqwydywgqgtqvtvs sb#27 qvqlvesggglvqaggslrlscaasgfpvyntwmewyrqapgkerewvaaitshgyktyyadsvk grftisrdnakntvylqmnslkpedtavyycnvkdegdmftaydywgqgtqvtvs sb#28 qvqlvesggglvqaggslrlscaasgfpvyhstmfwyrqapgkerewvaaiyssgqhtyyadsvk grftisrdnakntvylqmnslkpedtavyycnvkdsgqwrqeydywgqgtqvtvs sb#29 qvqlvesggglvqaggslrlscaasgfpvehemawyrqapgkerewvaairsmgrktlyadsvkg rftisrdnakntvylqmnslkpedtavyycnvkdfgytwheydywgqgtqvtvs sb#30 qvqlvesggglvqaggslrlscaasgfpvtmawmwwyrqapgkerewvaairsegvrtyyadsvk grftisrdnakntvylqmnslkpedtavyycnvkdygqahayydywgqgtqvtvs sb#31 qvqlvesggglvqaggslrlscaasgfpvnshfmewyrqapgkerewvaaiqhssgfhtyyadsv kgrftisrdnakntvylqmnslkpedtavyycnvkdtgttedydywgqgtqvtvs sb#32 qvqldesggglvqaggslrlscaasgfpvyhawmewyrqapgkerewvaaitssgrhtyyadsvk grftisrdnakntvylqmnslkpedtavyycnvkdagrvynsydywgqgtqvtvs sb#33 qvqlvesggglvqaggslrlscaasgfpvahawmewyrqapgkerewvaaitsygyktyyadsvk 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grftvsldnakntvylqmnslkpedtalyycaaateghahalyrlhyywgqgtqvtvs sb#51 qvqlvesggglvqaggslrlscaasgfpvyqgemhwyrqapgkerewvaairstgvqtwyadsvk grftisrdnakntvylqmnslkpedtavyycrvwvgthyfgqgtqvtvs sb#52 qvqlvesgggsvqaggslrlscaasgniqriyylgwfrqapgkeregvaalmtytghtyyadsvk grftvsldnakntvylqmnslkpedtalyycaaayvgaenplpysmygywgqgtqvtvs sb#53 qvqlvesgggsvqaggslrlscaasgqishikylgwfrqapgkeregvaalitrwgqtyyadsvk grftvsldnakntvylqmnslkpedtalyycaaadygasdplwfihylywgqgtqvtvs sb#55 qvqlvesgggsvqaggslrlscaasgkiwtikylgwfrqapgkeregvaalmtrwgytyyadsvk grftvsldnakntvylqmnslkpedtalyycaaanygsnfplaeedywywgqgtqvtvs sb#56 qvqlvesgggsvqaggslrlscaasgnisqihylgwfrqapgkeregvaalntdygytyyadsvk grftvsldnakntvylqmnslkpedtalyycaaayyfgddiplwweaysywgqgtqvtvs sb#58 qvqlvesgggsvqaggslrlscaasgnistieylgwfrqapgkeregvaalytwhgqtyyadsvk grftvsldnakntvylqmnslkpedtalyycaaarwgrhmplsateysywgqgtqvtvs sb#59 qvqlvesgggsvqaggslrlscaasgniesiyylgwfrqapgkeregvaalwtgdgetyyadsvk grftvsldnakntvylqmnslkpedtalyycaaaawgnsaplttyryyywgqgtqvtvs sb#61 qvqlvesgggsvqaggslrlscaasgfiygitylgwfrqapgkeregvaalvtwngqtyyadsvk grftvsldnakntvylqmnslkpedtalyycaaadwgydwplwdewywywgqgtqvtvs sb#62 qvqlvesgggsvqaggslrlscaasgtiadikylgwfrqapgkeregvaalmtrwgstyyadsvk grftvsldnakntvylqmnslkpedtalyycaaanyganyplysqqysywgqgtqvtvs sb#63 qvqlvesgggsvqaggslrlscaasgsissikylgwfrqapgkeregvaalmtrwgmtyyadsvk grftvsldnakntvylqmnslkpedtalyycaaanyganeplqythynywgqgtqvtvs sb#64 qvqlvesgggsvqaggslrlscaasgeiesifylgwfrqapgkeregvaalytyvgqtyyadsvk grftvsldnakntvylqmnslkpedtalyycaaasygaahplsimryyywgqgtqvtvs sb#65 qvqlvesgggsvqaggslrlscaasgtiahikylgwfrqapgkeregvaalmtkwgqtyyadsvk grftvsldnakntvylqmnslkpedtalyycaaasyganfplkasdysywgqgtqvtvs sb#66 qvqlvesgggsvqaggslrlscaasgsiqaitylgwfrqapgkeregvaalvtwngqtyyadsvk grftvsldnakntvylqmnslkpedtalyycaaadwgydwplwdewywywgqgtqvtvs sb#67 qvqlvesgggsvqaggslrlscaasgsissitylgwfrqapgkeregvaalvtysgntyyadsvk grftvsldnakntvylqmnslkpedtalyycaaatwghswplyndeywywgqgsqvtvs sb#68 qvqlvesgggsvqaggslrlscaasgsissitylgwfrqapgkeregvaalitvnghtyyadsvk grftvsldnakntvylqmnslkpedtalyycaaaawgyawplhqddywywgqgtqvtvs sb#69 qvqlvesgggsvqaggslrlscaasgsissitylgwfrqapgkeregvaalntfngttyyadsvk grftvsldnakntvylqmnslkpedtalyycaaatwgyswpliaeynwywgqgtqvtvs sb#71 qvqlvesgggsvqaggslrlscaasgsissitylgwfrqapgkeregvaalktqagftyyadsvk grftvsldnakntvylqmnslkpedtalyycaaanwgyswplyeaddwywgqgtqvtvs the plasmids encoding for the six highest affinity binders will very soon be available through addgene (addgene #153522 -#153527). for each of the six independent selec on reac ons, 47 clones were picked at random and analyzed by elisa. a non-randomized sybody was used as nega ve control (wells h6 and h12, respec vely). sybodies that were sequenced are marked with the respec ve sybody name (sb#1-72). please note that iden cal sybodies that were found 2-3 mes are marked with the same sybody name (e.g. sb#41) . elisa analyses shown in these graphs were performed on three different days: (1) rbd and mbp, (2) ecd, (3) . . . . concave adsvkgrftisrdnakntvylqmnslkpedtavyycx-vxvgxxyxgqgtqvtvs phylogene c tree of rbd sybodies. a radial tree was generated in clc 8.1.3. sybodies inhibit rbd binding to ace2. the effect of sybodies on rbd associa on with human ace2 was assessed with an elisa. individual sybodies (500 nm, sybody number shown on x-axis) were incubated with bio nylated rbd-vyfp (25 nm) and the mixtures were exposed to immobilized ace2. bound rbd-vyfp was detected with streptavidin-peroxidase/tmb. each column indicates background-subtracted absorbance at 405 nm, normalized to the signal corresponding to rbd-vyfp in the absence of sybody (dashed red line). simultaneous binding of sb#15 and sb#68. (a) simultaneous binding of sybodies was analyzed using gra ng-coupled interferometry on the wave system (creop x ag, switzerland). bio nylated ecd was immobilized and the binders were injected alone and simultaneously at satura ng concentra ons (sb#15: 200 nm, sb#45: 500 nm, sb#68: 500 nm). superimposed sensorgrams are shown. (b) compe on elisa. title of the graphs indicate the sybody which was directly coated on the plate at a concentra on of 25 nm. the labels on the x-axes depict the sybody used for compe on. to determine the background signal, buffer devoid of protein was added. herd immunity -estimating the level required to halt the covid-19 epidemics in affected countries the reproductive number of covid-19 is higher compared to sars coronavirus estimation of the reproductive number of novel coronavirus (covid-19) and the probable outbreak size on the diamond princess cruise ship: a data-driven analysis presumed asymptomatic carrier transmission of covid-19 estimating clinical severity of covid-19 from the transmission dynamics in wuhan, china predicting the future trajectory of covid-19 preliminary identification of potential vaccine targets for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies. viruses the sars-cov-2 vaccine pipeline: an overview use of antiviral drugs to reduce covid-19 transmission a novel coronavirus outbreak of global health concern a sars-like cluster of circulating bat coronaviruses shows potential for human emergence structure, function, and evolution of coronavirus spike proteins cryo-em structure of the 2019-ncov spike in the prefusion conformation structure, function, and antigenicity of the sars-cov-2 spike glycoprotein sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor we thank rony nehmé and andré heuer (creoptix ag, wädeswil, switzerland) for the acquisition, fitting and interpretation of gci measurements using the wavesystem. we thank florence projer, david hacker and kelvin lau (protein production and structure core facility, epfl, switzerland) for the production of the pre-fusion spike protein. we are grateful to jason mclellan (the university of texas at austin, u.s.) for having provided the pre-fusion-stabilized soluble spike expression vector. kine c characteriza on of the top six sybodies. (a) binding kine cs were measured by gra ng-coupled interferometry on the wave system (creop x ag, switzerland). rbd-vyfp and ecd were immobilized and the sybodies were injected at increasing concentra ons ranging from 1.37 nm to 1 μm. data were fi ed using a langmuir 1:1 model. key: cord-353161-mtq6yh25 authors: rodrigues, joão pglm; barrera-vilarmau, susana; teixeira, joão mc; seckel, elizabeth; kastritis, panagiotis; levitt, michael title: insights on cross-species transmission of sars-cov-2 from structural modeling date: 2020-06-05 journal: biorxiv doi: 10.1101/2020.06.05.136861 sha: doc_id: 353161 cord_uid: mtq6yh25 severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is responsible for the ongoing global pandemic that has infected more than 6 million people in more than 180 countries worldwide. like other coronaviruses, sars-cov-2 is thought to have been transmitted to humans from wild animals. given the scale and widespread geographical distribution of the current pandemic, the question emerges whether human-to-animal transmission is possible and if so, which animal species are most at risk. here, we investigated the structural properties of several ace2 orthologs bound to the sars-cov-2 spike protein. we found that species known not to be susceptible to sars-cov-2 infection have non-conservative mutations in several ace2 amino acid residues that disrupt key polar and charged contacts with the viral spike protein. our models also predict affinity-enhancing mutations that could be used to design ace2 variants for therapeutic purposes. finally, our study provides a blueprint for modeling viral-host protein interactions and highlights several important considerations when designing these computational studies and analyzing their results. introduction sars-cov-2, a novel betacoronavirus first identified in china in late 2019, is responsible for the ongoing global pandemic that has infected more than 6 million people worldwide and killed nearly 400.000 [1] . based on comparative genomics, sars-cov-2 is thought to have been transmitted to humans from an animal host, most likely bats or pangolins [2] . given the widespread human-to-human transmission across the globe, the question emerges whether humans can infect other animal species with sars-cov-2, namely domestic and farm animals. identifying potential intermediate hosts that can act as reservoirs for the virus has both important global health, animal welfare, and ecological implications. during the course of this pandemic, there have been several news reports of domestic, farm, and zoo animals testing positive for sars-cov-2 infection. belgium [3] and new york [4] reported positive symptomatic cases in cats, the netherlands reported infection of minks in farms [5] , and the bronx zoo in new york reported infections in lions and tigers [6] . in all these cases, the vehicle of transmission appears to be an infected human owner or handler. more importantly, in the case of the mink farms in the netherlands, there is evidence of human-to-animal-to-human transmission. in addition to these reported cases, several groups put forward both pre-prints and peer-reviewed studies on animal susceptibility to sars-cov-2 under controlled laboratory conditions [7] [8] [9] , two of which are of particular interest. the first study showed that cats, civets, and ferrets are susceptible to infection; pigs, chickens, and ducks are not, while the results for dogs were inconclusive [7] . a second study, using human cells expressing recombinant sars-cov-2 receptor proteins showed that camels, cattle, cats, horses, sheep, and rabbit can be infected with the virus, but not chicken, ducks, guinea pigs, pigs, mice, and rats [8] . together, these studies provide a dataset of confirmed susceptible and non-susceptible species that we can use to find molecular discriminants between the two groups. for simplicity, from here on we will refer to susceptible and non-susceptible species as sars-cov-2 pos and sars-cov-2 neg , respectively. like sars-cov-1 before, sars-cov-2 infection starts with the binding of the viral spike protein to the extracellular protease domain of angiotensin-converting enzyme 2 (ace2) [10] , a single-pass transmembrane protein expressed on the surface of a variety of tissues, including along the respiratory tract and the intestine. several biophysical and structural studies identified helices α1 and α2, as well as a short loop between strands β3 and β4 in ace2 as the interface for the viral spike protein [10] [11] [12] [13] . these studies also identified key differences between the sequences of the receptor binding domains (rbd) of sars-cov-1 and sars-cov-2, which explain the stronger interaction of the latter with human ace2. as such, we can reasonably assume that sequence variation across ace2 orthologs might explain why some animal species are susceptible to infection while others are not. in addition, combining structural and binding data with the natural diversity of ace2 across species is likely to shine a light on the key aspects that drive ace2 interaction to viral rbds and ultimately help guide the development of therapeutic molecules against sars-cov-2. unsurprisingly, several groups already published, or made available as preprints, multiple sequence and structure-based analyses of how sequence variation affects ace2 binding to sars-cov-2 rbd [14] [15] [16] [17] . two recent preprints, specifically, focus on the effects of ace2 variation on rbd binding. the first used an ace2 sequence library to select for mutants that bind rbd with high affinity, identifying several mutants that enhance or decrease affinity to the viral protein and providing a blueprint for engineering proteins and peptides with therapeutic purposes [14] . while useful, we note that the authors carried out a single round of selection as opposed to the multiple rounds commonly carried out in similar studies. the second study used computational modeling to predict δδg of mutations in 215 animal species and assess their risk for infection [15] . in addition, the authors also identified a number of locations on ace2 that contribute to binding the viral rbd, in particular residues 31, 38, 353, as well as a cluster of n-terminal hydrophobic amino acid residues. in this study, we aimed to leverage structural, binding, and sequence data to investigate how different ace2 orthologs bind to sars-cov-2 rbd. we selected 29 animal species likely to encounter humans in a variety of residential, industrial, and commercial settings. for each of these species, we generated 3d models of ace2 bound to rbd and refined these models using short molecular dynamic simulations. after refinement, we found that models of sars-cov-2 pos species generally have a lower (better) score than those of sars-cov-2 neg species. further, we carried out a per-residue energy analysis that identified key locations in ace2 that are consistently mutated across sars-cov-2 neg species. collectively, our results provide a structural framework that explains why certain animal species are not susceptible to sars-cov-2 infection, and also suggests potential mutations that can enhance binding to the viral rbd. sequence conservation of ace2 orthologs we analyzed the sequence conservation of ace2 across our dataset, with respect to the entire sequence (591 residues) and to the interface residues computed from a structure of ace2 bound to rbd (pdb id: 6m17) (22 residues) ( table 1 ). all orthologs are reasonably conserved, with global similarity values to the human ace2 sequence (hace2) ranging from 72% (goldfish) to 99.5% (chimpanzee) (figure 1 , left panel). all species coarsely cluster in three classes consistent with evolutionary distance to humans: primates have the highest similarity values, followed by other mammals, birds and reptiles, and finally fish. zooming in on the interface residues, we find substantially more variation (figure 1, right panel) . similarity values for these residues range from 50% (crocodile) to 100% (all 3 primates) but, despite an overall correlation (pearson r 2 of 0.69), do not always match global similarities. hedgehogs and sheep, for example, share 86.7% and 86.4% global similarity with hace2, respectively, but 59% and 95.5% for the interface region. in absolute numbers, these similarities mean that sheep share 21 out of 22 residues with hace2 at the interface with rbd, while hedgehogs share 13. the horseshoe bat, one of the proposed animal reservoirs for sars-cov-2, shares 72.2% interface similarity with hace2, a comparable value to the 77.3% of the sars-cov-2 neg mouse sequence. altogether, these results prompt two observations. first, neither global nor interface sequence similarity is predictive of sars-cov-2 susceptibility. second, that the interface of the viral rbd is substantially plastic and able to bind to sufficiently different ace2 orthologs. refinement of the hace2:rbd complex in order to validate the refinement protocol used in our analysis, we created and refined models of human ace2 (hace2) bound to sars-cov-2 rbd. we used the cryo-em structure of full-length human ace2 bound to the rbd, in the presence of the amino acid transporter b 0 at1 (pdb id: 6m17). compared to a high-resolution crystal structure of the same complex (pdb id: 6m0j), the cryo-em structure lacks several key contacts between our two proteins of interest, which we attribute to poor density for side-chain atoms at the interface region. our refinement protocol restores the majority of these contacts (table s1) , yielding an average haddock score of -116.2 (arbitrary units) for the 10 best models of the best cluster. see materials and methods for further details on the protocol. these negative haddock scores suggest a favorable interface and agree with scores calculated for a reference set of transient protein-protein interactions (n=144, haddock score=-124.9 ± 53.4) [18] . the interfaces in our models are dominated by hydrogen bond interactions involving the ace2 α1 helix and a small loop between strands β3 and β4. there is one single salt-bridge involving hace2 d30 and rbd k417 consistently present in all our hace2 models. these observations all agree with the published crystal structure. further, the buried surface area of the refined models is also in agreement with published crystal structures (~1800 å 2 ). as such, considering the low quality of the interface region in our template structure, we are confident that our modeling and refinement protocol is robust enough to model all ace2 orthologs. refinement of orthologous ace2:rbd complexes we modeled and refined complexes for all 29 ace2 orthologs in our dataset (table 1 ) using the same protocol as above. the representative models for each species (10 best models of the best cluster) are available for visualization and download at https://joaorodrigues.github.io/ace2-animal-models/. the haddock scores of all 30 ace2 complexes (including hace2) range from -137.5 (dog) to -93.2 (mouse), a significant range that indicates substantial differences between these interfaces (table 2 and figure 2 ). the average haddock score is -116.4, very close to that of the human complex (-116.2). overall, models of sars-cov-2 pos species have consistently lower (better) scores than those of sars-cov-2 neg species. although it is well-known that docking scores do not quantitatively correlate with experimental binding affinities [19] , these scores suggest that sars-cov-2 neg species lack one or more key ace2 residues that contribute significantly to the interaction with rbd. to understand what forces drive the interactions between ace2 and sars-cov-2 rbd, we quantified the contribution of each component of the haddock scoring function to the overall score ( figure 3 ). the haddock score is a linear combination of van der waals, electrostatics, and desolvation energy terms. in our models, electrostatics are the most discriminatory component (pearson r 2 of 0.60), followed by desolvation (0.31), and finally van der waals (0.08). these correlations suggest that differences between the models of the different species originate primarily in polar and charged residues, in agreement with observations from experimental structures. in addition, the buried surface area of the models also correlates quite strongly with the haddock score (pearson r 2 of 0.66), which is unsurprising since larger interfaces tend to make more contacts. most models bury between 1700 and 1850 å 2 , in agreement with the crystal and cryo-em structures, while the topscoring species (dog and goldfish) bury nearly 2000 å 2 and the lowest-scoring (mouse) bury only 1600 å 2 . finally, there is a weak correlation between the average haddock score of the representative models and the sequence similarity of the ace2 interface residues (pearson r 2 of 0.18) ( figure s1 ). per-residue energetics of the ace2:rbd interface to gain further insight on how ace2 sequence variation across the different orthologs affects binding to sars-cov-2 rbd, we calculated haddock scores for each interface residue in the refined models ( figure 4 ). this high-resolution analysis reveals several sites that discriminate between sars-cov-2 pos and sars-cov-2 neg species. the first and most relevant of these sites is amino acid 30, which in hace2 (d30) interacts with rbd k417 to form the only intermolecular salt-bridge of the interface. in all 12 sars-cov-2 pos species, this site is occupied by a negatively charged amino acid residue. in contrast, 4 out of 5 sars-cov-2 neg species have a hydrophobic or polar residue at this position. the goldfish ace2 sequence is an interesting outlier, with the second-best haddock score despite having a lysine at position 30 that breaks the intermolecular salt-bridge. the loss of such an important site is compensated by the introduction of an alternative salt-bridge between e34 and rbd r403. finally, the sequences of the top-scoring models also suggest that between aspartate and glutamate, the latter results in a stronger interaction, likely due to a stabilizing effect of the longer side-chain. the second site is amino acid 31, a lysine in hace2, and in nearly all of the sars-cov-2 pos species, that interacts both with ace2 e35 and rbd q493. the only exceptions are the civet and dromedary sequences, mutated to threonine and glutamate, respectively. in the case of the civet, our models show that t31 can still hydrogen bond with both e35 and rbd q493. dromedaries, on the other hand, share e31 with chickens, guinea pigs, and ducks, all sars-cov-2 neg species. however, and quite beautifully, dromedaries compensate the possible electrostatic repulsion between e31 and e35 with a lysine at position 76 (q76 in hace2) leading to the formation of an additional intramolecular salt-bridge that stabilizes the fold of ace2 and frees e35 to hydrogen bond with q493 in 90% of our models. all three sars-cov-2 neg species have an additional charge-reversal mutation at position 35, although with different outcomes in our models. in both chicken and duck ace2, e31 is locked in an intramolecular salt-bridge with r35 in all of our models, losing the intermolecular hydrogen bond with rbd q493. lastly, guinea pigs compensate k31e with e35k and remain able to hydrogen bond with rbd. the third discriminatory site between sars-cov-2 pos and sars-cov-2 neg species is amino acid 34, a histidine in hace2 and a polar residue in all sars-cov-2 pos species. in our hace2 models, h34 is doubly-protonated and forms an intramolecular salt-bridge/hydrogen bond with e37 and an intermolecular hydrogen bond with the hydroxyl group of rbd y453. in addition, in most of our models, the aromatic ring of h34 is close enough (<4.5 å) to the aliphatic side-chain of rbd l455 to form productive hydrophobic interactions. our energetic analysis shows that substituting h34 by polar (serine, threonine) or hydrophobic (leucine, valine) residues destabilizes the interface, while substitution by a tyrosine substantially contributes to a stronger interaction. sars-cov-2 neg species except mouse and rat have hydrophobic residues at position 34, losing the ability to hydrogen bond with rbd y453. in addition, the side-chain of rbd l455 is often out of range of hydrophobic interactions. in contrast, the h34y substitution in the dog, ferret, and civet sequences loses the intramolecular hydrogen bond with e37 but compensates by hydrophobic interactions with nearby rbd residues and hydrogen bonds with rbd r403 (ferret), s494 (civet) or y495 (dog). in addition, the loss of aromatic residues at position 34 leads to a steep decrease in desolvation energy of the models( figure s2 ). . cc-by-nc-nd 4.0 international license was not certified by peer review) is the author/funder. it is made available under a the copyright holder for this preprint (which this version posted june 5, 2020. . besides these three major discriminatory sites, we identified three other sites that are systematically mutated in sars-cov-2 neg species. the first of these sites is k353 (in hace2), which is involved in an intramolecular saltbridge with d38 and an additional backbone hydrogen bond with rbd g502. in rat and mouse ace2, both sars-cov-2 neg species, this residue is mutated to a histidine, which weakens the interaction with d38, possibly leading to increased conformational dynamics of the β3-β4 loop and consequently lower binding affinity. then, position 42, a glutamine in hace2 and in most other species, hydrogen bonds with rbd y449 in the majority of our models. in canary, chicken, pigeon, hedgehog, duck, and crocodile ace2 sequences, this amino acid is mutated to a glutamate. this substitution introduces the possibility of an additional intramolecular salt-bridge with k68, in ace2 helix α2, which we observe in some of our models, preventing the formation of the intermolecular hydrogen bond. finally, y83 in hace2 is mutated to phenylalanine in canary, chicken, rat, duck, and mouse ace2, mostly sars-cov-2 neg species. although our models do not offer a clear reason as to why this mutation could be damaging to rbd binding, the loss of the terminal hydroxyl group could have two negative consequences. first, there is the clear loss of two possible hydrogen bonds, with ace2 q24 and rbd n487. then, the gain in hydrophobicity could lead the aromatic moiety to bury between both α1 and α2 helices, causing rbd f486 to lose a valuable interaction partner. all our models and the scoring statistics are available for visualization and download at https://joaorodrigues.github.io/ace2-animal-models/. can structural modeling predict cross-species transmission of sars-cov-2? our computational modeling of 30 vertebrate ace2 orthologs bound to sars-cov-2 rbd discriminates between previously reported sars-cov-2 pos and sars-cov-2 neg species. models of sars-cov-2 neg species -chicken, duck, guinea pig, mouse, and rat -generally have higher (worse) haddock scores than average (figure 2 ), suggesting that these species' non-susceptibility to infection could stem from deficient rbd binding to ace2. despite this clear trend, there are two notable outliers. our modeling ranks guinea pig ace2 (sars-cov-2 neg ) as a better receptor for sars-cov-2 rbd than for example, human, cat, horse, or rabbit ace2 (all sars-cov-2 pos species), despite experiments showing that there is negligible binding between the two proteins [8] . then, the goldfish ace2 sequence ranks second among all models, despite reports that fishes are unlikely to be susceptible to infection due to their physiology and environment [20] . these two results highlight the need for critical thinking when evaluating predictions from computational models. as noted earlier in the introduction, sars-cov-2 infection is a complex multi-step process [21] . thus, while we can assume that impaired ace2 binding decreases odds of infection, we cannot state that ace2 binding is predictive of infection. for instance, experiments with recombinant ace2 show that the pig ortholog binds sars-cov-2 rbd and leads to entry of the virus in host cells [8] , but tests in live animals returned negative results [7] . in addition, our modeling protocol makes assumptions about the bound state of the two proteins, starting from the cryo-em template structure. however, cryo-em structures of the full-length sars-cov-2 spike protein [22] highlight multiple unbound conformations for rbd, and coarse-grained simulations of the hace2:rbd complex show that there is substantial flexibility in some of the interfacial rbd loops [23]. altogether, these limitations show that computational models alone cannot predict whether certain animal species are at risk of infection. what our models do predict, however, is that there are distinctive molecular features characteristic of sars-cov-2 neg species. as the adage goes, 'all models are wrong, but some are useful.' sars-cov-2 neg species lack important polar and charged ace2 residues on further inspection, we find that sars-cov-2 neg models rank worse due to a substantial decrease in electrostatic energy (figure 3 ), indicating loss of polar interface contacts, namely hydrogen bonds and saltbridges ( figure 4) . indeed, models of mouse, duck, rat, and chicken lack the ability to form an intermolecular salt-bridge with rbd due to the loss of hace2 d30. these predictions are supported by experimental work, where mutants lacking a negative charge at this position are largely unable to bind rbd [14] . non-conservative mutations at other sites on ace2 also contribute negatively to the interface scores. residues k31 and h34 (hace2) engage multiple neighboring residues in both intra-and intermolecular hydrogen bonds, contributing both to ace2 fold stability and rbd binding, respectively. our models suggest that the introduction of a negatively charged residue at position 31 is disruptive to binding, in agreement with experiments [14] . in sars-cov-2 pos species, like dromedary camels, this mutation is more likely to be tolerated due to additional compensatory mutations that stabilize the ace2 fold and still allow for contacts with rbd. in all sars-cov-2 neg species except guinea pig however, there are no additional mutations to compensate for this substitution. as for position 34, our predictions contrast with experimental measurements [14] , which show that mutation to a hydrophobic residue improves binding between ace2 and rbd. in our models, the preference seems to be for aromatic residues (histidine, tyrosine) capable of both hydrogen bonds and hydrophobic interactions. we note, however, that our coverage of sequence space is limited to naturally occurring variants. unlike in the work referenced before [14] , where the selection driver is rbd binding, natural selection of ace2 might impose additional constraints on sequence variability. finally, our models suggest that reduced flexibility of ace2 might be a positive contributor to rbd binding affinity. disrupting an intramolecular salt-bridge between d38 and k353 by substituting k353 with a shorter polar amino acid residue is a consistent feature in mice and rats, both sars-cov-2 neg species. these results support other computational modeling work [17] that suggest that rbd mutants g496d bind worse to ace2 because of the disruption of this intramolecular salt-bridge. natural variants of ace2 encode potential affinity-enhancing mutations for sars-cov-2 rbd in addition to identifying mutations that impair binding of sars-cov-2 rbd, our models suggest several hace2 variants that could be used to enhance affinity between the two proteins. the clearest affinity enhancer seems to be d30e, a variant observed in 6 of the 8 best scoring species ( figure 4 ) and shown in experiments to increase binding to rbd [8, 14] . the longer side-chain of a glutamate residue can help strengthen and stabilize the intermolecular salt-bridge with rbd k417. the impact of such conservative mutations in stabilizing protein interactions has been reported previously for other systems [24] . the second predicted enhancer is h34y, which as we discussed above, contrasts with experimental measurements. in addition to maintaining hydrogen bonds and hydrophobic contacts, our models show that this mutation results in a substantial increase in desolvation energy ( figure s2 ). in summary, our protocol combines structural, sequence, and binding data to create a structure-based framework to understand sars-cov-2 susceptibility across different animal species. our models help rationalize the impact of naturally-occurring ace2 mutations on sars-cov-2 rbd binding and explain why certain species are not susceptible to infection with the virus. in addition, we propose possible affinityenhancing mutants that can help guide engineering efforts for the development of ace2-based antiviral therapeutics. despite the aforementioned limitations, our protocol and models can easily be replicated using freely-available tools and web servers and serve as a blueprint for future modeling studies on ace2 interactions with coronaviruses' rbds. finally, to prevent human-to-animal transmission, we recommend following the world organization for animal health guidelines: people infected with covid-19 should limit contact with their pets, as well as with other animals (including humans). sequence alignment of ace2 orthologs sequences of ace2 orthologs from 28 species were retrieved from ncbi using the human gene as a reference (gene id: 59272, updated on 20-apr-2020) and the query term "ortholog_gene_59272[group]". other species, such as rhinolophus sinicus, were manually included using custom queries. the sequences were aligned with mafft version 7 [25, 26] , using the alignment method fft-ns-i (standard). some sequences had undefined amino acids ('x'), which we converted to glycine to allow modeling without any bias for amino acid identity. all species and the respective protein identifiers are listed in table 1 . . cc-by-nc-nd 4.0 international license was not certified by peer review) is the author/funder. it is made available under a the copyright holder for this preprint (which this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.05.136861 doi: biorxiv preprint all calculations were based on the alignments from mafft, restricted to the region used for modeling (residues 21-600). to calculate sequence similarity, we considered the following groups based on physico-chemical properties: charged-positive (arg, lys, his), charged-negative (asp, glu), aromatic (phe, tyr, trp), polar (ser, thr, asn, gln), and apolar (ala, val, ile, met). cys, gly, and pro residues were considered individual classes. the modeling of ace2 orthologs was carried out using modeller 9.24 [27] and custom python scripts (available upon request). we used the cryo-em structure of the sars-cov-2 rbd bound to human ace2 (pdb id: 6m17) [12] as a template for all our subsequent models, including all glycans and the coordinates of rbd. to save computational resources, we modeled only the extracellular domain of ace2, specifically residues 21-600, which are known to be sufficient to bind to rbd. to avoid unwanted deviation from the initial cryo-em structure, we restricted the optimization and refinement of the models to the coordinates of atoms of mutated or inserted residues. we used the fastest library schedule for model optimization and the very_fast schedule for model refinement. for each species, we generated 10 backbone or loop models and selected the one with the lowest normalized dope score as a representative. these final models were then processed to remove any sugar molecules in species where the respective asparagine residue had been mutated. the initial complex models were prepared for refinement using the pdb-tools suite [28] . each chain was separated into a different pdb file (pdb_selchain) and standardized with ter and end statements (pdb_tidy). we used haddock 2.4 [29] to carry out the refinement of the models. the protein molecules were parameterized using the standard force field in haddock, while the sugars were parameterized using updated parameters for carbohydrates [30] . we used a modified version of the topology generation scripts to allow automatic detection of n-linked glycans and expand the range of the interface refinement (10 å distance cutoff). each initial homology model was refined through 50 independent short molecular dynamics simulations in explicit solvent (solvshell=true). these refined models were then clustered using the fcc algorithm [31] with default parameters and scored using the haddock score, a linear combination of van der waals, electrostatics, and desolvation. a lower haddock score is better. the top 10 models of the top scoring cluster, ranked by its average haddock score, were selected as representatives of the complex. analysis of interface contacts of refined ace2:rbd complexes we used the interfacea analysis library (version 0.1) (http://doi.org/10.5281/zenodo.3516439) to identify intermolecular contacts between hace2 and rbd, specifically hydrogen bonds, salt bridges, and aromatic ring stacking. hydrogen bonds were defined between any donor atom (nitrogen, oxygen, or sulfur bound to a hydrogen atom) within 2.5 å of an acceptor atom (nitrogen, oxygen, or sulfur), if the donor-hydrogen-acceptor angle was between 120 and 180 degrees. salt bridges were defined between two residues with a pair of cationic/anionic groups within 4 å of each other. finally, two aromatic residues were defined as stacking if the centers of mass of the aromatic groups were within 7.5 å (pi-stacking) or 5 å (t-stacking) and the angle between the planes of the rings was between 0 and 30 degrees (pi-stacking) or between 60 and 90 degrees (t-stacking). additionally, for pi-stacking interactions, the projected centers of both rings must fall inside the other ring. for each modelled species, we took the 10 best models of the best cluster, judged by their haddock score, and aggregated all their contacts together. contacts present in at least 5 models were considered representative. per-residue decomposition of haddock scores we used a custom cns [32] script to calculate the haddock score of each residue at the interface between ace2 and rbd. briefly, the protocol was the following. for each model, since haddock uses a united-atom force field, we first added missing hydrogen atoms and minimized their coordinates, keeping all other atoms fixed. we marked a residue of ace2 as part of the interface if any of its atoms were within 5 å of any atom of rbd, and vice-versa. we then calculated the electrostatics, van der waals, and desolvation energies for each of these residues, considering only atoms belonging to the other protein chain. note that this protocol does not account for intramolecular effects of mutations. finally, we calculated the haddock score per residue, using the default scoring function weights, and averaged per-residue values for the best 10 models of the best cluster of each species. . cc-by-nc-nd 4.0 international license was not certified by peer review) is the author/funder. it is made available under a the copyright holder for this preprint (which this version posted june 5, 2020. . . cc-by-nc-nd 4.0 international license was not certified by peer review) is the author/funder. it is made available under a the copyright holder for this preprint (which this version posted june 5, 2020. . figure 1 . sequence similarity of ace2 orthologs to human ace2. global sequence similarity values range from 70-95%, while similarity values for residues interacting with sars-cov-2 rbd (derived from 6m17) range from 50-100%. species are ordered by decreasing global sequence similarity to human ace2. colors indicate known susceptibility to infection: sars-cov-2 pos species in green, sars-cov-2 neg species in red, others in gray. . cc-by-nc-nd 4.0 international license was not certified by peer review) is the author/funder. it is made available under a the copyright holder for this preprint (which this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.05.136861 doi: biorxiv preprint figure 2 . haddock scores of modeled ace2 orthologs bound to sars-cov-2 rbd. the haddock score predicts the strength of the interaction between proteins. models of sars-cov-2 pos species (green) generally have better (more negative) scores than sars-cov-2 neg species (red), suggesting that impaired binding between the two proteins might explain differences in viral susceptibility. the scores shown here are the average of the 10 best models for each ace2 ortholog. . cc-by-nc-nd 4.0 international license was not certified by peer review) is the author/funder. it is made available under a the copyright holder for this preprint (which this version posted june 5, 2020. . figure 3 . correlation of haddock score with individual energy terms and structural features. differences in electrostatics energy contribute the most towards discriminating sars-cov-2 pos species (green) from sars-cov-2 neg species (red), supporting observations of hydrogen bonding networks and charged interactions in experimental structures. the buried surface area of the models is also correlated with their haddock score. the units for van der waals and electrostatics energies, desolvation, and buried surface area are kcal.mol -1 , arbitrary units, and å 2 , respectively. the human complex is shown in black for reference. . cc-by-nc-nd 4.0 international license was not certified by peer review) is the author/funder. it is made available under a the copyright holder for this preprint (which this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.05.136861 doi: biorxiv preprint figure 4 . haddock score of individual ace2 interface residues. amino acid residues at positions 30, 31, 34, and 353 are predicted to be the largest contributors to the stability of the interface. sars-cov-2 neg (red labels) species consistently show changes in these positions which could explain their non-susceptibility to the virus. the top-scoring sars-cov-2 pos (green labels) also suggest that hace2 d30e and h34y could potentially act as affinity enhancers. for each species, each block represents an interface residue of ace2. the identity of the amino acid is shown in one-letter codes. the colors represent the average haddock score of each particular residue over the best 10 models: lower scores (blue) indicate more favorable interactions. positive scores (dark red) indicate steric clashes or electrostatic repulsion. blank squares indicate that in that ortholog, that position is not part of the interface of the complex. residues marked with *, and ** are observed to form hydrogen bonds or salt-bridges in the hace2:rbd crystal structure, respectively. see materials and methods for additional details on definitions. . cc-by-nc-nd 4.0 international license was not certified by peer review) is the author/funder. it is made available under a the copyright holder for this preprint (which this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.05.136861 doi: biorxiv preprint figure s1 . correlation between interface sequence similarity to hace2 and haddock score. . cc-by-nc-nd 4.0 international license was not certified by peer review) is the author/funder. it is made available under a the copyright holder for this preprint (which this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.05.136861 doi: biorxiv preprint figure s2 . desolvation energy of individual ace2 interface residues. aromatic residues at position 34 contribute the most to the gain in desolvation energy across all species of the complex, indicating that h34y could be a potential affinity enhancing mutation in hace2. for each species, each block represents an interface residue of ace2. the identity of the amino acid is shown in one-letter codes. the colors represent the average desolvation energy of each particular residue over the best 10 models: lower scores (blue) indicate more favorable interactions. blank squares indicate that in that ortholog, that position is not part of the interface of the complex. residues marked with *, and ** are observed to form hydrogen bonds or salt-bridges in the hace2:rbd crystal structure, respectively. see materials and methods for additional details on definitions. . cc-by-nc-nd 4.0 international license was not certified by peer review) is the author/funder. it is made available under a the copyright holder for this preprint (which this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.05.136861 doi: biorxiv preprint an interactive web-based dashboard to track covid-19 in real time the proximal origin of sars-cov-2 a cat appears to have caught the coronavirus, but it's complicated mink infected two humans with coronavirus: dutch government. reuters. 25 seven more big cats test positive for coronavirus at bronx zoo. in: animals [internet susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2 potential host range of multiple sars-like coronaviruses and an improved ace2-fc variant that is potent against both sars-cov-2 and sars-cov-1. microbiology simulation of the clinical and pathological manifestations of coronavirus disease 2019 (covid-19) in golden syrian hamster model: implications for disease pathogenesis and transmissibility structural and functional basis of sars-cov-2 entry by using human ace2 structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor structural basis for the recognition of sars-cov-2 by fulllength human ace2 structural basis of receptor recognition by sars-cov-2 the sequence of human ace2 is suboptimal for binding the s spike protein of sars coronavirus 2. biochemistry sars-cov-2 spike protein predicted to form stable complexes with host receptor protein orthologues from mammals receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus sars-cov-2, an evolutionary perspective of interaction with human ace2 reveals undiscovered amino acids necessary for complex stability proteins feel more than they see: fine-tuning of binding affinity by properties of the non-interacting surface are scoring functions in protein−protein docking ready to predict interactomes? clues from a novel binding affinity benchmark viewpoint: sars-cov-2 (the cause of covid-19 in humans) is not known to infect aquatic food animals nor contaminate their products the trinity of covid-19: immunity, inflammation and intervention structure, function, and antigenicity of the sars-cov-2 spike glycoprotein mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform aleaves facilitates on-demand exploration of metazoan gene family trees on mafft sequence alignment server with enhanced interactivity comparative protein modelling by satisfaction of spatial restraints pdb-tools: a swiss army knife for molecular structures the haddock2.2 web server: user-friendly integrative modeling of biomolecular complexes compatible topologies and parameters for nmr structure determination of carbohydrates by simulated annealing clustering biomolecular complexes by residue contacts similarity version 1.2 of the crystallography and nmr system sheep -126 acknowledgements jpglmr acknowledges support from the molecular sciences software institute (aci-1547580). jpglmr and ml acknowledge funding from the national institutes of health usa (r35gm122543). plk acknowledges funding from the federal ministry for education and research (bmbf, zik program) (02z22hn23) and the european regional development funds for saxony-anhalt (efre: zs/2016/04/78115). the authors thank t. dots, k. lindorff-larsen, j. puglisi, and r. fernandes for feedback and encouragement during the course of the project. key: cord-285900-3rr0j5tk authors: du, lanying; tai, wanbo; yang, yang; zhao, guangyu; zhu, qing; sun, shihui; liu, chang; tao, xinrong; tseng, chien-te k.; perlman, stanley; jiang, shibo; zhou, yusen; li, fang title: introduction of neutralizing immunogenicity index to the rational design of mers coronavirus subunit vaccines date: 2016-11-22 journal: nat commun doi: 10.1038/ncomms13473 sha: doc_id: 285900 cord_uid: 3rr0j5tk viral subunit vaccines often contain immunodominant non-neutralizing epitopes that divert host immune responses. these epitopes should be eliminated in vaccine design, but there is no reliable method for evaluating an epitope's capacity to elicit neutralizing immune responses. here we introduce a new concept ‘neutralizing immunogenicity index' (nii) to evaluate an epitope's neutralizing immunogenicity. to determine the nii, we mask the epitope with a glycan probe and then assess the epitope's contribution to the vaccine's overall neutralizing immunogenicity. as proof-of-concept, we measure the nii for different epitopes on an immunogen comprised of the receptor-binding domain from mers coronavirus (mers-cov). further, we design a variant form of this vaccine by masking an epitope that has a negative nii score. this engineered vaccine demonstrates significantly enhanced efficacy in protecting transgenic mice from lethal mers-cov challenge. our study may guide the rational design of highly effective subunit vaccines to combat mers-cov and other life-threatening viruses. a major goal of viral subunit vaccine development is to rationally design immunogens that can elicit strong neutralizing immune responses in hosts [1] [2] [3] [4] . the receptorbinding domains (rbds) of virus surface spike proteins are the prime candidates for subunit vaccine design because they contain epitopes that can trigger strong immune responses 5 . in addition, viral rbds play essential roles in viral infection cycles by binding to their host receptor for viral attachment 6 . thus, part of the host immune responses elicited by viral rbds can target the receptorbinding region and thereby neutralize viral entry into host cells. however, two problems potentially hinder the development of viral rbds as subunit vaccines. first, viruses can evade the host immune responses elicited by their own spikes or rbd-based vaccines. one of the immune evasion mechanisms by viruses is to use immunodominant non-neutralizing epitopes on their rbds to divert host immune responses, which has been thoroughly illustrated in the case of the hiv receptor-binding subunit gp120 (refs 1,3) . second, when taken out of the context of the full-length spike proteins, recombinant viral rbd vaccines expose large areas of previously buried surfaces that likely contain immunodominant non-neutralizing epitopes. whether an outcome of viral evolution or vaccine design, these immunodominant non-neutralizing epitopes on viral rbds can outcompete other epitopes in triggering host immune responses, so that the resulting immune responses target these non-neutralizing epitopes while neglecting neutralizing epitopes on viral rbds (refs 7-10) . rational design of viral subunit vaccines aims to focus the immune responses on neutralizing epitopes through masking or deletion of immunodominant non-neutralizing epitopes [11] [12] [13] . a critical gap in subunit vaccine design is the lack of an effective way to evaluate an epitope's neutralizing immunogenicity (that is, its capacity to elicit neutralizing immune responses). there have been extensive efforts to predict epitopes' immunogenicity based on the physical and chemical properties of the epitopes 14 . however, these methods are not designed to predict epitopes' 'neutralizing' immunogenicity, which holds the key for subunit vaccine design. although some experimental methods are available to measure the neutralizing immunogenicity of linear epitopes by taking linear peptides out of the context of proteins 15, 16 , these methods do not work for conformational epitopes, which are prevalent on rbd-based viral vaccines 5 . finding a way to measure the neutralizing immunogenicity of different conformational epitopes on viral rbds will tremendously help rational design of viral subunit vaccines. rbd-based coronavirus vaccines have been extensively pursued due to the threat that coronaviruses pose to human health. coronaviruses are enveloped and positive-stranded rna viruses. in 2002-2003, sars coronavirus (sars-cov) infected over 8,000 people with b10% fatality rate 17, 18 . since 2012, mers coronavirus (mers-cov) has infected about 1700 people with b36% fatality rate 19, 20 . the rbds from sars-cov and mers-cov both contain a core structure and a receptor-binding motif (rbm). their core structures are highly similar, but their rbms are markedly different [21] [22] [23] [24] , leading to different receptor specificity: sars-cov recognizes angiotensin-converting enzyme 2 (ace2), whereas mers-cov recognizes dipeptidyl peptidase 4 (dpp4) 6, 25, 26 . both sars-cov and mers-cov rbds are capable of eliciting strong neutralizing antibody responses 5, [27] [28] [29] [30] . on the one hand, because of the enriched neutralizing epitopes in their rbm and their high-yield expression as recombinant proteins, coronavirus rbds are promising subunit vaccine candidates. moreover, because of their relatively simple structures compared with the intact spike proteins, coronavirus rbds provide an excellent model system for structure-based subunit vaccine design. on the other hand, recently determined cryo-em structures of coronavirus spike proteins revealed that whereas the rbm of coronavirus rbds is accessible, large surface areas of the rbd core structure are buried in the full-length spike proteins (supplementary fig. 1) 31, 32 . thus, when these previously buried areas on the surface of the rbd core become exposed in recombinant rbd vaccines, they likely contain immunodominant non-neutralizing epitopes that divert host immune responses. therefore, coronavirus rbds both hold promises and present challenges for vaccine development. it is critical to evaluate the neutralizing immunogenicity of different epitopes on coronavirus rbds, such that immunodominant neutralizing and non-neutralizing epitopes can be preserved and eliminated, respectively. in this study we introduce a novel concept 'neutralizing immunogenicity index' (nii) to evaluate the neutralizing immunogenicity of different epitopes on viral subunit vaccines. as proof-of-concept, we used nii as a tool to identify epitopes with different neutralizing immunogenicity on a mers-cov-rbdbased vaccine. furthermore, we successfully applied this tool and significantly enhanced the efficacy of the mers-cov rbd vaccine in protecting human-dpp4-transgenic mice from lethal mers-cov challenge. our study fills in a critical gap in subunit vaccine design, and can facilitate rational design of subunit vaccines against mers-cov and other life-threatening viruses. to evaluate the neutralizing immunogenicity of a specific epitope on viral rbd vaccines, we can either delete or mask the epitope and then measure the corresponding changes in the vaccine's capacity to elicit neutralizing immune responses. alanine scanning of vaccine-surface residues likely leads to changes in the vaccine's overall immunogenicity that are too subtle to be measurable using currently available experimental methods, while deletion of a whole epitope may disturb the tertiary structure of the viral rbd. instead, in this study we chose to mask the epitope of interest using a host-cell-derived glycan probe. this approach is effective and convenient because the glycan probe can impose steric interference for the access of antibodies and immune cells to the epitope, and also because the glycan probe is unlikely to interfere with the folding and solubility of the rbd. to place the glycan probe on an epitope, we introduced the n-linked glycosylation motif, asparagine-x-threonine (where x is any amino acid other than proline) 33 , onto different epitopes on viral rbd vaccines using site-directed mutagenesis. as proof-of-concept, we chose to study several epitopes on the mers-cov rbd vaccine. the fc-tagged rbd fragment containing residues from 377 to 588 was selected in this study because we previously showed that this fragment is a stable and effective vaccine candidate 34 . four distinct epitopes on this mers-cov rbd fragment were selected based on their location on the rbd surface and their possible functional role in receptor binding: (i) arg511 (located on a protruding loop and in the receptor-binding motif (rbm) region); (ii) ala562 (located on a b-strand and in the rbm region); (iii) val403 (located on a b-strand and in the core region); (iv) thr579 (located on a protruding loop and in the core region) (fig. 1a,b) . on the basis of three-dimensional protrusion index map ( supplementary fig. 2) 35 , the epitopes containing arg511 and thr579 both have a high protrusion index, whereas the epitopes containing ala562 and val403 both have a low protrusion index. we introduced a glycan probe onto each of the above four epitopes on mers-cov rbd. to this end, we introduced single mutations v403n, t579n and a562n to pair with the already existent thr405, thr581 and thr564, respectively, to generate three n-linked glycosylation sites. we also introduced double mutations r511n/e513t to generate the fourth n-linked glycosylation site. each of these glycosylation sites was located in an individual mers-cov rbd fragment. we expressed and purified each of the four mutant rbds in mammalian cells ( supplementary fig. 3a ). to test whether each of the above four epitopes on mers-cov rbd was actually glycosylated, we performed both sds gel electrophoresis and mass spectrometry. compared with the wild type rbd, each of the mutant rbds exhibited a slower electrophoretic mobility on the gel, consistent with additional glycosylation (supplementary fig. 3a ). mass spectrometry revealed that the molecular weights of the mutant rbds were b1-2 kda larger than that of the wild type rbd, which was also consistent with an introduced glycan probe in each of the mutant rbds ( supplementary fig. 3b-f) . for each of the purified mutant rbd samples, there was no visible presence of unglycosylated rbd on the sds gel or the mass spectrometry spectrum ( supplementary fig. 3) . thus, each of the four epitopes on mers-cov rbd had been successfully glycosylated. to understand the correlation between the epitopes' role in receptor binding and their potential to be recognized by immune responses, we examined whether these engineered glycan probes on mers-cov rbd interfered with receptor binding. to this end, we used two alternative approaches. one approach was an alphascreen assay, which analysed the interaction between recombinant rbds and recombinant human dpp4 in solution (fig. 1c) , and the other approach was fluorescence-activated cell sorting (facs), which examined the interaction between recombinant rbds and human dpp4 expressed on the huh-7 cell-surface (fig. 1d) . the results from both assays revealed that the glycan probe located at residue 562 reduced the binding of the rbd to dpp4, the glycan probe located at residue 511 reduced the binding of the rbd to dpp4 even more, and the ones located at residues 403 and 579 had no impact on dpp4 binding. structural analysis of the rbd/dpp4 interactions suggests that a glycan probe located at residue 511 would have serious steric clash with dpp4 binding, whereas a glycan probe located at residue 562 would have partial steric interference with dpp4 binding (fig. 1b) . glycan probes located at residues 403 and 579 would be too far away from the receptor-binding region to have any impact on dpp4 binding. hence, both the biochemical and structural analyses similarly elucidated the role of each of the glycan probes in the binding of the rbd to dpp4. to understand the epitopes' potential to interact with neutralizing monoclonal antibodies (mabs), we analysed how the engineered glycan probes interfered with the binding of the rbd to different neutralizing mabs. we had access to four humanized mabs (hms-1, m336-fab, m337-fab, and m338-fab). all of these mabs were previously shown to be highly potent in neutralizing mers-cov infection of human cells [36] [37] [38] [39] . elisa between each of the rbds and each of the mabs demonstrated that the glycan probe located at residue 511 abolished the binding of the rbd to hms-1 (fig. 2a) , reduced the binding of the rbd to m336-fab and m337-fab (fig. 2b,c) , and had no significant impact on the binding of the rbd to m338-fab (fig. 2d ). in contrast, the glycan probes located at the other three residues, 403, 562 and 579, did not interfere with the binding of the rbd to any of the mabs. the binding sites on the rbd for each of the mabs were previously characterized through mutagenesis and/or structural studies [36] [37] [38] [39] . three of the four mabs, hms-1, m336-fab and m337-fab, bind at or near the epitope containing arg511, whereas all of the mabs bind away from the epitopes containing ala562, val403, and thr579 ( fig. 2e) . overall, among the four selected epitopes, the epitope containing arg511 played the most important role in the binding of neutralizing mabs, and consequently the glycan probe covering this epitope interfered most with the binding of neutralizing mabs. this study thus far has characterized the structural features, receptor binding, and neutralizing mab binding for four selected rbd epitopes using a glycan probe strategy. each of the glycan probes introduced to one of the rbd epitopes only interfered with the binding of dpp4 or mabs that interact with this specific epitope, but had no impact on the binding of dpp4 or mabs to distant epitopes. this observation suggests that each of the glycan probes only shielded the epitope where the glycan probe was attached to, but did not affect the structures of other antigenic sites. it is consistent with findings obtained in studies on another viral spike protein, respiratory syncytial (rsv) virus f protein 40 . to evaluate how the glycan probes altered the neutralizing immunogenicity (that is, the capacity to induce neutralizing immune responses) of mers-cov rbds, we immunized balb/c mice with each of the four rbds containing one of the glycan probes. the immunization schedule is shown in supplementary fig. 4a . sera were collected from mice immunized with each of the rbds, and tested for mers-cov-neutralizing antibodies. compared to the wild type rbd vaccine, the rbds containing a glycan probe at residues 579 and 511 induced significantly higher and lower neutralizing antibody titres, respectively, in mouse sera, whereas the rbds containing a glycan probe at residues 403 and 562 failed to induce significant changes in neutralizing antibody titres in mouse sera ( fig. 3a ; supplementary table 1) . thus, masking the epitope containing arg511 led to reduced neutralizing antibody titres in the immunized mice, demonstrating that this epitope made a positive contribution to the vaccine's overall neutralizing immunogenicity. based on the same rationale, the epitope containing thr579 made a negative contribution and the epitopes containing val403 and ala562 made insignificant contributions to the vaccine's overall neutralizing immunogenicity. the experiments were further repeated twice and similar results were obtained. these results provided a qualitative evaluation of the neutralizing immunogenicity for each of these epitopes. how can we quantitatively evaluate the epitopes' neutralizing immunogenicity? here we introduce a novel concept nii to describe an epitope's neutralizing immunogenicity. nii is defined as the contribution of an epitope to the vaccine's overall neutralizing immunogenicity. it can be determined by masking the epitope with a glycan probe and then measuring the relative change of the vaccine's overall capacity to elicit neutralizing antibody titres (fig. 3b) . based on this definition, we calculated the nii for each of the four epitopes on the rbd ( fig. 3c ; supplementary table 1 ). the epitope containing thr579 had an nii of à 3.0. the negative sign of the nii suggests a negative contribution from this epitope to the vaccine's overall neutralizing immunogenicity, and the value of the nii implicates that masking this epitope using a glycan probe increased the vaccine's overall neutralizing immunogenicity by three fold. conversely, the epitope containing arg511 had an nii of 0.6, suggesting that this epitope made a positive contribution to the vaccine's overall neutralizing immunogenicity and that masking this epitope using a glycan probe reduced the vaccine's overall neutralizing immunogenicity to 60% of that of the wild type vaccine. therefore, the nii can serve as an effective tool to quantitatively evaluate the neutralizing immunogenicity of any epitope on the mers-cov rbd vaccine. to investigate why masking a negative epitope led to enhanced neutralizing immunogenicity of the mers-cov rbd vaccine, we performed a competition assay between neutralizing mabs and mutant-rbd-induced mouse serum for the binding of wild type mers-cov rbd. more specifically, elisa was carried out between a neutralizing mab and mers-cov rbd in the presence of mouse serum induced by the 579-glycosylated mers-cov rbd (fig. 4a,b) . as a comparison, the mouse serum induced by the wild type mers-cov rbd was also included. two different mabs were used in the competition binding assay: hms-1, which binds to the rbm epitope containing arg511 (refs 36,39) , and m336-fab, which binds to the rbm epitope surrounding glu536-asp539 (refs 37,38) . the result showed that the serum induced by the 579-glycosylated rbd inhibited the mab-rbd binding significantly better than the serum induced by the wild type rbd, revealing enhanced neutralizing capability of the mouse serum due to the glycosylation at the 579 position. moreover, the mouse serum induced by the 579-glycosylated rbd demonstrated enhanced binding for at least two separate neutralizing epitopes on the rbm, one surrounding arg511 and the other glu536-asp539. thus, masking an epitope on the rbd core structure with a high negative nii refocuses the host immune response on neutralizing epitopes on the rbm, leading to enhanced neutralizing immunogenicity of the rbd vaccine. rational design of rbd vaccine with enhanced efficacy. to prove that highly effective mers-cov rbd vaccines can be rationally designed based on epitopes' neutralizing immunogenicity, we investigated the efficacy of two engineered mers-cov rbd vaccines using virus challenge studies. these engineered rbd vaccines have a negative epitope (that is, the epitope containing thr579 and with an nii of à 3.0) and a positive epitope (that is, the epitope containing arg511 and with an nii of 0.6) masked, respectively, by a glycan probe. we chose to mask the epitopes rather than deleting them or mutating all of their residues to alanines because introducing a glycan is more convenient in practice and less disruptive to the immunogen's tertiary structure. the wild type rbd vaccine was used as a control. the animal model for vaccine testing was the lethal transgenic mouse model expressing human dpp4 (hdpp4-tg mice) 41, 42 . these mice were chosen for analysis because they are very susceptible to mers-cov and also because preventing disease in these mice is a stringent test of efficacy. the immunization schedule is shown in supplementary fig. 4b . briefly, hdpp4-tg mice were immunized with each of the rbd vaccines and challenged with mers-cov, and the survival rate and weight changes of the mice were recorded. the efficacies of the rbd vaccines were evaluated based on the morbidity and mortality of the immunized and challenged mice. first, hdpp4-tg mice immunized with the negative-epitopemasked rbd vaccine (that is, rbd containing t579n mutation) all survived mers-cov challenge (100% survival rate), whereas hdpp4-tg mice immunized with the wild type rbd vaccine and with the positive-epitope-masked rbd vaccine (that is, rbd containing r511n/e513t mutations) demonstrated survival rates of 67 and 17%, respectively, after mers-cov challenge (fig. 5a) . second, mers-cov challenge did not cause any weight loss in hdpp4-tg mice immunized with the negative-epitope-masked rbd vaccine, but led to significant weight loss in hdpp4-tg mice immunized with either the wild type rbd vaccine or the positive-epitope-masked rbd vaccine (fig. 5b) . the experiments were further repeated twice and similar results were obtained. these results revealed the enhanced efficacy of the hms-1 mab current vaccine design lacks an effective approach to evaluate the neutralizing immunogenicity of epitopes on viral subunit vaccines. in this study, we have developed a novel approach to measure vaccine epitopes' neutralizing immunogenicity. using the mers-cov rbd as a model, we singly mask selected epitopes using host-derived glycan probes, and then measure the corresponding changes in the vaccine's overall neutralizing immunogenicity. we have also developed a method for calculating the nii for the selected epitopes. an epitope's neutralizing immunogenicity contains two parts: the neutralization capacity and immunogenicity. on the one hand, an epitope's neutralizing capacity is determined by the physical overlap of the epitope with the receptor-binding region and the potential role of the epitope in receptor binding. on the other hand, an epitope's immunogenicity is determined by its immune selfness (that is, how similar or dissimilar the viral epitope is to a host-originated epitope), protrusion, and other physical and chemical properties of the epitope. logically, an epitope's nii is correlated with a combination of factors such as immune selfness, protrusion, potential overlap with receptor-binding region, and more. because of the complex nature of nii, it is unlikely that the nii can be reliably predicted by software; instead, this study demonstrates that nii can be experimentally measured using the glycan probe approach. as proof-of-concept, we measured the nii for four distinct epitopes on the mers-cov rbd vaccine, and also characterized the protrusion index, receptor binding, and monoclonal antibody binding of the rbds each with an epitope masked by a glycan probe. the results revealed that the epitopes with a high and low protrusion index tend to have an nii with a high and low absolute value, respectively. in addition, epitopes within the receptor-binding region tend to have a positive nii, and the epitopes located outside the receptor-binding region tend to have a negative nii. we cannot correlate the immune selfness of epitopes with nii because there is no good method to evaluate the immune selfness of conformational epitopes. overall, in rational design of viral subunit vaccines, the epitopes with a high positive nii should be preserved and exposed, while those with a high negative nii should be eliminated via deletion or masking. indeed, our study has identified an epitope containing thr579 as one with a high negative nii on mers-cov rbd. thr579 is located on a protruding loop and away from the receptor-binding region, both of which contribute to its high negative nii. importantly, thr579 is buried inside the full-length coronavirus spike proteins, and only becomes exposed on the surface of the recombinant mers-cov rbd vaccine as an outcome of subunit vaccine design ( supplementary fig. 1 ). to overcome this limitation of subunit vaccine design, the newly exposed epitopes with a high negative nii need to be masked or deleted. to apply the nii strategy to vaccine design, we successfully enhanced the efficacy of the mers-cov rbd vaccine in virus challenge studies by masking its strong negative epitope (that is, the epitope containing thr579, with an nii of à 3.0) with a glycan probe. this engineered vaccine effectively protected hdpp4-transgenic mice from a lethal mers-cov infection. compared with the wild type rbd vaccine, mice immunized with the engineered rbd vaccine showed increased neutralizing antibody responses in their sera; when challenged by mers-cov, they also demonstrated higher survival rate and less weight loss. these results prove that negative epitopes should be eliminated in vaccine design. in contrast, another engineered vaccine with a positive epitope masked (that is, the epitope containing arg511, with an nii of 0.6) showed reduced efficacy in virus challenge studies, confirming that positive epitopes should be preserved and exposed in vaccine design. taken altogether, we validated both the significance and feasibility of the nii strategy in vaccine design by successfully engineering a variant form of the mers-cov rbd vaccine with significantly enhanced efficacy. overall, our study contributes to viral subunit vaccine design in the following ways. first, our study introduces a new concept nii for the evaluation of how individual epitopes contribute to the overall neutralizing immunogenicity of subunit vaccines. previous studies could not evaluate the neutralizing immunogenicity of conformational b-cell epitopes that dominate coronavirus rbd vaccines. second, using the nii strategy our study identified an immunodominant non-neutralizing epitope on the surface of the mers-cov rbd core structure. this result shows that exposure of previously buried epitopes on viral subunit vaccines poses a challenge for subunit vaccine design. this concept, although needing further investigations, may be critical for the development of many viral rbd-based vaccines. third, our study demonstrates that masking an immunodominant non-neutralizing epitope with a negative nii value on the surface of the mers-cov rbd core structure can shift host immune responses towards the neutralizing epitopes in the rbm region, providing means to overcome the limitation of viral subunit vaccines from vaccine design. previous studies showed that hypervariable regions on hiv gp120 divert host immune responses and that masking these regions can shift host immune responses towards conserved neutralizing epitopes 11, 12 , providing means to overcome the limitation of viral subunit vaccines from viral evolution. fourth, although the nii strategy was used in the current study to improve the efficacy of viral subunit vaccines, it can also be potentially helpful in other epitope-based vaccine research. for example, previous studies masked or resurfaced non-neutralizing epitopes on viral immunogens, and used the engineered immunogens as baits to screen from neutralizing sera for monoclonal antibodies that bind to conserved neutralizing epitopes [43] [44] [45] [46] . it is conceivable that the nii strategy can help identify immunodominant non-neutralizing epitopes on immunogens, allowing more targeted epitope modifications for efficient antibody screening. finally, our study suggests that a three-dimensional 'neutralizing immunogenicity map' (nim) can be drawn to describe the distribution of epitopes with different neutralizing immunogenicity on the surface of viral subunit vaccines. such an nim can guide targeted masking of multiple strong negative epitopes, further enhancing the efficacy of viral subunit vaccines. we believe that our approach can facilitate the rational subunit vaccine design not only for coronaviruses such as mers-cov and sars-cov, but also for other life-threatening viruses such as hiv, influenza virus, and ebola virus. cell lines. hek293t (human embryonic kidney) and vero e6 (monkey kidney) cells were obtained from american type culture collection. huh-7 (human hepatoma) cells were kindly provided by dr charles m. rice at rockefeller university. these cell lines were cultured in dulbecco's modified eagle medium (dmem) supplemented with 10% fetal bovine serum (fbs), 2 mm l-glutamine, 100 units ml à 1 penicillin, and 100 mg ml à 1 streptomycin (life technologies inc.). sf9 insect cells were purchased from life technologies inc., and cultured in sf-900 iii sfm medium supplemented with 100 units ml à 1 penicillin and 100 mg ml à 1 streptomycin (life technologies inc.) expression and purification of recombinant proteins. the expression and purification of recombinant mers-cov rbd was carried out as previously described 34 . briefly, wild type (wt) rbd (residues 377-588; genbank accession number: afs88936.1) containing a c-terminal human igg 1 fc tag was expressed in hek293t cells, secreted into the cell culture supernatant, and purified by protein a affinity chromatography (ge healthcare). mutant rbd fragments containing engineered glycan probes were constructed via site-directed mutagenesis, and expressed and purified in the same way as the wild type rbd. the expression and purification of recombinant human dpp4 was carried out as previously described 47 . briefly, human dpp4 ectodomain (residues 39-766; genbank accession no. np_001926.2) containing an n-terminal human cd5 signal peptide and a c-terminal his 6 tag was expressed in insect sf9 cells using the bac-to-bac expression system (life technologies inc.), secreted to cell culture medium, and purified sequentially on hitrap nickel chelating hp column and superdex 200 gel filtration column (ge healthcare). sds gel electrophoresis. 5 mg wild type or mutant mers-cov rbds were subjected to sds gel electrophoresis under denatured condition. protein bands were stained using coomassie brilliant blue r (sigma-aldrich), and image captured using myecl imager (life technologies inc.). mass spectrometry. wild type or mutant mers-cov rbds at 100 mm concentration in 20 mm tris-cl, ph 7.4, 200 mm nacl was ultrafiltrated with deionized water five times using an amicon ultra centrifugal filter with a 10 kda molecular weight cutoff. the desalted protein samples were subjected to maldi-tof mass spectrometry at tufts university core facility. mass spectrometry was performed in linear mode for molecular weight screening. alphascreen protein-protein binding assay. binding between recombinant mers-cov rbds and recombinant human dpp4 was measured using an alphascreen assay as previously described 34, 36 . briefly, 3 nm wild type or mutant mers-cov rbd with a c-terminal fc tag was incubated with 300 nm human dpp4 with a c-terminal his 6 tag at room temperature for 1 h. alphascreen protein a acceptor beads and nickel chelate donor beads (perkinelmer life sciences) were added to the mixture at a final concentration of 5 mg ml à 1 each. after incubation at room temperature for 1 h, the alphascreen signal was measured using an enspire plate reader (perkinelmer life sciences), reflecting the binding affinity between the two proteins. facs. the binding between recombinant mers-cov rbds and human dpp4 expressed on the huh-7 cell-surface was measured using fluorescence-activated cell sorting (facs) as previously described 28, 36 . briefly, huh-7 cells were incubated with wild type or mutant mers-cov rbd (1.25 mg ml à 1 ) at room temperature for 30 min, followed by addition of fitc-conjugated anti-human-igg-fc polyclonal antibody (1:50 dilution) (sigma-aldrich) for 30 min. the amounts of rbd-bound huh-7 cells were measured using flow cytometry, and the binding affinity between rbd and cell-surface dpp4 was characterized as median fluorescence intensity. animal immunization and sample collection. animal immunization and sample collection were carried out as previously described 34 . briefly, balb/c mice were subcutaneously immunized with wild type or mutant mers-cov rbd (10 mg per mouse) in the presence of montanide isa51 adjuvant 34, 48 . pbs plus montanide isa51 was included as a negative control. immunized mice were boosted twice with the same immunogen and adjuvant at a 3-week interval, and sera were collected 10 days after the last immunization for detection of neutralizing antibodies. elisa. the binding between recombinant mers-cov rbd and neutralizing mabs was measured using elisa as previously described 36 . briefly, elisa plates were pre-coated with the same amount of wild type or mutant rbd (1 mg ml à 1 ) overnight at 4°c. after blocking with 2% non-fat milk at 37°c for 2 h, serially diluted mabs were added to the plates and incubated at 37°c for 1 h. after washes, the plates were incubated at 37°c for 1 h with horseradish-peroxidaseconjugated anti-human-igg-fab polyclonal antibody (1:5,000 dilution) (sigma-aldrich). enzymatic reaction was carried out using substrate 3,3 0 ,5,5 0tetramethylbenzidine (life technologies inc.) and stopped with 1 n h 2 so 4 . absorbance at 450 nm (a 450 ) was measured using elisa plate reader (tecan group ltd.). the competition between neutralizing mabs and mutant-rbd-induced mouse serum for the binding of wild type mers-cov rbd was carried out using elisa as described above, except that the binding between wild type rbd and the neutralizing mab (hms-1 or m336-fab at 5 mg ml à 1 concentration) was performed in the presence of serially diluted mouse serum (t579n-rbdinduced, wild-type-rbd-induced, or pbs-induced). the rbd-mab binding was detected by addition of horseradish-peroxidase-conjugated anti-human-igg-fab polyclonal antibody (1:5,000 dilution) and subsequent enzymatic reaction. live mers-cov neutralization assay. a micro-neutralization assay was carried out to test neutralizing antibodies against live mers-cov as previously described 36 . briefly, serially diluted mouse sera were incubated at room temperature for 1 h with b100 infectious mers-cov virions (emc-2012 strain), and were then incubated with vero e6 cells at 37°c for 72 h. the neutralizing capability of the mouse sera was measured by determining the presence or absence of virus-induced cytopathic effect (cpe). neutralizing antibody titres were expressed as the reciprocal of the highest dilution of sera that completely inhibited virus-induced cpe in at least 50% of the wells (nt 50 ). mers-cov challenge studies. mers-cov challenge studies were carried out using human-dpp4-transgenic mice as previously described 41 . briefly, mice were intramuscularly immunized with wild type or mutant mers-cov rbd (5 mg per mouse) in the presence of aluminium adjuvant 49 , and boosted once 4 weeks after the initial immunization. 12 weeks after the second immunization, mice were challenged with mers-cov (emc-2012 strain, 10 4 tcid 50 ), and observed for 21 days for detection of survival rate and weight changes. statistical analyses. in fig. 1c -d, comparisons between wt rbd and each of the mutant rbds in their binding to recombinant dpp4 by alphascreen (fig. 1c) or to cell-surface dpp4 by facs (fig. 1d) were done using two-tailed t-test (***, po0.001; 3 measurements for each rbd in figs 1c and 4 measurements for each rbd in fig. 1d) . in figs 2a-d, nonlinear regression was performed using a log(inhibitor) versus normalized response-variable slope model. r 2 of curve fit is larger than 0.97 for all curves in fig. 2athe extra sum-of-squares f test (***, po0.001; 12 different dilutions of each mab, 4 measurements at each dilution for each mab). in fig. 3a , comparisons between wt rbd and each of the mutant rbds in their capacity to induce neutralizing serum in mice were done using two-tailed t-test (*, po0.05; 4 measurements for each rbd). in fig. 4 , nonlinear regression was performed using a log(inhibitor) versus normalized response-variable slope model. r 2 of curve fit is larger than 0.98 for all curves in fig. 4 . comparisons between wt-rbd-induced serum and t579n-rbd-induced serum in their inhibition of rbd/mab binding by elisa were done using the extra sum-of-squares f test (***, po0.001; 4 different dilutions of each serum, 4 measurements at each dilution for each serum). all statistical analyses were performed using graphpad prism 6 software. data availability. all relevant data are available from the authors. rational design of vaccines to elicit broadly neutralizing antibodies to hiv-1. cold spring harb structure-based antigen design: a strategy for next generation vaccines advances in structure-based vaccine design structural vaccinology starts to deliver the spike protein of sars-cov-a target for vaccine and therapeutic development receptor recognition mechanisms of coronaviruses: a decade of structural studies immunodominance of cd4 t cells to foreign antigens is peptide intrinsic and independent of molecular context: implications for vaccine design immunodominance: a pivotal principle in host response to viral infections immunodominance with progenitor b cell diversity in the neutralizing antibody repertoire to influenza infection immunodominance of conformation-dependent b-cell epitopes of protein antigens refocusing neutralizing antibody response by targeted dampening of an immunodominant epitope immune focusing and enhanced neutralization induced by hiv-1 gp140 chemical cross-linking designing immunogens to elicit broadly neutralizing antibodies to the hiv-1 envelope glycoprotein prediction of immunogenicity for therapeutic proteins: state of the art bcr-abl fusion regions as a source of multiple leukemiaspecific cd8 þ t-cell epitopes ctl responses of hla-a2.1-transgenic mice specific for hepatitis c viral peptides predict epitopes for ctl of humans carrying hla-a2.1 a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 structure of sars coronavirus spike receptor-binding domain complexed with receptor crystal structure of the receptor-binding domain from newly emerged middle east respiratory syndrome coronavirus angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc receptor-binding domain of severe acute respiratory syndrome coronavirus spike protein contains multiple conformation-dependent epitopes that induce highly potent neutralizing antibodies identification of a receptor-binding domain in the s protein of the novel human coronavirus middle east respiratory syndrome coronavirus as an essential target for vaccine development receptor-binding domain of sars-cov spike protein induces long-term protective immunity in an animal model current advancements and potential strategies in the development of mers-cov vaccines pre-fusion structure of a human coronavirus spike protein cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer assembly of asparagine-linked oligosaccharides searching for an ideal vaccine candidate among different mers coronavirus receptor-binding fragments-the importance of immunofocusing in subunit vaccine design an algorithm that identifies protruding atoms in proteins a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies junctional and allele-specific residues are critical for mers-cov neutralization by an exceptionally potent germline-like antibody single-dose treatment with a humanized neutralizing antibody affords full protection of a human transgenic mouse model from lethal middle east respiratory syndrome (mers)-coronavirus infection prefusion f-specific antibodies determine the magnitude of rsv neutralizing activity in human sera multi-organ damage in human dipeptidyl peptidase 4 transgenic mice infected with middle east respiratory syndrome-coronavirus characterization and demonstration of the value of a lethal mouse model of middle east respiratory syndrome coronavirus infection and disease rational design of envelope identifies broadly neutralizing human monoclonal antibodies to hiv-1 a combinatorial mutagenesis approach for functional epitope mapping on phage-displayed target antigen: application to antibodies against epidermal growth factor high throughput functional epitope mapping: revisiting phage display platform to scan target antigen surface defining a protective epitope on factor h binding protein, a key meningococcal virulence factor and vaccine antigen receptor usage and cell entry of bat coronavirus hku4 provide insight into bat-to-human transmission of mers coronavirus 720 and 51: a new generation of water in oil emulsions as adjuvants for human vaccines current adjuvants and new perspectives in vaccine formulation we thank drs. dimiter s. dimitrov and tianlei ying at the national institutes of health for providing m336, m337 and m338 mabs this work was supported by nih grants r01ai089728 and r01ai110700 (f.l.), nih grant po1ai060699 (s.p.), nih grants r01ai098775, u01ai124260, r21ai109094 and the intramural fund of new york blood center nyb000348 (l.d. and s.j.), nih grant r21ai113206-01 and pilot grants from the center for biodefense and emerging infectious diseases and from the galveston national laboratory (c.k.t), china national l.d., s.j., y.z., f.l. designed the experiments; l.d., w.t., y.y., g.z., q.z., s.s., c.l. and x.t. performed the experiments; l.d., c.k.t., s.p., s.j., y.z., f.l. analysed the data; l.d., s.p., s.j., y.z. and f.l. wrote the paper. key: cord-329011-spiuqngp authors: huang, yuan; yang, chan; xu, xin-feng; xu, wei; liu, shu-wen title: structural and functional properties of sars-cov-2 spike protein: potential antivirus drug development for covid-19 date: 2020-08-03 journal: acta pharmacol sin doi: 10.1038/s41401-020-0485-4 sha: doc_id: 329011 cord_uid: spiuqngp coronavirus disease 2019 is a newly emerging infectious disease currently spreading across the world. it is caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the spike (s) protein of sars-cov-2, which plays a key role in the receptor recognition and cell membrane fusion process, is composed of two subunits, s1 and s2. the s1 subunit contains a receptor-binding domain that recognizes and binds to the host receptor angiotensin-converting enzyme 2, while the s2 subunit mediates viral cell membrane fusion by forming a six-helical bundle via the two-heptad repeat domain. in this review, we highlight recent research advance in the structure, function and development of antivirus drugs targeting the s protein. the epidemic of novel coronavirus disease 2019 (covid-19) was caused by a new coronavirus occurred in december 2019, and now has spread worldwide and turned into a global pandemic [1] . the covid-19 was quickly discovered to be caused by a coronavirus later named severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [1] , which belongs to the β coronavirus family. it is the seventh known coronavirus to infect humans; four of these coronaviruses (229e, nl63, oc43, and hku1) only cause slight symptoms of the common cold. conversely, the other three, sars-cov, mers-cov, and sars-cov-2, are able to cause severe symptoms and even death, with fatality rates of 10%, 37%, and 5%, respectively. although a large number of studies and clinical trials are being launched on covid-19 around the world [2, 3] , no evidence from randomized clinical trials has shown that any potential therapy improves outcomes in patients [4] . as the epidemic spreads, it is critical to find a specific therapeutic for covid-19, and vaccines targeting various sars-cov-2 proteins are under development. sars-cov-2 is a single-stranded rna-enveloped virus [5] . an rna-based metagenomic next-generation sequencing approach has been applied to characterize its entire genome, which is 29,881 bp in length (genbank no. mn908947), encoding 9860 amino acids [6] . gene fragments express structural and nonstructural proteins. the s, e, m, and n genes encode structural proteins, whereas nonstructural proteins, such as 3-chymotrypsinlike protease, papain-like protease, and rna-dependent rna polymerase, are encoded by the orf region [7] . a large number of glycosylated s proteins cover the surface of sars-cov-2 and bind to the host cell receptor angiotensinconverting enzyme 2 (ace2), mediating viral cell entry [8] . when the s protein binds to the receptor, tm protease serine 2 (tmprss2), a type 2 tm serine protease located on the host cell membrane, promotes virus entry into the cell by activating the s protein. once the virus enters the cell, the viral rna is released, polyproteins are translated from the rna genome, and replication and transcription of the viral rna genome occur via protein cleavage and assembly of the replicase-transcriptase complex. viral rna is replicated, and structural proteins are synthesized, assembled, and packaged in the host cell, after which viral particles are released (fig. 1d) [9] . these proteins are critical to the viral life cycle and provide potential targets for drug therapies. for example, ace2-based peptide, 3clpro inhibitor (3clpro-1), and a novel vinylsulfone protease inhibitor have been experimentally demonstrated to be effective against sars-cov-2 [10] . the sars-cov-2 s protein is highly conserved among all human coronaviruses (hcovs) and is involved in receptor recognition, viral attachment, and entry into host cells. due to its indispensable functions, it represents one of the most important targets for covid-19 vaccine and therapeutic research. in this review, we summarize advances in research of the sars-cov-2 s protein and its therapeutic targeting. with a size of 180-200 kda, the s protein consists of an extracellular n-terminus, a transmembrane (tm) domain anchored in the viral membrane, and a short intracellular c-terminal segment [11] . s normally exists in a metastable, prefusion conformation; once the virus interacts with the host cell, extensive structural rearrangement of the s protein occurs, allowing the virus to fuse with the host cell membrane. the spikes are coated with polysaccharide molecules to camouflage them, evading surveillance of the host immune system during entry [12] . the total length of sars-cov-2 s is 1273 aa and consists of a signal peptide (amino acids 1-13) located at the n-terminus, the s1 subunit (14-685 residues), and the s2 subunit (686-1273 residues); the last two regions are responsible for receptor binding and membrane fusion, respectively. in the s1 subunit, there is an n-terminal domain (14-305 residues) and a receptor-binding domain (rbd, 319-541 residues); the fusion peptide (fp) (788-806 residues), heptapeptide repeat sequence 1 (hr1) (912-984 residues), hr2 (1163-1213 residues), tm domain (1213-1237 residues), and cytoplasm domain (1237-1273 residues) comprise the s2 subunit ( fig. 2a) [13] . s protein trimers visually form a characteristic bulbous, crown-like halo surrounding the viral particle (fig. 1a) . based on the structure of coronavirus s protein monomers, the s1 and s2 subunits form the bulbous head and stalk region [14] . the structure of the sars-cov-2 trimeric s protein has been determined by cryo-electron microscopy at the atomic level, revealing different conformations of the s rbd domain in opened and closed states and its corresponding functions (fig. 2b , c) [15, 16] . in the native state, the cov s protein exists as an inactive precursor. during viral infection, target cell proteases activate the s protein by cleaving it into s1 and s2 subunits [17] , which is necessary for activating the membrane fusion domain after viral entry into target cells [18] . similar to other coronaviruses, the s protein of sars-cov-2 is cleaved into s1 and s2 subunits by cellular proteases, and the serine protease tmprss2 is used as a protein primer. although the cleavage site of sars-cov is known, that of sars-cov-2 s has not yet been reported [18, 19] . structure of the s1 subunit the binding of virus particles to cell receptors on the surface of the host cell is the initiation of virus infection; therefore, receptor recognition is an important determinant of viral entry and a drug design target. rbd situated in the s1 subunit binds to the cell receptor ace2 in the region of aminopeptidase n. the s1 region contains the ntd and ctd, and atomic details at the binding interface demonstrate key residue substitutions in sars-cov-2-ctd. in addition, the sars-cov-2 s ctd binding interface has more residues that directly interact with the receptor ace2 than does sars-rbd (21 versus 17), and a larger surface area is buried with sars-cov-2 s ctd in complex with ace2 than with sars s rbd. mutations of key residues play an important role in enhancing the interaction with ace2. f486 in sars-cov-2, instead of i472 in sars rbd, forms strong aromatic-aromatic interactions with ace2 y83, and e484 in sars-cov-2-ctd, instead of p470 in sars rbd, forms ionic interactions with k31, which leads to higher affinity for receptor binding than rbd of sars-cov ( fig. 2d) [15, 16, 20, 21] . the rbd region is a critical target for neutralizing antibodies (nabs), and sars-cov-2 and sars-cov rbd are~73%-76% similar in sequence. nine ace2-contacting residues in cov rbd are fully conserved, and four are partially conserved. analysis of the rbm (receptor-binding motif, a portion of rbd making direct contacts with ace2) of sars-cov and sars-cov-2 revealed that most residues essential for ace2 binding in the sars-cov s protein are conserved in the sars-cov-2 s protein. however, some studies showed that murine monoclonal antibodies (mabs) and polyclonal antibodies against sars-rbd are unable to interact with the sars-cov-2 s protein, revealing differences in antigenicity between sars-cov and sars-cov-2 [20] . similarly, a sars-cov rbd-specific antibody failed to block infection mediated by the s protein of sl-cov-shc014 [22] , which suggests that the s1 rbd may not be an ideal drug target due to the highly mutable characteristic of broad-spectrum anti-cov drugs. structure of the s2 subunit the s2 subunit, composed successively of a fp, hr1, hr2, tm domain, and cytoplasmic domain fusion (ct), is responsible for viral fusion and entry. fp is a short segment of 15-20 conserved amino acids of the viral family, composed mainly of hydrophobic residues, such as glycine (g) or alanine (a), which anchor to the target membrane when the s protein adopts the prehairpin conformation. previous research has shown that fp plays an essential role in mediating b-c the s protein rbd closed and opened status. d the s protein binds to ace2 with opened rbd in the s1 subunit. e the six-helix structure formed by hr1 and hr2 of the s2 subunit. membrane fusion by disrupting and connecting lipid bilayers of the host cell membrane [23] . hr1 and hr2 are composed of a repetitive heptapeptide: hpphcpc, where h is a hydrophobic or traditionally bulky residue, p is a polar or hydrophilic residue, and c is another charged residue [24] . hr1 and hr2 form the six-helical bundle (6-hb) (fig. 2e) , which is essential for the viral fusion and entry function of the s2 subunit [13] . hr1 is located at the c-terminus of a hydrophobic fp, and hr2 is located at the n-terminus of the tm domain [25] . the downstream tm domain anchors the s protein to the viral membrane, and the s2 subunit ends in a ct tail [14] . rbd binds to ace2, and s2 changes conformation by inserting fp into the target cell membrane, exposing the prehairpin coiledcoil of the hr1 domain and triggering interaction between the hr2 domain and hr1 trimer to form 6-hb, thus bringing the viral envelope and cell membrane into proximity for viral fusion and entry [26] . hr1 forms a homotrimeric assembly in which three highly conserved hydrophobic grooves on the surface that bind to hr2 are exposed. the hr2 domain forms both a rigid helix and a flexible loop to interact with the hr1 domain. in the postfusion hairpin conformation of covs, there are many strong interactions between the hr1 and hr2 domains inside the helical region, which is designated the "fusion core region" (hr1core and hr2core regions, respectively). targeting the heptad repeat (hr) has attracted the greatest interest in therapeutic drug discovery. the s protein is an important target protein for the development of specific drugs, while the s1 rbd domain is part of a highly mutable region and is not an ideal target site for broad-spectrum antiviral inhibitor development [27] . in contrast, the hr region of the s2 subunit plays an essential role in hcov infections and is conserved among hcovs, as is the mode of interaction between hr1 and hr2 [28] . a synthetic peptide derived from the stem region of the zikv envelope protein was demonstrated in 2017 to potently inhibit infection by zikv and other flaviviruses in vitro [29] , implying antiviral efficiency of peptides derived from conserved regions of viral proteins. peptides derived from the hr2 region of class i viral fusion proteins of enveloped viruses competitively bind to viral hr1 and effectively inhibit viral infection [22] . therefore, hr1 is a promising target for the development of fusion inhibitors against sars-cov-2 infection. the s protein on the surface of the virus is a key factor involved in infection. it is a trimeric class i tm glycoprotein responsible for viral entry, and it is present in all kinds of hcovs, as well as in other viruses such as hiv (hiv glycoprotein 160, env), influenza virus (influenza hemagglutinin, ha), paramyxovirus (paramyxovirus f), and ebola (ebola virus glycoprotein) [30] . similar to other coronaviruses, the s protein of sars-cov-2 mediates receptor recognition, cell attachment, and fusion during viral infection [16, 20, 21, [31] [32] [33] . the trimer of the s protein located on the surface of the viral envelope is the basic unit by which the s protein binds to the receptor [16, 33] . the s1 domain contains the rbd, which is mainly responsible for binding of the virus to the receptor, while the s2 domain mainly contains the hr domain, including hr1 and hr2, which is closely related to virus fusion [34] . receptor binding as mentioned above, the sars-cov-2 s protein binds to the host cell by recognizing the receptor ace2 [33] . ace2 is a homolog of ace, which converts angiotensin i to angiotensin 1-9 [35] . ace2 is distributed mainly in the lung, intestine, heart, and kidney, and alveolar epithelial type ii cells are the major expressing cells [36] . ace2 is also a known receptor for sars-cov. the s1 subunit of the sars-cov s protein binds with ace2 to promote the formation of endosomes, which triggers viral fusion activity under low ph (fig. 1a, b) [37] . interaction between the s protein and ace2 can be used to identify intermediate hosts of sars-cov-2, as ace2 from different species, such as amphibians, birds, and mammals, has a conserved primary structure [38] . luan et al. compared the binding affinities between ace2 and sars-cov-2 s from mammals, birds, snakes, and turtles and found that the ace2 of bovidae and cricetidae interacted well with sars-cov-2 s rbd but that ace2 from snakes and turtles could not. the s protein binds to ace2 through the rbd region of the s1 subunit, mediating viral attachment to host cells in the form of a trimer [15] . sars-cov-2 s binds to human ace2 with a dissociation constant (k d ) of 14.7 nm, though that of sars-cov s is 325.8 nm [15] , indicating that sars-cov-2 s is more sensitive to ace2 than is sars-cov s. through the identification of sars-cov-2 proteins, researchers found~24% difference in s between sars-cov-2 and sars-cov, whereas that of rbd is~23% [39] . viral fusion viral fusion refers to fusion of the viral membrane and host cell membrane, resulting in the release of the viral genome into the host cell. cleavage of the sars-cov-2 s1 and s2 subunits is the basis of fusion. the s protein is cleaved into two parts, the s1 subunit and s2 subunit, by host proteases, and the subunits exist in a noncovalent form until viral fusion occurs [40] . researchers have found that the specific furin cleavage site is located in the cleavage site of sars-cov-2 but not in other sarslike covs [41, 42] . mutation of the cleavage site in sars-cov-2 or sars-like covs has revealed that the s protein of sars-cov-2 exists in an uncleaved state but that the others are mainly in a cleaved state. sars-cov-2 s has multiple furin cleavage sites, which increases the probability of being cleaved by furin-like proteases and thereby enhances its infectivity [43, 44] . the furin-like cleavage domain is also present in highly pathogenic influenza virus and is related to its pathogenicity, as observed in the avian influenza outbreak in hong kong in 1997 [45, 46] . in addition, host cell proteases such as tmprss2 are essential for s protein priming, and they have been shown to be activated in the entry of sars-cov and influenza a virus [18, 47, 48] . another host cell protease that has been proven to cleave viral s protein is trypsin [49] . in summary, the s protein of sars-cov-2 is similar to that of sars-cov, and host cell proteases are essential for promoting s protein cleavage of both sars-cov-2 and sars-cov. the presence of a specific furin cleavage site on sars-cov-2 s might be one reason that sars-cov-2 is more contagious than sars-cov. the formation of 6-hb is essential for viral fusion. the fp in the n-terminus of sars-cov-2 and the two hr domains on s2 is essential for viral fusion [50] . after cleavage of the s protein, the fp of sars-cov-2 is exposed and triggers viral fusion. under the action of some special ligands, the fusion protein undergoes a conformational change and then inserts into the host cell membrane (fig. 1c) [51] . for example, the ligand for influenza a virus is h + , while the ligand for hiv is a coreceptor such as ccr5 or cxcr4 [14] . the distance between the viral membrane and host cell membrane is shortened, and the hr1 domain of the s protein is in close proximity to the host cell membrane, whereas the hr2 domain is closer to the viral membrane side. then, hr2 folds back to hr1, the two hr domains form a six-helix structure in an antiparallel format of the fusion core, the viral membrane is pulled toward the host cell membrane and tightly binds to it, and the two membranes fuse [52] . the fundamental role of the s protein in viral infection indicates that it is a potential target for vaccine development, drug development targeting sars-cov-2 s y huang et al. antibody-blocking therapy, and small molecule inhibitors. considering the similarity with sars-cov and mers-cov, potential nabs and inhibitors targeting sars-cov-2 s are summarized below (fig. 3) . antibodies based on the sars-cov-2 s protein the s protein is the main antigen component in all structural proteins of sars-cov-2. unlike other functional proteins of sas-cov-2, it is responsible for inducing the host immune response, and nabs targeting the s protein can induce protective immunity against viral infection. similar to sars-cov and mers-cov, research on nabs of sars-cov-2 mainly includes mabs, antigenbinding fragments, single-chain variable region fragments, and single-domain antibodies (nbs), which target s1 rbd, s1-ntd, or s2 regions to prevent s2-mediated fusion [53, 54] . on the other hand, multiple sars-cov-2 vaccine types are under development, including rna/dna-based formulations, recombinant viral epitopes, adenovirus-based vectors, and purified inactivated virus [55] . the sequence and striking structural similarity between the sars-cov-2 and sars-cov s proteins emphasize the close relationship between these two viruses, which provides the possibility to treat covid-19 with antibodies targeting the sars-cov s protein [56] . compared with sars-cov-2 rbd, sars-cov-2 interacts with hace2 via the c-terminal domain (sars-cov-2-ctd), showing higher affinity for receptor binding. rbd can induce highly potent nab responses and has the potential to be developed as an effective and safe subunit vaccine against sars-cov-2. sars-cov s polyclonal antibodies obtained from immunized mice completely inhibited the invasion of sars-cov s-mlv (murine leukemia virus), whereas the invasion rate of sars-cov-2 s-mlv was reduced to~10% [20] . the polyclonal anti-sars s1 antibody t62 inhibits the entry of sars-cov s but not that of sars-cov-2 s pseudovirus particles [49] . consistently, recent studies have reported similar results, showing that three sars rbd-directed mabs, s230, m396, and 80r, were unable to bind to sars-cov-2 rbd [16, 20, 21] . on the other hand, several mabs have shown promising results in neutralizing sars-cov-2. cr3022, a sars-cov-specific human mab, binds potently with sars-cov-2 (k d of 6.3 nm, measured by bli in octetred96), suggesting that cr3022 has the potential to be developed as candidate therapeutic, alone or in combination with other nabs, for the prevention and treatment of sars-cov-2 infection [57] . a mab targeting s1 prepared from immunized transgenic mice expressing human ig variable heavy and light chains has recently been shown to neutralize both sars-cov-2 and sars-cov infections via an unknown mechanism that is independent of the blockade of rbd-hace2 interaction [58] . recently, many human blocking mabs (311mab-31b5, 311mab-32d4, 47d11, n3130, n3088, s309, p2c-1f11, p2b-2f6, b38, h4) have been successfully cloned from single memory b cells from recovered covid-19 patients [58] [59] [60] [61] [62] [63] . these mabs specifically bind to sars-cov-2 s to effectively neutralize infection. in addition, sera from sars patients during rehabilitation or animals specifically immunized with sars-cov s1 may cross-neutralize sars-cov-2 and reduce s protein-mediated sars-cov-2 entry (fig. 3) [18] . the stability of the sars-cov-2 s protein is lower than that of sars-cov s [42] . the mapping of multiple s sequences of the subgenus sarbecovirus underscores that the s2 fusion region is more conserved than the s1 subunit and that the s1 subunit is more exposed at the viral surface [16] . the sars-cov s2 subunit plays a key role in mediating virus-cell fusion and its integration into host cells, where hr1 and hr2 interact to form 6-hb, thus enabling the virus to bind to and fuse with the cell membrane [28] . sequence alignment shows that sars-cov-2 hr2 has the same sequence as sars-cov hr2. therefore, sars-cov-2 hr2p (1168-1203 residues) was designed to inhibit sars-cov-2 fusion and entry into a target cell. surprisingly, hr2p showed inhibitory activity against sars-cov-2 s-mediated fusion and sars-cov-2 pseudovirus, with ic 50 values of 0.18 and 0.98 μm, respectively [13] . notably, ek1 is a pancoronavirus fusion inhibitor targeting the hr1 domain of hcov s [22] . the x-ray crystal structure of the 6-hb core of the sars-cov-2 s2 subunit hr1 and hr2 domains has been solved, indicating that several mutant residues in the hr1 region may be related to enhanced interaction in the hr2 region [64] . subsequently, ek1c4, a lipopeptide derived from ek1, was generated and verified to inhibit sars-cov-2 s-mediated cell-cell fusion. as expected, the entry of sars-cov-2 s pseudovirus was also inhibited by ek1c4, with an ic 50 of 15.8 nm,~149-fold more potent than the original ek1 peptide. another sequence-based lipopeptide fusion inhibitor, ipb02, potently inhibits sars-cov-2 s protein-mediated cell-cell fusion and pseudovirus infection [65] . in addition to peptide fusion inhibitors, nelfinavir mesylate (viracept), a currently prescribed anti-hiv protease inhibitor, suppresses both sars-cov-2 s and sars-cov s-mediated cell-cell fusion. viracept is the first reported small molecule fusion inhibitor in addition to peptide fusion inhibitors. moreover, nelfinavir may inhibit the function of tmprss2 involved in activation of the s protein [66] . this discovery makes possible clinical applications of anti-sars-cov-2 therapeutics, especially in the early stage of infection. protease inhibitors targeting sars-cov-2 s cleavage sites sars-cov-2 entry requires cleavage of the s protein at the s1/s2 and s2 sites. proteolysis by tmprss2 and cathepsin b and l plays an important role in priming sars-cov-2 s for entry. camostat mesilate is a potent serine protease inhibitor of tmprss2. utilizing research on the sars-cov and sars-cov-2 cell entry mechanism, it has been demonstrated that sars-cov-2 cellular entry can be blocked by camostat mesilate [18, 67] . there are currently five clinical trials registered to evaluate the efficacy of camostat mesilate (clinicaltrials.gov identifier: nct04321096, nct04353284, nct04338906, nct04355052, nct04374019). in addition, cathepsins in lysosomes are crucial for sars-cov entry via endocytosis. e-64d, an inhibitor of cathepsin l, blocks infection with sars-cov and sars-cov-2 psv [68] [69] [70] . future trials with covid-19 patients may help to confirm the efficacy of e-64d therapy. phosphatidylinositol 3-phosphate 5-kinase (pikfyve) is the main enzyme synthesizing pi (3, 5) p2 in early endosomes [71] . apilimod, a potent inhibitor of pikfyve35, can significantly reduce the entry of sars-cov s pseudovirus into 293/hace2 cells via early endosomes in a dose-dependent manner [49] . treating 293/ hace2 cells with another pikfyve inhibitor, ym201636 [72] , also had a similar effect. moreover, a major downstream effector of pi (3,5)p2, two-pore channel subtype 2 (tpc2) [73] , is important for sars-cov-2 entry, and tetrandrine (an inhibitor of tpc2) inhibits the activity of sars-cov-2 s pseudovirus. furin (proprotein convertase (pc) subtilisin kexin 3, pcsk3), as a member of the pc family, catalyzes the hydrolysis of peptide and protein substrates at paired basic residues [74] . strikingly, sars-cov-2 s harbors a furin cleavage site (682-685 residues) at the s1/ s2 boundary, which may increase the efficiency of sars-cov-2 transmission [75] . the furin-like cleavage site in the s protein of sars-cov-2 may have implications for the viral life cycle and pathogenicity. therefore, furin inhibitors can be used as a drug therapy for sars-cov-2 [41] . patent literature since 1994 describes the use of furin or its inhibitors in the treatment of diseases, and some furin inhibitors that have been reported, including α-1-pdx (α1-antitrypsin portland) [76] , hexa-d-arginine(d6r) [77] , serpin proteinase inhibitor 8 (pi8) [78] , and a peptidomimetic furin inhibitor [79] . the sars-cov-2 s protein binds to the host cell receptor and induces virus-cell membrane fusion, which plays a vital role in the process of virus invasion. moreover, the high affinity between the s protein and ace2 increases the infectivity of sars-cov-2. mammals including pangolins, pets (dogs and cats), and members of cricetidae may be important for determining key residues for association with s from sars-cov and sars-cov-2 [80] . further drug development targeting sars-cov-2 s y huang et al. understanding of the structure and function of sars-cov-2 s will allow for additional information regarding invasion and pathogenesis of the virus, which will support the discovery of antiviral therapeutics and precision vaccine design. structural information will also assist in evaluating mutations of the sars-cov-2 s protein and will help in determining whether these residues have surface exposure and map to known antibody epitopes of s proteins from other coronaviruses. in addition, structural knowledge ensures that the proteins produced by constructs are homogeneous and participate in the prefusion conformation, which should maintain the most neutralizationsensitive epitopes when used as a candidate vaccine or b-cell probe for isolating neutralizing human mabs. furthermore, atomic-level details will enable the design and screening of small molecules that inhibit fusion. since sars-cov-2 and sars-cov rbd domains share 75% amino acid sequence identity, future work will be necessary to evaluate whether any of these abs neutralize newly emerged coronavirus. overall, interaction between the s protein of sars-cov-2 and ace2 should be further studied to contribute elucidation of the mechanism of sars-cov-2 infection. similarly, focusing on high expression of the s protein or its receptor binding region is also of great significance for the development of vaccines. the s2 subunit of sars-cov-2 shows 88% sequence homology with the sars-cov s2 domain and is structurally conserved. therefore, the development of antibodies targeting this functional motif may cross-bind and neutralize these two viruses and related covs. antiviral peptides prevent sars-cov-2 membrane fusion and can potentially be used for the prevention and treatment of infection. it is worth mentioning that ek1c4, which targets the highly conserved hr1 domain of the s2 subunit, is expected to have therapeutic potential against sars-cov-2. more importantly, ek1c4 can be used as a nasal drop, which increases its medicinal properties, it possesses a high genetic barrier to resistance, and does not easily induce drug-resistant mutations. on the other hand, peptide fusion inhibitors may not be widely used clinically and have low bioavailability. therefore, the development of oral small molecule fusion inhibitors is a major direction. in the course of virus epidemics, the ability to adapt to external pressure is an important factor affecting the spread of the virus. regarding the envelope s protein, recombination or mutation in the gene of its rbd can occur to promote transmission between different hosts and lead to a higher fatality rate [81] . mutation of the aspartate (d) at position 614 to glycine (g614) results in a more pathogenic strain of sars-cov-2 [82] , which makes it more difficult to develop antibodies or vaccines that target nonconservative regions. to effectively prevent disease, combinations of different mabs that identify different epitopes on the sars-cov-2 s surface can be assessed to neutralize a wide range of isolates, including escape mutants [83] . currently, no specific therapeutic or prophylactic has been used clinically to treat or prevent sars-cov-2 infection. nonspecific antiviral drugs, such as ifn-α (recombinant human ifn-α1b, ifn-α2a), remdesivir, chloroquine, favipiravir, and lopinavir-ritonavir (aluvia), have been clinically used to treat covid-19 in china [84] . nevertheless, niaid-vrc scientists are developing a candidate vaccine expressing sars-cov-2 s protein in mrna vaccine 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inhibits mers-and sars-coronavirus replication in cell culture, but not sars-coronavirus infection in a mouse model but not hcov-nl63, utilizes cathepsins to infect cells-viral entry. nidoviruses: toward control of sars and other nidovirus glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) the phosphatidylinositol-3-phosphate 5-kinase inhibitor apilimod blocks filoviral entry and infection inhibition of pikfyve using ym201636 suppresses the growth of liver cancer via the induction of autophagy two-pore channels control ebola virus host cell entry and are drug targets for disease treatment proprotein convertases in health and disease drug development targeting sars-cov-2 clinical features of patients infected with 2019 novel coronavirus in wuhan furin inhibition reduces vascular remodeling and atherosclerotic lesion progression in mice furin inhibitor d6r suppresses epithelial-mesenchymal transition in sw1990 and patu8988 cells via the hippo-yap signaling pathway the serpin proteinase inhibitor 8: an endogenous furin inhibitor released from human platelets peptidomimetic furin inhibitor mi-701 in combination with oseltamivir and ribavirin efficiently blocks propagation of highly pathogenic avian influenza viruses and delays high level oseltamivir resistance in mdck cells alteration of brain network topology in hiv-associated neurocognitive disorder: a novel functional connectivity perspective the establishment of reference sequence for sars-cov-2 and variation analysis sars-cov-2 viral spike g614 mutation exhibits higher case fatality rate perspectives on therapeutic neutralizing antibodies against the novel coronavirus sars-cov-2 remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro this project was supported by grants from guangzhou science and technology program (#201803040006 to wx), the fund of natural science foundation of guangdong province (#2018a030313056 to wx), and grants from major scientific and technological projects of guangdong province (#2019b020202002 to swl). competing interests: the authors declare no competing interests. key: cord-332855-u0amf1oh authors: parsons, lisa m.; bouwman, kim m.; azurmendi, hugo; de vries, robert p.; cipollo, john f.; verheije, monique h. title: glycosylation of the viral attachment protein of avian coronavirus is essential for host cell and receptor binding date: 2019-03-22 journal: journal of biological chemistry doi: 10.1074/jbc.ra119.007532 sha: doc_id: 332855 cord_uid: u0amf1oh avian coronaviruses, including infectious bronchitis virus (ibv), are important respiratory pathogens of poultry. the heavily glycosylated ibv spike protein is responsible for binding to host tissues. glycosylation sites in the spike protein are highly conserved across viral genotypes, suggesting an important role for this modification in the virus life cycle. here, we analyzed the n-glycosylation of the receptor-binding domain (rbd) of ibv strain m41 spike protein and assessed the role of this modification in host receptor binding. ten single asn–to–ala substitutions at the predicted n-glycosylation sites of the m41–rbd were evaluated along with two control val–to–ala substitutions. cd analysis revealed that the secondary structure of all variants was retained compared with the unmodified m41–rbd construct. six of the 10 glycosylation variants lost binding to chicken trachea tissue and an elisa-presented α2,3-linked sialic acid oligosaccharide ligand. lc/ms(e) glycomics analysis revealed that glycosylation sites have specific proportions of n-glycan subtypes. overall, the glycosylation patterns of most variant rbds were highly similar to those of the unmodified m41–rbd construct. in silico docking experiments with the recently published cryo-em structure of the m41 ibv spike protein and our glycosylation results revealed a potential ligand receptor site that is ringed by four glycosylation sites that dramatically impact ligand binding. combined with the results of previous array studies, the glycosylation and mutational analyses presented here suggest a unique glycosylation-dependent binding modality for the m41 spike protein. avian coronaviruses of poultry cause significant disease with subsequent economic losses in several commercially farmed bird species. avian infectious bronchitis virus (ibv) 2 is a gam-macoronavirus that predominantly affects domestic fowl, primarily chickens (gallus gallus). the virus initially infects upper airway epithelium tissues, and depending on the ibv strain, disease outcomes range from mild respiratory disease to kidney failure and death (1) . the viral envelope of ibv contains the highly-glycosylated spike (s) protein that is post-translationally cleaved into two domains, s1 and s2. this s glycoprotein is the major adhesion molecule of the virus. it is a class i viral fusion protein, in which the variable s1 domain is involved in host cell receptor binding, and the more conserved s2 domain mediates the fusion of the virion with the cellular membrane (2, 3) . the role of spike in host cell attachment and the induction of protective immunity has been reviewed (4) . the spike protein monomer is a transmembrane glycoprotein with a molecular mass of 128 kda before glycosylation (3) . a cleavable n-terminal signal peptide (5) directs the s protein toward the endoplasmic reticulum (er), where it is extensively modified with n-linked glycosylation (6, 7) . after glycosylation in the er, the monomers oligomerize to form trimers (6 -9) . the n-terminal 253 amino acids of s1 were shown to encompass the receptor-binding domain (rbd) of ibv strain m41 (10) , which interacts with sialyl-␣2,3-substituted glycans present on the host's cell surface (11, 12) . ten n-linked glycosylation sites are predicted to exist on the m41-rbd (5), of which most are highly conserved (fig. s1 ). it is interesting that 8 of the 10 sites are 95-100% conserved. sites asn-33 and asn-59 were less conserved at 80 and 25%. however, each had a nearby alternative site that was also highly conserved. alternative site asn-36 was conserved 50% of the time, and one or both asn-33 and asn-36 was present in 94% of the sequences. site asn-57 was conserved at 73%. in 97% of the sequences, either asn-59 or asn-57 was present but never together. therefore, all 10 sites, including the alternatives, likely serve important functions. the n-glycosylation of viral glycoproteins is known to modulate the ability of viruses to infect host cells and to be recognized by the host's immune system (13) . recently, zheng et al. (14) studied extracted spike proteins and mutant viruses with asn-to-asp (asparagine to aspartate) and asn-to-gln (aspara-gine to glutamine) mutations at 13 predicted glycosylation sites in the s protein of the beaudette ibv strain (14) . their results indicate that glycosylation at some sites on the beaudette s1-rbd was important for viral fusion and infectivity, which may include host recognition. however, the beaudette strain is a cell culture-adapted strain, is nonvirulent in chickens (15) , and does not bind chicken tissues known to be important for infectivity (11) , making it difficult to extrapolate these results to clinically relevant ibvs. to characterize and assess the role that glycosylation plays when interacting with host tissues through the rbd of pathogenic ibv strain m41, we used a combination of molecular and analytical techniques, including histochemistry, elisa, circular dichroism (cd), ms, and docking analyses as listed in table 1 . systematic deletion of each glycosylation site and histochemical analysis of each variant revealed which of the 10 glycosylation sites affect the binding of ibv s protein to host epithelial tissue. site occupancy analysis by lc/ms e indicated that at least 9 of 10 predicted n-glycosylation sites in the m41-rbd domain are glycosylated. analysis of site occupancy and signature n-glycan patterns at each site in combination with single glycosylation site deletions provided insight toward the biological relevance of each of those sites in binding to host tissue receptors. overall, our data confirm that n-glycosylation plays a critical and likely unique role in binding of the ibv spike domain to its host tissue receptors. to analyze the role of glycosylation of m41-rbd in receptor binding, missense mutants (asn-to-ala) were generated on a site-by-site basis at each of the predicted n-glycosylation sites. recombinantly produced glycovariant rbd proteins migrated with the same electrophoretic mobility as unmodified m41-rbd (fig. 1) . the rbd proteins were evaluated by cd spectroscopy to assess similarity to the wt secondary structure. wt m41-rbd, all 10 glycosylation-site variants, and two nonglycosylation variants, v57a and v58a, were analyzed for secondary structure differences at 25°c. thermal melts were performed on each construct from 25 to 95°c followed by full scans collected at 95°c and again at 25°c after the melt. overlays of all the cd spectra can be found in fig. s2 . visually, all spectra at all temperatures follow the same curve. the n85a spectra were generated at higher protein concentrations but aligned well to cd spectra of all other variants when normal-ized to the percent of maximum signal. likewise, all the proteins had analogous broad melting curves suggesting the proteins were similarly stable. protein folding was reversible for all proteins, with comparable recovery rates (see cd-25°c-aftermelt-normalized in fig. s2 ). dichroweb (16) was used to calculate the percent of ␣-helix, ␤-strand, turn, and unordered portions of the protein in the initial 25°c spectra to estimate secondary structure differences between the proteins (fig. 2) . the percent of ␣-helix varied with the extremes being unmodified rbd and n145a. n145a exhibited 19.5 ϯ 0.3% ␣-helix character as compared with wt, which has 31.6 ϯ 2.4%. interestingly, n145a gave a very strong signal in the histochemical assay ( fig. 3a ) and had the most notably different released glycans' signature compared with the other constructs. we conclude that all proteins maintained a very similar structure and therefore suggest that single n-glycosylation sites are by themselves not indispensable for protein folding or stability. because we established that all variant m41-rbd proteins are folded, we investigated their abilities to bind tissue receptors. recombinant proteins were incubated with chicken trachea tissue sections and examined by histochemical analysis. n145a, n219a, n229a, n246a, v57a, and v58a bound ciliated epithelial cells of the chicken trachea with similar staining intensity as the unmodified rbd with the most intense staining associated with the n145a construct (fig. 3a ). in contrast, binding of constructs n33a, n59a, n85a, n126a, n160a, and n194a to trachea tissue was not detectable. removal of sialic acids by treatment of the trachea tissues with arthrobacter ureafaciens neuraminidase (auna) abrogated binding of all constructs as shown in fig. s4 . these results demonstrate that glycosylation on the rbd affects binding to sialyl ligands on chicken trachea tissue. the interaction of the variants with neu5ac(␣2-3)gal (␤1-3)glcnac, a previously established ligand for m41 (11) , was assayed by elisa. n145a, n219a, n229a, and n246a variants were able to bind the ligand in a concentration-dependent manner (fig. 3b ) like unmodified rbd. binding affinities of n33a, n59a, n85a, n126a, n160a, and n194a were significantly reduced compared with unmodified rbd and comparable with that of a negative control protein, the s1 of turkey coronavirus, with specificity for nonsialylated dilacnac glycans (17) . fig. 3c shows the elisa absorbance at the 75 nmol of ligand concentration for each construct. no significant difference was observed for variants n145a, n219a, n229a, and n246a compared with unmodified rbd (shown in dark gray bars in fig. 3c ). all other variants (shown in light gray bars in fig. 3c ) demonstrated significantly lower affinity for the receptor, consistent with histochemistry and ligand titration plot results. six of the 10 single glycosylation site variants lost the ability to bind ligand. to investigate whether global changes in glycosylation may have affected binding, we analyzed release glycans from each protein. matrix-assisted laser desorption/ionizationtime of flight (maldi-tof) mass spectrometry (ms) analysis of enzymatically released and permethylated glycans allows for semi-quantitative analysis of glycan compositions. the method is particularly useful for samples containing sialylated glycans because they are stabilized by permethylation. the percent abundances of glycans identified in each sample are shown in fig. 4 . the majority of the asn-to-ala variants, as well as the v57a and v58a control variants, had similar maldi-tof-ms permethylation profiles (fig. 4) . over 100 glycan compositions were identified ranging from high-mannose glycans to large complex ones. nearly half of the glycans contained at least one and up to three sialic acid molecules in all samples. the most intense glycoforms clustered in five groups with increasing amounts of complexity as reflected by the number of n-acetyl glucosamines (hexnacs). these include high-mannose, complex, and hybrid forms as follows: i, hex 5-9 hexnac 2 (high mannose); ii, neuac 0 -1 hex 5-6 dhex 0 -1 hexnac 3 (complex and hybrid); iii, neuac 0 -2 hex 5 dhex 1 hexnac 4 (complex); iv, neuac 0 -1 hex 6 dhex 1 hexnac 5 (complex); and v, neuac 2 hex 7 dhex 1 hexnac 6 (complex). high-mannose glycans were less abundant in unmodified m41 than in variant rbds. the n194a, n219a, and n229a variants contained diminished amounts of the group v high-mass complex glycans. the n145a variant was the most atypical with less defined clustering in the common clustering regions of the spectrum and higher abundances in spectral regions where compositions had less hex and more hexnac overall. for instance, cluster iv was shifted from glycans with 6 hexoses (neuac 0 -1 hex6 dhex 1 hexnac 5 ) to glycoforms with 3-4 hexoses (neuac 0 -1 hex3-4 dhex 1 hexnac 5 ). more abundance was observed in regions containing 6 hexnac residues (neuac 0 -2 hex 3-6 dhex 1 hexnac 6 ). to better understand the difference between n145a and the other constructs, we calculated the monosaccharide percent mass and average mass for each construct. the average mass percent for glycans across all released glycan pools was hex (45.8%), hexnac (42.0%), dhex (5.0%), and neuac (7.2%). the n145a construct had the lowest amount of hex (38.6%) and the highest amounts of hexnac (46.0%) and neuac (9.8%). the former two were 2 s.d. or greater from the mean (see table s2 ). this indicates that the n145a construct likely had shorter, more branched, and more highly-charged glycans on average than the other constructs. two other variants had values more than 2 s.d. from the mean. n229a (normal binding) was most abundant in hex (53.6%) and least abundant in hexnac (37.5%) and dhex (3.8%), probably due to its higher high-mannose content. n246a (normal binding) had the lowest amount of neuac (3.6%). this is perhaps a reflection of the missing sugars in this variant because site asn-246 in other variants was populated with many sialylated glycoforms based on site-specific analysis (table s1 ). to assess the differences in glycosylation on a site-to-site basis, glycopeptide lc/ms analysis was carried out on unmodified m41 and two single glycosylation site variants, n59a and n145a, that represented a nonbinder and a binder of trachea tissue, respectively. m41-rbd had 10 predicted glycosylation sites, whereas the variant rbds had nine each. n145a was also of specific interest due to the unique glycosylation pattern observed in its free glycan profile. as cleavage with trypsin alone resulted in glycopeptides with more than one glycosylation site, we also analyzed glycopeptides after an additional treatment with chymotrypsin, which resulted in one glycosite per peptide, the identification of more glycopeptides, and decreased ambiguity concerning glycosylation site assignment. although a protein may contain the sequence (nx(s/t)), where n-glycosylation is known to occur, it may not actually be glycosylated, or it may be glycosylated only part of the time. potential glycosylation sites, their predicted glycosylation state, and their measured site occupancy are shown in table 2 . of the 10 glycosites, all but asn-246 were predicted to be glycosylated (occupied) based on netnglyc analysis (http://www.cbs. dtu.dk/services/netnglyc-1.0/). 3 percent occupancy was analyzed by lc/ms; however, a poor signal was obtained for the asn-219 site in m41 and n59a, and therefore, occupancies were not calculated. all other sites were estimated to be occufigure 3 . tissue-binding assay and elisas. histochemical assays of recombinant unmodified m41-rbd and single asn-to-ala and val-to-ala glycosylation variants to trachea tissue (a) and elisa-presented neu5ac␣2-3gal␤1-3glcnac (b and c). b, concentration dependence of binding. c, absorbance for each protein at the 75-nmol concentration. two-way anova showed significantly less binding by variant n33a, n59a, n85a, n126a, n160a, and n194a rbd proteins compared with unmodified rbd (compare light gray bars (variant) to unmodified (black bar)). no significant (n.s.) difference was observed for variants with dark gray bars. data points are averaged from three separate assays. ****, p ͻ 0.0001. pied at 89% or greater in m41 and n59a. the n145a variant exhibited site occupancy at all expected sites, including asn-219, although signal intensity at that site was low. two sites had much lower occupancy in n145a as compared with the other samples. site asn-126 dropped to 61% occupancy and site asn-246 to 79% occupancy compared with nearly complete occupancy in the n59a and m41 proteins. overall site occupancy was high for all sites. the difficulty in detecting some of the peptides, particularly asn-219, may be due to hydrophobicity. ionization is partially driven by hydrophobicity, and asn-219 only had 20% hydrophobic character after the two digestions, which may, in part, explain its low detectability. by comparison, glycopeptides containing asn-85, asn-145, and asn-160 were short and between 21 and 33% hydrophobicity, whereas glycopeptides containing other sites had predicted hydrophobicity ranging from 37 to 61% and tended to produce higher intensity spectra. glycoform relative abundances at each site are listed in table s1 . fig. 5 shows the location of each glycosylation site on the rbd of m41. overall compositions at each site were similar in charge and size across the three constructs. a representative glycan is shown at each site based on peak intensity. the n145a construct had glycoforms like those identified by maldi-tof ms with more hexnac and fewer hex compared with m41 and n59a. fewer overall glycan compositions were detected on glycopeptides by lc/ms compared with the free glycans observed by maldi-tof ms (63 versus 100 compositions). this can be expected because the technology of instrumentation used and the physiochemical characteristics of permethylated glycans and glycopeptides differ significantly. the forms detected overlapped between the two analyses. during our investigation, the first structure of the m41 spike protein was solved using electron microscopy (em) (18) . mapping the glycosylation sites onto the structure did not lead to a clear understanding of how the mutations affect binding. although em structural resolution is limited, and the precise coordinates for the attached glycans are not known, an attempt was made to dock a series of potentially sialylated ligands to a glycan-stripped structure of the rbd and a structure that was populated with glycans based on our data. the glycan chosen for each site on the rbd was based on the predominant glycans identified at each site by lc/ms (see fig. 5 ). seventeen oligosaccharide ligands were chosen based on a previous glycan array study of m41 (11) and elisa data (this work). both strong and weak binders were selected (fig. 6) . each ligand was docked 20 times against both the sugarstripped and in silico glycosylated m41-rbd coordinates. there was no statistically significant difference between the docked binding energies of ligands that did and did not bind on the array. all oligosaccharide ligands, except for 1, 3, 9, 13, 15, and 17, docked seven or more times to one or more of the four sites on the m41 sugar-stripped structure with no clear pattern differentiating between them (fig. 6 ). in the sugar-stripped structure, all binding occurred at sites a and b. site a is under the galectin fold near site asn-194, and site b encompasses asn-85 and asn-59. all three glycosylation sites are required for binding to trachea tissue. the docking pattern changed dramatically when glycans were modeled onto the structure. the most dramatic change was seen at site d where eight ligands bound seven or more times, whereas interactions at all other sites decreased. there were no binders at site a, only two at site c (3 and 16) and three at site b (6, 9, and 17) . all of the ligand oligosaccharides that docked at site d were sialylated, consistent with ligands identified by array and elisa. no control ligand (1 and 2 uncharged; 3 and 4 kdn-charged) bound at site d. the interaction at site d involved both sugar-protein and sugar-sugar contacts, and in some docking runs, the interaction was completely sugar-sugar. site d is in the center of a circle of glycosylation sites that showed altered binding profiles when mutated; n59a, n85a, and n160a lost the ability to bind, whereas n145a gave a very strong signal in the histochemical assay. of note, no ligands docked in the site at the top of the galectin fold where many structural homologs of m41 are thought to bind sugars, such as the bovine coronavirus rbd (19) . for comparison, we docked neu5ac(␣2-6)gal(␤1-3)glcnac (␤-ome) against the crystal structure of the bovine rbd. twenty five of 25 times the glycan docked in the proposed binding site at the top of the galectin fold in the negatively-charged area of the bovine rbd control near asn-198 (fig. 7b ). previously, we established that the ibv m41 s1 protein binds sialic acid-substituted glycoconjugate ligands in chicken trachea and lung tissue (11) . intriguingly, the m41 rbd is highlyglycosylated with 10 potential glycosylation sites, and glycosylation appears to be necessary for binding to host tissues because treating the protein with a neuraminidase diminishes binding (11) . this study extends our investigation toward determining the role of glycosylation in the function of the rbd, which encompasses the n-terminal region of the native protein. each of the potential glycosylation sites was individually ablated, and each construct was examined for its ability to bind tissue and an elisa-presented ligand. in addition, the global glycosylation profile of every construct was surveyed, and glycosylation of three representative constructs was examined on a site-specific basis. six of the 10 glycosylation sites in the rbd domain of ibv m41 were essential for binding to chicken trachea tissue and an elisa-presented sialylated oligosaccharide ligand. cd analysis demonstrated that both secondary structure and stability were similar across all the rbd constructs indicating the proper fold was likely retained for all. globally, percent abundances of sialylated glycans differed across mutants, but the differences were not associated with loss of binding. for example, 51 and 20% of the glycans in binding mutants n145a and n246a, respectively, and 46 and 51% of the glycans in the nonbinders n126a and n160a, respectively, were sialylated (summed from fig. 4) . by comparison, 40% of the glycans in the unmodified rbd construct were sialylated. on a site-specific basis, some glycosylation sites had more sialylation than others (table s1 ). on average, each of glycosites asn-126, asn-194, asn-229, and asn-246 were sialylated at least 50% of the time. sites asn-229 and asn-246 were in the less-ordered region of the protein figure 5 . site-specific glycosylation of m41, n59a, and n145a. the s1-n-terminal receptor binding domain residues 21-268 from pdb entry 6cv0 is represented as gray ribbons. the asparagines of glycosylation sites that could still bind trachea tissue after mutation to alanine are in cyan, and those that could not are in dark red. glcnac residues from the structure are dark blue balls and sticks. the most predominant glycan for each site across all three constructs is shown to the right. glycoforms shown on the right are based on our data, and inferred structural detail is based on accepted knowledge of the cell type used in protein production. monosaccharides are represented as follows: mannose (green circles); galactose (yellow circles); glcnac (blue squares); fucose (red triangles); and sialic acid (purple diamonds). numbering of the sites is based on the mature sequence. the figure was made with ccp4mg (38) and gimp. (11) and referenced in the figure as array score 1 . white columns were against structure without sugars, and gray columns were lc/ms-identified where the sugars were modeled. bottom, rbd-binding domain of m41 from pdb structure 6cv0. glycosylation sites are shown as cyan balls. sites where two or more oligosaccharides docked seven or more times are indicated as colored space-filled amino acids. colors and labels match the table above. b is a turned 90°toward the user. structure representations were made in ccp4-mg (38) . sugar symbols were rendered with drawglycan-snfg (www.virtualglycome.org/drawglycan/) 3 (39) . away from the galectin fold where binding is associated in the docking study. site asn-194 is at the bottom of the galectin fold and is required for ligand binding. site asn-126 is at the top of the galectin fold and is also required for binding. although we cannot conclude that sialylation is required at asn-194 and asn-126, it is clear that glycosylation at these sites serves a role in ligand binding. the publication of the cryo-em structure of m41 (18), the first structure of a spike protein from a gammacoronavirus, made it possible to visualize the distribution of the glycosylation sites in the tertiary structure of the protein. the study verified the site occupancy we observed on m41-rbd because 9 of 10 of the glycosylation sites in the em structure were occupied. site asn-246, not occupied in the em structure, is on a ␤-strand in the em structure, and it forms close contacts with the s1 c-terminal domain in the native protein. the c-terminal domain was not part of our construct. therefore, asn-246 in the recombinant constructs was likely in an environment much different from that found in the full-length protein. many human galectins, and also the bovine ␤-coronavirus spike protein (18) , bind sugars at what is the top of the ␤-sandwich near site asn-126 in the rbd constructs (see fig. 5 ). the bovine rbd site asn-198 closely aligns with site asn-126 of m41 (see fig. 7 ). in the bovine protein, this demarks the region of proposed ligand binding. loss of asn-126 in the m41 rbd abrogates binding to trachea tissue. although ablation of asn-126 diminishes ligand binding, our docking study gave no evidence that this is the sialyl ligand-binding site in m41. evaluation of the charge distribution in the proposed binding sites indicates that the bovine site is negatively charged, whereas the negative charge in the same region in m41 is sparse (fig. 7) . this difference in charge near asn-126 may explain the lack of ligand docking in this region (gray ␤-strands in fig. 6b ) during docking simulations. the precise ligand-binding region of proteins with a galectin fold varies. rotavirus protein vp4, for example, binds sialic acid in a groove between the ␤-sheets of the sandwich (20) . the clustering of five of six required n-glycosylation sites suggests the location of the ligand-binding site may be on the right of the galectin fold as shown in fig. 5 . our docking experiments studying 17 possible oligosaccharide ligands to m41 were not conclusive in terms of binding energies but did identify four potential saccharide-binding regions (fig. 6) . docking also demonstrated that glycosylation affects binding in silico because one potential site (site a; see fig. 6 ) lost favor, whereas another one, site d, dramatically gained favor when the protein was glycosylated. site d is in the center of three glycosylated asparagines required for binding (asn-59, asn-85, and asn-160), and one whose loss results in a very strong histochemical signal and has a protein-wide effect on glycosylation with increased sialylation (asn-145) . in addition, the site d region is negatively charged (see fig. 7a ) like the proposed sialyl ligandbinding site on the bovine protein (fig. 7b) (19) . all the ligands that interacted with site d were sialylated and included the glycan that bound in our elisa studies. interestingly, carbohydrate-carbohydrate contacts were detected in the rbd-ligand interactions at site d. this is an intriguing result because carbohydrate-carbohydrate interactions, although not common, have been reported between nonfucosylated antibodies and their receptor, in cell-cell adhesion interactions, between tumor antigens, and between bacterial receptors and mucin (21) (22) (23) (24) (25) . a literature search did not uncover any reported carbohydrate-carbohydrate interactions between virus and host. although our docking study must be evaluated in the context of the higher root mean square deviations typical of em structures, and the inexactness of modeled oligosaccharides, results suggest that a combination of carbohydratecarbohydrate and carbohydrate-protein interactions should be considered in the binding mechanism. in conclusion, we have shown that glycosylation of six sites on the m41 ibv rbd are necessary for the interaction of m41 with both trachea tissue and neu5ac(␣2-3)gal(␤1-3) glcnac ligand in elisa. based on occupancy data, at least nine sites were glycosylated in the recombinant m41-rbd. deletion of individual glycosylation sites had little effect on secondary structure, but it did have some effect on overall glycosylation profiles of some variants, especially n145a. some differences can be expected because one site, with specific glycans, is lost from each variant, thus mildly altering overall profiles. in silico docking suggests that glycosylation may guide ligand binding. especially intriguing is site d, where glycosylation is required for in silico docking at that site. the interaction of m41 ibv with sialyl ligand may prove to be a unique interaction involving both carbohydrates and protein. further investigation is warranted. the tissues used for this study were obtained from the tissue archive of the veterinary pathologic diagnostic center (department of pathobiology, faculty of veterinary medicine, utrecht university, the netherlands). this archive is composed of paraffin blocks with tissues maintained for diagnostic purposes; no permission from the committee on the ethics of animal experiment is required. the pcd5 vector containing ibv m41-rbd in-frame with a c-terminal gcn4 trimerization motif and strep-tag has been a and pink boxes on b. y162, e182, w184, and h185 in b are involved in binding to sialic acid. the large asterisk in a indicates possible binding site based on structural comparison between the two proteins. images were made with ccp4-mg (38) . bovine coordinates are from pdb code 4h14. described previously (10) . site-directed mutagenesis using the q5 technology (new england biolabs) was performed to mutate the asparagine-encoding residues of the n-linked glycosylation sequence motif nx(s/t) into alanine or valine using the primers in table 3 . sequences of the resulting rbds were confirmed by sanger sequencing (macrogen, the netherlands). hek293t (atcc crl-3216) cells were transfected with pcd5 plasmids using polyethyleneimine at a 1:12 ratio. the recombinant proteins were purified using strep-tactin-sepharose beads, as described previously (11) , and their production was confirmed by western blotting using strep-tactin hrp antibody (iba, germany). recombinant m41 and its variants were prepared for cd spectroscopy by buffer exchange and concentration with four centrifugation cycles through 10-kda mwco amicon ultra 0.5-ml centrifugal filters (ufc 501024) into 10 mm sodium phosphate, ph 7.75. final concentrations were measured with a thermo fisher scientific nanodrop 2000 spectrophotometer. cd spectra were collected on a jasco j-810 spectropolarimeter with a peltier thermostated fluorescence temperature controller module. samples were diluted to 0.06 mg/ml and four scans accumulated from 285 to 190 nm with a scanning speed of 10 nm/min, digital integrated time 1-s, bandwidth 1 nm, and standard sensitivity at 25°c. a thermal melt was done from 25 to 95°c with a ramp rate of 1°c/min. measurements were taken every 2°at 222, 218, 215, 212, 208, 205, 196, and 194 nm. a full cd scan was collected at 95°c. the temperature was then lowered to 25°c. after allowing the protein to refold for 20 min at 25°c, a third cd scan was taken at 25°c to measure recov-ery. a savitzky-golay filter was used to smooth cd data at different temperatures for visual comparison (fig. s2) . secondary structure calculations for the cd data collected at 25°c before the thermal melt were processed by dichroweb (16) using the cdsstr (26), selcon3 (27) , and contill (28) algorithms with protein reference set 7. results from the three algorithms were averaged and plotted in fig. 2 . histochemistry was performed as described previously (11) . briefly, chicken trachea tissues from a 7-week-old broiler chicken were sectioned at 4 m before incubation with rbd proteins at 100 g/ml. desialylated tissues were prepared by pre-treatment with 2 milliunits of neuraminidase (sialidase) from a. ureafaciens (auna, sigma, germany) in 10 mm potassium acetate, 2.5 mg/ml triton x-100, ph 4.2, at 37°c overnight before protein application. chicken trachea tissues were from a 7-week-old broiler chicken (g. gallus) obtained from the tissue archive of the veterinary pathologic diagnostic center (department of pathobiology, faculty of veterinary medicine, utrecht university, the netherlands). sialic acids (neu5ac␣2-3gal␤1-3glcnac-paa, 3-sialc-paa, glyconz, russia) were coated (1 g/well) in a 96-well maxisorp plate (nunc, sigma) at 4°c overnight, followed by blocking with 3% bsa (sigma) in pbs-0, 1% tween. rbd proteins (100 g/ml) were preincubated with strep-tactin-hrpo (1:200) for 30 min on ice, before applying them to the plates for 2 h at room temperature. 3,3ј,5,5ј-tetramethylbenzidine substrate was used as a peroxidase substrate to visualize binding, after which the reaction was terminated using 2 n h 2 so 4 . absorbances (a 450 nm ) were measured in a fluostar omega (bmg labtech) microplate reader, and mars data analysis software was used for analysis. protein samples of each recombinant protein were measured at each concentration in triplicate. statistical analysis was performed by comparing each protein to the unmodified rbd using two-way anova with dunnett's multiple comparisons test where ␣ was set to 0.05. the workflow is shown in fig. s3 . aliquots between 200 and 400 g of m41, n59a, and n145a and 50 g of the remaining proteins were digested with trypsin as per an and cipollo (29) . approximately 25-100-g aliquots of protease-digested proteins were processed for deglycosylated glycopeptide and permethylated glycan analyses. samples were resuspended in 50 mm ammonium bicarbonate, ph 8.0. glycans were released by digestion with 10 units/l pngase f (glycerol-free from new england biolabs) for 3 h at 37°c. the samples were adjusted to ph 5.0 with 2-4 l of 125 mm hcl. to maximize glycan release, samples were further digested with 0.15 milliunits/l pngase a overnight at 37°c. free glycans and deglycosylated peptides were separated using c18 spe cartridges (thermo fisher scientific). intact glycopeptide analyses were performed using 175-300 g of hilic-enriched glycopeptides as per an and cipollo (29) . following data collection on the trypsinized glycopeptides, the remainder of the m41, n59a, and n145a sam lc/ms e data were collected on trypsinized peptides deglycosylated with pngase f as described under n-glycan release. asparagines that are deglycosylated by pngase f are converted to aspartate with a mass gain of 0.984 da due to the replacement of -nh 2 with -oh. the percent occupancy for each site is calculated by comparing the intensity of peptides with asn to those with asp. however, spontaneous deamidation of unmodified asn to asp can also occur. 18 o-water, which results in mass shift of 2.984 da, was used to ensure calculated percent occupancy was not skewed due to spontaneous deamidation. this experiment allows for examination of both spontaneous and enzymatically catalyzed deamidation, and therefore, accurate estimations of percent occupancy of glycosites can be determined. percent occupancy was calculated by comparing the intensities of the deglycosylated (dg) and nonglycosylated (ng) peptides using the equation: dg/(dg ϩ ng)⅐100. pngase-released n-glycans were applied to c18 spe and eluted with 0.1% formic acid leaving the deglycosylated peptides bound to the c18 column. the glycan eluate fractions were combined, and butanol was added to a final concentration of 1%. the samples were then loaded onto 100-mg porous graphite columns prepared first by sequential washes of 1 ml of 100% acetonitrile (acn), 1 ml of 60% acn in water, 1 ml of 30% acn in water, and 1 ml of water. all solutions contained 0.1% trifluoroacetic acid (tfa). the loaded columns were washed three times with 1 ml of 0.1% tfa in water, then eluted with 30% acn, 0.1% tfa, water, followed by 60% acn, 0.1% tfa, and water. the eluents were pooled and dried in glass vials by rotary evaporation. permethylation was done following the method of cincanu and costello (30) and cincanu and kerek (31) . maldi-tof analysis of permethylated n-glycans was performed on a bruker autoflex tm speed mass spectrometer in positive polarity reflectron mode. 2,5-dihydroxybenzoic acid was used as a matrix, and malto-oligosaccharides were used as an external calibrant. data were processed using flexanalysis tm . each sample was spotted three times, and scans were collected in positive reflectron mode. peaks were picked and assigned, and intensities were averaged across each set of spots using in-house software. assignments were based on glycans known to be present in hek293t cells. each peptide or glycopeptide sample was analyzed three times. a c18 column (beh nanocolumn 100 m inner diameter ϫ 100 mm, 1.7-m particle, waters corp.) was used for nanolc/ms e analyses. a nanoacquity uplc system (waters corp.) was used for automatic sample loading and flow control. load buffer was 3% acn, 97% water. peptides were eluted via a 60-min gradient from 3 to 50% acn with a flow of 0.4 l/min. all chromatography solutions included 0.1% formic acid. the eluent flowed to an uncoated 20-m inner diameter picotip emitter (new objective inc., woburn, ma). the mass spectrometer was a synapt g2 hdms system (waters corp.). applied source voltage was 3000 v. data were collected in positive polarity mode using data-independent ms e acquisition, which consists of a starting 4-v scan followed by a scan ramping from 20 to 50 v in 0.9 s. to calibrate internally, every 30 s 400 fmol/l glu-fibrinopeptide b with 1 pmol/l leucine enkephalin in 25% acetonitrile, 0.1% formic acid, 74.9% water was injected through the lockmass channel at a flow rate of 500 nl/min. initial calibration of the mass spectrometer was performed in ms 2 mode using glu-fibrinopeptide b and tuned for a minimum resolution of 20,000 full-width at half-maximum. nanolc/ms e data were processed using biopharmalynx 1.3 (waters corp.) and glymps (in-house software) (32, 33) to identify specific glycans on each peptide. the search settings included trypsin digest with up to one missed cleavage, fixed cysteine carbamidomethylation, variable methionine oxidation, and variable n-glycan modifications based on a building block glycan library. assignment inclusion criteria were as follows: 1) the presence of a core fragment (peptide, peptide ϩ hexnac, peptide ϩ hexnac 2 , peptide ϩ dhex 1 hexnac 1 , and peptide ϩ hex 1 hexnac 2 ); 2) the presence of three or more peptide fragments; 3) the presence of three or more assigned glycopeptide fragments; 4) assignment is made in at least 2 of 3 injections; and 5) the existence of the glycan in glyconnect (https://glyconnect.expasy.org). 3 residues 21-268 of the m41 spike em structure were extracted from the published structure (pdb code 6cv0) (18) . this corresponds to the m41-rbd used in this paper. glycamweb's glycoprotein-builder program (34) was used to add the major oligosaccharide found at each glycosylation site onto the protein in silico. all glycosites in the m41 em structure were occupied except asn-246; however, asn-246 was occupied in our data and was populated accordingly. all glycosites were glycosylated in the new pdb file based on best evidence from our ms data. the coordinates of m41-rbd without glycans, m41-rbd with modeled glycans, and bovine rbd (pdb code 4h14) were used in docking experiments. a virtual library of 17 oligosaccharides representing a variety of binding epitopes was created based on the cfg array version 4.2 (see fig. 6 for a list). raw models of the oligosaccharide ligands were created with the amber tool tleap (www.ambermd.org) 3 utilizing the glycam06 force field (35), then energy minimized using yasara (36) . dock screening of the library was performed with the yasara implementation of autodock vina (37) with default parameters. a molecular dynamics simulation with explicit water (tp3) but with fixed coordinates for the backbone atoms was run on the glycosylated m41 rbd model to allow the amino acid side chains to accommodate the added glycans and to find low energy conformations. two models were extracted from the glycosylated md rbd run at 5 and 10 ns, which were used for dock screening with the virtual library. each oligosaccharide ligand was docked against the structures 20 times. docking results shown in fig. 6 are for the 10-ns model. results were similar in the 5-ns models. the long view: 40 years of infectious bronchitis research the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex coronaviridae the avian coronavirus spike protein cloning and sequencing of the gene encoding the spike protein of the coronavirus ibv coronavirus ibv: structural characterization of the spike protein coronavirus ibv glycopolypeptides: size of their polypeptide moieties and nature of their 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databases novel receptor specificity of avian gammacoronaviruses that cause enteritis cryo-em structure of infectious bronchitis coronavirus spike protein reveals structural and functional evolution of coronavirus spike proteins crystal structure of bovine coronavirus spike protein lectin domain the rhesus rotavirus vp4 sialic acid binding domain has a galectin fold with a novel carbohydrate binding site unique carbohydrate-carbohydrate interactions are required for high affinity binding between fc␥riii and antibodies lacking core fucose model system for cell adhesion mediated by weak carbohydrate-carbohydrate interactions carbohydrate-carbohydrate interaction as a major force initiating cell-cell recognition tn and stn are members of a family of carbohydrate tumor antigens that possess carbohydrate-carbohydrate interactions are lewis b and h type 1 on helicobacter pylori involved in binding of bacteria to muc1 mucin? adv variable selection method improves the prediction of protein secondary structure from circular dichroism spectra a self-consistent method for the analysis of protein secondary structure from circular dichroism an unbiased approach for analysis of protein glycosylation and application to influenza vaccine hemagglutinin elimination of oxidative degradation during the per-o-methylation of carbohydrates asimpleandrapidmethodforthepermethylation of carbohydrates glycosylation analysis of engineered h3n2 influenza a virus hemagglutinins with sequentially added historically relevant glycosylation sites glycosylation characterization of an influenza h5n7 hemagglutinin series with engineered glycosylation patterns: implications for avian coronavirus glycosylation 7797-7809 structure-function relationships glycam06: a generalizable biomolecular force field. carbohydrates autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading presenting your structures: the ccp4mg molecular-graphics software drawglycan-snfg: a robust tool to render glycans and glycopeptides with fragmentation information key: cord-333089-ufyzqgqk authors: aguilar-pineda, jorge alberto; albaghdadi, mazen; jiang, wanlin; lopez, karin j. vera; del-carpio, gonzalo davila; valdez, badhin gómez; lindsay, mark e.; malhotra, rajeev; lino cardenas, christian l. title: structural and functional analysis of female sex hormones against sars-cov2 cell entry date: 2020-07-29 journal: biorxiv doi: 10.1101/2020.07.29.227249 sha: doc_id: 333089 cord_uid: ufyzqgqk emerging evidence suggests that males are more susceptible to severe infection by the sars-cov-2 virus than females. a variety of mechanisms may underlie the observed gender-related disparities including differences in sex hormones. however, the precise mechanisms by which female sex hormones may provide protection against sars-cov-2 infectivity remains unknown. here we report new insights into the molecular basis of the interactions between the sars-cov-2 spike (s) protein and the human ace2 receptor. we further observed that glycosylation of the ace2 receptor enhances sars-cov-2 infectivity. importantly estrogens can disrupt glycan-glycan interactions and glycan-protein interactions between the human ace2 and the sars-cov2 thereby blocking its entry into cells. in a mouse model, estrogens reduced ace2 glycosylation and thereby alveolar uptake of the sars-cov-2 spike protein. these results shed light on a putative mechanism whereby female sex hormones may provide protection from developing severe infection and could inform the development of future therapies against covid-19. the novel coronavirus disease 2019 (covid-19) global pandemic caused by infection with the severe acute respiratory syndrome coronavirus 2 (sars-cov2) virus has infected nearly 15 million people worldwide resulting in nearly 600,000 deaths as july 21, 2020 1 . emerging data suggests that males are more susceptible to covid-19 infection and are at higher risk of critical illness and death than females [2] [3] [4] . there has been consistent evidence of an increased case fatality rate (cfr) among males in nearly every country with available sex-disaggregated data including peru, france, greece, italy, mexico, pakistan, philippines and spain amounting to a 1.7 times higher cfr than females 5 . understanding the mechanisms underlying enhanced covid-19 susceptibility and disease severity in males is key to developing new therapies and guiding vaccine development. changes in sex hormone concentration over an individual's lifetime and associated risk of comorbid conditions, such as cardiovascular diseases, may also contribute to variability in disease susceptibility and severity 6 . it has been postulated that the male-biased sex divergence in covid-19 deaths could be, in part, explained by the strict relationship between sex hormones and the expression of the entry receptor for sars-cov2, the angiotensin converting enzyme 2 (ace2) receptor 3, 7 . molecular studies have demonstrated that the male hormone testosterone regulates the expression of ace2 and the transmembrane serine protease 2 (tmprss2) which is an androgen-responsive serine protease that cleaves the sars-cov-2 spike (s) protein and facilitates viral entry via ace2 binding [8] [9] [10] . androgen-driven upregulation of ace2 levels may therefore be associated with increased vulnerability to severe infections in male patients with covid-19. paradoxically, ace2 plays an important role in lung protection during injury which is attenuated by the binding of sars-cov-2 11 . the presence of a male-biased dependence in covid-19 susceptibility may imply the presence of a protective factor against sars-cov-2 infectivity in women. in addition to the ability of sex hormones to modulate expression of proteins related to entry into host cells, both estrogens and androgens are also able to directly modulate immune cell function via receptor-mediated effects 12, 13 . additionally, sex chromosomes may mediate more favorable outcomes among women compared to men affected with covid-19 14 . x-linked genes associated with immune function tend to be expressed more often in females who generally have two x chromosomes compared to males 15 . additional clues to the possible protective effects of estrogens have been suggested by differences in dietary patterns among countries with different cfrs 16 . interestingly, countries with the lowest cfrs including japan and korea are the largest consumers of isoflavones-based foods, also known as phytoestrogens, that may also mediate favorable effects on ace2 expression and therefore covid-19 risk [17] [18] [19] . the observation that females and those individuals consuming higher levels of isoflavones may be protected from covid-19 infection and adverse consequences indicates a potential protective role of estrogens against sars-cov-2. here, we examine the role of two estrogen molecules (17β-diol and s-equol) to modulate the ace2-dependent membrane fusion protein and reduce cell entry of the sars-cov2 spike protein into lung cells. to the best of our knowledge, we report new findings regarding the importance of molecular interactions between hace2 and the viral spike (s) protein. furthermore, we provide insights into the molecular basis for our observations that estrogens impair sars-cov2 entry and highlight the potential for estrogens as an agent in patients with covid-19. glycosylation site-mapping of human ace2 and sars-cov-2 spike interactions. recent studies 20, 21 have shown the ability of the sars-cov2 virus to utilize a highly glycosylated spike (s) protein to elude the host's immune system and bind to its target membrane receptor, ace2, thus enabling entry into human cells. based on the structural complementarity and steric impediments between the s protein and human ace2 (hace2) protein membranes, we mapped the glycosylation sites of both models [21] [22] [23] [24] and performed molecular dynamics simulations (mds) by 250 ns to stabilize the glycosylated sars-cov2 spike (s) and hace2 complex (suppl. table 1 ., suppl. figure 1 and figure 1a ). these analyses revealed that glycosylation of the ace2 protein increases the affinity of the virus s protein to interact with the receptor via glycan-glycan interactions, glycan-protein interactions, hydrogen, and hydrophobic bonds (suppl. table 2 . and figure 1b) . notably, glycan-glycan interactions occur between the ace2 glycan at n322 and n546 and glycans found on the spike's receptor binding domain (s-rbd) at n165 and n343 (figure 1c , left panel). despite the close interaction between ace2 and s-rbd glycans, their affinity to anchor with highly negatively charged molecules such as the ace2 protein remains unalterable (figure 1c right panel) suggesting that glycan and electrostatic-dependent surface tethering may represent a plausible mechanism for ace-s-rbd binding and cell infection. the glycan-protein interactions occur between the ace2 glycan at n53 and the residues of the s-rbd at n437, s438, n439, l441, v445, g446, v483, q498, t500 and q506 (figure 1d ). while ace2 residues at d38, y41, w48 and g326 form hydrogen bonds with residues of the s-rbd at n440, d442, s443, n450 and e484 (figure 1f ). multiple distinct clusters of hydrophobic residues at the ace2 surface were also found to interact with the s-rbd protein (suppl. figure 2 ). importantly one key hydrophobic region on the ace2 surface at t334 interacts with five residues of the s-rbd (p479, g485, f486, g488 and y489), (figure 1g ). given the insights afforded by our in silico mds experiments, we sought to explore the impact of ace2 glycosylation on s-rbd cell entry using cultured human umbilical vein endothelial cells (huvecs). a variety of saccharide substrates were utilized for their ability to modulate glycosylation profiles in cells. the glycosylation pattern of the endogenous ace2 was increased in nearly all treated cells ( figure 1h ). notably co-incubation of huvecs with 10ug of recombinant s-rbd (rs-rbd) protein revealed that glucose (25mm) pre-treatment was associated with the greatest degree of rs-rbd entry into the cells by ~8 fold compared with hypoglycemic media (hbss or optimen) cells ( figure 1g ). this model indicates that glycosylated residues surrounding the cavity at the top of the ace2 molecule could increase binding by the s-rbd. given the possibility that occupancy at glycosylated residues or s-rbd binding sites by estrogens could modify the affinity of the sars-cov2 virus and alter entry into the cell thereby reducing infectivity, we sought to further examine these interactions using a range of complementary experimental approaches (see table s1 ). estrogens bind to hace2 and stimulate its stabilization and internalization. in an effort to explore the potential protective effects of female sex hormones against sars-cov-2 infection, we examined the impact of estradiol (17β-diol) and a dietary-derived phytoestrogen (s-equol) on hace2 structure and protein expression by a combination of in silico modeling, in vitro, and in vivo analysis. specifically, in light of the importance of glycan-glycan interactions that mediate virus-ace2 interactions, we sought to analyze the effect of estrogens on key molecular viral and receptor binding sites. in agreement with yan et al. 22 , we identified three important regions on the ace2 surface that are utilized for sars-cov-2 binding. the environment of these regions is composed of a high density of glycans, including a helix α1 from residues i21 to t52, a helix α2 from residues v59 to m82, and one loop from residues k353 to g354 (suppl. figure 3 and figure 2a ). we then homogenously solvated the glycosylated hace2 structure with 26.6mm of 17β-diol or 26.5mm of s-equol followed by 100 ns of mds. remarkably we found that the 17βdiol molecules interact with residues at f28, y41, q76, t78, q81, m82 and the s-equol molecules interacts with residues at q24, k26, t27, f28, k31, e35, l39, n64, d67, k68, a71, f72, e75, q76 and l79 (figure 2b , supp. table # ). both estrogen molecules energetically stabilized the α1 and α2 helices by physical interactions and thereby minimized the fluctuation of the ace2 chains a and b (figure 2c , supp 3#). importantly , our calculation of free-energy landscape (fel), demonstrated that the surface of chain b of ace2 (s-rbd's preferred interaction region) loses its interaction energy with the s-rbd protein from 10.2 kj/mol to 8.58 kj/mol (16%) for the 17β-diol system and to 9.18kj/mol (10%) for the s-equol system ( figure 2d ). in addition, binding of either estrogen molecules to the surrounding hydrophobic pocket of ace2 at the residue t334 promotes a decrease in energy by ~12% which may have a negative impact on the attachment of the s-rbd protein to the receptor (suppl. figure 4) . we also observed estrogen-glycan interactions particularly at the glycan-protein interactions between the ace2 (n53) and the s-rbd (n432) ( figure. 3a) . indeed, glycans are highly polar structures due to their high content of hydroxyl groups which make them suitable for attachment to the ace2 protein (mostly negatively charged) or the sars-cov-2 s-protein (polarly charged). the density functional theory (dft) calculation shows an important decrease of the glycan's molecular electrostatic potential (mep) due to the interactions with either estrogen molecules. therefore, estrogen-glycan interactions could decrease the adhesive effect of glycans that enhance s-rbd and ace2 receptor interactions (suppl. figure 5 and figure. 3b). these structural analyses suggest that estrogens could act as putative ace2 ligands due to their ability to bind to highly energetic pockets at the top of the ace2 surface protein which may increase its conformational equilibria and potentially boost its internalization to the cytoplasm. to support our in-silico analyses, we treated huvecs with 17β-diol (3nm) or s-equol (10nm) overnight under normal physiologic conditions. immunofluorescent staining demonstrated that estrogen-treated cells have less ace2 membrane cellular localization ( figure 3c ). immunoblot analysis revealed that endogenous and dietary estrogens promote ace2 internalization and degradation through the endocytosis process as assessed by lc3b 25 and lamp1 26 protein activation in treated cells ( figure. 3d). to test the hypothesis that lower levels of estrogens are associated with increased levels of ace2 protein in the respiratory tract, we administrated intratracheally either 17β-diol (0.3μm) or s-equol (1μm) to male mice. histologic analysis of lung sections demonstrated that both forms of estrogens decrease ace2 membrane expression levels in lung alveoli and also reduced the glycosylation of the ace2 receptor ( figure 3e & 3f) estrogens interfere with sars-cov-2 receptor binding and block entry into the cell. to determine if the decline of conformational gibbs free energy and gain in stabilization of ace2 due to estrogen binding could affect the ability of the s protein to interact with the ace2 receptor and thereby its entry into cells, we performed a refinement step of ace2-free or ace2-estrogen models with 100 ns of mds followed by molecular docking with the sars-cov-2 s protein. from 241 structures obtained, 57 with top scores were chosen for further analysis (suppl. table 4 ). the ace2-17β-diol model promoted the shift of s-rbds from the binding surface toward the lateral side of the ace2 protein decreasing the number of contact residues. notably s-rbds completely lose contact with key ace2-glycosylated residues at n53, n103, n432 and n690. we also observed that the contact between the s-rbd and the helix α1 and α2 of ace2 moved toward the n-terminal of the helix and thus affected the ability to bind the receptor. in the same manner, the ace2-s-equol model demonstrated that s-equol blocks the contact between the s-rbds and the receptor's surface, notably promoting novel interactions at the c-terminal of the helix α2 causing nonspecific contacts with the receptor at residues q429-i436 and p590-n601. interestingly, we found that the 17β-diol interacts with 66 residues on the surface of the receptor and notably forms a cluster on glycans at n546 (chain a) and n322 (chain b). on the other hand, the s-equol molecules tend to interact more widely accounting for a total of 145 interactions, including on 63 residues on the chain a and 82 residues on the chain b. (for better visualization, only the 5 top scored s-rdbs structures are shown in figure 4a ). the nonspecific binding by the s-rbds could be explained by the susceptibility of ace2 to interact with polar molecules and especially to electrophilic attacks. the fact that the 17β-diol or s-equol contain few polar groups but are deficient in negative charge renders them more susceptible to attack the surface of hace2 thereby blocking s-rbd from binding correctly. in addition, we computed the binding score of these models using the atomic energy contact function and in agreement with our previous docking results observed that both estrogen molecules significantly reduced the atomic energy contact between virus and receptor. remarkably, the 17β-diol reduced the atomic contact by 80% and the s-equol by 65% indicating that the entry of the virus may be affected by the presence of either estrogen molecules (figure 4b ). to validate our in-silico prediction, we pre-treated huvecs with either estrogen molecules followed by incubation with 10μg of rs-rbd protein overnight. importantly either low or high concentration of 17β-diol (low=1nm) & high=5nm) or s-equol (low=5nm & high=20nm) blocked more than 90% of the rs-rbd protein entry into the cell as assessed by immunofluorescence and colocalization with lamp1, a lysosome marker ( figure 4c ). in addition, immunoblot demonstrated a decrease of rs-rbd levels in the cytoplasm of huvecs for both estrogen-based treatments (figure 4d ). together these results suggest a potential molecular mechanism by which estrogens may provide protection against severe infection in covid19 among women and individuals with phytoestrogen intake. next, we sought to test the ability of estrogens to block key interactions between ace2 and the sars-cov-2 s protein and thereby infection of the respiratory tract. male wild-type mice were treated with 17β-diol (0.3μm) or s-equol (1μm) via intratracheal instillation for 24hrs before tissue collection. elisa-based binding assay showed a significant decrease of ace2 affinity to sars-cov-2 s protein in lungs from mice treated with either estrogen molecules compared with the control group (figure 5a ). we then evaluated in vivo whether intratracheal estrogen treatment would reduce internalization of the s protein in male mice. we observed that intratracheal instillation of both estrogen molecules 24hrs before intratracheal instillation of rs-rbd (20μg, overnight treatment) increased signal for the rs-rbd on the surface of lung cells which likely results from reduced binding to ace2 in estrogen-treated mice compared to the untreated group. in contrast control (dmso-treated) lungs showed normal ace2 membrane localization and cytoplasmic r-s-rbd signal indicating the unperturbed intake of the rs-rbd protein. the observed increase in extracellular rs-rbd in alveolar cells from lungs of estrogen-treated mice suggests estrogen-mediated reduction in internalization of the s-protein. indeed, we observed that pretreatment with estrogen resulted in rs-rbd protein accumulation on the surface of the alveolar cells (figure 5b ) rather than being internalized into the cytoplasm which would thereby support viral replication and disease progression. our data show that estrogens may interfere with sars-cov-2 infection in the respiratory tract through direct interaction with the ace2 receptor in vivo. increased susceptibility and risk of adverse clinical outcomes among males affected by covid-19 has been reported in multiple epidemiological studies [5] [6] [7] [8] . androgens can effectively upregulate viral target proteins that may increase viral entry and pathogenicity in patients following exposure to the sars-cov2 virus and sex-related hormones can modulate immune respose. a detailed understanding of the molecular and cellular mechanisms modulated by estrogen that contribute to viral pathogenicity is therefore critical to the development of new therapies to combat the covid-19 pandemic. beside the epidemiologic evidence suggesting that females are protected from severe infection, a recent study has demonstrated that the female reproductive tract, expresses very low levels of the ace2 receptor and almost undetectable tmprss2, suggesting that the virus is unlikely to infect the female reproductive tract, where female sex hormones are produced 27, 28 . in the current study, we utilized in silico, in vitro, and in vivo studies to characterize important glycosylation-mediated interactions between the sars-cov2 virus spike (s) protein and the human ace2 receptor that can be modulated by endogenous or dietary estrogens in a manner that may be protective against the sars-cov2 entry into human cells. previous studies have highlighted the critical role of glycosylation in viral pathobiology, host immune system evasion, and infectivity in a range of human viral illnesses 29 . in many of these viruses, the viral envelope and secreted proteins are extensively glycosylated which is necessary for structural integrity and functionality of these proteins. viral proteins may be glycosylated by the host cell as viruses are able to hijack cellular glycosylation processes. however little data exists on the impact of glycosylation of host proteins necessary for viral entry, such as ace2, on viral infectivity. using a novel molecular simulation approach, we demonstrated that ace2 glycosylation augments binding of the viral s protein by supporting multiple types of interactions including glycan-glycan and glycan-protein interactions, thereby facilitating the stability and affinity of viral binding to the target host receptor. we extend these in silico observations by also demonstrating that entry of the s-rbd can be augmented in vitro by exposure of cultured huvecs to a hyperglycemic environment that increases ace2 glycosylation. these observations provide insights into the enhanced susceptibility of diabetic patients to severe infections and death in covid-19 [30] [31] [32] . based on these findings that ace2 glycosylation enhances interaction with the viral s protein in silico, we explored whether the predominant endogenous form of estrogen, 17β-diol, may provide a protective effect as assessed using in silico modeling of viral s protein-ace2 interaction and in vitro and in vivo models of viral infectivity. in addition, we used an identical approach to understand the potential protective mechanisms of dietary phytoestrogens on sars-cov2 infectivity observed in populations with low cfrs where consumption of these foods is high. we found that both endogenous and dietary estrogens compete with the s-rbd protein to bind specific sites on hace2 that are used by the virus to bind the receptor. indeed, estrogens were found to bind at almost all sites including ace2 glycans causing a reduction of energy on the surface of the receptor rendering the receptor less susceptible to interact with other molecules via reduced cell surface expression including the viral s protein interactions. our findings that estrogens interfere with s protein and ace2 interactions in silico that is associated with reduced s protein uptake in an in vitro model of sars-cov-2 infectivity in cultured human endothelial cells are consistent with prior studies demonstrating that estrogens have antiviral properties against hiv, ebola and hepatitis viruses 33 . additionally, recent evidence indicates that decreased levels of estrogens in post-menopausal women are an independent risk factor for disease severity in female covid-19 patients 34 . the findings of the current study thus represent novel findings in our understanding of the molecular mechanisms underlying reduced susceptibility to sars-cov-2 among females or individuals with depressed estrogen levels and in countries where dietary estrogens are high. we then examined the ability of estrogen molecules to interfere with s protein uptake into pulmonary epithelial cells using an in vivo model of sars-cov2 infectivity. in agreement with our cellular experiments, lungs from mice treated with dietary or endogenous estrogens demonstrated a dramatic reduction in the uptake of s-rbd. in addition, we observed a remarkable reduction of ace2 binding possibly due to the low protein levels of ace2 in those lungs possibly in response to estrogen-mediated degradation. in conclusion we provide a molecular basis that helps elucidate the potential protective effect of estrogens in women infected by the sars-cov-2 virus which could inform the development of future therapeutic measures to protect against sars-cov-2 infection including the design of suitable blocking antibodies, estrogen-related treatments, and vaccine development. for immunefluorescence, huvec cells were cultured into 8-well lab-tektm ii chamber slides (nunctm) and were then treated with either 17β-diol at 3nm or s-equol at 10nm. cells were rinsed twice with ice-cold pbs, fixed with 4% paraformaldehyde in pbs (pfa, boston bioproducts) for 10 min at rt, and were permeabilized with 0.1% triton-x (sigma-aldrich) for 3 min. the slides were blocked with 10% donkey-serum, and 0.3 m glycine in pbs-tween 20 (0.1%) for 1 h at rt. subsequently, the antibodies anti-ace2 (1:100), s-rbd-his-tag (1:50), anti-lamp1 (1:50) and anti-lc3b (1:50) were added and slides were incubated overnight at 4°c. the slides were then washed 3 times for 5 min each with pbs-t and were incubated with secondary antibodies at 1:400 dilution for 1hr at room temperature. following immunostaining, slides were mounted with diamond mounting medium containing dapi (thermo fisher). slides were then visualized with the leica tcs sp8 confocal microscopy station and the pictures were digitized with the leica application suite x software. huvec cells were rinsed twice with ice-cold pbs and proteins were extracted with m-per for whole cell lysis, respectively (thermo fisher). these lysis buffers contained halt protease, phosphatase inhibitors and edta (thermo fisher). the protein concentration was determined by the colorimetric bicinchoninic acid assay (bca assay, thermo fisher). equal amounts of total protein from cell lysates were separated by sds-page (25μg or 40μg for ace2, lamp1, lc3b and rs-rbd-his-tag, respectively). proteins from the gel were then electro-transferred onto 0.45 μm nitrocellulose and 0.2 μm pvdf membranes. the membranes were then blocked for 1 h at room temperature, with either 5% non-fat powdered milk dissolved in tbs-t or 5% bovine serum albumin in tbs-t, for the nitrocellulose and pvdf membranes, respectively. following blocking, membranes were incubated overnight at 4°c with the primary antibodies anti-ace2 (1:1000), anti-lamp1 (1:2000), anti-lc3b (1:1000) and anti-his-tag (1:1000). the odyssey infrared western system was used to detect target proteins. band intensity was quantified using imagej software. all experiments involving mice were approved by the partners subcommittee on research animal care. personnel from the laboratory carried out all experimental protocols under strict guidelines to insure careful and consistent handling of the mice. mouse model of sars-cov-2 s protein entry. 9 weeks old male c57bl/6 were purchased from the jackson laboratories, usa. to induce the recombinant s-rbd protein. briefly, mice were anesthetized with sevoflurane inhalation (abbott) and placed in dorsal recumbency. transtracheal insertion of a 24-g animal feeding needle was used to instillate estrogen molecules, rs-rbd or vehicle (dmso), in a volume of 80 µl. mice were sacrificed 24 hrs after instillation of rs-rbs and lungs were removed for further analysis. histology. lungs were then fixed in formalin (10%) for 24 hours before transfer to 70% ethanol for photography prior to paraffinization and sectioning (7 μm) paraffin embedding. slides were produced for tissue staining) for quantitative analysis. saccharides treatment: hypoglycemic media was composed by hbss buffer or optiment media. normal media contained complete endothelial cell growth media. for hyperglycemic media, optiment was supplemented with d-glucose at 25mm, d-galactose at 50mm, d-ribose at 250μm, d-mannose at 300μm, or d-fructose at 20μm. huvecs at 60%-70% confluence were supplemented with hypoglycemic, normal or hyperglycemic media 24 hours before incubation with 10μg of recombinant s-rbd-his-tag overnight. estrogen treatment: huvecs at 60%-70% confluence were supplemented with opti-mem 24 hours before treatment with complete growing media containing 17β-diol at a concentration of 3nm or s-equol at a concentration of 10nm for 24 hrs. fresh media containing rs-rbd (10μg) was supplied the next day. prior to cellular collection, cells were washed with sterile pbs, protein extraction were performed as described above. rs-rbd-ace2 binding assay 500μg of total protein extracts from mouse lungs were cleaned up with iga/igg agarose beads for 1hr at 4c on a rotator followed by resuspension in assay diluent at 1x. then 100μl of each lysate containing 0, 5, 10, 15, 20, 25 or 30μg of total protein were placed into corresponding well of a covid19 s-protein microplate (cat#: cov-sace2, ray biotech,inc.) for overnight incubation at 4c on a rotator. then supernatant was removed, and wells were washed x 5 followed by incubation with 1x hrp-conjugated secondary antibody solution for 1hr at room temperature. then 100μl of tmb one-step substrate reagent was added to each well for 30 min at room temperature. before read 50μl of stop solution was added and microplate was read at 450nm. results are given as mean ± sd student's t test (2-tailed) was applied to determine the statistical significance of difference between control and treated groups (*p < 0.05, **p < 0.01 and ***p < 0.001). for all experiments, at least 3 experimental replicates were performed. violin plot graphs show mean ± sd. data were analyzed, and graphs were prepared with prism 6.0 (graphpad software). p values of less than 0.05 were considered statistically significant. the crystalline structures used in this work were pdb id:6vxx 23 for spike protein (sp, trimeric form) of virus sars-cov-2 and pdbid:6m17 22 (of rbd/ace2-b0at complex) for ace2 protein (dimeric form), both obtained in the rcsb protein data bank. the missing residues for sp located on n and c terminal domains (m-18 to p26 and f1148 to h1262, respectively) were not considered in the molecular simulations. therefore, each one sp chain was made up of 1121 residues (a27 to s1147). in our sp model, another disulfide bond was recognized between missing residues c499 and c507 and was considered in md simulations. for ace2, 29 residues on n-terminal domain were excluded (m-8 to t20) and on c-terminal domain only extramembrane residues were considered (to i21 to g732). ace2 structure contain two zinc ions in peptidase domain which were considered in this work. the remaining missing residues of both proteins were added using swissmodel server 34 (s-t1). the ace2 and sp structures are considered glycoproteins and the glycan-linked residues have already been reported [21] [22] [23] [24] . the oplsaa based doglycans software was used for building all models 35 (s-t1). for sp, there are 22 n-glycosylation residues on its surface, but n17, n1158, n1173 and n1194 sites were excluded due to residues considered in our sp model. oglycosylation sites was not included too. in ace2, all n-glycosylation sites were considered. the glycosylation process was carried out using the glycan glcnac2man3 model, a glycoside sequence composed of 2 n-acetyl glucosamines and 3 mannoses (s-f1b). this glycan type is the most common core sugar sequence on the n-glycans 36, 37 estrogen solvated systems. the systems were constructed with the average structure of glycosylated ace2, obtained in last 50 ns of mds trajectories. 17β-diol and s-equol structures were quantum optimized and their force fields were constructed using ligpargen server [38] [39] [40] . the previous simulation box of ace2 was augmented 0.4 nm in all directions and with the protein centered in box, was solvated two times using gmx solvate module. the first solvation was made homogeneous way with 60 17βdiol and s-equol molecules (26.6 and 26.5 mm solutions, respectively). in second solvation, explicit water molecules were added to fill the simulation box. in the solvation process, we made sure that the estrogen molecules were not close to the protein at the start of the md simulations. all quantum simulations were performed using density functional theory (dft) 41 at b3lyp/ tzvp level 42, 43 . the self-consistent reaction field (scrf) theory was used for describing the solvent effects on the molecules in water solutions. the calculations were performed in the electronic structure program gaussian 16 44 and results were visualized in gaussview v.6 45 . the molecular structures of 17β-diol and s-equol were optimized and it was ensured that they were at a global minimum through frequency analysis. these optimized structures were used in the molecular dynamic simulations. in meps analysis, single point calculations were carried out and total electron densities was mapped on molecular electrostatic potential surface. to address the structural interactions, we performed molecular dynamics simulations using gromacs (v.2019.4) 46 with the opls/aa force field parameters 47. the protein complexes were solvated with tip4p explicit water model 48 . in addition to na + counterions used to neutralize the total charge in the simulation box, we used a 150 mm nacl concentration to mimic physiological conditions. all molecular systems were built in a triclinic simulation box considering periodic boundary conditions (pbc) in all directions (x, y and z). minimum distance of the surface atoms of proteins to the edge of periodic box was 1.5 nm for ace2 receptor and sars-cov-19 spike protein, and 2.3 nm for ace2-estrogen solvated systems. the equations of motions were integrated with the leap-frog integrator 49 using a time step of 1 fs. temperature in the simulations was maintained at 309.65 k using modified berendsen thermostat (v-rescale algorithm) 50 with = 0.1 ps coupling constant with protein and water-ions coupled separately. pressure was maintained at 1bar using the parrinello-rahman barostat 51 with a compressibility of 4.5x10 −5 bar -1 and a coupling constant of = 2.0 ps. all simulations were carried out with a short-range non-bonded cut-off of 1.1 nm and the particle mesh ewald (pme) method 52 was used for computing long-range electrostatic interactions with a tolerance of 1x10 5 for contribution in real space. the verlet neighbor searching cut-off scheme was applied with a neighbor-list update frequency of 10 steps (20 fs). bonds involving hydrogen atoms are constrained using the linear constraint solver (lincs) algorithm 53 . simulations were first energy minimized using the steepest descent algorithm for a maximum of 100,000 steps. the equilibration was conducted by two steps. the equilibration was conducted by two steps. the first step, a 1 ns of dynamics in the nvt (isothermal-isochoric) ensemble and second step, was continued for another 2 ns in the npt (isothermal-isobaric) ensemble. production runs were performed in the npt ensemble for 300 ns for ace2 and sars-cov-19 spike protein and 150 ns for ace2-estrogen solvated systems. structure and data analysis. the structural interactions were obtained carried out a rigid-rigid body docking analysis using patchdock 54 server in order to obtain the contact residues between s-rbd and ace2 systems. the patchdock algorithm discard all unacceptable complex and results are assorted by geometry shape complementarity score. in addition, patchdock do calculate the effective atomic contact energies according to zhang et al. 55 the molecular docking was done take to ace2 protein as receptor molecule and spike protein as ligand molecule. clustering rmsd value and complex type they were selected according to the recommended parameters for protein-protein interactions (4.0ǻ and default mode). for ace2-estrogen systems, the docking was performed in the presence of the estrogen molecules bound to the ace2 structure, 40 17β-diol molecules and 47 s-equol molecules, respectively. from total results obtained in molecular docking, those structures that had steric impediments (intermembranal clashes) were discarded. the steric impediments were calculated based in sars-cov-2 virus size 56, 57 , whose diameter varies about 60 to 140 nm and its spike protein is about 9 to 12 nm length (100 and 10 nm on average, respectively). molecular interactions were analyzed with ligplot software 58 and the pdb files required was constructed with fortran based own computer programs and the statistical data results. statistical results, rmsd, rmsf, rg, sasa, hydrogen bonds, free energies, matches, structures, movies, b-factor maps, were obtained using gromacs modules and their different tool options. the analysis of structure properties was performed using md trajectories on the last 50 ns of each simulations and visualization of the md simulations was created using visual molecular dynamics (vmd) software 59 and the graphs were plotted by the xmgrace software 60 . each molecular conformation during an md simulation has an associated energy and this can be observed using fel maps. these maps are usually represented by two variables related to atomic position and one energetic variable, typically the gibbs free energy. this free energy can be estimated from probability distributions of the system with respect to the chosen variables that are then converted to a free-energy value by bolzmann inverting multi-dimensional histograms. when represented in three dimensions, the fel maps show the energy range of all possible conformations were obtained during a simulation. in this work, we considered two substructures of ace2 protein for fel map analysis, the alpha1-2 region (i21 to y83) and loops regions l2-3 and l3-4 (d303 to r357). the fel maps were plotted using gmx sham module while the rmsd and radius of gyration were considered as atomic position variables respect to its average structure and figures were constructed using wolfram mathematica 12.1 61 . . estrogens bind to ace2 glycans to promote its internalization. (a) glycanestrogen interactions stabilize ace2 structure through high-energy contacts involving ace2 glycan-residues at e57, n53, k341 and v339 (red color). (b) mep maps show the electrostatic impact of estrogen molecules on the surface of ace2 glycans. energy scale ranging from -0.075 μa (red) to 0.075 μa (blue). (c) immunofluorescence staining of human ace2 (magenta) and the lysosome marker lamp1 (green), shows loss of ace2 membrane levels in huvecs treated with 17β-diol or s-equol compared with control cells (dmso). (d) immunoblot of lysates isolated from huvecs showing decreased levels of total ace2 protein with estrogen treatment. reduced ace2 protein levels were associated with increased endocytosis activity as evidenced by immunoblot for lc3b and lamp1. (e) histologic analysis of mouse lungs after 48hrs of intratracheal installation with 17β-diol or s-equol shows loss of ace2 expression (red) on the membrane of alveolar cells. estrogen-treated lungs showed greater ace2-lamp1 colocalization (white arrows) indicating internalization of the receptor. (f) immunoblot showing decreased levels of total and glycosylated ace2 proteins in estrogen-treated lungs from male mice compared to control lungd. quantification of protein levels of three replicate experiments is shown. student's t-test, 2 tails. bar graphs are presented as mean with error bars (±sd). surface interacting with 5 top scored s-rbds (top 1-blue, 2-red, 3-orange, 4-purple and top 5yellow). s-rbds were scored based on shape complementarity principles. (b) heatmap of atomic contact energy between ace2 and 57 s-rbds, shows spontaneous energy structures from most favorable (green) to less favorable s-rbd structures (red). energy scale ranging from 500 kcal/mol to -500 kcal/mol. (c) immunofluorescence analysis of s-rbd entry into huvecs pretreated with 17β-diol or s-equol followed by treatment with 10μg/ml of recombinant s-rbd (red) demonstrate that estrogen-treated cells had reduced entry of s-rbd into cells in conjunction with a reduction in ace2 internalization as showed by colocalization with lamp1 (green). (d) immunoblot of isolated proteins from cultured huvecs shows a 90% reduction of s-rbd entry into cells in estrogen-treated cells. quantification of protein levels of three replicate experiments is shown. student's t-test, 2 tails. bar graphs are presented as mean with error bars (±sd). (a) elisa-based binding assay using lung protein lysates shows reduced sars-cov-2 s protein affinity for the ace2 receptor after treatment with either 17β-diol or s-equol. (b) immunofluorescence analysis of wild-type mouse lung treated with 17β-diol (0.3μm) or s-equol (1μm) compared with control lung (dmso) demonstrates rs-rbd protein accumulation on the surface of the alveolar cells rather than being internalized intracellularly where viral replication may occur. treatment with either estrogen also reduced ace2 prtoein expression (quantification in lower right panel). lamp1 (orange), ace2 (green) and rs-rbd (red). quantification levels of three replicate experiments is shown. student's t-test, 2 tails. bar graphs are presented as mean with error bars (±sd). a pneumonia outbreak associated with a new coronavirus of probable bat origin tripartite combination of candidate pandemic mitigation agents: vitamin d, quercetin, and estradiol manifest properties of medicinal agents for targeted mitigation of the covid-19 pandemic defined by genomics-guided tracing of sars-cov-2 targets in human cells circulating plasma concentrations of angiotensin-converting enzyme 2 in men and women with heart failure and effects of renin-angiotensin-aldosterone inhibitors predictors of mortality in hospitalized covid-19 patients: a systematic review and meta-analysis impact of sex and gender on covid-19 outcomes in europe are sex discordant outcomes in covid-19 related to sex hormones? sars-cov-2 and male infertility: possible multifaceted pathology covid-19 and androgen-targeted therapy for prostate cancer patients sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor structural and functional basis of sars-cov-2 entry by using human ace2 ace2, much more than just a receptor for sars-cov-2. front the x chromosome in immune functions: when a chromosome makes the difference the x-files in immunity: sex-based differences predispose immune responses considering how biological sex impacts immune responses and covid-19 outcomes correction: sex hormones promote opposite effects on ace and ace2 activity, hypertrophy and cardiac contractility in spontaneously hypertensive rats cross-country comparison of case fatality rates of covid-19/sars-cov-2. osong public health res whole versus the piecemeal approach to evaluating soy equol: history, chemistry, and formation beyond the cholesterol-lowering effect of soy protein: a review of the effects of dietary soy and its constituents on risk factors for cardiovascular disease developing a fully glycosylated full-length sars-cov-2 spike protein model in a viral membrane site-specific glycan analysis of the sars-cov-2 spike structural basis for the recognition of sars-cov-2 by full-length human ace2 structure, function, and antigenicity of the sars-cov-2 spike glycoprotein emerging covid-19 coronavirus: glycan shield and structure prediction of spike glycoprotein and its interaction with human cd26. emerging microbes & infections 9 lc3-associated endocytosis facilitates β-amyloid clearance and mitigates neurodegeneration in murine alzheimer's disease gangliosides are essential endosomal receptors for quasi-enveloped and naked hepatitis a virus coronavirus disease-19 and fertility: viral host entry protein expression in male and female reproductive tissues female reproductive tract has low concentration of sars-cov2 receptors impaired estrogen signaling underlies regulatory t cell loss-offunction in the chronically inflamed intestine molecular mechanisms of sex bias differences in covid-19 mortality differential regulation and targeting of estrogen receptor α turnover in invasive lobular breast carcinoma sars-cov-2 has a sweet tooth glycosylation in health and disease potential influence of menstrual status and sex hormones on female sars-cov-2 infection: a cross-sectional study from multicentre in wuhan, china swiss-model: homology modelling of protein structures and complexes doglycans-tools for preparing carbohydrate structures for atomistic simulations of glycoproteins, glycolipids, and carbohydrate polymers for gromacs probing the glycosidic linkage: secondary structures in the gas phase potential energy functions for atomic-level simulations of water and organic and biomolecular systems 1.14* cm1a-lbcc: localized bond-charge corrected cm1a charges for condensed-phase simulations ligpargen web server: an automatic opls-aa parameter generator for organic ligands density-functional thermochemistry. iv. a new dynamical correlation functional and implications for exact-exchange mixing fully optimized contracted gaussian basis sets for atoms li to kr gromacs: high performance molecular simulations through multilevel parallelism from laptops to supercomputers development and testing of the opls all-atom force field on conformational energetics and properties of organic liquids temperature and size dependence for monte carlo simulations of tip4p water quiet high-resolution computer models of a plasma molecular dynamics with coupling to an external bath canonical sampling through velocity rescaling die berechnung optischer und elektrostatischergitterpotentiale a parallel linear constraint solver for molecular simulation patchdock and symmdock: servers for rigid and symmetric docking determination of atomic desolvation energies from the structures of crystallized proteins a novel coronavirus from patients with pneumonia in china science forum: sars-cov-2 (covid-19) by the numbers ligplot: a program to generate schematic diagrams of protein-ligand interactions vmd: visual molecular dynamics version 5.1.19 mathematica, version 12 key: cord-346670-34wfy52f authors: gobeil, sophie m-c.; janowska, katarzyna; mcdowell, shana; mansouri, katayoun; parks, robert; manne, kartik; stalls, victoria; kopp, megan; henderson, rory; edwards, robert j; haynes, barton f.; acharya, priyamvada title: d614g mutation alters sars-cov-2 spike conformational dynamics and protease cleavage susceptibility at the s1/s2 junction date: 2020-10-12 journal: biorxiv doi: 10.1101/2020.10.11.335299 sha: doc_id: 346670 cord_uid: 34wfy52f the sars-cov-2 spike (s) protein is the target of vaccine design efforts to end the covid-19 pandemic. despite a low mutation rate, isolates with the d614g substitution in the s protein appeared early during the pandemic, and are now the dominant form worldwide. here, we analyze the d614g mutation in the context of a soluble s ectodomain construct. cryo-em structures, antigenicity and proteolysis experiments suggest altered conformational dynamics resulting in enhanced furin cleavage efficiency of the g614 variant. furthermore, furin cleavage altered the conformational dynamics of the receptor binding domains (rbd) in the g614 s ectodomain, demonstrating an allosteric effect on the rbd dynamics triggered by changes in the sd2 region, that harbors residue 614 and the furin cleavage site. our results elucidate sars-cov-2 spike conformational dynamics and allostery, and have implications for vaccine design. highlights sars-cov-2 s ectodomains with or without the k986p, v987p mutations have similar structures, antigenicity and stability. the d614g mutation alters s protein conformational dynamics. d614g enhances protease cleavage susceptibility at the s protein furin cleavage site. cryo-em structures reveal allosteric effect of changes at the s1/s2 junction on rbd dynamics. the severe acute respiratory coronavirus 2 (sars-cov-2) belongs to the b-coronavirus family of enveloped, positive-sense single stranded rna viruses, and has one of the largest genomes among rna viruses (de wit et al., 2016) . of the seven known coronaviruses that infect humans, four (hcov-229e, hcov-oc43, hcov-nl63, cov-hku1) circulate annually causing generally mild respiratory symptoms in otherwise healthy individuals, while the sars-cov-1 and middle east respiratory syndrome coronavirus (mers-cov) , that are closely related to sars-cov-2, have resulted in the 2002 -2003 sars and 2012 mers epidemics (zumla et al., 2016 , respectively. the ongoing pandemic of coronavirus disease of 2019 , is a global public health emergency with more than 37 million cases and 1 million deaths recorded worldwide (dong et al., 2020) (https://coronavirus.jhu.edu). the surface of the sars-cov-2 is decorated with the spike (s) glycoprotein turonova et al., 2020) that is the target of most current vaccine development efforts sempowski et al., 2020) . in its prefusion conformation the sars-cov-2 s protein is a large homo-trimeric glycoprotein forming a crown (from the latin corõna) at the surface of the virus capsid. each s protomer is subdivided into two domains, s1 and s2, which are delimited by a furin cleavage site at residue 682-685 (figure 1) . the s1 domain comprises the n-terminal domain (ntd), an ntd-to-rbd linker (n2r), the receptor binding domain (rbd), and subdomains 1 and 2 (sd1 and sd2). the s2 domain contains a second protease cleavage site (s2') followed by the fusion peptide (fp), heptad repeat 1 (hr1), the central helix (ch), the connector domain (cd), heptad repeat 2 (hr2), the transmembrane domain (tm) and a cytoplasmic tail (ct) (figure 1) . the s1 domain is responsible for recognition and binding to the host-cell angiotensin-converting enzyme 2 (ace2) receptor. the s2 domain is responsible for viral-host-cell membrane fusion and undergoes large conformational changes (hoffmann et al., 2020a) , but only upon furin cleavage and further essential processing by cleavage at the s2' site by tmprss2 and related proteases (bestle et al., 2020; hoffmann et al., 2020b; matsuyama et al., 2020) . previous reports have demonstrated the central role of the dynamics of the rbd domains between a "closed" (or all rbd-down receptor inaccessible conformation) and "open" (or rbd-up) conformations for recognition and binding to the host cell ace2 receptor (gui et al., 2017; shang et al., 2020; yuan et al., 2017) . since the early stages of the covid-19 pandemic, virus evolution has been followed by large-scale sequencing of the virus genomes isolated from patients, and several mutations that arose and propagated within different populations have been identified even though the virus has genetic proofreading mechanisms (elbe and buckland-merrett, 2017; korber et al., 2020) . the d614g mutation in particular has attracted attention since it has quickly become the dominant strain of sars-cov-2 circulating worldwide (korber et al., 2020) . the d614g mutation of the s protein has been associated in numerous reports with increased fitness and/or infectivity of the virus (korber et al., 2020; li et al., 2020; weissman et al., 2020) . cryo electron microscopy (cryo-em) structures of the s glycoprotein ectodomain have revealed that d614 is a surface residue in the vicinity of the furin cleavage site. mutation of this residue to a glycine is expected to disrupt a critical interprotomer hydrogen bond involving t859 of the s2 domain (korber et al., 2020) and resulting in a shift in the observed equilibrium between the open and closed state of the s protein ectodomain (johnson et al., 2020; weissman et al., 2020; yurkovetskiy et al., 2020) ( figure 1 ). most structures of the sars-cov-2 s ectodomain currently available include two mutations, one to disrupt the furin cleavage site (rrar to gsas = s-gsas), and a double proline mutation (pp) of residues 986-987, designed to prevent conformational change to the post-fusion state (wrapp et al., 2020) . originally designed for the mers s protein (pallesen et al., 2017) , insertion of two consecutive pro mutations at the junction of the hr1 and ch regions stabilized the pre-fusion conformation of the mers, sars and hcov-hku spikes, increased protein expression, and immunogenicity for the mers s protein (pallesen et al., 2017) . based on these prior data, introduction of two consecutive proline residues at the beginning of the central helix was postulated as a general strategy for retaining b-coronavirus s proteins in the prefusion conformation. thus, the pp mutations were carried over to the sars-cov-2 ectodomain (wrapp et al., 2020) that is currently widely used in the field for vaccine and structural studies, and is also the component of a vaccine candidate . although shown to stabilize the pre-fusion conformation of other coronaviruses, the effect of the pp insertion has not been systematically studied for the sars-cov-2 s ectodomain. with the goal of investigating the biophysical and structural consequences of the d614g mutation, and to prevent the engineered pp mutations from confounding our observations, we produced two sars-cov-2 s ectodomain constructs with the native k986 and v987 residues, incorporating either a d or a g at position 614 (figure 1) . the rrar sequence in the furin cleavage site was replaced by a gsas sequence thus rendering the s constructs furin-cleavage deficient. to probe the effect of the d614g substitution on furin cleavage of the s protein, we either reinstated the native furin sequence or replaced it with an exogeneous hrv3c proteolysis signal. we determined the cryo-em structures of the uncleaved d614 and g614 s ectodomains, as well as the structure of the fully cleaved g614 s ectodomain of the currently globally dominant sars-cov-2. our results demonstrate the effect of the d614g substitution on the conformational dynamics and furin cleavage susceptibility of the s ectodomain, and reveal insights into the allostery between the rbd and distal regions of the s protein. while the sars-cov-2 s ectodomain construct that includes mutations of residues k986 and v987, between the hr1 and ch subdomains (s2 domain), to prolines (pp) (named s-gsas/pp in this study) (figure 1 ) is widely used in the field, the origin of this pp construct was based upon the stabilization of the pre-fusion conformation of other coronavirus spikes (pallesen et al., 2017; walls et al., 2020; wrapp et al., 2020) . here, we generated an analogous s ectodomain construct that had the native k986 and v987 residues (named s-gsas) (figure 1 ). in our 293f expression system (see methods for details), both the s-gsas/pp and s-gsas constructs expressed at similar levels, yielding about 3 mg final protein per l of culture. both proteins also showed similar migration profiles on sds-page and by size exclusion chromatography (sec) on a superose 6 column (figure 2a, b) . similar to the s-gsas/pp construct henderson et al., 2020; wrapp et al., 2020) , s-gsas showed 80-90% intact prefusion spike trimers by negative stain electron microscopy (nsem) (figure 2c ). this finding is in contrast to previous observations for mers and the sars-cov-1 ectodomains, which showed a mixture of the prefusion and postfusion conformations unless the pp mutation was included (pallesen et al., 2017) . binding of s-gsas and s-gsas/pp measured by elisa to ace2 and cr3022 , both requiring an rbd-up conformation, ab712199 and ab511584, two antibodies isolated from a covid-19 convalescent donor with epitopes mapping to the ace2 binding site and s2 domain respectively, and 2g12, binding to a quaternary s2 glycan epitope, were all nearly identical demonstrating that both constructs showed similar antigenic behavior ( figure 2d ). using differential scanning fluorimetry to measure the spike thermostability, we found the s-gsas and s-gsas/pp ectodomains showed similar melting temperatures ( figure 2e ). we next solved cryo-em structures of the s-gsas ectodomain (figures 2f-h , s1-s2, table s1 ), to compare with the s-gsas/pp structures (walls et al., 2020; wrapp et al., 2020) and to visualize the impact that the engineered pp mutations had on the structure of the sars-cov-2 spike ectodomain. two populations of the s-gsas s ectodomain were identified in the cryo-em dataset -a 1-rbd-up (or open) and a 3-rbd-down (or closed) conformation ( figure 2f and table s1 ). both structures were similar to the corresponding structures of s-gsas/pp (walls et al., 2020) , with overall rmsds of 0.45 å and 0.54 å for the 1-up and 3-down structures, respectively. in the region around the pp mutations, we found the s-gsas structures to be similar to the corresponding s-gsas/pp structures ( figure 2h ). in the s-gsas 1-rbd-up structure, we observed that the k986 side chain was appropriately positioned to make an interprotomer salt bridge with the d427 residue of the rbd of the adjacent protomer, an interaction that would be abrogated in the pp construct. the corresponding residues in the mers s protein, v1060 and l1061 are non-polar, and the adjacent protomers too far to interact with these residues (figure s3 ). in the sars-cov-1 s protein cryo-em structure (pdb 5xlr, 5x5b) the residues d414-d415 (equivalent to sars-cov-2 d427-d428) lie further from k986 suggesting that this putative salt bridge interaction may be more transient in sars-cov-1. overall, our data show that for the sars-cov-2 s ectodomain, the s-gsas construct showed similar structural, antigenic and stability behavior as the s-gsass/pp construct that included the k986p and v987p mutations at the junction of the ch and hr1 regions. while these and analogous mutations had proved beneficial for the expression and stability of other covs (pallesen et al., 2017) , for the sars-cov-2 s protein other compensating interactions may help confer stability to the pre-fusion form in the absence of the pp mutations. for the rest of this study we have used the s-gsas construct as the platform for introducing mutations and other modifications of interest. to understand the molecular details of the spike d614g mutation that arose and quickly dominated circulating sars-cov-2 isolates globally, we sought to assess the impact of the d614g mutation on the structure and antigenicity of the sars-cov-2 s ectodomain. the d614g mutated s-gsas construct (s-gsas/d614g), yielded an average of ~ 2 mg of purified protein per l of culture (n = 4). the sds-page, sec and dsf profiles of the s-gsas/d614g ( figure 3a ) were similar to that of the s-gsas s ectodomain (figure 2a, b) . nsem of the s-gsas/d614g s ectodomain revealed typical and well-dispersed pre-fusion s particles ( figure 3b ). to visualize structural details at higher resolution, we determined the cryo-em structures of s-gsas/d614g construct ( figure 3c -e, table s1, figures s4-s7) . two major populations of the s ectodomain were identified in the cryo-em dataset -one population with one rbd in the "up" or ace2 receptor accessible conformation and the other with all three rbds in the figure 3c ). this is consistent with our previous observations made with nsem data that showed an increase in the rbd-up population for the s-gsas/d614g s ectodomain (weissman et al., 2020) . our results show that the d614g mutation in the sd2 domain, even though distal from the rbd region, has an allosteric effect on rbd dynamics leading to alteration of up/down rbd dispositions. to understand the nature of this allostery, we examined changes in the s protein that accompany the up and down rbd transition (figure 4 ) by comparing the rbd-up chain in the 1-rbd-up structure to the down chains in the 1-up and the 3-down structures ( figure 3c ). in each s protein protomer, the polypeptide chain folds into domains as it traverses the length of the s1 subunit and enters the s2 subunit i.e. the ntd (residues 27-305) followed by the rbd (residues 335-521), the sd1 (residues 529-591) and sd2 (residues 592-697) domains ( figure 4a ). the ntd and rbd are connected via a 28-residue linker spanning residues 306-334 (named n2r) that stacks against the sd1 and sd2 domains ( figure 4a-d) , as it makes its way from the ntd to the rbd, essentially connecting all the individual domains in the s1 subunit, and forming "super" subdomains sd1' and sd2', respectively . upon overlaying the protomers with the rbd in the up position with the protomers with their rbds in the down position by using the s2 subunit residues 908-1035 for superpositions, we found that the down-to-up rbd motion is accompanied by a rigid body movement of the sd1' domain resulting in a shift of up to ~4.5 å of the sd1 domain ( figure 4d ), relative to its position in the rbd-down protomers and a shift of up to a 7 å in the n2r linker as it hinges to enter into the rbd. this results in a ~20° tilt of residues 324-328 of the n2r linker region that forms part of the sd1' super subdomain, while residues 311-319 of the linker that associate with the sd2 subdomain remained virtually unmoved, showing only a slight tilt in the b beta strand that accompanied large movements in the rbd and adjoining sd1' domain ( figure 4d ). indeed, the sd2' super subdomain that harbors the d614g mutation appears to form a conformationally invariant anchor with the mobile rbd and ntd domains at either end ( figure 4d) . additionally, the s2 subunit remains invariant between the different protomers showing that the large movements that occur in the s1 subunit are effectively arrested by the sd2' super subdomain conformationally invariant anchor. these observations are mirrored in the difference distance matrices (ddm) comparing the rbd-up and down chains ( figure 4e and figure s8 ). ddm analyses (richards and kundrot, 1988) provide superposition-free comparisons between a pair of structures by calculating the differences between the distances of each pair of ca atoms in a structure and the corresponding pair of ca atoms in the second structure. the ddm analysis not only shows the large movement in the rbd region and the movement in the ntd, it also captures the movement in the n2r linker and the sd1 domain observed in the structures. overall, these analyses show that the d614g mutation is acquired within a key region encompassing the sd2 domain and an additional b-strand contributed by residues 311-319 of the n2r linker that forms a region of relative structural stillness separating the mobile ntd and rbd, as well isolating the motions in s1 from the s2 subunit. this distal mutation altering rbd conformational dynamics shows that small changes in this region can translate into large allosteric effects, and suggests a role for the sd2 domain in modulating rbd dynamics. in addition to the d614g mutation, the sd2 subdomain also harbors a furin cleavage site (residues 682-685) that separates the s1 and s2 subunits (figure 1) . cleavage of the s protein by furin at this site is essential for virus transmission (shang et al., 2020) . the proximity of the d614g mutation to the furin cleavage site and the increased flexibility observed in the cryo-em dataset of the s-gsas/d614g ectodomain ( figure 3c -e), prompted us to examine the effect of the d614g substitution on furin cleavage. since our expression system (i.e. 293freestyle cells) endogenously expresses furin, in order to obtain uncleaved spike that we could then test for protease cleavage in vitro, we engineered a hrv3c site (8 amino acids long) to replace the furin cleavage site (4 amino acids long) at the s1/s2 junction, resulting in the s-hrv3c and s-hrv3c/d614g s ectodomain constructs ( figure 1a ). both proteins expressed in 293f cells but at lower yields compared to the s-gsas constructs (36 µg/l and 410 µg/l for the s-hrv3c and s-hrv3c/d614g proteins, respectively). sec and sds-page profiles were similar to the s-gsas and s-gsas/d614g proteins confirming well-folded and homogeneous spike preparations ( figure 5a , b). nsem micrographs showed characteristic kite-shaped particles for the pre-fusion s protein, and 2d-classification of particles from nsem revealed well folded spikes, further confirming that s-hrv3c spikes retained the overall fold and structure of the s-gsas spikes (figure 5c, d) . to test the susceptibility of the hrv3c site engineered at the junction of the s1 and s2 subunits to protease cleavage, we incubated the purified s-hrv3c and s-hrv3c/d614g spikes with the hrv3c enzyme and followed the digestion by analyzing aliquots taken at different time-points by sds-page ( figure 5e -g). we found that the digestion of the s-hrv3c/d614g spike ( figure 5f -g) proceeded at a faster rate than that of the s-hrv3c spike ( figure 5e-g) with the s-hrv3c/d614g spike almost 100% digested within the first 10 minutes of incubation, whereas, the s-hrv3c constructs only achieved 50% of cleavage after 24 hours, and a substantial portion remained uncleaved even upon addition of more enzyme followed by 4 additional hours of incubation. these results suggested that the d614g mutation increased the susceptibility of protease cleavage at the s1/s2 junction. to study the effect of the d614g substitution on protease cleavage at the s1/s2 junction with the native furin site, we next generated spike ectodomains constructs where the furin site was restored to the native sequence, resulting in two constructs named s-rrar and s-rrar/d614g ( figure 1a) . the proteins were expressed and purified using our usual methodology for the furin cleavage-deficient constructs (see methods). the sec profiles ( figure 6a ) showed a higher proportion of the first higher molecular weight peak. a second peak eluting at a similar molecular weight as the s-gsas spike (at ~13.8 ml elution volume) was used for further characterization. the sec profile of the s-rrar spike preparation showed small populations of lower molecular weight peaks that were not observed for the s-rrar/d614g protein ( figure 6a ). on sds-page (figure 6b) , the peak corresponding to the s ectodomain showed the s-rrar construct as having one major band at the molecular weight corresponding to the spike monomer and some fainter bands corresponding to the s1 and s2 subunits while the s-rrar/d614g protein showed a band corresponding to the spike monomer and the two bands corresponding to the molecular weights of the s1 and s2 subunits. the smaller molecular weight bands corresponding to the s1 and s2 subunits were in higher proportions in the s-rrar/d614g spike preparation compared to the s-rrar preparation. in summary the sec and sds-page profiles showed that, although both the s-rrar and s-rrar/d614g constructs were cleaved by endogeneous furin (figure 6b ) during protein expression the s1 and s2 subunits remained together in solution ( figure 6a) . consistent with the enhanced cleavage observed for the s-hrv3c/d614g spike relative to the s-hrv3c spike, in the furin-site restored spikes we observed a higher proportion of cleaved spike in s-rrar/d614g relative to s-rrar, suggesting that the d614g mutation makes the spike more susceptible to furin cleavage. nsem of the purified s-rrar ( figure 6c ) and s-rrar/d614g ( figure 6d) confirmed that both of these furin site-restored spikes formed well-folded spike ectodomains. we next digested the sec isolated fractions of the s-rrar and s-rrar/d614g ectodomains ( figure 6a-d) in vitro by adding furin ( figure 6e ). as observed for the s-hrv3c constructs, the d614 version of the spike was less susceptible to cleavage than the g614 mutant for the same incubation time with the enzyme. sec purification of the fully digested s-rrar/d614g ectodomain revealed a peak corresponding to the ectodomain ( figure 6f) . on sds-page, this peak migrated as two distinct bands corresponding to the s1 and s2 domains thus confirming isolation of only the cleaved portion of the protein ( figure 6g ). nsem showed fully folded ectodomains for the furin digested and sec purified s-rrar/d614g protein ( figure 6h ). in summary, these results show that acquisition of the d614g mutation the s protein sd2 domain resulted in increased furin cleavage of the s ectodomain. to visualize the structure of the furin-cleaved s ectodomain at atomic level resolution, we obtained a cryo-em dataset, and resolved two populations of the furin-cleaved s ectodomain -a 1-rbd-up and a 3-rbd-down population ( figure 7a, figure s9 and s10 and table s1 ). we observed an increased proportion of the 3-rbd-down s compared to the uncleaved d614g s ectodomain, thus reporting a change in the rbd conformational dynamics upon furin cleavage. consistent with this result, we observed reduced binding to ligands such as ace-2 and cr3022 that require the rbd to be in the up conformation for binding ( figure 7b ). as expected, decrease in binding was also observed with antibody 712199, isolated from a convalescent covid-19 donor, with an epitope overlapping with the ace2 binding site. antibody 2g12 that binds a quaternary glycan epitope in the s2 subunit showed a small decrease in binding with the furin-cleaved s ectodomain, whereas another covid-19-derived s2 antibody 511584 showed increase in binding with the furin-cleaved s ectodomain. we compared the different protomers in the two structures by overlaying three protomers in the asymmetric 1-rbd-up structure and one protomer from the symmetric 3-rbd-down structure using residues 908-1035 (comprising the ch and hr1 regions) for superposition ( figure 7c ). similar to observations made with the s-gsas/d614g s ectodomain structure, the rbd up/down motion in the furin-cleaved g614 s ectodomain was associated with a movement in the sd1 domain and in the region of the rbd-to-ntd linker that joined the sd1 b sheet ( figure 7c, s8b) . as observed for s-gsas/d614g, the sd2 domain showed little conformational change and formed a stable motif anchoring the mobile ntd and rbd domains. these observations reinforce the divergent roles of the sd1 and sd2 domain in rbd motion. we next examined the region of the sd2 domain proximal to the ntd, and asked whether we could detect any structural changes in this region and if yes, could these be related to ntd motion. in the symmetric 3-rbd-down s ectodomain, all ntds are identical, each stacking against the down rbd of the adjacent promoter. in the asymmetric 1-rbd-up structure, each ntd were distinct. to distinguish between these, we named the ntds as follows: the ntd that was part of the protomer with the rbd in the up conformation was named ntd1. ntd1 stacked against a down rbd that contacted the up-rbd at one end and the second down-rbd at the other. the ntd2 stacked against a down-rbd that contacted a down-rbd at one end, and the ntd3 had the least amount of rbd contact by virtue of contacting the up-rbd ( figure 7a ). observing the ntd-proximal region on the sd2 domain (marked by a dotted square on figure 7c ) that also contacted the rbd-to-ntd linker, we noted shifts in the t602-606 loop between the different protomers. while the shifts were modest (with a maximal displacement of 2.2 å), interestingly, identical trends were observed in the 1-rbd-up structures of the s-gsas, s-gsas/d614g and furin-cleaved s-gsas/d614g s ectodomains, suggesting that this region of the sd2 domain responds to ntd motion and adopts a different conformation depending on the ntd environment ( figure 7d ). thus, these data provide further evidence for allostery in the s protein, with changes in the sd2 domain impacting the rbd conformational dynamics. while the sd2 domain remains almost structurally invariant, we observe small but reproducible changes in sd2 loops in response to rbd/ntd movement suggesting that small changes in the sd2 region may translate to large motions in the rbd/ntd region. stabilized ectodomain constructs have proven to be useful tools to understand conformational dynamics of cov s proteins. in particular, these have enabled high-resolution structural determination and atomic level understanding of the s ectodomain. they also are key components in vaccine development pipelines. the structural similarities in the s proteins of diverse covs have often enabled quick translation of structural rules and ideas from one cov s ectodomain to another. indeed, after the onset of the recent and ongoing covid-19 pandemic, the sars-cov-2 s ectodomain could be rapidly stabilized and structurally characterized by exploiting its similarities with other covs and following strategies that had proved successful previously pallesen et al., 2017; wrapp et al., 2020) . some of these stabilization strategies, such as introduction of proline residues in the fusion subunit to prevent transition from pre-to post-fusion, have been successful in stabilizing the pre-fusion conformation of diverse class i fusion proteins including rsv f (krarup et al., 2015) , hiv-1 env (sanders et al., 2002) , ebola and marburg gp (rutten et al., 2020) , influenza ha (qiao et al., 1998) and lassa gpc (hastie et al., 2017) . while the underlying hypothesis for the stabilization of the s ectodomain was that introduction of 2 proline residues at the junction of the ch and hr1 helices would arrest conformational transition to the post-fusion form, we found that even without the pp mutations, the sars-cov-2 s ectodomain retained its pre-fusion form. not only so, even following furin cleavage the s ectodomain retained its pre-fusion conformation. these differences between the observed behavior of the sars-cov-2 s relative to other covs suggests that even though they retain similar overall topology and structural folds, there are differences between these covs that profoundly affect their structural and biological properties. studying and accounting for these will be essential not only to understand sars-cov-2 but also to appreciate the nature and origin of these differences between covs for anticipating, preparing for and rapidly combating future cov pandemics. viral surface proteins that are involved in receptor binding mediated cellular entry typically consist of flexible and moving parts that exhibit large conformational changes. while this conformational flexibility is necessary for function, structural checkpoints are required to prevent premature activation and destabilization or unfolding of the protein structure. conformationally-silent structural islands provide the necessary stabilizing anchors for adjacent regions undergoing large motions. in this study we have identified the sd2 domain in the sars-cov-2 s protein as such a conformational anchor that is spatially interspersed between the highly mobile ntd and rbd regions, while itself remaining relatively invariant in its conformation. this conformational invariability of the sd2 subdomain is reminiscent of the beta sandwich structure in the hiv-1 envelope glycoprotein that connects and anchors a mobile layered architecture of the gp120 inner domain (pancera et al., 2010) . the conformationally invariant sd2 also serves to contain the movements of the rbd and ntd to the s1 subunit, such that the s2 subunit was unchanged between the various rbd "up" and "down" protomers ( figure 4 and figure s8 ). this suggests a role for the sd2 domain in preventing premature triggering due to the stochastic up/down rbd motions in the sars-cov-2 s protein, as well as the importance of downstream events such as ace2 receptor engagement and tmprss2 protease cleavage (bestle et al., 2020; hoffmann et al., 2020b; matsuyama et al., 2020) in orchestrating the full extent of pre-to post-fusion transformation. in this study, we also assigned a key role to the n2r linker that connects the ntd to the rbd within a protomer. rather than just being a connector, this 28-residue linker is also a modulator of conformational changes that are critical for receptor engagement. the linker contributes a beta strand to each of the sd1 and sd2 subdomains thus connecting all the structural domains in the s1 subunit. in addition to the much discussed d614g mutation, the sd2 subdomain also houses the multibasic furin cleavage site that demarcates the s1 and s2 subunits. furin cleavage is an essential processing step for the s protein and is necessary for viral infection and transmission (hoffmann et al., 2020a; shang et al., 2020) . we provide evidence in this study that the d614g mutation enhances susceptibility of the sars-cov-2 s ectodomain to furin cleavage, thus raising the possibility that this is a contributor to increased fitness and transmissibility of d614g isolates. in this paper, we study the effect of the d614g mutation on rbd dynamics and susceptibility to furin cleavage. we find that the d614g mutation results in increased furin cleavage susceptibility, which could be responsible for the increased transmissibility of the sars-cov-2 with the d614g mutation. it is important to consider though that these results are further information and requests for resources and reagents should be directed to priyamvada acharya (priyamvada.acharya@duke.edu) . data and code availability cryo-em reconstructions and atomic models generated during this study are available at wwpdb and embd (https://www.rcsb.org; http://emsearch.rutgers.edu) under the accession codes pdb ids 7kdg, 7kdh, 7kdk, 7kdl, 7kdi, 7kdj, 7ke4, 7ke6, 7ke7, 7ke8, 7ke9, 7kea, 7keb, 7kec and emdb ids emdb-22821, emd-22822, emd-22825, emd-22826, emd-22823, emd-22824, emd-22831, emd-22832, emd-22833, emd-22834, emd-22835, emd-22835, emd-22837, emd-22838 . gibco freestyle 293-f cells (embryonal, human kidney) were incubated at 37°c and 9% co2 in a humidified atmosphere. cells were incubated in freestyle 293 expression medium (gibco) with agitation at 120 rpm. plasmids were transiently transfected into cells using turbo293 (speedbiosystems) and incubated at 37 °c, 9% co2, 120 rpm for 6 days. on the day following transfection, hyclone cdm4hek293 media (cytiva, ma) was added to the cells. antibodies were produced in expi293 cells (embryonal, human kidney). cells were incubated in expi293 expression medium at 37°c, 120 rpm and 8% co2 in a humidified atmosphere. plasmids were transiently transfected into cells using the expifectamine 293 transfection kit and protocol (gibco). all genes in this study were synthesized and sequenced by geneimmune biotechnology (rockville, md). the sars-cov-2 spike protein ectodomain constructs used comprised the protein residues 1−1208 (genbank: mn908947) with or without the d614g mutation, with or without the furin cleavage site rrar (residue 682-685) mutated to gsas or levlfqgp (hrv3c protease site), a c-terminal t4 fibritin trimerization motif, a c-terminal hrv3c protease cleavage site (except for the constructs where the furin site was mutated to an hrv3c site), a twinstreptag and an 8xhistag. all spike ectodomain constructs were cloned into the mammalian expression vector pαh (wrapp et al., 2020) . for the ace-2 construct, the c-terminus was fused a human fc region. protein purification spike ectodomains were harvested from filtered and concentrated supernatant using streptactin resin (iba) and further purified by sec using a superose 6 10/300 gl increase column preequilibrated in 2mm tris, ph 8.0, 200 mm nacl, 0.02% sodium azide. all protein purification steps were performed at room temperature in a single day. the purified proteins were flash frozen and stored at -80 °c in single-use aliquots. each aliquot were thawed by incubation (~20 min) at 37 °c before use. antibodies were produced in expi293f cells and purified by protein a affinity. ace-2 with human fc tag was purified by protein a affinity chromatography. negative-stain electron microscopy samples were diluted to 100 µg/ml in 20 mm hepes ph 7.4, 150 mm nacl, 5% glycerol, 7.5 mm glutaraldehyde and incubated for 5 minutes before quenching the glutaraldehyde by the addition of 1 m tris (to a final concentration of 75 mm) and 5 minutes incubation. a 5-µl drop of sample was then applied to a glow-discharged carbon-coated grid for 10-15 seconds, blotted, stained with 2% uranyl formate, blotted and air-dried. images were obtained using a philips em420 electron microscope at 120 kv, 82,000× magnification, and a 4.02 å pixel size. the relion (scheres, 2012) program was used for particle picking, 2d and 3d class averaging. differential scanning fluorimetry dsf assay was performed using tycho nt. 6 (nanotemper technologies). spike ectodomains were diluted to approximatively 0.15 mg/ml. intrinsic fluorescence was measured at 330 nm and 350 nm while the sample was heated from 35 to 95 °c at a rate of 30°c/min. the ratio of fluorescence (350/330 nm) and inflection temperatures (ti) were calculated by the tycho nt. 6 apparatus. elisa assays spike samples were pre-incubated at different temperatures then tested for antibody-or ace-2binding in elisa assays as previously described . assays were run in two formats. in the first format antibodies or ace2 protein were coated on 384-well plates at 2 µg/ml overnight at 4°c, washed, blocked and followed by two-fold serially diluted spike protein starting at 25 µg/ml. binding was detected with polyclonal anti-sars-cov-2 spike rabbit serum (developed in our lab), followed by goat anti-rabbit-hrp and tmb substrate. absorbance was read at 450 nm. in the second format, serially diluted spike protein was bound in individual wells of 384-well plates, which were previously coated with streptavidin at 2 µg/ml and blocked. proteins were incubated at room temperature for 1 hour, washed, then human mabs were added at 10 µg/ml. antibodies were incubated at room temperature for 1 hour, washed and binding detected with goat anti-human-hrp and tmb substrate. cryo-em purified sars-cov-2 spike preparations were diluted to a concentration of ~1.5 mg/ml in 2 mm tris ph 8.0, 200 mm nacl and 0.02% nan3. a 2.5-µl drop of protein was deposited on a quantifoil-1.2/1.3 grid that had been glow discharged for 10 seconds in a pelco easiglow™ glow discharge cleaning system. after a 30 seconds incubation in >95% humidity, excess protein was blotted away for 2.5 seconds before being plunge frozen into liquid ethane using a leica em gp2 plunge freezer (leica microsystems). frozen grids were imaged in a titan krios (thermo fisher) equipped with a k3 detector (gatan). no statistical analysis were performed in this study. table reagent or resource source identifier antibodies ace2 n/a cr3022 n/a 2g12 n/a ab712199 n/a ab511584 n/a goat anti-rabbit-hrp abcam ab97080 goat anti-human-hrp jackson immunoresearch laboratories first derivative (ratio) figure 3c with the s1 subunit colored by domain and the s2 subunit colored grey. rbd is colored red, ntd green, sd1 dark blue, sd2 orange and the linker between the ntd and rbd colored cyan. b. overlay of the individual protomers in the 1-rbd-up structure and a protomer in the c3 symmetric 3-rbd-down structure shown in figure 3c . the structures were superimposed using s2 subunit residues 908-1035 (spanning the hr1 and ch regions). the domain colors of the up-rbd chain are as described in panel a. the down-rbds are colored salmon, the sd1 domains from the down rbd chains are colored light blue. the linker between the ntd and rbd in the down rbd chains are colored deep teal. c. zoomed-in view showing the association of the linker connecting the ntd and rbd with the sd1 and sd2 domains. d. zoomed-in views of individual domains marked in panel b. the n2r linker spanning residues 306-334 connects the ntd and the rbd. residues 324-328 of the n2r linker contribute a b-strand to the sd1 subdomain together forming the sd1' "super" subdomain. residues 311-319 of the n2r linker contribute a b-strand to the sd2 subdomain together forming the sd2' "super" subdomain. e. difference distance matrices (ddm) showing structural changes between different protomers for the structures shown in figure 3c . the blue to white to red coloring scheme is illustrated at the bottom. ab712199 (rbd-directed neutralizing antibody) and ab511584 (s2-directed non-neutralizing antibody) to s-gsas/d614g (in blue) and the furin-cleaved s-rrar/d614g ectodomain (in green) measured by elisa. the assay format was the same as in figure 2d . c. overlay of the individual protomers in the 1-rbd-up structure and a protomer in the c3 symmetric 3-down-rbd structure shown in panel a. rbd-up chain with the s1 subunit colored by domain and the s2 subunit colored grey. rbd is colored red, ntd colored green, sd1 dark blue, sd2 orange and the linker between the ntd and rbd colored cyan. the down rbds are colored salmon, the sd1 domains from the down rbd chains are colored light blue. the linker between the ntd and rbd in the down rbd chains are colored deep teal. insets show zoomed-in views of individual domains similar to the depiction in figure 4d . d. (left) the protomers of the 1-rbd-up structure of the furin-cleaved s-rrar/d614g ectodomain superimposed using residues 908-1035 and colored by the color of their ntd as depicted in panel a. zoomed-in views show region of the sd2 domain proximal to the ntd. a glycan cluster on the sars-cov-2 spike ectodomain is recognized by fab-dimerized glycan-reactive antibodies. biorxiv real-space refinement inphenixfor cryo-em and crystallography tmprss2 and furin are both essential for proteolytic activation of sars-cov-2 in human airway cells sars-cov-2 mrna vaccine design enabled by prototype pathogen preparedness sars and mers: recent insights into emerging coronaviruses an interactive web-based dashboard to track covid-19 in real time cold sensitivity of the sars-cov-2 spike ectodomain data, disease and diplomacy: gisaid's innovative contribution to global health features and development ofcoot ucsf chimerax: meeting modern challenges in visualization and analysis the bio3d packages for structural bioinformatics cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding structural basis for antibody-mediated neutralization of lassa virus controlling the sars-cov-2 spike glycoprotein conformation a multibasic cleavage site in the spike protein of sars-cov-2 is essential for infection of human lung cells sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor structures and distributions of sars-cov-2 spike proteins on intact virions tracking changes in sars-cov-2 spike: evidence that d614g increases infectivity of the covid-19 virus a highly stable prefusion rsv f vaccine derived from structural analysis of the fusion mechanism the impact of mutations in sars-cov-2 spike on viral infectivity and antigenicity macromolecular structure determination using x-rays, neutrons and electrons: recent developments in phenix enhanced isolation of sars-cov-2 by tmprss2-expressing cells immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen structure of hiv-1 gp120 with gp41-interactive region reveals layered envelope architecture and basis of conformational mobility ucsf chimera?a visualization system for exploratory research and analysis cryosparc: algorithms for rapid unsupervised cryo-em structure determination specific single or double proline substitutions in the "spring-loaded" coiled-coil region of the influenza hemagglutinin impair or abolish membrane fusion activity identification of structural motifs from protein coordinate data: secondary structure and first-level supersecondary structure structure-based design of prefusion-stabilized filovirus glycoprotein trimers stabilization of the soluble, cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1 a bayesian view on cryo-em structure determination processing of structurally heterogeneous cryo-em data in relion nih image to imagej: 25 years of image analysis pandemic preparedness: developing vaccines and therapeutic antibodies for covid-19 cell entry mechanisms of sars-cov-2 situ structural analysis of sars-cov-2 spike reveals flexibility mediated by three hinges. science function, and antigenicity of the sars-cov-2 spike glycoprotein d614g spike mutation increases sars cov-2 susceptibility to neutralization. medrxiv cryo-em structure of the 2019-ncov spike in the prefusion conformation cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains structural and functional analysis of the d614g sars-cov-2 spike protein variant coronaviruses -drug discovery and therapeutic options rbd "up" (1-rbd-up s) vs rbd "down" protomer #2 (1-rbd-up s) rbd "up" (1-rbd-up s) vs rbd "down" protomer (3-rbd-down s) rbd "up" (1-rbd-up s) vs rbd "down" protomer #1 (1-rbd-up s) rbd "down" protomer #1(1-rbd-up s) vs rbd "down" protomer #2 (1-rbd-up s) rbd "down" protomer #1(1-rbd-up s) vs rbd "down" protomer (3-rbd-down s) cryo-em data were collected at the national center for cryo-em access and training (nccat) and the simons electron microscopy center located at the new york structural biology center, supported by the nih common fund transformative high resolution cryo-electron microscopy program (u24 gm129539) and by grants from the simons foundation key: cord-347587-auook38y authors: zhao, guangyu; he, lei; sun, shihui; qiu, hongjie; tai, wanbo; chen, jiawei; li, jiangfan; chen, yuehong; guo, yan; wang, yufei; shang, jian; ji, kaiyuan; fan, ruiwen; du, enqi; jiang, shibo; li, fang; du, lanying; zhou, yusen title: a novel nanobody targeting middle east respiratory syndrome coronavirus (mers-cov) receptor-binding domain has potent cross-neutralizing activity and protective efficacy against mers-cov date: 2018-08-29 journal: j virol doi: 10.1128/jvi.00837-18 sha: doc_id: 347587 cord_uid: auook38y the newly emerged middle east respiratory syndrome coronavirus (mers-cov) continues to infect humans and camels, calling for efficient, cost-effective, and broad-spectrum strategies to control its spread. nanobodies (nbs) are single-domain antibodies derived from camelids and sharks and are potentially cost-effective antivirals with small size and great expression yield. in this study, we developed a novel neutralizing nb (nbms10) and its human-fc-fused version (nbms10-fc), both of which target the mers-cov spike protein receptor-binding domain (rbd). we further tested their receptor-binding affinity, recognizing epitopes, cross-neutralizing activity, half-life, and efficacy against mers-cov infection. both nbs can be expressed in yeasts with high yield, bind to mers-cov rbd with high affinity, and block the binding of mers-cov rbd to the mers-cov receptor. the binding site of the nbs on the rbd was mapped to be around residue asp539, which is part of a conserved conformational epitope at the receptor-binding interface. nbms10 and nbms10-fc maintained strong cross-neutralizing activity against divergent mers-cov strains isolated from humans and camels. particularly, nbms10-fc had significantly extended half-life in vivo; a single-dose treatment of nbms10-fc exhibited high prophylactic and therapeutic efficacy by completely protecting humanized mice from lethal mers-cov challenge. overall, this study proves the feasibility of producing cost-effective, potent, and broad-spectrum nbs against mers-cov and has produced nbs with great potentials as anti-mers-cov therapeutics. importance therapeutic development is critical for preventing and treating continual mers-cov infections in humans and camels. because of their small size, nanobodies (nbs) have advantages as antiviral therapeutics (e.g., high expression yield and robustness for storage and transportation) and also potential limitations (e.g., low antigen-binding affinity and fast renal clearance). here, we have developed novel nbs that specifically target the receptor-binding domain (rbd) of mers-cov spike protein. they bind to a conserved site on mers-cov rbd with high affinity, blocking rbd's binding to mers-cov receptor. through engineering a c-terminal human fc tag, the in vivo half-life of the nbs is significantly extended. moreover, the nbs can potently cross-neutralize the infections of diverse mers-cov strains isolated from humans and camels. the fc-tagged nb also completely protects humanized mice from lethal mers-cov challenge. taken together, our study has discovered novel nbs that hold promise as potent, cost-effective, and broad-spectrum anti-mers-cov therapeutic agents. ities, neutralization mechanisms, cross-neutralizing activity against divergent mers-cov strains, half-life, and protective efficacy against lethal mers-cov infection in an established hdpp4-tg mouse model (38) . this study reveals that efficacious, robust, and broad-spectrum nbs can be produced to target mers-cov s protein rbd and that they hold great promise as potential anti-mers-cov therapeutics. identification and characterization of mers-cov-rbd-specific nbs. to construct the nb (i.e., vhh) library, we immunized llama with recombinant mers-cov rbd (residues 377 to 588, emc2012 strain) containing a c-terminal human igg1 fc tag (i.e., rbd-fc) and isolated peripheral blood mononuclear cells (pbmcs) from the immunized llama. after four rounds of bio-panning and screening using mers-cov rbd-fc, we isolated a positive clone with the highest binding affinity for the rbd. the gene encoding this rbd-specific nb was subcloned into yeast expression vector to construct nbms10 (which contains a c-terminal his 6 tag) and nbms10-fc (which contains a c-terminal human igg1 fc tag) nbs (fig. 1) . both nbms10 and nbms10-fc were nbms10-fc nbs. blood was collected from mers-cov rbd-fc protein-immunized alpaca after the last immunization to isolate pbmcs. rna was then extracted to synthesize cdna via rt-pcr. this was followed by pcr amplification of the n-terminal igg heavy-chain fragment (ϳ700 bp), including the vhh gene, while the latter was used as the template to amplify the vhh gene fragment (ϳ300 to 450 bp). the vhh dna sequence was further ligated into phagemid vector pcantab5e and transformed into e. coli tg1 competent cells to construct vhh library. vhh phage display was carried out to isolate rbd-specific clones. after four rounds of bio-panning, the rbd-specific vhh coding sequence was confirmed from the selected positive clones. the identified vhh coding gene containing a c-terminal his 6 or human igg1 fc was inserted into pichia pastoris yeast expression vector ppicz␣a to construct nbms10 and nbms10-fc, respectively, for further soluble expression and purification. expressed in yeast cells, secreted into the cell culture supernatants, and purified to homogeneity ( fig. 2a, left) . the estimated molecular weights were about 16 kda for nbms10 and 50 kda for nbms10-fc, since the latter formed a dimer. these mers-cov rbd-specific nbs from llama, but not severe acute respiratory syndrome coronavirus (sars-cov) rbd-specific mabs from mice, were recognized by anti-llama antibodies ( fig. 2a, right) . thus, the yeast-expressed nbs maintained their native conformation and antigenicity. to characterize their functions, we examined how the nbs interact with mers-cov rbds. first, we evaluated the binding between the nbs and mers-cov rbd using elisa. the result showed that both nbs bound strongly to recombinant mers-cov rbd containing a c-terminal folden tag (rbd-fd) and mers-cov s1 containing a c-terminal his 6 tag (s1-his) in a dose-dependent manner (fig. 2b) . second, we determined the binding affinity of the two nbs for mers-cov rbd using surface plasmon resonance (spr). the result showed that the k d between nbms10 and rbd-fc was 0.87 nm, the nbs were subjected to sds-page (left) or western blotting (right), followed by detection using anti-llama antibody. the molecular weight marker (in kda) is indicated on the left. (b) detection of binding between nbms10 or nbms10-fc and mers-cov s1 (mers-s1) or rbd (mers-rbd) protein by elisa. the plates were coated with mers-cov s1-his or rbd-fd protein (2 g/ml), followed by sequential incubation with respective nbs and goat anti-llama and hrp-conjugated anti-goat igg antibodies. the data are presented as mean a 450 values ϯ the standard deviation (sds) (n ϭ 2). significant differences (*; **, and ***) are shown in the binding of nbs to mers-s1 or mers-rbd at various concentrations. (c) the binding kinetics between nbms10 or nbms10-fc and mers-cov rbd or s1 protein were measured by spr. mers-cov rbd-fc protein was used for binding to nbms10 (containing a c-terminal his 6 ), and s1-his protein was used for binding to nbms10-fc (containing a c-terminal human fc). (d) detection of nbms10 and nbms10-fc neutralizing activity against mers-cov infection (emc2012 strain) by a microneutralization assay. the nb-mers-cov mixtures were incubated with vero e6 cells and observed for the presence or absence of cpe. neutralizing activity of nbs was recorded as the concentration of nbs in complete inhibition of mers-cov-induced cpe in at least 50% of the wells (nd 50 ). the data are expressed as mean nd 50 ϯ the sd (n ϭ 3). the experiments were repeated twice, and similar results were obtained. the "(ϫ) control" in panels a, b, and d refers to sars-cov 33g4 mouse mab. whereas the k d between nbms10-fc and s1-his was 0.35 nm (fig. 2c) . third, we carried out mers-cov neutralization assay. the result showed that the nbs efficiently neutralized the infection of live mers-cov (emc2012 strain) in vero cells. the measured 50% neutralization doses (nd 50 ) were 3.52 g/ml for nbms10 and 2.33 g/ml for nbms10-fc (fig. 2d ). taken together, the nbs strongly bound to mers-cov rbd and neutralized mers-cov infection. molecular mechanism underlying the neutralizing activities of nbs. to investigate the mechanism underlying the neutralizing activities of nbs, we evaluated the competition between the nbs and hdpp4 for the binding to mers-cov rbd. first, we carried out a flow cytometry assay where recombinant mers-cov rbd interacted with cell-surface-expressed dpp4 in the presence or absence of recombinant nbs. the result showed that both nbs significantly blocked the binding of rbd to cell-surface dpp4 in a dose-dependent manner ( fig. 3a and b) . as a negative control, sars-cov-rbdspecific 33g4 mab did not block the binding between mers-cov rbd and cell surface dpp4 ( fig. 3a and b) . second, we carried out an enzyme-linked immunosorbent assay (elisa) where recombinant mers-cov rbd and recombinant hdpp4 interacted in the presence or absence of recombinant nbs. the result showed that both nbs, but not were coated with mers-cov rbd-fc protein (2 g/ml), followed by sequential incubation with serial dilutions of nbs or hdpp4 protein (2 g/ml), goat anti-hdpp4, and hrp-conjugated anti-goat igg antibodies. the percent inhibition was calculated as the rbd-hdpp4 binding in the presence or absence of nbs according to the following formula: (1 ϫ rbd-hdpp4-nb/rbd-hdpp4) ϫ 100. a significant difference (***) occurred between nbms10 and nbms10-fc in inhibition of rbd-hdpp4 binding. the "(ϫ) control" in panels b and c refers to sars-cov 33g4 mab. the data are presented as the mean percent inhibition ϯ the sd (n ϭ 2). the experiments were repeated twice, and similar results were obtained. 33g4 mab, blocked the binding between mers-cov rbd and dpp4 in a dosedependent manner. moreover, compared to nbms10, nbms10-fc blocked the rbd-dpp4 binding more efficiently (fig. 3c) . these data reveal that the nbs can compete with hdpp4 for the binding to mers-cov rbd, suggesting that the nb-binding site and the dpp4-binding site overlap on the mers-cov rbd. to map the binding site of the nbs on mers-cov rbd, we performed alanine scanning on the surface of mers-cov rbd and detected the binding of nbs to the alanine-containing rbd mutants. the results showed that nbms10 demonstrated tight binding to mers-cov rbd containing the single mutations l506a, d510a, r511a, e513a, e536a, w553a, v555a, and e565a and slightly reduced binding to rbd containing triple mutations l506f-d509g-v534a, suggesting that these rbd residues do not play significant roles in nb binding. instead, single mutation d539a and double mutations e536a-d539a on mers-cov rbd both ablated the binding of nbms10 to the rbd (fig. 4a) , suggesting that rbd residue asp539 plays an important role in nb binding. we further investigated the role of asp539 in nb binding using the mers-cov pseudovirus entry assay. neither nbms10 nor nbms10-fc could neutralize the cell entry of mers-cov pseudovirus bearing the d539a mutation, again confirming that residue asp539 is critical for nb binding (fig. 4b ). to examine of the role of the d539a mutation in dpp4 binding, we carried out an elisa to detect the binding between dpp4 and the plates were coated with rbd-fd protein (2 g/ml) and treated with or without dtt, followed by sequential incubation with serial dilutions of nbms10 or nbms10-fc and goat anti-llama and hrp-conjugated anti-goat igg antibodies. the data are presented as mean a 450 values ϯ the sd (n ϭ 2). the "(ϫ) control" in panels b and d refers to sars-cov 33g4 mab. the above-described experiments were repeated twice, and similar results were obtained. mers-cov rbd bearing the d539a mutation. the result showed that the d539a mutation significantly reduced the binding of the rbd to dpp4 (fig. 4c ). overall, these results demonstrate that nbs recognize the asp539-containing epitope on mers-cov rbd and that this epitope also plays an important role in dpp4 binding. therefore, the nbs and dpp4 compete for the same region on mers-cov rbd, and mutations in this region can reduce the binding of both the nbs and dpp4. to investigate whether nb-recognized epitopes on mers-cov rbd are conformational or linear, we detected the binding of nbs to mers-cov rbd with its conformational structure disrupted. to this end, we treated mers-cov rbd with reducing agent dithiothreitol (dtt) to break the disulfide bonds in the protein, and performed an elisa on the binding between nbs and dtt-treated rbd. the result showed that neither nbms10 nor nbms10-fc bound to the dtt-treated rbd (fig. 4d) . as a control, both nbs bound to untreated rbd with high affinity. thus, the nbs recognize the conformational epitope on the rbd. to understand the structural mechanism underlying the neutralizing activities of the nbs, we examined the competitive interactions among the nbs, dpp4, and mers-cov rbd using structural modeling (fig. 5 ). in the absence of the nbs, mers-cov rbd binds tightly to the dpp4 receptor, with d539 of rbd serving as a key residue at the binding interface (fig. 5a) . here, rbd residue d539 forms a critical salt bridge with dpp4, and it interacts with the surrounding key rbd residues via van der waals contacts and hydrogen bonds (fig. 5b) , enabling rbd and dpp4 to maintain strong binding interactions. the nbs bind tightly to the rbd in the same d539-containing region, abolishing the binding between rbd and dpp4 (fig. 5c) . cross-neutralizing activity of nbs against divergent mers-cov strains. to investigate the cross-neutralizing activity of nbs against divergent mers-cov isolates, we performed mers-cov pseudovirus entry assay in the presence of the nbs where the pseudoviruses encode the s gene of various mers-cov isolates from different countries (saudi arabia, qatar, and south korea), hosts (human and camels), and time periods (2012 to 2015). these mers-cov strains all contain mutations in their rbds. the results showed that both nbs potently neutralized the cell entry of all of the mers-cov pseudoviruses, with the nd 50 values ranging from 0.003 to 0.979 g/ml (for nbms10) and from 0.003 to 0.067 g/ml (for nbms10-fc) ( table 1) . therefore, although the nbs were developed using the rbd from one mers-cov strain (emc2012), they have broad-spectrum cross-neutralizing activity against existing mers-cov strains, as well as potentially future emerging mers-cov strains. in vivo half-life of nbs. to evaluate the in vivo half-life of the nbs, we injected the nbs into mice, collected the sera from the mice after different time intervals, and measured the binding between the sera and recombinant mers-cov s1 using elisa. the results showed that the sera collected from nbms10-injected mice gradually lost their binding affinity for mers-cov s1, and completely lost their binding for mers-cov s1 10 days postinjection (fig. 6a ). in comparison, nbms10-fc demonstrated stable binding for recombinant mers-cov s1 at 10 days postinjection (fig. 6b) . as a control experiment, sera collected from pbs-injected mice showed no binding for recombinant mers-cov s1 (fig. 6c) . thus, compared to monomeric nb, fc-fused nb has a significantly extended in vivo half-life likely due to its dimeric structure, which increases the molecular weight of nb from 16 to 50 kda and hence may slow down its renal clearance. prophylactic and therapeutic efficacy of nb in transgenic mice. because mers-cov does not infect wild-type mice, we previously developed hdpp4-tg mice (38) as the susceptible animal model for mers-cov research. to evaluate the prophylactic efficacy of nbms10-fc, mice were injected with a single dose of nbms10-fc 3 days before they were infected with a lethal dose of mers-cov and were subsequently monitored for their weight and survival. trastuzumab, an antibody used for treating breast cancer, was used as a control. the result showed that after mers-cov infection, mice treated with nbms10-fc had a 100% survival rate (fig. 7a, above) and steady weight (fig. 7a, below) . in comparison, mice treated with trastuzumab all died on day 8 postinfection, and their weight also sharply decreased starting from day 4 postinfection (fig. 7a) . to evaluate the therapeutic efficacy of nbms10-fc, mice were first infected with mers-cov and then treated with single-dose nbms10-fc either 1 or 3 days postinfection. the result showed that mice treated with nbms10-fc on day 1 postinfection had a 100% survival rate and steady weight (fig. 7b ). in addition, mice treated with nbms10-fc on day 3 postinfection also had a 100% survival rate (fig. 7c , above); although their weights first decreased on day 5 postinfection, it rebounded on day 7 postinfection (fig. 7c, below) . in comparison, mice receiving trastuzumab all died on day 8 after infection, and their weights continuously decreased ( fig. 7b and c) . overall, nbms10-fc has potent prophylactic and therapeutic efficacy in protecting susceptible animal models against lethal mers-cov challenge. . virus-challenged mice were monitored for 14 days to evaluate survival rate (above) and body weight changes (below). the body weight data are presented as means ϯ the sd of mice in each group (n ϭ 6). significant differences (** and ***) are indicated between the nbms10-fc and control groups. mers-cov continues to infect humans with a high fatality rate. because camels likely serve as the transmission hosts for mers-cov and also because humans have contact with camels, the constant and continuing transmissions of mers-cov from camels to humans make it difficult to eradicate mers-cov from the human population. thus, efficacious, cost-effective, and broad-spectrum anti-mers-cov therapeutic agents are needed to prevent and treat mers-cov infections in both humans and camels. nbs have been gaining acceptance as antiviral agents because of their small size, good tissue permeability, and cost-effective production, storage, and transportation. however, their small size may also lead to relative low antigen-binding affinity and quick clearance from the host body. in this study, we have developed a novel mers-covtargeting nb, nbms10, and its fc-fused version, nbms10-fc, both of which demonstrate great promise as anti-mers-cov therapeutic agents. nbms10 and nbms10-fc present superior characteristics common to other nbs. they target the mers-cov rbd, which plays an essential role in cell entry of mers-cov by binding to its receptor hdpp4. both nbs can be expressed in yeast cells with high purity and yields and are soluble in solutions. all of these properties suggest costeffective production, easy storage, and convenient transportation of these nbs in potential commercial applications. the mers-cov rbd-targeting nbs developed also demonstrate good qualities comparable to previously reported mers-cov rbd-specific conventional iggs. first, the nbs bind to mers-cov rbd with high affinities. the k d values for nbms10 and nbms10-fc to bind mers-cov rbd were 8.71 ϫ 10 ϫ10 m and 3.46 ϫ 10 ϫ10 m, respectively. the k d values for rbd-targeting conventional iggs to bind mers-cov rbd range from 7.12 ϫ 10 ϫ8 m to 4.47 ϫ 10 ϫ11 m (29, 35, 36) . moreover, the nd 50 values for nbms10 and nbms10-fc to neutralize mers-cov (emc2012 strain) infection in cultured cells were 3.52 and 2.33 g/ml, respectively. the nd 50 values for rbd-specific conventional iggs to neutralize various mers-cov strains ranged from micrograms/ml to nanograms/ml (30, 32, 35, 39, 40) . thus, the nbs developed in this study and conventional iggs reported previously have comparable mers-cov rbd-binding affinities and mers-covneutralizing activities. structural comparisons of conventional iggs and nbs have shown that the antigen-binding site of iggs consists of paired heavy-chain and lightchain variable (vh-vl) domains, whereas nbs lack the light chain and hence cannot form the paired vh-vl domains (8, 41) . instead, nbs have an extended cdr3 region (ͼ16 amino acid residues), longer than that of the vhs of conventional iggs (average length 12 amino acid residues) (42) (43) (44) . moreover, the nbs developed here contain a 22-amino-acid cdr3; the extended cdr3 enables the nbs to bind to the antigens with higher affinity (37) . furthermore, although the single-domain nb (i.e., nbms10) is small and can be cleared from the serum relatively quickly, the fc-fused nb (i.e., nbms10-fc) with relatively increased size demonstrates extended in vivo half-life. therefore, the potential short half-life of nbs can be overcome by adding the appropriate tag to the nbs to increase their half-life. overall, the present study has shown the feasibility of overcoming the potential limitations of nbs. the mers-cov rbd-targeting nbs potently neutralize mers-cov entry into host cells. the k d values between the nbs and mers-cov rbd are significantly lower than that between mers-cov rbd and hdpp4 receptor. as a result, the nbs can outcompete hdpp4 for the binding of mers-cov rbd, thereby blocking the binding of mers-cov to dpp4, as well as mers-cov entry into host cells. it is worth noting that the rbd on the mers-cov s trimer frequently undergoes conformational changes, switching between a lying down, receptor-inaccessible conformation and a standing-up, receptoraccessible conformation. hence, in the context of the virus particles where the rbd is part of the s protein, the nbs would need to bind the rbd when the rbd is in the standing-up conformation (45) . importantly, the nbs demonstrate strong crossneutralizing activities against various mers-cov strains isolated from different hosts (humans and camels) and from different time points during mers-cov circulation in humans (from years 2012 to 2015). nbms10 had a relatively high nd 50 against the agv08584/2012 strain containing a v534a mutation, which is consistent with the slightly reduced binding affinity between nbms10 and mers-cov rbd containing the v534a mutation (fig. 4a) . the broad neutralizing spectrum of the nbs results from the binding site of the nbs on mers-cov rbd, which is located in the asp539containing region that plays a critical role in dpp4 binding. interestingly, several mers-cov rbd-specific conventional iggs also bind to the same epitope (39, 46) , suggesting that this region is a hot spot for immune recognition. although mutations in this region can eliminate the binding of the nbs to mers-cov rbd and hence lead to viral immune evasion, they also reduce the binding of mers-cov rbd to receptor dpp4 and hence decrease the efficiency of viral entry. thus, viral immune evasion from the inhibition of the nbs through mutations can be costly to mers-cov itself. indeed, residue asp539 in s protein rbd is highly conserved in almost all of the natural mers-cov strains published to date (fig. 8) . therefore, the mers-cov-specific nbs can potentially be developed into broad-spectrum anti-mers-cov therapeutic agents. despite the above analysis, this study did not examine all possible mutations in the nb-binding region (since the atomic structures of mers-cov rbd complexed with the nbs are still unknown), and thus it is possible that future escape mutations may occur to residues that this study did not cover. in that case, a combination of the current nbs and other antibodies targeting other s regions or various rbd epitopes may be helpful in battling the emergence of immune escape mers-cov strains. in sum, the mers-cov-specific nbs developed in the present study possess superior qualities common to all nbs such as their small size and cost-effective production. they also overcome potential limitations of other nbs by maintaining a high binding affinity for their target mers-cov rbd and an optimized half-life. moreover, they recognize a functionally important region on mers-cov rbd, rendering viral immune evasion costly and at the same time making themselves good candidates as broad-spectrum anti-mers-cov therapeutics. we have confirmed the effectiveness of the nbs by showing that the fc-fused nb completely protected animal models from lethal mers-cov challenge. thus, the nbs can potentially be used in both humans and camels to prevent and treat mers-cov infections in either of these hosts and also block the camel-tohuman transmission of mers-cov. overall, our study proves the feasibility of developing highly effective nbs as anti-mers-cov therapeutic agents and points out strategies to preserve the advantages of nbs, as well as to overcome the potential limitations of nbs. construction of vhh library and screening for mers-cov-rbd-specific nbs. construction of the nb (i.e., vhh) library and screening of mers-cov-rbd-specific nbs were performed as previously described (47) . briefly, male and female alpacas (llama pacos, 1 year) were subcutaneously immunized with recombinant rbd-fc (260 g/alpaca) (48) plus freund complete adjuvant, and boosted three times with the same immunogen plus freund incomplete adjuvant (invivogen). blood was collected 10 days after the last immunization, and then pbmcs were isolated using ficoll-paque gradient centrifugation (ge healthcare). total rna was extracted with trizol reagent (invitrogen). cdna was synthesized by reverse transcription-pcr (rt-pcr) using a transscript cdna synthesis supermix (transgen biotech, china), followed by pcr amplification of the n-terminal igg heavy-chain fragment (ϳ700 bp), using the forward primer vhh-l-f (5=-ggtggtcctggctgc-3=) and the reverse primer ch2-r (5=-ggtacgtgctgttgaact gttcc-3=). the vhh gene (ϳ300 to 450 bp) was further amplified using the above dna fragment as the template and the forward primer vhh-fr1-d-f (5=-tttctattactaggcccagccggccgagtctggaggrr gcttggtgca-3=) and the reverse primer vhh-fr4-d-r (5=-aaaccgttggccataatggcctgaggagacgr tgacstsggtc-3=) (the sfii restriction site is underlined). the sfii-digested vhh dna fragment was then inserted into phagemid vector pcantab5e (bio-view shine biotechnology, china) to construct the vhh phage display library (49) . phage particles were analyzed by elisa using recombinant mers-cov rbd-fc and fc of human igg1 proteins as the positive and negative target proteins, respectively, to screen for rbd-specific nbs. after four rounds of bio-panning, one of five positive clones, cab10, with the highest binding to mers-cov rbd, was selected for further analyses (fig. 1) . expression of mers-cov-rbd-specific nbs in yeast cells. nbms10 and nbms10-fc nbs containing a c-terminal his 6 and fc of human igg1, respectively, were constructed based on the aforementioned cab10 vhh. the dna sequences encoding nbab10 and nbab10-fc were synthesized (genscript) and inserted into the pichia pastoris secretory expression vector, ppicz␣a (invitrogen) (fig. 1) . the recombinant nbms10 and nbms-fc were expressed in pichia pastoris gs115 cells and purified using a ni-nta column (for nbms10; ge healthcare) and a protein a sepharose 4 fast flow column (for nbms10-fc; ge healthcare), respectively. sds-page and western blotting. the purified anti-mers-cov-rbd nbs were analyzed using sds-page and western blotting (23, 48) . briefly, nbs (3 g) were loaded onto 10% tris-glycine sds-page gels and stained using coomassie brilliant blue or transferred to nitrocellulose membranes. after being blocked overnight at 4°c with 5% nonfat milk/phosphate-buffered saline-tween 20 (5% pbst), the membranes were incubated sequentially with goat anti-llama igg (1:3,000; abcam) and horseradish peroxidase (hrp)-conjugated anti-goat igg (1:1,000; r&d systems) antibodies for 1 h at room temperature and then with ecl western blot substrate reagents. finally, the membranes were visualized using amersham hyperfilm (ge healthcare). a sars-cov-rbd-specific mab, 33g4 (50) , was used as a control. elisa. elisa was performed to detect the binding between nbs and mers-cov s1 or rbd proteins (23, 51) . briefly, elisa plates were coated overnight at 4°c, respectively, with recombinant mers-cov s1-his (48), rbd-fc (48), rbd-fd (51), or one of the mutant rbds containing a c-terminal human fc tag (28) . after being blocked with 2% pbst for 2 h at 37°c, the plates were further incubated sequentially with serially diluted nbs (containing a c-terminal his 6 or fc tag), either goat anti-llama (1:5,000) or mouse anti-his (1:3,000) antibody (sigma) and either hrp-conjugated anti-goat igg (1:3,000) or hrp-conjugated anti-mouse igg (1:5,000) antibody (ge healthcare) for 1 h at 37°c. elisa substrate (3,3=,5,5=tetramethylbenzidine [tmb]; invitrogen) was added to the plates, and the reactions were stopped with 1 n h 2 so 4 . the absorbance at 450 nm (a 450 ) was measured using a tecan infinite 200 pro microplate reader (tecan). to detect the binding between nbs and denatured mers-cov rbd protein, elisa plates were coated with rbd-fd protein (2 g/ml) overnight at 4°c and then sequentially incubated with dtt (10 mm) and iodoacetamide (50 mm) (sigma) for 1 h at 37°c (28) . after three washes using pbst, elisa was performed as described above. inhibition of the binding between mers-cov rbd and hdpp4 proteins by nbs was performed using elisa as described above, except that recombinant hdpp4 protein (2 g/ml; r&d systems), and serially diluted nbs were added simultaneously to the rbd-fc-coated plates. the binding between rbd and dpp4 was detected using goat anti-hdpp4 antibody (1:1,000; r&d systems) and hrp-conjugated anti-goat igg (1:3,000). the percent inhibition was calculated based on the a 450 values of rbd-hdpp4 binding in the presence or absence of nbs. sars-cov 33g4 mab was used as a negative control to nbs. surface plasmon resonance. the binding between nbs and mers-cov s1 or rbd protein was detected using a biacores200 instrument (ge healthcare) as previously described (29) . briefly, recombinant fc-fused mers-cov rbd-fc protein or nbms10-fc nb (5 g/ml) was captured using a sensor chip protein a (ge healthcare), and recombinant his 6 -tagged mers-cov s1-his protein or nbms10 nb at various concentrations was flown over the chip surface in a running buffer containing 10 mm hepes (ph 7.4), 150 mm nacl, 3 mm edta, and 0.05% surfactant p20. the sensorgram was analyzed using biacore s200 software, and the data were fitted to a 1:1 binding model. flow cytometry. this assay was performed to detect the inhibition of the binding between mers-cov rbd and cell surface hdpp4 by nbs (28) . briefly, huh-7 cells expressing hdpp4 were incubated with mers-cov rbd-fc protein (20 g/ml) for 30 min at room temperature in the absence or presence of nbs at various concentrations. cells were incubated with fluorescein isothiocyanate-labeled antihuman igg antibody (1:50, sigma) for 30 min and then analyzed by flow cytometry. the percent inhibition was calculated based on the fluorescence intensity of rbd-huh-7 cell binding in the presence or absence of nbs. mers pseudovirus neutralization assay. neutralization of mers pseudovirus entry by nbs was performed as previously described (23, 52) . briefly, 293t cells were cotransfected with a plasmid encoding env-defective, luciferase-expressing hiv-1 genome (pnl4-3.luc.re) and a plasmid encoding mers-cov s protein. the mers pseudoviruses were harvested from supernatants at 72 h posttransfection and then incubated with nbs at 37°c for 1 h before being added to huh-7 cells. after 72 h, the cells were lysed in cell lysis buffer (promega), incubated with luciferase substrate (promega), and assayed for relative luciferase activity using tecan infinite 200 pro luminator (tecan). the nd 50 of the nbs was calculated as previously described (53) . mers-cov microneutralization assay. neutralization of mers-cov infection by nbs was performed as previously described (28, 54) . briefly, mers-cov (emc2012 strain) at an amount equal to 100 median tissue culture infective doses (tcid 50 ) was incubated with nbs at different concentrations for 1 h at 37°c. the nb-virus mixture was then incubated with vero e6 cells for 72 h at 37°c in the presence of 5% co 2 . the cytopathic effect (cpe) was observed daily. the neutralizing activity of nbs was reported as the nd 50 . the reed-muench method was used to calculate the nd 50 value for each nb (55) . measurement of half-life of nbs. male and female c57bl/6 mice (6 to 8 weeks old) were intravenously injected with nbs (50 g in 200 l per mouse) into the tail vein. sera were collected at different time points (30 min, 2 h, 6 h, 1 day, 5 days, and 10 days postinjection). the concentrations of nbs in the sera were detected by elisa, as described above. briefly, mers-cov s1-his protein (2 g/ml) was used to coat elisa plates, and then sera, goat anti-llama antibodies (1:5,000), and hrp-conjugated anti-goat igg antibodies (1:3,000) were sequentially added for elisa reactions. evaluation of protective efficacy of nbms10-fc nb. the prophylactic and therapeutic efficacy of nbms10-fc was evaluated in hdpp4-tg mice as previously described (29) . briefly, male and female mice (8 to 10 weeks old) were intraperitoneally anesthetized with sodium pentobarbital (5 mg/kg of body weight) before being intranasally inoculated with lethal dose of mers-cov (emc2012 strain, 10 5.3 tcid 50 ) in 20 l of dulbecco modified eagle medium. either 3 days preinfection or 1 or 3 days postinfection, the mice were intraperitoneally injected with nbms10-fc (10 mg/kg). trastuzumab mab was used as a control to the nb. the infected mice were observed daily for 14 days, and their body weights and survivals were recorded. statistical analysis. statistical analysis was performed using graphpad prism version 5.01. to compare the binding of nbs to mers-cov s1 or rbd protein, as well as the rbds with or without d539a mutation to hdpp4 receptor, a two-tailed student t test was used. one-way analysis of variance was used to compare the inhibition of nbs to rbd-hdpp4 binding. statistical significance between survival curves was analyzed using kaplan-meier survival analysis with a log-rank test. p values lower than 0.05 were considered statistically significant. in the figures, "*," "**," and "***" indicate p ͻ 0.05, p ͻ 0.01, and p ͻ 0.001, respectively. data availability. all data needed to evaluate the conclusions presented here are included. additional data related to this study may be requested from the authors. camelid and shark single domain antibodies: structural features and therapeutic potential nanobody-based products as research and diagnostic tools global analysis of vhhs framework regions with a structural alphabet application of camelid heavy-chain variable domains (vhhs) in prevention and treatment of bacterial and viral infections nanobodies® as inhaled biotherapeutics for lung diseases generation and characterization of alx-0171, a potent novel therapeutic nanobody for the treatment of respiratory syncytial virus infection nanobodies as therapeutics: big opportunities for small antibodies nanobodies: natural single-domain antibodies caplacizumab for acquired thrombotic thrombocytopenic purpura phase i study of 68ga-her2-nanobody for pet/ct 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the novel human coronavirus mers-cov theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies receptor-binding domain of mers-cov with optimal immunogen dosage and immunization interval protects human transgenic mice from mers-cov infection rapid human metapneumovirus microneutralization assay based on green fluorescent protein expression this study was supported by the national key plan for scientific research and summarized and analyzed the data. j.s. and f.l. performed the structural analysis. g.z., f.l., l.d., and y.z. wrote the manuscript. s.j., f.l., l.d., and y.z. revised the manuscript. key: cord-352527-eeyqh9nc authors: zhou, yusen; yang, yang; huang, jingwei; jiang, shibo; du, lanying title: advances in mers-cov vaccines and therapeutics based on the receptor-binding domain date: 2019-01-14 journal: viruses doi: 10.3390/v11010060 sha: doc_id: 352527 cord_uid: eeyqh9nc middle east respiratory syndrome (mers) coronavirus (mers-cov) is an infectious virus that was first reported in 2012. the mers-cov genome encodes four major structural proteins, among which the spike (s) protein has a key role in viral infection and pathogenesis. the receptor-binding domain (rbd) of the s protein contains a critical neutralizing domain and is an important target for development of mers vaccines and therapeutics. in this review, we describe the relevant features of the mers-cov s-protein rbd, summarize recent advances in the development of mers-cov rbd-based vaccines and therapeutic antibodies, and illustrate potential challenges and strategies to further improve their efficacy. middle east respiratory syndrome (mers) coronavirus (cov) is an infectious virus that was first reported in june 2012 [1] . mers-cov may infect people of any age, but older age, underlying comorbidity (such as diabetes mellitus, renal disease, respiratory disease, heart disease, and hypertension), and delayed confirmation or late diagnosis are all factors that affect mers disease outcomes and mortality [2] [3] [4] [5] [6] [7] . sex could be a factor in mers epidemiology, as more males seem to be affected than females [8] [9] [10] . mers-cov infection of women during pregnancy has adverse outcomes, with fetal mortality of~27%; however, only a limited number of pediatric mers-cov infections occur [11] [12] [13] [14] . at the end of december 2018, 2,279 laboratory-confirmed mers infections were reported globally (in 27 countries), leading to 806 deaths, and a mortality of 35.3%. among these infections, 1,901 (83.4%) were reported in saudi arabia, with mortality in 732 individuals (38.5%) (http://www.emro.who.int/health-topics/mers-cov/mers-outbreaks.html). the largest mers outbreak outside saudi arabia occurred in south korea in 2015, with 186 cases and 38 deaths [9, 15, 16] . the most recent mers cases were reported in 2018 in south korea, the united kingdom, and malaysia, in addition to saudi arabia, the united arab emirates, and oman (http://www.who.int/emergencies/mers-cov/en/). mers-cov is thought to have originated in bats [17] [18] [19] [20] . mers-like viruses have been isolated from bats that use (at lower efficiency) the same receptor for cell entry as the mers-cov isolated from humans [21] [22] [23] . dromedary camels are potential intermediates for long-term evolution of mers-cov and seasonal zoonotic transfer of virus to humans [24] [25] [26] [27] . antibodies specific to host cellular proteases for its activity in viral entry, but although evidence initially indicated that cellular furin activates s protein, subsequent results have demonstrated no evidence for the involvement of furin during viral entry [71, 78] . the dpp4 receptor varies among different host species, and mers-cov is thought to use multiple pathways to enable rapid adaptation to speciesspecific variations [79] [80] [81] . in addition to dpp4, mers-cov can bind to sialic acid via the s1 subunit of s protein, or utilize the membrane-associated 78 kda glucose-regulated protein (grp78) to attach to target cells, suggesting that these proteins may also have roles in virion attachment [82, 83] . the structures of mers-cov rbd alone and complexed with dpp4 have been determined ( figure 2 ) [77, 84, 85] . the rbd has a fold-rich tertiary structure, which consists of a core and a receptor-binding motif (rbm), with stabilization provided by four disulfide bonds and two glycans [77] . a number of rbd residues are located at the dpp4-binding interface, and they have a critical role in rbd-dpp4 binding [77, 84, 85] . structural analysis of mers-cov trimeric s protein has identified specific features of the rbd and its complex with dpp4. notably, in the prefusion conformation of the s trimer, individual rbds are either buried (lying state) or exposed (standing state), and this flexibility presumably facilitates recognition by dpp4 [86] . other structural studies have revealed four s-trimer conformational states, in which each rbd is either tightly packed at the membrane-distal apex or rotated into a receptor-accessible conformation, suggesting fusion initiation through sequential rbd events [87] . in configurations with one, two, or three rbds rotated out, rbd determinants are exposed at the apex of the rbd-dpp4 complex, and they are accessible for interaction with dpp4 ( figure 3 ) [87] . mers-cov s protein has an important role in viral pathogenesis, determining host tropism and entry into host cells [58, 70, 71] . the s protein contains an s1 subunit at the n terminus and an s2 subunit at the c terminus. the s1 subunit is composed of the n-terminal domain (ntd) and rbd [58, 72, 73] . the rbd has a key role in the mediation of binding of mers-cov to cells expressing dipeptidyl peptidase 4 (dpp4) receptor, enabling the virus to enter into target cells by fusing with cell membranes through the formation of a fusion core ( figure 1c ) [74] [75] [76] [77] . the s protein requires host cellular proteases for its activity in viral entry, but although evidence initially indicated that cellular furin activates s protein, subsequent results have demonstrated no evidence for the involvement of furin during viral entry [71, 78] . the dpp4 receptor varies among different host species, and mers-cov is thought to use multiple pathways to enable rapid adaptation to species-specific variations [79] [80] [81] . in addition to dpp4, mers-cov can bind to sialic acid via the s1 subunit of s protein, or utilize the membrane-associated 78 kda glucose-regulated protein (grp78) to attach to target cells, suggesting that these proteins may also have roles in virion attachment [82, 83] . the structures of mers-cov rbd alone and complexed with dpp4 have been determined ( figure 2 ) [77, 84, 85] . the rbd has a fold-rich tertiary structure, which consists of a core and a receptor-binding motif (rbm), with stabilization provided by four disulfide bonds and two glycans [77] . a number of rbd residues are located at the dpp4-binding interface, and they have a critical role in rbd-dpp4 binding [77, 84, 85] . structural analysis of mers-cov trimeric s protein has identified specific features of the rbd and its complex with dpp4. notably, in the prefusion conformation of the s trimer, individual rbds are either buried (lying state) or exposed (standing state), and this flexibility presumably facilitates recognition by dpp4 [86] . other structural studies have revealed four s-trimer conformational states, in which each rbd is either tightly packed at the membrane-distal apex or rotated into a receptor-accessible conformation, suggesting fusion initiation through sequential rbd the mers-cov rbd core is colored in blue, the rbm is colored in red, and dpp4 is colored in green. the rbm residues directly involved in dpp4 binding are shown as sticks. dpp4, dipeptidyl peptidase 4; rbd, receptor-binding domain; rbm, receptor-binding motif; s, spike protein. the function and structure of the s-protein rbd demonstrate that it is an important target for development of vaccines and therapeutic agents against mers-cov. a number of mers vaccines have been developed based on viral rbd, including nanoparticles, virus-like particles (vlps), and recombinant proteins, and their protective efficacy has been evaluated in animal models, including mice with adenovirus 5 (ad5)-directed expression of human dpp4 (ad5/hdpp4), hdpp4-transgenic (hdpp4-tg) mice, and non-human primates (nhps) [88] [89] [90] [91] [92] [93] [94] . features of these rbd-based vaccines, in terms of functionality, antigenicity, immunogenicity, and protective ability, are shown in table 1 . the function and structure of the s-protein rbd demonstrate that it is an important target for development of vaccines and therapeutic agents against mers-cov. a number of mers vaccines have been developed based on viral rbd, including nanoparticles, virus-like particles (vlps), and recombinant proteins, and their protective efficacy has been evaluated in animal models, including mice with adenovirus 5 (ad5)-directed expression of human dpp4 (ad5/hdpp4), hdpp4-transgenic (hdpp4-tg) mice, and non-human primates (nhps) [88] [89] [90] [91] [92] [93] [94] . features of these rbd-based vaccines, in terms of functionality, antigenicity, immunogenicity, and protective ability, are shown in table 1 . a soluble nanoparticle vaccine formed in escherichia coli by the rna-mediated folding of a rbd-ferritin (fr) hybrid elicits robust rbd-specific antibody and cellular immune responses in mice, producing antisera that effectively block the binding of rbd to hdpp4 in vitro [89] . the adjuvants alum and the squalene-based mf59 significantly augment the antibody titers and t-cell responses induced by rbd-fr nanoparticle vaccines engineered with or without a ssg linker [89] . similarly, a chimeric, spherical vlp (svlp) vaccine expressing mers-cov rbd induces specific antibody and cellular immune responses in mice, preventing pseudotyped mers-cov entry into susceptible cells [90] . the protective efficacy of these two types of mers vaccine does not yet seem to have been investigated in a viral-challenge animal model. recombinant vaccines involving rbd subunits have been extensively studied for protection against mers-cov infection in mers-cov-susceptible animal models [93, [95] [96] [97] 100, 101] . a recombinant rbd (rrbd) fragment (residues 367-606) expressed in insect cells elicits an antibody response and the production of neutralizing antibodies in mice and nhps [88, 91] . it gives incomplete protection in mers-cov-challenged nhps, with the alleviation of pneumonia and clinical manifestations, as well as the reduction of viral load in lung, trachea, and oropharyngeal swabs [91] . a mers-cov s-protein rbd fragment containing residues 377-588 has been identified as a critical neutralizing domain [95] . a treatment regimen involving two doses of a fusion of this fragment and the fc region of human igg (s377-588-fc) four weeks apart is able to induce strong, long-term antibody responses (including production of neutralizing antibodies) in mice [98] . these responses are significantly greater than those with a single dose or two doses at intervals of one, two, or three weeks [98] . rrbds with single or multiple mutations corresponding to s-protein sequences of mers-cov strains isolated from humans or camels from 2012 to 2015 have also been studied [100] . all these rrbds bind rbd-specific neutralizing monoclonal antibodies (mabs) and dpp4, and are highly immunogenic, eliciting the production of s1-specific antibodies in mice, which cross-neutralizes multiple mers pseudoviruses and live mers-cov [100] . a trimeric rbd-fd protein formed by fusing a mers-cov rbd fragment (residues 377-588) to the foldon trimerization motif, binds strongly to dpp4, and elicits robust and long-term responses with the production of mers-cov s1-specific antibodies and neutralizing antibodies in mice, and protects hdpp4-tg mice against mers-cov infection [94] . the protection provided by existing subunit vaccines based on wild-type mers-cov rbd is not complete, with survival rates in hdpp4-tg mice after a mers-cov challenge of~67% for s377-588-fc and 83% for rbd-fd [94, 98] . however, a variant rbd (t579n) vaccine produced by masking a non-neutralizing epitope at residue 579 with a glycan probe has both functionality in binding dpp4, and antigenicity in binding four potent mers-cov rbd-specific neutralizing mabs (hhs-1, m336, m337, and m338) [93] . the t579n vaccine has significantly greater efficacy than the wild-type rbd vaccine, and it fully protects against a lethal mers-cov challenge in immunized hdpp4-tg mice [93] , demonstrating the possibility of developing rbd-based mers-cov vaccines with high efficacy. [102] [103] [104] [105] [106] [107] [108] [109] . these antibodies generally have greater neutralizing activity against mers-cov infection than non-rbd s1-based or s2-based antibodies [58, 103, 110, 111] . the prophylactic and therapeutic efficacies of rbd-targeting antibodies have been tested in ad5/hdpp4 mice, hdpp4-tg mice, and nhps [102, 104, [112] [113] [114] . in an earlier review, we described the antiviral mechanisms, in vivo protection, and crystal structures of previously reported mers-cov rbd-specific mabs, including mouse mabs mersmab1, 2e6, 4c2, f11, and d12, and human mabs lca60, mers-4, mers-27, regn3048, regn3051, 1e9, 1f8, 3a1, 3b11, 3b12, 3b11-n, 3c12, m14d3, m336, m337, m338, hms-1, and 4c2h [58] . in this review, we focus on newly reported antibodies targeting mers-cov s-protein rbd, or on newly identified features of existing mabs that were not described previously (table 2 ) [102, [112] [113] [114] [115] . rbd-targeting human mabs have been extensively reported. most of these mabs can neutralize pseudotyped or live mers-cov in vitro, and some have shown protection against mers-cov infection in animal models in vivo [102, [112] [113] [114] [115] . the structures of several of these mabs with their antigen-binding fragments (fabs) or single-chain variable fragments (scfvs) complexed with rbd are known ( figure 4) [102, [112] [113] [114] [115] . binding of these mabs to rbd involves two major recognition modes, with binding to rbd residues contacted by or overlapping with dpp4 (as is the case for gd-27, mca1, and cdc2-c2), or with binding to the rbd residues outside of the dpp4-binding interface (as seen with mers-4) ( table 2) . infection in animal models in vivo [102, [112] [113] [114] [115] . the structures of several of these mabs with their antigen-binding fragments (fabs) or single-chain variable fragments (scfvs) complexed with rbd are known ( figure 4) [102, [112] [113] [114] [115] . binding of these mabs to rbd involves two major recognition modes, with binding to rbd residues contacted by or overlapping with dpp4 (as is the case for gd-27, mca1, and cdc2-c2), or with binding to the rbd residues outside of the dpp4-binding interface (as seen with mers-4) ( table 2) . the human mabs mers-gd27 and mers-gd33 each recognize distinct regions of the rbd [113] . these mabs have a synergistic effect in the neutralization of pseudotyped mers-cov in vitro, with a much lower half-maximal inhibitory concentration (ic50) for their use in combination than separately [113] . an analysis of crystal structures has indicated that mers-gd27 binds rbd at the dpp4-binding site, and that the neutralization and recognition epitopes almost completely overlap this site, as seen previously for mers-cov rbd-targeting neutralizing mabs, such as m336 [106, 113] . the mers-gd27 mab protects hdpp4-tg mice from mers-cov challenge, both preventively and therapeutically, with significantly lower lung virus titers and rna copy numbers at day 5 postchallenge, and higher survival rates (60% for pre-challenge vaccination and 40% for post-challenge vaccination) relative to control mice treated with an irrelevant mab [112] . the human mab mca1 was isolated from a mers survivor via the construction of a phagedisplay antibody library from peripheral b cells [114] . crystal structure analysis indicates that mca1 binds mers-cov s-protein rbd at residues involved in receptor binding, thus interfering with rbd binding to hdpp4 ( figure 4a ) [114] . this mab prophylactically and therapeutically inhibits mers-cov replication in common marmosets, resulting in significantly improved outcomes and reduced the human mabs mers-gd27 and mers-gd33 each recognize distinct regions of the rbd [113] . these mabs have a synergistic effect in the neutralization of pseudotyped mers-cov in vitro, with a much lower half-maximal inhibitory concentration (ic 50 ) for their use in combination than separately [113] . an analysis of crystal structures has indicated that mers-gd27 binds rbd at the dpp4-binding site, and that the neutralization and recognition epitopes almost completely overlap this site, as seen previously for mers-cov rbd-targeting neutralizing mabs, such as m336 [106, 113] . the mers-gd27 mab protects hdpp4-tg mice from mers-cov challenge, both preventively and therapeutically, with significantly lower lung virus titers and rna copy numbers at day 5 post-challenge, and higher survival rates (60% for pre-challenge vaccination and 40% for post-challenge vaccination) relative to control mice treated with an irrelevant mab [112] . the human mab mca1 was isolated from a mers survivor via the construction of a phage-display antibody library from peripheral b cells [114] . crystal structure analysis indicates that mca1 binds mers-cov s-protein rbd at residues involved in receptor binding, thus interfering with rbd binding to hdpp4 ( figure 4a ) [114] . this mab prophylactically and therapeutically inhibits mers-cov replication in common marmosets, resulting in significantly improved outcomes and reduced lung disease, compared with unvaccinated controls, and undetectable virus titers 3 days post-challenge [114] . a probe-based single-b-cell cloning strategy has been used for the isolation of cdc2-c2 and cdc2-c5 mabs from a patient convalescing from mers, as well as for the isolation of jc57-11 and jc57-14 mabs from nhps immunized with mers-cov full-length s dna and protein [102] . all these antibodies have neutralizing activities against both pseudotyped and live mers-cov. among them, cdc2-c2 is the most potent against 10 pseudotyped mers-cov strains, with neutralization ic 50 values ranging from 0.002 âµg/ml to 0.011 âµg/ml [102] . crystal-structure analysis of the cdc2-c2 and jc57-14 fab-rbd complexes indicates that both mabs bind rbd in the "out" (exposed) position, with the cdc2-c2 rbd binding overlapping with the dpp4-contacting residues ( figure 4b ,c) [102] . in addition, cdc2-c2 prophylactically protects hdpp4-tg mice from mers-cov infection, resulting in no detectable viral replication in the lungs three days post-challenge, and no fatalities over 28 days of observation [102] . the human mab mers-4 also neutralizes pseudotyped mers-cov and, notably, displays synergistic neutralization in combination with the mers-cov s-protein rbd-targeting mers-27 and m336 mabs [106, 118] , as well as the s-protein ntd-targeting 5f9 mab, in each case with dramatic reduction of the ic 50 compared with individual mabs [115] . structural analysis of a mers-4-fab-rbd complex revealed that mers-4 binds the rbd from outside the dpp4-binding interface, rather than competing with dpp4 ( figure 4d ). unlike mers-27, which binds rbd regardless of its conformational state within the s trimer, mers-4 binds rbd in the "standing" position where its epitopes are readily exposed and accessible [115] . thus, mers-4 displays unique epitope specificity, and an unusual mechanism of action involving indirect interference with dpp4 binding through conformational changes, which may explain the observation of synergistic neutralization in combination with other mabs [115] . single-domain antibody fragments (vhhs), or nanobodies, are the antigen-recognition regions of camelid heavy-chain-only antibodies (hcabs), which do not contain light chains. vhhs are easily expressed with high yield, and they have intrinsic stability, strong binding affinity, and specificity to target antigens, and they have therefore been developed as important therapeutic tools against viral infection, including that of mers-cov [116, 117, [119] [120] [121] [122] [123] . four vhhs (vhh-1, vhh-4, vhh-83, and vhh-101) have been identified from bone marrow cells of dromedary camels immunized with modified vaccinia virus (mva) expressing mers-cov s protein, and challenged with mers-cov [116] . these vhhs bind mers-cov s protein with low k d values (0.1-1 nm), recognize an epitope at residue d539 of rbd, and neutralize mers-cov (prnt 50 , 0.0014-0.012 âµg/ml) [116] . these four monomeric vhhs have each been fused with a c-terminal human igg2 tag to generate four hcabs (hcab-1, hcab-4, hcab-83, and hcab-101), with a higher binding affinity and a longer half-life than the free vhhs [116] . studies of protective efficacy show that hdpp4-tg mice (k18) injected with monomeric vhh-83 (20 or 200 âµg per mouse) lose weight, and die within seven days post-infection, possibly because of the short half-life of the vhh. however, when the mice are injected with hcab-83 (200 âµg per mouse), which has an extended half-life (~4.5 days), protection against mers-cov is complete, with no viral titers or pathological changes in the lungs of virally challenged mice [116] . by immunizing llamas with a recombinant rbd fragment (residues 377-588) fused to a c-terminal human igg fc tag (s377-588-fc), we constructed a vhh library, and we used it to generate a monomeric vhh, nbms10, and a human fc-fused vhh, nbms10-fc [117] . both vhhs can be expressed in a yeast expression system to high purity, and bind rbd with high affinity, recognizing a conformational epitope (residue 539) at the rbd-dpp4 interface, and blocking the binding of rbd to dpp4. these vhhs, particularly nbms10-fc, potently cross-neutralize pseudotyped mers-cov strains isolated from different countries, hosts, and time periods [117] . importantly, the fc-fused nbms10-fc significantly improves the serum half-life of nbms10, and a single-dose treatment of hdpp4-tg mice with this agent completely protects them against lethal mers-cov challenge [117] . these single-domain vhhs demonstrate the feasibility of developing cost-effective, potent, and broad-spectrum therapeutic antibodies against mers-cov infection. compared with vaccines based on mers-cov full-length s protein, which have the potential to attenuate neutralizing activity or enhance immune pathology, vaccines developed from mers-cov s-protein rbd are safer, and they do not cause immunological toxicity or eosinophilic immune enhancement [55, 99, 110, 124] . moreover, rbd-based therapeutic antibodies are generally more potent than non-rbd s1-based or s2-based antibodies [58, 104, 111] . hence, rbd-based vaccines and therapeutic antibodies have the potential for further development as effective tools to prevent and treat mers-cov infection. despite their acknowledged advantages, there are some issues associated with rbd-based interventions that need to be addressed. for example, rbd is under a high level of pressure of positive selection, and mutations occur in the rbd-dpp4 binding interface that might reduce the efficacy of these treatments [100, [125] [126] [127] . one possible way to avoid this effect, and to delay the emergence of escape mutants is to combine rbd-targeting therapeutics with those targeting other regions of the s protein, or to combine antibodies recognizing distinct epitopes within the rbd [102, 128] . such combinatorial strategies could also dramatically reduce antibody neutralization doses, providing feasible means to combat the continual threat of mers-cov. some recent advances have been made in the structure-guided design of anti-mers-cov interventions. structurally designed inhibitors of the 3cl protease have demonstrated potency against mers-cov [129] . also, a structurally designed s-protein trimer in the optimal prefusion conformation is shown to elicit production of high titers of anti-mers-cov neutralizing antibodies [87] . indeed, based on the previous studies on the structural design of mers-cov rbd, non-neutralizing epitopes in the rbd can be masked, to refocus the immunogenicity of the rbd on the neutralizing epitopes, and thus to enhance its ability to confer immune protection [93] . results from these structure-based studies will help to inform the design of innovative rbd-based anti-mers-cov vaccines and therapeutics with improved efficacy. isolation of a novel coronavirus from a man 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spread of mutant middle east respiratory syndrome coronavirus with reduced affinity to human cd26 during the south korean outbreak mutations in the spike protein of middle east respiratory syndrome coronavirus transmitted in korea increase resistance to antibody-mediated neutralization combining a fusion inhibitory peptide targeting the mers-cov s2 protein hr1 domain and a neutralizing antibody specific for the s1 protein receptor-binding domain (rbd) showed potent synergism against pseudotyped mers-cov with or without mutations in rbd structure-guided design of potent and permeable inhibitors of mers coronavirus 3cl protease that utilize a piperidine moiety as a novel design element this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: this study was supported by the nsfc grant 81571983, and the nih grants r21ai128311, r01ai137472, and r01ai139092. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. the authors declare no competing interests. key: cord-280939-d478p8u6 authors: abe, kento t.; li, zhijie; samson, reuben; samavarchi-tehrani, payman; valcourt, emelissa j.; wood, heidi; budylowski, patrick; dupuis, alan p.; girardin, roxie c.; rathod, bhavisha; wang, jenny h.; barrios-rodiles, miriam; colwill, karen; mcgeer, allison j.; mubareka, samira; gommerman, jennifer l.; durocher, yves; ostrowski, mario; mcdonough, kathleen a.; drebot, michael a.; drews, steven j.; rini, james m.; gingras, anne-claude title: a simple protein-based surrogate neutralization assay for sars-cov-2 date: 2020-10-02 journal: jci insight doi: 10.1172/jci.insight.142362 sha: doc_id: 280939 cord_uid: d478p8u6 most of the patients infected with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. while elisa-based assays to detect and quantitate antibodies to sars-cov-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the sars-cov-2 spike protein receptor binding domain (rbd) with its receptor, angiotensin-converting enzyme 2 (ace2). the assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an elisa for the detection of antibodies against the rbd, enabling a direct comparison. the results obtained with our assay correlate with those of 2 viral-based assays, a plaque reduction neutralization test (prnt) that uses live sars-cov-2 virus and a spike pseudotyped viral vector–based assay. the coronavirus s-protein (spike) is responsible for both receptor binding and fusion of the virus and host cell membranes. within the spike protein, the receptor binding domain (rbd) mediates the interaction with the host cell receptor, and sequence/structural variation in the rbd is responsible for the receptor binding specificity shown by those coronaviruses that use host proteins as receptors (1) . most of the patients infected with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. while elisa-based assays to detect and quantitate antibodies to sars-cov-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the sars-cov-2 spike protein receptor binding domain (rbd) with its receptor, angiotensin-converting enzyme 2 (ace2). the assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an elisa for the detection of antibodies against the rbd, enabling a direct comparison. the results obtained with our assay correlate with those of 2 viral-based assays, a plaque reduction neutralization test (prnt) that uses live sars-cov-2 virus and a spike pseudotyped viral vector-based assay. sars-cov-2, like sars-cov, uses the cell surface carboxypeptidase angiotensin-converting enzyme 2 (ace2) as a receptor for viral entry ( figure 1a) . the use of a common receptor is consistent with the fact that the 2 viruses share a high degree of sequence similarity and that their rbds are ~74% identical, though the rbd of sars-cov-2 binds ace2 with higher affinity than does that of sars-cov (2) . the spike proteins of both viruses are also both primed by the host protease, tmprss2, but unlike sars-cov-2, the spike protein of sars-cov does not contain a furin recognition motif that can be cleaved during viral biogenesis (2, 3) . the coronavirus spike protein is also a major target of the host immune system, and antibodies directed against it play a central role in host-mediated neutralization (4) . among neutralizing antibodies, those that block the interaction between viruses and their receptors represent the most common route to neutralization (5) . for this reason, both the spike protein and the rbd form the basis for most of the sars-cov-2 vaccines currently in development. the detection and study of neutralizing antibody activity following natural infection (or vaccination) can, therefore, support research aimed at the development of novel therapeutics and vaccine candidates. it can also aid in the identification of acceptable donors for convalescent plasma therapy (6) and, more generally, to establish immune correlates of infection. for sars-cov-2, viral neutralization assays are performed using either live virus (7) or viral vectors pseudotyped with the spike protein (8) . however, these cell culture-based assays are challenging to implement and time-consuming to run -factors that limit scalability. the conventional plaque reduction neutralization test (prnt) that uses live sars-cov-2 virus is further complicated by the need for containment level 3 (cl-3) and a specialized laboratory setup. although the pseudotyped viral vector-based assays do not require biosafety level 3 (bsl-3) containment (8) , they are complicated multistep procedures (9) . by contrast, the detection and quantitation of antigen-specific antibodies in patient samples can be easily assayed by elisa (10) . sars-cov-2 elisas are performed by immobilizing a recombinantly produced viral antigen (such as the spike trimer or rbd) ( figure 1b and supplemental figures 1 and 2; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.142362ds1) (see methods) onto multiwell plastic plates that are then incubated with diluted patient serum or plasma samples. the detection of antibodies that bind to the antigen involves a second incubation with enzyme-conjugated antihuman antibodies, where the enzyme is often horseradish peroxidase (hrp). this enables the detection of a color change when an hrp substrate such as 3,3′,5,5′-tetramethylbenzidine (tmb) is used. in direct binding assays of this type ( figure 1c ), the presence of patient antibodies against the viral antigen leads to a dose-dependent increase in the signal observed. elisa-based profiling has been developed by multiple groups and has been used to measure the kinetics of the antibody response in patient cohorts following sars-cov-2 infection. in several recent studies, including ours, this has revealed the relative stability in the igg response to the spike and rbd over several months, along with a more transient igm and iga response that wanes as patients convalesce (11) (12) (13) (14) (15) . however, the levels of neutralizing antibodies are not typically measured in large cohorts over time (with a few notable exceptions, as seen in refs. 12, 15) , as current assays have relatively low throughput. the relative lack of neutralizing antibody data represent a significant gap in our understanding of the immune response to sars-cov-2. here, we describe a modified elisa-type assay that serves as a surrogate neutralization assay. it measures the presence of antibodies capable of blocking the rbd-ace2 interaction, and like the direct binding elisa, it is easily scaled to allow for the analysis of large patient cohorts over time. we show that the results obtained by this assay correlate with those of both the sars-cov-2 prnt and a spike pseudotyped viral vector neutralization assay in a cohort of convalescent patients and on purified antibodies. we aimed to develop a simple protein-based assay to monitor the ability of antibodies, present in the serum or plasma of patients, to block the interaction between the rbd and the host receptor ace2. to do so, we elected for an elisa-type assay, since such assays are already widely used to detect antibodies that recognize sars-cov-2 antigens such as the spike trimer and its rbd. as with the standard direct elisa, the antigen (here, the rbd or the spike trimer) is first immobilized on multi-well plates and then incubated with patient plasma or serum ( figure 1b) . however, because we were interested in detecting functional antibodies that can prevent the interaction between the rbd (or spike) and ace2, we replaced the hrp-conjugated secondary antibody used in the direct elisa by a detection method involving human ace2. in our assay, recombinantly expressed soluble ace2 bearing a biotinylated c-terminal avitag is added to the antigen-bound plate after the plate has been incubated with the patient plasma or serum (see methods). bound ace2 is then detected by the addition of streptavidin-poly hrp and its colorimetric substrate tmb. the presence of patient antibodies that can block the rbd-ace2 interaction leads to a dose-dependent decrease in the signal observed, and as such, we refer to it as a surrogate neutralization elisa (snelisa; figure 1d ). we explored 2 different versions of the assay: the configuration described above and one involving immobilized ace2 and soluble biotinylated rbd, a configuration similar to that previously reported (16) . the assay with immobilized rbd and soluble biotinylated ace2 was more sensitive than its counterpart (supplemental figure 3 ). moreover, with the rbd immobilized, the same overall protocol and colorimetric detection can be used for both the direct binding elisa and the snelisa, thereby facilitating a direct comparison. although the snelisa worked well with either the rbd or the spike ectodomain trimer immobilized ( figure 1e and supplemental figure 4a ), we focused on the rbd, since it is easier to produce and provides a simple one-to-one binding interaction with ace2. using a small test set (supplemental figure 4b) , we first showed that the serum/plasma from positive but not negative control patients inhibited the interaction between ace2 and the immobilized rbd ( figure 1f ). the technical reproducibility of the assay was within 5%-10% coefficient of variation (cv). the total time required to perform the assay (once the plates are coated with the antigen) is 3.5-4 hours, and the assay can be performed using the same equipment and biosafety protocols as a standard elisa. using both the surrogate neutralization and direct binding (with a dilution series) elisas, we then profiled a set of 58 serum samples acquired at the canadian blood services as part of a screen for convalescent plasma therapy donors ( table 1 ). with reference to the direct binding results, the snelisa showed that samples with high levels of igg against the rbd were typically the most potent at blocking the rbd-ace2 interaction (e.g., cbs13, which is included as a positive control). conversely, samples lacking detectable rbd-binding antibodies were not able to block the interaction. to more systematically evaluate the relationship between the rbd-binding antibody levels and the ability to block the rbd-ace2 interaction (as determined by the snelisa), we calculated the auc for both assays and plotted the rbd-binding auc versus the snelisa auc ( figure 2c and supplemental figure 9 ). the plot showed a clear correlation (r 2 = 0.823), with the sera containing the highest rbd-binding antibody levels being the most effective at blocking the rbd-ace2 interaction ( figure 2c ; compare figure 2b with figure 2a ; supplemental table 1 ). nevertheless, there are samples with similar rbd-binding antibody concentrations that differ in their ability to block the rbd-ace2 interaction (figure 2d and supplemental figure 10 ). differences in antibody isotype, affinities, and abundance, as well as the rbd epitopes bound, are all factors that could explain these outliers. while it is reasonable to expect that antibodies that block the rbd-ace2 interaction would be neutralizing, we validated this using cell-based viral infectivity and entry assays. fifty-seven of the 58 samples analyzed by the snelisa were analyzed by prnt, the gold standard in the field. prnt50 is defined as the concentration of patient serum or plasma capable of reducing the formation of viral plaques by 50%; prnt90 is the concentration that reduces plaque formation by 90%. as shown in figure 3a , most of the samples displaying high values in the direct binding and snelisas were also positive by prnt90 (and those with low titers were negatives). both elisas also gave an overall agreement with the prnt50 titers (see supplemental figure 11 , with a coefficient of determination of 0.6). we also adapted and optimized a spike-pseudotyped lentiviral-based entry assay (8) , and we reprofiled the neutralization potential of a subset of samples. there was also a high correlation (r 2 = 0.76) between the snelisa results and the titers obtained with this spike-pseudotyped lentiviral-based entry assay ( figure 3b and supplemental figure 12 ). taken together, these results indicate that our snelisa is a good surrogate neutralization assay, particularly for distinguishing between samples with high versus low neutralization activity. as such, the assay should be of value in the selection of candidate donors for convalescent plasma therapy and for monitoring immune correlates of patient outcomes. future work will focus on providing a better understanding of the outliers observed across all assays. indeed, rare but potent neutralizing antibodies in patient samples with low pseudovirus neutralization titers have recently been reported (17) . to assess whether our snelisa might also be of value for screening the neutralization potential of monoclonal antibodies, we tested it using a number of neutralizing and nonneutralizing monoclonal antibodies and compared the results with the results obtained with the pseudotyped lentiviral-based entry assay or cytopathic effect-reduction neutralization assay with sars-cov-2. the llama vhh72 monoclonal antibody (expressed as a human fc fusion), previously shown to neutralize in a sars-cov-2 spike pseudotyped entry assay (18) , blocked the rbd-ace2 interaction in our snelisa and viral entry in our spike pseudotyped lentivirus assay; similar results were obtained for the active motif 414-1 antibody, which was isolated from a convalescent patient and was shown to be neutralizing (19) (figure 3 , c and d, and supplemental figure 13 ). in contrast, other antibodies, such as an igg derived from the monoclonal anti-sars1 cr3022 or a commercial antibody (hc2001) from genscript, had a much more moderate effect in the snelisa (supplemental figure 14) , and the genscript antibody had no effect in the cytopathic effectreduction neutralization assay (supplemental figure 15 ). the active motif 414-2 antibody was previously shown to be incapable of neutralizing live sars-cov-2 virus (8, 19) . in our assays, it efficiently bound the rbd in the direct elisa but did not block the rbd-ace2 interaction in the snelisa. the same antibody partially prevented entry in our lentivirus entry assay. taken together, these observations suggest that our snelisa is a good complement to more complex cell-based assays for the discovery and screening of neutralizing monoclonal antibodies. in summary, we have developed a simple and safe snelisa for sars-cov-2. it can be readily incorporated into existing testing platforms and may be of particular value in the selection of donors for convalescent plasma therapy and as a means of monitoring the immune response to vaccination. given that neutralizing antibody titres have recently been shown to wane fairly rapidly in some (12) (13) (14) 20) but not all (15, 20) studies, the assay may also be useful for broad serosurveillance, especially as it should be more scalable than the approaches requiring viral infection assays. when coupled with epidemiological studies, it might also be used to assess the risk of infection/reinfection. we also note that the optimized conditions used here for the direct rbd-binding elisa are similar to those reported in ref. 10 using rbd-expression constructs that have been widely distributed. their rbd can be obtained from bei resources (10), and we found that it generates similar results when used with our biotinylated ace2 in the snelisa (supplemental figure 16 ). this should further facilitate the broad implementation of our assay across multiple laboratories. there are limitations to the assay, however, that need to be acknowledged. first, the snelisa is limited to the detection of neutralizing antibodies that function by blocking the interaction between the rbd and ace2. while by no means dominant, examples of antibodies that neutralize by other mechanisms are beginning to emerge (21) (22) (23) . the snelisa, in conjunction with a neutralization assay, could be used to identify further such examples. as with those identified in this work, the outliers (e.g., those with high viral neutralization titers but low snelisa levels) provide a starting point for further work aimed at understanding the mechanisms of antibody-mediated neutralization. another limitation of our approach is that the current assay cannot directly map the epitopes targeted by the various antibodies. undoubtedly, the antibodies detected by the snelisa bind to different sites on the rbd, a suggestion supported by the structures of neutralizing antibody fragment antigen binding (fabs) in complex with the sars-cov-2 rbd. in one example, 2 different neutralizing antibodies that bind to different epitopes on the rbd were found to synergistically mediate viral neutralization (24) . while, in the current study, we simply wanted to provide evidence of antibodies that could block the rbd-ace2 interaction, the snelisa could be adapted to provide information on the site of antibody binding. as recently shown, a series of structure-guided point mutants in the rbd could be used to infer where on the rbd the antibodies are binding (25) . this type of approach would likely be more important in the characterization of monoclonal antibodies, such as those presented in figure 3c , and would set the stage for in-depth biophysical and structural studies. while the direct binding elisa described here employed an anti-igg secondary antibody (the predominant isotype in convalescent serum), we note that the snelisa measures the ability of any antibody isotype (or even antibodies from different species or any other molecule) to block the rdb-ace2 interaction. in this regard, it is similar to that of a viral-based neutralization assay. while we have not performed a detailed analysis, we did show that single-point direct binding elisas performed for igm, and to a lesser extent iga, are also correlated with the results obtained by the snelisa (supplemental figure 17) . the safety and simplicity of the snelisa should make it a valuable addition to the arsenal of assays for monitoring the immune response to sars-cov-2 infection. for all elisas, inactivation of potential infectious viruses in plasma or serum was performed by incubation with triton x-100 to a final concentration of 1% for 1 hour before use (26) . for the pseudotyped lentiviral assays, the serum was heat inactivated for 1 hour at 56°c (10). the expression plasmid generated is a derivative of those previously reported in our piggybac transposonbased mammalian cell expression system (27) . two versions of the plasmid were constructed: one contains the cmv promoter (pb-cmv) and the other the tre promoter (pb-tre). the vectors are otherwise identical and can be used to generate stable cell lines for constitutive or inducible protein expression. the protein cloning region contains several optional elements separated by restriction sites as follows: an n-terminal human cystatin-s secretion signal, the protein of interest, a foldon trimerization motif (28), a 6xhis purification tag, and an avitag biotinylation motif (29) (supplemental figure 1) . a woodchuck hepatitis virus posttranscriptional regulatory element (wpre) follows the orf to facilitate nuclear export of the mrna. a pair of piggybac transposon terminal repeats flank the expression cassette and an attenuated puromycin resistance marker (bioshop canada inc., pur333), thereby allowing for the generation of stable cell lines using the piggybac transposase. the human codon optimized cdna of the sars-cov-2 spike protein (mc_0101081) was purchased from genscript. the human ace2 cdna was derived from mgc clone 47598. to stabilize the soluble spike ectodomain trimer, 2 regions of the spike protein were mutated. residues 682-685 (rrar) were mutated to ssas to remove the furin cleavage site, and residues 986-987 (kv) were each mutated to a proline residue to stabilize the prefusion form as previously described (30) . the soluble spike protein ectodomain construct includes residues 1-1211 (mfvf...qyik), followed by the foldon trimerization motif, a 6xhis tag, and an avitag. both the sars-cov-2 rbd and the human ace2 constructs are preceded by the human cystatin-s secretion signal and followed by the 6xhis and avitag. the rbd and ace2 constructs contain residues 328-528 (rfpn...cgpk) and 19-615 (stie...pyad), respectively. the cdna of the human cr3022 fab was synthesized by genscript based on its previously reported sequence (31). the light chain and heavy chains were individually cloned into the pb-tre expression plasmid. for fab production, a 6xhis tag was added to the c-terminal end of the fab heavy chain. an igg form was generated by fusing the human igg1 fc coding sequence to the c-terminal end of the fab heavy chain. freestyle 293-f suspension cells (thermo fisher scientific, r79007) were grown in shaker flasks (125 rpm) in freestyle 293 expression medium (thermo fisher scientific, 12338018) in a humidified 37°c incubator filled with 3% (v/v) co 2 . the cell density and viability were monitored by manual counting using a hemocytometer and trypan blue staining. for transfection, cells of > 90% viability were counted and seeded at a density of approximately 1 × 10 6 cells/ml into 300 ml freestyle 293 medium supplemented with 1 μg/ml aprotinin (bioshop canada inc., apr600). the pb-cmv plasmid dna (300 μg) and 293fectin (400 μl; thermo fisher scientific, 12347019) were each added to separate tubes containing 15 ml of opti-mem medium (thermo fisher scientific, 31985062). the 2 solutions were then mixed and incubated for 5 minutes before being added to the 300 ml cell culture. two days after transfection, the 300 ml culture was expanded into three 1 l shaker flasks each containing 300 ml of culture medium. protein expression was continued for an additional 4 days. the stable cells were scaled up in 1 l shaker flasks containing 300 ml freestyle 293 medium without supplements. when the cell densities reached approximately 1 × 10 6 cells/ml, 1 μg/ml doxycycline (milliporesigma, d3447), and 1 μg/ml aprotinin were added to initiate protein expression. during the expression phase, 150 ml of the medium was removed, and fresh medium added every other day. the harvested expression medium was centrifuged at 10,000g for 30 minutes at 10°c to remove the cells and debris. for the 6xhis tagged proteins, the clarified media were passed through an ni-nta column (qiagen, 30230). for the spike ectodomain, 3 ml of ni-nta resin was used for each liter of medium. for the rbd, ace2, and cr3022 fab, 8 ml of ni-nta resin was used for each liter of medium. the ni-nta resin was washed with 20 column volumes of phosphate buffered saline (pbs), followed by 3-5 column volumes of pbs containing 10 mm imidazole. the protein was eluted with pbs containing 300 mm imidazole (bio basic, ib0277) and 0.1% (v/v) protease inhibitor cocktail (milliporesigma, p-8849). for the cr3022 antibody, the harvested medium was incubated with rprotein a sepharose ff resin (ge healthcare, 17127903). the resin was then washed with 20 column volumes of pbs, and the antibody was eluted with 50 mm glycine, ph 3.0, containing 150 mm nacl. the acid-eluted antibody was immediately neutralized by the addition of 1/20 volume of 1m tris, ph 8.5. protease inhibitor cocktail was also added to a final concentration of 0.1% (v/v). the approximate purified yields of the various proteins are as follows: rbd, 70 mg/l; spike trimer, 3 mg/l; ace2, 50 mg/l; cr3022 fab, 80 mg/l; and cr3022 igg, 20 mg/l. the protein samples were stored in 40% glycerol at -12°c. shortly before use, the glycerol stocks were further purified using size-exclusion chromatography. for the rbd, ace2, and cr3022 fab/igg, a superdex 200 increase (ge healthcare, 28990944) column was used. for the spike ectodomain, a superose 6 increase (ge healthcare, 29091596) column was used (supplemental figure 2 ). each biotinylation reaction contained 200 μm biotin, 500 μm atp (milliporesigma, cat # a2383), 500 μm mgcl 2 , 30 μg/ml bira (produced from e. coli; a gift from walid houry, university of toronto), 0.1% (v/v) protease inhibitor cocktail, and no more than 100 μm of the protein-avitag substrate. the mixture was incubated at 30°c for 2 hours, followed by size-exclusion chromatography to remove unreacted biotin (bioshop canada inc., bio302). for the rbd, the degree of biotinylation was assessed using a band-shift assay. a total of 5 μg of the biotinylated rbd was heated to 95°c for 30 seconds in sds-page loading buffer (containing 2% sds, 50 mm dtt); after cooling, 1 μl of a 5 mg/ml streptavidin solution was added. the mixture was then analyzed by sds-page to assess the formation of the rbd-streptavidin complex (supplemental figure 2) . the llama single domain antibody vhh72 sequence (pdb entry 6waq_1) was obtained from wrapp et al. (18) . a cdna encoding vhh72 fused to an adcc-attenuated human igg1 fc domain (hfc1x7, from patent us 2019 352 383a1) was codon optimized for expression in cho cells, synthesized by genscript, and cloned into the ptt5 plasmid (33) . the ptt5-vhh72hfc1x7 plasmid was transiently expressed in cho 55e1 cells (34) using pei max transfection reagent (polysciences) and a slightly modified protocol as described previously (35) . the cell culture was harvested at day 7 after transfection, centrifuged 20 minutes at 3000g at room temperature, and filter sterilized using a 0.22 μm membrane vacuum filter (express plus, milliporesigma). filtered supernatant was loaded on a 5 ml mabselect sure column (ge healthcare) equilibrated in pbs. the column was washed with pbs, and the antibody eluted with 100 mm citrate buffer ph 3.6. the fractions containing the antibody were pooled, and elution buffer was exchanged for pbs using nap-25 columns (ge healthcare). purified vhh72h-fc1x7 in pbs was quantified by absorbance at 280 nm using a nanodrop spectrophotometer (thermo fisher scientific) and the calculated extinction coefficient of the protein. overall volumetric yield after protein a purification was 275 mg/l. the purified protein was analyzed by analytical size-exclusion ultra high-performance liquid chromatography coupled to a mals detector and eluted as a major (>98% integrated area) symmetrical peak of 102 kda with less than 2% aggregates (not shown). an alternative source for rbd was bei resources nr-52306 (contributors f. krammer, f. amanat, s. strohmeier; icahn school of medicine, mount sinai, new york, usa; lot 7034437). commercial antibodies tested also included a human igg chimeric antibody from genscript (sars-cov-2 spike s1 antibody, hc2001; genscript, a02038) and 2 sars-cov-2 spike antibodies from active motif (am002414, 91349; am001414, 91361). manual single-point elisas in 96-well format. for the manual single-point elisas in 96-well format, concentrations and incubation times were optimized to maximize the separation between anti-rbd levels in convalescent plasma or serum from that of pre-covid-era banked serum while maintaining the required levels of antigens as low as possible. a total of 1 μl of serum or plasma was used for the detection of antibodies on 96-well plates coated with 75 ng/well of recombinant purified rbd. single-point elisas are expressed as ratios to a positive control convalescent plasma sample. multipoint elisas. for the multipoint elisas, the rbd amount was fixed to 100 ng/well to match the design of the snelisa, and 2-fold serial dilutions of the serum or plasma sample from 1 μl to 0.06 μl were employed. both cases. in both cases, the rbd antigen (diluted to 2 μg/ml in pbs) was first adsorbed to 96-well clear immulon 4 hbx plates (thermo fisher scientific, 3855) in pbs overnight at 4°c and then washed 3 times with 200 μl pbs plus 0.1% tween-20 (pbs-t; milliporesigma). plates were blocked for 1 hour at room temperature with 200 μl 5% blocker blotto (thermo fisher scientific, 37530) and washed 3 times with 200 μl pbs-t. in the single-point elisas, plate blocking was performed with 3% w/v milk powder (bioshop canada inc., alb005.250, lot 9h61718) in pbs for 1-2 hours. patient samples (pretreated with 1% final triton x-100 for viral inactivation) diluted in pbs-t containing 1% w/v milk powder (1:50 for the single-point elisa) were then added to the plates and incubated for 2 hours at room temperature (50 μl total volume); technical duplicates were performed unless otherwise indicated. a chimeric human anti-spike antibody (sars-cov-2 spike s1 antibody, hc2001; genscript, a02038) was added to a set of wells on each plate as a serial dilution (1:5,000-1:80,000 or 10-0.63 ng per well in 4 steps) to enable cross-plate comparisons. positive (convalescent plasma from a single patient) and negative controls (pre-covid-era banked serum) were also added to each plate, at 1 μl. wells were washed 3 times with 200 μl pbs-t. goat anti-human anti-igg (goat anti-human igg fcy hrp, jackson immunoresearch, 109-035-098) at a 1:60,000 dilution (0.67 ng/well) in 1% blotto was added and incubated for 1 hour. wells were washed 3 times with 200 μl pbs-t, and 50 μl of 1-step ultra tmb-elisa substrate solution (thermo fisher scientific, 34029) was added for 15 minutes at room temperature. the reaction was quenched with 50 μl stop solution containing 0.16n sulfuric acid (thermo fisher scientific, n600). the plates were read in a spectrophotometer (biotek instruments inc., cytation 3) at 450 nm. for all elisa-based assays, raw od values had blank values subtracted before analysis. for the single-point direct binding assay, the average cv across cbs samples is 3.3% (mean) and 1.8% (median) (supplemental table 1 ). for single-point assays, all data were normalized to the positive serum control (single point) on each plate and expressed as a ratio to this control. for the multipoint dose responses, blank-adjusted reads were used. variations. variations to this protocol included the following. (a) replacement of the rbd on the plate by the bei resources nr-52306. the assay was set up identically to and in parallel with our in-house produced rbd (supplemental figure 16 ). (b) replacement of rbd (100 ng) on plate by the spike trimer purified above (667 ng) (supplemental figure 3) . (c) performing the single-point elisas using an automated platform with chemiluminescent detection for anti-igg, -iga, and -igm, exactly as described in (11) (supplemental figure 17) . our final optimized snelisa used 100 ng immobilized recombinant rbd on 96-well immulon hbx plates incubated overnight at 4°c (2 μg/ml). all volumes added to the well were 50 μl, unless specified otherwise. plates were washed 3 times with 200 μl pbs-t and blocked for 1-1.5 hours at room temperature with 200 μl 3% bsa (bioshop canada inc., ski400.1, lot 9h61850). after washing as above, a 4-step, 2-fold serial dilution series of patient serum or plasma (0.5-4 μl of sample) was incubated for 1 hour. the wells were washed as above and incubated with 50 ng biotinylated recombinant ace2 for 1 hour. after washing as above, the wells were incubated with 44 ng streptavidin-peroxidase polymer (milliporesigma, s2438). the resultant signal was developed and quantified with tmb in an identical manner to the direct elisas. due to day-to-day variation in signal, all od 450 values are normalized to the od 450 of the well where no patient serum/antibody was added for each sample. all values are expressed in this ratio space. variations. variations of this protocol included using a different source of rbd (bei resources, nr-52306) and using spike trimer as shown above (670 ng/well) ( figure 1c and supplemental figure 16 ). another variation of the assay was to bind nonbiotinylated ace2 to the plate (100 ng) and to use biotinylated rbd (50 ng) for detection (supplemental figure 3 ). neutralization assays on the canadian blood services samples used in figure 2 were performed by 2 independent laboratories, the nml of the public health agency of canada, and the wadsworth center, new york state department of health. the cytopathic effect-reduction neutralization assay on the recombinant genscript antibody was performed in toronto. for the prnt assay at nml, sars-cov-2 (canada/on_on-vido-01-2/2020, epi_isl_42517) stocks were titrated (7) for use in a prnt adapted from a previously described method for sars-cov (36) . briefly, serological specimens were diluted 2-fold from 1:20 to 1:640 in dmem supplemented with 2% fbs and incubated with 50 pfu of sars-cov-2 at 37°c and 5% co 2 for 1 hour. the sera-virus mixtures were added to 12-well plates containing vero e6 cells at 100% confluence, followed by incubation at 37°c and 5% co 2 for 1 hour. after adsorption, a liquid overlay composed of 1.5% carboxymethylcellulose diluted in mem, supplemented with 4% fbs, l-glutamine, nonessential amino acids, and sodium bicarbonate, was added to each well; the plates were incubated at 37°c and 5% co 2 for 72 hours. the liquid overlay was removed, and the cells were fixed with 10% neutral-buffered formalin for 1 hour at room temperature. the monolayers were stained with 0.5% crystal violet for 10 minutes and washed with 20% ethanol. plaques were enumerated and compared with controls. the highest serum dilution resulting in 50% and 90% reduction in plaques compared with controls were defined as the prnt50 and prnt90 endpoint titers, respectively. prnt50 titers ≥ 1:160 and prnt90 titers ≥ 1:20 were considered positive. for the prnt assay at wadsworth, the assay for the detection of sars-cov-2 neutralizing antibodies was a modified version of previously described methods (37) (38) (39) . patient sera and sars-cov-2 (usa/wa-1/2020, bei resources, nr-52281) were diluted in vero e6 cell culture maintenance medium (emem, 2% heat-inactivated fbs, 200 u/ml penicillin g, 200 u/ml streptomycin). patient samples were serially diluted 1:10-1:320 and mixed with an equal volume of virus containing 150 pfus. virus and serum mixtures were incubated at 37°c and 5% co 2 for 1 hour. following the initial incubation, 0.1 ml of each dilution was plated in a single well of a 6-well plate containing confluent monolayers of vero e6 cells (atcc, crl-1586) and allowed to adsorb for 1 hour at 37°c and 5% co 2 . following adsorption, cell cultures were overlaid with 0.6% agar in cell culture medium and returned to the incubator. at 2 days after infection, a second overlay containing 0.2% neutral red was added. monolayers were inspected for 2 days, and plaques were counted. antibody titers were reported as the inverse of the serum dilution resulting in 50% (prnt50) and 90% (prnt90) reduction in plaques as compared with the virus inoculum control. for the cytopathic effect-reduction neutralization assay in toronto, 200 μl of 0.2 × 10 6 veroe6 cells/ml were seeded into a 96-well flat-bottom plate to adhere overnight. all plasma and serum samples were heat inactivated at 56°c for 30 minutes. in a separate 96-well plate, the serum, plasma, or antibody (1 μg/ml) samples were serially diluted 2-fold 8 times in serum-free dmem starting from a dilution of 1:20 to 1:2560 in a volume of 25 μl. to all wells, 25 μl of sars-cov-2 sb2 clone 1 was added, ensuring that each well had a dose of 100 issue culture infectious dose (tcid). for the cell control, 50 μl of serum-free dmem was added. for the virus control, 25 μl of sars-cov-2 sb2 clone 1 was added with a dose of 100 tcid and topped off with 25 μl of serum free dmem. the plate was incubated for 1 hour at 37°c, 5% co 2 with shaking every 15 minutes. after incubation, all the media from the veroe6 culture were removed, and the full 50 μl of serum/sars-cov-2 coculture was layered on the cells. the plate was again incubated for 1 hour at 37°c, 5% co 2 , with shaking every 15 minutes. after the incubation, the inoculum was removed, and 200 μl of dmem containing 2% fbs was added. the plate was incubated for 5 days and cytopathic effect was tracked. the assay was established using constructs previously described (8) (constructs obtained through a gift from jesse bloom and katharine crawford, fred hutchison cancer research centre, seattle, washington, usa, and now available through bei resources) and optimized in-house. major changes to the reported protocol included: (a) use of a second-generation pspax2 (addgene, 12260) lentivirus packaging system instead of the third-generation system used by the bloom lab, (b) production of spike pseudotyped virus-like particles (vlps) at 33°c, (c) a neutralization assay plate layout that increases throughput, (d) adjustments to the luciferase protocol to minimize variability in readings, and (e) use of a cell line that coexpresses ace2 and tmprss2. to generate this cell line, entry vectors for ace2 and tmprss2 coding sequences were cloned into plenti cmv puro dest (addgene, 17452) and plenti cmv hygro dest (addgene, 17454), respectively. the resulting transfer vectors were used to generate lentivirus via the second-generation pspax2 and vsv-g (addgene, 8454). hek293t cells were transduced with ace2 lentivirus at an moi < 1 and selected with puromycin (1 μg/ml) to generate a stable population. these cells were subsequently transduced with tmprss2 lentivirus and selected with hygromycin (200 μg/ml) in a similar fashion. for vlp generation, hek293t cells were transiently cotransfected in a 6-well-plate format containing 2 ml growth medium (10% fbs, 1% penicillin/streptomycin [pen/strep] in dmem) with 1.3 μg pspax2, 1.3 μg phage-cmv-luc2-ires-zsgreen-w (bei, nr-52516; a gift from jesse bloom and katharine crawford; lentiviral backbone plasmid that uses a cmv promoter to express luciferase followed by an ires and zsgreen), and 0.4 μg hdm-idtspike-fixk (bei, nr-52514; a gift from jesse bloom and katharine crawford; expressed under a cmv promoter a codon-optimized wuhan-hu-1 spike; genbank, nc_045512) using 8 μl jetprime (polyplus-transfection sa, 114-01)in 500 μl jetprime buffer. after 8 hours of transfection, the medium was replaced by 3 ml of dmem containing 5% heat-inactivated fbs and 1% pen/strep, and the cells were incubated for 16 hours at 37°c and 5% co 2 ; they were then transferred to 33°c and 5% co 2 for an additional 24 hours. at 48 hours after transfection, the supernatant was collected, spun at 500g for 5 minutes at room temperature, filtered through a 0.45 μm filter, and frozen at -80°c. the virus titers were evaluated using hek293t-ace2/tmprss2 cells at 10,000 cells per well on a poly-l-lysine-coated (5-10 μg/ml) 96-well plate using hi10 media (10% heat-inactivated fbs, 1% pen/strep), along with a virus dilution resulting in > 1000 relative luciferase units (rlu) over control (~1:100 virus stock dilution). for the neutralization assay, 2.5-fold serial dilutions of the serum samples were incubated with diluted virus at a 1:1 ratio for 1 hour at 37°c before being transferred to plated hek293-ace2/tmprss2 cells and incubated for an additional 48 hours at 37°c and 5% co 2 . after 48 hours, cells were lysed, and bright-glo luciferase reagent (promega, e2620) was added for 2 minutes before reading with a perkinelmer envision instrument. auc values were tabulated for both the direct binding elisa and the snelisa using r version 4.0.1 and r package pracma. for the snelisa, the ratios (normalized values) are used in the auc calculations. to identify outliers, we calculated the distance of each point from the regression line using total least squares and labeled points with distances > 0.4. for the lentiviral pseudotyping assays, 50% inhibitory concentration or dilution (ic 50 or id 50 ) were calculated with nonlinear regression (log[inhibitor] versus normalized response -variable slope) using graph-pad prism 8 (graphpad software inc.). the "variable slope" option is a parameter selected in graphpad prism 8 for nonlinear regression that does not assume a standard slope of -1.0 with each dose-response curve but, instead, determines the slope of the curve based on the data generated. for the extended direct binding dilution series, titres were calculated by taking the dilution of serum that produced 50% of the maximum response in the elisa as determined by the nonlinear regression line (sigmoidal, 4pl, x is log[dilution]) using graphpad prism 8. the assay reproducibility was estimated across experiments by comparing the auc values for those samples profiled across different batches. cbs13 (n = 3) cv for displacement was 5.1% and direct binding was 5.5%; cbs16 (n = 3) cv for displacement was 3.4% and 11.5% for binding; cbs50 (n = 2) cv for displacement was 9.9% and binding was 0.7%. when applicable, graphical data from experiments with 3 or more replicates are presented as mean ± sem. all samples were collected after research ethics board (reb) review. the elisas were performed at the lunenfeld-tanenbaum research institute with mount sinai hospital (msh; toronto, ontario, canada) reb approval (study no. 20-0078-e). external samples were transferred through material transfer agreements. all research has been performed in accordance with relevant guidelines and regulations. all participants have provided informed consent. the samples were deidentified before transfer to the assay laboratory. kta and acg designed the snelisa and the direct rbd antibody assay. zl and jmr designed the protein expression, biotinylation, and purification procedures. rs and pst optimized the lentiviral pseudotyping assay. ejv, hw, mad, apd, rcg, kam, pb, and mo developed, performed and analyzed the prnt and/or cytopathic effect-reduction neutralization assays. kta and br performed direct elisa experiments. yd designed the vhh72hfc1x7 construct, expression, and purification procedures. jhw and mbr implemented the automated direct binding elisa. kc helped coordinate the project. sjd provided samples, coordinated neutralization testing, and integrated prnt and snelisa data. kta and acg analyzed the snelisa data. jlg, ajm, sm, mo, and sjd contributed essential patient samples. kta, jmr, and acg wrote the manuscript with input from all authors. the order of authors for the 2 co-first author was determined by the contribution of kta in the overall study design, as well as data analysis and manuscript preparation. coronavirus spike protein and tropism changes structure, function, and antigenicity of the sars-cov-2 spike glycoprotein sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor neutralizing antibodies against sars-cov-2 and other human coronaviruses broadly neutralizing antiviral antibodies deployment of convalescent plasma for the prevention and treatment of covid-19 two detailed plaque assay protocols for the quantification of infectious sars-cov-2 protocol and reagents for pseudotyping lentiviral particles with sars-cov-2 spike protein for neutralization assays pseudotype neutralization assays: from laboratory bench to data analysis a serological assay to detect sars-cov-2 seroconversion in humans evidence for sustained mucosal and systemic antibody responses to sars-cov-2 antigens in covid-19 patients dynamics of neutralizing antibody titers in the months after sars-cov-2 infection longitudinal evaluation and decline of antibody responses in sars-cov-2 infection neutralizing and binding antibody kinetics of covid-19 patients during hospital and convalescent phases sars-cov-2 infection induces robust, neutralizing antibody responses that are stable for at least 3 months a sars-cov-2 surrogate virus neutralization test based on antibody-mediated blockage of ace2-spike protein-protein interaction convergent antibody responses to sars-cov-2 in convalescent individuals structural basis for potent neutralization of betacoronaviruses by single-domain camelid antibodies clinical and immunological assessment of asymptomatic sars-cov-2 infections potent neutralizing antibodies from covid-19 patients define multiple targets of vulnerability a neutralizing human antibody binds to the n-terminal domain of the spike protein of sars-cov-2 a human monoclonal antibody blocking sars-cov-2 infection a noncompeting pair of human neutralizing antibodies block covid-19 virus binding to its receptor ace2. science human-igg-neutralizing monoclonal antibodies block the sars-cov-2 infection evaluation of inactivation methods for severe acute respiratory syndrome coronavirus in noncellular blood products simple piggybac transposon-based mammalian cell expression system for inducible protein production structure of bacteriophage t4 fibritin: a segmented coiled coil and the role of the c-terminal domain site-specific biotinylation of purified proteins using bira immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen human monoclonal antibody combination against sars coronavirus: synergy and coverage of escape mutants chromosomal transposition of piggybac in mouse embryonic stem cells purification and characterization of a recombinant g-protein-coupled receptor, saccharomyces cerevisiae ste2p, transiently expressed in hek293 ebna1 cells rapid protein production from stable cho cell pools using plasmid vector and the cumate gene-switch optimization of a high-cell-density polyethylenimine transfection method for rapid protein production in cho-ebna1 cells assays for the assessment of neutralizing antibody activities against severe acute respiratory syndrome (sars) associated coronavirus (scv) a plaque reduction test for dengue virus neutralizing antibodies serum dilution neutralization test for california group virus identification and serology antigenic relationships between flaviviruses as determined by cross-neutralization tests with polyclonal antisera we thank janet mcmanus at canadian blood services for her technical and logistical expertise and the wadsworth center media and tissue culture core. we thank joan wither for the lupus patient samples, and jesse bloom and katharine crawford for sharing protocols and reagents for the lentiviral s pseudotyping assay. key: cord-328003-yovp8squ authors: duan, liangwei; zheng, qianqian; zhang, hongxia; niu, yuna; lou, yunwei; wang, hui title: the sars-cov-2 spike glycoprotein biosynthesis, structure, function, and antigenicity: implications for the design of spike-based vaccine immunogens date: 2020-10-07 journal: front immunol doi: 10.3389/fimmu.2020.576622 sha: doc_id: 328003 cord_uid: yovp8squ the ongoing pandemic of coronavirus disease 2019 (covid-19), caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), poses a grave threat to global public health and imposes a severe burden on the entire human society. like other coronaviruses, the sars-cov-2 genome encodes spike (s) glycoproteins, which protrude from the surface of mature virions. the s glycoprotein plays essential roles in virus attachment, fusion and entry into the host cell. surface location of the s glycoprotein renders it a direct target for host immune responses, making it the main target of neutralizing antibodies. in the light of its crucial roles in viral infection and adaptive immunity, the s protein is the focus of most vaccine strategies as well as therapeutic interventions. in this review, we highlight and describe the recent progress that has been made in the biosynthesis, structure, function, and antigenicity of the sars-cov-2 s glycoprotein, aiming to provide valuable insights into the design and development of the s protein-based vaccines as well as therapeutics. the coronavirus disease 2019 (covid-19) global pandemic represents an unprecedented public health, social and economic challenge (1, 2) . the etiological agent of covid-19 is a new member of the coronaviridae family that is closely related to severe acute respiratory syndrome coronavirus (sars-cov) and was recently referred to as sars-cov-2 by the coronavirus study group of the international committee on taxonomy of viruses (3) . the virus has spread rapidly and sustainably around the global resulting in over twenty-one million cases and more than 750,000 deaths as of august 15, 2020 (4) . coronaviruses (covs) are enveloped positive-sense rna viruses (5) . enveloped covs entering host cells and initiating infection is achieved through the fusion of viral and cellular membranes (6, 7) . membrane fusion is mediated by the large type i transmembrane s glycoprotein on the viral envelope and the cognate receptor on the surface of host cells (8) (9) (10) . the surfaceexposed location of the s glycoprotein not only allows it to carry out membrane fusion but also renders it a direct target for host immune responses, making it the major target of neutralizing antibodies (11) . because of its central roles in viral infection and eliciting protective humoral and cell-mediated immune responses in hosts during infection (10) , the s protein is the primary target for vaccine design as well as antiviral therapeutics (12) . here, we provide a comprehensive overview of the wealth of research related to the sars-cov-2 s glycoprotein biosynthesis, structure, function, and antigenicity, aiming to provide useful insights into the design and development of the s protein-based vaccines as well as therapeutics to prevent or treat the ongoing global spread of sars-cov-2/covid-19. the sars-cov-2 s glycoprotein is synthesized as a 1273-amino acid polyprotein precursor on the rough endoplasmic reticulum (rer) (figure 1 ) (13) . the unprocessed precursor harbors an endoplasmic reticulum (er) signal sequence located at the n terminus, which targets the s glycoprotein to the rer membrane and is removed by cellular signal peptidases in the lumen of the er (14, 15) . a single stop-transfer, membrane-spanning sequence located at the c terminus of the s protein prevents it from being fully released into the lumen of the er and subsequent secretion from the infected cell (16, 17) . co-translationally, n-linked, highmannose oligosaccharide side chains are added during synthesis (18, 19) . shortly after synthesis, the s glycoprotein monomers trimerize, which might be thought to facilitate the transport from the er to the golgi complex. once in the golgi complex, most of the high-mannose oligosaccharide side chains are modified to more complex forms (20, 21) , and o-linked oligosaccharide side chains are also added (22, 23) . in the trans-golgi network, the sars-cov-2 s glycoprotein is proteolytically cleaved by cellular furin or furin-like proteases at the s1/s2 cleavage site, comprising multiple arginine residues that are not found in the closely related sars-cov (24, 25) . cleavage at the s1/s2 site yields a surface subunit s1, which attaches the virus to the host cell surface receptor, and a transmembrane subunit s2, which mediates the fusion of viral and host cell membranes (10) . the s1 and s2 subunits remain associated through noncovalent interactions in a metastable prefusion state (11) . furin-like cleavage is essential for the sprotein mediated cell-cell fusion and viral infectivity, and is required for efficient sars-cov-2 infection of human lung cells (24) and airway epithelial cells (26) . following cleavage, an er retrieval signal (errs) consisting of a conserved kxhxx motif (27) located at the extreme c terminus ensures that the mature sars-cov-2 s protein accumulates near the er-golgi intermediate compartment (ergic) (27, 28) , where driven by interactions with another structural protein, the membrane (m) protein, the s protein participates in virus particle assembly and is incorporated into virus envelope ( figure 1 ) (29, 30) . besides, a fraction of mature sars-cov-2 s proteins travel through the secretory pathway to the plasma membrane, where they can mediate fusion of infected with uninfected cells to form multinucleated giant cells (syncytia) (24, 31) . this may allow direct spreading of the virus between cells and potentially alter the virulence of sars-cov-2 (24) . notably, a deletion of~20 amino acid containing the errs from the cytoplasmic tail of the sars-cov-2 s protein has been shown to increase the infectivity of single-cycle vesicular stomatitis virus (vsv)-s pseudotypes (9) and replicationcompetent recombinant vsvs bearing the s glycoprotein (32, 33) , which likely could be translated to single-cycle human immunodeficiency virus (hiv)-s or other retrovirus-s pseudotypes straightforward (33) . presumably, this deletion may enhance the cell surface expression of the sars-cov-2 s glycoprotein (32) , thereby facilitating the s protein incorporation into pseudovirions and replication-competent virions. as mentioned above, the sars-cov-2 s glycoprotein plays pivotal roles in viral infection and pathogenesis. mature s glycoprotein on the viral surface is a heavily glycosylated trimer, each protomer of which is composed of 1260 amino acids (residues 14-1273) ( figure 2a) . the surface subunit s1 is composed of 672 amino acids (residues 14-685) and organized into four domains: an n-terminal domain (ntd), a c-terminal domain (ctd, also known as the receptor-binding domain, rbd), and two subdomains (sd1 and sd2) ( figure 2a ) (34) . the transmembrane s2 subunit is composed of 588 amino acids (residues 686-1273) and contains an n-terminal hydrophobic fusion peptide (fp), two heptad repeats (hr1 and hr2), a transmembrane domain (tm), and a cytoplasmic tail (ct), arranged as fp-hr1-hr2-tm-ct ( figure 2a ) (34) . as a typical class i viral fusion protein (35) , the sars-cov-2 s glycoprotein shares common structural, topological and mechanistic features with other class i fusion proteins, including hiv envelope (env) glycoprotein and influenza virus haemagglutinin (ha) (36) (37) (38) . like other class i viral fusion proteins, the sars-cov-2 s glycoprotein is also a conformational machine that mediates viral entry by rearranging from a metastable unliganded state, through a prehairpin intermediate state, to a stable postfusion state (38, 39) . since the first genome sequence of sars-cov-2 became publicly available (40) , a number of structures have been determined for the sars-cov-2 s glycoprotein trimer fragments in both the prefusion and postfusion states ( figures 2b-d) (11, 34, 41) . the overall architecture of the prefusion sars-cov-2 s ectodomain stabilized by two consecutive proline mutations in two conformations determined by single particle cryo-electron microscopy (cryo-em) is a~160 å long trimer with a triangular cross-section, with the s1 subunit adopting a "v" shape contributing to the overall triangular appearance and the s2 subunit forming the stalk ( figures 2b, c) (11, 34) . the structural difference between these two conformations only lies in the position of one of the three s1 rbds ( figures 2b, c) (11) . when all three rbds are in the "down" position, the resulting s ectodomain trimer assumes a closed conformation, in which the receptor-binding surface of the s1 rbd is buried at the interface between protomers and cannot be accessible by its receptor ( figure 2b ) (11) . the s ectodomain trimer with one single rbd in the "up" position assumes a partially open conformation and represents the functional state, as the receptorbinding surface of the "up" rbd can be fully exposed ( figure 2c ) (11, 34) . the structural information provides a blueprint for structure-based design of vaccine immunogens and entry inhibitors of sars-cov-2. in the closed sars-cov-2 s ectodomain trimer, interprotomer interactions occur through the s1 ctd packed against the other two s1 ctds and one ntd from an adjacent protomer because of domain swapping and through s2, primarily between helical interactions formed by the upstream and central helices from each subunit around the trimer axis ( figure 2b ) (11) . the s1 subunits rest above the s2 trimer, the life cycle of sars-cov-2 begins with membrane fusion occurring at the plasma membrane or within acidified endosomes after endocytosis, which is mediated by conformational changes in the s glycoprotein triggered by angiotensin-converting enzyme 2 (ace2) binding. following viral entry, sars-cov-2 releases its genomic rna into the host cell cytoplasm. genome rna is first translated into viral replicase polyproteins (pp1a and 1ab), which are further cleaved by viral proteases into a total of 16 nonstructural proteins. a replication-transcription complex (rtc) is formed based on many of these nonstructural proteins. in the process of genome replication and transcription mediated by rtc, the negative-sense (− sense) genomic rna is synthesized and used as a template to produce positive-sense (+ sense) genomic rna and subgenomic rnas. the nucleocapsid (n) structural protein and viral rna are replicated, transcribed, and synthesized in the cytoplasm, whereas other viral structural proteins, including the s protein, membrane (m) protein and envelope (e) protein, are transcribed and then translated in the rough endoplasmic reticulum (rer) and transported to the golgi complex. in the rer and golgi complex, the sars-cov-2 glycoprotein is subjected to co-translational and post-translational processing, including signal peptide removal, trimerization, extensive glycosylation and subunit cleavage. the n protein is subsequently associated with the positive sense genomic rna to become a nucleoprotein complex (nucleocapsid), which together with s, m, and e proteins as well as other viral proteins, is further assembled and followed by budding into the lumen of the er-golgi intermediate compartment (ergic) to form mature virions. finally, the mature virions are released from the host cell, waiting for a new life cycle to start. this figure is adapted from the template in biorender (https://biorender.com/). stabilizing the later in the prefusion conformation ( figure 2b ) (11) . when the s ectodomain trimer adopts a partially open conformation, the rbd in the "up" position will abolish the contacts with the s2 subunit of an adjacent protomer, destabilizing the partially open conformation ( figure 2c ) (11, 34) . this will be beneficial to the dissociation of the s1 subunit and facilitate conformational rearrangements that the s2 trimer undergoes to mediate viral entry. prefusion structures of human coronavirus hku1 (hcov-hku1) and mouse hepatitis virus s protein ectodomains without two consecutive proline mutations reveal only fully closed conformation (37, 42) , similar to that observed for a full-length, wild-type prefusion form of the sars-cov-2 s glycoprotein (41) . notably, it is well established that trimeric prefusion hiv-1 env primarily resides in a closed configuration that is conformationally masked to evade antibody-mediated neutralization (43, 44) and can spontaneously sample a transient, functional configuration (45) . it can thus be speculated that native cov s glycoproteins on mature and infectious virions share a similar conformational masking feature (46) , concealing the receptor-binding surface (for those utilizing ctds as rbds) ( figure 2c ), which is further discussed below. several lines of research have established that angiotensinconverting enzyme 2 (ace2) is an entry receptor for sars-cov-2 (47) (48) (49) . detailed interactions between the sars-cov-2 rbd and its receptor ace have been revealed by several structures of ace2 in complex with rbd (50) (51) (52) (53) . structurally, rbd consists of two subdomains: a core and an external subdomain (51, 52) . an extended loop (residues 438-506), which lies on one edge of the core subdomain, presents a gently concave surface to cradle the n-terminal helix (a1) of ace2. analysis of the interface between the sars-cov-2 rbd and ace2 reveals that a total of 17 residues in rbd are in contact with 20 amino acids in ace2, forming a network of hydrophilic interactions that are suggested to predominate the virus-receptor engagement (51) . outside this extended loop, residue lys417 located in helix a3 of the core subdomain, was shown to form ionic interactions with asp30 of ace2. as the extended loop contains almost all the amino acids of the sars-cov-2 rbd that contact ace2, it is referred to as the receptor-binding motif (rbm) (51) . it has been proposed that inhibiting the interaction between rbd and ace2 might be useful in treating sars-cov-2 infection. recombinant soluble ace2 (54) and ace2-fc (55, 56) have been shown to have potential applications in the prevention and treatment of sars-cov-2 infection in vitro. as the interaction between the rbd and ace2 is extensive, small molecules probably cannot be used as entry inhibitors to effectively block the virus entry by targeting the interaction interface. however, peptides would be able to engage most of the residues belonging to rbm (57) . a pioneering study demonstrated that a 23-amino acid peptide (residues 21-43), derived from the n-terminal helix (a1) of ace2, specifically associates with the sars-cov-2 rbd with low nanomolar affinity and disables receptor interactions (57), representing a promising strategy for preventing the virus from invading human cells. in another study, a 65-amino acid peptide (residues 19-83), derived from the n-terminal back-to-back helices (a1 and 2) and composed of most of the residues of ace2 that mediate interactions with the s protein, shows a similar but probably more potent inhibitory effect (58) . the formation of a trimer-of-hairpins structure (also known as six-helix bundle) comprising hr1 and hr2 in the postfusion conformation is a unifying feature of class i viral fusion proteins (37) . the crystal structure of a protein construct in which sars-cov-2 hr1 and hr2 were connected by a six-residue hydrophilic flexible linker was determined to be a canonical six-helix bundle structure with a rod-like shape ∼115 å in length and ∼25 å in diameter (59) . three hr1 helices form a parallel central coiled-coil with three hr2 helices packing in an oblique, antiparallel manner against deep hydrophobic grooves on the surface of the central coiled-coil (59) . notably, when a full-length s protein construct bearing the native furin-like cleavage site was transiently expressed by expi293f cells, the purified s proteins contained the dissociated s2 trimer in the postfusion conformation (41) . the cryo-em structure of this trimeric postfusion s2 shows that the central helix (ch) extended regular helices from the central coiled-coil, oriented toward target cells ( figure 2d ) (41) , which forms the longest central triple helical coiled-coil (~180å) among all known class i transmembrane subunit structures. the sars-cov-2 s trimer in the pre-hairpin intermediate state is very unstable and is just transiently present in vivo after triggering by ace2 engagement, stymieing structural characterization of the s protein in this state (60) . however, although this fusion-intermediate phase is very short, it is enough for inhibitory peptides to associate with the prehairpin intermediate and block the six-helix bundle formation (39) . furthermore, it has already been shown that the hr1 regions in various human covs are highly conserved (61) , and therefore could serve as an attractive target for the design and development of potent and broad-spectrum inhibitors of pan-covs, including sars-cov-2. a highly potent pan-coronavirus fusion inhibitor, ek1c4, has been reported to have good prophylactic and therapeutic potential against sars-cov-2 infection (59). as mentioned earlier, the sars-cov-2 s proteins are heavily decorated by heterogeneous n-linked glycans projecting from the s trimer surface. the sars-cov-2 s sequence encodes up to 22 n-linked glycan sequons per protomer, which likely plays an important role in protein folding (19) and host immune evasion as a glycan shield (62) . of the 22 potential n-linked glycosylation sites on the s protein, 14 were identified to be predominantly occupied by processed, complex-type glycans (63) . the remaining eight sites were found to be dominated by oligomannose-type glycans, which are divergent from those founded on host glycoproteins (63) . although glycosylation sites (n165, n234, n343) proximal to the receptor-binding sites on the sars-cov-2 s protein can be observed, ace2 bound to the glycosylated and deglycosylated s ectodomains with nearly identical affinity (1.7 nm vs 1.5 nm) determined by a biolayer interferometry binding assay (64) . this observation suggests that the high binding affinity between the sars-cov-2 s protein and ace2 does not depend on the s protein glycosylation. when the site-specific n-linked glycans are mapped onto the prefusion structure of the sars-cov-2 s ectodomain (63), the resulting model exhibited substantially higher levels of glycanfree surface than that revealed by structures of fully glycosylated, trimeric hiv-1 env ectodomains (65, 66) . this suggests that the sars-cov-2 s protein is covered by a less dense and less effective glycan shield compared to viral glycoproteins from hiv-1 (36, 66) and lassa virus (67) , which may be beneficial for the induction of humoral immunity and could be good news for a sars-cov-2 vaccine (68) . notably, it has been shown that multiple major viral surface antigens have neutralizing epitopes that are partly or even exclusively composed of carbohydrate moieties (69, 70) , exemplified by the hiv-1 env spike, which could be recognized by a large number of carbohydrate-binding antibodies, including 2g12, pg9, pg16, ch04, pgt121, pgt128, pgt135, and pgt145 (70, 71) . in the case of sars-cov-2, more recently a potent neutralizing antibody against both sars-cov and sars-cov-2, s309, has been shown to recognize a highly conserved glycan-containing rbd epitope (72) . these observations suggest that carbohydrate moieties could be immunogenic and highlight the need for immunogens to display the glycans important for the recognition of neutralizing antibodies (73) ; in support of this, specific n-linked glycans on hemagglutinin has been shown to be essential for the elicitation of broadly neutralizing antibodies against influenza (74) . accordingly, there has been mounting interest in exploring the potential of immunogenic glycan moieties as vaccine candidates against multiple viruses, including sars-cov-2 (75, 76) . membrane fusion and viral entry of sars-cov-2 is initiated by binding of rbd in the viral s glycoprotein transiently sampling the functional conformation to ace2 on the surface of target cells (figure 1 ) (10). after receptor engagement at the plasma membrane or ensuing virus endocytosis by the host cell (8), a second cleavage (s2′ cleavage site) is generated, which is mediated by a cellular serine protease tmprss2 (48) or endosomal cysteine proteases cathepsins b and l (10) (figure 1) . protease cleavage at s2′ site frees the fusion peptide from the new s2 n-terminal region, further destabilizes the sars-cov-2 s glycoprotein and may initiate s2-mediated membrane fusion cascade. following the second cleavage, the fusion peptide at the n terminus of the s2 trimer is inserted into the host membrane (8) , forming the pre-hairpin intermediate state (39) . since the pre-hairpin intermediate state is extremely unstable, the s2 fusion protein is refolded quickly and irreversibly into the stable postfusion state (39, 77) . these large conformational rearrangements pull the viral and host cell membrane into close proximity, leading ultimately to the membrane fusion (8, 39) . since sars-cov-2 was identified as the causative agent of covid-19, and its first genome sequence was released immediately and freely by a chinese research group (40), sars-cov-2 vaccine candidates based on various vaccine platforms, such as inactivated or live attenuated vaccines, dna and mrna vaccines, viral vector-based vaccines, and recombinant protein-based vaccines, have been developed (12, 78) . most of these vaccine strategies are based on the full-length s glycoprotein, the major viral surface antigen (12) . when a vaccine strategy requires that the sars-cov-2 s protein be recombinantly expressed in the human body, the errs should be omitted to enhance the cell surface expression level of the resulting protein. theoretically, the native hiv-1 env trimer present on the surface of intact virions is thought to be a most ideal immunogen (60) , as most of the neutralizing antibodies thus far described could recognize and bind to the prefusion form of trimeric hiv-1 env, although it is with great difficulty that such neutralizing antibodies against this glycan-covered, sequence-variable native form are induced (36) . for sars-cov-2, different lines of research have shown that convalescent sera from sars-cov and sars-cov-2 patients showed no or limited crossneutralization activity against these two viruses by pseudotyped and authentic viral infection assays, despite significant crossreactivity in binding to the s glycoproteins of both viruses (9, (79) (80) (81) . similar results were also observed in infected or immunized animals (48, 79, 81) . together with the finding that although the sars-cov-2 s protein shares a high degree of amino acid sequence identity with that of sars-cov (~76% overall), the rbm is less conserved (~47% identity) than any other functional region or domain (82) , it can thus been surmised that the rbm has the most immunodominant neutralizing epitope(s) of the whole s protein, capable of readily eliciting strong neutralizing antibody responses. however, the native trimeric sars-cov-2 s protein could conceal each of its immunodominant rbms by adopting the closed conformation (41, 83) . therefore, sars-cov-2 evades immune surveillance also through conformational masking, which is well-documented for hiv-1 (43, 44) ; while at the same time, the s protein could transiently sample the functional state to engage ace2, consistent with the notion that the fusion glycoprotein of highly pathogenic viruses have evolved to perform its functions while evading host neutralizing antibody responses. another concern for vaccine candidates based on the fulllength s glycoprotein of sars-cov-2 is raised by the observation that the s1 subunit could spontaneously dissociate from the s glycoprotein probably as a trimer that still assumes the rbd closed conformation, leaving only the postfusion s2 trimer (41) . the resulting s1 and s2 subunits might expose immunodominant, nonneutralizing epitopes that are utilized by sars-cov-2 to serve as decoys to distract the host immune system, inducing a large proportion of ineffective antibody responses, as documented for hiv-1 (60) and respiratory syncytial virus (rsv) (84) . it should be noted that although vaccine candidates based on the full-length s protein of the closely related sars-cov could elicit neutralizing antibody responses against infection of sars-cov, they may also induce harmful immune responses, including liver damage of the vaccinated animals, infection of human immune cells by sars-cov, and antibody-dependent enhancement of sars-cov infection (85) (86) (87) (88) (89) . therefore, although the s proteins of both sars-cov and sars-cov-2 are thought to be promising vaccine immunogens for generating protective immunity, optimizing antigen design is critical to ensure an optimal immune response through exposing more neutralizing epitopes and displaying fewer potentially weakly or non-neutralizing epitopes (90) . vaccines containing or expressing the full-length s protein or its soluble ectodomain form should thus be engineered to sample a rbd(s) "up" conformation while the rest is still kept in the prefusion state (91, 92) . apart from recombinant, soluble, stabilized ectodomains that are engineered to expose the immunodominant rbd by adapting the rbd(s) "up" conformation, rbd proteins of sars-cov and sars-cov-2 have also been widely used as recombinant protein-based vaccines (85, (93) (94) (95) . the rbd of sars-cov is highly immunogenic (96, 97) and is targeted by most of the neutralizing monoclonal antibodies that have been characterized (98) . based on the observation that a 193-amino acid fragment (residues 318-510) was previously identified to be the minimal rbd region of sars-cov (99), a corresponding 194-amino acid fragment (residues 331-524) can be readily selected as the minimal rbd region of sars-cov-2 and has already been characterized (100) . this minimal form of rbds of both viruses could serve as a vaccine candidate (100) . however, a conserved cysteine residue is located immediately upstream of the minimal rbd fragments of both viruses and always forms a disulfide bond in nearly all published structures containing this residue (101, 102) ; this is also the case for middle east respiratory syndrome coronavirus (mers-cov) (103, 104) and hcov-hku1 (37), consistent with the observation that all rbds of these viruses share a conserved structural core. the disulfide bond contributes to stabilization of the rbd structure and likely modulates the protein immunogenicity. this notion is consistent with the observation that mice immunized with a longer form of the sars-cov rbd (residues 318-536) produced a higher titer of neutralizing antibodies compared with mice immunized with the minimal rbd region (residues 318-510) (105) . therefore, when each of the minimal rbd fragments of sars-cov and sars-cov-2 is used as vaccine candidates, the critical cysteine residue should not be ignored and thus should be included (106) . besides the rbd, which has been shown to a major target for human neutralizing antibody responses (107), the ntd was recently identified to be a new vulnerable site of the sars-cov-2 s protein for antibody neutralizing and therefore could also serve as a recombinant protein-based vaccine (108) (109) (110) . as expected, ntd-specific neutralizing antibodies could target the s protein in both closed and open conformations (108) . in addition, the apparent accessibility of the fusion peptide and hr1 region in published structures of the sars-cov-2 s ectodomain trimer as well as their high sequence conservation among covs suggests that they would be good immunogen candidates for epitope-focused vaccine design aimed at raising broadly cov neutralizing antibodies (46) . the epitope-focused vaccine design has proven to be successful in generating neutralizing antibodies against rsv fusion glycoprotein (111). however, neutralizing antibodies targeted against these two regions still need to be isolated in infected individuals to support this notion. unlike wild-type full-length s protein of sars-cov-2, the above monomeric fragments do not induce any infection-enhancing antibodies or harmful immune or inflammatory responses (106, 112) , all of which could be potentially avoided through structure-based immunogen design to improve immunogenicity (113, 114) . however, wide-type full-length or soluble ectodomain form of the sars-cov-2 s protein could trigger stronger cellular immune responses (115) , which have been demonstrated to play an important role in controlling diseases caused by covs (116, 117) , including sars-cov-2 (118) , and are probably also an important determinant of effective vaccines against sars-cov-2 (115, 119) . additionally, when more than one rbd of the s protein trimer is engineered to be locked in the "up" conformation (120, 121) , the antigenicity and immunogenicity of the resulting rbds would be significantly enhanced compared to monomeric rbd form (97, 122) . moreover, improved protection is likely to be achieved when vaccinated with full-length or soluble ectodomain form of the sars-cov-2 s protein in that both forms can elicit neutralizing antibodies directed against non-rbd sites, as observed for mers-cov (123) . genetic variation has been used by many viruses that have rna genomes (124) , including hiv and influenza, as a mechanism to avoid antibody-mediated immunity, and is partially responsible for the great difficulty in developing effective and durable vaccines against these viruses (36) . as an rna virus, however, sars-cov-2 has a very low mutation rate overall (125) likely because covs have a genetic proofreading mechanism (126) . all reported variations occurred in the sars-cov-2 s glycoprotein have a prevalence of no more than 1% (127) , with an exception of d614g, which has become the most prevalent genotype in the global covid-19 pandemic (127) . fortunately, although the d614g mutation of the sars-cov-2 s protein has been shown to enhance viral infectivity (128) (129) (130) , until now there is no evidence that infection with sars-cov-2 carrying the g614 mutant will be associated with disease severity (127, 131) . furthermore, assays using both monoclonal and polyclonal antibodies generated from individuals naturally infected with d614-or g614-carrying viruses demonstrated that the d614g mutation retains or even increases viral susceptibility to neutralization (127, 130, 132, 133) . this suggests that the d614g mutant maintains or favors an open, functional conformational state (134) . although at an extremely low frequency, natural variations, including l452r a475v, v483a, and f490l that render the s glycoprotein resistant to certain neutralizing antibodies targeting the rbd, emerged under no selection pressure exerted by approved vaccines or neutralizing antibodies or entry inhibitors (127, 132) . however, it has been shown that sars-cov-2 escape mutants could be easily selected and quickly amplified under the selection pressure of single antibody treatment (135) . these observations suggest that a combination of at least two neutralizing antibodies that recognize and bind to distinct and non-overlapping epitopes on the sars-cov-2 s glycoprotein (e.g., rbd and ntd, as well as hr and glycan) is required to restrict the possible occurrence of viral escape mutants and potential subsequent loss of single antibody-mediated neutralization (135) (136) (137) (138) . when these observations are taken into consideration for vaccine design and development, an ideal sars-cov-2 immunogen should contain as many exposed neutralizing epitopes as possible, although the rbd also possesses extra epitope(s) besides the epitope in the rbm region (72, (139) (140) (141) . sars-cov-2 is a highly contagious pathogen that continues to spread quickly around the globe, causing covid-19 to be one of the worst pandemics in recorded history. a safe and efficacious vaccine represents one of the best ways to reduce or eliminate the covid-19 pandemic (142) . unfortunately, no vaccines for any of the known human covs have been licensed (143, 144) , although several potential sars-cov and mers-cov vaccines have advanced into human clinical trials for years (117, 145) , suggesting the development of effective vaccines against human covs has always been challenging. however, it has been shown that both sars-cov and sars-cov-2 could readily induce neutralizing antibodies following natural infection or immunization (146) (147) (148) (149) . moreover, a growing number of neutralizing monoclonal antibodies targeting the sars-cov-2 s glycoprotein with high potency have been isolated from plenty of convalescent donors (33) as well as 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neutralization. medrxiv the sars-cov-2 spike variant d614g favors an open conformational state antibody cocktail to sars-cov-2 spike protein prevents rapid mutational escape seen with individual antibodies studies in humanized mice and convalescent humans yield a sars-cov-2 antibody cocktail neutralizing antibodies against sars-cov-2 and other human coronaviruses perspectives on the development of neutralizing antibodies against sars-cov-2 a highly conserved cryptic epitope in the receptor binding domains of sars-cov-2 and sars-cov structural basis for neutralization of sars-cov-2 and sars-cov by a potent therapeutic antibody a human monoclonal antibody blocking sars-cov-2 infection updated approaches against sars-cov-2 vaccines for covid-19: perspectives, prospects, and challenges based on candidate sars, mers, and animal coronavirus vaccines antibodies and vaccines against middle east respiratory syndrome coronavirus vaccines against coronaviruses: the state of the art. vaccines (basel) (2020) 8:309 immunogenic profile of sars-cov-2 spike in individuals recovered from covid-19. medrxiv immunogenicity of a dna vaccine candidate for covid-19 dna vaccine protection against sars-cov-2 in rhesus macaques antibody signature induced by sars-cov-2 spike protein immunogens in rabbits progress and prospects on vaccine development against sars-cov-2. vaccines (basel) (2020) 8:153 what are the most powerful immunogen design vaccine strategies? reverse vaccinology 2.0 shows great promise structure-based vaccine antigen design all authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication. we would like to thank prof. xinqi liu for critical reading of the manuscript; and drs. yanbin feng, mengyuan xu, jing ma and jianrong feng for helpful comments and discussions on the manuscript. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 duan, zheng, zhang, niu, lou and wang. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-354868-pqn59ojj authors: yao, hebang; cai, hongmin; li, tingting; zhou, bingjie; qin, wenming; lavillette, dimitri; li, dianfan title: a high-affinity rbd-targeting nanobody improves fusion partner’s potency against sars-cov-2 date: 2020-09-25 journal: biorxiv doi: 10.1101/2020.09.24.312595 sha: doc_id: 354868 cord_uid: pqn59ojj a key step to the sars-cov-2 infection is the attachment of its spike receptor-binding domain (s rbd) to the host receptor ace2. considerable research have been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (rbm) and to block ace2-rbd binding. simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. here, we identify and characterize a high-affinity synthetic nanobody (sybody, sr31) as a fusion partner to improve the potency of rbm-antibodies. crystallographic studies reveal that sr31 binds to rbd at a conserved and ‘greasy’ site distal to rbm. although sr31 distorts rbd at the interface, it does not perturb the rbm conformation, hence displaying no neutralizing activities itself. however, fusing sr31 to two modestly neutralizing sybodies dramatically increases their affinity for rbd and neutralization activity against sars-cov-2 pseudovirus. our work presents a tool protein and an efficient strategy to improve nanobody potency. sars-cov-2, the pathogenic virus for covid-19, has caused a global pandemic since its first report in early december 2019 in wuhan china (1), posing a gravely crisis for health and economic and social order. sars-cov-2 is heavily decorated by its surface spike (s) (2, 3) , a single-pass membrane protein that is key for the host-virus interactions. during the infection, s is cleaved by host proteases (4, 5) , yielding the nterminal s1 and the c-terminal s2 subunit. s1 binds to angiotensin-converting enzyme 45 2 (ace2) (6-10) on the host cell membrane via its receptor-binding domain (rbd), causing conformational changes that trigger a secondary cleavage needed for the s2mediated membrane fusion at the plasma membrane or in the endosome. because of this essential role, rbd has been a hot spot for the development of therapeutic monoclonal antibodies (mabs) and vaccine (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) . 50 llama-derived heavy chain-only antibodies (nanobodies) are attractive biotherapeutics (29) . these small (~14 kda) proteins are robust, straightforward to produce, and amenable to engineering such as mutation and fusion. owing to their ultra-stability, nanobodies have been reported to survive nebulization, a feature that has been explored 55 for the development of inhaled nanobodies to treat respiratory viral diseases (30, 31) which categorizes covid-19. owing to their high sequence similarities with human type 3 vh domains (vh3), nanobodies are known to cause little immunogenicity (29) . for the same reason, they can be humanized with relative ease to reduce immunogenicity when needed. therefore, nanobodies as biotherapeutics are being 60 increasingly recognized. examples of nanobody drugs include caplacizumab (32) for the treatment of acquired thrombotic thrombocytopenic purpura, and ozoralizumab and vobarilizumab that are in the clinical trials for rheumatoid arthritis (29, 33) . recently, several groups have independently reported neutralizing nanobodies (22, (34) (35) (36) (37) (38) (39) or single-chain vh antibodies (40) against sars-cov-2 with variable potencies. 65 we have recently reported several synthetic nanobodies (sybodies) which bind rbd with various affinity and neutralizing activity (35) . affinity and neutralizing activity are very important characteristics for therapeutic antibodies, and they can be improved by a number of ways such as random mutagenesis (22, 36) and structure-70 based design. previously, in the case of one modestly-neutralizing sybody mr17, we have determined its structure and designed a single mutant that improves its potency by over 23 folds (35) . the rational design approach, while very effective, inevitably requires high-resolution structural information which are non-trivial to obtain. generally applicable tools will be welcome. here, we report a strategy to increase sybody potency by biparatopic fusion with sr31, a sybody that binds rbd tightly with a kd of 5.6 nm. as revealed by crystal structure, sr31 engages the rbd at a conserved site that is distal to the rbm. as such, it does not neutralize sars-cov-2 but forms non-competing pairs with several other rbm-binders and increases their neutralization potency when conjugated. sr31 may 80 be used as a general affinity-enhancer for both detection and therapeutic applications. a high-affinity rbd binder without neutralizing activity 85 previously, we generated 99 sybodies from three highly diverse synthetic libraries by ribosome and phage display with in vitro selections against the sars-cov-2 rbd. most of the sybody binders showed neutralizing activity. interestingly, about 10 sybodies bind rbd but showed no neutralizing activities (35) even at 1 m concentration. one such sybodies, named sr31, was characterized in this study. in analytic fluorescence-detection size exclusion chromatography (fsec), sr31 caused earlier retention of rbd (fig. 1a) which was included at a low concentration (0.5 m), suggesting nanomolar affinity for sr31-rbd binding. this was confirmed by bio-layer 95 interferometry analysis (fig. 1b) which showed a kd of 5.6 nm and an off-rate of 1 × 10 -3 s -1 . consistent with its inability to neutralize sars-cov-2 pseudovirus, sr31 did not affect rbd-ace2 binding (fig. 1c) . to characterize the sr31-rbd interactions in detail, we purified the complex (fig. 1d ), and obtained crystals (fig. 1d ) that diffracted to 1.97 å resolution ( table 1) . the structure was solved by molecular replacement using the published rbd and sybody 110 structures (pdb ids 6m0j and 5m13) (6, 41) as search models. the structure was refined to rwork/rfree of 0.182/0.207 ( table 1) . the asymmetric unit contained one molecule each for the rbd and sr31, indicating an expected 1:1 stoichiometry. sr31 binds to the rbd sideways at a buried surface area of 1,386.3 å 2 ( fig. 2a) , which is significantly larger than that for the previously reported sybodies sr4 (727.4 å 2 ) and mr17 (853.944 å 2 ) (35) . the binding surface is near a heavily decorated glycosylation site, asn343 ( fig. 2a-2c) , which, although at an apparent strategic 130 position to possibly divide the accessible surfaces for immune surveillance, does not show clashes with sr31. all three cdrs participated in the interaction by providing five (cdr1), three (cdr2), and nine h-bonds (cdr3) (fig. 2e-2g) . peculiarly, the cdr3, which contains a cluster of hydrophobic side chains that include met99, val100, phe102, trp103, and tyr104, inserted into a greasy pocket (fig. 2b ) in the rbd that 135 was lined with twelve hydrophobic/aromatic residues (fig. 2f) . unlike salt bridges, hydrophobic interactions are more tolerant to environment such as change of ph and ionic strength. in addition, they are less specific and thus less likely to be affected by mutations. this binding mode thus makes sr31 an attractive candidate for detection purposes. most rbd-targeting neutralizing antibodies, including neutralizing nanobodies characterized so far (8, 13-15, 19, 20, 22-24, 26-28, 34, 35, 37) , engage the rbd at the receptor-binding motif (rbm) (fig. 3a) , thus competing off ace2 and preventing viral entry. aligning the ace2 structure to the sr31-rbd structure showed that the sr31-145 binding epitope is distant from the rbm (fig. 3a) . comparing the epitopes of existing monoclonal antibodies showed that the sr31 epitope partly overlaps with cr3022 (12) and the recently identified ey6a (22) (fig. 3b, 3c ). it has been established that the binding of the bulky cr3022 and ey6a at the interface between rbd and the n. taken together, the structural data rationalize the high-affinity binding between sr31 and rbd, and its inability to neutralize sars-cov-2. because nanobodies are relatively easy to produce, the availability of nanobodies that recognize a wide spectrum of epitopes can be a useful toolkit to probe binding 160 mode of uncharacterized antibodies using competitive binding assays. they may also be used to select binders with new epitopes by including them as pre-formed sybody-rbd complexes during in vitro selection (and thus excluding binders at the same site). other rbd-targeting nanobodies (22, 35, 36, 39) and mabs (13-15, 19, 20, 23, 24, 26-28) . red, the collective epitope of rbm-binders; blue, the sr31 epitope; magenta, the collective epitope of cr3022 and ey6a; orange, the overlap between the structure alignment of sr31-rbd with ace2-rbd revealed that the two rbd structures were overall very similar with a c rmsd of 0.452 å (fig. 4a) . nevertheless, significant structural rearrangements at the binding interface were observed (fig. 4a, 4b) . specifically, the small -helix 364-370 (numbers mark start-end) 185 moves towards the direction of rbm by a dramatic ~8.0 å and transforms to a short sheet (367-370) which in turn forms a parallel -sheet pair with 102-104 in the cdr3 region. in addition, nudged by the cdr1, the short helix 383-388 swings towards the rbd core by ~4.0 å. remarkable, the dramatic rearrangements did not cause noticeable conformational change of rbm (fig. 4a) nor did it affect ace2 binding (fig. 1c) . given that rbd is 210 a relatively small entity, and that the two surfaces are relatively close (~25 å), this was somewhat unexpected. a probable explanation is that rbd is very rigid and hence stable. indeed, as shown in fig. 4c , rbd showed ultra-stability, with an apparent melting temperature of greater than 95 º c (20-min heating). intriguingly, the rearrangement happens at a region that is rich in disulfide bonds. specifically, 367-370 is tethered between the disulfide pairs cys379-cys432 and cys336-cys361, and 383-388 bridges cys379-cys432 and cys-391-cys525 (fig. 4d) . thus, the three disulfide bonds segregate the two local motifs from the rest of rbd, preventing these conformational changes from propagating through the domain. the neutral feature of sr31 so far suggests it could bind to rbd in addition to rbm binders such as mr17 and sr4 (35) . indeed, bli assays showed no competition 225 between sr31 and mr17 (fig. 5a) , indicating a 'sandwich complex' where the rbd is bound with both sybodies. this non-competing feature was also observed in the case of mr6 (fig. 5b) which has also been shown to have neutralizing activities (35) . as a further proof for the simultaneous binding, we determined the structure of the sandwich complex sr31-rbd-mr17 (fig. 5c, table 1 ) to 2.10 å resolution. the sandwich 230 complex was similar to the individual mr17-and sr31-rbd complexes, with an overall c rmsd of 0.667 and 0.375 å, respectively. aligning the sandwich complex with the mr17-rbd structure revealed no noticeable changes at the mr17-binding surface (fig. 5c) , reinforcing the idea that sr31-binding does not allosterically change the rbm surface nor affect rbm binders. to the two-component complex structure (rbd (green) and mr17, pdb id 7c8w) (35) . although sr31 does not neutralize sars-cov-2 pseudovirus itself, its highaffinity may help increase the affinity of other neutralizing nanobodies through avidity 250 effect by fusion. indeed, the biparatopic fusion sr31-mr17 displayed remarkable increase in binding affinity compared to sr31 or mr17 alone. its kd of 0.3 nm (fig. 6a ) was lower than mr17 (kd = 83.7 nm) (35) by 230 folds and lower than sr31 (kd = 5.6 nm) by 17 folds. consistently, sr31-mr17 neutralized sars-cov-2 pseudovirus 13 times more effectively (in molarity) than mr17 alone (fig. 6b) . 255 that sr31 can enhance potency of its fusion partner was also demonstrated in the case for mr6. at its free form, mr6 bound to rbd with a kd of 23.2 nm (fig. 6c) , and showed modest neutralizing activity with an ic50 of 1.32 g ml -1 (77.5 nm). fusing it to sr31 increased its affinity by over 40 folds, displaying a kd of 0.5 nm (fig. 6d) . 260 in line with this, sr31-mr6 showed a 27-fold higher neutralization activity compared to mr6, with an ic50 of 2.7 nm (0.08 g ml -1 ) (fig. 6e) . interestingly, when fused to mr3, a neutralizing antibody that had higher affinity (kd = 1.0 nm) than sr31, the neutralizing activity decreased by 2 folds (fig. 6f) . possible reasons include steric incompatibility caused by improper link length, and allosteric effects. such hypothesis 265 warrants future structural investigation. binding affinity and neutralizing activity are important characteristics of therapeutic antibodies. for modestly neutralizing nanobodies, the potency can be increased in a number of ways, including random mutagenesis (22) , structure-based 280 design (35) , and fusion (35, 36, 42) . compared with the other two approaches, the fusion technique is more rapid, less involving and does not rely on prior structural information. depending on whether the two fusion partners are the same, divalent nanobodies 285 can be categorized into two types: monoparatopic and biparatopic. biparatopic fusions recognize two distinct epitopes on the same target. therefore, they are more likely to be resistant to escape mutants because simultaneous mutations at two epitopes should occur at a much lower rate than at a single epitope. because of the minute size, sr31 could be used as an 'add-on' to monoclonal antibodies, scfv fragments, and other nanobodies to enhance their affinity and potency, especially for those with modest neutralizing activities. in addition, due to its small size and high stability, sr31 may be chemically modified as a vector to deliver smallmolecule inhibitors specifically targeting sars-cov-2. in summary, we have structurally characterized sr31, a high-affinity nanobody against sars-cov-2 rbd. although lacking neutralizing activity alone, sr31 is an attractive biparatopic partner for rbm-binders owing to its distinct epitope from rbm. our work presents a generally useful strategy and offers a simple and fast approach to 300 enhance potency of modestly active antibodies against sars-cov2. the authors claim no conflict of interest. sars-cov-2 rbd was expressed essentially as described (35) . briefly, a dna 330 fragment encoding, from n-to c-terminus, residues 330-541 of sars-cov2 s, a gly-thr linker, the 3c protease site (levlfqgp), a gly-ser linker, the avi tag (glndifeaqkiewhe), a ser-gly linker, and a deca-his tag were cloned into the pfastbac-based vector. baculovirus was generated in sf9 cells following the invitrogen bac-to-bac transfection protocol. high five insect cells were infected with p3 virus. for crystallization, sr31 or sr31-mr17 was mixed with rbd at a 1:1.5molar ratio. the mixture was then loaded onto a superdex 200 column for gel filtration. fractions containing the complex were pooled and concentrated to 10 mg ml -1 . to screen rbd binders by size exclusion chromatography (sec) using unpurified sybodies, rbd was fluorescently labelled as follows. first the avi-tagged rbd was for 200-300 s, before moving into sybody-free buffer for dissociation. bli signal was monitored during the whole process. data were fitted with a 1:1 stoichiometry using the build-in software analysis 10.0 for kinetic parameters. for competitive assay of the 390 rbd between sr31 and ace2, the rbd-coated sensor was saturated in 200 nm of sr31, before soaked in 25 nm sr31 with or without 25 nm of ace2. as a control, bli assays were also carried out by soaking the rbd-coated sensor in ace2 without sr31. for competitive rbd-binding assays for different sybodies, the assays were carried out the same manner as described above. desired crystals were cryo-protected, harvested using a mitegen loop under a microscope, and flash-cooled in liquid nitrogen before diffraction. x-ray diffraction data were collected at beamline bl19u1 (44) at shanghai synchrotron radiation facility with a 50 x 50 μm beam on a pilatus 6m detector, with 440 oscillation of 0.5° and a wavelength of 0.97853 å. data were integrated using the software xds (45) , and scaled and merged using aimless (46) . the sr31-rbd structure was solved by molecular replacement using phaser (47) with pdb ids 6m0j and 5m13 (41) as the search model. the sr31-mr17-rbd structure was solved using the sr31-rbd and mr17 structure (35) as search models. the models were manually 445 adjusted as guided by the 2fo-fc maps in coot (48) , and refined using phenix (49) . structures were visualized using pymol (50). the structure factors and coordinates were deposited in the protein data bank (pdb) under accession codes 7d2z (sr31+rbd) and 7d30 (sr31-mr17+rbd). a novel coronavirus outbreak of global health concern cryo-em structure of the 2019-ncov spike in the prefusion conformation structure, function, and antigenicity of the sars-cov-2 spike glycoprotein cell entry mechanisms of sars-cov-2 sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor structural basis of receptor recognition by sars-cov-2 structural and functional basis of sars-cov-2 entry by using human ace2 structural basis for the recognition of sars-cov-2 by full-length human ace2 conformational dynamics of sars-cov-2 trimeric spike glycoprotein in complex with receptor ace2 revealed by cryo-em. biorxiv structural basis for the neutralization of sars-cov-2 by an antibody from a convalescent patient a highly conserved cryptic epitope in the receptor binding domains of sars-480 a noncompeting pair of human neutralizing antibodies block covid-19 virus binding to its receptor ace2 a human neutralizing antibody targets the receptor-binding site of sars-cov-2 isolation of potent sars-cov-2 neutralizing antibodies and protection from disease in a small animal model convergent antibody responses to sars-cov-2 in convalescent individuals the receptor-binding domain of the viral spike protein is an 490 immunodominant and highly specific target of antibodies in sars-cov-2 patients cross-neutralization of sars-cov-2 by a human monoclonal sars-cov antibody potent neutralizing antibodies against multiple epitopes on sars-cov-2 spike. microbe neutralizing nanobodies bind sars-cov-2 spike rbd and block interaction with ace2 studies in humanized mice and convalescent humans yield a sars-cov-2 antibody cocktail potent neutralizing antibodies against sars-cov-2 identified by high-throughput single-cell sequencing of convalescent patients' b cells a potent neutralizing human antibody reveals the n-terminal domain of the spike protein of sars-cov-2 as a site of vulnerability. biorxiv potent neutralizing antibodies from covid-19 patients define multiple targets of vulnerability structures of human antibodies bound to sars-cov-2 spike reveal 510 structural basis for potent neutralization of sars-cov-2 and role of antibody affinity maturation. biorxiv : the preprint server for biology the therapeutic potential of nanobodies delivery of alx-0171 by inhalation greatly reduces respiratory syncytial virus disease in newborn lambs nanobodies® as inhaled biotherapeutics for lung diseases caplacizumab treatment for acquired thrombotic thrombocytopenic purpura emerging therapies in rheumatoid arthritis: focus on monoclonal antibodies. f1000res 8, f1000 faculty rev-1549 structural basis for potent neutralization of betacoronaviruses by single-525 potent synthetic nanobodies against sars-cov-2 and molecular basis for neutralization. biorxiv an ultra-high affinity synthetic nanobody blocks sars-cov-2 infection by locking spike into an inactive conformation. biorxiv an alpaca nanobody neutralizes sars-cov-2 by blocking receptor interaction synthetic nanobodies targeting the sars-cov-2 receptor-binding domain. biorxiv selection, biophysical and structural analysis of synthetic nanobodies that 535 effectively neutralize sars-cov-2. biorxiv identification of human single-domain antibodies against sars-cov-2 synthetic single domain antibodies for the conformational trapping of membrane proteins generation of synthetic nanobodies against delicate proteins the protein complex crystallography beamline (bl19u1) at the shanghai 545 synchrotron radiation facility how good are my data and what is the resolution? phaser crystallographic software features and development of coot phenix: a comprehensive python-based system for macromolecular structure solution key: cord-332948-h297ukuu authors: olotu, fisayo a.; omolabi, kehinde f.; soliman, mahmoud e.s. title: leaving no stone unturned: allosteric targeting of sars-cov-2 spike protein at putative druggable sites disrupts human angiotensin-converting enzyme interactions at the receptor binding domain. date: 2020-10-16 journal: inform med unlocked doi: 10.1016/j.imu.2020.100451 sha: doc_id: 332948 cord_uid: h297ukuu the systematic entry of sars-cov-2 into host cells, as mediated by its spike (s) protein, is highly essential for pathogenicity in humans. hence, targeting the viral entry mechanisms remains a major strategy for covid-19 treatment. although recent efforts have focused on the direct inhibition of s-protein receptor-binding domain (rbd) interactions with human angiotensin-converting enzyme 2 (hace2), allosteric targeting remains an unexplored possibility. therefore, in this study, for the first time, we employed an integrative meta-analytical approach to investigate the allosteric inhibitory mechanisms of sars-cov-2 s-protein and its association with hace2. findings revealed two druggable sites (sites 1 and 2) located at the n-terminal domain (ntd) and s2 regions of the protein. two high-affinity binders; zinc3939013 (fosaprepitant – site 1) and zinc27990463 (lomitapide – site 2) were discovered via site-directed high-throughput screening against a library of ∼1500 fda approved drugs. interestingly, we observed that allosteric binding of both compounds perturbed the prefusion s-protein conformations, which in turn, resulted in unprecedented hace2 displacement from the rbd. estimated δg(binds) for both compounds were highly favorable due to high-affinity interactions at the target sites. in addition, site 1 residues; r190, h207, k206 and k187, i101, r102, i119, f192, l226, v126 and w104 were identified for their crucial involvement in the binding and stability of zinc3939013. likewise, energy contributions of q957, n953, q954, l303, y313, q314, l858, v952, n953, and a956 corroborated their importance to zinc27990463 binding at the predicted site 2. we believe these findings would pave way for the structure-based discovery of allosteric sars-cov-2 s-protein inhibitors for covid-19 treatment. the novel coronavirus disease also referred to as covid-19 is caused by the sars-cov-2 (severe acute respiratory syndrome coronavirus 2), with incidences first reported in wuhan china in december 2019. 1 this disease has, however, persisted till mid-2020, spreading across 212 countries with over 3,513,507 cases reported coupled with increasingly high casualties numbering over 245,544 globally. 2 sars-cov-2 belongs to a large group of coronaviruses which are known to cause respiratory infections and related complications. these rna viruses are spherical, pleomorphic, positive-sensed, single-stranded and polyadenylated. 3 of all known viruses, coronaviruses (covs) have the largest rna genome 4 , with diverse pathogenic effects in animals and humans. this virus class is divided into four genera namely: alpha-cov, beta-cov, gamma-cov and delta cov [5] [6] [7] , with the beta-cov class prominent for their disease-causing effects in humans (hcovs). seven hcovs have been characterized to date [6] [7] [8] ; among which four (hcov-hku1, hcov-oc43, hcov-nl63 and hcov-229e) cause very mild respiratory symptoms. 9, 10 on the other hand, mers-cov, sars-cov, and sars-cov-2 cause severe respiratory and gastrointestinal infections which, in most cases, can be fatal. 11 although sars-cov-related infections were zoonotically transmitted into human populations, 12, 13 human to human transmissions has further contributed towards viral super-spread via respiratory aerosols. 14 the entry of sars-cov-2 coupled with its replication process in target human cells is achieved by the functionalities of a cohort of components, majorly non-structural and structural proteins, that make up the virus. generally, about 16 non-structural proteins (nsps) mediate diverse pro-pathogenic functions such as replication, processing and proof-reading of genomic frames, host immune evasion among many others, as previously reported. [15] [16] [17] more so, covs comprises of four major structural proteins that are integral to their pathogenesis. [18] [19] [20] these are the nucleocapsid (n), envelope (e), membrane (m) and spike (s) proteins. the n protein makes up the nucleocapsid and other viral genome-related processes 21 while the m protein is the most abundant of the four, playing major roles in maintaining viral structural integrity as well as coordinating other structural proteins. 22 e protein, on the other hand, is crucial to the maturation of the virus [23] [24] [25] [26] [27] while the trimeric s protein mediates viral entry into the host cell via the endosomal or non-endosomal route. 28 two domains make up the s protein namely the n-terminal s1 domain and the c-terminal s2membrane-anchored domain. the s2 region is extensively conserved in covs while constituent s1 region residues are highly diverge across the cov strains. 29 these domains have been further characterized into subdomains due to specific functionalities with respect to host receptor recognition and binding (s1), coupled with membrane fusion and entry (s2) (figure 1 ). similar to sars-cov architecture, some recent reports have sub-categorized the sars-cov-2 s1 ectodomain into the n-terminal domain (ntd), a conserved receptor-binding domain (rbd) which recognizes the human angiotensin-converting enzyme 2 (hace2), 30 and subdomains 1 and 2 (sd1 and sd2). during infection, proteolytic cleavage or priming of the s protein is crucial for viral fusion and entry into host cells, a process mediated by host cell proteases such as the transmembrane serine protease 2 (tmprss2) and cathepsin l, [31] [32] [33] at the s1/s2 (boundary between s1 and s2 subunits) and s2' (immediately upstream s2 fusion peptide -fp) cleavage sites. [34] [35] [36] the s protein primarily exists in a metastable prefusion complex prior to cleavage, after which notable conformational arrangements occur in order to fuse the viral membrane into j o u r n a l p r e -p r o o f the host cell. [37] [38] [39] in addition, the rbd adopts disparate conformational motions to engage the host cell receptor. 40, 41 conformations. [42] [43] [44] the up conformation corresponds to the hace2 accessible state while the down state cannot engage the host cell receptor. 44 the s2 domain, on the other hand, consists of the functionally important fusion peptide (fp), which is critical for viral fusion and formation of the post-fusion complex; heptad repeats 1 and 2 (hr1 and hr2); transmembrane domain (tm) and cytoplasmic tail (ct). the hrs of the s-protein trimer interact to form a fusion core of sixhelical bundle which helps bring the membranes of the virus and host cell in close proximity for fusion and entry. 42 therefore, the roles of sars-cov-2 s-protein present it as an important therapeutic target, which would enable the prevention of viral entry and fusion in host cells. numerous studies have been reported over the past months with regards to the possibility of blocking direct interactions between sars-cov-2 s-protein and hace2. most of these studies were aimed at targeting the s protein rbd domain with antibodies, peptide-based or small molecule compounds that binds with a much higher affinity to block s-protein-hace2 interactions. [45] [46] [47] [48] [49] [50] also, targeting host proteases such as tmprss2 was explored in a recent study, with consequential impediments on sars-cov-2 entry. 30 identification of other functional (allosteric) sites on the prefusion s protein could present another dynamic and effective approach of preventing sars-cov-2 infectivity relative to its interaction with the host cell ace2 and proteases. this alternative target approach for sars-cov-2 s protein is important because its rbd (similar to other covs) has been associated with a high mutational propensity which may in turn alter the affinity of small molecule inhibitors or peptide designed to bind therein. 51 allosteric targeting was explored in a recent study wherein the cov-conserved s2 hr1 region was identified as an important target site for the development of broad-spectrum inhibitors of human covs. the resulting peptide inhibitor (ek1) was evaluated in vivo and exhibited desirable safety and efficacy 52 . more so, the protein contact network (pcn) paradigm was used to map functional allosteric loci on sars-cov s protein. 53 relatively, this study was implemented to (i) identify potential druggable sites across the s1 and s2 domains of the sars-cov-2 s protein other than the rbd-hace2 interface (ii) perform high-throughput (virtual) screening of ~1500 fda approved drugs against the most druggable site(s) (iii) investigate the binding dynamics and interaction mechanisms of the compounds and their consequential effects on the s-protein rbd-ace2 complex. we believe this systematic study will be able to provide structural and molecular insights into possible allosteric sites on sars-cov-2 s protein suitable for selective targeting and structure computational methodologies the three-dimensional structure of sars-cov-2 s-protein (prefusion) was retrieved from pdb with entry 6vsb. 44 this, as previously reported, represents the s-protein rbd conformation in its up (open) state, which is most suitable for hace2 binding. also, to model binding interactions between the prefusion sars-cov-2 s-protein (s1/s2) and the hace2, a crystalized structure with pdb entry 6m0j 54 was separately retrieved. this complex depicts binding between the rbd domain (truncated) of sars-cov-2 s-protein and the protease domain (pd) of hace2. co-crystallized molecules not relevant to this study were removed while missing residues (gaps) in the structures were filled using the modeller algorithm. 55 this preparation was performed on the ucsf chimera graphic user interface (gui). 56 subsequently, using the structural superposition method, we were able to model a complex between prefusion s-protein (s1/s2) monomer (rbd -up conformation) and the hace2 protein ( figure 2 ). j o u r n a l p r e -p r o o f possible druggable sites other than the sars-cov-2 rbd interface were predicted using approaches previously reported. [57] [58] [59] [60] herein, we employed multiple tools for site identification and validation, which include sitemap 61 , fpocket 62 , discovery studio 2016 client 63 and prankweb. 64 sitemap is an exhaustive tool which ranks protein pockets based on properties such as druggability, surface exposure, hydrophobicity and hydrophilicity among others [65] [66] [67] . these details were then used to characterize the predicted pockets after which other predictive algorithms were used complementarily for cross-validation. two highly ranked sites were then selected for further analyses. furthering on the rationale of the study, we mapped out the two most druggable sites on the target protein and virtually screened against them a large chemical library of fda approved drugs (~1500 compounds) derived from the zinc repository (http://zinc.docking.org/substances/subsets/fda/). this screening was performed using highperformance computing-integrated autodock vina 68 prior to which coordinates of the predicted sites were mapped using gridboxes. corresponding binding scores were retrieved from the resulting .pdbqt files and were used to filter down to the topmost 20 compounds for each predicted sites 1 and 2. subsequently, two compounds with the highest binding scores (most negative) were selected for the two predicted sites yielding complexes that were subjected to further simulation studies. as explained in 2.1, the prefusion s-proteins (ligand-bound and unbound) were superimposed with the rbd-hace2 complex (6m0j) after which the single j o u r n a l p r e -p r o o f truncated rbd was removed. by so doing, we obtained models of allosterically-bound and unbound pre-fusion s-protein-ace2 complex. this, as aimed in this study, would provide structural and dynamical insights into the mechanistic effects of allosteric targeting on sars-cov-2 host entry machinery. although computationally expensive (1673 residues), we proceeded with long-timescale md simulation runs for the systems on amber18 graphical processing unit (gpu) using its embedded modules. 69 protein parameters were defined using the ff14sb forcefield while ligand parameters were generated with the antechamber and parmchk modules. likewise, the leap program was used to define coordinate and topology files for the ligand-bound and unbound protein complexes. this program, also, was used to neutralize (addition of counter-ions; na + and cl -) and solvate the systems in a tip3p water box of size 10å. structural minimization was first carried out partially for 5000steps with a restraint potential of 500kcal mol -1 . å 2 followed by another 100000 steps of full minimization with no restraints. a canonical (nvt) ensemble with a 5kcal mol -1 å 2 harmonic restraints was used to heat the systems gradually from 0 -300k for 50ps, after which the systems were equilibrated for 10000ps at a constant 300k temperature without restraints in an npt ensemble. atmospheric pressure was maintained at 1bar with a berendsen barostat 70 while each protein system was subjected to a production run of 350ns. studied systems include zinc3939013-s-protein-hace2 (allosteric site 1), zinc27990463-sprotein-hace2 (allosteric site 2), and unbound s-protein-hace2. corresponding trajectories were saved at every 1ps time-frame until the end of the simulation followed by data plot analyses using microcal origin software. 71 snapshots were also taken and analyzed to monitor structural events and ligand interaction dynamics across the trajectories on the ucsf chimera user j o u r n a l p r e -p r o o f interface (gui) and discovery studio client. 63 the molecular mechanics/generalized born surface area (mm/gbsa) method was used to evaluate binding affinities of the predicted allosteric s-protein binders at their target sites. binding energy profiles for both compounds, inclusive of their energy components, were estimated using 1000 snapshots from the terminal 30ns of md trajectories where conformational stabilities were visible. this approach was important in order to minimize the effects of conformational disorder or entropy on ligand interactions. the equations below mathematically express binding energy calculations: as shown, internal (∆e int ), electrostatic (∆e ele ) and van der waals (∆e vdw ) energies sum up the gas-phase energy (∆g gas ) while the solvation free energy (∆g sol ) is defined by the polar solvation (∆g ele,sol ) and non-polar contribution to solvation (∆g np,sol ) terms. the mm/gbsa method was used to estimate the generalized born (gb) for ∆g ele,sol while the linear relationship between the surface tension proportionality constant (γ = 0.0072 mol -1 å -2 ), solvent accessible surface area (sasa, å 2 ), and β constant was used to solve ∆g np,sol . furthermore, estimated ∆g bind was decomposed into individual residue energies, most especially those that constitute the predicted allosteric pockets where the ligands were bound. this method was essential to identify specific residues that contribute crucially to the stability and inhibitory activities of potential allosteric inhibitors. j o u r n a l p r e -p r o o f based on the study rationale, we set out to identify possible sites for drugging the target protein table 1 ). the architectures of these pockets are shown in figure 3 . furthermore, defining the druggability of a site on target proteins depends on the size (volume) and hydrophobicity (with minimal hydrophilicity) while, on the other hand, high hydrophilicity, reduced hydrophobicity, small pocket size and shallowness characterize "difficult-to-drug" and undruggable pockets 61, [65] [66] [67] . while large hydrophilicity could have repulsive effects on ligand mobility at the binding site, a small or shallow cavity would impede ligand access, fitness, optimal binding and stability. j o u r n a l p r e -p r o o f from table 2 , sites 1 → 3 ranks above the 0.83 halgren dscore threshold making them suitable for therapeutic targeting. relatively, site 1 appears to be highly surface-exposed with a score of 0.933 while a large pocket size and volume for site 2 could favor the use of large-molecule compounds. taken together, high surface-exposure coupled with relatively large volumes, hydrophobicity and favorable donor/acceptor properties for sites 1 and 2 could account for their suitability as targetable allosteric regions on the s-protein other than the rbd (figure 3 ). these presumptions are also reflected by the estimated dscore and sitescore values. in addition, since these predicted sites are highly functional, particularly the overlapping fp, hr1 and cr, targeting them could high-throughput screening and identification of potential allosteric binders to the predicted sites 1 and 2 high-throughput screening using a library of ~1500 fda approved drug compounds (http://zinc.docking.org/substances/subsets/fda/) were performed against the two predicted allosteric sites. results for the top 20 compounds with the highest binding scores are presented in supplementary table s1 and supplementary table s2 for sites 1 and 2 respectively. from the screening results, overall highest scores were estimated for zinc3939013 (-10 j o u r n a l p r e -p r o o f kcal/mol) at site 1 and zinc27990463 (-9.3 kcal/mol) at site 2. as highlighted in our methods, md simulations were performed for the prefusion s-protein-hace2 complexes bound distinctly at two potential allosteric sites. this approach was essential to investigate the likely effects of allosteric targeting on the entry/fusion mechanisms of sars-cov-2 via host hace2. however, this conformation appeared distorted the allosterically-bound s-proteins and could account for displacement motions of the interacting hace2 from the rbd interface. therefore, the allosteric-mediated disruption of sars-cov-2 s-protein rbd and its interaction with hace2, as reported herein, is a major finding that could indicate the viability of allosteric targeting in sars-cov-2 therapy. furthermore, we measured structural stabilities across the ligand-protein complexes relative to the unbound system using the rmsd metrics. as shown in figure 6 , structural instability was highest in the unbound s-protein while its associated hace2 was relatively stable compared to this could indicate the structural effects of allosteric targeting on the s-protein and its interaction with hace2. estimated mean rmsds, as presented in table 2 , corroborates conformational variations among the unbound and bound protein complexes. to minimize the effects of structural disorderliness (entropy) in our calculations, we selected, from the md trajectories, terminal time-frames (270-300ns) from which the systems appeared to relatively stabilize. these were defined as the finally equilibrated (fe) time-frames and were used for subsequent structural analyses ( table 2 ). from the resulting fe-rmsd plots, unbound s-protein was highly unstable while its associated hace2 exhibited low structural motion in line with the rmsd calculations, which could also imply that the binding of s-protein stabilized hace2. in contrast, the allosterically-bound sproteins (sites 1 and 2) were notably stable while their corresponding hace2 showed high structural instability that could correlate with their systemic motions at the s-protein rbd as earlier mentioned. structural analyses of ligand orientations at the respective allosteric sites of sars-cov-2 sprotein were performed using averaged structures from the md trajectories ( figure 9 ). findings reveal that the allosteric binding of zinc3939013 (fosaprepitant) was stabilized at the ntd. fosaprepitant contains a terminal triphosphate group that orients towards residues such as n99, k187, n188, r190 and h207. likewise, its trifluoromethyl group oriented towards d102 while constituent -o and -nh groups mediate interactions with q173 and n121, among others. these altogether could facilitate high-affinity interactions accountable for its stability and allosteric inhibitory effects against the sars-cov-2 and associated hace2. j o u r n a l p r e -p r o o f binding affinities of the compounds were determined using the mm/pbsa technique, which also allowed us to measure the energy contributions of interactive residues at the predicted allosteric sites. energy calculations, as presented in table 4 were performed using relatively stable time-frames (270-300ns) to minimize entropical effects that may interfere with ligand binding activities. in addition, we observed that electrostatic effects contributed most notably to the allosteric binding of zinc3939013 at the ntd region while van der waals contributions had the highest effect on the binding of zinc27990463 at the predicted site 2 pocket. electrostatic contributions at site 1 could be due to the high number of electropositive residues that constitute the pocket, as shown in figure 10 , which may form high-affinity interactions with electronegative moieties of the compound. calculations further revealed that ∆e vdw and ∆e ele were more favorable in the gas phase for zinc3939013 while polar solvation energies were more favorable for zinc27990463 j o u r n a l p r e -p r o o f at the s2 region of the s-protein. this could imply that while the former was buried in the deep hydrophobic pocket of the ntd, the latter was surface exposed due to its trans-domain binding activity as earlier reported. to understand the mechanistic binding of the compounds at both predicted sites, we decomposed the binding free energies into individual contributions of the interacting residues. these were juxtaposed with structural analysis that showed the type and (π-alkyl) interactions. more so, π-π stacked interaction between y313 and a benzene ring (of the 4-tri-fluoromethyl-1,1'-biphenyl group) could be highly crucial for the stability of the compound. taken together, electrostatic energies favored the binding of zinc3939013 at site 1 while vdw energies favored zinc27990463 binding at site 2, which consequentially, were able to perturb the s-protein rbd and allosterically disrupt hace2 interactions. the systemic entry of sars-cov-2 into the human host cell is a crucial process that underlies its virulence and pathogenicity in humans and other animals it infects. this mechanism is mediated by its interaction with the host ace2 (hace2) via attachment and fusion. potential intervention approaches in sars-cov-2 treatment include therapeutic strategies that could prevent sars-cov-2 s-protein binding to hace2. in this study, we implemented an exhaustive approach to identify drug molecules that could potentially bind to sars-cov-2 s-protein at other sites other than the rbd. pertinent to the allosteric targeting approach implemented herein j o u r n a l p r e -p r o o f was the identification of highly druggable sites inherent in the s-protein (s1/s2), which was carried out using multiple pocket prediction algorithms for identification and validation of possible allosteric sites. predicted pockets were then characterized based on their attributes after which two highly probable pockets were selected. these were then screened distinctly against a library of ~1500 fda approved drugs retrieved from the zinc database. amongst all, thermophoresis (mst) can be employed for further validation. these implementations will provide additional insights into the targetability and suitability of these pockets for novel covid-19 therapeutics. findings from this study paves way for novelty in the structure-based design of high-affinity allosteric inhibitors or disruptors of sars-cov-2 association with host hace2 thereby preventing viral entry. authors thank the college of health sciences, university of kwazulu-natal, south africa for providing infrastructural support and we also acknowledge the center for high performance computing (chpc), capetown, south africa, for providing computational resources. authors declare no conflict of 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conformers autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading amber 18 molecular dynamics with coupling to an external bath originpro 9.1: scientific data analysis and graphing software-software review structural basis for the recognition of sars-cov-2 by full-length human ace2 mapping allosteric communications within individual proteins the following information is required for submission. please note that failure to respond to these questions/statements will mean your submission will be returned. if you have nothing to declare in any of these categories then this should be stated. all sources of funding should be declared as an acknowledgement at the end of the text. authors should declare the role of study sponsors, if any, in the collection, analysis and interpretation of data; in the writing of the manuscript; and in the decision to submit the manuscript for publication. if the study sponsors had no such involvement, the authors should so state. studies on patients or volunteers require ethics committee approval and fully informed written consent which should be documented in the paper.authors must obtain written and signed consent to publish the case report from the patient (or, where applicable, the patient's guardian or next of kin) prior to submission. we ask authors to confirm as part of the submission process that such consent has been obtained, and the manuscript must include a statement to this effect in a consent section at the end of the manuscript, as follows: "written informed consent was obtained from the patient for publication of this case report and accompanying images. a copy of the written consent is available for review by the editor-in-chief of this journal on request".patients have a right to privacy. patients' and volunteers' names, initials, or hospital numbers should not be used. images of patients or volunteers should not be used unless the information is essential for scientific purposes and explicit permission has been given as part of the consent. if such consent is made subject to any conditions, the editor in chief must be made aware of all such conditions. even where consent has been given, identifying details should be omitted if they are not essential. if identifying characteristics are altered to protect anonymity, such as in genetic pedigrees, authors should provide assurance that alterations do not distort scientific meaning and editors should so note. please specify the contribution of each author to the paper, e.g. study design, data collections, data analysis, writing, others, who have contributed in other ways should be listed as contributors.this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.not applicable to this study.fao conceptualized, implemented, analyzed, interpreted and wrote the manuscript, kfo performed molecular dynamics simulation, while mes revised and approved the manuscript for submission. ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests:j o u r n a l p r e -p r o o f key: cord-331786-wgt7kg6f authors: diego-martin, borja; gonzález, beatriz; vazquez-vilar, marta; selma, sara; mateos-fernández, rubén; gianoglio, silvia; fernández-del-carmen, asun; orzáez, diego title: pilot production of sars-cov-2 related proteins in plants: a proof of concept for rapid repurposing of indoors farms into biomanufacturing facilities date: 2020-10-13 journal: biorxiv doi: 10.1101/2020.10.13.331306 sha: doc_id: 331786 cord_uid: wgt7kg6f the current covid-19 crisis is revealing the strengths and the weaknesses of the world’s capacity to respond to a global health crisis. a critical weakness has resulted from the excessive centralization of the current biomanufacturing capacities, a matter of great concern, if not a source of nationalistic tensions. on the positive side, scientific data and information have been shared at an unprecedented speed fuelled by the preprint phenomena, and this has considerably strengthened our ability to develop new technology-based solutions. in this work we explore how, in a context of rapid exchange of scientific information, plant biofactories can serve as a rapid and easily adaptable solution for local manufacturing of bioreagents, more specifically recombinant antibodies. for this purpose, we tested our ability to produce, in the framework of an academic lab and in a matter of weeks, milligram amounts of six different recombinant monoclonal antibodies against sars-cov-2 in nicotiana benthamiana. for the design of the antibodies we took advantage, among other data sources, of the dna sequence information made rapidly available by other groups in preprint publications. mabs were all engineered as single-chain fragments fused to a human gamma fc and transiently expressed using a viral vector. in parallel, we also produced the recombinant sars-cov-2 n protein and its receptor binding domain (rbd) in planta and used them to test the binding specificity of the recombinant mabs. finally, for two of the antibodies we assayed a simple scale-up production protocol based on the extraction of apoplastic fluid. our results indicate that gram amounts of anti-sars-cov-2 antibodies could be easily produced in little more than 6 weeks in repurposed greenhouses with little infrastructure requirements using n. benthamiana as production platform. similar procedures could be easily deployed to produce diagnostic reagents and, eventually, could be adapted for rapid therapeutic responses. the current pandemic is evidencing several weaknesses in our ability to respond to a global crisis, one of which is the insufficient and heavily centralized distribution of the world manufacturing capacity of bioproducts such as antibodies, vaccines and other biological reagents, specially proteins. since it is economically impracticable to ensure readiness by maintaining inactive infrastructures during large periods of normality, the development of dualuse systems has been proposed, which would serve regular production needs in normal times but could be rapidly repurposed to strategic manufacturing requirements in times of crisis. ideally, such adaptable infrastructures should be widespread to serve local demand in case of emergency. recombinant protein production in plants is a technologically mature bioengineering discipline, with most current plant-based bioproduction platforms making use of non-food crops, mainly the nicotiana species tabacum and n. benthamiana as biomanufacturing chassis (moon et al., 2019; capell et al., 2020) . n. benthamiana is most frequently used in association with agrobacterium-mediated transient expression, also known as agroinfiltration, a technology that dramatically reduces the time required for product development. briefly, agroinfiltration consists in the massive delivery of an agrobacterium suspension culture to the intercellular space of plant leaves, either by pressure, using a syringe (small scale), or applying vacuum to plants whose aerial parts have been submerged in a diluted agrobacterium culture (large scale). agrobacterium transfers its t-dna to the cell nucleus, therefore massively reprogramming the plant cell machinery towards the synthesis of the t-dna-encoded protein(s)-of-interest. transient expression of the transgene is often assisted by self-replicating deconstructed virus vectors that amplify the transgene dose, thus boosting protein production by several orders of magnitude (gleba et al., 2004) . other systems, such as the peaq system, rely on viral genetic elements for boosting expression without recurring to viral replication (sainsbury et al., 2009 ). transient expression in n. benthamiana has become the standard in plant-based recombinant protein production due to a unique combination of advantages, with speed and high yield as the most obvious ones. maximum production levels in the g/kg fresh weight (fw) range for certain highly stable proteins such as antibodies have been reported (marillonnet et al., 2005) . regarding speed, the in-planta incubation times required to obtain maximum yield of recombinant protein are no more than two weeks. an important, often insufficiently highlighted feature of n. benthamiana transient expression is its relatively small infrastructure requirements, partially overlapping with those employed in more conventional, medium/high-tech indoors agriculture, such as hydroponics, vertical farming, etc. (buyel, 2019) . in this context, when confronted with the covid-19 crisis, we decided to exercise a partial reorientation of the activities in our academic lab, which is equipped with a multipurpose glass greenhouse facility, towards the production of sars-cov-2 antigens and antibodies against the virus. here we describe the recombinant production, purification and analysis of six anti-sars-cov-2 monoclonal antibodies at laboratory scale, plus a pilot upscaling of two of those six antibodies. next to production scale, a critical parameter to assess was the response time. the process described here started in mid-april 2020 with the selection of literature-available antibody variable sequences and finalized nine weeks later with approximately 0.2 g of anti-sars-cov-2 antibody (ab) produced in modular badges of 56 n. benthamiana plants and formulated as one litre of ab-enriched plant apoplastic fluid. based on this experience, we estimate that the same process can be reduced up to 6-7 weeks with small pre-adaptations, a remarkably short reaction time for a de novo antibody production system. absolutely key for this fast reaction is the immediate availability of scientific data including antibody sequences in pre-print repositories. this is in our opinion one of the most positive lessons that can be extracted from the covid-19 crisis. we discuss here the possible applications of the fast plant-produced antigens and antibodies in diagnostics and therapy and propose the repurpose of high-tech agricultural facilities as an alternative for decentralized biomanufacturing in times of crisis. both, nicotiana benthamiana wild type plants and 1,2-xylosyltransferase/alpha1,3fucosyltransferase (δxt/ft) rnai knock down lines (strasser et al., 2008) were grown in the greenhouse. growing conditions were 24 °c (light)/20 °c (darkness) with a 16-h-light/8-hdark photoperiod. all sequences were cloned and assembled using the goldenbraid (gb) assembly system https://gbcloning.upv.es) . antibody sequences were obtained from literature (see table 1 ). all antibodies were cloned as single chain antibodies fused to the human igg1 fc domain. those antibodies derived from synthetic or camelid single domain vhh libraries (sybody 3, sybody 17 and nanobody 72) were designed as direct fusions. cr3009, cr3018 and cr3022 human monoclonal antibodies were redesigned as single chain variable fragment (scfv) by connecting the variable light (vl) and heavy (vh) chains with a ggggsggggsggggssgggs peptide linker. antibody sequences were codon optimised for n benthamiana with the idt optimization tool at http://eu.idtdna.com/codonopt . the sars-cov-2 antigen sequences used (n protein, yp_009724397.2; and s-protein rbd domain, yp_009724390.1, aa 319-541) derive from the wuhan strain nc_045512. four different versions of rbd were designed corresponding to (i) the native sequence with a cterminal 6xhis-tag or (ii) an n-terminal 6xhis-tag and a c-terminal kdel sequence for er retention, and (iii and iv) their corresponding n. benthamiana codon optimised counterparts, using the same tool as above. dna sequences were domesticated as level 0 phytobricks for gb cloning and ordered for synthesis as double-stranded dna fragments (gblocks, integrated dna technologies). gblocks were first cloned into the domestication vector pupd2 (vazquez-vilar et al., 2017) in a bsmbi golden gate restriction/ligation reaction (37 °c -10 min, 50x (37 °c -3 min / 16 °c -4 min), 50 °c -10 min, 80 °c -10 min). the ligation product was transformed into e. coli top 10 electrocompetent cells and positive clones were verified by restriction digestion analysis and sequencing. pupd2 level 0 phytobricks were then cloned into the expression vectors pgreen sp-higg1 (antibody sequences), pcambiav1 (rbd sequences) or pcambiav2 (n sequences). pgreen sp-higg1 is a pgreen vector based adaptation of the the magnicon® 3' provector pich7410 (icon genetics) that is designed for bsai cloning of gb (b4-b5) standard parts as in-frame fusions with the tobacco (1-3)-beta-glucanase signal peptide and the human igg1 fc domain. similarly, pcambiav1 and pcambiav2 are pcambia based adaptations of the magnicon® 3' provector pich7410 that are designed for bsai cloning of gb standard parts as in-frame fusions with tobacco (1-3)-beta-glucanase signal peptide (pcambiav1, for expression of secreted proteins) or without any subcellular localization signal (pcambiav2, for expression of cytoplasmic proteins). assembly reactions were performed as above, and the ligation reactions were transformed into e. coli top 10 electrocompetent cells. positive clones were verified by restriction digestion analysis. all level 0 parts generated in this work are listed in supplementary table 1 . for transient expression in n. benthamiana, the plasmids were transformed into agrobacterium tumefaciens strain gv3101 c58c1 by electroporation. the same strain but carrying the psoup helper plasmid was employed to allow the replication of the pgreen vectors which encode the antibodies. overnight grown exponential cultures were collected by centrifugation and the bacterial pellets were resuspended in agroinfiltration solution (10 mm mes, 20 mm mgcl2, 200 µm acetosyringone, ph 5.6) and incubated for 2 h at rt in a horizontal rolling mixer. for small scale agroinfiltration, culture optical density at 600 nm was adjusted to 0.1 with agroinfiltration solution and the bacterial suspensions harbouring the 3' antibody or antigen modules, the integrase (pich14011), and the 5' module (pich17388) were mixed in equal volumes. control samples were agroinfiltrated with pich11599_dsred and integrase module. agroinfiltration of 5 to 6-week-old n. benthamiana plants was carried out through the abaxial leaf surface using a 1 ml needle-free syringe (becton dickinson s.a.). for pilot scale production, the bacterial suspensions were prepared as above except that a lower od600 was used (0.005 for sybody17 agroinfiltration and 0.01 for nanobody72 agroinfiltration). additionally, for sybody17 agroinfiltration, a bacterial suspension of pich11599_dsred, a magnicon® 3´module encoding the fluorescent protein dsred, was added to the final agrobacterium infiltration solution in a ratio 1:1:0.9:0.1 (pich17388:pich14011:pgreensp-sybody17-higg1:pich11599_dsred). delivery of agrobacterium to the plant cells was carried out by vacuum infiltration in a vacuum degassing chamber (model dp118, applied vacuum engineering) provided with a 30 l infiltration tank. the aerial part of whole plants (seven plants at a time) was immersed into the agrobacterium infiltration solution; vacuum was applied for 1 min at a vacuum pressure of 0.8 bar and then slowly released. 14 days post-vacuum agroinfiltration leaves were excised and then infiltrated with 20 mm phosphate buffer (7.4 mm nah2po4, 12.6 mm na2hpo4.7h2o, ph 7), without (sybody17) or with (nanobody72) 0.5 mm pmsf (sigma-aldrich, #78830), following the same procedure as the vacuum agroinfiltration. after eliminating the buffer excess with tissue paper, the leaves were introduced into mesh zipped bags and then centrifuged using a portable cloth dryer orbegozo sc4500. thus, the apoplastic fluid was obtained from the drain tube. the apoplastic fluid was centrifuged (10 min, 11000 x g, at 4 °c) to remove any cell debris and agrobacterium, the supernatant was collected and then 4 fractions of 4ml were concentrated 8 times using 10 kda amicon ultra-4 10k centrifugal filters (millipore) after centrifugation (20 min, 3700 x g, at 4 °c). protein crude extracts were obtained by homogenizing ground frozen leaf tissue with cold pbs buffer (20 mm nah2po4, 80 mm na2hpo4.7h2o, 100 mm nacl, ph 7.4) in a 1:3 (w/v) and were centrifuged at 13000 rpm for 15 min at 4 °c. for antibody purification, 4 g of ground agroinfiltrated tissue were extracted in 12 ml of cold 20 mm phosphate buffer. samples were centrifuged at 10000 x g for 15 min and the supernatant was transferred to a clean tube and further clarified by filtration through a 0.22 µm membrane filter. the recombinant antibodies were purified by affinity chromatography with protein a agarose resin (abt technology) following a gravity-flow procedure according to the manufacturer's instructions. 100 mm citrate buffer ph 3 was used for elution and 1 m tris-hcl ph 9 was used for neutralization of the eluted sample (37.5 µl for each 250 µl elution fraction). purified antibodies were quantified using the bio-rad protein assay following the manufacturer's instructions and using bsa for standard curve preparation. the n. benthamiana leaves infiltrated with the different sars-cov-2 proteins were collected 5 (rbd) or -7 (n protein) days post infiltration (dpi). leaves were frozen in liquid nitrogen and stored at -80 °c until used. protein extraction was performed using 3-6 g of ground frozen tissue in 3 volumes of coldextraction buffer. three different buffers were tested as a first approach, in order to optimize the purification yields. buffer a: pbs buffer with 10 mm imidazole, ph 8. buffer b: buffer a supplemented with 1% triton x-100, and buffer c: buffer b supplemented with 20% glycerol, 10% sucrose and 0.05% 2-β-mercaptoethanol. samples were vigorously vortexed and centrifuged at 10000 x g for 15 min at 4 °c. the supernatant was carefully transferred to a clean tube and filtered through a 0.22 µm syringe filter. protein purification was carried out by ni-nta affinity chromatography as described in (fernandez-del-carmen et al., 2013) . purified proteins were quantified using the bio-rad protein assay following the manufacturer's instructions and using bsa for standard curve preparation. proteins were separated by sds-page electrophoresis on nupage 10% bis-tris gels (invitrogen) using mes-sds running buffer (50 mm mes, 50 mm tris-base, 3.5 mm sds, 1 mm edta, ph 7.3) under reducing conditions. gels were visualized by coomassie blue staining. for western blot analysis, proteins were transferred to pvdf membranes (amersham hybond™-p, ge healthcare) by semi-wet blotting (xcell ii™ blot module, invitrogen, life technologies) following the manufacturer's instructions. blots were blocked with 2% ecl prime blocking agent (ge healthcare) in pbs-t (pbs buffer supplemented with 0.1% (v/v) tween-20). for anti-sars-cov-2 antibody detection, the blots were incubated with 1:20000 hrp-conjugated rabbit anti-human igg (sigma-aldrich, #a8792). for sars-cov-2 antigen detection the blots were incubated with 1:2000 anti-his mouse monoclonal primary antibody (qiagen, #34660) and then incubated with 1:10000 peroxidase labelled anti-mouse igg secondary antibody (ge healthcare). blots were developed with ecl prime western blotting detection reagent (ge healthcare) and visualised using a fujifilm las-3000 imager. the overnight coating of costar 96 well eia/ria plates (corning) was carried out at 4 °c with 100 µl of 4 µg/ml rbd (raybiotech, #230-30162) or bsa (used as control) in coating buffer (15 mm na2co3, 35 mm nahco3, ph 9.8). after 4 washes with 300 µl of pbs, the plate was blocked with 200 µl of a 2% (w/v) ecl advance blocking reagent (ge healthcare) solution in pbs-t (pbs supplemented with 0.1% (v/v) tween-20) for 2 h at rt. the plate was washed 4 times with pbs, and then, starting at 2 µg of the purified antibody per well (100 µl), 1:5 serial dilutions in blocking solution were incubated for 1 h 30 min at rt. after 4 washing steps with pbs-t (pbs buffer supplemented with 0.05% tween-20), 1:2000 hrp-labelled rabbit anti-human igg (sigma-aldrich, #a8792) in blocking solution was added. after 1 h, the plate was washed with pbs and the substrate o-phenylenediamine dihydrochloride sigmafast™ opd tablet (sigma-aldrich, #p9187) was added (following manufacturer's instructions). reactions were stopped with 50 µl 3 m hcl per well and absorbance was measured at 492 nm. the endpoint titer was determined as the last concentration of each purified antibody showing an absorbance value higher than the value defined as cutoff (mean blank + 3sd). blank is defined as the values from each elisa test against bsa (zrein et al., 1986; armbruster and pry, 2008) . the sandwich elisas were performed as described in the antigen elisa section with a few changes. the plates were coated with 100 µl of 4 µg/ml murine anti-his mab (qiagen, #34660), and after blocking, the plates were incubated with 100 µl of the crude extracts of the (rbd/n) antigen expressing leaves serially diluted (1:2) in bsa 0.5%. wt crude extracts were used as negative control. the crude extracts were prepared by adding a volume of pbs buffer corresponding to 3 times the mass of the ground tissue in liquid nitrogen. then the mix was centrifuged (13000 rpm, 4 °c, 15 min) and the supernatant was subjected to sonication before use. the antigens were sandwiched with 1 µg of the corresponding purified antibody (or 100 µl of the apoplastic fluid in 2% blocking reagent) per well (1 h 30 min incubation, rt). the same procedure as in the antigen elisa was followed for the incubation with the conjugated secondary antibody, colorimetric reaction and measurement. six different antibody sequences were selected for recombinant production in n. benthamiana, following a plant deconstructed viral strategy based on magnifection technology, as described earlier (marillonnet et al., 2005 ) (see table 1 ). four of those were directed against the receptor binding domain (rbd) of the sars-cov-2 spike (s) protein, whereas the remaining two were directed against the n protein. all six antibodies were engineered as single polypeptide chains fused to the human cɣ2-cɣ3 constant immunoglobulin domains. three of them, those derived from single chain camelid or synthetic vhh antibody libraries, were produced as direct fusions. the other three, derived from full-size human monoclonal antibodies, were redesigned as scfvs, using a linker peptide that connects vh and vl regions (see fig 1a) . the nucleotide sequences of the different variable regions were all obtained from the literature, then chemically synthesized with appropriate extensions and cloned into a destination magnifection-adapted vector using a type iis restriction enzyme strategy. the cloning cassette was flanked by a β-endoglucanase signal peptide for apoplastic localization in n-terminal and the human cɣ2-cɣ3 domains of the human igg1 in the c-terminal side. the resulting vectors were transferred to agrobacterium cultures and agroinfiltrated in n. benthamiana leaves in combination with a 5´ magnicon® module, containing the rna polymerase and movement protein, and with an integrase module (fig 1b) . for antibody production we used wild-type and rnai δxt/ft glycoengineered n. benthamiana plant lines, the latter lacking plantspecific xylose and fucose glycosylation (strasser et al., 2008) . infiltrated leaves were examined daily, and only minimal damage was observed in the agroinfiltrated tissues during the incubation period. after seven days, leaf samples were collected, ground, and crude extracts were analyzed in sds-page. as can be observed in fig 2a-b (upper panel) , all samples produced coomassie-detectable bands of the expected antibody size. scfv-igg1 55-56 kda antibodies migrated slightly above the 50-55 kda rubisco large subunit, partially masking its detection. vhh-igg1 antibodies migrated at the expected 42-43 kda size. the identity of the coomassie bands was confirmed by western blot using an anti-human igg1 antibody for detection (fig 2a-b , lower panel). as shown in fig 2a-b , under reducing conditions lower molecular weight (mw) bands were also detected, probably as a result of partial proteolytic degradation. small-scale affinity purification was carried out for all six antibodies produced in δxt/ft plants using protein a affinity chromatography (fig 2c-d) . the resulting purified antibodies were used to estimate the yield of the final product, which ranged between 73.06 µg/g fw (cr3022 antibody) to 192.63 µg/g fw (nanobody72 antibody) (see table 2 ). the in-planta production of sars-cov-2 rbd and n protein antigens was also assayed in parallel using a similar strategy as described for antibody production. for this purpose, two versions of the expression vector were designed for rbd, one with the native viral sequence and the other with a n. benthamiana codon-optimized sequence. for the n protein, only the n. benthamiana codon-optimized sequence was employed. for rbd, native and codon optimized versions were targeted to the apoplast with the tobacco glucan endo-1,3-beta-glucosidase signal peptide and versions containing a kdel peptide for er retention were also generated. all nucleotide sequences were chemically synthesized with a small nucleotide extension coding for a histidine tag for detection ( fig 3a) . as described for antibody production, magnicon®-derived 3´ vector modules encoding rbd and n proteins were agroinfiltrated in combination with an integrase module and a 5´-module lacking any additional subcellular localization signal. shorter incubation times were decided in antigen production as compared to antibodies because antigen constructs produced different degrees of necrotic lesions in the leaves, ranging from mild symptoms in n protein to severe necrosis after four days in native rbd. for those constructs producing more severe lesions, incubation time was reduced to five days, and for the rest the incubation period was extended to seven days. rbd was extracted and purified using small-scale affinity-chromatography with niquel columns and the resulting coomassie and a western blot analysis are shown in fig 3b. rbd can be detected as a major estimated 30 kda band, with the presence of higher mw bands that suggest multimerization. er retention did not improve expression levels of rbd for the native version, nor for the n. benthamiana optimized one (data not shown). addition of 1% triton x-100 to the standard extraction buffer (see materials and methods) did not improve the yield, which was estimated as 2-4 µg/g fw (table 2) . n protein was extracted from agroinfiltrated leaves and affinity purified following the same procedure described for rbd. a major 49 kda band was detected both on the crude extract and upon purification (fig 3b) . small-scale affinity-chromatography with niquel columns gave an estimated yield of 30 µg/g fw for n protein ( table 2) . binding activities of affinity purified anti-rbd antibodies were analysed by antigen elisa as shown in fig 4a. as expected, all assayed antibodies were active in binding their respective antigen. endpoint dilution titers were calculated for anti-rbd antibodies using a commercial antigen. sybodies 3 and 17 and nanobody72 showed high dilution titers, (0.75 µm, 0.03 µm and 0.03 µm, respectively), but the performance of cr3022 was significantly lower (2.85 µm). in a parallel experiment, we tested the ability of plant-made antibodies to selectively detect our own plant-made antigens, including here also the n protein, using a sandwich elisa approach. for this analysis, elisa plates were coated with a murine anti-his mab, incubated with serial dilutions of crude plant extracts from antigen-producing plants and sandwiched with purified plant-made antibodies. as shown in fig 4bc, all antigen-producing plant extracts gave sandwich-elisa signals significantly above the background when assayed using their cognate antibodies, thus evidencing the capacity of both, antibodies and antigens, to function as potent diagnostic tools. background signals in this experiment are likely due to cross-reaction of the anti-human igg secondary antibody with the murine anti-his mab, and could be easily reduced for more potent diagnostic applications by employing recombinant antibody formats other than igg. in the design of a pilot upscaling experiment, we favoured modularity and tried to maximize the affordability and adaptability of the process by reducing the requirements for highly specialized lab equipment. we carried out a final agroinfiltration for recombinant antibody production using a total of 112 plants, equivalent to approximately 2.5 kilograms of fresh plant material. the plants were divided in two batches of 56 plants each, and used to produce sybody17 and nanobody72 respectively, as these antibodies showed the most promising binding activities and yields. to facilitate the upscaling of the agroinfiltration process, plant seedlings were transplanted in growth modules, each module comprising seven pots kept together in a double layer of disposable plastic-board hexagons as shown in fig 5a. each production batch consisted in eight hexagonal modules. when plants were six weeks old, they were agroinfiltrated by submerging each hexagon upside down into a 40 cm diameter cylindrical tank filled with 30 l of an agrobacterium suspension, set inside a cylindrical vacuum degas chamber (fig 5a) . in this way, seven plants at a time were vacuumagroinfiltrated by slowly releasing vacuum while leaves remained submerged in the solution. next, plants were rinsed, brought back to the growth chamber and incubated for 14 days before harvest. two different concentrations of the agrobacterium suspension were used in this experiment. one of them (sybody17) consisted in an od600 0.005 final mix containing plasmids pich14011, pich17388, pgreensybody17-igg1, and pich11599_dsred at 1:1:0.9:0.1 ratio, where pich11599_dsred is a magnicon® 3´module encoding dsred. the fluorescent marker was added to the infiltration mix to monitor the extension of the viral infection foci. as described elsewhere (julve et al., 2013; julve parreño et al., 2018) superinfection exclusion among virial clones yields mosaic-like expression patterns of individual clones, therefore the tiles produced by red fluorescent proteins were used as an indication of the extension and distribution of the unlabelled foci producing the recombinant antibody. in parallel, nanobody72 upscaled production was undertaken by agroinfiltration of an od600 0.01 agrobacterium culture mix containing pich14011, pich17388 and pgreennanobody72-igg1 at 1:1:1 ratio. after 14 days, dsred tiles in sybody17 experiment, clearly visible with the naked eye, finalized their expansion in most agroinfiltrated leaves, an indicator that the expression tiles had covered the whole leaf surface (fig 5b) . at this stage, leaves were harvested and submitted to an apoplastic fluid recovery assay, where >0.5 kg batches of detached leaves were vacuum infiltrated in 20 mm phosphate buffer using the same vacuum device as described above. once rinsed to remove the excess of buffer, leaves were packed in mesh zipped bags, spinned down in a spin portable cloth dryer, and the intercellular apoplastic fluid was recovered from the drain tube. with this simple procedure, between 940 and 1200 millilitres of apoplastic fluid (sybody 17 and nanobody 72, respectively) was recovered from 1.2 kg of detached leaves. a fraction of the apoplastic fluid of both antibodies was concentrated 8 times in 10 kda centricons, and the rest was kept refrigerated for further analysis. fig 5c-d show the coomassie-staining and western blot analysis of crude extracts as well as apoplastic fluid preparations, and their corresponding purifications. crude extracts in this pilot experiment showed a vhh-igg1 band similar in intensity to that obtained in small scale experiments (data not shown). interestingly, apoplastic fluid consisted in a very simplified mix of proteins, with the recombinant antibody being among the most predominant ones. as shown, the different optical density of the agrobacterium culture, together with the presence of a competing dsred clone clearly influenced the accumulation levels, with the yields of nanobody72 clearly outperforming those of its sybody counterpart. unfortunately, the antibodies seemed partially degraded as indicated by the presence of two bands smaller than the expected vhh-cɣ2-cɣ3 size, which could be compatible with degradation fragments. degradation was only partially solved with the addition of the protease inhibitor pmsf into the recovered phosphate buffer, as shown with nanobody72 production (fig 5d) . despite degradation, in a densitometric analysis we estimate that the recovered apoplastic fluid contains 0.2 g per liter of intact mab full-size. finally, we performed sandwich elisa tests of sybody17 and nanobody72 ( fig 5e and fig 5f, respectively) using the total and concentrated apoplastic fluid as detection reagent against serial dilutions of crude plant extracts from rbd-producing plants, showing that this simple antibody preparation can be directly employed in detection procedures without the need of additional purification steps. several n. benthamiana-dedicated bioproduction facilities are functioning worldwide, as those from leaf expression systems in uk (dobon, 2019) , icon genetics (giritch et al., 2006) and fraunhofer in germany (wirz et al., 2012) or kentucky bioprocessing in us (olinger et al., 2012) , among others. notably, medicago recently announced the building a new 44000 sqm facility with capacity for around 40-50 million of planned doses of flu vaccine per year. such facilities usually involve separated modules for upstream processing, namely a wet-lab module for preparation of the bacterial inoculum, a regular plant growth chamber, and agroinfiltration room, and a post-infiltration growth chamber. in addition, downstream processing facilities are often situated next or to the production ones to minimize the handling time of fresh tissues. whereas installed capacity of plant-dedicated biofactories is in continue growth, they are clearly insufficient to respond to global or even regional demands in times of crisis. we reasoned that, at least for upstream processes, the infrastructures required for medium scale n. benthamiana-based production are not radically different to those employed in high-tech agriculture practices as hydroponics, aeroponics or vertical farming, and thus high-tech agriculture facilities could be easily repurposed as biomanufacturing facilities in a matter of days or weeks (mcdonald and holtz, 2020) . as an exercise to practically test the repurposing requirements, we describe here the partial adaptation of our research laboratory and greenhouse facilities to the production of sars-cov-2-related antigens and antibodies using n. benthamiana agroinfiltration as manufacturing platform. in figure 6 we show a chronogram of the activities undertaken by our team towards the production of sars-cov-2 antigens and antibodies, from the initial selection of the nucleotide sequences of the genes-of-interest to the production of one litre of plant apoplastic fluid of recombinant sybody17 and nanobody72. in our hands, the whole process took a total of nine weeks with non-exclusive personnel dedication and partially restricted access to our facilities. the process can be divided in three periods: the first step (design), taking approximately ten days, was dedicated to construct design and gene synthesis. it was pivotal in this step to have open access to viral and antibody sequences deposited in pre-print repositories. particularly remarkable was the openness of academic labs that immediately released primary sequence information of partially characterized anti-sars-cov-2 monoclonal antibodies, an exercise that should serve as an example in the future. due to our limited testing capacity, the number of parallel designs per product was maintained relatively low, and several design decisions (e.g. codon optimization, purification tags) were taken based in a best-guess approach. ideally, proper crisis preparedness should involve a centralized automated equipment such as a biofoundry (hillson et al., 2019) , with which the design space could be extended dramatically without causing delay. the second phase (build) was dedicated to cloning and construct building and lasted less than three weeks. our lab counts with adapted plasmids and cloning procedures from previous projects (sarrion-perdigones et al., 2011; vazquez-vilar et al., 2017) , therefore no significant time lag occurred in this step. importantly, this period also involved seeding a new plant batch at the scale required for pilot production in week seven (112 plants distributed in 16 hexagonal modules in this case). in a third phase (test), starting on week 5, all constructs were infiltrated at a small scale (three replicate leaves each), shortly incubated (5 or 7 days) and then tested functionally in parallel analyses. this small-scale assay took two additional weeks, summing a total of approximately 50 days for the complete process. the synthetic biology-inspired design-build-test (dbt) process described above is conceived as an iterative one, so that new dbt cycles can be run fuelled by the conclusion of previous cycles to generate new optimized versions of the product. based on this experience, we estimate that the whole dbt cycle could be shortened to 30 days or less by optimising the pipeline (e.g. introducing centralized, automatized design and build phases), and by improving preparation and anticipation in the facilities (fig 6) . for instance, note that moving from step 2 to step 3 without delay requires a small batch of plants be always maintained in the facility, as it was in our case to supply our research requirements. this only involves transplanting 10-15 seedlings every three weeks, and then disposing of them every other three weeks once they start flowering. if a continuous plant supply is not maintained, a minimum of three extra weeks needs to be considered to have plants ready for the first test iteration. whereas the first version of products shown here lack iterative optimization, it would serve eventually to respond to the most urgent demands. in our case, as the results of the first dbt process arose, the best performing version (v1.0) of two of the products were taken to production phase. in the exercise shown here, the upscaling was relatively small (112 plants, approximately 2.5 kg fw). post-agroinfiltration incubation time was extended to 14 days to maximize yields. in the meantime, optimization of the purification/extraction methods were undertaken at small scale, so that the new knowledge acquired could be applied in the batch purification of the pilot experiment. in a crisis-scenario, and given the modularity of the proposed scheme, several medium-size production modules can be replicated in a farming facility, and reproduced in several farms, allowing easy scalability. successive iterations with small scale agroinfiltration could be an effective way to maximize yields and reduce development times by comparing different small-scale strategies. it should be mentioned that the basic apoplast-based downstream processing proposed here could only be used, with the necessary adaptations, in a limited number of crisis-related applications, mainly related with detection and diagnosis. other uses, certainly therapeutic ones, would involve additional regulatory considerations including gmp downstream facilities, which are beyond the scope of this exercise. as a result of this experience, several improvements can be envisioned. we employed the magnicon® vector system with few adaptations for all the attempted proteins. although magnicon® produces maximum yields for many products, some proteins, particularly viral antigens may express better with other (e.g. non-viral) systems. in our experiments, antigens showed rather low expression levels despite optimization attempts using codon optimization and different localization signals. in adapting to an emergency, it would be advisable to perform initial expression tests using different production platforms also involving nonreplicative methods (sainsbury and lomonossoff, 2008) or dna viruses (yamamoto et al., 2018; zhang and mason, 2006) , and to incorporate them to the initial optimization test. as mentioned, this could be done in a centralized manner, later distributing expression clones to several repurposed production facilities. in contrast to antigens, recombinant single-chain antibodies showed in general higher and more uniform expression levels, as could be expected from their more similar structure. we chose to adapt full human iggs to a scfv-igg1 format to facilitate cloning and expression procedures, since it has been earlier described in plants that single chain formats reproduce the binding activities of the original full-size antibodies from which they derive. this format also facilitates comparisons with vhh antibodies, also produced as igg1 fusions. the plant-made sars-cov-2 products described here have several potential applications in the diagnosis area. both rbd and n proteins can be used as reagents for serological assays (amanat et al., 2020; liu et al., 2020) , although further yield optimizations should be required. for those assays where antigen glycosylation is an important factor, glycoengineered plants (strasser et al., 2008) can provide a competitive alternative to mammalian cells cultures (o'flaherty et al., 2020) . regarding antibodies, they can serve as internal references for the quantification of serological responses. with small modifications, the same antibodies can be adapted for sandwich elisa and employed in the detection and quantification of viral particles, a better proxy for infectiveness than rna. we also show here that apoplastic fluid is an inexpensive antibody preparation suitable for certain applications that require low-cost preparations, e.g. the concentration of the virus from environmental samples. as shown here, the protein complexity in the apoplast is greatly reduced, therefore the apoplast could be regarded as a plant-equivalent of hybridoma supernatant or ascited fluid, although at much lower cost. unfortunately, apoplastic preparations are prone to partial antibody degradation, probably due to endogenous proteases, however this can be minimized using extraction buffers with appropriate protease inhibitors, as it was shown for nanobody72. the current pandemic crisis has evidenced the power of new antibody selection procedures, either based on single-cell selection from human peripheral blood mononuclear cells, in the case of full-size antibodies, or based on ultra-high throughput selection of synthetic libraries (sybodies) in the case of camelid-derived nanobodies (zimmermann et al., 2018; walter et al., 2020) . large collections of anti-sars-cov-2, potentially neutralizing antibody sequences were made available to the scientific community in a question of weeks rather than months. it does not go unnoticed that the combination of rapid antibody selection procedures with fast, modular and scalable plant expression also has implications in the therapeutic arena as an ideal system for passive immunization. intravenous polyclonal immunoglobulins (ivig) from recovered patients have been shown a very effective covid-19 treatment in several studies (montelongo-jauregui et al., 2020 , and references herein) however the limited availability of patient sera hampers its application in practice. interestingly, we showed in a recent work that large recombinant polyclonal antibody cocktails (pluribodies), mimicking a mammalian immune response can be produced in n. benthamiana with high batch-to-batch reproducibility (julve parreño et al., 2018) . passive immunization with recombinant antibody cocktails resembles a natural response more than a monoclonal therapy, requires shorter developmental times and is probably more robust against the development of resistances. in conclusion, based on the results of the exercise described here, we propose the repurposing of indoors farms into plant-based biomanufacturing facilities as a viable option to respond to local and global shortages of bioproducts such as diagnostics and therapeutic reagents in times of crisis. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. all authors designed and performed the experiments, and analyzed the data. d.o. wrote this manuscript. all authors revised and edited the written manuscript. this work was supported by h2020 eu projects 760331 newcotiana and 774078 pharmafactory. s.s. is recipient of a fpi fellowship bio2016-78601-r from the spanish zimmermann, i., egloff, p., hutter, c. a., arnold, f. m., stohler, p., bocquet, n., et al. (2018 the upper timeline represents the actual timespan of the experiments. note that the time points represent approximately the days required to produce and initially characterize the designated products; notwithstanding, some of the results shown in previous figures correspond to extended analysis obtained at a later stage, during the preparation of this manuscript. the lower timeline describes the estimated minimal timespan that would result by introducing some of the optimizations described in the text. a serological assay to detect sars-cov-2 seroconversion in humans limit of blank, limit of detection and limit of quantitation plant molecular farming -integration and exploitation of side streams to potential applications of plant biotechnology against sars-cov-2 transient gene expression seeds plant-based bioproduction systems: leaf expression systems' hypertrans technology promises low costs and high yields recombinant jacalin-like plant lectins are produced at high levels in nicotiana benthamiana and retain agglutination activity and sugar specificity rapid high-yield expression of full-size igg antibodies in plants coinfected with noncompeting viral vectors engineering viral expression vectors for plants: the 'full virus' and the 'deconstructed virus' strategies building a global alliance of biofoundries a coat-independent superinfection exclusion rapidly imposed in nicotiana benthamiana cells by tobacco mosaic virus is not prevented by depletion of the movement protein a synthetic biology approach for consistent production of plant-made recombinant polyclonal antibodies against snake venom toxins evaluation of nucleocapsid and spike protein-based enzyme-linked immunosorbent assays for detecting antibodies against sars-cov-2 systemic agrobacterium tumefaciens-mediated transfection of viral replicons for efficient transient expression in plants from farm to finger prick-a perspective on how plants can help in the fight against covid-19 convalescent serum therapy for covid-19: a 19th century remedy for a 21st century disease development of systems for the production of plant-derived biopharmaceuticals. plants 9 mammalian cell culture for production of recombinant proteins: a review of the critical steps in their biomanufacturing delayed treatment of ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques extremely high-level and rapid transient protein production in plants without the use of viral replication peaq: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants goldenbraid: an iterative cloning system for standardized assembly of reusable genetic modules goldenbraid 2.0: a comprehensive dna assembly framework for plant synthetic biology generation of glyco-engineered nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like n-glycan structure: xylt and fuct down-regulation in n. benthamiana potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody molecular and biological characterization of human monoclonal antibodies binding to the spike and nucleocapsid proteins of severe acute respiratory syndrome coronavirus gb3.0: a platform for plant bio-design that connects functional dna elements with associated biological data sybodies targeting the sars-cov-2 receptor-binding domain automated production of plant-based vaccines and pharmaceuticals structural basis for potent neutralization of betacoronaviruses by single-domain camelid antibodies improvement of the transient expression system for production of recombinant proteins in plants bean yellow dwarf virus replicons for high-level transgene expression in transgenic plants and cell cultures the authors want to thank to the staff of the ibmcp and the polytechnic university of valencia who help us to access safely to our laboratory and greenhouses during the covid-19 lockdown in spain, and specially eugenio grau for his readiness to help us with sanger sequencing during that period. we are grateful to prof. steinkellner and strasser for providing glycoengineered plant lines and to prof. gleba for sharing magnifection plasmids. ministry of science and competitiveness and r.m. is recipient of a gva fellowship (acif/2019/226). key: cord-338517-1mxcssjj authors: ishay, yuval; kessler, asa; schwarts, asaf; ilan, yaron title: antibody response to sars‐co‐v‐2, diagnostic and therapeutic implications date: 2020-08-26 journal: hepatol commun doi: 10.1002/hep4.1600 sha: doc_id: 338517 cord_uid: 1mxcssjj the immune response against sars‐cov‐2 is comprised of both cellular and humoral arms. while current diagnostic methods are mainly based on pcr, they suffer from insensitivity. therefore, antibody‐based serological tests are being developed to achieve higher sensitivity and specificity. current efforts in treating sars‐cov‐2 infection include blocking of viral entry into the host cells, prohibiting viral replication and survival in the host cells, or reducing the exaggerated host immune response. administration of convalescent plasma containing anti‐viral antibodies was proposed to improve the outcome in severe cases. in this paper, we review some of the aspects associated with the development of antibodies against sars‐cov‐2 and their potential use for improved diagnosis and therapy. sars-cov-2 is an infectious rna virus responsible for causing the covid-19 disease (1) . while current diagnostic methods for covid-19 diagnosis are mainly based on pcr, they suffer from insensitivity. widespread reports of both false positive tests and false negative tests have been reported. therefore, serological tests are being developed to identify patients suffering from covid-19, and to assist in identifying subjects who have been diseased and may now be immune to re-infection or to severe disease. the host immune response mounted towards the virus contributes to disease severity. the immune response towards sars-cov-2 is comprised of both the cellular and humoral arms. current evidence points to the severe manifestation of covid-19 disease as being driven by inappropriate hyperactivation of the immune system, associated cytokine storm, and end organ damage (2, 3) . current efforts for the treatment of covid-19 include blocking of viral entry into the host cells, prohibiting viral replication and survival in the host cells, or reducing the exaggerated host immune response. however, these strategies have shown limited efficacy (4) . administration of convalescent plasma was proposed to improve patient outcomes in severe cases. in this paper, we review some of the aspects associated with the development of antibodies against sars-cov-2, their biology, potential uses, expected advantage, and disadvantages. sars-cov-2 is an enveloped, single-stranded rna virus. the viral genome encodes four structural proteins including the spike (s), envelope (e), membrane (m), and nucleocapsid (n), as well as other non-structural proteins. the s protein of the sars-cov-2 consists of two subunits, s1 and s2. acting as a homotrimer, the heavily glycosylated s protein binds its cellular receptor, angiotensin converting enzyme 2 (ace2), present on the pneumocytes and enterocytes, via the c-terminal domain of the s1 subunit, in the receptor binding domain (rbd) region (5, 6) . extending from the viral membrane, the s protein extends outward from the virion. while the s1 subunit extends furthest beyond from the virus membrane, the inner s2 subunits consists of a mostly helical structure, leading towards the viral membrane. the interaction of the s1-ace receptor leads to conformational changes in the helical s2 subunit. the next event in viral binding and entry includes cleavage of the s1/s2 protein subunits by cellular proteases. this proteolytic activity may be performed via furin protease, a feature not unique to the sars-cov-2 among the coronaviruses, but absent in sars-cov (7) . the cleaving protease, dictating the exact exposed viral amino acid sequence, also determines the pattern of viralcell fusion (8, 9) . the release of newly constructed virions, and the later activities of these new virions are also dependent on specific protease activity (6) . among the sites enumerated in this description, several appear as attractive targets for biologically active antibodies. of note, while new data is continuously and vigorously obtained, specifically regarding sars-cov-2, much of the functional data regarding coronavirus activity and mechanisms come from the research on sars-cov and mers-cov. this appears particularly poignant where homologies in the structure and function between these viruses are sought. while sequence and biological similarities are common, major differences exist, influencing virus function and antibody biology. these range from matters such as cleavage by similar proteases, though sars-cov-2 shows unique furin sensitivity, to receptor binding, where it shares the affinity towards ace2 with sars-cov, through highly conserved rbd residues(10). the final event of protective and effective antibody production is the differentiation of b-cells into plasma cells, a change accompanied by robust antibody production. a fraction of these cells will differentiate into memory bcells, allowing for an early antibody response upon re-infection, and have been demonstrated after sars-cov infection (11) . presumably, the "first contact" of the sars-cov-2 with the immune system occurs upon introduction of viable viral particles into the airways. the very first responding part of the immune system may be the epithelial cells themselves, both acting as antigen presenting cells (apcs) (12) and internally expressing antiviral proteins, specifically type-i interferons (13) . type-i interferon signaling is usually initiated via toll like receptors (tlrs). variance in the vulnerability to the virus, namely men being more vulnerable, has been attributed partially to a superior tlr7 signaling in women, possibly resulting in the enhanced antibody production (14) . notably, tlr7 functions in b-cells as well,and may contribute to enhanced function and differentiation of plasma cells (15) . following initial contact with epithelium, innate immune cells come in contact with the virus and with infected cells. the superficial intraepithelial dendritic cells (dcs) in the lungs adjacent to the airways are required for antibody production(16). after antigen encounter, they will move to the regional lymph nodes and help trigger robust antibody production by activation of cd4 "follicular helper" t-cells, supporting b-cell function (17) . some this article is protected by copyright. all rights reserved dc functions, including type-i interferon secretion in response to viral stimulation, is also dependent on tlr signaling (18) . while existing research is focused on the endogenic immune response to sars-cov-2, and its possible beneficial manipulations, isolation of neutralizing antibodies (nabs) from the infected persons or laboratory manufacturing of these antibodies is another subject of intense interest. monoclonal antibodies (mabs) with some neutralizing activities were demonstrated to occur in the infected human sera (19) . nabs may be defined in various ways; commonly as the antibody concentration required to prevent or decrease the infectivity (20) . the most attractive antibodies are those targeting the s protein, whether in the rbd or other regions, including the s1/s2 proteolytic cleavage site (21) . it is plausible that the antibodies targeting these sites will block the essential viral functions including viral-antigen binding (expected from s1-rbd antibodies) and/or interfere with s protein-mediated viral fusion or cell entry (21) (22) (23) . multiple specific regions in sars-cov-2 show high homology to the sars-cov virus, suggesting potential b and t cell epitopes for sars-cov-2 (24) . a set of b cell and t cell epitopes were derived from the s and n proteins which, excluding notable differences, are generally conserved between sars-cov and sars-cov-2. the lack of mutations in these identified epitopes allows assessment of possible sars-cov-2 immune targets (25) . this study showed no mutations occurred between sars-cov and sars-cov-2 in these sequences, confirming the possibility of antibody cross reactivity and humoral immunity. in spite of this high homology, cross-reactivity of sars-cov antibody is limited between two viral s proteins (26, 27) . murine polyclonal sars-cov antibodies directed against the s protein inhibited sars-cov-2 entry into cells, indicating that the cross-nabs targeting conserved s epitopes can be produced (6) . s1-targeting mabs from immunized transgenic mice expressing human ig variable heavy and light chains can neutralize sars-cov-2 and sars-cov infections (28, 29) . in a previously mentioned trial, 206 sars-cov-2 rbd-specific mabs were generated, among which, only two clones showed significant blocking of viral entry, associated with a high competitive capacity against ace2 receptor binding (19) . similar results were observed in the studies using sera from recovered sars and covid-19 patients, where limited crossneutralization occurred, suggesting that cross nabs are either incompletely reactive or insufficient for disease prevention (28) . prior to and concurrently with the isolation of specific antibodies, sars-cov s1-specific serum from convalescent sars patients or from animals was proposed to cross-neutralize the sars-cov-2 infection by reducing s proteinmediated sars-cov-2 entry (30) . cross reactivity of the antibodies from sars-cov-2 patients against the s proteins, but not against the rbd of sars-cov and mers-cov, has been documented. the roles played by rbd in the invasion of sars-cov-2 into host cells make it a potential target for nabs. blocking of binding between the rbd and its respective receptor may restrict the conformational change of s, or hamper the s2-mediated membrane fusion, thereby inhibiting the viral infection of host cells (21) . the human nabs s230.15 and m396 were isolated from sars-covinfected patients. they neutralize sars-cov infection by interacting with the rbd and by blocking the binding between viral rbd and ace2 receptor (31) . the sars-cov rbd-specific human nabs, cr3022 binds sars-cov-2 rbd with high affinity and recognizes an epitope on the rbd that does not overlap with the ace2-binding site (27) . the s109.8 and s227.14 mabs can neutralize the infectious clones of sars-cov, and protect the mice against four different homologous and heterologous sars-cov strains (32, 33) . of note, such mabs produced in the chimeric mouse cells and originating from sars-cov patients were shown to neutralize sars-cov-2 virus particles by an ace2-independent mechanism, which probably has to do with s protein fusion or proteolysis and preventing viral fusion (28) . while these studies hold both promise and interest, isolation and analysis of neutralizing antibodies remains a difficult task. a majority of 26 patients recovered from covid-19 showed high titers of sars-cov-2 s1-specific igg antibodies when tested by enzyme-linked immunosorbent assay (elisa) (29) . however, only 3 out of these 26 patients manifested effective blockade of sars-cov-2 rbd binding to hace2 when tested in vitro (29) . the transient and dynamic conformational states of the s protein have been suggested to provide a narrow window for an exposure of the immunogenic epitopes of rbd to b lymphocytes (34) . early and transient peak levels of anti-s antibody response were associated with a less favorable outcome for the patients, compared with a more delayed and sustained response (35) . the phage display method, allowing rapid and wide display of proteins directly correlated to their associated genes, can detect nabs against sars-cov from both naïve and immune antibody libraries, capable of blocking the binding of s1 domain, thereby showing virus neutralization and prophylaxis capability either in vitro or in the animal models (31, 33, 36) . another method, possibly allowing the production and utilization of existing nabs, may include the use of epstein-barr virus (ebv) transformation of human b cells to improve the isolation of nabs from the memory b cells harvested from the sars-cov infected patients (11) . transgenic mice with human immunoglobulin genes are being developed to produce nabs against sars-cov by antigen immunization, which are effective for virus prophylaxis in animal models (37, 38) . cloning of human mabs using samples from covid-19-recovered patients whose sera showed hace2 receptor binding inhibition has been reported (39) . following antibody cloning, three pairs of igg variable heavy chain and light chain inserted expression plasmids were expressed and named as 311mab-31b5, 311mab-32d4, and 311mab-31b9. all three mabs bind to the rbd protein. mab-31b5 and 311mab-32d4 blocked sars-cov-2 rbd-hace2 interaction and neutralized a sars-cov-2 s pseudotyped lentiviral particle (28) . the 311mab-31b5 and 311mab-32d4 neutralized pseudovirus entry into host cells ectopically expressing hace2 (29) . several nabs, such as b1, 1f8, and 5e9, towards epitopes on sars-cov s2 manifested neutralization properties (40, 41) . this article is protected by copyright. all rights reserved nucleocapsid-specific antibodies have been also demonstrated in the sera of infected patients. most studies assessing nucleocapsid antibodies have not differentiated these antibodies from other antibodies directed against sars-cov-2 studies that have seem to show similar kinetics to that of the general antibody response (42) . no studies have shown the occurrence of definitive nabs directed at the n protein or the nature of the immune response triggered by such antibodies. serum igg, igm, and iga antibodies against sars-cov appeared in the patients after primary sars infection (43) . data on the production of igg and igm is important for improved diagnosis of covid-19 (44) . several studies have described the dynamics of antibody production in these patients. while it is too early to definitively summarize the characteristics of antibody dynamics, certain conclusions seem consistent across these studies. broadly, antibody titers increase and the prevalence of viral rna decrease as time progresses from the symptomatic disease onset (45, 46) . elisa-based diagnostic kits often report a specificity of ~90% (47), with some trials reporting higher percentage (45) . while this is an impressive figure by itself, it may yield a relatively poor positive predictive value (ppv) when employed on a large scale to a disease with relatively low prevalence. elisa tests were argued to be efficient when trying to augment the sensitivity of testing of close contacts (46, 48) , or deciding to allow a person to leave from quarantine. this specificity may be further reduced when testing a person recently exposed to the milder coronaviruses circulating within humans and livestock. however, to our knowledge, this question has not been directly assessed. igg and igm antibodies may appear simultaneously or sequentially, with cases of igm antibodies appearing last being described in some of the studies (48) . conversion from seronegativity to seropositivity is likely to occur between 14-21 days after the onset of symptoms. data from some of these studies show that the patients with more severe illness were more likely to mount a high-titer and high-affinity antibody response, which was not necessarily associated with a reduction in the viral rna assayed from their blood (49) . this is supported by the reports of recurring pcr positivity after igg seroconversion (50) . if these studies become the prevalent findings, they may stand in a stark contrast to well established viral disease behaviors where high igg levels are thought to denote virtual immunity to the disease, allowing, at most, a mild manifestation upon re-exposure. it seems that in covid-19, as our current understanding stands, antibody titers should be thought of as the disease markers and not as the definitive markers of immunity or disease resolution in the actively ill. in the antibody detection, different elisa kits, based on the recombinant sars-cov-2 nucleocapsid protein (rn) and recombinant spike protein (rs), show variable results. in a study of 214 patients with confirmed covid-19, 68% were diagnosed with rn-based igm, 70% with an igg, 77% with rs-based igm, and 74% with igg tests. the positive rates for rn-based and rs-based elisa detections were 80% for igm and 82% for igg. the sensitivity of the rs-based elisa for igm was higher than that of the rn-based test. here also, the antibody positivity increased as disease time progressed (51) . another stratum of results expected from antibodies is the identification of immune and recovered persons who may be able to work in the critical locations during the times of pandemic. the ability to definitively identify specific nabs in the serum of recovered patients could also allow identifying the potential plasma donors for the development of passive immunization, and may assist in evaluating the effectiveness of various treatments in addition to assisting in determining the prognosis (52) . most convalescent plasmas obtained from individuals who recover from covid-19 do not contain high levels of nabs. a recent analysis of 149 covid-19 convalescent individuals evaluated plasmas collected an average of 39 days after the onset of symptoms showing variable half-maximal pseudovirus neutralizing titers below 1:50 in 33% and below 1:1,000 in 79%. only 1% showed titers above 1:5,000. expanded clones of rbd-specific memory b cells expressing closely related abs in different individuals were identified. the abs were directed against three distinct epitopes on rbd. rare but recurring rbdspecific antibodies with potent antiviral activity were identified in all subjects recovered (53) . the relevance of the titers for the clinical effect are yet to be determined. a recent review analyzed the diagnostic accuracy of antibody tests for sars-cov-2 infection, for assessing past infections and for use in seroprevalence surveys (54) . a total of 57 publications reporting cohorts with 15,976 samples, of which 8526 were from cases of sars-cov-2 infection, were evaluated. substantial heterogeneity in sensitivities of iga, igm and igg abs, or combinations thereof, for results aggregated across different time periods postsymptom onset. pooled results for igg, igm, iga, total antibodies and igg/igm showed low sensitivity during the first week since onset of symptoms, rising in the second week and reaching their highest values in the third week. the sensitivity of antibody tests was proposed to be too low in the first week since symptom onset, to have a primary role for the diagnosis, but were suggested to have a role complementing other testing in individuals presenting later, when rt-pcr tests are negative. antibody tests are useful for detecting previous sars-cov-2 infection if used 15 or more days after the onset of symptoms (54) . several currently-available covid-19 antibody tests that are used in diagnostics and epidemiology, with a focus on their strengths and weaknesses are summarized in table 1 . the lack of specific sars-cov-2-targeted treatments and vaccines poses great challenges for the management of patients with severe illness. igg levels against sars-cov, drawn from affected patients, reach peak serum concentration during the convalescent phase and are reduced following recovery (55) . while the capacities of antibodies to neutralize the virus were highly variable in the required concentration, some of them indeed showed such capability, and have been shown to provide protection against re-infection in a mouse model (11) . use of convalescent plasma and development of nabs are attractive methods for the treatment of viral infections (56, 57) . blocking mabs with high antigen specificity were proposed as potential candidates for neutralizing infections (58) (59) (60) . convalescent plasma has intermittently emerged this article is protected by copyright. all rights reserved during the last few decades as a treatment for various infectious diseases (61) (62) (63) , enjoying attention whenever diseases prove resistant to more conventional treatment methods. plasma-derived nabs can provide passive immune responses to viral infections and were effective in patients with severe illnesses caused by other viruses (64, 65) . a meta-analysis showed that the mortality was reduced after receiving various doses of convalescent plasma in the patients with severe acute respiratory infections, with no adverse events or complications after treatment (66) . antibodies from convalescent plasma were proposed to reduce the viremia by enhancing viral clearance, blocking infection of new cells, and contributing in the clearance of infected cells (57, 67, 68) . during the 2003 sars epidemic, severely ill patients who deteriorated despite the treatment with methylprednisolone were given convalescent plasma at around 14 th day of the disease onset. earlier plasma administration correlated with a better prognosis and higher rate of hospital discharge at day 22 (69) . convalescent plasma or immunoglobulins were effective in sars patients whose condition continued to deteriorate. some studies suggested a shorter hospital stay and lower mortality rate following convalescent plasma administration (57, 64, (70) (71) (72) (73) (74) . a similar trend for the treatment timing was described in 27 patients with lassa fever in nigeria treated with convalescent plasma (75) . the empirical use of convalescent plasma for ebola virus disease showed some positive results (76) (77) (78) . experimental and clinical data on the use of convalescent plasma products and humanized monoclonal antibodies for h5n1 influenza infection have also shown positive outcomes, and this treatment was proposed as a mean for overcoming anti-viral drug resistance (62, 79, 80) . in a study involving 20 patients with severe pandemic influenza a (h1n1) 2009 virus infection, administration of convalescent plasma reduced respiratory tract viral load, serum cytokine response, and mortality (81) . a prospective cohort study during the 2009 pandemic showed reduction in the relative risk of mortality in the patients treated with convalescent plasma, demonstrating reduction of the viral loads without any adverse effects (60) . a randomized trial of convalescent plasma failed to achieve its primary end point, a reduction of mortality; however, a subgroup multivariate analysis performed on 22 of the 35 patients enrolled in the trial demonstrated that h-ivig treatment was the only factor independently associated with reduced mortality (82) . development of nabs against sars-cov-2 was proposed as a method for developing therapeutic agents for covid-19 (23, 36, 83, 84) . several sars-cov-2 proteins (discussed above) prove attractive targets for nabs. the sars-cov-2 s protein is a target for developing nabs to block its binding and fusion (36) . currently, no sars-cov-2-specific nabs have been reported (21) . however, polyclonal antibodies from recovered sars-cov-2-infected patients are being used to treat the patients with severe infections. while many patients will develop an antibody response following their illness, specific characterization of these antibodies and their properties as nabs has yet to be determined (48, 85) . early administration of convalescent plasma was advised in order to maximize its viral clearance effect (86) . this article is protected by copyright. all rights reserved plasma collection is done via apheresis. in order to qualify for the donation, the donor must meet several conditions: diagnosis of prior covid-19 infection confirmed by pcr, donation needs to take place 14-28 days after resolution of the symptoms followed by two consecutives negative pcr results, donors need to be tested for absence of transmissible pathogens, donation should be done from male or nulliparous female donors, with no previous exposure to blood products in order to minimize the risk of transfusion associated acute lung injury (trali). plasma (200-600 ml) is donated according to the abo compatibility. pathogen inactivation measures need to be undertaken (87) . it is advised to administer up to two units of plasma, possibly from two different donors (87) . several studies that described the administration of convalescent plasma to critically ill covid-19 patients suggested post transfusion viral elimination and clinical improvement. a study of critically ill patients (n=5) reported clinical improvement in patients' status and laboratory indication of viral clearance for up to 12 days post transfusion of two consecutive doses of convalescent plasma (total 400 ml).(88) three of the patients were on mechanical ventilation and two on ecmo, and the treatment was provided between 10-20 days after hospitalization, following which improvement in fever, pao2/fio2 ratio, and viral clearance were noted. three patients were discharged from the hospital, and two were in stable condition at the end of the follow-up period. although a clinical effect was obtained, the delay of up to three weeks in the administration, and the concurrent use of other therapies, make it difficult to assess the effect of plasma (89) . administration of convalescent plasma in six critically ill patients was followed by discontinued viral shedding three days after infusion without reducing the mortality (90) . a study in six covid-19 patients showed clinical, radiological, and laboratory improvement following administration of abo-compatible convalescent plasma indicating that this therapy is effective and specific (91) . in a study of 10 severe patients administration with 200 ml of convalescent plasma, showed improved clinical, laboratory, and radiological status without severe adverse effects (92) . in this study, the antibody titers of donor's plasma were assessed and found to be elevated in the majority of donors, along with a concurrent increase in nabs titers in the patients' sera following transfusion. treatment within two weeks of initial symptoms has improved the response (93) . differences in the outcomes between the studies may reflect temporal variations of administration including the time lag between plasma donation and administration as well as the time from disease detection to treatment. safety evaluation of candidate antibodies must not be overlooked. although antibodies are generally protective, the antibody-dependent enhancement (ade) phenomenon of viral infections is documented for dengue virus and other viruses (94) . in sars-cov infection, ade is mediated by the engagement of fc receptors (fcrs) expressed on various immune cells, including monocytes, macrophages and b cells (95) . pre-existing sars-covspecific antibodies were proposed to promote viral entry into fcr-expressing cells. internalization of virus-antibody immune complexes may induce inflammation and tissue injury by activating myeloid cells via fcrs (95) . this article is protected by copyright. all rights reserved figure 2 presents several putative and proven nabs interactions in covid-19, such as antibody targets and functions including those associated with disruption and non-disruption binding mechanisms, and those targeting the virus itself. in addition, non-neutralizing antibodies, cross reactive antibodies, and antibodies with low specificity or low titers, which are unable to act as nabs, are also generated. several large trials using convalescent plasma are being conducted (96) . identifying and cloning mabs that target viral proteins to block the entry into host cells is being explored for preventing and treating covid-19 (29, 36) . computational simulation of antibody-antigen complexes can improve the design of these therapies. key residues between rbd and nabs can be identified, and models are being used to assess the interaction between s protein and human ace2 or antibodies (27, 36, (97) (98) (99) (100) . several methods for improving the effectiveness of convalescent plasma or nabs are being considered. the outcomes of passive convalescent plasma therapy from recovered donors are unpredictable due to variability among the donors in both the levels and types of antibodies (101) . appropriate selection of the donors is required for improving the quality of the collected plasma. assessment of the antibody titers needs to be performed prior to harvesting due to a marked variability in titers among the donors. titers correlate with the disease severity, timing of donation, use of steroids during acute illness (102, 103) , and quality of antibodies (i.e. whether they are nabs or not) nonwithstanding. timing of plasmapheresis is a major factor as lower levels of antibodies are detected within the first two weeks following recovery (48) . more data is required on the amount of virus neutralization by antibodies upon exposure to convalescent plasma. in vitro testing for neutralizing and/or crossneutralizing activity, and in vivo evaluation in available covid-19 animal models for protective efficacy, along with preclinical studies and clinical trials testing the safety and efficacy, are needed for optimizing this therapeutic option (21) . the gender of the donor also plays a role in mounting a significant response. the degree of activation of the immune cells is higher in women than in men, which correlates with the triggering of tlr7 and production of proinflammatory cytokines. tlr7 is expressed in innate immune cells, which recognize single strand rna virus by promoting the production of viral antibodies and generation of il-6 and il-1 inflammatory cytokines. tlr7 is higher in women than in men and its expression may lead to better immune responses and increased resistance to viral infections (14) . pairing hla-typing with covid-19 was proposed to improve the assessment of disease severity and assist in preferred donor selection (104) . the use of hyper-immune globulin rather than whole plasma was proposed for improving efficacy and validity of the therapy. the main advantages are associated with an ability to provide the patients with controlled quantities of antibodies in lower volumes (105) . similar techniques for concentrating this article is protected by copyright. all rights reserved antibodies are being used for the treatment and prevention of other diseases (106) . this is similar to the concept of using hyper-immune globulin for various indications, including viral diseases in immunocompetent and immunocompromised hosts (107) (108) (109) . a "cocktail antibody approach" for sars-cov-2 was proposed based on the studies suggesting that the combination of antibodies from diverse donors may exert a synergistic neutralization effect (36) . a mixture of two antibodies showed a synergistic neutralization effect due to recognition of different epitopes on rbd (110) . the use of immune adjuvants may also improve the response to the antibodies (111) . sphingolipid-based adjuvants, when administered with antibodies, augmented the anti-viral response (112) and improved the systemic anti-inflammatory effects of antibodies (113) . the use of hyper-immune bovine colostrum comprised of antibodies and sphingolipids was effective in reducing systemic inflammation (114) (115) (116) . mode of antibody administration may also have an impact on the effect of antibody-based therapy. oral administration of antibodies ameliorated viral-mediated chronic inflammation via promotion of regulatory t cells (117) , and oral administration of viral antigens augmented an anti-viral immunity while reducing the inflammation (118, 119) . the data on the possible harmful effects of antibody-mediated immune response in the development of pulmonary complications of sars-cov is controversial. several patients died of sars manifested strong nabs responses and pulmonary inflammation, suggesting that the nabs could be associated with the deterioration of the lung disease (36, 120) . similar notions have been proposed as explaining the more severe phenotype of covid-19 prevalent in china. this may be related to the higher degree of exposure to milder coronaviruses and a "priming" of the immune system by pre-existing antibodies, leading to immune dysfunction and over-function (121) . this notion is supported by the mild disease manifestations in the patients with agammaglobulinemia (122) . previous exposure to coronaviruses may also explain a relatively high prevalence of spike protein-reactive cd4 cells in the healthy donors in a study (123) . a major obstacle for implementing immune-based therapies for the viruses, including the administration of mabs, is associated with the development of viral resistance due to the immune evasion mechanisms, which the virus generates in response to the immune-pressure imposed on it by the immunomodulatory agents (124) . prolonged exposure to the anti-viral drugs is associated with drug resistance, leading to persistent viremia or severe disease. in cases where anti-viral treatment is highly effective leading to viral elimination, resistance is less likely to occur. however, immunotherapy, including the administration of antibodies, are associated with selective pressure that may result in rapid viral and host adaptations leading to resistance to the therapy (125) . both host and viral factors are associated with the development of resistance. viral-related tools include mechanisms of viral replication, genomic inference, and high rates of viral mutations (125, 126) , (127) . an immune adaptation process towards antibody-induced pressure on the virus or on anti-viral humoral and cellular responses may limit the efficacy and longevity of these therapies. combination of several potent nabs could improve the sensitivity to neutralization (36) . methods for overcoming resistance by implementing host-tailored variability are being developed based on the data generated from the use of these methods for improving the effects of other immunomodulatory drugs (128) (129) (130) (131) . these include implementing artificial intelligence methods for overcoming host compensatory responses in sepsis and its sequela (128) , and for improving the effects of adjuvants (130) . algorithmcontrolled treatment regimens are now being used in several clinical trials for overcoming drug resistance (nct03843697; nct03747705). the lack of accurate diagnostic and effective therapeutic methods for the sars-cov-2-infected patients led to the need of developing humoral-based approaches. while this approach holds promise, more data is needed for optimizing the antibody-based diagnosis, and for improving the implementation of convalescent plasma and other antibody-based therapies. the potential development of effective vaccines will benefit from the results achieved from these diagnostic and therapeutic attempts. immunotherapeutic methods are expected to require targeting the cellular arm of the immune system either in addition or as part of the design of antibody-based approaches, mainly for alleviating the immune-mediated target organ damage in covid-19. this article is protected by copyright. all rights reserved sars-cov-2: severe acute respiratory syndrome coronavirus 2; s protein: spike protein; s1 and s2: s protein subunits; rbd: receptor binding domain; abs: antibodies; nabs: neutralizing antibodies; and ace2: angiotensin-converting enzyme 2. how to reduce the likelihood of coronavirus-19 (cov-19 or sars-cov infection and lung inflammation mediated by il-1 sars-cov-2: a storm is raging 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and colloidal goldimmunochromatographic assay kit for detection of novel coronavirus (sars-cov-2) causing an outbreak of pneumonia (covid-19) this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved accepted article key: cord-348455-vcxalkeo authors: graham, n. r.; whitaker, a. n.; strother, c. a.; miles, a. k.; grier, d.; mcelvany, b. d.; bruce, e. a.; poynter, m. e.; pierce, k. k.; kirkpatrick, b. d.; stapleton, r. d.; an, g.; botten, j. w.; crothers, j. w.; diehl, s. a. title: kinetics and isotype assessment of antibodies targeting the spike protein receptor binding domain of sars-cov-2 in covid-19 patients as a function of age and biological sex. date: 2020-07-22 journal: medrxiv : the preprint server for health sciences doi: 10.1101/2020.07.15.20154443 sha: doc_id: 348455 cord_uid: vcxalkeo sars-cov-2 is the newly emerged virus responsible for the global covid-19 pandemic. there is an incomplete understanding of the host humoral immune response to sars-cov-2 during acute infection. host factors such as age and sex as well the kinetics and functionality of antibody responses are important factors to consider as vaccine development proceeds. the receptor-binding domain of the cov spike (rbd-s) protein is important in host cell recognition and infection and antibodies targeting this domain are often neutralizing. in a cross-sectional study of anti-rbd-s antibodies in covid-19 patients we found equivalent levels in male and female patients and no age-related deficiencies even out to 93 years of age. the anti-rbd-s response was evident as little as 6 days after onset of symptoms and for at least 5 weeks after symptom onset. anti-rbd-s igg, igm, and iga responses were simultaneously induced within 10 days after onset, but isotype-specific kinetics differed such that anti-rbd-s igg was most sustained over a 5-week period. the kinetics and magnitude of neutralizing antibody formation strongly correlated with that seen for anti-rbd-s antibodies. our results suggest ageand sexrelated disparities in covid-19 fatalities are not explained by anti-rbd-s responses. the multi-isotype anti-rbd-s response induced by live virus infection could serve as a potential marker by which to monitor vaccine-induced responses. and infection and antibodies targeting this domain are often neutralizing. in a cross-sectional 23 study of anti-rbd-s antibodies in covid-19 patients we found equivalent levels in male and 24 female patients and no age-related deficiencies even out to 93 years of age. the anti-rbd-s 25 response was evident as little as 6 days after onset of symptoms and for at least 5 weeks after 26 symptom onset. anti-rbd-s igg, igm, and iga responses were simultaneously induced within 27 10 days after onset, but isotype-specific kinetics differed such that anti-rbd-s igg was most 28 sustained over a 5-week period. the kinetics and magnitude of neutralizing antibody formation 29 strongly correlated with that seen for anti-rbd-s antibodies. our results suggest age-and sex-30 related disparities in covid-19 fatalities are not explained by anti-rbd-s responses. the multi-31 isotype anti-rbd-s response induced by live virus infection could serve as a potential marker by 32 which to monitor vaccine-induced responses. 33 human pathogenic coronaviruses (cov) such as severe acute respiratory syndrome (sars)-37 cov-1, middle east respiratory syndrome (mers)-cov, and sars-cov-2 (all b-covs) have 38 resulted from zoonoses and utilize cellular receptors to bind and access host cells for productive 39 infection (1-3). cov spike (s) proteins are large (>200 kda) glycosylated trimeric structures that 40 protrude from viral particles and enable binding of cov to cellular receptors. sars-cov-2 41 interacts with angiotensin converting enzyme-2 (ace2) via a flexible receptor-binding domain 42 (rbd) located on the distal tip of the s protein (4-7). after binding, several proteases act upon 43 s, priming it to adopt large conformational shifts that facilitate entry into host cells(8). first the 44 s1 domain (which contains rbd) is cleaved from the c-terminal s2 domain. for sars-cov-2 45 this process may involve furin in the host cell membrane due to a novel furin-recognition site in 46 the s1/s2 region (9-11). the s2 domain is further processed by other serine and cysteine-47 proteases such as trypsin, cathepsin, and tmprss2 to facilitate viral entry into the host cell (4, 48 12). 49 neutralizing antibodies to sars cov-1 have been isolated and were found to target 50 rbd-s (13). one of these mabs cr3022 was also found to bind sars-cov-2 rbd-s(14). at 51 the polyclonal level, the quantity of anti-rbd s igg antibodies against sars-cov-2 correlate 52 well with neutralizing activity(15-18). cross-neutralization amongst sars viruses by rbd-s-53 targeting antibodies can occur (18-21). however, sequence homology for rbd-s is low for non-54 sars b-covs (such as mers) and for a-covs such as nl63, oc43, 229e, and hku1(16, 17). 55 for these reasons serology for sars-cov-2 rbd-s is being used to help identify recovered 56 covid-19 patients as plasma donors for passive immunotherapy (22). 57 there are several risk factors for covid-19 mortality but whether two of these -age and 58 biological sex -are associated with the sars-cov-2 rbd-s immune response has to our 59 knowledge not been addressed in the peer-reviewed literature. furthermore, most serology 60 studies have been done in the setting of severe covid-19 disease and, save for one study 61 (17), without the benefit of detailed kinetics. herein we tracked the kinetics and magnitude of 62 neutralizing and anti-sars-cov-2 s and rbd-s antibodies in a cross-sectional cohort of pcr-63 confirmed covid-19 patients. 64 65 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. we chose a two-step elisa-based rbd-s-focused approach to serology in our study 67 population. reagents and pre-print protocols were available in mid-march 2020, which indicated 68 that rbd-s screening and full-s confirmation could identify specific and functional antibodies 69 and be quickly operationalized. using the established protocol (23) we confirmed the expected 70 protein size of mammalian-expressed rbd-s ( figure 1a ) and trimerized spike ( figure 1b ) 71 produced from dna plasmids (gift from florian krammer, mt sinai school of medicine). rbd-s 72 antibodies were specific and correlated with neutralization (15), findings that have been 73 validated using similar rbd-s-focused assays(16, 17). we confirmed rbd-s and s protein 74 conformation by binding of cr3022 human igg1 (figure 1c, d) . cr3022 was isolated as a 75 sars-s1 domain-binding single chain antibody fragment by phage display and is neutralizing 76 as an igg1(13). cr3022 binds adjacent to rbd-s in trimeric s of sars-cov-2 in a 77 glycosylation-sensitive manner(14). mammalian expression of appropriate size proteins and 78 recognition by cr3022 together confirm that our protein preparations exhibited the expected 79 characteristics. 80 we first piloted our antigen preps for the rbd-s igg screening assay using serum 81 samples from a pcr-confirmed severe covid-19 patient (defined as admission to the intensive 82 care unit, icu) who was admitted to the hospital 10 days following symptom onset and based 83 on an early report suggesting that sars-cov-2 could trigger antibody responses in this 84 timeframe (24). we compared igg reactivity in this sample to decreasing amounts of our rbd-s 85 antigen preparations against a fixed, recommended amount of commercially produced rbd-s 86 protein derived from the protocol we used (23). we found that a wide range of locally produced 87 rbd-s antigen yielded igg reactivity equivalent to 100 ng of commercial antigen in an acute 88 serum sample from this covid-19-positive patient ( figure 1e) . no signal was observed in a 89 pre-2019 serum sample or in the absence of serum ( figure 1e ). using the standard 100 ng 90 amount hereafter, we found that rbd-s-binding igm and igg were present at 10-13 days after 91 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 22, 2020. . https://doi.org/10.1101/2020.07.15.20154443 doi: medrxiv preprint symptom onset. we did not detect any rbd-s-binding in healthy pre-2019 sera (figure 1f) , in 92 agreement with extensive testing of this assay in pre-covid-19 serum performed elsewhere 93 (15). due to different secondary antibodies for igm and igg detection we cannot conclude 94 whether absolute levels of rbd-s igg were higher than rbd-s igm. total igg and igm were 95 readily detected in both covid-19 and in healthy non-covid-19 serum ( figure 1g) . 96 for a cross-sectional covid-19 serological survey we collected serum samples from 32 97 patients that tested covid-19 positive by nasopharyngeal swab rt-qpcr testing. all patients 98 had been admitted to the hospital and 13/32 (40%) were admitted to the icu. twenty-five 99 patients were subsequently discharged and 7 died. one to five serum samples were collected 100 from each patient with the first sample being taken within approximately 9 days after diagnosis, 101 in which diagnosis occurred around 5 days after symptom onset ( table 1) . there was a 102 53%:47% male: female distribution and patients were on average 68 ± 14 years of age (range 103 30 -93 years) ( table 1) . 104 a male bias in covid-19 mortality was reported early during the pandemic (25-27) and 105 has been confirmed worldwide in a recent meta-analysis (28). one of the hypotheses to explain 106 this is differences in adaptive immunity between males and females. although the mean serum 107 rbd-s igg reactivity level appeared higher in male samples (o.d. = 1.8, n = 40) versus female 108 samples (o.d. = 1.0, n = 37) this difference was not significant and the same maximum 109 reactivity values were found in males and females (figure 2a) . 110 although not absolute, it appears that irrespective of comorbidities, there is a higher risk 111 of covid-19 mortality and morbidity in older individuals (60 years of age and over) (29-31). we 112 therefore assessed rbd-s igg antibodies by age. there was a broad range of rbd-s igg 113 responses that did not differ as a function of age as assessed by correlation analysis (r 2 < 0.01, 114 figure 2b ). notably, one of the highest rbd-s igg responses was from a 93-year old patient. a 115 serum sample from a 30-year old covid-19 patient was negative for rbd-s igg, but this 116 sample was taken just three days after symptom onset, which may be too early for induction of 117 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 22, 2020. . https://doi.org/10.1101/2020.07.15.20154443 doi: medrxiv preprint robust igg responses. taken together, we did not find evidence of biological sex-or age-related 118 deficiencies in rbd-s igg responses in covid-19 patients. 119 rdb-s-reactive serum igg was detected in 5 of 12 (42%) samples that were taken 120 within 10 days of symptom onset ( figure 2c ). after day 10 of symptoms, 98% of samples were 121 positive for rbd igg ( figure 2c ). there were small variations in positive threshold for rbd by 122 assay date ( figure s1 ). we therefore confirmed each sample (whether rbd-positive or not) 123 with an endpoint titration and area under the curve calculation for reactivity against the full spike 124 ectodomain trimer (15). samples that were rbd-s-negative were also low for spike total 125 reactivity (auc) and titer (figures 2d, e) . furthermore, we found a very strong correlation 126 between rbd and spike igg ( figure in the patient-specific rbd igg data ( figure s2a ) we found several patterns: initial 134 seroconversion (e.g. patients 0003, and 0017), rapid increases (e.g. patients 0005, 0006, 0009, 135 0011, 0020, occurring between days 10-20), and plateaued responses (e.g. patients 0012 and 136 0021, occurring mainly after day 20). these responses were concordant with temporal patient-137 specific s igg titers ( figure s2b ). anti-s titers in patients with a negative rbd-s test were 138 generally low and in rbd-positive samples, followed the same trends as rbd-reactivity, 139 providing further confirmation of robust serological responses to sars-cov-2 during acute 140 covid-19. at the patient level, neutralizing activity was observed after as few as five days after 141 symptom onset and throughout the study period and was predominantly found in those samples 142 with positive rbd-s igg ( figure s3) . 143 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 22, 2020. . https://doi.org/10.1101/2020.07.15.20154443 doi: medrxiv preprint to assess antibody isotype dynamics during acute sars-cov-2 we followed rbd-s 144 and full spike-specific igm and iga levels in the same samples for which rbd-s and spike igg 145 was determined. at the patient level we found robust co-occurrence of igm, igg, and iga 146 antibodies reactive to rbd-s in most samples, particularly in post-day 10 samples (figure s4) . 147 pooling all the data revealed that all pre-day 10 rbd-s responses for all isotypes were low. 148 around day 10, igm targeting rbd-s as well as the switched isotypes igg and iga 149 simultaneously rose. while rbd-reactive igm and iga responses tapered after 3 weeks post-150 onset (though remained higher than baseline), those for igg continued to rise to a plateau that 151 was sustained up to 5 weeks after symptoms onset (the most protracted timepoint measured, 152 figure 3a ). similar patterns were obtained for full spike-reactive antibodies ( figure 3b ). these 153 results suggesting that during acute infection covid-19 patients undergo a seroconversion 154 across isotypes to sars-cov-2 rather than an expansion of pre-existing anti-cov antibodies. 155 lastly, we assessed anti-rbd-igg responses by clinical severity. all the patients in this 156 study were hospitalized and 40% of were admitted to the intensive care unit. when we stratified 157 by icu admission and compared rbs-s igg levels, we found a trend towards higher levels in 158 those requiring icu-level care (p = 0.09) ( figure 4a) . additionally, we observed a significant 159 association between rbd-s igg and duration of icu admission ( figure 4b ). lastly 7 of 32 160 (22%) patients succumbed to covid-19. while a significant difference in the median rbd-s 161 igg was not observed between survivors and decedents, a smaller range trending towards 162 higher rbd-s reactivity was observed in those patients that died ( figure 4c ). although we did 163 not have continuous monitoring of viral load in these patients during hospitalizations it is 164 possible that rbd-s igg levels reflect ongoing viral replication during more severe disease and 165 in conjunction with other factors may allow for recovery. 166 taken together, our results provide the first comprehensive survey of sars-cov-2 spike 167 rbd antibodies that accounts for two key risk factors for covid-19. neither rbd-s nor s 168 antibodies were significantly different as a function of biological sex. anti-rbd-s and spike igg 169 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 22, 2020. . https://doi.org/10.1101/2020.07.15.20154443 doi: medrxiv preprint responses were induced across 6 decades of age with robust responses found in several 170 samples from patients ≥ 80 years old. these results also extend kinetic analyses and confirm 171 the paucity of anti-sars-cov-2 anti-spike responses in very early blood samples taken prior to 172 day 10 after symptoms onset (17, 24). we also assessed protective anti-spike rbd responses 173 as a function of level of hospital care and disease severity and found that duration of icu-level 174 care was associated with higher responses, possibly due to an extended period of sars-cov-2 175 replication during severe disease. a limitation of our study is that we only followed symptomatic 176 patients admitted to hospital; it is unclear whether antibody responses differ in asymptomatic or 177 mildly symptomatic patients. we also did not directly assess whether the rbd-specific 178 antibodies we studied were neutralizing at the clonal level, though we did observe a strong 179 association with polyclonal rbd-s igg responses and sars-cov-2 neutralizing activity. this is 180 in agreement with other reports which confirm that rbd-s igg levels correlate with neutralizing 181 activity and that the rbd of sars-cov-2 is a potent target for neutralizing antibodies (16-18, 182 20, 21, 33). it will be important to determine whether anti-rbd iga or even igm antibodies 183 contribute to blocking activity. 184 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 22, 2020. was also used as a positive control during assay set up and this reagent was produced in 208 hek293t cells under hhsn272201400008c and obtained through bei resources, niaid, nih: 209 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 22, 2020. . https://doi.org/10.1101/2020.07.15.20154443 doi: medrxiv preprint wuhan-hu-1, recombinant from nr-52306. 211 cr3022 is a sars-cov s-specific antibody originally isolated by single chain variable region 213 phage display and then cloned as an igg1/kappa monoclonal human igg1/k (13). we received 214 cr3022 heavy chain (hc) and light chain (lc) cloned into pfusess-chig-hg1 and pfuse2ss-215 clig-hk, respectively (invivogen) from florian krammer spotted on filter paper. we (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 22, 2020. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 22, 2020. graphics and statistical testing. 285 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 22, 2020. . https://doi.org/10.1101/2020.07.15.20154443 doi: medrxiv preprint all statistics and graphics were performed using r version 3.6.1 using standard packages or 286 graphpad prism 8.4.3. non-parametric loess (local regression) was used for smoothing. 287 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 22, 2020. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 22, 2020. detection of serum igg from a covid-19 patient (left), but not from pre-2020 serum (center) or 421 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 22, 2020. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 22, 2020. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 22, 2020. . https://doi.org/10.1101/2020.07.15.20154443 doi: medrxiv preprint and 95% confidence intervals are shown for each isotype. 443 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 22, 2020. spearman's rho coefficient (r 2 ), 95% confidence interval, and p-value are shown. (c) rbd-s 449 igg in patients that were deceased or discharged were analyzed by student's t-test and p-value 450 is shown. boxplots show the median, 95% confidence level, and all individual samples. 451 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 22, 2020. . https://doi.org/10.1101/2020.07.15.20154443 doi: medrxiv preprint origin and evolution of pathogenic coronaviruses genomic characterisation and 314 epidemiology of 2019 novel coronavirus: implications for virus origins and receptor 315 binding a pneumonia outbreak 317 associated with a new coronavirus of probable bat origin sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically 320 proven protease inhibitor receptor recognition by the novel 322 coronavirus from wuhan: an analysis based on decade-long structural studies of 323 sars coronavirus site-specific glycan 325 analysis of the sars-cov-2 spike cryo-em 327 structure of the 2019-ncov spike in the prefusion conformation coronavirus spike protein and tropism 330 changes cleavage site in the spike 332 protein of sars-cov-2 is essential for infection of human lung cells phylogenetic analysis 335 and structural modeling of sars-cov-2 spike protein reveals an evolutionary distinct 336 and proteolytically sensitive activation loop proteolytic cleavage of the sars-cov-2 spike 338 protein and the role of the novel s1/s2 site. iscience characterization of spike glycoprotein of 340 sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov a highly conserved cryptic epitope 346 in the receptor binding domains of sars-cov-2 and sars-cov 349 a serological assay to detect sars-cov-2 seroconversion in humans the receptor binding domain of the viral spike protein is an immunodominant and highly 353 specific target of antibodies in sars-cov-2 patients rapid generation of neutralizing antibody responses in covid-19 patients human neutralizing antibodies elicited 358 by sars-cov-2 infection antigenicity of the sars-cov-2 spike glycoprotein broad 366 neutralization of sars-related viruses by human monoclonal antibodies convalescent 368 plasma treatment of severe covid-19: a matched control study temporal profiles of 374 viral load in posterior oropharyngeal saliva samples and serum antibody responses 375 during infection by sars-cov-2: an observational cohort study characteristics of and important lessons from the 378 coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 379 72314 cases from the chinese center for disease control and prevention epidemiological and clinical 382 characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a 383 descriptive study clinical characteristics of 385 coronavirus disease 2019 in china considering how 387 biological sex impacts immune responses and covid-19 outcomes estimates of the 390 severity of coronavirus disease 2019: a model-based analysis age and 393 multimorbidity predict death among covid-19 patients: results of the sars-ras 394 study of the italian society of hypertension disease and healthcare burden of 397 covid-19 in the united states serological assays for emerging coronaviruses: 399 challenges and pitfalls :8,100) and plotted against days of symptoms. (e) spike igg endpoint titer or (f) auc 433 is plotted against rbd-igg reactivity. (g) sars-cov-2 microneutralization titers are plotted 434 against rbd-s igg reactivity. cutoff values no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted key: cord-315437-h6xjudm0 authors: nyon, mun peak; du, lanying; tseng, chien-te kent; seid, christopher a.; pollet, jeroen; naceanceno, kevin s.; agrawal, anurodh; algaissi, abdullah; peng, bi-hung; tai, wanbo; jiang, shibo; bottazzi, maria elena; strych, ulrich; hotez, peter j. title: engineering a stable cho cell line for the expression of a mers-coronavirus vaccine antigen date: 2018-03-27 journal: vaccine doi: 10.1016/j.vaccine.2018.02.065 sha: doc_id: 315437 cord_uid: h6xjudm0 abstract middle east respiratory syndrome coronavirus (mers-cov) has infected at least 2040 patients and caused 712 deaths since its first appearance in 2012, yet neither pathogen-specific therapeutics nor approved vaccines are available. to address this need, we are developing a subunit recombinant protein vaccine comprising residues 377–588 of the mers-cov spike protein receptor-binding domain (rbd), which, when formulated with the addavax adjuvant, it induces a significant neutralizing antibody response and protection against mers-cov challenge in vaccinated animals. to prepare for the manufacture and first-in-human testing of the vaccine, we have developed a process to stably produce the recombinant mers s377-588 protein in chinese hamster ovary (cho) cells. to accomplish this, we transfected an adherent dihydrofolate reductase-deficient cho cell line (adcho) with a plasmid encoding s377-588 fused with the human igg fc fragment (s377-588-fc). we then demonstrated the interleukin-2 signal peptide-directed secretion of the recombinant protein into extracellular milieu. using a gradually increasing methotrexate (mtx) concentration to 5 μm, we increased protein yield by a factor of 40. the adcho-expressed s377-588-fc recombinant protein demonstrated functionality and binding specificity identical to those of the protein from transiently transfected hek293t cells. in addition, hcd26/dipeptidyl peptidase-4 (dpp4) transgenic mice vaccinated with addavax-adjuvanted s377-588-fc could produce neutralizing antibodies against mers-cov and survived for at least 21 days after challenge with live mers-cov with no evidence of immunological toxicity or eosinophilic immune enhancement. to prepare for large scale-manufacture of the vaccine antigen, we have further developed a high-yield monoclonal suspension cho cell line. with over 712 deaths and 2040 confirmed cases since its original appearance on the arabian peninsula in 2012 [1] , middle east respiratory syndrome (mers) coronavirus (mers-cov) has emerged as an important global pathogen and potential pandemic threat. there remains a critical need for a vaccine targeting mers-cov [2] , and the newly established coalition for epidemic preparedness innovation (cepi) has now designated research and development for the mers-cov vaccine as a global priority [3] . recently, phase i studies of dna-based vaccines against mers-cov showed that 98% of vaccinated volunteers generated antibody against mers cov [4] . however, to date there is no licensed dna vaccine for humans due in part to questions about their long-term safety, and their ability to induce high titers of protective or neutralizing antibodies relative to recombinant protein-based vaccines [5, 6] . a lead candidate for such a protein-based vaccine is the receptor-binding domain (rbd) of the mers-cov spike (s) protein. the mers-cov rbd plays an essential role in host receptor binding, membrane fusion, and cell entry [7] [8] [9] , thus making it an ideal vaccine target. moreover, focusing on the rbd component, rather than the full-length s protein, reduces the likelihood of eosinophilic-or antibody-dependent immune enhancement [10, 11] . expressed and purified as a recombinant fusion protein with the human fc fragment, the addavax(mf59-like)-adjuvanted rbd (residues 377-588) of mers-cov has been shown to elicit high neutralizing antibody titers in both mice and rabbits [9, [12] [13] [14] [15] [16] . these antibodies displayed potent neutralizing activity against almost 20 human and camel mers-cov strains, including those with amino acid mutations in the rbd region of their spike proteins [15, 17, 18] . in a complementary approach, recombinant rbd mutants representing different human and camel virus isolates were all able to elicit broad-spectrum neutralizing antibodies against a wide range of human and camel mers-cov strains [15] . antibodies against the rbd were able to block the binding of the rbd to the mers-cov's cellular receptor, dpp4 [17] , and thus block the viral entry into permissive human cells [18] . most importantly, recent studies found that these neutralizing antibodies were indeed associated with protection, when vaccinated ad5-hdpp4-transduced mice and hdpp4-transgenic mice were found to be immune against lethal mers-cov challenge [14, 19, 20] . although the vaccine antigen has been produced at small, laboratory scale in a transient hek293 cell system [17] , little effort has been put forth to develop and scale up of mers-cov rbd suitable for future vaccine manufacture. therefore, we have now engineered a stable cho cell line suitable for producing this mers-cov protein vaccine antigen. the codon-optimized dna sequence encoding the human igg fc-fused s377-588 and the signaling peptide of interleukin-2 (il2) residing at the n-terminus were synthesized and cloned into pjet2.1 using xbai and noti restriction sites (genescript) (pjet2.1_il2_s377-588-fc). sequences encoding signal peptides derived from igk light chain (igk), human serum albumin (sa), and azurocidin (azu) were incorporated into the mers s377-588-fc by touchdown pcr with ultramer oligomers (table 1, supp. table 1 ). the pcr products were gel purified using a qiaquick pcr purification kit (qiagen) and subsequently cloned into the poptivec-topo vector (invitrogen), followed by escherichia coli top10 transformation. ampicillin-resistant transformants were selected on lb agar plates containing 100 lg/ml of ampicillin and subsequently grown in lb broth. plasmid dna prepared from isolated colonies were sequenced. to construct the expression plasmid with the il2 signal peptide, pjet2.1_il2_s377-588-fc was digested with xbai and noti restriction enzymes, and the gene cassette was gel purified. the expression plasmid poptivec was digested with the same enzymes and gel purified, followed by ligation with t4 dna ligase. adherent, dihydrofolate reductase (dhfr)-deficient cho cells (adcho) (atcc ò crl-9096 tm , cho-dhfr) were cultured in iscove's modified dulbecco's medium (imdm, gibco) supplemented with 2 mm l-glutamine (gibco), 5% fetal bovine serum (fbs, gibco) and 0.1 mm sodium hypoxanthine/0.016 mm thymidine (h/t, gibco). to establish s377-588-fc-expressing adcho cell lines, 1 x 10 6 cells were transfected with 10 lg plasmid dna with different signal peptides using lipofectamine ò 2000 (invitrogen), according to the manufacturer's instructions. all plasmid dnas were linearized with ahdi prior to transfection. stable transfectants were selected by culturing in selective medium (same medium as previously described without h/t supplementation). cells were passaged every 6 days after reaching a maximum cell density of 1.2 â 10 6 viable cells per ml (split ratio 1:15). to investigate the effect of the different signal peptides on protein expression, conditioned medium from each transfection was collected for quantitative analysis. for gene amplification, transfected adcho cells were grown to confluence (6 days) in the selective medium supplemented with mtx. the concentration of mtx was increased gradually during each passage (6 days per passage) of adcho cells (20 nm > 100 nm > 200 nm > 400 nm > 600 nm > 800 nm > 1500 nm > 2000 nm > 3000 nm > 5000 nm, [21] ). the recombinant mers s377-588-fc was loaded onto a hitrap protein-a hp column (ge) at a flow rate of 1 ml/min. the column was washed with 1x pbs (ph 7.5, amesco) for 10 column volumes (cvs) and eluted with 3 cvs of 0-100% elution buffer (100 mm citric buffer ph 4.0), followed by 7 cvs of 100% elution buffer. the elution fractions (0.5 ml) were collected in tubes containing 0.2 ml of 1 m tris-hcl (ph 8.0) to elevate the ph of the eluted protein to ph 7.0. sds-page and western blotting analysis were performed with rabbit-anti-mers-cov rbd (1:2000, [13] ) and rabbitanti-bovine (fab) 2-biotin (1:4000, sigma) antibodies to identify the s377-588-fc protein in the elution fractions. the peak fractions containing the mers s377-588-fc protein were pooled and concentrated to 2 mg/ml with amicon ultra-15 centrifugal filter unit (mwco 10 kda) and buffer-exchanged into 20 mm tris-hcl and 20 mm nacl (ph 7.4). the protein secondary structure was predicted from circular dichroism (cd) spectra. samples for cd experiments were prepared in 20 mm citrate phosphate at a concentration of 0.2 mg/ml. cd spectra were recorded with a jasco j-1500 s spectrophotometer, scanning from 280 nm to 200 nm at 100 nm/min with a bandwidth of 1 nm and response time of 1 s. experiments were performed using one quartz cuvette with a path length of 0.1 cm, keeping a constant temperature of 25°c. the average value was determined after five scans, and the spectrum of the matching 'buffer alone' sample served as the control. the secondary structure was predicted using the cdpro software by comparing with three reference sets (sp43, sdp48 and smp56) and using two data fitting programs (contin and cdsstr). a real-time protein melt experiment was performed using protein thermal shift tm dye (thermo fisher scientific) in an applied biosystems viia 7 realtime pcr system (thermo fisher scientific), yielding a fluorescence table 1 overview of tested signal peptides in the adcho expression system. signal peptide signal peptide sequence ref. profile specific to purified mers s377-588-fc. to evaluate ptm, the purified protein was treated with peptide-n-glycosidase f (pngasef), o-glycosidase and neuraminidase in a nondenatured form and subsequently analyzed by gel electrophoresis. a co-ip assay was carried out to analyze the interactions between adcho-expressed mers s377-588-fc and human dpp4 (hdpp4) receptor in huh-7 cell lysates, using mers s377-588-fc expressed in hek293t cells [13] as a positive control and hek293t-expressing sars-cov rbd-fc as a negative control. the huh-7 cell lysates (5 â 10 7 /ml in 1 ml lysis buffer containing 0.3% n-decyl-d-maltopyranoside-phosphate-buffered saline [22] ) and sars-cov rbd-specific 33g4 mab (anti-sars-rbd, 1 lg/ml, [10] ). flow cytometry analysis was carried out to quantify the binding between mers-cov rbd and hdpp4-expressing huh-7 cells. cells were incubated with s377-588-fc (40 lg/ml), expressed either in adcho cells or hek293t cells, for 30 min at room temperature, followed by addition of fitc-labeled anti-human igg antibody (abcam) for 30 min. cells were then analyzed by flow cytometry. to detect binding between mers-cov rbd and hdpp4 protein, 96-well elisa plates were pre-coated overnight at 4°c with 50 ll of 2 lg/ml purified s377-588-fc protein, expressed either in hek293t cells [13] or in adcho cells, and blocked with 2% fatfree milk at 37°c for 2 h. serial dilutions of hdpp4 protein (histagged, 50 ll/well) were then added to the plates and incubated at 37°c for 1.5 h, followed by four washes with pbs in 0.05% tween-20 (pbst). subsequently, the plates were incubated with mouse anti-his primary antibody (1:2000, sigma) at 37°c for 1.5 h. after four washes with pbst, horseradish peroxidase (hrp)-conjugated anti-mouse igg antibody (1:5000, ge healthcare) was added to the wells and incubated at 37°c for 30 min. finally, plates were washed with pbst, and binding was visualized by adding the colorogenic substrate 3,3 0 ,5,5 0 -tetramethylbenzidine (tmb, sigma). the reaction was stopped after 10 min by adding 1 n h 2 so 4 , and absorbance at 450 nm (a450) was measured on an elisa plate reader (tecan). detection of the binding between mers-cov rbd and rbdspecific neutralizing mabs was performed following a protocol similar to that described above, except that the plates were precoated with 1 lg/ml of purified s377-588-fc proteins, followed by sequential incubation with serially diluted mouse mab (mers-mab1) or human mabs (m336-fab, m337-fab, and m338-fab) [23] [24] [25] and hrp-conjugated anti-mouse igg (1:3000, for mouse mab, ge healthcare) and anti-human-fab (1:5000, for human fab-mabs, sigma) antibodies. the binding between denatured mers-cov rbd and the aforementioned rbd-specific neutralizing mabs or rbd-immunized mouse sera was tested by elisa as described above, except that the plates were pre-coated with s377-588-fc (1 lg/ml) protein treated with dithiothreitol (dtt) (10 mm, sigma) at 37°c for 1 h, followed by incubation with iodoacetamide (50 mm, sigma) at 37°c for 1 h to stop the reaction [25] . after three washes, the elisa was carried out as described above. all in vitro and in vivo studies required the usage of infectious mers-cov (emc/2012 strain) and were conducted within approved biosafety level 3 (bsl-3) and animal bsl-3 laboratories at the galveston national laboratory, strictly following approved notification-of-usage (nou) and animal protocols and the guidelines and regulations of the national institutes of health and aaalac. for a ''proof-of-principal" study to confirm that adchoexpressed mers s377-588-fc is an effective and safe vaccine, two groups of five age-matched cd26/dpp4 transgenic (tg) mice were immunized twice, four weeks apart, via the intramuscular (i.m.) route, with either 10 lg of mers s377-588-fc formulated with addavax (invivogen) or pbs/addavax only (as control). this immunization protocol was selected because it is optimized for mers-cov rbd proteins [16] . the addavax adjuvant was chosen because it promoted the rbd-fc protein to generate the highest neutralizing antibodies among several adjuvants tested in our previous studies [14] . serum specimens were collected at day 28 after the second immunization through the retro-orbital bleeding route to determine the prospective capacity to neutralize infectious mers-cov by using the standard vero e6-based micro-neutralization test. immunized mice were subsequently challenged intranasally (i.n.) with 100x 50% lethal dose (ld 50 ) ($10 3 tcid 50 ) of mers-cov (emc/2012 strain), a gift of heinz feldmann (nih, hamilton, mt) and ron a. fouchier (erasmus medical center, rotterdam, the netherlands), followed by daily monitoring for the onset of clinical manifestations (e.g., weight loss and other clinical manifestations) and mortality. three mice from each group were euthanized at day 3 post-infection (p.i.) to assess lung viral loads by vero e6-based infection assay and quantitative (q) pcr analysis targeting the upe gene of mers-cov for quantifying infectious virus and viral rna, respectively. additionally, de-paraffinized lung tissues were hematoxylin-and-eosin (h&e)-stained for routine histopathologic evaluations, as described. we continued to monitor the remaining two mice in each group for their overall well-being for a total of 3 weeks until terminating the experiment. all methodologies required to assess the immunogenicity (neutralization antibody titers) and efficacy of mers s377-588-fc have been previously reported ( [19, 26] , supplementary methods). suspension cho (cho dg44, gibco, hereinafter termed sus-cho) cells were cultured in cd dg44 medium (gibco) supplemented with 8 mm l-glutamine (gibco) and 0.18% pluronic ò f-68 prior to transfection. transfection was performed by combining 18 lg ahdi-linearized plasmid popti_il2_s377-588-fc and 15 ll of freestyle tm max reagent (invitrogen) in 1.2 ml optipro sfm and incubated at room temperature for 10 min, followed by dropwise addition to 1.5 â 10 7 cells in 30 ml of cd dg44 culture medium (non-selective) according to the manufacturer's instructions. after 48 h, cells were transferred to selective medium (cd opti-cho, invitrogen), supplemented with 8 mm l-glutamine and 0.18% pluronic ò f-68 (gibco), and cultivated until cell viability reached 90%. after selection, stably transfected suscho cells underwent dna amplification by gradually increasing mtx concentration (20-5000 nm) in selective medium. all suspension culture flasks were maintained in a humidified incubator, 37°c/8% co 2 on a shaker, at a constant rotation rate of 135 rpm. the 5 lm mtx-adapted suscho cell pools from serum-free medium were used for single-cell cloning by limited dilution at 0.25-2 cells/well. cloning was performed in 96-well plates (falcon u-bottom untreated), utilizing a cloning medium composed of 80% hybridoma sfm (clonacell) and 20% conditioned media supplemented with 0.5x cho acf supplement (clonacell) at 37°c/5% co 2 for approximately 14 days. conditioned medium from wells with actively growing single colonies was assayed by elisa as follows. a mixture of 10 ll of conditioned medium and 90 ll of coating buffer were added to a 96-well elisa plate (thermo fisher scientific) and incubated at 4°c overnight. after washing the plate with pbst, rabbit anti-human igg was used to detect s377-588-fc protein, followed by a biotinylated goat antirabbit antibody and streptavidin hrp. tetramethylbenzidine (kpl inc., vwr) was added (100 ll) before the reaction was stopped with 100 ll 1 m hcl. elisa plates were read on an epoch microplate spectrophotometer (biotek instruments, inc) at 450 nm. clones which gave high absorbance reading were further propagated in 6-well plates, utilizing a cd opticho medium with 8 mm l-glutamine and 0.18% pluronic ò f-68. conditioned medium from 6-well plates was also screened by elisa for confirmation, and the highest expressing clone was selected and expanded by passaging in shake flasks (37°c, 8% co 2 in air on an orbital shaker platform rotating at 135 rpm). clonal cell lines and heterogeneous suscho cell pools were collected daily (up to 10 days) and counted using the acridine orange (ao) and propidium iodide (pi) nuclear staining dyes (nexcelom bioscience), which enter live cells and dead cells, respectively. conditioned medium from each time point was analyzed by sds-page, and the protein concentration was estimated by densitometry, comparing it to protein standards using the chemidoc tm imaging system (biorad). statistical significance was calculated by student's t test using graphpad prism statistical software. ⁄⁄⁄ indicates p < 0.001. in our previous studies, the human igg fc-fused s377-588 fragment of the mers-cov s protein (genbank afs88936.1) (hereinafter termed mers s377-588-fc) had already been expressed in transiently transfected hek293t cells [17] . however, to establish stably expressing cell clones, we transfected an adherent cho (adcho) with a poptivec construct containing an internal ribosome entry site (ires)-driven dhfr gene for selection and copy number amplification, as well as mers s377-588-fc gene fusion. signal peptides were added to the n-terminal end of the s377-588-fc gene in order to drive secretion into the culture medium (fig. 1a) . addition of gradually increasing methotrexate (mtx) to the culture medium of transfected cells resulted in binding to and inactivation of dihydrofolate reductase (dhfr) activity. transfected adcho cells compensated for this reduced dhfr activity by increasing the dhfr copy number in the genome to overcome inhibition by mtx. since the mers s377-588-fc fusion gene was integrated into the same genetic locus as that of the dhfr gene, the s377-588-fc gene was amplified, as well, leading to increased production of the protein. in the course of developing the adcho cell line expressing the s377-588-fc protein, optimization of a purification protocol using hitrap protein-a hp was performed. on the purification chromatogram, two protein peaks were observed in the elution step (fig. 1b) . denaturing sds-page and western blotting analysis with anti-mers-rbd-specific antibodies confirmed the second peak to be the s377-588-fc fragment (fig. 1c) . further analysis using non-denaturing sds-page and western blotting with rb-anti-bovine (fab)2 antibodies showed that the first peak was mainly contaminating bovine igg (fig. 1d-e) that originated from the fetal bovine serum (fbs) supplemented in the culture medium. we estimated relative productivity of adcho cells by measuring the ratio of the area of first peak and second peak. different signal peptides have been shown to result in different expression levels in cho cell systems [27] . therefore, we transfected adcho cells with linearized mers s377-588-fc expression plasmids with four different signal peptides at the n-terminus. peptides were derived from interleukin 2 (il2), igk light chain (igk), human serum albumin (sa), and azurocidin (azu). conditioned media from confluent monolayers of each transfected cell line were collected, followed by protein purification using protein a to establish yield and estimate relative productivity. in these studies, we found that adcho cells with the signal peptide derived from il2 showed 20-50% more secretion of s377-588-fc than cells with other signal peptides (fig. 2a) . elevated expression levels were achieved by gradually increasing mtx concentration during each cell passage from 20 nm to 5 lm. conditioned medium from transfected adcho cells was collected during the dna amplification process, followed by protein purification using protein a to estimate relative productivity (fig. 2b) . we observed the expected correlation between the expression of s377-588-fc and increased resistance to higher levels of mtx. compared to adcho cells with 20 nm mtx, expression of s377-588-fc was increased 39-fold in the presence of 3-5 lm of mtx (fig. 2c ). the purified protein was analyzed by circular dichroism (cd) spectroscopy. the mers-cov s377-588-fc consists mainly of beta-sheet (39.4%) and loop structures (53%) with limited helices (7.6%) (fig. 3a) . the secondary structure of the protein starts to unfold at 65°c. during thermal melt analysis, mers s377-588-fc had two endothermic transitions: 52.5°c and 67.5°c (fig. 3b) . a comparison between mers s377-588-fc expressed from hek293t cells and adcho cells was carried out using different page analyses. both proteins appeared to be identical following reduced and non-reduced sds-page. on native page and ief gels, adchoexpressed mers s377-588-fc had a lower isoelectric point (pi 6.6) when compared to the protein expressed in hek293t cells (pi 7.2) (fig. 3c) . after removal of n-linked glycans, the molecular size of mers s377-588-fc was slightly smaller than that of the n-linked glycosylated form (fig. 3d) . no change in molecular weight was observed after o-linked deglycosylation and desialylation (reduced sds-page). enzymatic treatment did not affect dimerization of mers s377-588-fc, as determined by nonreducing sds-page. the high pi (>7.4) isomers of s377-588 exhibited a lower shift (between pi 7.4 and pi 6.0) in electrophoretic mobility after n-linked deglycosylation, while no change of band pattern for the protein was seen after treatment with o-glycosidase. after removal of sialic acid through neuraminidase treatment, s377-588-fc isomers shifted to pi higher than 7.4 (fig. 3d) . three assays, including co-immunoprecipitation (co-ip), flow cytometry, and elisa, were performed to detect the binding of mers-cov rbd to its receptor, hdpp4. co-ip demonstrated that similar to the hek293t-expressed mers s377-588-fc protein, the rbd protein expressed in adcho bound strongly to hdpp4expressing huh-7 cells. two clear bands were identified from immunoprecipitated mixture of s377-588-fc and huh-7 cell lysates, and these bands were recognized by both anti-hdpp4 antibody and anti-mers-rbd antibody. in contrast, only one band was identified in huh-7 cells only and s377-588-fc protein only samples, and it was reactive with either anti-hdpp4 antibody or anti-mers-rbd antibody, but not with both antibodies. as expected, sars-cov rbd-fc protein was only recognized by sars-cov rbd-specific mab 33g4 (fig. 4a) . flow cytometry analysis further quantified the binding between mers-cov rbd protein and hdpp4 receptor in huh-7 cells. results showed a similar strong binding for all mers s377-588-fc proteins from adcho and hek293t, but not for sars-cov rbd-fc control (fig. 4b) . the elisa analysis demonstrated a dose-dependent binding between these mers-cov rbd proteins and hdpp4 protein, while no binding was observed between hfc control and hdpp4 (fig. 4c) . the antigenicity of mers-cov rbd proteins was carried out using elisa to test their binding with rbd-specific neutralizing antibodies (mersmab1, m336, m337, and m338), which recognize epitopes at rbd residues f506, d510, r511, w535, d539, y540, r542, or w553, and demonstrate strong activity to block rbd-hdpp4 receptor binding and neutralize mers-cov infection [23] [24] [25] 28] . similar to the s377-588-fc protein expressed in hek293t, the rbd proteins expressed in adcho bound strongly to mouse mab mersmab1 and human mabs m336, m337, and m338 in a dose-dependent manner (fig. 4d) , confirming their antigenicity. while these mabs bound strongly to the non-denatured (no dtt) s377-588-fc proteins expressed in both adcho and hek293t, they had significantly reduced affinity to rbd proteins treated with dtt, a reducing agent cleaving disulfide bonds of rbds and thus disrupting a protein's native conformation (fig. 4e) . these results demonstrate that the neutralizing mabs recognize conformational structures of mers-cov rbd [25] . transgenic mice expressing the human cd26/dpp4 receptor (hdpp4-tg) are a well-characterized animal model with which to evaluate the efficacy of vaccine candidates against mers-cov infection and disease [19, 20] . the immunogenicity of the mers s377-588-fc-based subunit vaccine was verified in hdpp4-tg mice. immune sera obtained from immunized mice were tested by elisa for the binding with denatured and non-denatured s377-588-fc protein or subjected to the vero e6-based micro-neutralization assay to quantify their capacity to neutralize infectious mers-cov. to evaluate protective efficacy against viral infection, viral loads and histopathology of the lungs of three mice in immunized and control groups were measured at day 3 after lethal challenge with 100x ld 50 of mers-cov. the remaining two mice in each group were monitored for morbidity (weight loss) and mortality to determine if this vaccine formulation would sufficiently protect against the disease and lethality caused by mers-cov infection. mers s377-588-fc induced high titers of rbd-specific igg antibodies, which reacted strongly with non-denatured s377-588-fc protein. nevertheless, these rbd-specific antibodies had significantly reduced activity with s377-588-fc treated by dtt (fig. 5) . this suggests that the rbd vaccine-induced antibody response was indeed directed towards one or more conformational epitopes. mice immunized with pbs/addavax uniformly failed to elicit any detectable neutralizing antibodies; however, those immunized with mers s377-588-fc/addavax consistently produced readily detectable titers of neutralizing antibody, ranging from 10-160 and 20-320 of neutralizing titer (nt)-100 (nt 100 ) and nt-50 (nt 50 ), respectively ( table 2) . consistent with the readily detectable neutralization antibodies, mers s377-588-fc/addavaximmunized mice were fully protected against viral infection and disease, as evidenced by the absence of recoverable infectious virus and negligible focal inflammatory responses, if any (supp. fig. 3) , within the infected lungs at 3 days post-infection (dpi). importantly, the remaining two immunized mice did not suffer any significant morbidity (weight loss) and survived until 21 dpi when the experiment was terminated. in contrast, pulmonary infectious viruses, albeit low in titers, were detected from all three unimmunized controls, with an average of 2.7 ± 0.1 tcid 50 /g of mers-cov recovered at 3 dpi, accompanied by mild inflammatory responses. the remaining two control mice suffered profound weight loss prior to succumbing to infection by 8 dpi. taken together, results of this ''proof-of-principle" pilot study indicated not only immunogenicity of mers-cov s377-588-fc, but also its efficacy and safety in the protection of hdpp4-tg mice against lethal challenge with mers-cov. similar to the adcho cell development described earlier, the dna copy number of stably transfected suspension cho dg44 dhfr-cells was amplified by gradual exposure to increased mtx concentrations of up to 5 lm. the resulting heterogeneous suscho cell pools were subsequently cloned by limited dilution to obtain a monoclonal cell population. elisa analysis was performed on the supernatants from individual clones, leading to the identification of clone 1b11 as the highest expressing clone. by comparing the growth curves of clone 1b11 to the heterogeneous (non-clonal) suscho cells, we discovered that clone 1b11 and heterogeneous suscho cells reached their maximum growth on the seventh day with viable cell counts of 8 â 10 6 cells/ml and 7 â 10 6 cells/ml, respectively (fig. 6) . through quantitative analysis using sds-page, we determined that the supernatant of clone 1b11 expressed approximately 97 mg/l of mers s377-588-fc, while the heterogeneous non-clonal cell pools expressed about 65 mg/l ( table 3 , supp. fig. 4 ) on the seventh day. the mers-cov rbd subunit fragment s377-588 has been identified to be a critical neutralizing receptor-binding fragment and an ideal candidate for the development of an effective mers-cov recombinant protein vaccine [13] . our aim here was to optimize expression and purification conditions suitable for pilot scale production of this rbd vaccine candidate. initially, both escherichia coli (bacteria) and pichia pastoris (yeast) expression systems proved well suited for recombinant vaccine production because of low production costs. however, our data showed that e. coli could not produce soluble mers s377-588, and yeast cells could not overexpress mers s377-588. thus, these two systems were not considered suitable to advance the mers vaccine candidate into process development and scale up production (see supplementary information for more detail). in previous reports, mers fc-fused s377-588 had been expressed in transiently transfected hek293t cells. however, transiently transfected cell lines may give low protein yield and potentially lose their production ability over time in continuously expressing recombinant proteins [29, 30] . unlike transient transfection, dna is integrated into cells long-term through stable transfection. despite the fact that the development of stably expressing cell lines is laborious and time-consuming, production with stable cell lines can be scaled up easily and would be suitable for use in manufacturing processes. hence, we generated a stably transfected adcho cell line by transfecting mtxdriven poptivec into cells to produce recombinant mers s377-588-fc protein. fc-fused gp120 protein was first constructed in 1989 as a potential candidate for aids therapy [31] . while there is no fda approved fc-fusion vaccine, vaccine development using fc-fusion proteins is active and ongoing. a number of studies have been initiated on the development of vaccines against ebola, hiv, influenza, as well as tuberculosis [32] [33] [34] [35] . it is noted that adverse side effects with vaccines are likely limited as biotherapeutic fc fusions have been repeatedly shown to be safe and biocompatible in humans. currently, all commercial therapeutics use the fc domain from human igg1, although other options, such as igg3, iga, and igm are also currently being explored [36] . furthermore, the fc domain is known to increase plasma half-life and simplify the purification process [36] . purification of s377-588-fc was performed using a protein a sepharose column, removing most of the impurities from the culture medium. bovine igg originates from fbs in the culture medium in constant amount and was used as a standard to gauge expression levels of the s377-588-fc protein. this estimation method allowed us to select the most suitable signal peptide and to evaluate the effect of the mtx-induced dna amplification process. since the poptivec plasmid has no signal peptide, four different signal peptides were subsequently tested at the n-terminus of the s377-588-fc sequence to drive secretion of the protein, leading to the identification of il2 signal peptide as the most suitable signal sequence. although signal peptides derived from human sa and human azu have been reported to improve production rates in other adcho systems [27] , the yield in our hands was lower than that with the il2 signal peptide. stable and highly productive cell pools were then isolated through a gradual increase in the mtx concentration in the culture [21] . analysis of the relative productivity of adcho cells indicated an increase in s377-588-fc in media proportional to the mtx concentration in the medium, reaching a plateau at 3 lm mtx. the biophysical and biochemical characterization of the mers s377-588-fc protein revealed that is was stable up to a temperature of 52.5°c. after the first major unfolding event at 52.5°c, another unfolding event occurred between 65 and 67.5°c, which could have resulted from destabilization of the ch2-ch2 bond of the fc domain of the recombinant protein [37] . no change in molecular weight was observed after removal of o-linked glycan on mers s377-588-fc, which suggested no o-linked glycosylation in the protein. after neuraminidase treatment, the recombinant protein band pattern showed fewer bands and a pi shift, suggesting that sialic acids might contribute to the charge of the protein. interestingly, although the complexities of the multiple protein bands were greatly reduced after glycosidase and sialidase treatments, the pattern representing charge heterogeneity remained, suggesting the existence of additional ptms, such as mannose-6phosphate, of the recombinant mers s377-588-fc protein. due to the charge differences between adcho-and hek293texpressed s377-588-fc proteins, we evaluated the functionality and antigenicity of the target protein. functionality studies, including co-ip assays, flow cytometry analyses, and elisa binding assay, confirmed that the adcho-expressed mers s377-588-fc protein maintained functionality equal to that expressed in hek293t cells in binding the dpp4 receptor of mers-cov, both of which showed dose-dependent binding with the soluble hdpp4 protein. in addition, mers s377-588-fc expressed in either adcho or hek293t demonstrated similar dose-dependent binding to rbd-specific neutralizing antibodies, an indicator that both s377-588-fc proteins could maintain sufficient antigenicity. we further investigated the protective efficacy of adcho-expressed s377-588-fc protein vaccine in protecting against mers-cov infection in the established transgenic mouse model expressing hdpp4 (hdpp4-tg). by formulating this s377-588-fc protein with adda-vax all vaccinated animals could produce neutralizing antibodies and survive a live viral challenge for 21 days. taken together, we confirmed the absence of functional, antigenic and immunogenic differences between adcho-and hek293t-expressed mers s377-588-fc proteins. moreover, mouse vaccinations with the rbd subunit vaccines did not appear to elicit eosinophilic or antibody-dependent immune enhancement. although we verified adcho-expressed mers s377-588-fc protein as an effective vaccine against mers-cov infection, the use of fbs in the growth medium proved unsuitable for a human vaccine antigen [38] . for both safety and compliance with future regulatory requirements, we therefore developed a stably transfected suspension cho cell line in serum-free medium. the adcho cell development process described here became the foundation for the establishment of the serum-free suspension cho cell line. subsequently, we transfected the poptivec expression plasmid with the il2 signal peptide into suscho cells and carried out the dna amplification as before. from the heterogeneous cell pools adapted to 5 lm mtx, we isolated clone 1b11, which was the highest expressing clone from a two-cycle screening process. in shake flasks, the growth of clone 1b11 was slightly slower than that from the heterogeneous cell pools, but 1b11 expressed 50% more mers s377-588-fc protein than the heterogeneous cell pool. typically, highly productive cell clones have lower growth rates since a significant portion of resources are used for expression of the recombinant protein [39] . additional experiments in the transgenic mice and non-human primate models will be needed to further determine the immunogenicity of mers s377-588-fc protein that produced by suscho. we envision that with a proper production process, the recombinant protein can be scaled up, manufactured, formulated and stockpiled as an efficient countermeasure against future mers-cov outbreaks. table 3 s377-588-fc expressed from heterogeneous non-clonal suscho cell pools and from the monoclonal clone 1b11. the protein concentration (mg protein per liter of culture supernatant) was determined by sds-page gel analysis (supp. fig. 3 ). who. middle east respiratory syndrome coronavirus the middle east respiratory syndrome coronavirus -a continuing risk to global health security vaccine development against prioritized epidemic infectious diseases inovio reports new positive clinical data on vaccine advances in the fight against emerging infectious diseases innate immune signaling by, and genetic adjuvants for dna vaccination the future of human dna 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polymerase chain reaction this study was supported through the us-malaysian vaccine development program, funded by the university of malaya, and grants from the nih (r01ai098775-03s1 and r21ai128311). we thank drs. dimiter s. dimitrov and tianlei ying for providing m336, m337, and m338 mabs. the authors are involved in the development of a vaccine against mers coronavirus. supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.vaccine.2018.02. 065. key: cord-323324-h2a25xym authors: armijos‐jaramillo, vinicio; yeager, justin; muslin, claire; perez‐castillo, yunierkis title: sars‐cov‐2, an evolutionary perspective of interaction with human ace2 reveals undiscovered amino acids necessary for complex stability date: 2020-05-07 journal: evol appl doi: 10.1111/eva.12980 sha: doc_id: 323324 cord_uid: h2a25xym the emergence of sars‐cov‐2 has resulted in nearly 1,280,000 infections and 73,000 deaths globally so far. this novel virus acquired the ability to infect human cells using the sars‐cov cell receptor hace2. because of this, it is essential to improve our understanding of the evolutionary dynamics surrounding the sars‐cov‐2 hace2 interaction. one way theory predicts selection pressures should shape viral evolution is to enhance binding with host cells. we first assessed evolutionary dynamics in select betacoronavirus spike protein genes to predict whether these genomic regions are under directional or purifying selection between divergent viral lineages, at various scales of relatedness. with this analysis, we determine a region inside the receptor‐binding domain with putative sites under positive selection interspersed among highly conserved sites, which are implicated in structural stability of the viral spike protein and its union with human receptor ace2. next, to gain further insights into factors associated with recognition of the human host receptor, we performed modeling studies of five different betacoronaviruses and their potential binding to hace2. modeling results indicate that interfering with the salt bridges at hot spot 353 could be an effective strategy for inhibiting binding, and hence for the prevention of sars‐cov‐2 infections. we also propose that a glycine residue at the receptor‐binding domain of the spike glycoprotein can have a critical role in permitting bat sars‐related coronaviruses to infect human cells. the recent emergence of the novel sars coronavirus 2 (sars-cov-2) marked the third introduction of a highly pathogenic coronavirus into the human population in the twenty-first century, following the severe acute respiratory syndrome coronavirus (sars-cov) (drosten et al., 2003; who, 2004) . mers-cov was the second emergence and was first detected in saudi arabia in 2012 and resulted in nearly 2,500 human infections and 858 deaths in 27 countries (fehr, channappanavar, & perlman, 2017; zaki, boheemen, bestebroer, osterhaus, & fouchier, 2012) . in december 2019, sars-cov-2, a previously unknown coronavirus capable of infecting humans was discovered in the chinese city of wuhan, in the hubei province (huang et al., 2020; zhu et al., 2020) . sars-cov-2 is associated with an ongoing pandemic of atypical pneumonia, now termed coronavirus disease 19 (covid-2019) that has affected over 1,280,000 people with 72,614 fatalities as of april 7, 2020 (who, 2020 . both sars-cov and mers-cov are thought to have originated in colonies of bats, eventually transmitted to humans, putatively facilitated by intermediate hosts such as palm civets and dromedary camels, respectively (cui, li, & shi, 2019) . the genome of sars-cov-2 shares about 80% nucleotide identity with that of sars-cov and is 96% identical to the bat coronavirus batcov ratg13 genome, reinforcing the probable bat origin of the virus . however, better assessing the evolutionary dynamics of sars-cov-2 is an active research priority worldwide. betacoronavirus within the subfamily coronavirinae of the family coronaviridae. members of this family are enveloped viruses containing a single positive-strand rna genome of 27-32 kb in length, the largest known rna virus genome. the coronavirus spherical virion consists of four structural proteins: the spike glycoprotein (s-protein), the envelope protein, membrane protein, and nucleocapsid. the transmembrane trimeric s-protein plays a critical role in virus entry into host cells (gallagher & buchmeier, 2001; tortorici & veesler, 2019) . it comprises two functional subunits: s 1 subunit, where the receptor-binding domain (rbd) is found, is responsible for binding host cell surface receptors and s 2 subunit mediates subsequent fusion between the viral and cellular membranes (kirchdoerfer et al., 2016; yuan et al., 2017) . both sars-cov and sars-cov-2 interact directly with angiotensin-converting enzyme 2 (ace2) to enter host target cells (hoffmann et al., 2020; li et al., 2003; walls et al., 2020; yan et al., 2020) . in the case of sars-cov, ace2 binding was found to be a critical determinant for the range of hosts the virus can infect, and key amino acid residues in the rbd were identified to be essential for ace2-mediated sars-cov infection and adaptation to humans (li et al., 2005 (li et al., , 2006 . understanding the dynamics that permits a virus to shift hosts is of considerable interest and can further be an essential preliminary step toward facilitating the development of vaccines and the discovery of specific drug therapies. we employ a multidisciplinary approach to look for evidence of diversifying selection on the s-protein gene and model the interactions between human ace2 (hace2) and the rbd of selected coronavirus strains, which ultimately afforded us novel insights detailing virus and host cell interactions. given the rapid pace of discovery, we aim to add clarity to evolutionary dynamics of diseases strains by more precisely understand the dynamics at the s-protein and its interaction with hace2. the most similar genomes to sars-cov-2 mn908947 were retrieved using blastp (altschul et al., 1997) versus the nr database of genbank (table 1) . genomes were then aligned using mauve (darling, mau, blattner, & perna, 2004) , and the s-protein gene was trimmed. the extracted genomic sections were aligned using the translation align option of geneious (kearse et al., 2012) with a mafft plugin (katoh & standley, 2013) . the phylogenetic reconstruction of s-protein genes was performed with phyml (guindon et al., 2010) , using a gtr + i + g model, using with 100 nonparametric bootstrap replicates. both, the alignment and the tree were used as inputs for paml codeml (yang, 2007) . the presence of sites under positive selection was tested by the comparison of m2 model (which it allows for a proportion of positive, neutral, and negative selection sites in the alignment) versus the m1 model (it which only allows a proportion of neutral and negative selection sites in the alignment) and m8 (ω follows a beta distribution plus a proportion of sites with ω > 1) versus m7 (ω follows a beta distribution) models using the ete toolkit 3.0 (huerta-cepas, (weaver et al., 2018) was used to perform the hyphy analyses. the crystal structure of the sars-cov s-protein rbd (genebank id nc_004718) in complex with hace2 was retrieved from the protein data bank (code 2ajf) (berman et al., 2000) . homology models were constructed using this structure as template for the rbds of sars-cov-2 (sars2, genebank id mn908947), the bat sars-like coronavirus isolate rm1 (rm1, genebank id dq412043), and the bat sars-like coronavirus isolate rs4231 (rs4231, genebank id ky417146). one additional homology model for the g496d mutant of the sars-cov-2 rbd (sars2-mut) was constructed. homology models were built with modeller v. 9 (webb & sali, 2016) using its ucsf chimera interface (pettersen et al., 2004) . five models were constructed for each target sequence and the one with the lowest discrete optimized protein energy (dope) score was selected for the final model. all nonamino acidic residues were removed from the sars-cov rbd-hace2 complex to obtain a clean complex. the homology models of the sars2, rm1, rs4231 rbds, and sars2-mut were superimposed into the sars-cov rbd to obtain their initial complexes with hace2. these complexes were then subject to molecular dynamics (md) simulations and estimation of their free energies of binding using amber 18 (case et al., 2018) . for the later, ace2 was considered as the receptor and the rbds as ligands. the protocol described below was employed for all complexes and otherwise noted default software parameters were employed. systems preparation was performed with the tleap program of the amber 18 suite. each complex was enclosed in a truncated octahedron box extending 10 å from any atom. next, the boxes were solvated with tip3p water molecules and na+ ions were added to neutralize the excess charge. systems were minimized in two steps, the first of which consisted in 500 steps of the steepest descent algorithm followed by 500 cycles of conjugate gradient with protein atoms restrained using a force constant of 500 kcal/mol.å 2 . the pme method with a cutoff of 12 å was used to treat long-range electrostatic interactions. during the second minimization step, the pme cutoff was set to 10 å and it proceeded for 1,500 steps of the steepest descent algorithm followed by 1,000 cycles of conjugate gradient with no restrains. the same pme cutoff of 10 å was used in all simulation steps from here on. both minimization stages were performed at constant volume. the minimized systems were heated from 0 to 300 k at constant volume constraining all protein atoms with a force constant of 10 kcal/mol.å 2 . the shake algorithm was used to constrain all bonds involving hydrogens and their interactions were omitted from this step on. heating took place for 10,000 steps, with a time step of 2 fs and a langevin thermostat with a collision frequency of 1.0 ps −1 was employed. all subsequent md steps utilized the same ta b l e 1 list of coronavirus isolates used for positive selection analysis (closer dataset) thermostat settings. afterward, the systems were equilibrated for 100 ps at a constant temperature of 300 k and a constant pressure of 1 bar. pressure was controlled with isotropic position scaling with a relaxation time of 2 ps. the equilibrated systems were used as input for 10 ns length production md simulations. the free energies of binding were computed under the mm-pbsa approach implemented in ambertools 18 (case et al., 2018) . a total of 100 md snapshots were evenly selected, one every 50 ps, from the last 5 ns of the production run for mm-pbsa calculations. the ionic strength was set to 100 mm and the solute dielectric factor was set to 4 for all systems. in order to detect branches and sites under positive/negative selection, two datasets were explored. the first ("closer" dataset) harbors the most similar genomes to wuhan-hu-1 coronavirus (sars-cov-2) (mn908947). for this dataset, several genomes were excluded from the analysis because they showed minimal variation to other sequences. we used a preliminary phylogeny to select a representative isolate of each clade (table 1) in both datasets, we observed evidence of purifying selection in the majority of nodes of the tree. specifically, in the "closer" dataset, we identified 38 nodes with evidence of negative selection, and 4 under positive selection when free ratios model of codeml model was applied. to confirm the four nodes under positive selection, we use ltr test for contrasting hypothesis using branch free, branch neutral, and m0 models of codeml. using these approximations, any node predicted by free ratios model with ω > 1 was significantly different to the purifying (ω < 1) or neutral (ω = 1) models. an equivalent analysis was performed using absrel of hyphy, observing episodic diversifying selection in at least 8 of 41 nodes of the phylogenetic tree reconstructed with the "closer" dataset ( figure 1 ). interestingly, one of the divisions detected with diversifying selection was the branch that contains sars-cov-2, pangolin coronavirus isolate mp789 and bat coronavirus ratg13 (called sars-cov-2 group) but not the specific branch that contains sars-cov-2. and 500 (using sars-cov-2 s-protein as a reference) have drastic amino acid changes for alpha-helical tendencies. in addition, the section between residues 448 and 485 shows radical changes in amino acids implicated in the equilibrium constant (ionization of cooh). in the structural analysis we performed, the section between 472 and 486 forms a loop that is not present in certain s-proteins of coronavirus isolated in bats. this loop extends the interaction area between rbd of s-protein and human ace2; in fact, the lack of this loop decreases the negative energy of interaction (increasing the binding) among these two molecules (see table 2 ). these results, obtained from independent analyses, strongly highlight the importance of 439-508 region. additionally, important hace2-binding residues in the rbd from sars-cov-2 obtained from the crystallography and structure determination performed by shang et al. (2020) are also present in the section we highlight here. we propose that this region is the most probable to contain the sites under positive selection due to predictions by our codeml and fubar models. in that sense, we refer to this section as region under positive selection (rps). it is important to additionally clarify that even inside the rps we found at least 20 aa highly conserved between coronaviruses, several of them are predicted as sites under purifying selection. this shows that it is necessary to maintain conserved sites which are located around polymorphic sites, probably to maintain the protein structure and at the same time to have the ability to colonize more than one host. with a list of broader observations related to the role of selection across viral genomes, we aimed to specifically understand how these regions could affect virus/host interactions. to understand more in more detail the importance of rps in the evolution of sars-cov-2, we quantified the relative importance of this region in the interaction between the rbds and hace2. in that sense, md simulations were run for five complexes (listed in methods). simulations were initially performed for four rbds corresponding to the sars2, sars, rs4231 and rm1 coronaviruses. as discussed below in this section, the g496d mutation is predicted to have a large negative influence in the stability of the rbd-hace2 complexes. to further clarify this influence, we added as fifth system a g496d sars2 mutant rbd-hace2 complex in our studies. in all cases, the systems were stable with root mean square deviations (rmsd) of their backbones between 1.11 and 3.30 å relative the contribution of each residue in the studied coronaviruses that interact with the hace2 receptor are shown in table 3 . rows are presented in such a way that each of them contains the residues occupying the same position in the viral rbds structures as in the sar2 rbd structure. from here on, residues numeration will take that of sars2 as reference. to better interpret the influence of the key interactions between the studied coronaviruses rbds and the hace2 receptor, their complexes were visually analyzed. the predicted rbd-hace2 complexes for sars2, sars, and sars2-mut are depicted in figure 3 . for each complex, the structure used to create figure 3 is the representative one of the largest cluster formed by the md snapshots previously used in mm-pbsa calculations. many studies have focused on coronaviruses mutations that favor adaptations for infecting human host infections. for f i g u r e 3 predicted interaction of sars2 (top), sars2-mut (middle) and sars (bottom) with the human ace2 receptor. hace2 in depicted in gray and the rbd of coronaviruses in cyan. oxygen atoms are colored red and nitrogen blue. the numbering of the residues corresponds to that of the sequence of each spike glycoprotein example, it has been shown that specific substitutions at positions 455, 486, 4983, 494, and 501 (442, 472, 479, 480, and 487 in sars) of the rbd of sars favors the interaction between the rbd of sars and hace2 (cui et al., 2019) . likewise, homology modeling studies found favorable interactions between the residues occupying these positions in the sars2 rbd and the human receptor (wan, shang, graham, baric, & li, 2020) . the cornerstone of these favorable interactions is the complementarity of the rbds with hot spots 31 and 353. these are salt bridges between k31 and e35 and between d38 and k353 of ace2 which are buried in a hydrophobic environment (see figure 2 ). in the cases of sars2 table 3 ) respectively, do not interact with any hot spot residue. instead, they interact with d30 of hace2 in the sars2 complex and with e329 of the human receptor in the sars complex. this could indicate that interactions additional to those previously identified with the hace2 hotspots could be critical for the stabilization of the rdb-human receptor complexes. finally, we analyzed the possible reasons for the predicted negative impact that the g496d mutation has on the predicted free energies of binding of the rbd to hace2. as depicted in figure 3 , g496 directly interacts with k353 in hot spot 353 and its mutation interferes with the d38-k353 salt bridge. specifically, d496 of the rdb point to d38 of hace yields a high electric repulsion between these amino acids. consequently, this portion of the rbd is pushed to a position further from hace2 than that observed in the wild type receptor, resulting in the reduction of its network of contacts with k353. as a result, the binding of the rbd to hace2 is considerably inhibited and unlikely to occur. propose that blocking its interaction with the receptor d30 could be a promising strategy for future drug discovery efforts. the authors would like to thank daniela santander for her valuable comments on the manuscript. this work was supported by the none declared. the phylogenetic tree presented in this manuscript was uploaded to dryad (https://doi.org/10.5061/dryad.w3r22 80mp). the full networks of interactions between the coronaviruses and the hace2 receptor are provided as supporting information. vinicio armijos-jaramillo https://orcid. org/0000-0003-2965-2515 justin yeager https://orcid.org/0000-0001-8692-6311 claire muslin https://orcid.org/0000-0002-2746-6914 yunierkis perez-castillo https://orcid. org/0000-0002-3710-0035 gapped blast and psi-blast: a new generation of protein database search programs the 2019-new coronavirus epidemic: evidence for virus evolution the protein data bank origin and evolution of pathogenic coronaviruses mauve: multiple alignment of conserved genomic sequence with rearrangements identification of a novel coronavirus in patients with severe acute respiratory syndrome middle east respiratory syndrome: emergence of a pathogenic human coronavirus coronavirus spike proteins in viral entry and pathogenesis new algorithms and methods to estimate maximum-likelihood phylogenies: assessing 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recombination in coronaviruses implicating pangolin origins of ncov-2019 treesaap: selection on amino acid properties using phylogenetic trees isolation and characterization of 2019-ncov-like coronavirus from malayan pangolins structural basis for the recognition of the sars-cov-2 by full-length human ace2 paml 4: phylogenetic analysis by maximum likelihood cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains isolation of a novel coronavirus from a man with pneumonia in saudi arabia a pneumonia outbreak associated with a new coronavirus of probable bat origin a novel coronavirus from patients with pneumonia in china key: cord-339093-mwxkvwaz authors: li, wei; schäfer, alexandra; kulkarni, swarali s.; liu, xianglei; martinez, david r.; chen, chuan; sun, zehua; leist, sarah r.; drelich, aleksandra; zhang, liyong; ura, marcin l.; berezuk, alison; chittori, sagar; leopold, karoline; mannar, dhiraj; srivastava, shanti s.; zhu, xing; peterson, eric c.; tseng, chien-te; mellors, john w.; falzarano, darryl; subramaniam, sriram; baric, ralph s.; dimitrov, dimiter s. title: high potency of a bivalent human vh domain in sars-cov-2 animal models date: 2020-09-04 journal: cell doi: 10.1016/j.cell.2020.09.007 sha: doc_id: 339093 cord_uid: mwxkvwaz novel covid-19 therapeutics are urgently needed. we generated a phage-displayed human antibody vh domain library from which we identified a high-affinity vh binder ab8. bivalent vh, vh-fc ab8 bound with high avidity to membrane-associated s glycoprotein and to mutants found in patients. it potently neutralized mouse adapted sars-cov-2 in wild type mice at a dose as low as 2 mg/kg and exhibited high prophylactic and therapeutic efficacy in a hamster model of sars-cov-2 infection, possibly enhanced by its relatively small size. electron microscopy combined with scanning mutagenesis identified ab8 interactions with all three s protomers and showed how ab8 neutralized the virus by directly interfering with ace2 binding. vh-fc ab8 did not aggregate and did not bind to 5300 human membrane-associated proteins. the potent neutralization activity of vh-fc ab8 combined with good developability properties and cross-reactivity to sars-cov-2 mutants provide a strong rationale for its evaluation as a covid-19 therapeutic. the global outbreak of a severe acute respiratory distress (sars) coronavirus 2 (sars-cov-2) associated disease 2019 requires rapid identification of therapeutics and vaccines. while many vaccines are in clinical development, the time to market can be relatively long and immunogenicity can be limited for high-risk groups (amanat and krammer, 2020) . alternatively and complementarily, antibodies can be used as safe and effective prophylactics and therapeutics (pelegrin et al., 2015) . convalescent plasma from covid-19 patients inhibited sars-cov-2 infection and alleviated symptoms of newly infected patients (casadevall and pirofski, 2020; rojas et al., 2020) suggesting that potent neutralizing monoclonal antibodies (mabs) may be even more effective. sars-cov-2 genome shares more than 80% homology to the sars-cov . similar to sars-cov, sars-cov-2 uses the spike (s) envelope glycoprotein to enter into host cells. the viral entry is initiated by the receptor binding domain (rbd) of the s protein binding to its receptor, angiotensin-converting enzyme 2 (ace2), leading to conformational change of the s2 subunit and formation of six helical-bundle resulting in membrane fusion between viral and host cells yan et al., 2020) . the sars-cov rbd contains immune-dominant epitopes that can elicit neutralizing antibodies conferring protection to sars-cov infection (he et al., 2005) . a recent bioinformatics study showed that sars-cov-2 rbd has several b cell epitopes (grifoni et al., 2020) . sars-cov-2 rbd based immunogens were able to elicit neutralizing sera in animals (quinlan et al., 2020) . thus, sars-cov-2 rbd is a good target for developing potent neutralizing mabs. we and others have identified such potent neutralizing human mabs targeting the rbd of sars-cov (zhu et al., 2007) and the middle east respiratory syndrome coronavirus (mers-cov) (ying et al., 2014a) . recently, several groups have reported the isolation of potent neutralizing antibodies from convalescent human donors but all are in an immunoglobulin g1 (igg1) format with a molecular mass of about 150 kda ju et al., 2020; rogers et al., 2020; shi et al., 2020; zost et al., 2020) . antibody domains and fragments such as fab (fragment antigen binding, molecular weight of 50 kda), scfv (singe-chain variable fragment, 30 kda) and v h (heavy chain variable domain, 15 kda) are attractive antibody formats as candidate therapeutics (nelson, 2010) . for example, isotope labeled antibody fragments are more suitable for bio-imaging due to their better tissue penetration and faster clearance compared to full-size antibodies (freise and wu, 2015) . single antibody domains (sabd), e.g., camelid v h h (15 kda) exhibit strong antigen binding and high stability (harmsen and de haard, 2007) . we and others have demonstrated that human igg1 heavy chain variable domain (v h ) can be engineered to achieve high stability and affinity to antigens (nilvebrant et al., 2016) , as exemplified by the v h , m36.4, targeting the human immunodeficiency virus type 1 (hiv-1) envelope glycoprotein co-receptor binding site (chen et al., 2008a) . the v h domains small size could improve therapeutic efficacy for infectious diseases, such as covid-19 because of greater penetration to sites of infection. the conformation of the sars-cov-2 s trimer is dynamic with only one rbd in the "up" conformation presenting neutralizing epitopes while epitopes in the other two rbds may be masked . small v h s may achieve binding to the cryptic rbd epitopes during the dynamic "breathing" of the s trimer . in addition, v h s may have j o u r n a l p r e -p r o o f an advantage for treatment of respiratory virus infections because v h s could efficiently penetrate tissue, especially when using direct delivery through inhalation (detalle et al., 2016) . to identify potent neutralizing v h s against sars-cov-2, we panned our large (10 11 clones) and diverse phage-displayed human v h antibody library against recombinant rbd. several v h binders were isolated and screened for their affinities, ace2 competition and stabilities. one of those v h s, ab8, in an fc (human igg1, crystallizable fragment) fusion format, showed potent neutralization activity and specificity against sars-cov-2 both in vitro and in two animal models. to our knowledge, this is the first report for high potency of a human antibody domain (v h ) in two animal models of infection. we generated a large phage-displayed human v h library where heavy chain complementarity-determining regions (hcdr1, 2, 3s) were grafted into their cognate positions of a stable scaffold based on the germline v h 3-23 ( figure s1a) . it was panned against recombinant rbd antigens with two different tags (avi-his and human igg1 fc tag) which were sequentially used to avoid phage enrichment to tags and related epitopes. the quality of the rbd used for panning was confirmed by ace2 binding (figure s1b and c). after three rounds of panning, a panel of v h binders was obtained. among the highest affinity binders, we selected one, v h ab8, which did not aggregate during a six-days incubation at 37°c as tested by dynamic light scattering (dls) (figure s1d ). to increase the v h ab8 avidity and extend its in vivo half-life, it was converted to a bivalent antibody domain by fusion to the human igg1 fc (v h -fc ab8) (figure s1e ). v h ab8 bound to sars-cov-2 rbd and s1 with half-maximal binding concentrations (ec 50 s) of 10 nm as measured by elisa (figure 1a and d) and an equilibrium dissociation constant (k d ) of 19 nm as measured by the biolayer interferometry (blitz system) ( figure 1b) . the relatively fast dissociation rate constant (k d = 4.1× 10 -3 s -1 ) was significantly (23-fold) decreased by the conversion to a bivalent fc fusion format (k d = 1.8× 10 -4 s -1 ) ( figure 1e ) resulting in high avidity. v h -fc ab8 bound to sars-cov-2 rbd and s1 subunit of s protein with ec 50 s of 0.40 nm and 0.20 nm, respectively, and a k d of 0.54 nm ( figure 1e ). it specifically bound to 293t cells expressing s, but not to control 293t cells ( figure 1c and figure s2a ). the binding of v h -fc ab8 was higher than that of igg1 cr3022, an anti-sars-cov antibody cross-reactive with sars-cov-2 (tian et al., 2020) . the v h -fc ab8's halfmaximal facs measured binding concentration (fc 50 ) of 0.07 nm was higher than that of recombinant human ace2-fc (fc 50 = 0.52 nm) ( figure 1f ). these data demonstrate that ab8 selected by an isolated rbd can bind to cell surface associated native s trimer. the binding of v h -fc ab8 to the s protein was significantly improved compared to that of the v h ab8 through avidity effect. competition with human ace2 for binding to rbd is a surrogate indicator for antibody neutralization activity. v h -fc ab8 outcompeted human ace2-fc with a half-maximal inhibitory concentration (ic 50 ) of 1.0 nm ( figure 2a ). note that the v h -fc ab8 was much more effective in outcompeting ace2-fc than v h ab8, consistent with its enhanced binding. ace2 can also block v h ab8 for binding to rbd ( figure s2b ) and cell surface associated s ( figure s2c) . v h -fc ab8 also significantly decreased the kinetics of ace2 binding as measured by blitz ( figure 2b ). v h -fc ab8 did not bind to the sars-cov rbd ( figure 2c ) and did not compete with cr3022 for binding to rbd ( figure 2d ). the cr3022 epitope is located in a conserved region on the rbd core domain distal from the ace2 binding interface, as seen in the crystal structure of the fab cr3022-rbd complex . these results indicate that the ab8 epitope may overlap with the ace2 binding site on rbd. currently, nine prevalent rbd mutants were found in covid-19 patients (priyanka et al., 2020) . six of these mutations (f342l, n354d. n354d/d364y, v367f, r408i, w436r) are located in the rbd core domain and three, k458r, g476s and v483a are in the receptor binding motif (rbm) (figure 3a) . v h -fc ab8 bound to all mutants similarly to wild type rbd as measured by elisa ( figure 3b ). to map the ab8 epitope, we also generated several mutations in non-conserved positions compared to sars-cov spanning the footprint of ace2 on rbm (n439a, g446l, l455a, f456a, a475i, f486a, q493a, q498a, n501a, y505a) ( figure 3c ). most of these mutants retained v h -fc ab8 binding except f486a, f456a and a475i ( figure 3d and 3e) . the f486a significantly decreased binding without affecting the overall rbd conformation (figure s2c and s2d) indicating that f486 directly interacts with ab8. the f456a and a475i mutations decreased the binding by 15% and 40%, respectively, but they also affected the rbd conformation ( figure s2c and s2d) . these results suggest that a portion of the v h ab8 epitope could be in the rbm distal loop tip where the f486 is located at ( figure 3f ). to explore structural aspects of sars-cov-2 neutralization by v h ab8, we performed negative stain electron microscopic analysis of the complex formed between the s protein ectodomain and v h ab8 or soluble ace2 (figure 4) . the density maps showed that both v h ab8 and ace2 were in a quaternary conformation in which two of the protomers in the trimer are in the "down" conformation with the third one in the "up" conformation ( figures 4a and 4b) , similar to the quaternary conformation of the reported ace2-bound s ectodomain (pdb id: 6vyb) (walls et al., 2020) . one molecule of the v h ab8 was observed bound to each rbd domain ( figure 4a ). in the ace2-s complex, one molecule of ace2 was bound to the s protein trimer, straddling one "up" and one "down" rbd region ( figure 4b ). there appears to be a noticeable shift of the "up" rbd domain when it is bound to v h ab8 ( figure 4a ). this shift is not observed when ace2 is bound to the trimer ( figure 4b) . superposition of the two density maps reveals that the binding site of v h ab8 directly overlaps with the ace2 one, precluding simultaneous occupancy on the s protein ectodomain ( figure 4c ). we also found that when ace2 was added subsequent to the j o u r n a l p r e -p r o o f addition of v h ab8, only the v h ab8 bound state was observed, further confirming the ace2 competition with v h ab8. to better understand the spatial relationship between the site of v h ab8 binding and that of ace2 binding, we created a molecular model for ace2 bound s trimer by aligning the rbd region of the crystal structure of sars-cov-2 rbd bound ace2 (pdb id: 6m0j) (lan et al., 2020) to the "up" rbd region in the cryo-em structure of the trimer (pdb id: 6yvb) (wrapp et al., 2020) . superposition of this chimeric structure with the density map of v h ab8-bound s protein trimers reveals that the bound ace2 has extensive overlap with the space occupied by bound v h ab8 ( figure 4d ). the direct spatial overlap between bound v h ab8 and ace2 provides a structural mechanism for the observed effect of ab8 on blocking ace2 binding. the structural findings also showed that the rbm distal loop, which has f486 at its tip, is directly covered by the footprint of the bound v h ab8, consistent with the epitope mapping results showing that f486 is a direct contacting residue for ab8. we used four different assays to evaluate v h -fc ab8 mediated inhibition of sars-cov-2 infection in vitro: a βgalactosidase (β-gal) reporter gene-based quantitative cell-cell fusion assay (xiao et al., 2003) ; an hiv-1 backbonebased sars-cov-2 pseudovirus assay ; and two different replication-competent virus neutralization assays (a luciferase reporter gene assay and a microneutralization (mn)-based assay) (scobey et al., 2013; yount et al., 2003) . v h -fc ab8 inhibited cell-cell fusion much more potently than v h ab8 ( figure 5a ). the inhibitory activity of v h -fc ab8 was also higher than that of ace2-fc. the control anti mers-cov antibody igg1 m336 did not show any inhibitory activity. v h -fc ab8 neutralized pseudotyped sars-cov-2 virus (ic 50 = 0.03 µg/ml) more potently than ace2-fc (ic 50 = 0.40 µg/ml) and v h ab8 (ic 50 = 0.65 µg/ml) ( figure 5b ). the pseudovirus neutralization ic 50 for ace2-fc in our assay is comparable to the one reported by changhai lei et al. (0.03-0.1 µg/ml) (lei et al., 2020) . interestingly, the maximum neutralization by v h ab8 was only 50% compared to the 100% by v h -fc ab8 and ace2-fc, which was also observed for another antibody s309 (pinto et al., 2020) . the complete neutralization by v h -fc ab8/ace2-fc emphasizes the role of bivalency and related avidity in neutralization (klasse and sattentau, 2002) . furthermore, in the reporter gene assay v h -fc ab8 neutralized live sars-cov-2 with an ic 50 of 0.04 µg/ml ( figure 5c ), which is much lower than that for ace2-fc (ic 50 of 6.1 µg/ml) and v h ab8 (ic 50 = 29 µg/ml). ace2-fc seemed to be much less potent against the live virus compared to the pseudovirus, which is also observed by others (ic 50 = 12.6 µg/ml) and may relate to the s expression levels and rbd/s conformation on the virus surface. we also confirmed the high v h -fc ab8 live virus neutralization potency by a microneutralization (mn) assay-100% neutralization (nt 100 ) at 0.1 µg/ml ( figure 5d ). the nt 100 from the mn assay (0.1 µg/ml) was close to the ic 100 (0.2 µg/ml) from the reporter gene assay suggesting consistency in the live virus neutralizing activity of v h -fc ab8 obtained with two independent assays at two different laboratories. these results suggest that v h -fc ab8 is a potent neutralizer of sars-cov-2, which correlates with its strong competition with ace2 for binding to rbd. to evaluate the prophylactic efficacy of v h -fc ab8 in vivo, we used a recently developed mouse ace2 adapted sars-cov-2 infection model, in which wild type balb/c mice are challenged with sars-cov-2 carrying two j o u r n a l p r e -p r o o f mutations q498t/p499y at the ace2 binding interface in the rbd . it was shown that in this model, the aged balb/c mice exhibited more clinically relevant phenotypes than those seen in hace2 transgenic mice . groups of 5 mice each were administered 36, 8, 2 mg/kg v h -fc ab8 prior to high titer (10 5 pfu) sars-cov-2 challenge followed by measurement of virus titer in lung tissue 2 days post infection. v h -fc ab8 effectively inhibited sars-cov-2 in the mouse lung tissue in a dose dependent manner ( figure 6a ). there was complete neutralization of infectious virus at the highest dose of 36 mg/kg, and statistically significant reduction by 1000-fold at 8 mg/kg. remarkably, even at the lowest dose of 2 mg/kg it significantly decreased virus titer by 10fold (two tailed, unpaired t test, p = 0.0075). to exclude possible effects of residual ab8 on viral titration, we performed another experiment in which mouse lungs were perfused with 10 ml of pbs before harvesting for titration. the perfusion did not affect to any significant degree the infectious virus in the lungs ( figure 6b ). the v h -fc ab8 completely neutralized the virus in the lungs at 36 mg/kg and significantly reduced infectious virus at 8 mg/kg. v h -fc ab8 also reduced viral rna in the lungs ( figure 6c ). these results demonstrate the neutralization potency of v h -fc ab8 in vivo. they also suggest that the double mutations q498t/p499y on rbd did not influence v h -fc ab8 binding and contribute to the validation of the mouse adapted sars-cov-2 model for evaluation of neutralizing antibody efficacy. recently hamsters were demonstrated to recapitulate clinical features of sars-cov-2 infection (chan et al., 2020) (imai et al., 2020) . to evaluate the v h -fc ab8 efficacy in hamsters, it was intraperitoneally administered either 24 hours before (prophylaxis) or 6 hours after (therapy) intranasal 10 5 tcid 50 virus challenge. in the therapeutic group, the rationale for administration of the antibody six hours post viral infection is based on the replication cycle length of 5-6 hours after initial infection for sars-cov in veroe6 cells (keyaerts et al., 2005) . six hours after challenge with a high dose of 10 5 tcid 50 , approximately the same number of susceptible cells could become infected and likely produce much more infectious virus, which would need to be neutralized by the antibody to prevent subsequent cycles of infection. nasal washes and oral swab at 1, 3, 5 days post infection (dpi) and different lung lobes at 5 dpi were collected. v h -fc ab8 decreased viral rna by 1.7 log in the lung when administered prophylactically. the lung viral rna decrease in the therapeutic groups was slightly lower (by 1.2 log) ( figure 6d) . interestingly, the viral rna load in the therapeutic groups was to some extent tissue location dependent ( figure 6f ). the variation of the viral load in different lung lobes may relate to nonuniform antibody transport and viral spread inside the lung. remarkably, v h -fc ab8 alleviated hamster pneumonia and reduced the viral antigen in the lung (h&e staining, figure 7a and c and immunohistochemistry figure 7b and d). the control hamsters exhibited severe interstitial pneumonia characterized by extensive inflammatory cell infiltration, presence of type ii pneumocytes, alveolar septal thickening and alveolar hemorrhage. both prophylactic and therapeutic treatment of v h -fc ab8 reduced the lesions of alveolar epithelial cells, focal hemorrhage and inflammatory cells infiltration. v h -fc ab8 also reduced the shedding from mucosal membranes including in nasal washes and oral swabs ( figure s4 ). the decrease in viral rna in nasal washes and oral swabs were not as large as the decrease observed in the lung tissue, similar to a recent finding in hamsters (imai et al., 2020) . overall, the j o u r n a l p r e -p r o o f prophylactic treatment was more effective than the therapeutic treatment in decreasing viral load in nasal washes and oral swabs. notably, prophylactic administration of v h -fc ab8 effectively reduced the infectious virus in the oral swab at 1 dpi, while the post-exposure treatment did not (figure s4c and g) . interestingly, viral reduction (except the viral titer in the oral swab at 1 dpi) was more effective at 3 and 5 dpi compared to that at 1 dpi, likely due to the infection peak occurring before day 3 as reported in hamsters (sia et al., 2020) . a striking finding is that v h -fc ab8 given therapeutically at as low dose as 3 mg/kg can still decrease viral loads in the lung, nasal washes and oral swabs ( figure s5 ). we measured the v h -fc ab8 concentrations at both doses (10 and 3 mg/kg) in the sera at 1 dpi and 5 dpi in the post-exposure treatment groups ( figure s5c ). the higher dose (10 mg/kg) resulted in higher antibody concentration and better inhibitory activity than the lower dose (3 mg/kg). the relatively high concentration of v h -fc ab8 five days after administration also indicates good pharmacokinetics. furthermore, we also compared the v h -fc ab8 concentration in both the sera and lung with that of igg1 ab1, which has a similar affinity to sars-cov-2 and similar degree of competition with the receptor ace2 as v h -fc ab8 . we found that the concentration of v h -fc ab8 in hamster sera is significantly higher than that of igg1 ab1 at 1 and 5 dpi after postexposure administration of the same dose of 10 mg/kg ( figure 7e ), possibly indicating more effective delivery of v h -fc ab8 from the peritoneal cavity to the blood than that of igg1 ab1. we also found that the v h -fc ab8 concentration in all hamster lung lobes was higher than that of the igg1 ab1 ( figure 7f ), suggesting that v h -fc ab8 appears to penetrate the lung tissue more effectively than igg1 ab1. these results indicate that the in vivo delivery of v h -fc ab8 may be more effective than that of full-size antibodies in an igg1 format. the v h -fc ab8 propensity for aggregation was measured at 37°c by dynamic light scattering (dls), which detects particle size distributions in the nanometer range (stetefeld et al., 2016) . it displayed a single peak at 11.5 nm which is the size of a monomeric v h -fc protein ( figure s6a ). the absence of large-size peaks corresponding to large molecular weight species (aggregates) in solution, indicates that v h -fc ab8 is highly resistant to aggregation at high concentration (4 mg/ml) and relatively long times of incubation (6 days) at 37°c. the v h -fc ab8 propensity for aggregation was also evaluated by size exclusion chromatography (sec), which showed that >96% of v h -fc ab8 was eluted in a peak at a position corresponding to a monomeric state with a molecular weight of 80 kda ( figure s6b ). antibody nonspecificity and polyreactivity can be an obstacle for developing an antibody into a clinically useful therapeutic. polyreactivity may not only cause off-target toxicities and interfere with normal cellular functions, but may also reduce antibody half-life (chuang et al., 2015) . to test for potential polyreactivity of v h -fc ab8, a membrane proteome array (mpa) platform was used, in which 5,300 different human membrane protein clones were separately overexpressed in 293t cells in a matrix array achieving a high-throughput detection of binding by facs. v h -fc ab8 did not bind to any of those proteins ( figure s6c ), demonstrating its lack of polyreactivity and nonspecificity. interestingly, we did not detect v h -fc ab8 binding to the human fcγria, which is probably due to the relatively low expression level of fcγria on hek-293t cell surface without concomitant expression of the common γ chain (van vugt et al., 1996) . in addition, we found that v h -fc ab8 bound to the fcγrs much weaker than igg1 (figure s7 ), likely due to the different conformation in the lower hinge region for fc fusion proteins compared to that of igg1s (ying et al., 2014b) . for the fc fusion proteins (even with the same hinge sequence as igg1), binding to fcγrs may be different from that of igg1, and can be affected by the fusion partners (lagassé et al., 2019) . the importance of antibody binding to fcγrs for therapeutic or prophylactic efficacy or toxicity in sars-cov-2 infection is unknown. neutralizing mabs are promising for prophylaxis and therapy of sars-cov-2 infections. recently, many potent neutralizing antibodies from covid19 patients were identified that neutralize pseudovirus with ic 50 s ranging from 1 to 300 ng/ml, and replication-competent sars-cov-2 with ic 50 s from 15 to 500 ng/ml ju et al., 2020; rogers et al., 2020; shi et al., 2020; zost et al., 2020) . by comparison, the v h -fcab8 reported here exhibited comparable or better neutralizing potency against sars-cov-2 pseudovirus and live virus (ic 50 s of 30 ng/ml and 40 ng/ml respectively). of note, ic 50 s can vary widely between different assays and laboratories because there is no generally accepted standardized assay. in addition, there are many factors that contribute to potency and efficacy in vivo. animal models are a more comprehensive and likely more reliable predictor of potential efficacy in humans than in vitro neutralization assays. to our knowledge v h -fc ab8 is the first human antibody domain whose activity was validated in two animal models. in the mouse ace2 adapted sars-cov-2 infection model, v h -fc ab8 significantly decreased infectious virus by 10-fold at 2 days post infection even at a very low dose of 2 mg/kg ( figure 6a ). it also exhibited both prophylactic and therapeutic efficacy in a hamster model. it not only reduced the viral load in the lung and alleviated pneumonia; but it also reduced shedding in the upper airway (nasal washes and oral swab), which could potentially reduce transmission of sars-cov-2. impressively, v h -fc ab8 was active therapeutically even at 3 mg/kg. the finding that v h -fc ab8 persisted for 4 days post administration at significant levels indicates that the pharmacokinetics of v h -fc ab8 is comparable to that of a full size antibody; the half-lives of fc fusion proteins were reported to vary from those of igg1s and can range from hours to days (unverdorben et al., 2016) . the molecular weight of v h -fc ab8 (80 kda) is half of that of full-size igg1 which suggests an advantage in terms of smaller quantities needed to be produced compared to those for igg1s to reach similar number of molecules and efficacy. in addition, it was shown that decreasing binder's size exponentially increases its diffusion through normal and tumor tissues (jain, 1990) . thus, decreasing the size two-fold can increase diffusion through tissues by four-fold. we found that after administration at the same dose, the concentration of v h -fc ab8 was higher than that of igg1 ab1 in both hamster sera and lung tissue. this result might suggest that the v h -fc ab8 diffusion from the peritoneal cavity to the blood and penetration of lung may be faster than that of igg1 ab1. this may further explain its efficacy at low doses in animals. although the low dose showed efficacy in the small animal models, it should be noted that in humans higher doses could be required to achieve comparable degree of efficacy. another caveat is that in the j o u r n a l p r e -p r o o f hamster post-exposure experiment, the v h -fc ab8 was administered at a time (six hours) when the first round of virus replication was likely completed (keyaerts et al., 2005) , but before the infection peak at 1-2 days (sia et al., 2020) . because it inhibits infection of new cells, its administration at around the infection peak or after may not be as effective unless it also kills infected cells in vivo which is under investigation. recently antibody domains including human v h and camelid v h h were reported having varying neutralization potency (chi et al., 2020; sun et al., 2020; wrapp et al., 2020; wu et al., 2020a) . compared to those domains, v h -fc ab8 is unique in terms of potency, aggregation resistance and specificity. v h -fc ab8 exhibited good developability properties including stability at high concentrations and long incubation at 37°c, as well as absence or very low aggregation. in addition, v h -fc ab8 did not bind to the human cell line 293t even at high concentration (1 µm) which is about 1754-fold higher than its k d indicating absence of non-specific binding to many membraneassociated human proteins. a similar result was obtained by the membrane protein array assay showing that v h -fc ab8 did not bind to any of 5,300 human membrane-associated proteins, indicating its lack of non-specificity and thus low potential for off-target toxicity when used in vivo. besides, unlike camel v h hs, the v h ab8 sequence is fully human and therefore likely less immunogenic than that of camelid v h hs. multiple structures are now available for the sars-cov-2 s protein trimer in complex with various neutralizing antibodies, offering insight into antigenic epitopes and inhibitory mechanisms critical for s protein neutralization. epitopes on the sars-cov-2 s protein rbd have emerged as effective targets, as evidenced by the action of several rbd binding antibodies including cr3022, b38, c105, cb6, h014, and s309 (barnes et al., 2020; lv et al., 2020; pinto et al., 2020; shi et al., 2020; wu et al., 2020b) . while b38, c105, and cb6 directly compete with ace2 for binding sites on the rbd surface, h014 occupies a position distinct from these binding sites, precluding ace2 binding via steric inhibition . s309 targets the rbd of the s protein both in closed and open s protein conformations, exhibiting a different mechanism of neutralization (pinto et al., 2020) . a recent study of the structure of the s protein trimer in complex with the nanobody h11-d4 (pdb id: 6z43) revealed full occupancy of the nanobody on all three rbds in a "one up and two down" conformation (huo et al., 2020) , similar to what we report here. our structural analysis demonstrates that the location of the v h ab8 bound to the trimeric s ectodomain directly overlaps the region that would be occupied by ace2 when bound to the s protein. the ace2 blocking is likely the major mechanism of the v h -fc ab8 neutralizing activity, which is significantly augmented by avidity effects due to its bivalency. the narrow neutralization concentration range in the live virus neutralization (10-200 ng/ml for 0%-100% neutralization) ( figure 5d ) indicates a plausible cooperative neutralization mechanism, probably due to the synergistic binding of v h molecules in v h -fc ab8 to rbds. due to its small size, v h may facilitate targeting occluded epitopes on rbd that are otherwise inaccessible to full-length iggs, which is important because the sars-cov-2 s protein is conformationally heterogenous, exposing neutralizing epitopes to varying degrees . the structural analysis shows that v h ab8 is able to simultaneously target all three rbd epitopes in both "up" and "down" conformations, which may provide a structural basis for a unique cooperative neutralization mechanism for v h -fc ab8. v h -fc ab8 with a long flexible linker between v h and fc may allow two j o u r n a l p r e -p r o o f v h molecules to bind simultaneously two protomers in the same s trimer or cross-link two different protomers from different s trimers. the ab8 epitope is distal to the cr3022 epitope, explaining its lack of competition with cr3022. the ab8 contact residue f486 (l472 in sars-cov) is not conserved which likely explains its lack of cross-reactivity to sars-cov. from the gisaid and ncbi databases, we found nine mutations in rbd with relatively high frequencies in current circulating sars-cov-2. six of them are in the core domain (f342l, n354d, n354d/d364y, v367f, r408i and w436r) and three in the rbm (k458r, g476s, v483a). the core domain mutations are far away from the ab8 epitope, thus these mutations do not affect v h -fc ab8 binding to rbd. those three rbm mutations also did not affect ab8 binding although they are close to the ab8 epitope, suggesting that these mutations may not affect ab8 neutralizing activity although neutralization of whole virus carrying these mutations is needed to definitely demonstrate this possibility. interestingly, v h -fc ab8 effectively inhibited the mouse ace2 adapted sars-cov-2 with a q498t/p499y mutation in rbd, indicating that this double mutation also does not affect v h -fc ab8 binding to rbd. these results suggest that v h -fc ab8 may be a broadly crossreactive sars-cov-2 neutralizing antibody. in conclusion, we identified a fully human antibody v h domain that shows strong competition with ace2 for binding to rbd and potent neutralization of sars-cov-2 in vitro and in two animal models. this potent neutralizing activity combined with its specificity and good developability properties warrants its further evaluation for prophylaxis and therapy of sars-cov-2 infection. our elucidation of its unique epitope and mechanism of neutralization could also help in the discovery of more potent inhibitors and vaccines. hamsters were bled at one and five dpi for measuring antibody concentrations in sera by sars-cov-2 s1 elisa. sera was diluted 1:100 and binding was detected by using the goat anti human igg-hrp. (f). viral rna levels in different lung lobes. rna quantity was presented as the tcid 50 equivalence. experiments were performed in duplicate and the error bars denote ± sd, n =2. detailed methods are provided in the online version of this paper and include the following: • key resources table further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, dimiter dimitrov (mit666666@pitt.edu). all requests for resources and reagents should be directed to and will be fulfilled by the lead contact author. this includes antibodies, viruses, plasmids and proteins. all reagents will be made available on request after completion of a material transfer agreement. antibody nucleotide sequence has been deposited to genbank with an accession number of mt943599. the antibody is only allowed for non-commercial use. all data supporting the findings of this study are available within the paper and are available from the corresponding author upon request. vero e6 (crl-1586, american type culture collection (atcc) and 293t (atcc) were cultured at 37°c in dulbecco's modified eagle medium (dmem) supplemented with 10% fetal bovine serum (fbs), 10 mm hepes ph 7.3, 1 mm sodium pyruvate, and 100 u/ml of penicillin-streptomycin. 293t stably expressing sars-cov-2 and human ace2 was cultured in dmem medium containing 200 µg/ml zeocin. hek293f and expi293f were cultured in freestyle 293 serum free medium (thermofisher, cat#12338018) and expi293™ expression medium j o u r n a l p r e -p r o o f (thermofisher, cat# a1435103), respectively. the sars-cov-2 spike pseudotyped hiv-1 backboned virus is packaged in 293t cells after transfecting pnl4-3.luc.re and pcdna3.1 s plasmids. the sars-cov-2 (us_wa-1/2020) and sars-cov2/canada/on/vido-01/2020 obtained from centers for disease control and prevention were propagated in vero e6 cells. the recombinant sars-cov-2-seattlenluc virus and the mouse ace2 adapted sar-cov-2 virus (carrying a q498t/p499y mutation in rbd) recovered by the reverse genetics was produced in veroe6 cells. all work with infectious sars-cov-2 was performed in institutional biosafety committee approved bsl3 facilities using appropriate positive pressure air respirators and protective equipment. the recombinant proteins sars-cov-2 rbd-his, rbd mutants, rbd-fc, ace2-hfc were subcloned into pcdna3.1 expression plasmids, and expressed in expi293f cells. proteins with his tag were purified by ni-nta affinity chromatography and protein with fc tag purified by protein a chromatography. protein purity was estimated as >95% by sds-page and protein concentration was measured spectrophotometrically (nanovue, ge healthcare). v h ab8 antibody was identified by panning of the phage library. v h -fc ab8 were constructed by fusing v h to human igg1 fc with the native igg1 hinge. igg1 ab1 was obtained by our lab through panning of a fab phage library. mers-cov-specific igg1 m336 and sars-cov antibody igg1 cr3022 sequences from other groups were subcloned into the pdr12 plasmid for expression. v h ab8 (in a phagemid pcomb3x with a flag tag) was expressed in hb2151 e. coli and purified by ni-nta affinity chromatography. all other igg1 were expressed in expi293 cells and purified with protein a chromatography. for the mouse model, balb/c mice purchased from envigo (balb/cannhsd, stock# 047, immunocompetent, 11-12 months of age, female) were used for all experiments. they are drug/test naïve and negative for pathogens. biofresh bedding with crinkle bedding added. hamsters have access to food and water ab libitum. food is lab diet 5p00 prolab rmh300. cages are changed weekly or as needed and spot cleaned. for experiment, hamsters were intraperitoneally treated with v h -fc ab8 either 24 hrs before (prophylaxis) or 6 hrs (therapy) after intranasal challenge of 1×10 5 tcid 50 of sars-cov-2. nasal washes and oral swabs were collected at day 1, 3 and 5 post infection (dpi). hamsters were bled at 1 and 5 dpi. all hamsters were euthanized on 5 dpi. at euthanasia, lungs were collected for rna isolation. for viral titer determination, veroe6 cells tcid 50 assay was used. for testing viral rna, viral rna rt-qpcr was used. for testing antibody concentration at sera and lung, sars-cov-2 s1 elisa was used. for histopathology, 10% formalin fixed and paraffin embedded tissues were processed with either hematoxylin and eosin stain (h&e) or immunohistochemistry (ihc). lung lobes were scored based on pathology using microscopy. cr3022. the sars-cov-2 s and the anti-sars-cov antibody igg1 cr3022 and genes were synthesized by idt (coralville, iowa). mers-cov-specific igg1 m336 antibody was expressed in human mammalian cell as described previously (ying et al., 2014a) . briefly, igg1 m336 light chain and heavy chain fd were subcloned into the pdr12 vector containing dual promoters and a igg1 fc cassette. the recombinant plasmid was sequenced and transfected into expi293 cells for expression. the human angiotensin converting enzyme 2 (ace2) gene was ordered from origene (rockville, md). the rbd domain (residues 330-532) and s1 domain (residues 14-675) and ace2 (residues 18-740) genes were cloned in frame to human igg1 fc in the mammalian cell expression plasmid pcdna3.1. the rbd protein with an avitag followed by a 6×his tag at c-terminal was subcloned similarly. these proteins were expressed with expi293 expression system (thermo fisher scientific) and purified with protein a resin (genscript) and by nickel-nitrilotriacetic acid (ni-nta) resin (thermo fisher scientific). the fab cr3022 antibody gene with a his tag was cloned into pcat2 plasmid (developed in house) for expression in hb2151 bacteria and purified with ni-nta resin. protein purity was estimated as >95% by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) and protein concentration was measured spectrophotometrically (nanovue, ge healthcare). unlike camel v h hs, which naturally evolved to be autonomously stable, human v h is usually unstable and easy to aggregate in the absence of v l (li et al., 2016; nguyen et al., 2000) . however, human v h can be selected or engineered with high stability and solubility. to facilitate identification of stable v h binders, we chose engineered germline v h 3-23 as our library scaffold (chen et al., 2008b) . our human v h phage display library was made by grafting heavy chain cdr1, 2, 3 genes derived from 12 healthy donors' peripheral blood monocytes (pbmcs) and j o u r n a l p r e -p r o o f splenocytes (takara, cat. no. 636525) into their cognate positions of a stable scaffold (based on the germline v h 3-23) in a manner similar to the method we previously described but without mutagenesis of cdr1 (chen et al., 2008a) . briefly, cdrs were pcr-amplified by using primers with degenerated adaptors covering cdrs edge regions from diverse v h families in one end, and with sequences annealing to the v h 3-23 framework (fr) regions in the other end. the pcr products were then assembled by overlapping extension pcr by using primers with homologous ending. the whole v h was assembled by overlapping fr1-cdr1-fr2-cdr2 and fr3-cdr3-fr4 fragments. after assembly, the v h fragment was sfi i digested followed by ligated into sfi i linearized pcomb3x phagemid. the recombinant phagemid was then purified, desalted and concentrated for electroporation of bacteria tg1, from which the v h phage particles were rescued and produced. the library size was determined by tittering transformants. the library quality (diversity) was checked by randomly sanger sequencing hundreds of v h clones and also evaluated by panning of diverse antigens. this library contains very large number of clones (10 11 ). for panning, the v h library was alternatively panned against biotinylated rbd-his and rbd-fc proteins. rbd biotinylation occurred through biotin ligase (bira) mediated enzymatic conjugation of a single biotin on avitag (glndifeaqkiewhe) (fairhead and howarth, 2015) . the panning was for 3 rounds with input antigens of 10 µg rbd-his, 2 µg rbd-fc and 0.5 µg rbd-his for the 1 st , 2 nd and 3 rd round, respectively. the panning process begun with incubation of antigens with 10 12 v h phage particles followed by washing with phosphate-buffered saline (pbs) containing 0.1% tween-20. bound phage pulled down by streptavidin-m280-dynabeads were rescued by log-phase tg1 cells with the m13ko7 helper phage. after the 3 rd round panning, positive clones were selected by soluble expression monoclonal (sem) elisa followed by sequencing (chen et al., 2008b) . v h binders were further screened for their binding affinity, stability and ace2 competition. for conversion to fc-fusion, the v h gene was subcloned into psectag b vector containing human igg1 fc fragment. v h -fc ab8 was expressed as described above. enzyme-linked immunosorbent assays (elisas). for detection of rbd biotinylation efficacy, horseradish peroxidase (hrp) conjugated streptavidin was used. for conformation of function of rbd-his after biotinylation, 100 ng ace2-fc was coated into the plates followed by addition of serially diluted biotinylated rbd-his. hrp conjugated streptavidin was used for detection. for other elisas, the sars-cov-2 rbd (residues 330-532) protein was coated on 96-well plates (costar) at 100 ng/well in pbs overnight at 4 o c. for screening sem elisa, clones randomly picked from the infected tg1 cells were incubated with immobilized antigen. bound phages were detected with hrp-conjugated mouse anti-flag tag ab (sigma-aldrich). for the v h -fc binding assay, hrpconjugated goat anti-human igg fc (sigma-aldrich) was used for detection. for the competition elisa with hace2, 2 nm of human ace2-mouse fc was incubated with serially diluted v h , or v h -fc, and the mixtures were added to rbd coated wells. after washing, bound ace2-mouse fc was detected by hrp-conjugated anti mouse igg (fc specific) (sigma-aldrich). for evaluation of ace2 blocking of v h ab8 binding to rbd, 10 nm v h ab8 was incubated with coated rbd in the presence of various concentration of ace2-his (sino biological), and the bound v h ab8 was detected by hrp conjugated anti flag antibody. for evaluation of conformational changes of the epitope mapping rbd mutants, we used a mouse polyclonal anti sars-cov-2 rbd antibody (sino biological, cat. no. 40592-mp01) and the human igg1 cr3022 antibody. for measuring the binding of v h -fc ab8 to rbd mutants, 100 ng rbd mutant was coated on 96-wells plates and incubated with v h -fc ab8 with binding detected by using j o u r n a l p r e -p r o o f hrp conjugated anti human fc antibody. to evaluate the binding of v h -fc ab8 and igg1 ab1 to human fcγrs, recombinant human fcγria, iia, iiia were coated on 96-wells plates followed by addition of biotinylated v h -fc ab8 and igg1 ab1. binding was detected by the streptavidin-hrp. all colors were developed by 3,3′,5,5′tetramethylbenzidine (tmb, sigma) and stopped by 1 m h 2 so 4 followed by recording absorbance at 450 nm. experiments were performed in duplicate and the error bars denote ± 1 sd. blitz. antibody affinities and avidities were analyzed by the biolayer interferometry blitz (fortebio, menlo park, ca). for measuring v h ab8 affinity, the rbd-fc was mounted on the protein a sensor (fortebio: 18-5010). 125 nm, 250 nm and 500 nm v h ab8 were used for association. for measuring avidity of v h -fc ab8, biotinylated rbd-fc was immobilized on streptavidin biosensors (fortebio: 18-5019) for 2 min and equilibrated with dulbecco's phosphate-buffered saline (dpbs) (ph = 7.4) to establish baselines. 50 nm, 100 nm and 200 nm v h -fc ab8 were chosen for association. the association was monitored for 2 min and then the antibody was allowed to dissociate in dpbs for 4 min. the k a and k d were derived from sensorgrams fittings and used for k d calculation. for the competitive blitz, 500 nm v h -fc ab8 was loaded onto the rbd-fc coated sensor for 300 s to reach saturation followed by dipping the sensor into a 100 nm ace2-fc or fab cr3022 solution in the presence of 500 nm v h -fc ab8. the association was monitored for 300 s. the signals from100 nm hace2 or cr3022 binding to the rbd-fc coated sensor in the absence of v h -fc ab8 was independently recorded in parallel. competition was determined by the percentage of signal in the presence of v h -fc ab8 to signal in the absence of v h -fc ab8 (< 0.7 is considered to be competitive) (wu et al., 2020a) . (agilent, cat. no. 200521) . mutants were expressed and purified according to the abovementioned rbd purification procedures. elisa was used to evaluate the binding of these mutants compared to the wild type rbd. a. expression and purification. the codon optimized sars-cov-2 2p s protein ectodomain construct (genbank: yp_009724390.1) was c-terminally tagged with 8xhis and a twin strep tag and cloned into the mammalian expression vector pcdna 3.1 (synbio). hek293f cells were grown in suspension culture using freestyle media (thermofisher) at 37 °c in a humidified co 2 incubator (8% co 2 ). cells were transiently transfected at a density of 1 x 10 6 cells/ml using branched polyethylenimine (pei) (sigma) (portolano et al., 2014) . media was exchanged after 24 h and supplemented with 2.2 mm valproic acid. supernatant was harvested by centrifugation after 4 days, filtered and loaded onto a 5 ml histrap hp column (cytiva). the column was washed with buffer (20 mm tris ph 8.0, 500 mm nacl, 20 mm imidazole) and the protein was eluted with buffer (20 mm tris ph 8.0, 500 mm nacl, 500 mm imidazole). purified protein was concentrated (amicon ultra 100 kda cut off, millipore sigma) and loaded onto a j o u r n a l p r e -p r o o f superose 6 column (cytiva) equilibrated with gf buffer (20 mm tris ph 8.0 and 150 mm nacl). peak fractions were pooled and concentrated to 1.3 mg/ml (amicon ultra 100 kda cut off, millipore sigma). purified s protein ectodomain (0.04 mg/ml) was mixed with v h ab8 (0.02 mg/ml) or soluble ace2 (0.02 mg/ml) and incubated on ice for 10 mins. for the competition experiment, the s protein (0.04 mg/ml) was first incubated on ice with v h ab8 (0.02 mg/ml) for 10 mins then followed by addition of ace2 (0.02 mg/ml) for another 10 mins. the mixtures (4.8 µl) were applied to 300mesh copper grids coated with continuous ultrathin carbon. grids were plasma cleaned using an h 2 /o 2 gas mixture for 15 s in a solarus plasma cleaner (gatan inc.) prior to adding the sample. samples were allowed to adsorb for 30 s before blotting away excess liquid, followed by a brief wash with milliq h 2 o. grids were stained by three successive applications of 2% (w/v) uranyl formate (20 s, 20 s, 60 s). grids containing s protein ectodomain with v h ab8, and s protein ectodomain mixed with both v h ab8 and soluble ace2 were imaged using a 200 kv glacios transmission electron microscope (thermofisher scientific) equipped with a falcon3 camera operated in linear mode. using epu automated acquisition software (thermofisher scientific), 15-frame movies were collected at 92,000x magnification (corresponding to a physical pixel size of 1.6 -) over a defocus range of -0.5 to -3.0 µm with an accumulated total dose of 40 e -/å 2 /movie. grids containing purified s protein ectodomain (0.04 mg/ml) with soluble ace2 (0.02 mg/ml) were imaged using a 200kv glacios transmission electron microscope equipped with a ceta 16m cmos camera (thermofisher scientific). micrographs were collected at 92,000x magnification (physical pixel 1.6 -) over a defocus range of -0.5 to -3.0 µm with a total dose of 50 e -/å 2 using epu automated acquisition software. c. image processing. motion correction and ctf estimation were performed in relion (3.1) (scheres, 2012) . particles were picked by cryolo (1.7.4) (wagner et al., 2019) with pre-trained model for negative stain data. after extraction, particles were imported to cryosparc live (v2.15.1) (punjani et al., 2017) and subjected to 2d classification and 3d heterogeneous classification. final density maps were obtained by 3d homogeneous refinement. figures were prepared using ucsf chimera (pettersen et al., 2004) . after washing, v h ab8 binding was detected by pe conjugated anti flag tag antibody. to test antibody mediated inhibition of cell fusion, the β-galactosidase (β-gal) reporter gene based quantitative cell fusion assay was used (xiao et al., 2003) . in this assay, 293t-s cell expression of t7 rna polymerase was achieved by infection with vaccinia virus vtf7.3, while 293t-ace2 cell expression of t7 promoter controlled β-gal was obtained by infection with vaccinia virus vcb21r. β-gal will be expressed only after fusion of the two types of cells, which can be monitored by chromogenic reactions using β-gal substrate. to assay cell-cell fusion, 293t cells stably expressing sars-cov-2 s (293t-s) cells were infected with t7 polymerase-expressing vaccinia virus (vtf7-3), and 293t cells stably expressing ace2 (293t-ace2) were infected with vaccinia virus (vcb21r lac-z) encoding t7 promotor controlled β-gal. two hours after infection, cells were incubated with fresh medium and transferred to 37 °c for overnight incubation. the next day, 293t-s cells were pre-mixed with serially diluted antibodies or ace2-fc at 37 °c for 1 h followed by incubation with 293t-ace2 cells at a 1:1 ratio for 3 h at 37°c. then cells were then lysed, and the β-gal activity was measured using βgalactosidase assay kit (substrate cprg, g-biosciences, st. louis, mo) following the manufacturer's protocol. fusion inhibition percentage (sample reading, f) was normalized by maximal fusion (reading, f max ) of 293t-s and 293t-ace2 cells in the absence of antibodies using this formula: fusion inhibition % = [(f max -f)/(f max -f blank )] × 100%, in which f blank refers to the od reading of 293t-s and 293t incubation wells. fusion inhibition percentage was plotted against antibody concentrations. experiments were performed in duplicate and the error bars denote ± 1 sd. pseudovirus neutralization assay. pseudovirus neutralization assay was performed based on previous protocols . briefly, hiv-1 backbone based pseudovirus was produced in 293t cells by co-transfection with plasmid encoding sars-cov-2 s protein and plasmid encoding luciferase expressing hiv-1 genome (pnl4-3.luc.re) using pei. pseudovirus-containing supernatants were collected 48 h later and concentrated using lenti-x™ concentrator kit (takara, ca). pseudovirus neutralization assay was then performed by incubation of sars-cov-2 pseudovirus with serially diluted antibodies or ace2-fc for 1 h at 37 °c, followed by addition of the mixture into pre-seeded 293t-ace2 cells. the mixture was then centrifuged at 1000 × g for 1 hour at room temperature. the medium was replaced 4 hrs later. after 24 h, luciferase expression was determined by bright-glo kits (promega, madison, wi) using biotek synergy multi-mode reader (winooski, vt). cells only and virus only wells were included and used for normalization. the 50% pseudovirus neutralizing antibody titer (ic 50 ) was calculated using graphpad prism 7. experiments were performed in duplicate and the error bars denote ± 1 sd. (mn) assay was used as previously described (agrawal et al., 2016a; agrawal et al., 2016b; du et al., 2013; du et al., 2014) . briefly, serially three-fold and duplicate dilutions of individual monoclonal antibodies (mabs) were incubated with 120 pfu of sars-cov or sars-cov-2 at room temperature for 2 h before transferring into designated wells of confluent vero e6 cells grown in 96-well microtiter plates. vero e6 cells cultured with medium with or without virus were included as positive and negative controls, respectively. mers-cov rbd-specific j o u r n a l p r e -p r o o f neutralizing m336 mab (ying et al., 2014a) were used as additional controls. after incubation at 37 o c for 4 days, individual wells were observed under the microcopy for the status of virus-induced formation of cytopathic effect. the efficacy of individual mabs was expressed as the lowest concentration capable of completely preventing virusinduced cytopathic effect in 100% of the wells. full-length viruses expressing luciferase were designed and recovered via reverse genetics as described previously (scobey et al., 2013; yount et al., 2003) . briefly, the sars-cov-2 rna from infected cell culture was reverse-transcribled and constructed into the seven contiguous genomic cdna subclones with interconnecting junctions, which were then bsai/bsmbi digested and ligated into a full-length sars-cov-2 genome cdna through the cohesive ends. a silent mutation of t15102a was introduced into a conserved region in nsp12 to differentiate our recombinant viruses from the circulating sars-cov-2 strains through sanger sequencing. the reporter viruse was synthesized by replacing a 276-bp region in orf7 with a gfp-fused nanoluciferase (nluc) gene. after assembly into full-length cdna, full-length rna was in vitro transcribed and was electroporated into vero e6 cells. virus stocks were propagated on vero e6 cells in minimal essential medium containing 10% fetal bovine serum (hyclone) and supplemented with penicillin/kanamycin (gibico). viruses were tittered in vero e6 usamrid cells to obtain a relative light units (rlu) signal of at least 20× the cell only control background. ab or ace2-fc were serially diluted 4-fold up to eight dilution spots with at a starting dilution 100 µg/ml, and were incubated with sars-cov-urbaninluc and sars-cov-2-seattlenluc viruses at 37°c with 5% co 2 for 1 hour. then virus-antibody dilution complexes were added to the pre-seeding e6 usamrid cells (20,000) in duplicate. virus-only controls and cell-only controls were included in each neutralization assay plate. following infection, plates were incubated at 37 °c with 5% co 2 for 48 hours. then cells were lysed and luciferase activity was measured via nano-glo luciferase assay system (promega) according to the manufacturer specifications. sars-cov and sars-cov-2 neutralization ic 50 were defined as the sample concentration at which a 50% reduction in rlu was observed relative to the average of the virus control wells. experiments were performed in duplicate and ic 50 was obtained by the non-linear fitting of neutralization curves in graphpad prism 7. mouse ace2 adapt sars-cov-2 variant was constructed by introduction of two amino acid changes (q498t/p499y) at the ace2 binding pocket in rbd. virus stocks were grown on vero e6 cells and viral titer was determined by plaque assay . groups of 5 each of 10 to 12-month old female balb/c mice (envigo, #047) were treated prophylactically (12 hours before infection) by intraperitoneal injection with 36, 8, or 2 mg/kg of v h -fc ab8, respectively. mice were challenged intranasally with 10 5 pfu of mouse-adapted sars-cov-2. two days post infection, mice were sacrificed and lung viral titer was determined by the plaque assay. to exclude the residual lung antibody impact on viral titration, mice were euthanized and perfused with 10 ml of pbs via cardiac puncture before lung harvest for viral titration. for virus titration, the caudal lobe of the right lung was homogenized in pbs. the resulting homogenate was serial-diluted and inoculated onto confluent monolayers of vero e6 cells, followed by agarose overlay. plaques were visualized via staining with neutral red on day 2 post j o u r n a l p r e -p r o o f infection. to measure the viral rna in the lung, tissue homogenate lysed in trizol ls (thermofischer) was then processed with thermofischer trizol rna isolation protocol followed by rt-qpcr using the quantifast probe rt-pcr kit (qiagen) to amplify a portion of upe gene. the 50% tissue culture infectious doses (tcid 50 ) equivalence were estimated by running serial dilutions of known tcid 50 standards. infection. sars-cov2/canada/on/vido-01/2020 was propagated on vero'76 cells using dmem with 2% fbs and 1µg/ml l-(tosylamido-2-phenyl) ethyl chloromethyl ketone (tcpk) trypsin. infectious work with sars-cov-2 was approved by the biosafety protocol approval committee (bpac) at the university of saskatchewan and performed in the high containment laboratories at vido-intervac. male hamsters (9-week-old) were obtained from charles river (montreal, qc). for evaluations of prophylactic efficacy, all hamsters (n=7) were injected intraperitoneally with 10 mg/kg of v h -fc ab8 24 hours prior to intranasal challenge of 50 µl/nare containing a total of 1×10 5 tcid 50 of sars-cov-2. for the therapeutic group, hamsters were infected as above and treated intraperitoneally with 10 mg/kg (n=3) or 3 mg/kg (n=4) of v h -fc ab8 6 hours post-infection. untreated hamsters were kept as a control. nasal washes and oral swabs were collected at day 1, 3 and 5 post infection (dpi). hamsters were bled at 1 and 5 dpi. all hamsters were euthanized on 5 dpi. at euthanasia, lung lobes were collected for virus titration and rna isolation. for viral titer determination, nasal washes were diluted in a 10-fold dilution series and absorbed on vero'76 cells in triplicates for 1 hour at 37°c. inoculum was removed and replaced with fresh dmem containing 2% fbs, penn/strep and 1µg/ml tpck. cytopathic effect was scored on day 3 and day 5 post infection. the limit of detection is 13.6 tcid 50 . for testing viral rna, viral rna isolated from nasal and oral swabs using the qiaamp viral rna mini kit (qiagen) and the quantifast probe rt-pcr kit (qiagen) to amplify a portion of upe gene. for rna levels in tissues, 30 mg of tissue homogenate in buffer rlt were processed with the rneasy kit (qiagen) followed by rt-qpcr as above. tcid 50 equivalence were estimated by running serial dilutions of known tcid 50 standards. for testing ab8 concentrations post injection at hamster sera and lung tissue, sars-cov-2 spike-1 elisa was used. s1 protein was coated at 1 µg/ml overnight at 4°c in pbs onto maxisorp plates (nunc). the following day plates were blocked with 5% skim milk and 0.05%tween20. serum collected on day 1 and day 5 post-challenge was diluted 1:100 and absorbed for 1 hour at 37 °c. plates were washed and goat anti human igg-hrp was added. plates were washed and subsequently developed with opd (o-phenylenediamine dihydrochloride) substrate. optical density was measured at 450 nm after 30 mins of incubation. for lung tissues, after blocking homogenates were diluted 1:10 and absorbed overnight at 4°c followed by detection with anti-human igg-hrp and substrate as stated above. the control hamster lung homogenate was used for background correction. for histopathology on day 5 p.i., 10% formalin fixed and paraffin embedded tissues were processed with either hematoxylin and eosin stain (h&e) or immunohistochemistry (ihc) for detection of sars-cov2 antigen; in ihc after blocking tissue slides were treated with anti-nucleocapsid rabbit polyclonal antibodies followed with anti-rabbit hrp antibody. (tucker et al., 2018) . the entire library of plasmids is arrayed in duplicate in a matrix format and transfected into hek-293t cells, followed by incubation for 36 h to allow protein expression. before specificity testing, optimal antibody concentrations for screening were determined by using cells expressing positive (membrane-tethered protein a) and negative (mock-transfected) binding controls, followed by flow cytometric detection with an alexa fluor-conjugated secondary antibody (jackson immunoresearch laboratories). based on the assay setup results, v h -fc ab8 (20 µg/ml) was added to the mpa. binding across the protein library was measured on an ique3 (ann arbor, mi) using the same fluorescently labeled secondary antibody. to ensure data validity, each array plate contained positive (fc-binding; sars-cov-2 s protein) and negative (empty vector) controls. identified targets were confirmed in a second flow cytometric experiment by using serial dilutions of the test antibody. the identity of each target was also confirmed by sequencing. for the mouse model, the statistical significance of difference between v h -fc ab8 treated and control mice lung virus titers was determined by the two-tailed, unpaired, student t test calculated using graphpad prism 7.0. a p value < 0.05 was considered significant. ** p < 0.01. for the mice lung viral titer after perfusion, viral rna and hamster lung viral rna, statistical significance was determined by the mann-whitney u test. a p value < 0.05 was considered significant. ns: p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. for comparing v h -fc ab8 and igg1 ab1 concentration, significance analysis was determined by the two-way anova followed by tukey test in graphpad prism 7.0. a p value < 0.05 was considered significant. ns: p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus 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sars-cov-2 key: cord-265697-bbvlowyo authors: sang, eric r.; tian, yun; gong, yuanying; miller, laura c.; sang, yongming title: integrate structural analysis, isoform diversity, and interferon-inductive propensity of ace2 to predict sars-cov2 susceptibility in vertebrates date: 2020-08-31 journal: heliyon doi: 10.1016/j.heliyon.2020.e04818 sha: doc_id: 265697 cord_uid: bbvlowyo the current new coronavirus disease (covid-19) has caused globally near 0.4/6 million confirmed deaths/infected cases across more than 200 countries. as the etiological coronavirus (a.k.a. sars-cov2) may putatively have a bat origin, our understanding about its intermediate reservoir between bats and humans, especially its tropism in wild and domestic animals are mostly unknown. this constitutes major concerns in public health for the current pandemics and potential zoonosis. previous reports using structural analysis of the viral spike protein (s) binding its cell receptor of angiotensin-converting enzyme 2 (ace2), indicate a broad potential of sars-cov2 susceptibility in wild and particularly domestic animals. through integration of key immunogenetic factors, including the existence of s-binding-void ace2 isoforms and the disparity of ace2 expression upon early innate immune response, we further refine the sars-cov2 susceptibility prediction to fit recent experimental validation. in addition to showing a broad susceptibility potential across mammalian species based on structural analysis, our results also reveal that domestic animals including dogs, pigs, cattle and goats may evolve ace2-related immunogenetic diversity to restrict sars-cov2 infections. thus, we propose that domestic animals may be unlikely to play a role as amplifying hosts unless the virus has further species-specific adaptation. findings may relieve relevant public concerns regarding covid-19-like risk in domestic animals, highlight virus-host coevolution, and evoke disease intervention through targeting ace2 molecular diversity and interferon optimization. erupting in china last december, the novel coronavirus disease has become a worldwide pandemic and caused near 0.4 million confirmed deaths and 6 million infected cases across 200 countries by the end of may 2020 [1, 2] . the etiological virus, designated as severe acute respiratory syndrome coronavirus 2 (sars-cov2) has been identified [3] and related to the viruses previously causing sars or middle east respiratory syndrome (mers) in humans in 2003 and 2012, respectively [4] . these three human-pathogenic coronaviruses putatively evolve from bat coronaviruses, but have different animal tropisms and intermediate reservoirs before transmission to humans [4, 5] . as civet cats and camels were retrospectively determined as reservoirs for sars and mers respectively, there is no conclusion about what animal species passing sars-cov2 to humans [4, 5] . investigations indicated that canivora animals including raccoon dogs, red foxes, badgers and minks as well swine, at a less extent, are susceptible to sars virus infections [6, 7] . although the viral nucleic acids and antibodies to mers were detectable in multiple ruminant species including sheep, goat, and donkeys, the virus inoculation studies did not result in a productive infection for mers disease in these domestic ruminants, nor in horses [8, 9] . as a group of obligate pathogens, viruses need to engage cell receptors for entering cells and race with the host immunity for effective replications and spreading to initiate a productive infection [10] . in this context, the spike proteins protruding on the coronavirus surface are responsible for cell receptor binding and mediating viral entry [5, 6, 7] . for example, mers-cov adopts the dipeptidyl peptidase 4 (dpp4, a.k.a cd26) and sars-cov uses angiotensin-converting enzyme 2 (ace2) as primary receptors for cell attachment and entry [4, 5, 6, 7, 8, 9] . several groups have reported that sars-cov2 uses the same ace2 receptor as sars-cov, but exerts higher receptor affinity to human ace2, which may ascribe to the efficacy of sars-cov2 infection in humans [11, 12] . after cell attachment via the receptor binding domain (rbd) in the n-terminal s1 region of the s protein, the c-terminal s2 region thus engages in membrane fusion. further cleavage of s2 from s1 by a furin-like protease will release and prime the virus entering the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 receipient cells. several furin-like proteases, especially a broadly expressed trans-membrane serine protease 2 (tmprss2), are adopted for priming sars-cov entry [11, 12] . compared with sars-cov, studies showed that sars-cov2 spike protein also evolutionarily obtains an additional furin-like proteinase cleavage site within the s1/s2 junction region for efficient release from the cell surface and entry into the cells [3, 11, 12, 13] . because tmprss2 is widely expressed, the tissue-specific expression of ace2 has been shown to determine sars-cov2 cell tropism in humans [11, 12] . namely, human nasal secretory cells, type ii pneumocytes, and absorptive enterocytes are ace2-tmprss2 double positive and highly permissive to sars-cov2 infection [14, 15] . for cross-species animal tropism, the potential infectivity of sars-cov2 in both wild and domestic animals raises a big public health concern after the prevalence of sars-cov2 infections in humans [16, 17] . this concern involves two aspects: (1) screening to identify the animal species that serve as a virus reservoir originally passing sars-cov2 to humans; and (2) the existing risk of infected people passing the virus to animals, particularly domestic species, thus potentially amplifying the zoonotic cycle to worsen sars-cov2 evolution and prevalence [16, 17] . by diagnosis of animals that in close contact with covid-19 patients or screening of animal samples in some covid-19 epidemic zones, studies detected that domestic cats and dogs could be virally or serologically positive for sars-cov2 [18, 19, 20, 21, 22, 23, 24] , as was a reported infection in a zoo tiger [25] . using controlled experimental infection of human sars-cov2 isolates, several studies demonstrated that ferrets, hamsters, domestic cats and some non-human primate species are susceptible to human sars-cov2 strains [18, 19, 20, 21, 22, 23, 24, 25] . obviously, it is impractical to test sars-cov2 susceptibility experimentally in all animal species. by adoption of a structural simulation based on published structures of the viral s-rbd/ace2 complex, studies have predicted a broad spectrum of vertebrate species with high potential for sars-cov2 susceptibility, which, if true, entails unexpected risks in both public and animal health, and warrants further critical evaluation [26, 27, 28] . ace2 is a key enzyme catalyzing angiotensin (agt) further conversion into numeral active forms of agt1-9, which are hormonal mediators in the body's renin-angiotensin system (ras) [29, 30] . thus, ace2 plays a regulatory role in the blood volume/pressure, body fluid balance, sodium and water retention, as well as immune effects on apoptosis, inflammation, and generation of reactive oxygen species (ros) [29, 30] . in this line, the expression of ace2 is also inter-regulated by immune mediators pertinent to its systemic function. multiple physio-pathological factors, including pathogenic inflammation, influence on ras through action on ace2 expression [29, 30, 31] . interferon (ifn) response, especially that mediated by type i and type iii ifns, comprises a frontline of antiviral immunity to restrict viral spreading from the initial infection sites, and therefore primarily determines if a viral exposure becomes controlled or a productive infection [32] . most recent studies indicated that sars-cov2 evolved viral antagonisms against ifn responses; however, the viral infections was significantly inhibited in vitro or at the early phase in vivo using human ifn-α, ifn-β or type iii inf-λ, indicating that ifns are potential anti-covid19 prophylactics [32, 33, 34] . several recent studies revealed that human ace2 gene behaves like an interferon-stimulated gene (isg) and is stimulated by a viral infection and ifn treatment; however, mouse ace2 gene is not [15, 33, 34] . therefore, to determine the cell tropism and animal susceptibility to sars-cov2, the cross-species ace2 genetic and especially epigenetic diversity in regulation of ace2 expression and functionality should be evaluated [26, 27, 28, 29, 30, 31, 32, 33, 34] . in this study, through integration of structural analysis and key immunogenetic factors that show species-dependent differences, we critically refine the sars-cov2 susceptibility prediction to fit recent experimental validation [16, 17, 18, 19, 20, 21, 22, 23, 24, 25] . along with showing a broad susceptibility potential across mammalian species based on structural analysis [26, 27, 28] , our results further reveal that domestic animals including dogs, pigs, cattle and goats may evolve previously unexamined immunogenetic diversity to restrict sars-cov2 infections. the amino acid sequences of ace2 proteins and dna sequences of the proximal promoters of each ace2 genes were extracted from ncbi gene and relevant databases (https://www.ncbi.nlm.nih.gov/gene). ace2 genes and corresponding transcripts have been well annotated in most representative vertebrate species. in most cases, the annotations were double verified through the same gene entries at ensembl (http s://www.ensembl.org). the protein sequences were collected from all non-redundant transcript variants and further verified for expression using relevant rna-seq data (ncbi geo profiles). the proximal promoter region spans~2.5 kb before the predicted transcription (or translation) start site (tss) of ace2 or other genes. the protein and dna sequences were aligned using the multiple sequence alignment tools of clustalw or muscle through an embl-ebi port (https://www.ebi.ac.uk/). other sequence management was conducted using programs at the sequence manipulation suite (http://www.bioinformatics.org). sequence alignments were visualized using jalview (http://www.ja lview.org) and megax (https://www.megasoftware.net). sequence similarity calculation and plotting were done using sdt1.2 (http://web.cbio.uct.ac.za/~brejnev). other than indicated, all programs were run with default parameters. the phylogenic analysis and tree visualization were performed using megax and an online program, evoview. the evolutionary history was inferred using the neighbor-joining method. percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) was also performed. the evolutionary distances were computed using the p-distance method and in units of the number of amino acid differences per site. other than indicated, all programs were run with default parameters as the programs suggested. the structure files of human ace2 protein and its interaction with sars-cov2 s-rbd were extracted from the protein data bank under the files of 6m17 and 6m0j. the residual mutation and structure simulation were performed using ucsf chimera and pymol available at http s://www.cgl.ucsf.edu/chimera/ and https://pymol.org/, respectively. structural visualization were using pymol. the binding affinity energy (δg), dissociation constant (kd) and interfacial contacts between s-rbd and each ace2 were calculated using a prodigy algorithm at https://bi anca.science.uu.nl/prodigy/. the regulatory elements (and pertinent binding factors) in the~2.5 kb proximal promoter regions were examined against both human/animal tfd database using a program nsite (version 5.2013, at http: //www.softberry.com). the mean position weight matrix (pwm) of key cis-elements in the proximal promoters were calculated using pwm tools through https://ccg.epfl.ch/cgi-bin/pwmtools, and the binding for expression confirmation, several sets of rna-seq data from ncbi gene databases, and one of ours generated from porcine alveolar macrophages (bioproject with an accession number of srp033717), were analyzed for verification of the differential expression of ace2 genes in most annotated animal species. especially, the expression of porcine ace2 isoforms and relevant other genes in the porcine lung macrophage datasets. significantly differentially expressed genes (degs) between two treatments were called using an edger package and visualized using heatmaps or bar charts as previously described [35] . the phylogenic tree of major identified ace2 orthologs/variants from different species was built with a neighbor-joining approach and visualized using an evoview program under default parameter setting. the prediction of sars-cov2 susceptibility is based on the sequence similarity of each ace2 to human ace2 in the s-rbd binding region and simulated using a published human ace2-rbd structure (6m0j) and refers to two recent publications using similar procedures but different structural models [27, 28] . compared with the currently available experimental data, incongruence of the predicted sars-cov2 susceptibility is clearly demonstrated in pangolin, ferret, tiger, cat and horseshoe bat, indicating that some other factors besides ace2-rbd affinity should be considered. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 figure 2 . prediction of sars-cov2 susceptibility in major livestock species based on the conservation of key interacting residues and binding capacity between the viral spike (s) protein on the host ace2 receptor. (a) sars-cov-2 uses the cell receptor, angiotensin-converting enzyme 2 (ace2) for entry and the serine protease tmprss2 and furin for s protein priming. (b) as tmprss2 is broadly expressed and active with a furin-like cleavage activity, the affinity adaption of the s receptor binding domain (rbd) and ace2 receptor determines the viral permissiveness. the contacting residues of human ace2 (a distance cutoff 4.5 å) at the sars-cov-2 rbd/ace2 interfaces are shown, and the contacting network involves at least 19 residues in ace2 (listed in the table cells and referred to the aligned residual positions in human ace2) and 10 residues in the sars-cov-2 rbd (blue circles with residue labels), which are listed and connected with black lines (indicating hydrogen bonds) and red line (represents salt-bridge interaction). the cross-species residual identity (%) of these interacting residues in ace2 are listed in a broad range (32-100%) [26, 27, 28] . (c) we also detected several short ace2 isoforms (underlined) in the domestic animals including dog, pig, goat and cattle, which have an n-terminal truncation spanning 10-13 key residues in the contacting network to s-rbd but keeping the enzyme active sites (indicated by yellow triangles), thus resulting in little engagement by the viral s protein and predicting an unexpected evolutionary advantage for relieving potential covid-19 risk caused by the viral engagement and functional distortion on the classical long ace2 isoforms in these animal species. the ncbi accession numbers of the ace2 orthologs are listed as in figure 1 . human ace2-rbd dog-ace2l-rbd table 1 . predicɵon of binding affinity energy (δg), dissociaɵon constant (kd) and interfacial contacts of the sars-cov2 s-rbd with ace2 orthologs of major livestock species simulated using the human ace2/cov2-rbd structure (6m0j). . most residues involved in binding are highlighted as magenta (ace2) or orange (s) sticks and labeled as one-letter amino-acid codes plus residual numbers in bold or regular font respectively for s or ace2 residues. the dotted/ blue lines indicate intermolecular salt bridge or hydrogen bonds between interacting residues (generated and visualized with ucsf chimera and pymol from protein data bank file 6m0j). (b) to (d) rbd interaction with the simulated structures of ace2 long isoforms from the dog, pig and cattle, respectively. amino acid exchanges in ace2 from another species compared with human ace2 are highlighted in red. (e) prediction of binding affinity energy (δg), dissociation constant (kd) and interfacial contacts of the sars-cov2 s-rbd with ace2 orthologs of major livestock species. most domestic animals ace2 including that from mouse and rat (species known not to be susceptible to sars-cov2) have a binding affinity (δg) at -11.2 to -12.8 kcal/mol that is within the range (11.2-12.9 kcal/mol) between the rbd and the ace2 from the known susceptible species (underlined in the left part of the table), indicating that some other factors, especially those from genetic divergence and natural immunity, contribute to the sars-cov2 susceptibility of different animal species. figure 4 . detection of several short ace2 isoforms (ace2-s) in the domestic animals including dog, pig, goat and cattle. (a) in contrast to most splicing isoforms such as in cats and humans, which share a common proximal promoter and encode ace2 proteins with similar sequences containing all 19 key rbd-interacting residues, these short ace2-s isoforms in domestic animals truncate for 71 (cattle/goat ace2-s) or 132 (dog/pig ace2-s) residues at their n-termini compared with human ace2 or the long ace2 isoforms in these species, thus destroying 10-13 key residues in the contacting network to s-rbd but retaining all enzyme active sites (yellow triangles in the blue ace2 domain bar). this results in little chance to be engaged by the viral s protein binding and predicts an unexpected evolutionary advantage to relieve potential covid-19 risk caused by the viral engagement and functional distortion on the classical long ace2 isoforms in these animal species. (b), (c) and (d) paired structural comparison between the human ace2 structure (6m17) with each simulated ace2-s structure from pig (b), dog (c) and cattle/goat (d). human ace2 structure are in green, and each compared animal ace2-s structure in magenta. the n-terminal residues of both compared structures are in cyan (arrows indicating n-termini of the ace2-s isoforms) and shared c-termini are in red. the 19 key s-interacting residues in human ace2 are shown in blue sticks. in general, all ace2-s orthologs, particular the porcine, show high structural similarity to the human ace2 except the n-terminal truncations. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 3. results and discussions 3.1. vertebrate ace2 orthologs share an functional constraint but experience intra-species diversification in livestock with unknown selective pressure sequence comparison among ace2 orthologs across 30 representative vertebrate species shows a pairwise identity range at 57-85% ( figure 1a and supplemental fig. s1 and excel sheet), which is 15-27% higher than the average value generated through a similarity analysis at 30-70% on gene orthologs at a genome-wide scale [37] . this indicates that ace2 exerts a similar and basic function cross-species, consistent with its systemic and regulatory role as a key enzyme in ras, an essential regulatory axis underlying the body circulatory and execratory systems in vertebrates [29, 30, 31] . a comparison of evolutionary rates of major genes within ras including angiotensinogen (agt), ace, and several receptors of the processed angiotensin hormones showed that ace2 actually evolves slightly faster than ace [ 36, and unpublished data] . this implies that ace2 may bear pressure for ras adapting evolution per a species-dependent physiological and pathological requirement [29, 30, 31] . this evolutionary adaptability of ace2 genes is demonstrated by the existence of numerical genetic polymorphisms [38] and several transcript isoforms particularly in humans and major livestock species ( figure 1b and supplemental fig. s1 and excel sheet). we identified (and verified by rna-seq data analysis) four transcripts of ace2 isoforms in humans ( figure 1b ) that primarily differ in the c-terminal 50 residues within the collectrin domain. particularly, 1-2 short ace2 isoforms were identified in dogs, pigs, cattle, and goats in addition to the longer ace2 consensus to the human's (designated as -s or -l, respectively after the animal common names in figure 1b and thereafter). these livestock ace2-s isoforms have a 70-130 residual truncation at their n-terminal peptidase domains, which also span the region interacting with sars-cov spike protein. the selective mechanisms driving the evolution of these short ace2 isoforms in livestock are unknown, but may relate to previous pathogenic exposure or unprecedented physio-pathological pressure. to support this reasoning, short ace2 isoforms are detected in both domestic bos taurus and hybrid cattle, but not in the wild buffalo and bison; and ace2 isoforms from each species are generally paralogous and sister each other within a clade in the phylogenic tree ( figure 1b ). phylogenic analysis of vertebrate ace2 orthologs/paralogs reveals a general relationship aligning to the animal cladistics ( figure 1b) . in this context, homologs from the fish, frog and chicken conform to a primitive clade. all ungulate homologs form into parallel clades next to each other. the homologs from the glires, primates and carnivores cluster into a big clade (marked with yellow triangle node), which contains all the sars-cov2 susceptible species that have been verified via natural exposure or experimental infections ( figure 1b , marked with red/orange circles). we examined and merged several previous studies about the prediction of sars-cov2 susceptibility in vertebrates based on the simulated structural analysis of s-rbd-ace2 complex [26, 27, 28] . as numerous vertebrate species were predicted to be high or low potential ( figure 1b , labeled as red h or green l) for sars-cov2 susceptibility, incongruence between the predicted sars-cov2 susceptibility and infected validation is apparent in pangolin, ferret, tiger, cat and horseshoe bat, indicating that some other factors besides ace2-rbd affinity should be considered [15, 32, 33, 34] . we, therefore, refined the prediction matrix to include the rbd-binding evasion of some ace2 orthologs identified in major livestock species and the interferon-stimulated ace2 expression in priming sars-cov2 infections [15, 32, 33, 34] . several recent studies have elegantly demonstrated the structural interaction of the viral s protein or its rbd in complex with human ace2 receptor [39, 40] . showing that the contacting residues at the rbd/ace2 interface (figure 2a ) involve at least 19 residues in ace2 ( figure 2b , listed in the table cells and referred to the aligned residual positions in human ace2) and 10 residues in the sars-cov-2 rbd ( figure 2b , blue circles with residue labels above the table) [28, 39, 40] . the cross-species residual identity (%) of these interacting residues in ace2 are dispersed in a broader range (32-100%) than the whole ace2 sequence identity rate at 57-85% [37] , indicating a faster evolution rate of this virus-interacting region. notably, the s-binding region spans a large part of the n-terminal peptidase domain, thus s-binding may competitively block a majority of active sites to inhibit the physiological action of ace2 ( figure 2c ). using a similar structural analysis procedure [27, 28] , we modeled the ace2 structures of animal species of interest and simulated their interaction with sars-cov2 s-rbd based on a published rbd-human ace2 structure (protein data bank file 6m0j) [39] . figure 3 demonstrates the s-rbd interaction with the simulated structures of ace2 long isoforms from the dog, pig and cattle, respectively. the major changes of the rbd-ace2 interacting interfaces are from the residual exchanges in ace2 from other species compared with human ace2 ( figure 2b -2d, highlighted in red). in addition, the exchange of n90t (in pigs) and n322y (in cattle and sheep) would destroy the n-glycosylation site in human ace2. ace2 from goat (supplement fig. s1 ) exhibits identical amino acid exchanges as in cattle in the rbd-ace2 interfacial contacts. in contrast, when compared with human ace2, ace2 from cats (supplement fig. s1 ) conserves all relevant glycosylation sites in human ace2 [28, 39] . we also calculated the interfacial contacts using parameters of protein-protein interaction including the predictable binding affinity energy (δg), dissociation constant (kd) and number of different interfacial contacts within the s-rbd and ace2 contact. although the exact numbers may differ from the previous reports [39] , they provide a very comparable matrix generated using the same algorithm ( figure 3e ) [41] . data show that the ace2 of most domestic animals, including that from mouse and rat (the species known to be unsusceptible to human sars-cov2) have a binding affinity (δg) at -11.2 to -12.8 kcal/mol. this is within the binding affinity range (11.2-12.9 kcal/mol) between the rbd and the ace2 from known susceptible species ( figure 3e , underlined in the left part of the table). this indicates that other factors, conceivably from genetic divergence and/or natural immunity, also contribute to sars-cov2 susceptibility in animal species. therefore, an effective prediction matrix should include the critical immunogenetic factors to further determine virus susceptibility in addition to the sequence/structural similarity of ace2 receptors (figure 1 and fig. s1 ) [15, 34, 38] . we detected several short ace2 isoforms in the domestic animals including dog, pig, goat and cattle that have an n-terminal truncation spanning 10-13 key residues in the contacting network to s-rbd but retain the enzyme active sites ( figure 4a ). most of the splicing isoforms from ace2 genes such as in zebrafish, cats and humans, share a common proximal promoter and encode ace2 proteins containing all 19 key rbdinteracting residues [39, 40] . however, these short ace2-s isoforms in domestic animals truncate for 71 (cattle/goat ace2-s) or 132 (dog/pig ace2-s) residues at their n-termini compared with the long ace2 isoforms in the same species (figure 1 and fig. s1 ). therefore, these short 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 ace2 isoforms destroy 10-13 key residues in the contacting network to s-rbd but likely retain ace2 enzymatic function in ras. paired structural comparison between the human ace2 structure (extracted from 6m17) with each simulated ace2-s structure from the pig, dog, and cattle/goat, reveals that all these ace2-s orthologs from domestic animals, particularly the porcine one, show high structural similarity to the human ace2 except for the n-terminal truncations ( figure 4b-4d ). this indicates that these short ace2 isoforms in domestic animals have little chance to be engaged by the viral s-binding, and predict an unexpected evolutionary advantage to allay potential covid-19 risk resulting from viral engagement and functional distortion on the classical long ace2 isoforms in these animal species [38, 42] . sars-cov2 infection induces a weak ifn response but a production of high amount of inflammatory cytokines including interleukin (il)-6 and chemokine cxcl10 in most severe covid-19 patients [43, 44, 45, 46] . studies of sars and mers showed that these pathogenic coronaviruses share similar viral antagonisms including the endoribonuclease (endou) encoded by nonstructural protein 15 (nsp15), which directly blunts cell receptors responding to viral dsrna and in turn weaken the acute antiviral response [47] . several recent studies revealed that sars-cov2 seems more cunning in not only evading or antagonizing but also in exploiting the ifn response for efficient cell attachment [15, 43, 44, 47] . as a key enzyme in ras, the expression of ace2 gene has been primarily investigated for physiological response to circulatory regulations, and a response to pathological inflammation is also expected [29, 30, 31] . however, the expression of human ace2 gene was highly responsive to both viral infection and host ifn response, i.e. human ace2 gene seems an unstudied ifn-stimulated gene (isg) [15, 33] . surprisingly, the isg propensity of ace2 genes is species-dependent, for examples, the mouse ace2 gene is less ifn responsive, which may partly explain the mouse insusceptibility to sars-cov2 infection [15] . to categorize the different ifn-inductive propensity of ace2 genes in vertebrates, particularly in major livestock species, we profiled the regulatory cis-elements and relevant transcription factors in the proximal promoter regions of each ace2 genes (2.5 kb before tss or atg). figure 5 illustrates major regulatory cis-elements located in ace2 genes from major livestock animals and several reference animal species. data show that animal ace2 gene promoters are evolutionally different in containing ifn-or virus-stimulated response elements (isre, prdi, ifrs, and/or stat1/3 factors) and cis-elements responsive to proinflammatory mediators. all these cis-elements recruit corresponding transcription factors (tf) to mediate differential ace2 responses to antiviral ifns and inflammation that is associated with covid-19 disease [2, 3, 48] . we discover that ace2 genes obtain species-different isg propensity responsive to ifn and inflammatory stimuli. in most (if not all) of the sars-cov2 susceptible species, the ace2 genes obtained the ifn-responsive elements between the typical robust and tunable ifn-stimulated genes (isg) [49] . in general, the robust isgs (isg15 as an example here) are stimulated in the acute phase of viral infection and play a more antiviral role; in contrast, the later responsive tunable isgs (irf1 as an example) contribute more to anti-proliferation of ifn activity [49] . in addition, unlike the promoter of the short ace2 isoforms in cattle and goats, which share most common promoter regions with their paralogous long isoforms, the short ace2 isoforms of dogs (dog-s) and pigs (pig-s) have distinct proximal promoter regions (and different ifn responsivity) to the paralogous long ace2 isoforms (figures 5 and 6) . results indicate that the short ace2 isoforms in pigs and dogs diversify from their long paralogs at both the levels of genetic coding and epigenetic regulation to adapt to some evolutionary pressure, such as that from pathogenic interaction (figure 7 ) [38, 50] . 3.6. matrix scores of interferon-inductive elements in ace2 gene promoters correspond to sars-cov2 susceptibility the position weight matrix (pwm) stands as a position-specific scoring model for the binding specificity of a transcription factor (tf) on the dna sequences [51] . using pwm toolsets online (https://ccg.epfl. ch/cgi-bin/pwmtools), we evaluate mean pwm of key cis-elements in the proximal promoters of ace2 genes that containing binding sites for canonical ifn-dependent transcription factors, which include isre/stat, irf1. irf3/7 and irf8, as well as c/ebp representing a core transcription factor for pro-inflammation. these ifn-dependent transcription factors, particularly irf3/7 and isre/stat for ifn stimulation, are differentially enriched in the promoter regions of ace2 genes in a species-dependent way. higher enrichment of isre/stat1/3 and/or irf3/7 binding sites are detected in most sars-cov2/covid19 susceptible species (indicated with solid orange or red circles, respectively). in contrast, the pwm for irf1 and c/ebp, which regulate inflammation, are less differential in ace2 promoters from different animal species, indicating that ace2 genes are more universally regulated by inflammation than that by the viral infection or ifn-induction in a species-dependent way ( figure 6 ). as compared with the promoters of a typical human robust isg15 and tunable irf1 genes, this data indicate that ace2 genes (particularly the primate ones) are not typical robust or tunable isgs as represented by isg15 or irf1, but respond differently to viral infection (through irf3/7) or ifn auto-induction (via isre/stat) in a species-dependent manner ( figure 6 ) [49] . higher enrichment of isre/stat1/3 and/or irf3/7 corresponds to sars-cov2 susceptibility in experimentally validated mammalian species especially primates, but not to the phylogenically distant species such as zebrafish, which has very low potential for sars-cov2 susceptibility due to the high disparity of ace2 structures (figure 1 and fig. s1 ). in addition, the proximal promoters of the pig and dog ace2-s genes differ much in their ifn-responsive elements to most ace2 promoters in mammalians (figures 5 and 6 ). however, they are phylogenically sister to the ace2 promoters from the primitive vertebrates (frog, chicken and zebrafish) (figure 7 , phylogenic tree). this indicates that the expression of these short ace2 isoforms is more conservative than the long ace2 paralogs, which represent a more recent evolution obtaining ace2 epigenetic regulation by ifn-signaling ( figure 7) [50]. studies show that affinity adaption of the viral s-rbd and ace2 receptor determines the cellular permissiveness to the virus [28, 39, 40] . sars-cov2 not only adapts a high binding affinity to human ace2 for cell attachment, but also antagonizes host antiviral interferon (ifn) response and utilizes ifn-stimulated property of human ace2 gene to boost spreading [15, 39, 40, 50] . in addition to structural analysis of simulated s-rbd-ace2 interaction, we propose that several immunogenetic factors, including the evolution of s-binding-void ace2 isoforms in some domestic animals, the species-specific ifn system, and epigenetic regulation of ifn-stimulated property of host ace2 genes, contribute to the viral susceptibility and the development of covid-19-like symptoms in certain animal species [15, 39, 40, 50] . a computational program in development that incorporates this multifactorial prediction matrix and in vitro validation of sars-cov2 susceptibility in major vertebrate species will be necessary to address public concerns relevant to sars-cov2 infections in animals (figure 7 ). it will also lead to the development of better animal models for anti-covid19 investigations [21] . in addition, several ifn-based therapies for treatment of covid19 have been proposed and are in the process of clinic trials [52, 53, 54, 55] . considering the viral stealth of ifn-stimulated property of human ace2, a timely and subtype-optimized ifn treatment should be delivered than a general injection of typical human ifn-α/β subtypes [52, 53, 54, 55] . in this line, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 domestic livestock like pigs and cattle have a most evolved ifn system containing numerous unconventional ifn subtypes. some of these unconventional ifn subtypes, such as some porcine ifn-ω exert much higher antiviral activity than ifn-α even in human cells and most ifn-λ retaining antiviral activity with less pro-inflammatory activity, could be utilized for developing effective antiviral therapies [56, 57] . in summary, a predication matrix, which integrates the structural analysis of s-rbd-ace2 interfacial interface and the species-specific immunogenetic diversity of ace2 genes, was used to predict the sars-cov2 susceptibility and fit current knowledge about the infectious potential already validated in different animal species (figure 7 ). more extensive validation experiments are needed to further improve this prediction matrix. our current results demonstrate several previously unstudied immunogenetic properties of animal ace2 genes and imply some domestic animals, including dogs, pigs and cattle/goats, may obtain some immunogenetic diversity to confront sars-cov2 infection and face a less figure 6 . scores of mean position weight matrix (pwm) of key cis-elements in the proximal promoters of ace2 genes that containing binding sites for canonical ifndependent transcription factors, which include isre/stat1/3, irf1, irf3/7 and irf8, as well as c/ebp as a core transcription factors for pro-inflammatory response. these ifn-dependent transcription factors, particularly irf3/7 and isre/stat critical for ifn stimulation, are differentially enriched in the promoter regions of ace2 genes in a species-dependent way. especially, increased enrichment of isre/stat1/3 and irf3/7 binding sites are detected in the sars-cov2/covid19 susceptible species (indicated with solid orange or red circles, respectively). in contrast, the pwm for irf1 and c/ebp, which regulate inflammation, are less differentially enriched in ace2 promoters from different animal species. the promoters of a typical human robust interferon-stimulated gene (isg) 15 and irf1 (a typical tunable isg) are used as references. higher enrichment of isre/stat1/3 and irf3/7 corresponds to sars-cov2 susceptibility in experimentally validated animal species and humans. abbreviations: c/ebp, ccaat/enhancer binding protein; irf, interferon-regulatory factor; isre, interferon-sensitive response element; stat, signal transducer and activator of transcription; pwm, position weight matrix. the pwm tools are used through https://ccg.epfl.ch/cgi-bin/pwmtools. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 covid19 risk than previously thought. however, immediate biosecurity practices should be applied in animal management to reduce animal exposure to the virus and prevent potential species-specific adaptation ( figure 7) . for livestock breeding programs that targeting disease resistance to respiratory viruses, the genetic and epigenetic diversity of ace2 genes as well antiviral isgs are highly recommended [49, 50, 56, 57] . in conclusion, sars-cov2 evolves to fit well with human (and nonhuman primates) ace2 receptor through the structural interfacial affinity, immunogenetic diversity and epigenetic expression regulation, which results in a highly infectious efficacy [1, 2, 3, 15, 28, 39, 40]. most mammalian animals, especially those that belong to glires, primates and carnivores, have a higher potential for sars-cov2 susceptibility but in a species-different manner based on the existence of s-binding-void ace2 isoforms and the difference of the ifn-inductive propensity of the major ace2 genes. most ungulate animals appear have a low susceptibility potential with horses and sheep having a high potential (figure 7) . current development of ifn-based anti-covid19 therapies should consider the isg property of human ace2 gene to optimize for timely application using a highly-antiviral subtype that potentially have less inflammatory (or even anti-inflammatory) activity [ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 evolution of the ifn complex and functional diversity in domestic animals (such as pigs and cattle) provides a natural model for optimizing ifn antiviral regulation and therapy development [56, 57] . eric r. sang, yongming sang: conceived and designed the experiments; performed the experiments; analyzed and interpreted the data; wrote the paper. yun tian: performed the experiments; analyzed 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https://doi.org/10.1016/j.heliyon.2020.e04818. key: cord-296187-nnv2e7gr authors: mulgaonkar, nirmitee; wang, haoqi; mallawarachchi, samavath; fernando, sandun; martina, byron; ruzek, daniel title: bcr-abl tyrosine kinase inhibitor imatinib as a potential drug for covid-19 date: 2020-08-18 journal: biorxiv doi: 10.1101/2020.06.18.158196 sha: doc_id: 296187 cord_uid: nnv2e7gr the rapid geographic expansion of severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the infectious agent of coronavirus disease 2019 (covid-19) pandemic, poses an immediate need for potent drugs. enveloped viruses infect the host cell by cellular membrane fusion, a crucial mechanism required for virus replication. the sars-cov-2 spike glycoprotein, due to its primary interaction with the human angiotensin-converting enzyme 2 (ace2) cell-surface receptor, is considered as a potential target for drug development. based on in silico screening followed by in vitro studies, here we report that the existing fda-approved bcr-abl tyrosine kinase inhibitor, imatinib, inhibits sars-cov-2 with an ic50 of 130 nm. we provide evidence that although imatinib binds to the receptor-binding domain (rbd) of sars-cov-2 spike protein with an affinity at micromolar, i.e., 2.32 ± 0.9 μm levels, imatinib does not directly inhibit the spike rbd:ace2 interaction – suggesting a bcr-abl kinase-mediated fusion inhibition mechanism is responsible for the inhibitory action. we also show that imatinib inhibits other coronaviruses, sars-cov, and mers-cov via fusion inhibition. based on promising in vitro results, we propose the abl tyrosine kinase inhibitor (atki), imatinib, to be a viable repurposable drug against covid-19. in early december 2019, the chinese health authorities reported several cases of pneumonia of unknown cause that had originated in wuhan, a city in the hubei province of china. the causative agent of this outbreak was identified to be a virus that belonged to the sarbecovirus subgenus, orthocoronavirinae subfamily which was previously referred to by its interim name 2019 novel coronavirus (2019-ncov) [1, 2] and was later named as sars-cov-2 [3] . due to the rapid spread of covid-19, the world health organization (who) declared it a global pandemic in march 2020 [4] . by mid-august 2020, over 21 million cases have been confirmed around the world, resulting in more than 768,000 deaths [5] . unfortunately, there is no approved antiviral treatment or preventive vaccine for coronaviruses in humans. since supportive care is the only recommended interim treatment, it is imperative to identify repurposable lead compounds to rapidly treat covid-19 patients until a sars-cov-2-specific drug and a vaccine is developed. although the coronavirus genome consists of numerous conserved druggable enzymes, including papain-like protease (plpro), 3c-like protease (3clpro), non-structural proteins rna-dependent rna polymerase (rdrp) and helicase, development of clinically approved antiviral therapies has proven to be a difficult task [6] . the surface structural spike glycoprotein (s), a key immunogenic cov antigen essential for virus and host cell-receptor interactions, is an important target for therapeutic development. the spike protein consists of an n-terminal s1 subunit (receptor binding) and a c-terminal s2 subunit (membrane fusion). the s1 subunit contains the receptor-binding domain (rbd) which attaches to the host membrane, thus playing an important role in viral entry. sars-cov-2 utilizes the ace2 receptor for entry and the transmembrane protease, serine 2 (tmprss2) for spike protein priming [7] . crystallographic studies have shown that sars-cov-2 binds to the ace2 receptor, with a binding mode nearly identical to that of sars-cov [8] [9] [10] [11] . the binding affinity of the ace2 receptor to the rbd of the sars-cov-2 spike protein is reported to be significantly higher as compared to sars-cov [10, 11] . based on the importance of virus membrane fusion events in the viral life cycle and its infectivity, the spike protein of sars-cov-2 was targeted for drug screening. this study utilizes in silico methodology followed by in vitro experimental validation to screen existing fda-approved small molecule drugs specific to the rbd of the spike protein of sars-cov-2 to identify repurposable drugs targeting further clinical validation. a model for sars-cov-2 spike protein was constructed using the crystal structure (6vsb_chain a) to correct missing residues. the amino acid sequence identity between the target sequence (genbank: qhd43416.1) and template (6vsb_chain a) was 99.58%. the sars-cov-2 model showed an rmsd of 0.4683 å relative to the crystal structure (6vsb_chain a). structure assessment of the predicted model using the ramachandran plot showed 90.04% residues in the most favored regions with 2.03% outliers. none of the outliers contained the residues present at the active site of the protein. the predicted model was further used for in-silico studies. in virtual screening, a library of approximately 5,800 compounds was docked against the sars-cov-2 spike rbd protein. the output was analyzed for common classes of drugs with highest (most negative) docking scores that resulted in seven compounds with three compounds, antiviral825, antiviral2038 and antiviral2981 with docking scores of -6.30 ± 0.00, -6.20 ± 0.20 and -6.00 ± 0.00 kcal/mol from the enamine antiviral library, and four compounds, ponatinib, imatinib, ergotamine, and glecaprevir with docking scores of -7.63 ± 0.06, -6.80 ± 0.17, -7.70 ± 0.00 and -7.20 ± 0.35 kcal/mol from the zinc15 fda library respectively. the above libraries were chosen to help identify a repurposable drug that can potentially inhibit the sars-cov-2. the screened compounds had the highest scores within their respective sets and had one or more binding conformations at the ace2 binding domain of the spike protein. the most common class of drugs was found to be abl tyrosine kinase inhibitors (atki), and hence two drugs (ponatinib and imatinib) with the highest scores were selected for in vitro testing. the binding scores for the seven screened compounds at the rbd are shown in fig. 1a and detailed description of the screened drugs is given in table s2 under supplementary data. the high affinity of the screened compounds is visible when compared with the negative control dimethyl sulfoxide (dmso), which is ineffective against coronaviruses [12] . based on promising in silico data, and initial viral plaque assay results (fig. 1a) , imatinib was chosen to be advanced for further experimental validation. (due to a supply-shortage ponatinib and ergotamine were unavailable for purchase and hence could not be included in the initial viral plaque assays). previous studies have shown imatinib to inhibit sars-cov and mers-cov by blocking endosomal fusion at the cell-culture level [6, [12] [13] [14] [15] . it has been suggested that tyrosinekinase inhibitors do not affect the cleavage of the spike protein but inhibit spike-mediated endosomal fusion [6, 12, 13] . the high affinity of tyrosine-kinase inhibitors towards the spike protein is deduced from the initial docking results, where both imatinib and ponatinib have shown highly negative binding free energies. first, we evaluated the toxicity of imatinib when incubating the compound on vero cells for one hour or eight hours. in the experiments where the compound remained on the cells for eight hours toxicity was measured at concentrations of 25 µm, 12.5 µm, 6.3 µm, and 3.2 µm. however, in the 1-hour design, no toxicity was observed. next, we evaluated the ability of imatinib to inhibit replication and entry. at concentrations as low as 0.2 µm the compound was effective in suppressing 50% of plaque formation in the 8-hr design, and the ic50 value determined using linear regression was 130 nm. consistent with the toxicity data, toxicity was observed between 25 and 3.2 µm. the compound also showed efficacy in the 1-hour design, with higher ic50 values. these data indicate that imatinib inhibits virus replication in vitro as shown in fig. 2a and 2b. to evaluate if imatinib inhibits viral entry, we performed two fusion assays: endosomal (vero) and plasma membrane (vero-tmprss2) as shown in fig. 2c and 2d, respectively. based on cytotoxicity, at concentrations below 15 nm, no toxicity was observed microscopically (red arrow in the graph). vsv-g control revealed 100% infectivity (cytopathic effect at every concentration below this, suggesting the inhibitor did not affect vsv-g entry. vsv-g particles cells do not carry spike proteins and thus, no significant entry inhibition occurred, suggesting that entry inhibition is likely mediated through the spike protein. however, the effect on vero-tmprss2 cells was less clear for any of the coronaviruses used when compared to the vsv-g control. a similar level of toxicity was observed in these cells. it is worth noting that toxicity is probably the result of incubating cells with imatinib for 16 hours in the assay. taken together, there is evidence that imatinib inhibits spike fusion and prevents viral entry, possibly by preventing endosomal entry. the binding kinetics of imatinib to the rbd of sars-cov-2 spike protein was evaluated using biolayer interferometry (bli), as shown in fig. 3a . the analysis showed that imatinib binds to the sars-cov-2 rbd protein with an on-rate (kon) as (3.22 ± 0.45) × 10 3 m -1 s -1 and dissociates with an off-rate (koff) as (7.07 ± 1.87) × 10 -3 s -1 . this resulted in an equilibrium affinity constant (kd) 2.32 ± 0.9 µm which is calculated as a ratio of the koff and kon rates. the affinity values indicate that 50% of the rbds on the surface spike glycoproteins will be occupied at micromolar concentrations of imatinib. however, this value is too close to the toxicity levels observed in the above assays and very high compared to the ic50 value, as well as the nanomolar affinity of ace2 on immobilized rbd (fig. s1 ) suggest that it is likely to inhibit spike fusion by the other previously suggested moa [12] . in vitro colorimetric assays were performed over a 160 pm to 500 nm range in imatinib concentration to assess the ability of imatinib to directly inhibit the rbd:ace2 interaction. the colorimetric signal of the positive control (no inhibitor) reaction was strong, and the blank wells exhibited an absorbance of ~ 0.05 at 450 nm as per the manufacturer's instructions. the test wells (with inhibitor) showed absorbance comparable to the positive control wells indicating that imatinib did not affect the sars-cov-2 rbd:ace2 interaction in the indirect competitive enzyme-linked immunosorbent assay (elisa), as shown in fig. 3b . a pharmacophore analysis was done to evaluate as to why imatinib showed promising in-silico results yet failed to directly inhibit the rbd:ace2 interaction. the primary binding site of the sars-cov-2 rbd was revealed via docking. pharmacophore analyses were done to further elucidate the interactions between the drug molecules and their receptors (fig. 4) . twenty-five pharmacophores were collected from the top five binding positions of imatinib at the primary active site of abl2 tyrosine kinase (native receptor), where each purple sphere represents a pharmacophore as depicted in fig 4a. similarly, an additional 25 pharmacophores were collected from the first five binding sites of imatinib at the primary active site of the sars-cov-2 rbd. the rbd pharmacophores were represented as yellow spheres in fig 4b. from the results of elixir-a alignment, it is evident that four pharmacophores between abl2 kinase and rbd overlapped (red spheres). this significant overlap reveals why the compounds that were originally screened using sars-cov-2 rbd also bound to abl2 kinase, ultimately ensuing in the inhibitory action. the above point is further explicated due to the 54.55% identity between the active sites of the abl2 (uniprotkb: p42684 aa 288-539) and sars-cov-2 spike rbd (uniprotkb: p0dtc2 aa 319-541) generated by a protein blast (blastp) [16, 17] search. there is an urgent need for finding a treatment against the current pandemic of the sars-cov-2. health experts across the globe are trying to use existing clinically approved drugs to treat patients until a specific drug is developed. the present study, using a combination of computational techniques followed by in vitro studies, identified imatinib, an fda approved anti-cancer drug as a potential treatment of sars-cov-2 infection. the data indicate inhibition of sars-cov-2 replication at ic50 of 130 nm. our results suggest that imatinib prevents viral replication by inhibiting the virus at the fusion stage, possibly by preventing endosomal entry. binding studies revealed that the affinity of imatinib for the sars-cov-2 spike rbd protein is still lower (higher kd value) than the previously published values of nanomolar range (ligand id: bdbm13530) [18] for imatinib on abl tyrosine kinase [19] and in range with the micromolar affinities of imatinib to the src-family kinases, frk and fyn [20] . although imatinib is not a promiscuous drug, it has been found to bind tightly to tyrosine kinases other than abl [20, 21] . pharmacophore mapping between abl2 and sars-cov-2 rbd and a 54.55% identity at the active site of the two proteins explains why imatinib binds to the sars-cov-2 rbd as well. however, imatinib failed to directly inhibit the sars-cov-2 spike rbd:ace2 interaction in the competitive elsa assays. therefore, it is likely that imatinib causes inhibition of virus fusion via cellular kinase pathway resulting in inhibition of virus replication, as previously described for other coronaviruses [12] . the results provide further evidence supporting the recent clinical trials (clinicaltrials.gov identifier: nct04346147, nct04357613, nct04356495, nct04394416, and nct04422678) for covid-19 patients with imatinib. a swiss-model server [22] was used to construct a homology model of the sars-cov-2 spike protein using the crystal structure of the sars-cov-2 spike protein (pdb:6vsb_chain a) as the template [10] . the genome sequence wuhan-hu-1 (genbank: mn908947.3) was used as a representative of the sars-cov-2. spike protein sequence (genbank: qhd43416.1) was used as the target sequence [23] . the swiss-model structure assessment tool was used to validate the quality of the predicted model. around 5,800 compounds, including 3,700 nucleoside-like compounds from the enamine targeted antiviral library (enamine.net) and 2,100 food and drug administration (fda)approved drugs from the zinc15 database [24] were used for molecular docking. all molecules were prepared with obabel [25] from .sdf or .mol2 format to .pdbqt format. the 3d compound structures from the enamine library were resolved by obabel --gen3d command. the docking file of the protein model was prepared with mgltools v1.5.4 [26] and the molecules were docked at the rbd of the spike protein via autodock vina 1.1.2 [27] . the grid box of 40×60×30 size with 1.0 å spacing was fixed around the rbd (thr323-val511) of the spike protein. each docking was done in three replicates, and the conformation with the highest binding score was recorded. the batch processing of docking and data collection was performed using an in-house python script which is deposited in github. data were analyzed statistically using r studio [28] and graphs were constructed with ggplot in r [29]. the ligand-receptor interactions were studied using schrödinger maestro [30] , and molecules with high docking scores were selected from each screening library for further studies. codon-optimized mers-cov (isolate emc, vg40069-g-n) and sars-cov (isolate cuhk-w1; vg40150-g-n) s expression plasmids (pcmv) were ordered from sino-biological and subcloned into pcaggs using the clai and kpni sites. the last 19 amino acids of the sars-cov spike protein were deleted to enhance pseudovirus production. codon-optimized cdna encoding sars-cov-2 s glycoprotein (isolate wuhan-hu-1) with a c-terminal 19 amino acid deletion was synthesized and cloned into pcagss in between the ecori and bglii sites. pvsv-egfp-dg (#31842), pmd2.g (#12259), pcag-vsv-p (#64088), pcag-vsv-l (#64085), pcag-vsv-n (#64087) and pcaggs-t7opt (#65974) were ordered from addgene. s expressing pcaggs vectors were used for the production of pseudoviruses, as described below. the cdna encoding human tmprss2 (nm_005656; ohu13675d) was obtained from genscript. the cdna fused to a c-terminal ha tag was subcloned into pqxcih (clontech) in between the noti and paci sites to obtain the pqxcih-tmprrs2-ha vector. vero-tmprss2 cells were produced by retroviral transduction. to produce the retrovirus, 10 μg pqxcih-tmprrs2-ha was co-transfected with polyethylenimine (pei) with 6.5 μg pbs-gagpol (addgene #35614) and 5 μg pmd2.g in a 10 cm dish of 70% confluent hek-293t cells in opti-mem i (1x) + glutamax. retroviral particles were harvested at 72 hours post-transfection, cleared by centrifugation at 2000 x g, filtered through a 0.45μm low protein-binding filter (millipore), and used to transduce vero cells. polybrene (sigma) was added at a concentration of 4 μg/ml to enhance transduction efficiency. transduced cells were selected with hygromycin b (invitrogen). hek-293t cells were maintained in dulbecco's modified eagle's medium (dmem, gibco) supplemented with 10% fetal bovine serum (fbs), 1x non-essential amino acids (lonza), 1mm sodium pyruvate (gibco), 2mm l-glutamine (lonza), 100 μg/ml streptomycin (lonza) and 100 u/ml penicillin. vero, vero-tmprss2, and veroe6 cells were maintained in dmem supplemented with 10% fbs, 1.5 mg/ml sodium bicarbonate (lonza), 10mm hepes (lonza), 2mm l-glutamine, 100 μg/ml streptomycin and 100 u/ml penicillin. all cell lines were maintained at 37°c in a 5% co2, humidified incubator. the protocol for vsv-g pseudovirus rescue was adapted from whelan and colleagues (1995). briefly, a 70% confluent 10 cm dish of hek-293t cells was transfected with 10µg pvsv-egfp-dg, 2µg pcag-vsv-n (nucleocapsid), 2µg pcag-vsv-l (polymerase), 2µg pmd2.g (glycoprotein, vsv-g), 2µg pcag-vsv-p (phosphoprotein) and 2µg pcaggs-t7opt (t7 rna polymerase) using pei at a ratio of 1:3 (dna:pei) in opti-mem i (1x) + glutamax. forty-eight hours post-transfection the supernatant was transferred onto new plates transfected 24 hours prior with vsv-g. after a further 48 hours, these plates were retransfected with vsv-g. after 24 hours the resulting pseudoviruses were collected, cleared by centrifugation at 2000 x g for 5 minutes, and stored at -80°c. subsequent vsv-g pseudovirus batches were produced by infecting vsv-g transfected hek-293t cells with vsv-g pseudovirus at a moi of 0.1. titres were determined by preparing 10-fold serial dilutions in opti-mem i (1x) + glutamax. aliquots of each dilution were added to monolayers of 2 × 10 4 vero cells in the same medium in a 96-well plate. three replicates were performed per pseudovirus stock. plates were incubated at 37°c overnight and then scanned using an amersham typhoon scanner (ge healthcare). individual infected cells were quantified using imagequant tl software (ge healthcare). all pseudovirus work was performed in a class ii biosafety cabinet under bsl-2 conditions at erasmus medical center. for the production of mers-cov, sars-cov, and sars-cov-2 s pseudovirus, hek-293t cells were transfected with 10 µg s expression plasmids. twenty-four hours post-transfection, the medium was replaced for in opti-mem i (1x) + glutamax, and cells were infected at a moi of 1 with vsv-g pseudotyped virus. two hours post-infection, cells were washed three times with optimem and replaced with medium containing anti-vsv-g neutralizing antibody (clone 8g5f11; absolute antibody) at a dilution of 1:50,000 to block remaining vsv-g pseudovirus. the supernatant was collected after 24 hours, cleared by centrifugation at 2000 x g for 5 minutes and stored at 4°c until use within 7 days. coronavirus s pseudovirus was titrated on veroe6 cells as described above. transduction experiments were carried out by incubating pseudovirus with imatinib at concentrations ranging from 0-125nm in opti-mem i (1x) + glutamax for 1 hour at 37°c. pseudovirus-imatinib mixes were added to monolayers of 2 × 10 4 vero or vero-tmprss2 cells in a 96-well plate. plates were incubated for 16 hours before quantifying gfp-positive cells using an amersham typhoon scanner and imagequant tl software. to determine the toxicity profile of imatinib, we performed the mtt assay using a 1-hr and an 8hr design. briefly, a serial dilution of imatinib was prepared and incubated on vero cells for 1 hr at 37 o c. subsequently, cells were washed, further cultured for eight hrs. in the 8-hr design, cells were incubated with a serial dilution of imatinib for eight hours without a washing step. we tested serial dilutions of imatinib for its ability to neutralize sars-cov-2 (german isolate; gisaid id epi_isl 406862; european virus archive global #026v-03883) using a plaque reduction neutralization test (prnt) as previously described [31] . fifty μl of the virus suspension (200 spot forming units) was added to each well and incubated at 37°c for either 1 hr. following incubation, the mixtures were added on vero cells and incubated at 37°c for either 1hr or 8 hrs. the cells incubated for 1 hr were then washed and further incubated in medium for 8 hrs. after the incubation, the cells were fixed and stained with a polyclonal rabbit anti-sars-cov antibody (sino biological; 1:500). staining was developed using a rabbit anti-sars-cov serum and a secondary alexa-fluor-labeled conjugate (dako). the number of infected cells per well were counted using the imagequant tl software. the binding kinetics of imatinib on sars-cov-2 rbd protein were studied using a blitz® system (fortébio). experiments were conducted using the advanced kinetics mode, at room temperature and a buffer system consisting of 1x kinetics buffer (fortébio), 5% anhydrous dimethyl sulfoxide (dmso; sigma aldrich). recombinant his-tagged sars-cov-2 rbd protein (40592-v08h; sino biological) at a concentration of 10 µg/ml was loaded on anti-penta-his (his1k) biosensors (fortébio), followed by a washing step with assay buffer to block the unoccupied sensor surface. the association and dissociation profiles of imatinib (sigma aldrich) were measured at various concentrations (four-point serial dilutions from 6.25 µm to 0.78 µm). a reference biosensor loaded in the same manner with 0 µm imatinib was used for baseline correction in each assay. the final binding curves were analyzed with the blitz pro 1.3 software (fortébio) using the 1:1 global-fitting model. the assay was repeated twice to validate the binding constants. here, data is represented as mean ± sd. similarly, sars-cov-2 rbd was immobilized on his1k biosensors to study the binding kinetics of mfc-tagged hace2 (10108-h05h; sino biological) before being dipped into tubes containing the 1x kinetics buffer. various concentrations of ace2 (four-point serial dilutions from 800 to 100 nm) were used to measure the association and dissociation profiles. data were reference subtracted and fit to a 1:1 binding model using the blitz pro 1.3 software. the ability of imatinib to inhibit the interaction of spike rbd:ace2 proteins was evaluated by using the spike rbd (sars-cov-2): ace2 inhibitor screening colorimetric assay kit (bps bioscience). the avi-his-tagged spike s1 rbd (sars-cov-2) protein 50 ng/well in pbs was coated onto 96-well microplate by overnight incubation at 4°c. blocking buffer 2 was used to block the nonspecific binding sites by incubation for 1 hour. different concentrations of imatinib were added and incubated for 1 hour at room temperature with slow shaking. for the wells designated "blank" and "positive control", inhibitor buffer (pbs with 0.5% dmso) was added. the reaction was initiated by adding ace2 his-avi-tagged biotin-labeled hip tm protein (50 ng/well) in 1x immuno buffer 1 to the "positive control" and "test inhibitor" wells by incubation for 1 hour at room temperature with slow shaking. streptavidin-hrp (dilution 1:1,000 in blocking buffer 2) was added to each well and incubated at room temperature for 1 hour with slow shaking. washing procedure (3 × 100 µl 1x immuno buffer 1) was performed after each step. the chromogenic reaction was initiated by adding colorimetric hrp substrate to each well and incubated at room temperature until blue color was developed (approximately 2 minutes) in the "positive control" well. after the blue color was developed, the reaction was terminated by adding 1n hcl, and absorbance at 450 nm was measured using synergy h1 hybrid multi-mode microplate reader (biotek instruments). the pharmacophore model was generated using pharmit [32] , an online interactive platform to elucidate pharmacophores from the receptor and ligand complex. top five binding conformations of drug-protein complexes were produced by autodock vina. the pharmacophores of the ligands interacting with the receptor were considered active pharmacophores while the rest were defined as inactive pharmacophores. the pharmacophores from the native receptor of imatinib (abl2 tyrosine kinase; pdb:3gvu) and rbd of sars cov-2 were generated using imatinib as the ligand. using the enhanced ligand exploration and interaction recognition algorithm (elixir-a), the two sets of pharmacophores were merged and processed to identify any overlap in 3d space. a detailed description of elixir-a can be found in our previous work [33] and the algorithm has been deposited in github. the python script 'elixir-a-vina-batch-screening-module' used for running docking jobs in batch mode and elixir-a, the algorithm used for pharmacophore mapping have been deposited in github and will be made public upon publication of the manuscript. with md simulations. we are thankful to mart lammers for allowing us to use the fusion assays. funding: dr was supported by the ministry of health of the czech republic (project no. 20-05-00472). author contributions: sf, bm, and dr conceived and designed the study. first author nm designed the experiments, performed bli studies and immunoassays, reviewed literature, and compiled the manuscript and figures. co-first author hw conducted in silico experiments and compiled figures. sm did literature review on the resulting compounds and compiled the manuscript and figures. bm performed virology experiments. sf directed and verified studies and authored the manuscript. all authors reviewed and edited the paper. competing interests: all the authors declare that there are no conflicts of interest. percent inhibition compared to the amount of plaques on cells. here, 2-fold dilution of the compounds were done in duplo. then 300 tcid50 of sars-cov-2 was added to each well, and plates were incubated at 37c for 1 hour. then, the mixes were added onto vero cells and incubated for 8 hours at 37 c. subsequently, cells were fixed for 15 min with 2% pfa, followed by another 15 min fixation with 70% ethanol. fixed cells were stained with a monoclonal antibody, followed by alexafluor 488. here imatinib shows significant % inhibition as compared to other compounds tested. (1 µg/ml) were incubated for 1 hour at room temperature with slow shaking in the presence of various imatinib concentrations. streptavidin hrp (1:1,000) was added to the reaction mixture. colorimetric substrate was added to initiate the chromogenic reaction, and 2 minutes were allowed for color development. the reaction was terminated with the addition of 1n hcl and absorbance was measured at 450 nm. positive control (no inhibitor) was assumed to represent 0% inhibition. values obtained from test wells (with imatinib) compared to the positive control showed 0% inhibition of rbd:ace2 interaction, indicating that imatinib does not inhibit spike fusion by direct inhibition. pharmacophore distribution of five most stable conformations on tyrosine kinase abl2 (purple spheres); and b] pharmacophore distribution (yellow spheres) on sars-cov-2 spike protein rbd with pharmacophores common to both receptors depicted in red. a novel coronavirus from patients with pneumonia in china the species 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leadlike you can dock. abstracts of papers of the open babel: an open chemical toolbox autodock4 and autodocktools4: automated docking with selective receptor flexibility software news and update autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading gplots: various r programming tools for plotting data. 2015. 30. release, s., 1: maestro. schrödinger, llc severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease pharmit: interactive exploration of chemical space a non-beta-lactam antibiotic inhibitor for enterohemorrhagic escherichia coli o104: h4 pharmacophore distribution on sars-cov-2 spike protein rbd with imatinib we gratefully acknowledge the support from texas a&m high performance research computing (hprc) and tamu laboratory for molecular simulation (lms). we would like to thank dr. lisa perez (associate director for advanced computing enablement, hprc tamu) for guidance key: cord-292578-co5essuw authors: johnson, marina; wagstaffe, helen r.; gilmour, kimberly c.; mai, annabelle lea; lewis, joanna; hunt, adam; sirr, jake; bengt, christopher; grandjean, louis; goldblatt, david title: evaluation of a novel multiplexed assay for determining igg levels and functional activity to sars-cov-2 date: 2020-08-02 journal: j clin virol doi: 10.1016/j.jcv.2020.104572 sha: doc_id: 292578 cord_uid: co5essuw background: the emergence of sars-cov-2 has led to the development of serological assays that could aid in an understanding of the burden of covid-19 disease. many available tests lack rigorous evaluation and therefore results may be misleading. objectives: the aim of this study was to assess the performance of a novel multiplexed immunoassay for the simultaneous detection of antibodies against sars-cov-2 trimeric spike (s), spike receptor binding domain (rbd), spike n terminal domain and nucleocapsid antigen and a novel pseudo-neutralisation assay. methods: a multiplexed solid-phase chemiluminescence assay (meso scale discovery) was evaluated for the simultaneous detection of igg binding to four sars-cov-2 antigens and the quantification of antibody-induced ace-2 binding inhibition (pseudo-neutralisation assay). sensitivity was evaluated with a total of 196 covid-19 serum samples (169 confirmed pcr positive and 27 anti-nucleocapsid igg positive) from individuals with mild symptomatic or asymptomatic disease. specificity was evaluated with 194 control serum samples collected from adults prior to december 2019. results: the specificity and sensitivity of the binding igg assay was highest for s protein with a specificity of 97.4 % and sensitivity of 96.2 % for samples taken 14 days and 97.9 % for samples taken 21 days following the onset of symptoms. igg concentration to s and rbd correlated strongly with percentage inhibition measured by the pseudo-neutralisation assay. conclusion: excellent sensitivity for igg detection was obtained over 14 days since onset of symptoms for three sars-cov-2 antigens (s, rbd and n) in this multiplexed assay which can also measure antibody functionality. severe acute respiratory syndrome-related coronavirus-2 (sars-cov-2) was first recognised in january 2020 and rapidly spread world-wide (1) . tests designed to measure antibodies to sars-cov-2 antigens were rapidly developed and are important for diagnostics and seroprevalence studies. the latter could help inform disease burden estimates, studies of transmission dynamics and modelling of the epidemic. antibody tests are particularly important in the context of mild or asymptomatic disease where a swab reverse transcriptase polymerase chain reaction (rt-pcr) test may be negative. for this reason, an understanding of the sensitivity and specificity of the tests being used is critical. the trimeric spike (s) protein of sars-cov-2 is present on the viral surface and in most cases is cleaved by host proteases into the s1 and s2 subunits, responsible for receptor recognition and membrane fusion respectively. s1 uses a region of the molecule, known as the receptor binding domain (rbd) to bind to host ace-2 receptor and thereby gain entry to the cell (2) . specific immunoglobulin-g (igg) and igm antibody responses to sars-cov-2 s, n and rbd of the spike protein develop between 6-15 days following disease-onset (4) . despite a rapid increase in the number and availability of sars-cov-2 serologic assays, most have undergone minimal external evaluation and validation (5) . a recent large scale spanish seroprevalence study used a point of care igg test with a stated sensitivity of 97.2% but on verification found it to have a sensitivity of either 82.1%, 89.7%, 99.6% or 100% depending on the sample sets used for evaluation (6) . all assays currently suffer from the absence of a defined standard serum so results are reported as positive or negative or as optical density readouts complicating the comparison between assays and studies and for many binding assays the relationship between antibody concentration and function is unclear. we have evaluated a novel assay designed to simultaneously measure igg to four sars-cov-2 antigens; full-length trimeric s, rbd and ntd of spike as well as n protein. the assay, based on meso scale discovery (msd) technology, utilises a 96-well based solid-phase antigen printed plate and an electrochemiluminescent detection system. in addition this assay can measure the ability of serum to inhibit the interaction between spike protein components and soluble ace-2, also called a pseudo-neutralisation assay (7) . to evaluate the sensitivity and specificity of the msd assay, we were able to utilise a relatively large number of samples obtained from sars-cov-2 rt-pcr positive health care workers or patients as well as antibody positive health care staff enrolling in a large sars-cov-2 cohort study. samples were screened for igg to sars-cov-2 n protein using a commercially available kit (epitope diagnostics inc, san diego, usa) as previously described (8) . to measure igg antibodies, plates were blocked with msd blocker a following which reference standard, controls and samples diluted 1:500 in diluent buffer were added. after incubation, detection antibody was added (msd sulfo-tag™ anti-human igg antibody) and then msd gold™ read buffer b was added and plates read using a meso® sector s 600 reader. plates were blocked and washed as above, assay calibrator (covid-19 neutralising antibody; monoclonal antibody against s protein; 200µg/ml), control sera and test sera samples diluted 1 in 10 in assay diluent were added to the plates. following incubation plates an 0.25µg/ml solution of msd sulfo-tag™ conjugated ace-2 was added after which plates were read as above. percentage inhibition was calculated relative to the assay calibrator (maximum 100% inhibition). statistical analysis was performed using msd discovery workbench and graphpad prism version 8.0 (graphpad, san diego, ca). antibody concentration in arbitrary units (au) was interpolated from the ecl signal of the internal standard sample using a 4-parameter logistic curve fit. roc curves showing the sensitivity and specificity (plotted as 100%-specificity %) calculated using each value in the data as a cut-off were plotted for each antigen. a cut-off antibody concentration was chosen based on the lowest value leading to a positive likelihood ratio (lr) of >10, in order to maximise sensitivity while providing strong evidence to rule-in j o u r n a l p r e -p r o o f infection (9) . for s antigen binding, all lr's were above 10, therefore the llod was used as the cut-off for this antigen. comparisons between groups were performed by kruskal-wallis one-way anova with dunn's correction for multiple comparisons. correlation analysis was performed using spearman correlation. p values of <0.05 were considered as significant. latent class models with two classes were fitted with the binary antibody responses as outcome variables, using the polca package in the r statistical environment. the code used for the latent class analysis is available on request. the lower limit of detection (llod) was assigned as 1% of the standard value in au, and upper limit of detection (ulod) was assigned for ntd and rbd only as the s and n antigen did not reach an upper limit (table 1) . for statistical purposes, ulod was assigned the highest calculated concentration plus 20% and llod as 0.5%. the mean coefficient of variation (cv) between duplicates was <15% for all except ntd (17.4%, data not shown). the mean intra-assay cv was 6.2% and inter-assay variation <15% across all sars-cov-2 antigens except ntd (19.0%) on one of four samples (supplementary the specificity for s, rbd and n assays are shown in table 2 table 2 : assay specificity calculated for each sars-cov-2 antigen from the control cohort. table 3 . sensitivity and specificity was calculated for groups 0-7d, >7d, >14d and >21d since the onset of symptom the s antigen was the most sensitive of the three, with a sensitivity of 96.2% and 97.9% >14 days and >21 days respectively (table 3) . rocs were plotted to visualise the trade-off between sensitivity and specificity for s and rbd neutralisation. cut-offs (lr>10) were 0.162% for s and 0.524% for rbd (shown by the dotted line on figure 5a -b). sensitivity and specificity for s were 97.8% and 97.9% respectively but lower for rbd (77.2% and 92.8% respectively). in the covid-19 cohort there were some igg positive sera that did not demonstrate neutralisation (below cut-off, n= 4 for s and 36 for rbd). these sera were predominantly those taken soon after the onset of symptoms; 22 between 0-7 days, 9 over 14 days and 5 over 21 days. using a carefully defined cohort of known sars-cov-2 exposed individuals and relevant controls we were able to show the sensitivity and specificity of the assay for the four antigens of interest. comparing the performance of s and rbd assays in a recently published systematic review and metanalysis of the diagnostic accuracy of serological tests for covid-19 (10) the s assay we evaluated had superior sensitivity to all of the assays included in the review while rbd performance was superior to most. the reason for this could be related to the technical aspects of the assay itself including the integrity of the antigen used and the sensitivity of the detection platform but also the use of a well-defined cohort of individuals with known exposure to sars-cov-2. only the n terminal domain of the spike protein did not perform well in this assay with poor sensitivity due to the overlap in antibody titres between the covid-19 cohort and controls. the assay format permitted the measurement of antibody against spike protein derived from sars-1, mers and two seasonal coronaviruses, but the results of antibody binding to these antigens could not be assessed in the same way as for the sars-cov-2 antigens due to the absence of defined negative and positive serum sets. an advantage of this assay is its ability to measure antibody induced inhibition of ace-2 receptor-spike interaction thought to be the major mechanism by which sars viruses, including sars-cov-2 attach to host cell surfaces (11, 12) . in the covid-19 cohort, there was a good correlation between anti-s and anti-rbd igg and function although a few sera bound antigen but did not neutralize. these were dominated by sera taken soon after infection and as recently described, could be non-neutralising and targeting epitopes outside the rbd (13). few of the control cohort sera had any pseudo-neutralisation activity despite pre-existing igg to seasonal coronavirus spike proteins suggesting season coronavirus exposure is unlikely to modify interaction with sars-cov-2. other cross reactive immunological mechanisms (eg t cells) cannot be ruled out and may explain the varied clinical response following exposure to sars-cov-2 (14) . this pseudo-neutralisation assay has been shown to correlate well with neutralisation assays using live sars-cov-2 (msd, personal communication). in summary, the msd multiplexed coronavirus panel assay evaluated in this study is highly reproducible, specific and sensitive for the detection of anti-sars-cov-2 antibody over 14 days since the onset of covid-19 symptoms. the assay can be adapted to measure antibody function which corelated well with spike protein antibody concentration. funding: this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. none world health organisation. 2020. coronavrius disease 2019 (covid-19) situation report -51 relationship between anti-spike protein antibody titers and sars-cov-2 in vitro virus neutralization in convalescent plasma structural proteins in severe acute respiratory syndrome coronavirus-2 temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study serodiagnostics for severe acute respiratory syndrome-related coronavirus-2: a narrative review prevalence of sars-cov-2 in spain (ene-covid): a nationwide, population-based seroepidemiological study a sars-cov-2 serological assay to determine the presence of blocking antibodies that compete for human ace2 binding comparison of four new commercial serologic assays for determination of sars-cov-2 igg diagnostic tests 4: likelihood ratios diagnostic accuracy of serological tests for covid-19: systematic review and metaanalysis angiotensin-converting enzyme 2: a functional receptor for sars coronavirus structural and functional basis of sars-cov-2 entry by using human ace2 characterization of neutralizing antibodies from a sars-cov-2 infected individual transmission, diagnosis, and treatment of coronavirus disease 2019 (covid-19): a review the study team would like to thank meso scale discovery for the donation of the plates and reagents that allowed us to complete the work, the costars study team at great ormond street children's hospital, staff in the great ormond street children's hospital clinical immunology laboratory for additional support and the nihr ucl great ormond street j o u r n a l p r e -p r o o f key: cord-335118-oa9jfots authors: taka, e.; yilmaz, s. z.; golcuk, m.; kilinc, c.; aktas, u.; yildiz, a.; gur, m. title: critical interactions between the sars-cov-2 spike glycoprotein and the human ace2 receptor date: 2020-09-21 journal: biorxiv doi: 10.1101/2020.09.21.305490 sha: doc_id: 335118 cord_uid: oa9jfots severe acute respiratory syndrome coronavirus 2 (sars-cov-2) enters human cells upon binding of its spike (s) glycoproteins to ace2 receptors and causes the coronavirus disease 2019 (covid-19). therapeutic approaches to prevent sars-cov-2 infection are mostly focused on blocking s-ace2 binding, but critical residues that stabilize this interaction are not well understood. by performing all-atom molecular dynamics (md) simulations, we identified an extended network of salt bridges, hydrophobic and electrostatic interactions, and hydrogen bonding between the receptor-binding domain (rbd) of the s protein and ace2. mutagenesis of these residues on the rbd was not sufficient to destabilize binding but reduced the average work to unbind the s protein from ace2. in particular, the hydrophobic end of rbd serves as the main anchor site and unbinds last from ace2 under force. we propose that blocking this site via neutralizing antibody or nanobody could prove an effective strategy to inhibit s-ace2 interactions. the covid-19 pandemic is caused by sars-cov-2, which is a positive-sense single-stranded rna betacoronavirus. phylogenetic analyses demonstrated that the sars-cov-2 genome shares ~79% sequence identity with severe acute respiratory syndrome coronavirus (sars-cov), and ~52% with the middle-east respiratory syndrome coronavirus (mers-cov) (1). despite these similarities, sars-cov-2 is much more infectious and fatal than sars-cov and mers-cov together (2) . sars-cov-2 consists of a 30 kb single-stranded rna genome that is encapsulated by a lipid bilayer and three distinct structural proteins that are embedded within the lipid membrane: envelope (e), membrane (m), and spike (s). host cell entry is primarily mediated by homotrimeric s glycoproteins located on the viral membrane ( fig. 1a) (3) . each s protomer consists of s1 and s2 subunits that mediate binding to the host cell receptor and fusion of the viral envelope, respectively (3, 4) . the receptor-binding domain (rbd) of s1 undergoes a large rigid body motion to bind to ace2. in the closed state, all rbds of the s trimer are in the down position, and the binding surface is inaccessible to ace2. the switching of one of the rbds into a semi-open intermediate state is sufficient to expose the ace2 binding surface and stabilize the rbd in its up position (fig. 1b) (5) . the s protein binds to the human angiotensin-converting enzyme 2 (ace2) receptor, a homodimeric integral membrane protein expressed in the epithelial cells of lungs, heart, kidneys, and intestines (6) . each ace2 protomer consists of an n-terminal peptidase domain (pd), which interacts with the rbd of the s protein through an extended surface (fig. 1a , c) (6) (7) (8) . upon ace2 binding, proteolytic cleavage of the s protein by the serine protease tmprss2 separates the s1 and s2 subunits (9) . the s2 protein exposes fusion peptides that insert into the host membrane and promote fusion with the viral membrane (4) . to prevent sars-cov-2 infection, there is a global effort to design neutralizing antibodies (10) , nanobodies (11) , peptide inhibitors (12) , and small molecules (13) that target the ace2 binding surface of the s protein. yet, only a limited number of studies were performed to investigate critical interactions that facilitate s-ace2 binding using md simulations. initial studies have constructed a homology model of sars-cov-2 rbd in complex with ace2, based on the sars-cov crystal structure (8, 14) and performed conventional md (cmd) simulations totaling 10 ns (15, 16) and 100 ns (17, 18) in length to estimate binding free energies (15, 16) and interaction scores (18) . more recent studies used the crystal structure of sars-cov-2 rbd in complex with ace2 to perform coarse-grained (19) and all-atom (20-23) md simulations. the effect of the mutations that disrupt close contact residues between sars-cov-2 rbd and ace2 on binding free energy was investigated by post-processing of the md trajectories (15, 16, 21, 22) or by using bioinformatic methods (20) . the work required to unbind the s protein from ace2 would provide a more accurate estimate of the binding strength, but this has not been performed under low pulling velocities using the structure of sars-cov-2 rbd in complex with ace2. in addition, systematic analysis of critical residues that stabilize s-ace2 binding and how mutagenesis of these interaction sites reduces the binding strength and alters the way the s protein detaches from ace2 under force have not yet been performed. in this study, we performed a comprehensive set of all-atom md simulations totaling 16.5 µs in length using the recently-solved structure of the rbd of the sars-cov-2 s protein in complex with the pd of ace2 (7) . simulations were performed in the absence and presence of external force to investigate the binding characteristics and estimate the binding strength. these simulations showed additional interactions between rbd and pd domains to those observed in the crystal structure (7) . an extensive set of alanine substitutions and charge reversal mutations of the rbd amino acids involved in ace2 binding were performed to quantify how mutagenesis of these residues weaken binding in the presence and absence of force in simulations. we showed that the hydrophobic end of rbd primarily stabilizes s-ace2 binding, and targeting this site could potentially serve as an effective strategy to prevent sars-cov-2 infection. to model the dynamic interactions of the s protein-ace2 binding interface, we used the costructure of rbd of the sars-cov-2 s protein in complex with the pd of human ace2 (7) (fig. 1c) . the structure was solvated in a water box that contains physiologically-relevant salt (150 mm nacl) concentration. two sets of cmd simulations, each of 100 ns in length, were performed to determine the formation of a salt bridge (24) and a hydrogen bond, as well as electrostatic and hydrophobic interactions between rbd and pd (table s1). a cutoff distance of 6 å between the basic nitrogens and acidic oxygens was used to score a salt bridge formation (24) . for hydrogen bond formation, a maximum 3.5 å distance between hydrogen bond donor and acceptor and a 30° angle between the hydrogen atom, the donor heavy atom, and the acceptor heavy atom was used (25) . interaction pairs that satisfy the distance, but not the angle criteria were analyzed as electrostatic interactions. for hydrophobic interactions, a cutoff distance of 8 å between the side chain carbon atoms was used (26) (27) (28) . using these criteria, we identified eleven hydrophobic interactions ( fig. 2a) , eight hydrogen bonds (fig. 2b) , two salt bridges and six electrostatic interactions (fig. 2c ) between rbd and pd. observation frequencies were classified as high and moderate for interactions that occur in 49% and above and between 15-48% of the total trajectory, respectively. f486 and y489 of rbd formed hydrophobic interactions with f28, l79, m82, and y83 of pd, while l455, f456, y473, and a475 of rbd formed hydrophobic interactions with t27 of pd at high frequencies (fig. 2d ). salt bridges between k417-d30 (rbd-pd) and e484-k31, and hydrogen bonds between n487-y83, t500-d355, and q493-e35 were observed at high frequencies, whereas hydrogen bonds y449-d38, q498-k353, t500-y41, y505-e37, and q493-e35 were observed at moderate frequencies (fig. 2d ). residue pairs y453-h34, n487-q24, t500-y41, n501-k353, q493-k31, and y449-q42 exhibited electrostatic interactions throughout the simulations (fig. 2d ). the interaction network we identified in our md simulations were mostly consistent with reported interactions in the rbd-pd crystal structure (7) . however, our simulations identified four hydrogen bonds (q498-k353, t500-d355, y505-e37, and q498-q42), one hydrophobic interaction (l455-t27), and two electrostatic interactions (y453-h34 and n501-k353) that are not present in the crystal structure. in turn, we did not detect frequent hydrogen bonding between g446-q42, g502-k353, and y505-r393 and an electrostatic interaction between g496-k353 observed in the crystal structure (7). this discrepancy may be due to radically different thermodynamic conditions between crystallization solutions and cmd simulations (29) . we divided the rbd-pd interaction surface into three contact regions (cr1-3, fig 2a the core region (cr2) comprised significantly fewer interactions than the ends of the rbd binding surface (cr1 and cr3). remarkably, 10 out of 13 interactions we detected in cr1 were hydrophobic, which were proposed to play a central role in anchoring of rbd to pd (23) . unlike cr1, cr2 formed only a single hydrophobic interaction with pd, whereas cr3 did not form any hydrophobic interactions. to estimate the binding strength of the s protein to ace2, we performed steered md (smd) simulations to pull rbd away from pd at a constant velocity of 2 å −1 along the vector pointing away from the binding interface (fig. 3a) . steering forces were applied to the cα atoms of the rbd residues on the binding interface, whereas cα atoms of pd residues at the binding interface were kept fixed. because part of the work applied is lost to the irreversible processes as we pull rbd away from pd at a finite velocity, the second law of thermodynamics indicates that unbinding free energy difference between the initial and final states cannot be larger than the average work required for unbinding. therefore, our calculations report relative changes in the binding free energy of wild-type (wt) and mutant rbd under the same velocity and thermodynamic conditions. in 20 smd simulations (each 15 ns, totaling 300 ns in length, table s1), the average work applied to unbind rbd from pd was 71.1 ± 12.7 kcal/mol (mean ± s.d.), demonstrating that the s protein binds stably to ace2 (fig. 3b) . to investigate the contribution of each of the 16 interactions we identified to the overall binding strength, we introduced point mutations on the rbd. salt bridges were eliminated by charge reversals (k417e and e484k). we also replaced each amino acid with alanine (table s1) to disrupt the pairwise interactions (30) , with minimal perturbations the protein backbone (31) . two sets of cmd simulations (a total of 3.4 µs in length) were performed for each point mutant. we first quantified the root mean square fluctuation (rmsf) of the cα atom of the rbd residues located on the pd binding surface (fig. 3c) . the rigid body motions were eliminated by aligning the rbd interaction surface of pd for each conformer (see methods). 13 out of 17 mutations increased the residue fluctuations compared to wt (fig. s1a), suggesting that disrupting the interactions between rbd and pd results in floppier binding. largest fluctuations were observed for 2 mutations in cr1 (f486a, and n487a), 2 mutations in cr3 (y449a and y505a) and 1 mutation in cr2 (l455a) (fig. 3c) . mutation of these residues also increased the fluctuations in their neighboring region. while mutations in cr1 increased fluctuations in cr3 significantly, mutations in cr3 had little to no effect on the fluctuations in cr1 ( fig. 3d and fig. s1b ). we next performed smd simulations modeling the unbinding of rbd of each point mutant from pd (20 simulations for each mutant, a total of 5.1 µs in length, table s1). f486a, y489a, y505a, n487a, and y473a mutations substantially decreased the work requirement to unbind rbd-pd by 15.0%, 12.1%, 11.1%, 10.8% and 9.3%, respectively ( fig. 3e-f, fig. s2 ). we note that most of these mutations also led to the largest increase in residue fluctuations on the binding surface (fig. 3c ). 3 of these residues (f486, n487, and y473) are located in cr1, whereas y505 is located in cr3. these results highlight the primary role of hydrophobic interactions in cr1 to stabilize the s-ace2 binding. to further characterize critical interactions of the s-ace2 binding interface, we introduced double mutants to neighboring residues of rbd that form critical interactions with pd. we performed a total of 2.8 µs of cmd and 4.2 µs of smd simulations for 14 double mutants (table s1). in particular, double mutants in cr1 resulted in 4 out of the 7 highest increase in rmsf ( fig. 4a and fig. s1 ). the f486a/n487a mutation at cr1 resulted in the largest increase in fluctuations in both cr1 and cr3 (fig. 4b and fig. s1 ). in smd simulations, 12 out of 14 double mutations also further decreased the average work to unbind rbd from pd ( fig. 4c-d, and fig. s3 ). similar to the rmsf analysis, double mutants in cr1 (f486a/n487a, e484a/y489a, e484a/f486a, and l455a/f456a) resulted in 4 out of the 6 largest decreases in average work (fig. 4d) . a charge reversal of k417e in combination with either q493a or y453a also resulted in a large decrease in work values (fig. 4d) . we also used jarzynski equality (32, 33) to construct the free energy profiles as a function of a reaction coordinate, referred to as the potential of mean force (pmf) (34) . based on the estimated pmf ( fig. s4 ), double mutants in cr1 resulted in the largest decrease in the binding energy by 35-21% compared to wt. collectively, these results show that two salt bridges (e484-k31 and k417-d30) and the network of hydrophobic interactions in cr1 involving f486, y489, and f456 residues are the most significant contributors of binding strength between the s protein and ace2. to test whether cr1 anchors rbd to pd (23), we investigated the order of events that result in detachment of rbd from pd in smd simulations. the unbinding process appears to perform a zipper-like detachment starting from cr3 and ending at cr1 in 85% of the simulations (fig. 5a) . in only 15% of the simulations, cr3 released last from pd (fig. 5a) . because unbinding simulations can reveal features characteristic for the reverse process of binding (35) (36) (37) (38) (39) , these results suggest that cr1 binding is the first and critical event for the s protein binding to ace2. mutagenesis of the critical residues in cr1, in general, resulted in a substantial decrease in the percentages of unbinding events that terminate with the release of cr1 from pd. in alanine replacement of the hydrophobic residues (f486a, f487, and y489), cr1 was released last for 60%, 55%, and 65% of the smd simulations, respectively (fig. 5b) . the probability of cr1 to release last under force was further reduced in double mutants of e484a/f486a (50%) and l455a/f456a (55%) (fig. 5b ). unlike these mutants, f456a and f486a/n487a mutants in cr1 increased the probability of cr1 to release last, but this could be attributed to a large increase in fluctuations in cr3 upon these mutations ( fig. s1b ). these results indicate that single and double mutants of the critical residues in cr1 substantially reduce the binding free energy of this region to ace2. it remains unclear whether higher infectivity of sars-cov-2 than sars-cov can be attributed to stronger interactions between s and ace2 in sars-cov-2 (2, 15) . to test this possibility, we performed two sets of md simulations for the rbd of sars-cov s protein bound to the pd of ace2 (pdb id: 2ajf (8)), and compared these results to that of sars-cov-2. similar to sars-cov-2, rbd of sars-cov makes an extensive network of interactions with pd. we identified eleven hydrophobic interactions (fig. 6a) , six hydrogen bonds (fig. 6b) , and seven electrostatic interactions (fig. 6c) . out of these 15 interactions, only 6 are conserved in sars-cov and the following mutations have taken place: l443/f456 (sars-cov/sars-cov-2), f460/y473, p462/a475, p470/e484, l472/f486 v404/k417, n479/q493, y484/q498, and t487/n501. similar to sars-cov-2, l472 and y475 of sars-cov rbd formed a total of seven hydrophobic interactions at a high frequency with the hydrophobic pocket of ace2 (fig. 6d) . unlike sars-cov-2, sars-cov rbd did not form any salt bridges with ace2. we next modeled the unbinding of rbd of sars-cov from pd by performing 20 smd simulations (totaling 300 ns in length, table s1). the average total unbinding work of sars-cov (71.6 ± 14.5 kcal/mol, mean ± s.d., fig. 6e ) was identical but more broadly distributed than that of sars-cov-2 (71.1 ± 12.7 kcal/mol, fig. 3b ). unlike sars-cov-2, cr1 released last from pd in only 40% of the unbinding events of rbd of sars-cov, whereas the unbinding of cr3 was the last event in the remaining 60% (fig. 6f) . these results indicate that the s protein binds stably to ace2 in both sars-cov and sars-cov-2 and the higher infectivity of sars-cov-2 cannot be explained by an increase in binding strength. higher variability in unbinding work values and the absence of a clear order in unbinding events of rbd of sars-cov suggest that sars-cov has a more variable binding mechanism to ace2 than sars-cov-2. we performed an extensive set of in silico analysis to identify critical residues that facilitate binding of the rbd of the sars-cov-2 s protein to the human ace2 receptor. mutagenesis of these residues and pulling the rbd away from pd at a low velocity enabled us to estimate the free energy of binding and the order of events that result in the unbinding of rbd from pd. our simulations showed that the pd interacting surface of rbd can be divided into three contact regions (cr1-3). hydrophobic residues of cr1 strongly interact with the hydrophobic pocket of pd in both sars-cov and sars-cov-2. cr1 of sars-cov-2 also forms a salt bridge with ace2 that is not present in sars-cov. based on our smd simulations, we did not observe a major difference in binding strength of the s protein to ace2 between sars-cov and sars-cov-2, indicating that higher infectivity of sars-cov-2 is not due to tighter binding of s to the ace2 receptor. these results are consistent with a recent md simulation that applied the generalized born and surface area continuum solvation approach (mm-gbsa) (22) , coarse-grained simulations (19) , and biolayer interferometry (2). our analysis suggests that cr1 is the main anchor site of the sars-cov-2 s protein to ace2, and blocking the cr1 residues f456, e484, f486, n487, and y489 could significantly reduce the binding affinity. consistent with this prediction, llama based nanobody h11-h4 that neutralizes sars-cov-2 (11), by interacting with 60% and 50% of the critical residues we identified in cr1and cr2, respectively. similarly, the human antibody ha001 (10), and vh-fc ab8 (40) neutralizes sars-cov-2 by interacting with f456, a475, and f486 residues on cr1, which were among the strongest interactions we detected between rbd and pd. experimental studies revealed that antibodies against sars-cov induce limited neutralizing activity against sars-cov-2 (10, 23) . this may be attributed to the low sequence conservation of the cr1 region between sars-cov and sars-cov-2. in particular, the s protein of sars-cov-2 contains critical phenylalanine (f486) and glutamate (e484) residues not present in sars-cov, that form hydrophobic interactions and a salt bridge with ace2, respectively. it remains to be determined whether this difference plays a role in higher infectivity of sars-cov-2 than sars-cov. our simulations show that single and double mutants of cr1 are not sufficient to disrupt the binding of rbd to ace2, but reduce the binding free energy of this region. because rbd makes multiple contacts with ace2 through an extended surface, small molecules or peptides that target a specific region in the rbd-ace2 interaction surface may not be sufficient to prevent binding of the s protein to ace2. instead, blocking of a larger surface of the cr1 region with a neutralizing antibody or nanobody is more likely to introduce steric constraints to prevent the s protein-ace2 interactions. materials and methods md simulations system preparation. for cmd simulations, the crystal structure of sars-cov-2 s protein rbd bound with ace2 at 2.45 å resolution (pdb id: 6m0j) (7) was used as a template. the chloride ion, zinc ion, glycans, and water molecules in the crystal structure were kept in their original positions. single and double point mutants were generated using the mutator plugin in vmd (41) . each system was solvated in a water box (using the tip3p water model) having 35 å cushion in the positive x-direction and 15 å cushions in other directions. this puts a 50 å water cushion between the rbd-pd complex and its periodic image in the xdirection, creating enough space for unbinding simulations. ions were added to neutralize the system and salt concentration was set to 150 mm to construct a physiologically relevant environment. the size of each solvated system was ~164,000 atoms. all system preparations steps were performed in vmd (41) . all md simulations were performed in namd 2.13 (42) using the charmm36 (43) force field with a time step of 2 fs. md simulations were performed under n, p, t conditions. the temperature was kept at 310 k using langevin dynamics with a damping coefficient of 1 ps -1 . the pressure was maintained at 1 atm using the langevin nosé-hoover method with an oscillation period of 100 fs and a damping time scale of 50 fs. periodic boundary conditions were applied. 12 å cutoff distance was used for van der waals interactions. long-range electrostatic interactions were calculated using the particle-mesh ewald method. for each system; first, 10,000 steps of minimization followed by 2 ns of equilibration was performed by keeping the protein fixed. the complete system was minimized for additional 10,000 steps, followed by 4 ns of equilibration by applying constraints on cα atoms. subsequently, these constraints were released and the system was equilibrated for an additional 4 ns before initiating the production runs. the length of the equilibrium steps is expected to account for the structural differences due to the radically different thermodynamic conditions of crystallization solutions and md simulations (29) . md simulations were performed in comet and stampede2 using ~8 million core-hours in total. rmsf calculations. rmsf values were calculated as 〈∆ 2 〉 1/2 = 〈( − 〈 〉 ) 2 〉 1/2 , where, 〈 〉 is the mean atomic coordinate of the i th cα atom and is its instantaneous coordinate. smd simulations. smd (44) simulations were used to explore the unbinding process of rbd from ace2 on time scales accessible to standard simulation lengths. smd simulations have been applied to explore a wide range of processes, including domain motion (5, 45) , molecule unbinding (46) , and protein unfolding (47) . in smd simulations, a dummy atom is attached to the center of mass of 'steered' atoms via a virtual spring and pulled at constant velocity along the 'pulling direction', resulting in force f to be applied to the smd atoms along the pulling vector (42), where is the guiding potential, is the spring constant, is the pulling velocity, is time, and 0 are the coordinates of the center of mass of steered atoms at time t and 0, respectively, and is the direction of pulling (42) . total work (w) performed for each simulation was evaluated by integrating f over displacement along the pulling direction as = ∫ ( ) 0 . in smd simulations of sars-cov-2, cα atoms of ace2 residues s19-s43, t78-p84, q325-n330, g352-i358, and p389-r393 were kept fixed, whereas cα atoms of rbd residues k417-i418, g446-f456, y473-a475, and n487-y505 were steered (fig. 3a) . steered atoms were selected as the region comprising the interacting residues. for sars-cov smd simulations the same ace2 residues were kept fixed. however, two slightly different steered atoms selections were applied: i) using the same residue positions as for sars-cov-2, which are v404-i405, t433-l443, f460-s461, and n473-y491, and ii) selecting the region comprising the interacting residues, which aret433-l443, f460-d463, and n473-y491. the total number of fixed and steered atoms were identical in all simulations. the pulling direction was selected as the distance between the center of mass of steered and fixed atoms. the pulling direction also serves as the reaction coordinate ξ for free energy calculations. each smd simulation was performed for 15 ns using a 2 å −1 pulling velocity. at a spring constant of 100 −1 å −2 , the center of mass of the steered atoms followed the dummy atom closely while the spring was still soft enough to allow small deviations. for each system, 20 conformations were sampled with a 10 ns frequency from their cmd simulations (10 conformers from each set of the cmd simulations listed in table s1 md1-33 a-b). these conformations served as 20 separate starting conformations, 0 , for each set of smd simulations (table s1 md1-33 c-d). potential of mean force for unbinding of rbd. work values to unbind rbd from ace2 at low pulling velocities along the reaction coordinate were analyzed using jarzynski equality, which provides a relation between equilibrium free energy differences and the work performed through non-equilibrium processes (32) (33) (34) : where δf is the helmholtz free energy, kb is the boltzmann constant and t is the temperature. because work values sampled in our smd simulations differ more than1 kbt ( fig. s2 and s3) , the average work calculated in eq. 3 will be dominated by small work values that are only rarely sampled. for a finite (n) number of smd simulations, the term − ln(∑ − / =1 ⁄ ) did not converge to 〈 − / 〉. thus, eq. 3 provides an upper bound on δf, which was used as an estimate of the pmf. fig. s1 . rmsf values of single and double mutants of rbd of sars-cov-2. fig. s2 . distribution of work values obtained from smd simulations for each single point mutant system of rbd of sars-cov-2. fig. s3 . distribution of work values obtained from smd simulations for each double point mutant system of rbd of sars-cov-2. fig. s4 . pmf and δf values of wt and six mutants of rbd of sars-cov-2. table s1 . starting conformations and durations of the md simulations performed. movie s1. cr1 releasing last when sars-cov-2 rbd was pulled away from ace2 pd. movie s2. cr3 releasing last when sars-cov-2 rbd was pulled away from ace2 pd. identification of a novel coronavirus causing severe pneumonia in human: a descriptive study structure, function, and antigenicity of the sars-cov-2 spike glycoprotein mechanisms of coronavirus cell entry mediated by the viral spike protein stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis conformational transition of sars-cov-2 spike glycoprotein between its closed and open states structural basis for the recognition of the sars-cov-2 by full-length human ace2 structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor structure of sars coronavirus spike receptorbinding domain complexed with receptor sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor key residues of the receptor binding motif in the spike protein of sars-cov-2 that interact with ace2 and neutralizing antibodies neutralizing nanobodies bind sars-cov-2 spike rbd and block interaction with ace2 computational design of ace2-based peptide inhibitors of sars-cov-2 repurposing approved drugs as inhibitors of sars-cov-2 s-protein from molecular modeling and virtual screening cryo-em structure of the sars coronavirus spike glycoprotein in complex with its host cell receptor ace2 molecular mechanism of evolution and human infection with sars-cov-2 sars-cov-2, an evolutionary perspective of interaction with human ace2 reveals undiscovered amino acids necessary for complex stability computational prediction of mutational effects on the sars-cov-2 binding by relative free energy calculations the sars-cov-2 exerts a distinctive strategy for interacting with the ace2 human receptor critical differences between the binding features of the spike proteins of sars-cov-2 and sars-cov effect of mutation on structure, function and dynamics of receptor binding domain of human sars-cov-2 with host cell receptor ace2: a molecular dynamics simulations study dynamics of the ace2-sars-cov-2/sars-cov spike protein interface reveal unique mechanisms the mers-cov receptor dpp4 as a candidate binding target of the sars-cov-2 spike enhanced receptor binding of sars-cov-2 through networks of hydrogen-bonding and hydrophobic interactions zipping and unzipping of adenylate kinase: atomistic insights into the ensemble of open↔ closed transitions hbonanza: a computer algorithm for moleculardynamics-trajectory hydrogen-bond analysis direct and quantitative afm measurements of the concentration and temperature dependence of the hydrophobic force law at nanoscopic contacts a study of the preferred environment of amino acid residues in globular proteins molecular dynamics simulation of antimicrobial peptide arenicin-2: b-hairpin stabilization by noncovalent interactions why protein conformers in molecular dynamics simulations differ from their crystal structures: a thermodynamic insight rapid mapping of protein functional epitopes by combinatorial alanine scanning comparing experimental and computational alanine scanning techniques for probing a prototypical protein-protein interaction equilibrium free-energy differences from nonequilibrium measurements: a master-equation approach nonequilibrium equality for free energy differences free energy calculation from steered molecular dynamics simulations using jarzynski's equality molecular dynamics simulations suggest that electrostatic funnel directs binding of tamiflu to influenza n1 neuraminidases molecular dynamics study of unbinding of the avidin-biotin complex steered molecular dynamics simulations reveal the likelier dissociation pathway of imatinib from its targeting kinases c-kit and abl unbinding of nicotine from the acetylcholine binding protein: steered molecular dynamics simulations computational insights into the mechanism of ligand unbinding and selectivity of estrogen receptors high potency of a bivalent human vh domain in sars-cov-2 animal models vmd: visual molecular dynamics scalable molecular dynamics with namd optimization of the additive charmm all-atom protein force field targeting improved sampling of the backbone ϕ, ψ and side-chain χ1 and χ2 dihedral angles steered molecular dynamics and mechanical functions of proteins steered molecular dynamics simulation of the rieske subunit motion in the cytochrome bc1 complex computational design of new peptide inhibitors for amyloid beta (aβ) aggregation in alzheimer's disease: application of a novel methodology unfolding of titin immunoglobulin domains by steered molecular dynamics simulation shielding and beyond: the roles of glycans in sars-cov-2 spike protein. biorxiv data and materials availability: data and the analysis software are available from the corresponding author upon request. the structure of the full-length s protein in complex with ace2. the s protein is a homotrimer (green, purple, and grey) and embedded into the viral membrane. ace2 is a homodimer (blue and orange) and embedded into the host cell membrane. the full length structure of the s protein in complex with ace2 was modeled using the full length s protein model (48) and the crystal structure of the s protein rbd in complex with ace2 (pdb id: 6m17). both proteins were manually inserted into the membrane by their transmembrane domains. (b) the structure of an s protomer in the down and up position of its rbd. s1/s2 and s2' are the cleavage sites of the s protomer upon ace2 binding. (c) md simulations were performed for rbd of the s protein in complex with the pd of ace2. catalytic residues of ace2, glycans, and zn +2 and clions are shown in brown, red, yellow and purple, respectively. hydrophobic interactions (b) hydrogen bonds, and (c) salt bridges and electrostatic interactions, between rbd (green) and pd (blue) are shown on a conformation obtained from md simulations in the left panels. the interaction surface is divided into three distinct regions (cr1-3). normalized distributions of the distances between the amino-acid pairs that form hydrophobic interactions (red), hydrogen bonds (purple), salt bridges (orange), and electrostatic interactions (green) are shown in the right panels. lines with colored numbers represent maximum cutoff distances for these interactions. key: cord-344871-486sk4wc authors: wu, jianping; mok, chee-keng; chow, vincent tak kwong; yuan, y. adam; tan, yee-joo title: biochemical and structural characterization of the interface mediating interaction between the influenza a virus non-structural protein-1 and a monoclonal antibody date: 2016-09-16 journal: sci rep doi: 10.1038/srep33382 sha: doc_id: 344871 cord_uid: 486sk4wc we have previously shown that a non-structural protein 1 (ns1)-binding monoclonal antibody, termed as 2h6, can significantly reduce influenza a virus (iav) replication when expressed intracellularly. in this study, we further showed that 2h6 binds stronger to the ns1 of h5n1 than a/puerto rico/8/1934(h1n1) because of an amino acid difference at residue 48. a crystal structure of 2h6 fragment antigen-binding (fab) has also been solved and docked onto the ns1 structure to reveal the contacts between specific residues at the interface of antibody-antigen complex. in one of the models, the predicted molecular contacts between residues in ns1 and 2h6-fab correlate well with biochemical results. taken together, residues n48 and t49 in h5n1 ns1 act cooperatively to maintain a strong interaction with mab 2h6 by forming hydrogen bonds with residues found in the heavy chain of the antibody. interestingly, the pandemic h1n1-2009 and the majority of seasonal h3n2 circulating in humans since 1968 has n48 in ns1, suggesting that mab 2h6 could bind to most of the currently circulating seasonal influenza a virus strains. consistent with the involvement of residue t49, which is well-conserved, in rna binding, mab 2h6 was also found to inhibit the interaction between ns1 and double-stranded rna. residues 30-53 in h5n1-ns1 are sufficient for its interaction with mab 2h6. as described previously 28 , the deletion of residues 42-53 of ns1 abolished its interaction with mab 2h6. these residues lie in the helix α 2 (residues 30-53) of h5n1-ns1(rbd) and are well conserved between h5n1, h1n1 and h3n2 viruses (fig. 1a) . this is consistent with the ability of mab 2h6 to bind to both avian h5n1 and seasonal iavs 28 . in order to determine if the helix α 2 of h5n1-ns1(rbd) is sufficient for the interaction with mab 2h6, enzyme-linked immunosorbent assay (elisa) was performed by using a synthetic peptide h5n1-ns1-24mer corresponding to helix α 2. as shown in fig. 1b , mab 2h6 bound to h5n1-ns1-24mer in a dose dependent manner, indicating that the helix α 2 of ns1(rbd) is sufficient for its interaction with mab 2h6. in contrast, there was no binding between mab 2h6 and an irrelevant control peptide of similar molecular weight. within helix α 2, there is only one amino acid difference between h5n1 and h1n1-pr8, namely n48 in h5n1-ns1 and s48 in h1n1-pr8-ns1 (fig. 1a) . to determine if residue 48 in ns1 is involved in its interaction with mab 2h6, recombinant h5n1-ns1(rbd) and h1n1-pr8-ns1(rbd) proteins were bacterially expressed and purified for elisa. the elisa readings were similar for h5n1-ns1(rbd) and h1n1-pr8-ns1(rbd) when high concentrations of proteins were coated on the plate (fig. 1c) . however, the readings were significantly higher for h5n1-ns1(rbd) at lower protein concentrations, indicating that that the single amino acid difference between helix α 2 of h5n1-ns1(rbd) and h1n1-pr8-ns1(rbd) affects their interactions with mab 2h6 (fig. 1c ). residue 48 in ns1 is critical for its interaction with mab 2h6. to further define the contribution of residue 48 in h5n1-ns1 to the interaction with mab 2h6, two mutant proteins in which n was mutated to a and s respectively, were generated. comparative elisa showed that mab 2h6 bound to ns1(rbd)-wild-type (wt) stronger than ns1(rbd)-n48s when different amounts of protein were coated on the plate and analyzed with 5 μ g/ml of mab 2h6 ( fig. 2a) . similarly, when a fixed amount of protein (125 μ g/ml) was coated on the plate and analyzed with different concentrations of mab 2h6, the binding to ns1(rbd)-n48s was significantly lower than ns1(rbd)-wt (fig. 2b ). furthermore, when n was substituted with a at residue 48, the interaction between mab 2h6 and ns1(rbd) was totally abolished in all the conditions tested ( fig. 2a,b) . this result suggests that the difference at residue 48 is the main reason for the stronger binding of mab 2h6 to h5n1-ns1(rbd) when compared to h1n1-pr8-ns1(rbd) (fig. 1 ). since both n48 and s48 are polar amino acids while a48 has no side chain, it is probable that the formation of hydrogen bonds is important for the interaction between mab 2h6 and ns1. mab 2h6 binds differently to h1n1-pr8 virus when compared with mutant viruses carrying substitution at residue 48 in ns1. since mab 2h6 binds to bacterially-expressed ns1 protein of h5n1 and h1n1-pr8 with different affinities, it is important to investigate whether this holds true for ns1 expressed in infected cells. thus, recombinant pr8 (rgpr8) viruses expressing the ns1 protein containing a single amino acid substitution at residue 48 (rgpr8-ns1-s48a and rgpr8-ns1-s48n) were generated using a reverse genetics system. subsequently, a549 cells were infected with each virus at a low multiplicity of infection (moi) of 0.01 respectively and plaque assay was used to determine the amount of virus secreted at different time points post-infection. as shown in fig. 3a , although the viral titer was slightly lower in the case of rgpr8-ns1-s48n infection, the overall growth rates of wt and mutant viruses were similar from 12 to 60 hours post-infection (h.p.i.). this is consistent with a previous report showing that substitution of residue 48 in ns1 does not affect viral replication in vitro 30 . next, 293t cells were infected with 2 moi of viruses and cell lysates were collected at 12 and 24 h.p.i. to determine the rate of viral protein synthesis. consistent with the virus growth kinetics, the expressions of structural proteins np and m1 were similar for all 3 viruses at both time-points (fig. 3b ). the level of ns1, as determined by using a rabbit anti-ns1 polyclonal antibody, was also comparable for all 3 viruses. however, mab 2h6 bound to rgpr8-ns1-s48n virus significantly stronger than rgpr8-ns1-wt virus containing s48 residue in mismatches are shown in white letters. ns1(rbd) is composed of 3 α -helices as shown above the sequence. the region corresponding to helix α 2 (residues 30-53) is boxed. (b) peptide elisa was performed to determine the region of ns1 sufficient for binding to mab 2h6. wells were coated with serially diluted h5n1-ns1-24mer or a negative control peptide and probed with 5 μ g/ml mab 2h6. * indicates statistically significant difference of p < 0. 05 when compared with control peptide. (c) comparative elisa was performed to determine the ability of ns1(rbd) of h1n1-pr8 and h5n1 to bind to mab 2h6. wells were coated with serially diluted proteins and probed with 5 μ g/ml mab 2h6. data shown represents result from three independent experiments and error bars represent standard deviation (sd) of the experiment carried out in duplicates. *indicates statistically significant difference of p < 0.05 when compared with h1n1-pr8-ns1(rbd). scientific reports | 6:33382 | doi: 10.1038/srep33382 ns1, supporting the above results that n48 in ns1 is preferred over s48 for the interaction with mab 2h6. as expected, mab 2h6 did not bind to rgpr8-ns1-s48a virus. to further define the interaction interface between mab 2h6 and ns1, attempts were made to co-crystallize the complex but failed. however, the 2h6-fab alone was found to crystallize in space group p2(1) and the structure was solved by molecular replacement using the structure of bl3-6 (pdbid: 4q9q) as the starting model and refined to the crystallographic r-factor of 28.5% (deposited in the pdb under accession code 5b4m). the refinement statistics are shown in table 1 . previously, we demonstrated that the intracellular expression of 2h6-scfv in mammalian cells reduced the replication of pr8 virus. since the three dimensional structure of 2h6-fab has been solved, a commercially available lipodin-ab reagent (abbiotec), which is a protein transfection reagent dedicated to the transport of antibodies into living cells, was used to deliver 2h6-fab into a549 cells. the cells were then subjected to infection so as to determine if 2h6-fab has an impact on viral replication. when either 2h6-fab or 1a9-fab (which is derived from a negative-control antibody binding to the spike glycoprotein of sars coronavirus) 31 was transfected into a549 cells using lipodin-ab reagent, intracellular accumulation of fab was observed even up to 24 h post transfection (fig. 4a) . the intracellular accumulation of fab was also observed at as early as 4 h post transfection (data not shown). the transfection efficiency was about 70% and most of the fab molecules were evenly distributed in the cytoplasm but there were some punctate staining which could be due to aggregation of fab inside a549 cells. in contrast, no intracellular accumulation of fab was observed when 1a9-fab or 2h6-fab was added to the cells without lipodin-ab reagent. as shown in fig. 4b , the delivery of fab did not have any significant effect on cell viability at either 12 or 24 h post transfection. upon successful delivery of fab, its biological function was assessed in influenza a virus infected cells. as mab 2h6 binds strongly to rgpr8-ns1-s48n virus (fig. 3) , a549 cells were infected with rgpr8-ns1-s48n virus after transfection of 2h6-fab. cell lysates were collected at 6, 12, 24 h.p.i. to determine the rate of viral protein synthesis. as shown in fig. 4c ,d, the level of viral m1 protein at 24 h.p.i. was significantly reduced in 2h6-fab transfected cells when compared to 1a9-fab transfected cells. at 24 h.p.i., the average reduction in normalized m1 expression in 2h6-fab transfected cells was 50% when compared to 1a9-fab transfected cells. at 12 h.p.i., a slight reduction in m1 expression (~25%) was also observed in 2h6-fab transfected cells but it is not statistically significant. this result suggests that the successful delivery of 2h6-fab into living cells could reduce viral replication by affecting certain function(s) of ns1 in the infected cells. next, computational modelling was used to study the complex between this high resolution 2h6-fab structure and the published structure of h5n1-ns1(rbd) of h5n1/a/crow/kyoto/t1/2004 strain (pdb id: 2z0a). this was conducted by using haddock on 192 water-refined models, including an analysis of energy contributions from van der waals interaction, electrostatic interaction, restraints violation and buried surface area 32 . as comparative elisa in this and previous studies 29 showed that residues n48 and t49 in ns1(rbd) are important for the interaction with mab 2h6, they were defined as active residues involved in the binding interaction to generate a series of models of the ns1(rbd) and 2h6-fab complex. all the models of ns1(rbd) and 2h6-fab complex were found to cluster into 3 groups, in which there were at least two conformations of the ensemble showing backbone root-mean-square deviations at the interface of less than 1.0 å. as additional elisa results showed that 3 residues, namely s42, r44, and g47, in ns1 are not involved in interaction with mab 2h6 ( figure s1 ), this information was used to distinguish between the models in these 3 clusters. of all energetically best models generated, 9 predicted models were grouped into cluster 2. the average buried surface area was 1670.7 ± 52.6 å 2 , and rmsd from the overall lowest-energy structure was 4.9 ± 0.3 å. among them, the best predicted model from this cluster showed good agreement with our comparative elisa data, since only residues n48 and t49 were predicted to be involved in the interaction, while the side chains of s42, r44 and g47 were either distal from the interface (s42 and r44) or inaccessible (g47) to the binding partner of 2h6-fab (fig. 5) . by analyzing the polarity of the amino acids and distance between them in this model, it is predicted that residues n52 and n54 in the variable domain of heavy chain (vh)-complementarity determining region 2 (cdr2) of 2h6-fab could form hydrogen bonds with the side chain of n48 in ns1(rbd). in addition, the side chain of t49 in ns1(rbd) could form hydrogen bonds with residue r57 in vh-cdr2. in contrast, vh-cdr1, vh-cdr3 and all the cdrs in the variable domain of light chain (vl) are unlikely to be involved in the interaction as they are distal to helix α 2 of ns1(rbd). on the other hand, 179 predicted models were grouped into cluster 1. for these refined structures analyzed, the average buried surface area was 1234.9 ± 22.5 å 2 , and rmsd from the overall lowest-energy structure was 1.1 ± 0.8 å. based on the best predicted model from this cluster ( figure s2 ), it was predicted that t49 of ns1(rbd) could form the hydrogen bond with n52 of vh-cdr2 while n48 of ns1(rbd) could form the hydrogen bond with n96 of vl-cdr3. these predictions are in agreement with the results shown in fig. 2 and in our previous publication 29 . however, this model also predicted that r44 of ns1(rbd) could be involved in the interaction with 2h6-fab because it was in close proximity to two residues in vl-cdr1. in this model, the distance between r44 of ns1(rbd) and y31 of vl-cdr1 was 2.1 å while the distance between r44 of ns1(rbd) and s35 of vl-cdr1 was 2.9 å. thus, this model does not agree with the results from comparative elisa which showed that substitution of r44 of ns1(rbd) with k did not affect its interaction with mab 2h6 ( figure s1 ). lastly, another 4 predicted models were grouped into cluster 3. the average buried surface area was 1174.4 ± 22.1 å 2 , and rmsd from the overall lowest-energy structure was 7.3 ± 0.6 å. based on the best predicted model from this cluster ( figure s2 ), the contacts between n48 and t49 with residues in 2h6-fab were the same as described above for the model from cluster 1. however, this model also predicted that r44 of ns1(rbd) could be involved in the interaction with 2h6-fab because it was in close proximity to three residues in vl-cdr1. in this model, y31, s35 and y36 of vl-cdr1 were found to be at 1.7 å, 3.3 å and 2.8 å from r44 of ns1(rbd) respectively. thus, this model does not agree with the results from comparative elisa which showed that substitution of r44 of ns1(rbd) with k did not affect its interaction with mab 2h6 ( figure s1 ). overall, the predicted model from cluster 2 is consistent with our comparative elisa data and suggests that residues n48 and t49 are important for the binding between ns1(rbd) and 2h6-fab because their side-chains could make hydrogen bonds with residues in the vh-cdr2 of the fab. in addition, r44 of ns1(rbd) was distal from the antibody-antigen interface, which is consistent with the results from comparative elisa ( figure s1 ) showing that substitution of r44 of ns1(rbd) with k did not affect its interaction with mab 2h6. in contrast, the predicted models from cluster 1 and cluster 3 do not agree with the results from comparative elisa. mab 2h6 disrupts ns1 and dsrna interaction. ns1(rbd) forms a symmetric six-helical homodimer, which binds to dsrna. the key residues in ns1 involved in the interaction with dsrna are t5, d29, d34, r35, r37, r38, l41, s42 and t49, most of which are positively charged residues and mainly clustered in the middle of helices α 2/α 2′ of the rbd 33, 34 . to investigate whether mab 2h6 hampers dsrna-ns1 interaction in vitro, an alphascreen assay was carried out. in this experiment, glutathione s-transferease (gst)-tagged h5n1-ns1(rbd) protein, which was produced in e. coli, was incubated with synthetic 21-nucleotide sirna (21nt-sirna) followed by addition of streptavidin coated donor beads and anti-gst-conjugated acceptor beads which recognize biotinylated rna and gst-tagged protein respectively. if the interaction between ns1 and 21nt-sirna brings both beads to close proximity, transfer of excitation energy from donor beads into acceptor beads will yield a luminescent signal (fig. 6a ) 35 . when ns1(rbd) was pre-incubated with different concentrations of mab 2h6 followed by addition of 21nt-sirna, acceptor and donor beads, the luminescent signal decreased at high concentration of mab 2h6 (fig. 6b ). the luminescent signal was reduced by ~30% and ~70% at mab 2h6 concentrations of 1 and 5 μ m respectively (fig. 6b) , suggesting that the binding of mab 2h6 to ns1(rbd) can block the interaction of ns1 with dsrna. on the other hand, the negative control mab 1a9 did not reduce the luminescent signal at all the concentrations tested. furthermore, when mab 2h6, ns1 and 21nt-sirna were mixed simultaneously, 5 μ m of mab 2h6 could reduce the signal by about 30% (fig. 6c) , which suggest that mab 2h6 also directly competes with dsrna to bind to ns1. the ns1 protein of influenza virus is a multi-functional protein that is involved in key aspects of the virus replication cycle 36 . the ns1(rbd) consists of the first 73 amino acids and contains residues critical for dsrna binding 14 . the dimerization of the rbd is a prerequisite for its rna binding activity 13 . since the ns1 protein is an intracellularly expressed viral protein, which is subjected to less host selective immune response and thus has lower mutation rate, antibodies targeting this protein could be helpful for the development of therapeutic treatment of influenza a infection. indeed, our previous study showed that mab 2h6 binds to the highly conserved t49 residue in ns1 and reduces viral replication of h1n1-pr8 in mammalian cell lines 29 . in this study, comparative elisa showed that helix α 2 of ns1(rbd) is sufficient for its interaction with mab 2h6 (fig. 1) . while mab 2h6 binds to ns1(rbd) of both h5n1 and h1n1-pr8, the binding affinity to the homologous h5n1 viral protein is higher. interestingly, a single amino acid difference at residue 48 in helix α 2 of ns1(rbd) of h5n1 and h1n1-pr8 is found to be critical for the interaction with mab 2h6 (figs 2 and 3 ). by using either purified ns1 protein or ns1 expressed in infected cells, our results showed that the interaction of mab 2h6 with ns1 is stronger when residue 48 is n than when it is s and is abolished when the residue is an a. to understand the pattern of polymorphism at residue 48 in ns1, sequences were retrieved from niaid influenza research database (http://www.fludb.org) and analyzed ( table 2 ). sequence analysis of avian h5n1 isolates revealed that 87% of them have n48 and 13% have s48 suggesting that mab 2h6 has the ability to bind to the majority of avian h5n1 isolates. interestingly, a recent computational study compared the viral proteins of highly and lowly pathogenic h5 viruses and identified s48 to n substitution as a potential marker of pathogenicity of avian influenza virus subtype h5 37 . as for human isolates, the majority (93%) of seasonal h1n1 isolated before 2009 have s48 in ns1 like h1n1-pr8. however, almost all the pandemic 2009 h1n1 (pdmh1n1) isolates have n48 in ns1. as for seasonal h3n2, 91% of viruses isolated before 2009 have n48 and this percentage increased to 99% for those isolated from 2009 to 2015. thus, mab 2h6 is expected to bind to the majority of circulating seasonal influenza viruses. by solving a crystal structure of 2h6-fab and docking it onto the ns1(rbd) of h5n1 with the haddock program, molecular contacts made by residues t49 and n48 at the interface of antibody-antigen complex were predicted (fig. 5) . based on one of the energetically best models generated, the side chain of residue n48 in ns1(rbd) could form hydrogen bonds with the side chains of residues n52 and n54 of vh-cdr2 of 2h6-fab. meanwhile, residue t49 in ns1(rbd) seems to interact with residue r57 in vh-cdr2. these predictions are consistent with the results of binding assays performed using substitution mutants of ns1 as described above and in our previous study 29 . while the predicted 3d model of the antibody-antigen complex gives us some clues on how residues 48 and 49 in ns1(rbd) interact with 2h6-fab, it may be able to accurately reveal the contributions of all molecular contacts between ns1(rbd) and 2h6-fab. hence, further studies could focus on the use of other epitope mapping methodologies 38 , besides crystallography, to obtain a more precise map of the molecular contacts at the antigen and antibody interface. furthermore, an alphascreen assay showed that mab 2h6 could inhibit the interaction between ns1 and dsrna (fig. 6) . while both residues 48 and 49 are located in helix α 2 of the ns1(rbd), it has been shown that t49, but not s48, is one of the key residues involved in dsrna binding 34 . thus, the interaction between t49 and 2h6-fab predicted in the docked model is consistent with the ability of mab 2h6 to inhibit the interaction between ns1 and dsrna. although a previous study has demonstrated that mutation at residue 48 did not affect the affinity of ns1(rbd) for dsrna 30 , our study suggests that the side chain of n48 makes crucial contacts with mab 2h6 to stabilize the antibody-antigen complex. as such, when the side chain is not present in the case of gst-tagged ns1 protein was incubated with biotinylated dsrna and the interaction between ns1 and dsrna could bring streptavidin-coated donor bead and anti-gst-conjugated acceptor bead close to each other. when excited at 680 nm, singlet oxygen molecules ( 1 o 2 ) are produced from donor beads, which react with acceptor beads to produce light emission measured at 520-620 nm. (b) inhibition activity of mab 2h6 was determined by pre-incubating 10 nm gst-tagged ns1 protein with serially diluted mab 2h6 or a negative control mab 1a9. following that, luminescent signal was measured after the addition of 10 nm biotinylated dsrna, acceptor and donor beads. (c) inhibition activity of mab 2h6 was determined by incubating 10 nm gst-tagged ns1 protein, 10 nm biotinylated-dsrna and serially diluted mab 2h6 or 1a9 simultaneously. following that, luminescent signal was measured after the addition of acceptor and donor beads. all readings obtained were normalized against that of samples in the absence of antibody. error bars represent sd of the experiment carried out in triplicates. *indicates statistically significant difference of p < 0.05 when compared to mab 1a9. the n48a substitution mutant, interaction with mab 2h6 is completely abolished (figs 2 and 3 ). this indicates that the interaction between the side chains of residue t49 in ns1 and residue r57 in 2h6-fab is not sufficient to maintain the antibody-antigen interface. on the other hand, the t49v and t49a substitution mutants still retained some binding to mab 2h6 ( figure s1 ), presumably because of the hydrogen bonds between residue n48 and the heavy chain of the antibody. based the alphascreen assay, a high concentration of mab 2h6 (~5 μ m) was required to reduce the interaction between ns1(rbd) and dsrna by > 50% (fig. 6) . on the other hand, the concentration of mab 2h6 required for binding ns1(rbd) in elisa was in the nanomolar range (fig. 2) . this discrepancy could be due to the differences between the two assays but it also suggests that the binding of mab 2h6 to ns1 could have other effects on ns1 besides disrupting its interaction with dsrna. indeed, our previous gel filtration and dynamic light scattering results suggest that the complex between 2h6-fab and ns1(rbd) is multimeric in nature and each oligomer could consist of 6 molecules of ns1(rbd) and 6 molecules of 2h6-fab 29 . in contrast, ns1(rbd) eluted out the gel filtration column as a dimer, as would be expected, in the absence of 2h6-fab 29 . in addition, the delivery of 2h6-fab into a549 cells also caused a reduction in the replication of rgpr8-ns1-s48n recombinant virus (fig. 4) . while it is difficult to precisely define the biologically relevant quaternary structures of ns1, several studies have shown that the ns1 has conformational plasticity and dynamic changes in the quaternary structure of ns1 are likely to be important for the different functions of ns1 in infected cells 39 . hence, it is possible that the binding of 2h6-fab to the ns1 expressed in infected a549 cells could affect the conformational plasticity of ns1 and/or its ability to interact with certain cellular factor, thus resulting in a reduction in viral replication. in future studies, advanced fluorescence microscopic techniques could be used to determine the effect of 2h6-fab on the dynamic of ns1 inside infected cells. in summary, we have used biochemical and structural methods to characterize the interaction between ns1 and mab 2h6. our results showed that helix α 2 in ns1(rbd) is sufficient for interacting with mab 2h6 and residues n48 and t49 in this helix are likely to make hydrogen bonds with the cdr2 of the antibody heavy chain. helix α 2 is highly conserved and this is consistent with the ability of mab 2h6 to bind to different subtypes of iav. after solving a high resolution crystal structure of 2h6-fab, a haddock-derived model of the antibody-antigen complex has been obtained and the molecular contacts predicted from this model are in agreement with results obtained from comparative elisa performed using ns1 mutants. this model may be used in antibody engineering experiments to increase the affinity of interaction between ns1 and mab 2h6 so as to increase mab 2h6's potency in viral inhibition. in addition, it may be useful in structure-based rational drug design to identify small molecule inhibitors of ns1. cells. a549, 293t and mdck cells were purchased from american type culture collection (manassas, va, usa). a549 cells were cultured in minimum essential medium (mem) (gibco). 293t and mdck cells were cultured in dulbecco's modified eagle's medium (invitrogen). both media were supplemented with 10% fetal bovine serum (hyclone), penicillin (10,000 units/ml)-streptomycin (10 mg/ml) solution (sigma aldrich). all cell lines were maintained at 37 °c with 5% co 2 . ascites and rabbit polyclonal antibodies production. ascites were produced by injecting hybridoma cells into peritoneal cavities of pristine-primed balb/c mice. the protocol was approved by institutional animal care and use committee (iacuc) of the biological resource center, a*star, singapore (protocol number: 110694). in order to generate rabbit anti-h5n1-ns1 polyclonal antibody, gst-fusion ns1 protein was purified using the method as previously described 28 . new zealand white rabbits were then immunized with this protein and bled as previously described 40 . the protocol was approved by iacuc of the biological resource center, a*star, singapore (protocol number: 110693). all the animal procedures were performed in strict compliance with the recommendations of the naclar guideline in singapore. all efforts were made to minimize the suffering and euthanasia was performed using carbon dioxide. protein expression and purification. ns1(rbd) (residues 1-73) of a/chicken/hatay/2004(h5n1) was pcr amplified from a full-length ns1 gene (accession no.: caj01906.1) and the ns1(rbd) (residues 1-70) of a/puerto rico/8/1934(h1n1) was pcr amplified from a full-length ns1 gene (accession no.: abd77680.1). the expression constructs were generated by inserting pcr product into pet sumo expression vector (invitrogen). ns1 mutants expression construct was generated by overlap pcr as described previously 41 . all proteins were expressed and purified as previously described 29 . the purified proteins were dialyzed against dialysis buffer (20 mm tris-hcl, ph 7.4, 100 mm nacl) and concentrated to 3 mg/ml in a centrprep-10 (amicon) for subsequent assays. crystallization of 2h6-fab. mab 2h6 was purified from the ascites by using affinity chromatography and 2h6-fab was obtained by papain cleavage as described previously 29 . the purified 2h6-fab was dialyzed against dialysis buffer and concentrated to 10 mg/ml for subsequent crystallization. crystallizations were performed with the hanging drop vapor diffusion method at 20 °c by mixing 1 μ l of 2h6-fab with 1 μ l of reservoir solution and the mixture was equilibrated against 600 μ l of reservoir solution. crystals of 2h6-fab were grown against crystallization buffer containing 25% peg 3350, 0.2 m nacl and 0.1 m bis-tris, ph 6.5. these crystals grew to a maximum size of 0.3 mm × 0.1 mm × 0.1 mm over the course of 10 days. single crystals were obtained by dissecting from multiple crystals. crystals were flash frozen (100 k) in the above reservoir solution supplemented with 30% glycerol. a total of 360 frames of a native data set with 1 oscillation at 1.5418 å wavelength were collected for 2h6-fab. all data sets were processed by hkl2000. most of the crystals diffracted rather weak and the scaled data sets were anisotropic with strong ice rings and high mosaicities. nevertheless, one of the data sets displaying scientific reports | 6:33382 | doi: 10.1038/srep33382 weak ice ring was able to scale to 2.4 å at the mosaicity of 1.8°. this data was used for structure determination and refinement with the statistic table listed as table 1 . of 2h6-fab were predicted using the online igblast tool (http://www.ncbi.nlm.nih.gov/igblast/). as the three-dimensional structure of ns1(rbd) of a/chicken/hatay/2004(h5n1) has not been solved, the structure of ns1(rbd) of a/crow/kyoto/t1/2004(h5n1) (pdb id: 2z0a) was used instead. as shown in fig. 1a , there are 3 amino acid differences between the ns1(rbd) of these two strains but the sequence of helix α 2 in the ns1(rbd) is 100% identical. mutagenesis data from comparative elisa was used to generate a series of models for the complex between 2h6-fab and ns1(rbd) by using version 2.2 of haddock webserver 32, 42 . along with the available individual structure, haddock utilizes the experimentally derived data to predict the complex structure. to achieve this, ns1(rbd) was docked to 2h6-fab using the easy interface of haddock webserver, where both residues n48 and t49 of ns1(rbd) and amino acids in the cdrs of 2h6-fab were defined as active residues involved in the interaction. in this docking experiment, only the variable domains of 2h6-fab were used. enzyme-linked immunosorbent assay (elisa). elisa was performed as described previously 29 . briefly, a synthetic peptide corresponding to residues 30-53 in h5n1-ns1 (apfldrlrrdqkslrgrgntlgld, chemically synthesized by gl biochem) or purified wt and mutant ns1(rbd) proteins were serially diluted into 0.05 m carbonate-bicarbonate buffer, ph 9.6. a 29-mer peptide corresponding to a fragment of the hepatitis c virus core protein (rpswgpidprrrsknlgkvidtltcgfap, chemically synthesized by genway biotech) was used as a negative control. proteins or peptides (50 μ l) were then coated onto 96-well elisa plates (nunc) overnight at 4 °c. the wells were blocked in 5% milk in pbs with 0.1% tween 20 (pbst) for 1 h at 37 °c followed by addition of 100 μ l of mab 2h6 as primary antibody to each well and incubated at 37 °c for 2-3 h. the wells were then washed in pbst followed by the addition of goat anti-mouse horse-radish peroxidase (hrp)-conjugated antibody (pierce) as secondary antibody and incubated at 37 °c for 1 h. tetramethylbenzidine substrate (pierce) was added and reaction was stopped using 0.2 m sulfuric acid. absorbance at 450 nm was recorded using an absorbance reader (tecan infinite m200). recombinant viruses were generated with phw2000 reverse genetic system as described previously 43 . residue 48 in ns1 was changed from s to a or n by pcr mutagenesis and the resulting dna were inserted into phw2000 vector. this plasmid was co-transfected with another seven phw2000 plasmids containing the other pr8 genomic dnas into 293t cells. 2 days post transfection, culture supernatant was collected and used to infect mdck cells. when cytopathic effect (cpe) was visibly detected, culture supernatant was collected and used to infect naïve mdck cells. individual plaque was then amplified and viral titer was determined by plaque assay. virus infection and western blot analysis. 80% confluent 293t cells were infected with 2 moi of wt or mutant viruses at 37 °c for 1 h. the medium was discarded and cells were rinsed with pbs. cell lysates were harvested at 12 and 24 h.p.i. in ripa buffer (50 mm tris-hcl ph 8.0, 150 mm nacl, 0.5% np40, 0.5% sodium deoxycholate, 0.005% sds). then, 25 μ g of total lysate were resolved using electrophoresis on an sds-polyacrylamide gel and transferred to a nitrocellulose membrane (bio-rad). antibodies against gapdh (santa cruz), np (millipore), and m1 (as described previously) 29 were used. mab 2h6 and rabbit anti-h5n1-ns1 polyclonal antibody (as described above) were also used. after washing, the membrane was incubated with a hrp-conjugated secondary antibody (pierce) at room temperature for 1 h. the membranes were then washed and detected with enhanced chemiluminescence substrate (pierce) using chemidoc ™ mp imaging system (bio-rad). multiple-cycle growth kinetics of recombinant virus. plaque assay was applied to determine the growth kinetics of rgpr8-ns1-wt, rgpr8-ns1-s48a and rgpr8-ns1-s48n recombinant viruses. 90% confluent monolayers of a549 cells were rinsed with pbs and subsequently adsorbed with 0.01 moi of wt and mutant viruses respectively for 1 h at 37 °c. the medium was discarded and the cells were rinsed using pbs and cultured in mem without serum at 37 °c. supernatant containing virus was collected at 12, 24, 36, 48 and 60 h.p.i. respectively and subjected to plaque assay to determine viral titer. plaque assay in mdck cells. 90% confluent mdck cells were adsorbed with serially diluted supernatants containing viruses for 1 h at 37 °c. the medium was discarded and the cells were rinsed using pbs. the cells were overlaid with 2 ml of dmem supplemented by 0.3% agar and 2 μ g/ml tpck-trypsin (thermo scientific). after incubation at 37 °c for 2 days, the cells were fixed using 10% formalin for 1 h and stained using 0.1% crystal violet solution. delivery of 1a9-fab and 2h6-fab into a549 cells. a549 cells were cultured in 12-well plate. the 1a9-fab and 2h6-fab were then transfected into 80% confluent cells by using lipodin-ab reagent (abbiotec) according to manufacturer's protocol with slight modification. briefly, 40 μ l of fab solution (4 μ g) was mixed thoroughly with 4 μ l lipodin-ab solution and incubated for 15 min at room temperature. 100 μ l of serum-free mem medium was added to fab/lipodin-ab solution and then immediately added to the cells. the cells were washed with pbs once before the fab/lipodin-ab solution was added. the cells were incubated at 37 °c in 5% co 2 for 12 h, washed once with pbs and subjected to viral infection and western blot analysis as described above. cell viability assay. cell viability was determined using wst-1 reagent (roche) according to manufacturer's protocol. briefly, 10 μ l of wst-1 reagent was diluted in the 90 μ l of culture media and added to a549 cells cultured in a transparent 96-well microplate and incubated for 1 h, followed by measuring absorbance at 440 nm. immunofluorescence assay. a549 cells grown on coverslip were transfected with fab as described above. approximately 12 h after transfection, the medium was aspirated, washed with pbs once and replaced with serum-free mem medium and incubated at 37 °c in 5% co 2 for another 24 h. the cells were then washed with pbs once and fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% tritonx-100 for 10 min, followed by blocking with 1% bsa in pbs for 30 min. the cells were incubated with alexa fluor 488-conjugated goat anti-mouse igg antibody (invitrogen) for 1 h. after washing, cells were stained with dapi before mounting. images were captured using an olympus fluoview fv1000 laser-scanning confocal microscope. alphascreen-based dsrna binding inhibition assay. a 21nt-sirna previously reported to form complex with ns1(rbd) 34 was purchased from thermo scientific dharmacon (dharmacon, lafayette, co). the sirna was biotinylated at the 5′ end of the sense strand and the sirna sequences were as follows: 5′ -biotin-agacaccauuaugcugucuuu-3′ (sense) and 5′ -agacagcauaauggugucuuu-3′ (antisense). the lyophilized sirnas were reconstituted in rnase-free water to a final concentration of 50 nm. to express gst-tagged ns1(rbd) of a/chicken/hatay/2004(h5n1) was pcr amplified from a full-length ns1 gene (accession no.: caj01906.1) and cloned into pgex-6p-1 vector (ge healthcare). the expression and purification were performed as described previously 28 . the purified gst-fusion protein was dialysed against pbs and the final concentration was determined using the coomassie plus protein assay reagent (thermo scientific). in vitro rna binding inhibition assay was carried out in 384-well proxiplate by using the alphascreen anti-gst kit (perkinelmer). in the first experiment, 5 μ l of 50 nm gst-tagged proteins were mixed with same volume of serially diluted mab 2h6 and incubated at room temperature for 1 h. then, 5 μ l of 50 nm biotinylated 21nt-sirna was added into the binding mixture and incubated at room temperature for 1 h before the addition of 10 μ l of detection mixture containing 0.1 μ l of anti-gst (glutathione s-transferase) acceptor beads and 0.1 μ l of streptavidin-coated donor beads (perkinelmer). after another incubation at room temperature for 1 h, luminescent signal was measured using an enspire multimode plate reader (perkinelmer) 35 . in the second experiment, 5 μ l serially diluted mab 2h6 was mixed with 5 μ l of 50 nm gst-tagged proteins and 5 μ l of 50 nm biotinylated 21nt-sirna simultaneously. after incubation at room temperature for 1 h, detection mixture was added and measurement was made similarly. statistical analysis. two-tailed student's t test was applied to evaluate the statistical significance of differences measured from the data sets obtained in 3 independent experiments. p < 0.05 was considered statistically significant. influenza-who cares orthomyxoviridae: the viruses and their replication new world bats harbor diverse influenza a viruses mixed infections of pandemic h1n1 and seasonal h3n2 viruses in 1 outbreak advances in the development of influenza virus vaccines toward a universal influenza virus vaccine: prospects and challenges evasion of influenza a viruses from innate and adaptive immune responses viral m2 ion channel protein: a promising target for anti-influenza drug discovery influenza neuraminidase inhibitors: antiviral action and mechanisms of resistance avian flu: isolation of drug-resistant h5n1 virus influenza a virus strains that circulate in humans differ in the ability of their ns1 proteins to block the activation of irf3 and interferon-beta transcription virulence of h5n1 avian influenza virus enhanced by a 15-nucleotide deletion in the viral nonstructural gene rna binding by the novel helical domain of the influenza virus ns1 protein requires its dimer structure and a small number of specific basic amino acids binding of influenza a virus ns1 protein to dsrna in vitro the influenza virus ns1 protein is a poly(a)-binding protein that inhibits nuclear export of mrnas containing poly(a) the influenza virus ns1 protein binds to a specific region in human u6 snrna and inhibits u6-u2 and u6-u4 snrna interactions during splicing the primary function of rna binding by the influenza a virus ns1 protein in infected cells: inhibiting the 2′ -5′ oligo (a) synthetase/rnase l pathway influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i structural basis for a novel interaction between the ns1 protein derived from the 1918 influenza virus and rig-i binding of the influenza a virus ns1 protein to pkr mediates the inhibition of its activation by either pact or doublestranded rna loss of function of the influenza a virus ns1 protein promotes apoptosis but this is not due to a failure to activate phosphatidylinositol 3-kinase (pi3k) influenza a virus ns1 protein activates the pi3k/akt pathway to mediate antiapoptotic signaling responses identification of influenza virus inhibitors targeting ns1a utilizing fluorescence polarization-based high-throughput assay synthesis and evaluation of quinoxaline derivatives as potential influenza ns1a protein inhibitors novel influenza virus ns1 antagonists block replication and restore innate immune function antiviral activity of baicalin against influenza virus h1n1-pdm09 is due to modulation of ns1-mediated cellular innate immune responses small interfering rna targeting the nonstructural gene 1 transcript inhibits influenza a virus replication in experimental mice a new panel of ns1 antibodies for easy detection and titration of influenza a virus a monoclonal antibody binds to threonine 49 in the non-structural 1 protein of influenza a virus and interferes with its ability to modulate viral replication roles of the phosphorylation of specific serines and threonines in the ns1 protein of human influenza a viruses monoclonal antibodies targeting the hr2 domain and the region immediately upstream of the hr2 of the s protein neutralize in vitro infection of severe acute respiratory syndrome coronavirus haddock: a protein-protein docking approach based on biochemical or biophysical information conserved surface features form the double-stranded rna binding site of non-structural protein 1 (ns1) from influenza a and b viruses structural basis for dsrna recognition by ns1 protein of influenza a virus assay optimization and screening of rna-protein interactions by alphascreen the multifunctional ns1 protein of influenza a viruses a complete map of potential pathogenicity markers of avian influenza virus subtype h5 predicted from 11 expressed proteins current approaches to fine mapping of antigen-antibody interactions conformational plasticity of the influenza a virus ns1 protein comparing the antibody responses against recombinant hemagglutinin proteins of avian influenza a (h5n1) virus expressed in insect cells and bacteria simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies haddock versus haddock: new features and performance of haddock2.0 on the capri targets a dna transfection system for generation of influenza a virus from eight plasmids the pymol molecular graphics system, version we are grateful to r.g. webster for the pr8 based reverse genetics system. we thank members of the monoclonal antibody unit at institute of molecular and cell biology for technical assistance. this work was supported by the singapore ministry of health's national medical research council under its nmrc-cbrg scheme [grant no. nmrc/cbrg/0017/2012]. key: cord-305742-wf6qxplf authors: gomez, santiago a.; rojas-valencia, natalia; gomez, sara; egidi, franco; cappelli, chiara; restrepo, albeiro title: binding of sars–cov–2 to cell receptors: a tale of molecular evolution date: 2020-09-28 journal: chembiochem doi: 10.1002/cbic.202000618 sha: doc_id: 305742 cord_uid: wf6qxplf the magnified infectious power of the sars–cov–2 virus compared to its precursor sars–cov is intimately linked to an enhanced ability in the mutated virus to find available hydrogen bond sites in the host cells. this characteristic is acquired during virus evolution because of the selective pressure exerted at the molecular level. we pinpoint the specific residue (in the virus) to residue (in the cell) contacts during the initial recognition and binding and show that the virus· · · cell interaction is mainly due to an extensive network of hydrogen bonds and to a large surface of non–covalent interactions. in addition to the formal quantum characterization of bonding interactions, computation of absorption spectra for the specific virus· · · cell interacting residues yields significant shifts of ∆λ max = 47 and 66 nm in the wavelength for maximum absorption in the complex with respect to the isolated host and virus, respectively. at the time of the writing of this manuscript, the situation regarding the global pandemic produced by the spread of the sars-cov-2 virus (over 17.7 million confirmed cases, over 675000 deaths, with no end in sight), with dire consequences in all aspects of life, from social interactions, to the overwhelming of health and economic systems, is changing fast. because this is a critical problem, just as the rate of virus transmission on the early stages of dissemination, the number of scientific papers on the subject (mostly preprints) increases exponentially. sars-cov-2 is an enveloped virus of the coronaviridae family with a single-stranded rna genome. [1] figure 1 highlights the most important structural features of the virus: besides the nucleocapsid (n) proteins, the only proteins in direct contact with the genetic material (the n-rna core is embedded in a lipid environment), there are membrane (m), envelope (e), and spike (s) proteins. it is the spike proteins which lead to the now familiar external morphology of the virus, but more importantly, s proteins are responsible for the interactions with receptors in the host membrane (epithelial cells in humans). these s· · · receptors contacts initiate the infectious cycle of the virus. [2] each spike consists of a trimer of s proteins. individual s proteins have been divided into two clear s1, s2 sections, [3] with s1 containing the n-terminal domain (ntd), and the receptor binding domain (rbd), the domain ultimately responsible for the interactions with the coupling factors present in cell membranes. [4] coupling factors include a variety of proteins, carbohydrates, or other types of biomolecules expressed on the surface of the cell membrane and in charge of signaling and transport, among other functions. viruses take advantage of these molecules during the infection process. it seems well established that initial virus↔host recognition and binding is driven by s1, and that further changes in the conformation of the s2 section mediate the viral envelope fusion to the host cell membrane. we also show a cell with internal organelles and with a few enzymes that act as a virus receptors. 4 10.1002/cbic.202000618 accepted manuscript chembiochem this article is protected by copyright. all rights reserved. the most commonly invoked culprit (with plenty of experimental evidence [5] ) for the reception of sars-cov-2 is the angiotensin converting enzyme 2 (ace2). this receptor is the subject of intensive studies aiming at finding effective therapies, and is the central focus of the ongoing race to find a vaccine. in this work, we are interested in two crucial aspects of the initial virus· · · cell interaction problem: to pinpoint the specific residue to residue binding sites between the structurally known spike proteins of the virus [6] and the structurally known ace2 receptor in cell membranes, [5] and to understand, from a fundamental, quantum perspective, the molecular factors driving the virus· · · cell binding. we expect this knowledge to considerably better our understanding of the problem and to hopefully contribute to a rational design of drugs and vaccines to fight the virus. see the computational methods section for details of our calculations. our data shows that the rbd(s)· · · ace2 complex reached well defined persistent equilibrium states long before the 600 ns of the molecular dynamics (md) simulation time are consumed in each of the three replicas. this stability is especially encouraging in the interaction region as clearly shown in the highlighted areas of the bottom panels in figure 2 . we obtained an interaction energy ∆g int = −419.91 kcal/mol, which is in excellent agreement with calculations reported in closely related systems. [7] 2.1 virus· · · cell contacts in all cases, only hydrogen bonds (hbs) were found as responsible for explicit virus· · · cell pair-wise interactions. naturally, this does not mean that other weak, long range cumulative for the later steps of the simulations. table 1 lists all individual binary contacts in the form of hydrogen bonds between residues in the rbd(s)· · · receptor complex found in our md simulations with an arbitrary threshold average of 15% occupancy on the triplicate runs. notice that this procedure intends to extract a representative sample from the simulations, thus, there are considerably more contacts not explicitly shown because they have lower occupancies or, because while having high occupancies, are not persistent in the 3 replicas. these hb contacts, whose bonding interactions are dissected below, are responsible for the attachment of the virus to epithelial cells in humans, initiating the infection process. number of fragment to fragment hydrogen bonds in the rbd(s)· · · ace2 complex averaged over three independent replicas as the md progresses. a summary of the quantum descriptors for the virus· · · cell interactions is listed in table 1 , the corresponding pictures are shown in figures 4, 5. without exception, despite being weak organic acids, residue to residue hydrogen bonds are stronger than the archetypal hb in the water dimer, this is seen in the larger binding energies, smaller distances, orbital interaction energies, e d→a of comparable magnitudes, larger bond indices, and in the properties of the bond critical points. the lys417→asp30 is an exceptional case because it corresponds to table 1 : properties of virus· · · cell hydrogen bonds. persistent hydrogen bonds in the rbd(s)· · · receptor complex exceeding the 15% occupancy threshold during the entire 600 ns md simulation of each replica. the arrows state the directionality of the donor → acceptor interaction in the corresponding hydrogen bond according to the classical electrostatic x δ− − h δ+ → y δ− description. all occupancies averaged over the three md replicas. wbi are the wiberg bond indices. [8] the archetypal hydrogen bond in the water dimer is included for comparison purposes. see the specialized literature [9] [10] [11] [12] [13] [14] for the formalism on how nbo and qtaim descriptors are related to bonding. bridge in a previous work. [15] all hydrogen bonds are well characterized long range interactions. this is clearly seen in (i) there is never a formal σ orbital between the fragments, on the contrary, orbital in other words, in the nbo picture ( figure 4 ), all explicit virus· · · cell contacts are stabilized by charge transfer from one lone pair in an oxygen atom in the donor residue to an antibonding orbital in the acceptor residue. (ii) all properties of the bond critical points support the same picture: small ρ (r c ), small bond orders, virial ratios smaller than 1, and positive bond degree parameters. again, is the exception, with all the calculated descriptors indicating a highly ionic contact. the non covalent interactions (nci) calculation for the interaction region uncovers a large discontinuous non-covalent wall separating rbd(s) from the receptor. therefore, we characterize the virus· · · cell binding as due to a large number of non-covalent contacts between the two proteins, enhanced by the water molecules, acting in conjunction with the specific residue to residue hydrogen bonds. the ace2 receptor (in blue). the snapshot was randomly extracted from the late stages of one of the three md replicas. [16] all persistent virus· · · cell hydrogen bonds listed in table 1 are explicitly highlighted on the right frames. the non covalent [17, 18] virus· · · cell interaction surface is explicitly shown in green, including the water molecules. notice that both fragments contain glycosylated glycoproteins and that all fine structure is accounted for during the calculations, however, the glycans are not shown in this picture for clarity. bottom: nbo donor→acceptor interactions responsible for the persistent hydrogen bonds. we concentrate on simulating the spectra of the aminoacids involved to investigate whether measurable changes in their spectral response occur upon binding, while residues that are not involved in the interaction can safely be viewed as changing very little as the two structures connect. for this purpose we propose a qm/mm approach where the aminoacid pairs involved in the binding are treated quantum mechanically by means of density functional theory (dft), while the rest of the protein environment is modelled classically, through the use of the amber force field. [19] in this way, the electronic structure of the qm portion is influenced by its environment by means of an electrostatic embedding paradigm, [20] where fixed charges are assigned to the mm atoms and directly affect the qm density and computed electronic excitations. figure 5 shows that in all six cases, binding events set off drastic changes in the spectral response of the system, explicitly seen in red-shifted absorptions. the red-shift of the absorption bands is clearly visible in the convoluted spectrum and therefore provides unequivocal evidence of virus· · · cell bonding. the results listed here are quite encouraging and constitute an initial step that will hopefully motivate the design of experimental protocols to detect virus infection. however, it is clear that a number of details need to be worked out before practical applications can be devised. in particular, the potential interference of signals arising from functional groups in the same region of λ max , and the ability of the dimer model to accurately mimic physiological environments, should be addressed. the most serious problems faced when developing vaccines and therapies, and is particularly true for sars-cov-2. [21] we argue that this view of evolution as driven by environment induced molecular responses at the virus/biomolecules scales helps explain many aspects of evolution that are difficult to rationalize otherwise, namely, (i) in most cases evolution is a highly localized process (ii) because of the large number of mutation possibilities, which occur no matter how small the individual probabilities, an increase in entropy of the universe is the ultimate factor driving evolution (iii) evolution is a deterministic process driven by cumulative random changes (iv) in that sense, life itself is a deterministic process that only requires a large increase in the entropy of the universe (in other words, a long time), such that it will emerge in local environments capable of sustaining it. see for example early arguments by schrodinger [22] stating the the apparent macroscopic stability is due to the microscopic chaos resulting from random events, and invoking a net entropy gain by the universe due to the continual energy transformation despite the heavy entropy investment in maintaining highly organized living organisms. more recently, england has discussed the statistical physics of self-replication. [23] in the context of this work, the previous discussion of molecular and virus evolution leads us to hypothesize that one of the key factors in the molecular evolution problem faced by the precursor sars-cov virus on its way to mutating into sars-cov-2 was solved by favoring those changes in rbd(s) that lead to an improved ability to locate available sites for hydrogen bonding in the host cell, ability that is further enhanced by the slightly basic ph ≈ 7.4 found in physiological environments. this improved hydrogen bonding capabilities may be achieved in a number of ways, for example, incorporating aminoacids with more acidic protons, or incorporating larger aminoacids whose hydrogen bonding regions are simply closer to the receptor, among others. we support the need for improved hydrogen bonding as the selective pressure in virus mutation hypothesis in the following evidence: 1. table 1 shows that fragment to fragment contacts are all in the form of hydrogen bonds. for the specific case of the sars-cov-2 virus, in five of the six identified contacts, including the most persistent hbs, the residues in rbd(s) act as donors to the corresponding hydrogen bond 2. besides a small sheet and a small helix (figure 4 ), there is no secondary structure in rbd(s), thus, the receptor binding domain of the spike protein has a high structural flexibility which allows the virus to probe for available hydrogen bonding sites in the receptor, which in contrast has well defined secondary and tertiary structures in the interaction region 3. we obtained from the genbank the sequences of aminoacids for the precursor sars-cov (id afr58742.1) and for the mutated sars-cov-2 (id qhd43416.1) viruses. [24] we compare below only the 196 aminoacids in the rbd(s) and highlight in red the receptor binding motif (rbm). we also underline aminoacid substitution in the muproteins. [25] [26] [27] [28] [29] here, we take a pragmatic approach to determine relative acidities between the precursor sars-cov and the mutated sars-cov2 viruses. we took averages of the experimental isoelectric points [30] (iep) for the 49 aminoacids involved in the mutation, that is, we calculated the average ipe in the replaced aminoacids in rbd(s) and found 6.50 and 6.44 for the precursor and for the mutated viruses, respectively. we also calculated the same averages for the interaction motifs only and obtained 6.44 and 5.98 over the 34 mutations. thus the mutated virus is collectively considerably more acidic in the interaction region, which improves its ability to donate protons to hydrogen bonds the most important contributions from this work may be summarized as follows: 1. we pinpoint the specific residue (in the virus) to residue (in the cell) interactions during the initial virus· · · cell binding 2. we characterize the virus· · · cell molecular attachment as the result of a large number the starting point of our calculations was the complex between the receptor binding domain of the s protein, rbd(s), and the ace2 receptor. cartesian coordinates for the rbd(s)· · · ace2 complex were taken from the protein data bank (pdb id 6lzg [31] ) and then treated with charmm-gui, the graphical user interface of charmm [32, 33] to include missing hydrogen atoms at ph = 7.4, to ensure that all glycans are included, and to construct the force field. the entire system was enclosed by a truncated octahedral box such that the smallest atom· · · wall distance was set to 9å, then the available volume in the box was filled with tip3p [34] water molecules. nacl molecules were added until a physiological 0.154 m concentration was attained and, finally, counterions were added to restore charge neutrality. this procedure lead to a system comprising a total of 171161 atoms, with 52735 water molecules, 9585 atoms in the receptor, and 3034 atoms in rbd(s). the system was subjected to a steepest descent energy minimization in order to correct for potential inconsistencies in atom coordinates that may arise during the procedure of randomly filling the available space with water, nacl, and counterions. once minimized, we ran triplicate all-atom md simulations under the conditions summarized in table 2 and described next. first, there were three equilibration steps lasting a total of 0.625 nanoseconds (ns) with 1 femtosecond (fs) time intervals, during which the structural constraints were progressively relaxed until finally being totally lifted. these structural constraints were imposed by harmonic constants that prevent deformation of the backbone (k bb ), side chain (k sc ), and dihedrals (k d ). then, the system underwent a production step lasting 600 ns with time intervals of 2 fs. for all md runs, the lennard-jones potential was softened starting at 0.8 nm until eventually vanishing at 1.0 nm. also, the cutoff radius for electrostatic interactions was set to 1.0 nm. all these simulations were conducted using the charmm36m force field [35, 36] as implemented in gromacs 2019.4 [37] at 310.15 k and 1 bar. the rbd(s)· · · ace2 interaction energy (∆g int ) was estimated via the mm/pbsa [38] as implemented in gromacs. [37] dielectric constants were set to solute = 1, solvent = 80 at the simulation temperature. in short, ∆g int = ∆e virus···cell + ∆g p + ∆g np − t ∆s. here, ∆e virus···cell is the gas phase energy of the rbd(s)· · · ace2 complex, ∆g p , ∆g np are the solvation energies due to the polar and non-polar interactions, respectively, and t ∆s is the entropy contribution. more precisely, ∆g p was computed under the poisson-boltzmann model, ∆g np was estimated using the solvent accessible surface area, and the entropy term was obtained from the model of duan and coworkers. [39] to finally estimate ∆g int , we took 300 points in the 140-170 ns interval of one of the md replicas. it has been recently shown [16] that randomly chosen configurations from late stages of md simulations are adequate sources to obtain deep insight into interfragment bonding. accordingly, aiming at understanding the fundamental forces driving the attachment of rbd(s) to host cells, virus· · · cell bonding interactions were dissected following these steps: 1. persistent residue (in the virus) to residue (in the host cell) contacts during the 600 ns of the md simulations were identified using the vmd program [40] with a cutoff radius of 3.5å 2. one frame was randomly chosen from the late stages of one md run 3. we extracted all extended interacting pairs in the chosen frame, kept them in the configurations they had in the interacting system (this is more accurate to understand the virus· · · cell bonding interactions than reoptimizing the isolated pairs), and (a) computed accurate interaction energies using highly correlated domain based local pair-natural orbital coupled-cluster (dlpno-ccsd(t)/aug-cc-pvdz) single point energy calculations [41, 42] on the dimers and in the monomers. the orca suite of programs, version 4.0.1.2, was used to this end [43] (b) dissected the intermolecular interactions using the tools provided by the natural bond orbitals (nbo [12] [13] [14] as implemented in nbo7.0 [44] ) and by the quantum theory of atoms in molecules (qtaim [9] [10] [11] as implemented in aimall [45] ) (c) calculated qm/mm absorption spectra for the monomers and for the dimers. all td-dft calculations were carried out using the b3lyp/aug-cc-pvdz model chemistry [46] [47] [48] [49] (tests using the dispersion corrected b3lyp-d3, ωb97xd, functionals yielded essentially identical results). qm/mm electrostatic embedding was exploited, [20] in which only the extended dimers were considered as the quantum region, and the rest of the system as the mm region, which was modelled by the amber force field [19] and by assigning to atom types the same charges used in the md runs. a large number of excited states are needed to guarantee that both the intensities and shapes of the absorption spectra are accurately reproduced. therefore, in this work the first 20 excited states were computed at the td-dft/qm-mm level in each case. vertical excitations were shifted by -0.7 ev to account for the systematic error due to the choice of functional. this value was chosen in order to match the experimental absorption maximum for tyrosine. [50, 51] all spectra were then convoluted with gaussian lineshapes with full width half maximum (fwhm) of 0.6 ev. all qm/mm calculations were carried out with gaussian16 [52] 4. we isolated the interaction region by including everything within a 3.0å radius from the last atom at the end of each aminoacid (1325 atoms in total) and calculated the interfragment non covalent interaction (nci as implemented in nciplot [53] ) surface using the promolecular densities approximation. [17, 18] atoms in molecules: a quantum theory discovering chemistry with natural bond orbitals proceedings of the national academy of sciences 2020 what is life? the physical aspect of the living cell electrophoresis crc handbook of chemistry and physics wires computational molecular science aimall (version 19.10.12) gaussian 16 revision b.01 internal support from universidad de antioquia via "estrategia para la sostenibilidad" is acknowledged. partial funding for this project from h2020-msca-itn-2017 european training network "computational spectroscopy in natural sciences and engineering" (co-sine), grant number 765739 is also acknowledged. n.r. thanks colciencias for her doctoral scholarship. cartesian coordinates for all dimer pairs listed in table 1 and for the fragment taken for the nci surfaces. a figure with the minimum interacting distances used to determine the interaction regions is included as well. a video of one of the trajectories is also provided. key: cord-319590-f9qcabcx authors: han, yanxiao; král, petr title: computational design of ace2-based peptide inhibitors of sars-cov-2 date: 2020-04-14 journal: acs nano doi: 10.1021/acsnano.0c02857 sha: doc_id: 319590 cord_uid: f9qcabcx [image: see text] peptide inhibitors against the sars-cov-2 coronavirus, currently causing a worldwide pandemic, are designed and simulated. the inhibitors are mostly formed by two sequential self-supporting α-helices (bundle) extracted from the protease domain (pd) of angiotensin-converting enzyme 2 (ace2), which bind to the sars-cov-2 receptor binding domains. molecular dynamics simulations revealed that the α-helical peptides maintain their secondary structure and provide a highly specific and stable binding (blocking) to sars-cov-2. to provide a multivalent binding to the sars-cov-2 receptors, many such peptides could be attached to the surfaces of nanoparticle carriers. the proposed peptide inhibitors could provide simple and efficient therapeutics against the covid-19 disease. s evere acute respiratory syndrome coronavirus 2 (sars-cov-2), previously known as 2019 novel coronavirus (2019-ncov), 1 is causing a pandemic of coronavirus disease. 2,3 sars-cov-2 shares about 80% of its genome identity with sars-cov, which emerged in 2002−2003. 4 sars-cov-2 is highly contagious in humans, which has rapidly caused an unprecedented pandemic, with a large number of fatalities worldwide. the sars-cov-2 virion, 50−200 nm in diameter, contains four structural proteins, known as the s (spike), e (envelope), m (membrane), and n (nucleocapsid) proteins. 2 the s protein, imaged at the atomic level using cryo-electron microscopy, 5 is responsible for the host attachment and fusion of the viral and host-cell membranes. 6, 7 this process is triggered when the s1 subunit of s protein binds to a host-cell receptor. to engage a host-cell receptor, the receptor-binding domain (rbd) of s1 undergoes transient hinge-like conformational motions (receptor-accessible or receptor-inaccessible states). 8 the angiotensin-converting enzyme 2 (ace2) is the host cellular receptor with a higher affinity to sars-cov-2 than to sars-cov. 5 in the recognition of rbd, the protease domain (pd) of ace2 mainly engages the α 1 -helix with a minor contribution from the α 2 -helix and the linker of the β 3and β 4 -sheets. 8, 9 in addition to a hectic search for vaccines against covid-19, there is a very fast ongoing search for therapeutics acting on sars-cov-2. depending on the activity, the therapies can be divided into several main categories: (1) preventing the viral rna synthesis and replication, (2) blocking the virus from binding to human cell receptors, (3) restoring the host's innate immunity, and (4) blocking the host's specific receptors or enzymes. 10 despite many experimental and computational studies currently exploring all of these categories, to date, there is no confirmed effective treatment specifically available for covid-19. computational approaches have been used to search potential therapeutics against sars-cov-2 protease (category 1). 11 analogous screening of potential drugs against the s protein of sars-cov-2 (category 2) provided small molecular compounds with a high binding affinity. unfortunately, most of these compounds do not attach with the binding interface of the rbd−ace2 complex. hesperidin was predicted to lie on the surface of rbd, but it did not cover the whole interface. 10 in the early attempts of sars-cov blocking, short peptide inhibitors were studied and amino acid mutations were implemented to the s protein of sars-cov. 12, 13 however, the proposed peptide was too short (8 residues) to maintain secondary structure, so it was unable to block the whole sars-cov binding surface. 12 broad-spectrum antiviral nanoparticles and cyclodextrins were designed, simulated, and implemented in blocking of other viruses. 14−16 they are category 2 or 3 inhibitors, but their applicability to sars-cov-2 is unknown. proteins or rigid peptides with specific (multivalent) binding domains and conformations matching rbd could be promising therapeutics for covid-19. overall, protein therapies show a high specificity, small interference with biological processes, good tolerance to human organisms, and faster fda approval times. 17 in this work, we design and simulate several peptide inhibitors against sars-cov-2, which included components from the virus-binding domains of ace2; based on the recently released crystal structure (pdb code: 6m17 9 ). the inhibitors, which have relatively low molecular weights, are structurally stable, they conformationally match the s protein, and are highly specific to sars-cov-2. this study could provide a potential guidance in antigen recognition and structure-based designs of antibodies with high affinities. the proposed small peptides could be used as inhaled therapeutics for topical lung delivery, providing an efficient way to combat covid-19. 18 preparation of inhibitors. in the crystal structure of ace2 and rbd of sars-cov-2 (pdb: 6m17 9 ), we first analyzed the interacting amino acids at the ace2 and rbd interface. in total, 15 residues from ace2 interact with rbd: residues 24(q), 27(t), 30(d), 31(k), 34(h), 35(e), 37(e), 38(d), 41(y), and 42(q) are in α 1 , one residue (residue 82 m) comes from α 2 , residues 353(k), 354(g), 355(d), and 357(r) come from the linker between β 3 and β 4 . therefore, the 15 amino acids can be labeled as critical amino acids and α 1 , α 2 , β 3 , and β 4 as critical binding components. because most of the interacting residues are from α 1 , we picked as inhibitor 1 the α 1 -helix alone. in particular, the 21− 55 residues, shown in figure 1a , were selected. realizing that α 1 (alone) might not even be stable, we next picked as inhibitor 2 both α 1 -and α 2 -helices (residues 21 to 88) and the residues 349 to 357 (residues between β 3 and β 4 shown in orange in figure 1b ). this selection included all 15 interacting residues from the crystal structure 6m17. 9 as the two α-helices are closely joined on one side (figure 1b) , they stabilize each other. to connect the two helices (red) with the β-sheets with residues 349 to 357 (orange), as shown in figure 1b , residues 45 (leu) and 351 (leu) were linked together by a side chain with a carbon−carbon bond, as shown in figure 2b . we have also designed other inhibitors that are closer to the ace2 protein, whose parts are connected by peptide bonds, and which contain all 15 residues that initially bind to rbd in the 6m17 crystal structure. 9 figure 1c (detail in figure 1e ) shows inhibitor 3, where residues 323 to 362 (orange) include the two β-sheets and a random coil (residues 323 to 348), whereas residues 21 to 105 (red) include the two α-helices with another random coil (residues 89 to 105). the two sequences are joined together by a peptide bond between residues 105 and 323, and the two pieces of random coils were moved close to each other. finally, figure 1d (detail in figure 1f ) shows inhibitor 4, where two sequences including residues 21 to 95 (red) and residues 335 to 500 (orange) were selected. an extra peptide bond was made between residue 21 and residue 400 by adjusting the position of the corresponding sequences. the sequences of all inhibitors are shown in table s1 . to examine how these potential inhibitors bind to rbd of sars-cov-2, we prepared these systems in the initial position known from the crystal structure (pdb: 6m17) and simulated them in physiological solution (methods), as shown in figure 2a −d. as a control, the pd of ace2 (residues 19 to 615) and rbd of sars-cov-2 were also simulated ( figure 2e) . binding conformations. in figure 2a , 200 ns long simulations showed that the helical structure of inhibitor 1 deforms from the left sideloose end unfolding, although it still binds to the rbd of sars-cov-2. in figure 2b −d, 120− 300 ns long simulations revealed that inhibitors 2−4 bind in a stable way to the rbd of sars-cov-2, without α 1 losing its structure. due to different linkages among the critical binding components, the overall conformations of inhibitors 2−4 vary. specifically, the α 1 -helix, which mostly contributes to the complementary sequence and conformational matching to rbd, is maintained in inhibitors 2−4 with different degrees of bending. the β-sheets in the structures of inhibitors 3 and 4 are also preserved. overall, the critical binding components in inhibitors 2−4 bind to rbd in a manner very similar to that of the crystal structure. the simulated stable conformation of inhibitors 2, 3, and 4 correspond to their energy minima of folding, which would drive the folding process toward the stable direction. energies. to further quantify the binding of these inhibitors to rbd, we calculated the rmsd for the 15 critical amino acids in each inhibitor and for the whole inhibitors. figure 2f shows the average rmsd at the end of our simulations (see also figure s1 ). inhibitor 1 has larger rmsd for the critical amino acids compared to that of the control and the largest fluctuations for both the critical amino acids and the overall rmsd ( figure s1a,b) . this can be attributed to unfolding of α 1 , shown in figure 2a . a highly promising inhibitor 2 has a rmsd of the critical amino acids and the overall rmsd similar to those in the control (lowest). inhibitor 3 has a rmsd of the critical amino acids and the overall rmsd higher than that of the control and inhibitors 1 and 2. however, figure s1b shows that inhibitor 3 has a very smooth overall rmsd at later times. this may be due to a poor adaptation of their added connections at early times. inhibitor 4 shows slightly bigger fluctuation for the overall rmsd but steady rmsd ( figure s1a ) for the critical amino acids at later times, which indicates fluctuation shown in the overall structure comes from nonessential connection parts. the interaction energies have van der waals (vdw) and electrostatic components, calculated by the namd energy plugin. the total energies are shown in figure 2 g and figure s2 (detail). the residues which contribute to the interaction energies between inhibitors and sars-cov-2 are selected with a cutoff of 3 å. the selections are updated in every frame. inhibitors 1 and 4 show interaction energies similar to those of the control, with inhibitor 3 having slightly stronger binding than the control; however, inhibitor 2 shows an interaction energy slightly lower than that of the control. the larger interaction energy in inhibitor 1 might be due to nonspecific interactions caused by the deformed helix. the lower interaction energy in inhibitor 2 could be attributed to the total number of residues, which is less than those of inhibitor 3 and 4. in summary, using classical molecular dynamics simulations, we have shown that peptide inhibitors extracted from ace2 provide highly promising trails for sars-cov-2 blocking. the single α 1 -helix used in inhibitor 1 is less stable, whereas the α 1,2 -helices used in inhibitors 2−4 support each other and retain their bent shape, which provides a conformational matching to the rbd of sars-cov-2 and a full cover of the rbd surface. precise conformational matching between the designed peptides and the virus provides room for improving the binding affinity, which should be considered in future inhibitor design protocols. suitable inhibitors should have a selective binding with lower rmsd for critical amino acids and relatively high binding energies. the binding affinity could be further enhanced by a multivalent binding of multiple peptides attached to surfaces of nanoparticles, dendrimers, and clusters. in analogy to nanoparticle-based inhibitors, 14 we could attach to the α 1 helix a sulphonated ligand mimicking a heparane sulfate, which can attach to positively charged residues at the bottom of rbd. these inhibitors could be used as inhaled therapeutics, preventing the virus activation in lungs. the inhibitors and rbd of the virus were simulated by namd 19 and the charmm36 protein force field. 20 the particle mesh ewald (pme) method was used for the evaluation of long-range coulombic interactions. 21 the time step was set to 2 fs. the simulations were performed in the npt ensemble (p = 1 bar and t = 310 k), using the langevin dynamics with a damping constant of 1 ps −1 . after 2000 steps of minimization, ions and water molecules were equilibrated for 2 ns around proteins, which were restrained using harmonic forces with a spring constant of 2 kcal/(mol å 2 ). the last frames of restrained equilibration were used to start simulations of free inhibitors and partially constrained pd of ace2 (two residues on the bottom). the simulations last for 120−300 ns due to different atom numbers in different systems and different computer power used. calculation of rmsd. the time-dependent rmsd for the critical amino acids and the whole inhibitors ( figure s1 ) were calculated from where n α is the number of atoms whose positions are being compared, r⃗ α (t j ) is the position of atom α at time t j , and r⃗ α (t 0 ) is the initial coordinate. the selection of coordinates contains all of the atoms in the inhibitors or critical amino acids, excluding hydrogens. the time-dependent rmsd was averaged over the last 50 ns of simulation time, which corresponds to the last 50 frames of each trajectory, as shown in figure 2f . the standard deviations are shown by the error bars. calculation of binding energy. the interacting residues from inhibitors and rbd of sars-cov-2 were first selected with a 3 å cutoff distance. the electrostatic and vdw energy contributions between the interacting residues are calculated by the namd energy plugin. the electrostatic contribution is given by where |r⃗ i − r⃗ j | is the distance between the two charges, q i and q j ; ε is the dielectric constant of the solvent which is set to 1. to increase the efficiency of the simulations, pairwise interaction calculations are not performed beyond a cutoff distance. long-range electrostatic interactions are calculated by the pme method. 21 the lennard-jones (lj) 6−12 potential energies are used to describe the vdw interactions and close distance atomic repulsions: where ε ij is the maximum stabilization energy for the ith and the jth atoms, σ ij is the distance between ith and jth atoms at the minimum of the potential, and r ij is the actual distance between the two atoms. the lj parameters between different atom types are calculated using a mixing rule, such as σ ij = (σ ii + σ jj )/2 and ε εε = ij ii jj (lorentz− berthelot rules). the time evolution of the interaction energy is shown in figure s2 , and the time-averaged interaction energy over the last 50 ns (50 frames) is shown in figure 2g , with standard deviation shown by the error bar. the supporting information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.0c02857. sequences 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declare no competing financial interest. we would like to thank lela vuković(utep) for useful discussions. y.h. acknowledges the support from the dean's scholar fellowship (uic). key: cord-291790-z5rwznmv authors: li, qianqian; wu, jiajing; nie, jianhui; zhang, li; hao, huan; liu, shuo; zhao, chenyan; zhang, qi; liu, huan; nie, lingling; qin, haiyang; wang, meng; lu, qiong; li, xiaoyu; sun, qiyu; liu, junkai; zhang, linqi; li, xuguang; huang, weijin; wang, youchun title: the impact of mutations in sars-cov-2 spike on viral infectivity and antigenicity date: 2020-07-17 journal: cell doi: 10.1016/j.cell.2020.07.012 sha: doc_id: 291790 cord_uid: z5rwznmv summary the spike protein of sars-cov-2 has been undergoing mutations and is highly glycosylated. it is critically important to investigate the biological significance of these mutations. here we investigated 80 variants and 26 glycosylation site modifications for the infectivity and reactivity to a panel of neutralizing antibodies and sera from convalescent patients. d614g, along with several variants containing both d614g and another amino acid change, were significantly more infectious. most variants with amino acid change at receptor binding domain were less infectious but variants including a475v, l452r, v483a and f490l became resistant to some neutralizing antibodies. moreover, the majority of glycosylation deletions were less infectious whilst deletion of both n331 and n343 glycosylation drastically reduced infectivity, revealing the importance of glycosylation for viral infectivity. interestingly, n234q was markedly resistant to neutralizing antibodies, whereas n165q became more sensitive. these findings could be of value in the development of vaccine and therapeutic antibodies. the spike protein of sars-cov-2 has been undergoing mutations and is highly glycosylated. it is 25 critically important to investigate the biological significance of these mutations. here we 26 investigated 80 variants and 26 glycosylation site modifications for the infectivity and reactivity to 27 a panel of neutralizing antibodies and sera from convalescent patients. d614g, along with several 28 variants containing both d614g and another amino acid change, were significantly more 29 infectious. most variants with amino acid change at receptor binding domain were less infectious 30 but variants including a475v, l452r, v483a and f490l became resistant to some neutralizing 31 antibodies. moreover, the majority of glycosylation deletions were less infectious whilst deletion 32 of both n331 and n343 glycosylation drastically reduced infectivity, revealing the importance of 33 glycosylation for viral infectivity. interestingly, n234q was markedly resistant to neutralizing 34 antibodies, whereas n165q became more sensitive. these findings could be of value in the 35 development of vaccine and therapeutic antibodies. 36 covid-19 pandemic is a tremendous threat globally. as of july 3, 2020, 216 countries have 38 reported covid-19 cases, with more than 10 million confirmed cases and approximately 518,000 39 deaths (https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/). 40 the causative agent of covid-19, sars-cov-2 causes a lower respiratory tract infection that 41 can progress to severe acute respiratory syndrome and even multiple organ failure (lv et al., 42 2020a; yang et al., 2020) . 43 sars-cov-2 is a single-stranded positive-strand rna virus whose genome encodes four 44 structural proteins: spike (s), small protein (e), matrix (m) and nucleocapsid (n) (chan et al., 45 2020) . the s protein is a type i fusion protein that forms trimers on the surface of the virion. it is 46 composed of two subunits, with s1 responsible for receptor binding and s2 for membrane fusion 47 7 single mutants were also constructed to compare with the double mutants with d614g. group c is 112 comprised of 26 mutants at the putative glycosylation sites (22 sites). this group includes both 113 variants (n74k, n149h and t719a) and investigational mutants that we made for the analyses of 114 the effects of glycosylation. specifically, all 22 sites (n to q) were made in the lab to generate 22 115 individual mutants; we also made a combination by deleting the two glycosylation sites in rbd. 116 in total, we have generated 106 pseudotyped viruses, i.e., 80 variants and 26 glycosylation 117 mutants ( figure 1 ). these viruses were prepared as described previously (nie et al., 2020 ) (see 118 star methods). 119 to determine the infectivity of these variants and mutants, we first infected 26 cell lines with 121 pseudotyped viruses with either sars-cov-2 s protein or vsvg protein (see star methods). 122 as expected, the two types of pseudotyped viruses are different in the infection efficiency in the 123 26 cell lines ( figure 2) . while almost all cell lines were generally susceptible to infection by vsv 124 g pseudotyped virus, sars-cov-2 pseudotyped virus could efficiently infect certain cell lines 125 including three human cell lines (293t-hace2, 293t and huh-7) and three non-human primate 126 cell lines (vero, veroe6 and llc-mk2). as such, we selected these four out of the six cell lines 127 in subsequent experiments, including 293t-hace2, huh-7, vero and llc-mk2. 128 we first tested the infectivity of 106 pseudotyped viruses (80 natural variants and 26 129 glycosylation mutants) in 293t-hace2 cells, where a difference by 4 -fold in rlu compared 130 with the reference wuhan-1 strain (genbank: mn908947) was deemed as being significant 131 ( figure s1 ). of all 106 pseudotyped viruses, 22 were determined as low-infectivity (16 natural 132 mutants and 6 glycosylation mutants), with rlu reading decreased by 4 to 100 folds ( figure 3a) . 133 among them, 13 were located in the rbd region. variant v341i and investigational glycosylation 134 mutant (n331q +n343q) were deemed as no-infectivity as demonstrated by over 100-fold 135 8 decrease in rlu values compared with the reference strain. both of them were located in rbd. it 136 is worth noting that double glycosylation deletions at n331 and n343 resulted in a drastic 137 reduction in viral infectivity (1200-fold), whereas single deletion at each site caused modest 138 reduction in viral infectivity, with the infectivity of n331q reduced by only 3-fold and n343q by 139 20-fold. moreover, the non-natural double glycosylation mutations in rbd (n331q and n343q) 140 resulted in significantly reduced infectivity, suggesting that the two glycosylation sites in the rbd 141 region may participate in the binding of the receptor or maintain the conformation of the rbd 142 region. 143 the remaining 63 variants were tested further with other three cell lines for infectivity 144 suggesting that the enhanced infectivity was more likely ascribed to d614g itself. 149 antibodies 151 having identified the variants with altered infectivity, we next set out to investigate the 152 antigenicity of the infectious mutants using 13 neutralizing monoclonal antibodies (mabs) (see 153 star methods). it was noted that some changes in rbd region demonstrated altered sensitivity 154 to neutralizing mabs (figure 4 and figure s2 ). specifically, a475v reduced the sensitivity to 155 mabs 157, 247, cb6, p2c-1f11, b38 and ca1, while f490l reduced the sensitivity to mabs 156 x593, 261-262, h4 and p2b-2f6. moreover, v483a became resistant to mabs x593 and 157 p2b-2f6, and l452r to mabs x593 and p2b-2f6. finally, y508h reduced the sensitivity to 158 mabs h014, n439k to mab h00s022, a831v to mab b38, d614g+i472v to mab x593 and 159 d614g+a435s to mab h014 by more than 4 times. in addition, some changes in the rbd region, 9 including v367f, q409e, q414e, i468f, i468t, y508h and a522v, were observed to be more 161 susceptible to neutralization mediated by mabs. 162 we next determine how infectious glycosylation mutants reacted to the same panel of mabs. 163 mutant n165q actually became more sensitive to mab p2b-2f6, whereas n234q reduced the 164 neutralization sensitivity to different set of mabs including 157, 247, cb6, p2c-1f11, h00s022, 165 b38, ab35 and h014. these results confirmed that these two glycosylation sites are important for 166 receptor binding. 167 these mabs have proven to be valuable in our analyses of the amino acid changes. as shown 168 in figure 4 , five mabs, i.e., 157, 247, cb6, p2c-1f11 and b38, were unable to effectively 169 neutralize both a475v and n234q. neither x593 nor p2b-2f6 was effective in neutralizing 170 l452r, v483a and f490l whilst p2b-2f6 was more effective in neutralizing n165q. in addition, 171 mab h014 was incapable of neutralizing n234q, y508h and d614g+a435s while mabs h4 172 and 261-262 were found not to neutralize f490l. furthermore, finally, h00s022 was unable to 173 neutralizing n439k and n234q. 174 finally, we determined the sensitivity of the strains with amino acid changes to ten 176 covid-19 convalescent sera (see star methods). none of the variants and mutants 177 demonstrated significantly altered sensitivity to all 10 convalescent sera, i.e., the ec50 values 178 were not altered by more than 4-fold, irrespective of an increase or decrease, when compared with 179 the reference strain ( figure 5a and figure s3 ). however, the neutralization sensitivity of both 180 f490l and h519p to three of ten patient sera were found to have decreased by more than 4 times, 181 while six variants and mutants (n149h, n149q, n165q, n354d, n709q and n1173q) became 182 over 4-fold sensitive to one or two of the ten tested sera. notably, five out of the six were glycan 183 deletion mutants. 184 as shown in figure 5b , when the data of individual convalescent sera were pooled together 185 to analyze the sensitivity of all variants, no marked difference was observed (>4 fold). however, 186 modest differences between some variants and reference strain (within 4-fold) were observed in 187 their reactivity to grouped convalescent sera. these differences were statistically significant 188 (p<0.05). it is worth mentioning that some variants including f338l, v367f, i468f, i468t and 189 v615l ( figure 5b ) were even more sensitive to the convalescent sera compared with reference 190 strain, whereas more variants were found to be resistant to the convalescent sera. these variants 191 include single amino acid change such as y145del, q414e, n439k, g446v, k458n, i472v, 192 a475v, t478i, v483i, f490l and a831v, as well as the double amino acid changes including 193 d614g + q321l, d614g +i472v, d614g +a831v, d614g +a879s and d614g +m1237i. 194 similar to natural variants, although the magnitude of some glycosylation deletions in 195 sensitivity to the sera is less than 4-fold, the differences between mutants and the reference strain 196 (wuhan-1) were found to be still several-folds and statistically significant, i.e., glycosylation 197 mutants n331q and n709q significantly increased the sensitivity to convalescent sera ( and ambiguous sequences, we narrowed down to 80 variants. moreover, as glycosylation of viral 206 protein is well documented to affect viral replication and immune response and sars-cov-2 s 207 protein is heavily glycosylated, we also made 26 substitutional mutations at all 22 putative 208 glycosylation sites. in total, we made 106 pseudotyped viruses, allowing us to characterize them 209 using the established method (nie et al., 2020) (see star methods). 210 table 1 summarize the characteristics of variants and investigational mutants. of all variants, 211 d614g is of particular note. this variant has been shown to rapidly accumulating since its 212 emergence and linked to more clinical presentations (korber et al., 2020) . at the beginning of this 213 study (may 6, 2020), it accounted for 62.8% of all circulating strains, but by july 3, it had reached 214 75.7%. this dominant strain could effectively infect the four cell lines tested, being 10-fold more 215 infectious than the original wuhan-1 strain ( figure 3) . 216 another important finding is that natural variants capable of affecting the reactivity to 217 neutralizing mabs were almost all located in the rbd region (except a831v and 218 d614g+a831v), as all antibodies used in this study were targeting the rbd ( with decreased sensitivity to neutralization by p2b-2f6 mab; as both l452r and f490l remain 225 sensitive to p2c-1f11, suggesting this mab is not derived from the same clone for p2b-2f6. 226 moreover, both mutants displayed decreased sensitivity to another neutralizing mab x593 by 227 10-fold compared with the reference strain (figure4). 228 while we identified multiple variants with decreased sensitivity to neutralizing mabs, we 229 need to look at how frequent these variants are in the field. v483a in rbd is one of the two 230 variants with a mutation frequency of over 0.1%. it showed decreased reactivity to the two mabs 231 (p2b-2f6 and x593) ( figure 6a and 6b) (ju et al., 2020) . another rbd variant a475v sits in the 232 binding epitope of rbd. it is significantly resistant to several neutralizing mabs including 233 p2c-1f11, ca1, 247 and cb6. it is noteworthy that cb6 mab targets the receptor binding 12 epitope ( figure 6c and 6d) (shi et al., 2020) . specifically, y508 was buried in the epitope 235 targeted by mab h014 (figure 6e and 6f) (lv et al., 2020b) . indeed, the y508h was found to be 236 resistant to this mab. 237 it is worth mentioning that d614g+i472v has shown increased infectivity and more 238 resistance to neutralizing antibodies (table 1 ), but only one sequence (originated from canada) 239 was reported in gisaid. moreover, some variants, including n439k, l452r, a475v, v483a, 240 f490l and y508h, do have decreased sensitivity to neutralizing mabs. however, only v483a 241 exceeded 0.1% in frequency at the beginning of the study, all of which were found in us, with 28 242 sequences reported as of may 6, 2020, and 36 up to july 3, 2020. variants containing n439k 243 showed a significant increase in circulation, i.e., with 5 case reported as of may 6, 2020 (all in uk) 244 to 47 by july 3, 2020 (45 in uk, 2 in romania). in addition, only one sequence from france 245 containing y508h was deposited in girsaid as of may 6, while four sequences reported as of 246 july 3, 2020, of which two originated from netherlands, one from sweden, and one from france. 247 only one or two isolates were reported for other variants, which have not been observed to have 248 increased during the time frame we have been monitoring. nevertheless, as rna viruses mutate 249 all the time and some variants may only appears during certain period of time, while others could 250 emerge in an unpredictable fashion, continued analyses of the circulating strains in terms of the 251 mutation frequency and temporal pattern are warranted. 252 our results suggest that the 13 mabs used in this study could be divided into seven groups as 253 they appear to be different in the inhibitory effects on the variants. as such, it would be interesting 254 to formulate a therapeutic regimen comprised of at least two mabs. for example, a combination 255 of p2c-1f11 and x593 should be effective to inhibit all variants in this study. it would be of 256 interest to test more neutralizing antibodies which could be targeting epitopes outside rbd. 257 with regard to the glycosylation mutants analyzed in this study, n165q increased the 258 sensitivity to mab p2b-2f6 whilst n234q displayed resistance to neutralizing mabs such ca1, 259 cb6, 157 and others. although neither of them is found in circulation, the reactivity of these two 260 13 mutants to neutralizing mab is still worth noting. as n165 and n234 are located near the rbd 261 region (watanabe et al., 2020) , these mutants may affect some epitopes targeted by neutralizing 262 mabs. specifically, n165 glycosylation site is involved in the binding of mab to the rbd region 263 of s protein (cao et al., 2020) . it is likely that the sugar chain can mask the epitope targeted by the 264 antibody. this type of glycan shield has been observed in other virus such as hiv-1. specifically the use of sera from 10 convalescent patients in neutralizing assay largely confirmed the 277 results obtained with the well characterized neutralizing mabs. it is understood that the magnitude 278 of altered reactivity is slightly smaller with human sera than that with mabs, given that polyclonal 279 antibodies from convalescent patents are directed against multi-epitopes on the full-length s 280 protein; as a result, these polyclonal antibodies could complement one another. however, the 281 differences in their reactivity to the human antibodies were found to be by several folds in most 282 cases and all determined as statistically significant. notably, some rbd variants such as a475v 283 and f490l have been confirmed to have decreased sensitivity to both human sera and multiple 284 neutralizing mabs. a475v reduced the sensitivity to 6 mabs out of the 13 mab used in this study, 285 while f490l reduced the sensitivity to neutralization by 3 mabs. it is possible that antibodies in 286 14 convalescent sera are able to neutralize these critical epitopes targeted by these mabs that are 287 known to disrupt the binding of the s protein to hace2 receptor (ju et serial dilutions of mab preparations were pre-incubated with the pseudotyped viruses at 37â°c for 355 one hour before they were added to huh-7 cells. luciferase activity was measured 24 hours later 356 to calculate ec50 of each antibody. the ratio of ec50 between the variant or mutant strains and 357 the reference strain (wuhan-1) was calculated and analyzed to generate heatmap using hem i 17 (deng et al., 2014) . the data were the results from 3-5 replicates. the red and blue boxes indicate 359 the increase or decrease of the neutralization activity as shown in the scale bar. see also figure s2 . serial dilutions of mab preparations were pre-incubated with the virus at 37â°c for one hour 393 before they were added to huh-7 cells. luciferase activity was measured 24 hours later to 394 calculate ec50 of each antibody. the y-axis represents the ratio of ec50 between the 395 variant/mutant strain and the reference strain (wuhan-1). the data were the results from 3-5 396 replicates. the horizontal dashed lines indicate the threshold of 4-fold difference. the significant 397 changes were marked with colored symbols, blue for decreased, red for increased. related to 398 further information and requests for resources and reagents should be directed to and will be 409 fulfilled by the lead contact, dr. youchun wang (wangyc@nifdc.org.cn). 410 all the unique reagents generated in this study are available from the lead contact with a 412 completed materials transfer agreement. 413 this study did not generate any unique datasets or code. primers. following site-directed mutagenesis pcr, the template chain was digested using dpni 452 restriction endonuclease (neb, usa). afterwards, the pcr product was directly used to 453 transform e. coli dh5î± competent cells; single clones were selected and then sequenced. the 454 primers designed for the specific mutation sites are listed in table s2 , and the frequency of 455 different variants in the epidemic population is listed in table s1 . 456 highlights over 100 mutations were selected for analyses on their infectivity and antigenicity the dominant d614g itself and combined with other mutations are more infectious ablation of both n331 and n343 glycosylation at rbd drastically reduced infectivity ten mutations such as n234q, l452r, a475v, v483a was markedly resistant to some mabs eighty natural variants and twenty-six glycosylation spike mutants of sars-cov-2 were analyzed in terms of infectivity and antigenicity using high throughput pseudovirus assay in conjunction with neutralizing antibodies. reference l5f l8v l8w h49y y145del f338l p384l n354d n354k s359n v367f k378r p384l r408i q409e q414e a435s n439k g446v l452r k458r k458n i468f i468t i472v a475v g476s t478i v483a v483i f490l y508h h519p h519q a520s a522s a522v v615l a831v d839e d936y s943t s943r g1124v y145del+r408i d614g+q239k d614g+q321l d614g+v341i d614g+a435s d614g+k458r d614g+i472v d614g+h519p d614g+a831v d614g+a845s d614g+a879s d614g+d936y d614g+s939f d614g+s943t d614g+m1229i d614g d614g+m1237i d614g+p1263l d614g+l5f n17q n61q n74q n149q n165q n234q n282q n331q n603q n616q n657q n709q n1098q n1134q n1158q n1173q n1194q n74k n149h a b reference cs1 cs2 cs10 cs3 cs86 cs7 cs4 cs87 cs6 cs8 l5f l8v l8w h49y y145del f338l a348t n354d n354k s359n v367f k378r p384l r408i q409e q414e a435s n439k g446v l452r k458r k458n i468f i468t i472v a475v g476s t478i v483a v483i f490l y508h h519p h519q a520s a522s a522v d614g v615l a831v d839e d936y s943t s943r g1124v y145del+r408i d614g+l5f d614g+q239k d614g+q321l d614g+v341i d614g+a435s d614g+k458r d614g+i472v d614g+h519p d614g+a831v d614g+a845s d614g+a879s d614g+d936y d614g+s939f d614g+s943t d614g+m1229i d614g+m1237i d614g+p1263l n17q n61q n74q n149q reference l5f l8v l8w h49y y145del f338l a348t n354d n354k s359n v367f k378r p384l r408i q409e q414e a435s n439k g446v l452r k458r k458n i468f i468t i472v a475v g476s t478i v483a v483i f490l y508h h519p h519q a520s a522s a522v d614g v615l a831v d839e d936y s943t s943r g1124v y145del+r408i d614g+l5f d614g+q239k d614g+q321l d614g+v341i d614g+a435s d614g+k458r d614g+i472v d614g+h519p d614g+a831v d614g+a845s d614g+a879s d614g+d936y d614g+s939f d614g+s943t d614g+m1229i d614g+m1237i d614g+p1263l n17q n61q n74q n149q n165q n234q n282q n331q n603q n616q n657q n709q n1098q n1134q n1158q n1173q n1194q n74k sars-cov-2 viral spike g614 mutation exhibits 526 higher case fatality rate potent neutralizing antibodies against sars-cov-2 identified by high-throughput 529 single-cell sequencing of convalescent patients' b cells genomic 532 characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with 533 atypical pneumonia after visiting wuhan mutated covid-19, may foretells mankind in a great risk in the future hemi: a toolkit for illustrating 537 heatmaps ebola virus glycoprotein with increased 540 infectivity dominated the 2013-2016 epidemic the hiv glycan shield as a target for broadly neutralizing antibodies the spike protein of 544 sars-cov--a target for vaccine and therapeutic development why are rna virus mutation rates so damn high? the highly conserved glycan at asparagine 260 of hiv-1 547 gp120 is indispensable for viral entry identification of 549 immunodominant sites on the spike protein of severe acute respiratory syndrome (sars) 550 coronavirus: implication for developing sars diagnostics and vaccines airborne transmission of influenza 554 a/h5n1 virus between ferrets n-linked glycans 556 and k147 residue on hemagglutinin synergize to elicit broadly reactive h1n1 influenza virus coronavirus spike protein and 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pseudovirus neutralization assay for antigenic 592 drift of influenza a(h7n9) virus hemagglutinin influenza a(h7n9) virus evolution: which genetic 594 mutations are antigenically important? a virus that has 596 gone viral: amino acid mutation in s protein of indian isolate of coronavirus covid-19 might 597 impact receptor binding, and thus, infectivity emerging genetic diversity among clinical isolates of 601 sars-cov-2: lessons for today a human neutralizing antibody targets the receptor binding site of sars-cov-2 a single mutation in 606 chikungunya virus affects vector specificity and epidemic potential human adaptation of ebola 609 virus during the west african outbreak two n-linked glycosylation sites in the v2 and c2 612 regions of human immunodeficiency virus type 1 crf01_ae envelope glycoprotein gp120 613 regulate viral neutralization susceptibility to the human monoclonal antibody specific for the cd4 614 binding domain emergence of genomic diversity and recurrent 617 mutations in sars-cov-2 emerging wuhan (covid-19) coronavirus: glycan shield 619 and structure prediction of spike glycoprotein and its interaction with human cd26 function, and antigenicity of the sars-cov-2 spike glycoprotein structural and functional basis of sars-cov-2 entry by using human ace2 a systematic study of the n-glycosylation sites of hiv-1 envelope protein on infectivity and 629 antibody-mediated neutralization n463 glycosylation site on v5 loop of a mutant gp120 regulates the sensitivity of 632 hiv-1 to neutralizing monoclonal antibodies vrc01/03 site-specific glycan 635 analysis of the sars-cov-2 spike cryo-em structure of the 2019-ncov spike in the prefusion conformation a noncompeting pair of human neutralizing antibodies block covid-19 virus binding to 641 its receptor ace2 clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in 644 china: a single-centered, retrospective, observational study characterization of a filovirus (mengla virus) from rousettus bats in 648 china role of stem 650 glycans attached to haemagglutinin in the biological characteristics of h5n1 avian influenza virus pseudotyped viruses incorporated with spike protein from either sars-cov-2, variants or 458 mutants were constructed using a procedure described by us recently (nie et al., 2020) . on day 459 before transfection, 293t cells were prepared and adjusted to the concentration of 5 -7 ã� 10 5 460 cell/ml, 15 ml of which were transferred into a t75 cell culture flask and incubated overnight at 461 37 0 c in an incubator conditioned with 5% co 2 . the cells generally reach 70-90% confluence after 462 overnight incubation. thirty microgram of dna plasmid expressing the spike protein was 463 transfected according to the user's instruction manual. the transfected cells were subsequently 464 infected with g*â��g-vsv (vsv g pseudotyped virus) at concentration of 7.0 ã� 10 4 tcid50/ml. 465these cells were incubated at 37â°c for 6-8 hours in the presence of in 5% co 2 . afterwards, cell 466 supernatant was discarded, followed by rinsing the cells gently with pbs +1% fbs. next, 15ml 467 fresh complete dmem was added to the flask and cultured for 24 h. twenty-four hours post 468 infection, sars-cov-2 pseudotyped viruses containing culture supernatants were harvested, 469 filtered (0.45-âµm pore size, millipore, cat#slhp033rb) and stored at â��70â°c in 2-ml aliquots 470 until use. 471the 50% tissue culture infectious dose (tcid50) of sars-cov-2 pseudovirus was 472 determined using a single-use aliquot from the pseudovirus bank to avoid inconsistencies resulted 473 from repeated freezing-thawing cycles. 474for titration of the pseudotyped virus, a 2-fold initial dilution with six replicates was made in 475 96-well culture plates followed by serial 3-fold dilutions. the last column was employed as the 476 cells control without pseudotyped virus. subsequently, the 96-well plates were seeded with huh-7 477 cells adjusted to 2ã�10 5 cells/ml. after 24 h incubation at 37â°c in a humidified atmosphere with 5% 478 co 2 , the supernatant was aspirated and discarded gently to leave 100 âµl in each well; next, 100 âµl 479 of luciferase substrate (perkinelmer, cat#6066769) was added to each well. after 2-min 480 incubation at room temperature in the dark, 150 âµl of lysate was transferred to white 96-well 481 plates for the detection of luminescence using a luminometer (perkinelmer, ensight). positive was 482 23 determined to be ten-fold higher than the negative (cells only) in terms of relative luminescence 483 unit (rlu) values. the 50% tissue culture infectious dose (tcid50) was calculated using the 484 reed-muench method (nie et al., 2020) . 485 before quantification, all the pseudotyped viruses were purified through a 25% sucrose cushion by 487 ultra-centrifugation at 100,000ã� g for 3 h (nie et al., 2020) resources table. 495 using the quantitative rt-pcr, we normalized the pseudotyped virus particles to the same 497 amount. after normalization, 100 âµl of the pseudotyped virus with 10-fold dilution was added to 498 wells in 96-well cell culture plate. after the cells were trypsin-digested, 2ã�10 4 /100 âµl cells were 499 added to each well in the 96-well plates. the plates were then incubated at 37â°c in a humidified 500 atmosphere with 5% co 2 . after incubation for 24 hours, chemiluminescence detection was 501 performed as described in the titration of pseudotyped viruses. each group contained 3-5 502replicates. 503 the virus neutralization assay was conducted as described previously (nie et al., 2020) . briefly, 505 100 âµl serial dilutions of human sera or monoclonal antibody preparations were added into 506 96-well plates. after that, 50 âµl pseudoviruses with concentration of 1300 tcid50/ml were added 507 into the plates, followed by incubation at 37â°c for 1 hour. afterwards, huh-7 cells were added 508 24 into the plates (2ã�10 4 cells/100 âµl cells per well), followed by incubation at 37â°c in a humidified 509 atmosphere with 5% co 2 . chemiluminescence detection was performed after 24 hours incubation. 510the reed-muench method was used to calculate the virus neutralization titer. the results are 511 based on 3-5 replicates unless specified. in order to validate the test operation process, the 512 coefficient of variance (cv) control of replicates is set within 30% of six wells, so is the cv for 513 the duplicate sample wells. 514 graphpad prism 8 was used for plotting and statistical analysis; the values were expressed as 517 mean â±sem. one-way anova and holm-sidak's multiple comparisons test was used to analyze 518 the differences between groups. a p-value of less than 0.05 was considered to be significant. * 519 p<0.05, ** p<0.01, *** p<0.005, **** p<0.001, ns represents no significant difference. 520 key: cord-300847-ycuiso0g authors: li, wei; drelich, aleksandra; martinez, david r.; gralinski, lisa; chen, chuan; sun, zehua; schäfer, alexandra; leist, sarah r.; liu, xianglei; zhelev, doncho; zhang, liyong; peterson, eric c.; conard, alex; mellors, john w.; tseng, chien-te; baric, ralph s.; dimitrov, dimiter s. title: rapid selection of a human monoclonal antibody that potently neutralizes sars-cov-2 in two animal models date: 2020-06-02 journal: biorxiv doi: 10.1101/2020.05.13.093088 sha: doc_id: 300847 cord_uid: ycuiso0g effective therapies are urgently needed for the sars-cov-2/covid19 pandemic. we identified panels of fully human monoclonal antibodies (mabs) from eight large phage-displayed fab, scfv and vh libraries by panning against the receptor binding domain (rbd) of the sars-cov-2 spike (s) glycoprotein. one high affinity mab, igg1 ab1, specifically neutralized replication competent sars-cov-2 with exceptional potency as measured by two different assays. there was no enhancement of pseudovirus infection in cells expressing fcγ receptors at any concentration. it competed with human angiotensin-converting enzyme 2 (hace2) for binding to rbd suggesting a competitive mechanism of virus neutralization. igg1 ab1 potently neutralized mouse ace2 adapted sars-cov-2 in wild type balb/c mice and native virus in hace2 expressing transgenic mice. the ab1 sequence has relatively low number of somatic mutations indicating that ab1-like antibodies could be quickly elicited during natural sars-cov-2 infection or by rbd-based vaccines. igg1 ab1 does not have developability liabilities, and thus has potential for therapy and prophylaxis of sars-cov-2 infections. the rapid identification (within 6 days) of potent mabs shows the value of large antibody libraries for response to public health threats from emerging microbes. the severe acute respiratory distress coronavirus 2 (sars-cov-2) (1) has spread worldwide thus requiring safe and effective prevention and therapy. inactivated serum from convalescent patients inhibited sars-cov-2 replication and decreased symptom severity of newly infected patients (2, 3) suggesting that monoclonal antibodies (mabs) could be even more effective. human mabs are typically highly target-specific and relatively non-toxic. by using phage display we have previously identified a number of potent fully human mabs (m396, m336, m102.4) against emerging viruses including severe acute respiratory syndrome coronavirus (sars-cov) (4) , middle east respiratory syndrome coronavirus (mers-cov) (5) and henipaviruses (6, 7) , respectively, which are also highly effective in animal models of infection (8) (9) (10) (11) ; one of them was administered on a compassionate basis to humans exposed to henipaviruses and successfully evaluated in a clinical trial (12) . size and diversity of phage-displayed libraries are critical for rapid selection of high affinity antibodies without the need for additional affinity maturation. our exceptionally potent antibody against the mers-cov, m336, was directly selected from very large (size ~10 11 clones) library from 50 individuals (5) . however, another potent antibody, m102.4, against henipavirusses was additionally affinity matured from its predecessor selected from smaller library (size ~10 10 clones) from 10 individuals (7, 13) . thus, to generate high affinity and safe mabs we used eight very large (size ~ 10 11 clones each) naive human antibody libraries in fab, scfv or vh format using pbmcs from 490 individuals total obtained before the sars-cov-2 outbreak. four of the libraries were based on single human vh domains where cdrs (except cdr1 which was mutagenized or grafted) from our other libraries were grafted as previously described (14) . another important factor to consider when selecting effective mabs is the appropriate antigen. similar to sars-cov, sars-cov-2 uses the spike glycoprotein (s) to enter into host cells. the s receptor binding domain (rbd) binds to its receptor, the human angiotensinconverting enzyme 2 (hace2), thus initiating series of events leading to virus entry into cells (15, 16) . we have previously characterized the function of the sars-cov s glycoprotein and identified its rbd which is stable in isolation (17) . the rbd was then used as an antigen to pan phage displayed antibody libraries; we identified potent antibodies (5, 8) more rapidly and the antibodies were more potent than when we used whole s protein or s2 (unpublished). in addition, the sars-cov rbd based immunogens are highly immunogenic and elicit neutralizing antibodies which protect against sars-cov infections (18). thus, to identify sars-cov-2 mabs, we generated two variants of the sars-cov-2 rbd (aa 330-532) (fig. s1 ) and used them as antigens for panning of our eight libraries. panels of high-affinity binders to rbd in fab, scfv and vh domain formats were identified. there was no preferential use of any antibody vh gene (an example for a panel of binders selected from the scfv library is shown in fig. s2a ) and the number of somatic mutations was relatively low (fig. s2b , for the same panel of binders as in fig. s2a ). for nine of the highest affinity mabs a provisional patent application was filed on march 12, 2020 by the university of pittsburgh. those high affinity mabs can be divided into two groups in terms of their competition with hace2. two representatives of each group are fab ab1 and vh ab5. to further increase their binding through avidity effects and extend their half-live in vivo they were converted to igg1 and vh-fc fusion formats, respectively. ab1 was characterized in more details because of its potential for prophylaxis and therapy of sars-cov-2 infection. the fab and igg1 ab1 bound strongly to the rbd (fig. 1a ) and the whole sars-cov-2 s1 protein (fig. 1b) as measured by elisa. the fab ab1 equilibrium dissociation constant, kd, as measured by the biolayer interferometry technology (blitz), was 1.5 nm (fig. 1c) . the igg1 ab1 bound with high (kd =160 pm) avidity to recombinant rbd (fig. 1d) . igg1 ab1 bound cell surface associated native s glycoprotein suggesting that the conformation of its epitope on the rbd in isolation is close to that in the native s protein (fig. 2, s3 ). the binding of igg1 ab1 was of higher avidity than that of hace2-fc (fig. 2b) . binding of ab1 was specific for the sars-cov-2 rbd; it did not bind to the sars-cov s1 (fig. 3a ) nor to cells that do not express sars-cov-2 s glycoprotein ( fig. 2a ). ab1 competed with hace2 for binding to the rbd ( fig. 3b and c) indicating possible neutralization of the virus by preventing binding to its receptor. it did not compete with the cr3022 (fig. 3d and e) , which also binds to sars-cov (19) and with ab5 ( fig. 3f ). igg1 ab1 potently neutralized sars-cov-2 pseudovirus with an ic50 of 10 ng/ml ( fig 4a) . it did not enhance pseudovirus infection of fcγria overexpressing 293t-hace2 cells at any concentration ( fig 4b) . it also did not mediate pseudovirus infection of fcγrii expressing k562 cells ( fig s4b) . importantly, igg1 ab1 exhibited potent neutralizing activity against authentic sars-cov-2 in two independent assays -a microneutralization-based assay (100% neutralization at < 400 ng/ml) (fig. 4c ) and a luciferase reporter gene assay (ic50 = 200 ng/ml) ( fig. 4d ). in agreement with the specificity of binding to the sars-cov-2 s1 and not to the sars-cov s1 the igg1 ab1 did not neutralize live sars-cov (fig. 4c ). the igg1 m336 (5) control which is a potent neutralizer of mers-cov, did not exhibit any neutralizing activity against sars-cov-2 (fig. 4c ). the vh ab5 and vh-fc ab5 bound the rbd with high affinity and avidity (fig. s5a .b) but did not compete with hace2 ( fig. s5c ) or neutralize sars-cov-2 ( fig. 4d) , indicating that not all antibodies targeting epitopes on the rbd affect virus replication. to evaluate the efficacy of igg1 ab1 in vivo we used two animal models. the first one is based on the recently developed mouse ace2 adapted sars-cov-2 which has two mutations q498t/p499y at the ace2 binding interface on rbd (20). igg1 ab1 protected mice from high titer intranasal sars-cov-2 challenge (10 5 pfu) of balb/c mice in a dose dependent manner ( fig 5a) . there was complete neutralization of infectious virus at the highest dose of 0.9 mg, and statistically significant reduction by 100-fold at 0.2 mg; there was a trend for reduction at 0.05 mg dose but did not reach statistical significance. the igg1 m336 which potently neutralizes the mers-cov in vivo was used as an isotype control because it did not have any activity in vitro. these results also suggest that the rbd double mutations q498t/p499y do not affect igg1 ab1 binding. the second model we used is the transgenic mice expressing human ace2 (hace2) (21). mice were administered 300 ug of igg1 ab1 prior to wild type sars-cov-2 challenge followed by detection of infectious virus in lung tissue 2 days later. replication competent virus was not detected in four of the five mice which were treated with igg1 ab1 ( fig 5b) . all six control mice and one of the treated mice had more than 10 3 pfu per lung. these results show clear evidence of a potent preventive effect of igg1 ab1 in vivo. the reason for absence of virus neutralization in one of the mice is unclear but may be due to individual variation in antibody transfer from the peritoneal cavity where it was administered to the upper and lower respiratory tract. our previous experiments with transgenic mice expressing human dpp4 and treated with two different doses of m336 (0.1 and 1 mg per mouse) showed similar lack of protection of one (out of four) mice at the lower dose but at the higher dose all four mice were protected (9) similarly to the results obtained with the mouse adapted sars-cov-2. the in vivo protection also indicates that igg1 ab1 can achieve neutralizing concentrations in the respiratory tract. this is the first report of in vivo activity of a human monoclonal antibody against sars-cov-2 by using two different mouse models. the results also show some similarity between the two models in terms of evaluation of antibody efficacy. in both models about the same dose of antibody (0.2-0.3 mg) reduced about 100-fold the infectious virus in the lungs. this result now suggests that testing of antibody efficacy could be performed at a larger scale than testing with the hace2 transgenic mice due to the availability of wild type mice. it also shows robust neutralizing activity of igg1 ab1 in two different models of infection. interestingly, fab ab1 had only several somatic mutations compared to the closest germline predecessor genes. this implies that ab1-like antibodies could be elicited relatively quickly by using rbd-based immunogens especially in some individuals with naïve mature b cells expressing the germline predecessors of ab1. this is in contrast to the highly mutated broadly neutralizing hiv-1 antibodies that require long maturation times, are difficult to elicit and their germline predecessors cannot bind native hiv-1 envelope glycoproteins (22, 23). the rbd of the mers-cov s protein was previously shown to elicit neutralizing antibodies (24, 25). for sars-cov-2 only a few somatic mutations would be sufficient to generate potent neutralizing antibodies against the sars-cov-2 rbd which is a major difference from the elicitation of broadly neutralizing antibodies against hiv-1 which requires complex maturation pathways (22, 26-29). the germline-like nature of the newly identified mab ab1 also suggests that it has excellent developability properties that could accelerate its development for prophylaxis and therapy of sars-cov-2 infection (30). to further assess the developability (drugability) of ab1 its sequence was analyzed online (opig.stats.ox.ac.uk/webapps/sabdab-sabpred/tap.php); no obvious liabilities were found. in addition, we used dynamic light scattering (dls) and size exclusion chromatography to evaluate its propensity for aggregation. igg1 ab1 at a concentration of 2 mg/ml did not aggregate for six days incubation at 37°c as measured by dls (fig. 6a) ; there were no high molecular weight species in freshly prepared igg1 ab1 also as measured by size exclusion chromatography (sec) (fig. 6b ). igg1 ab1 also did not bind to the human cell line 293t ( fig. 2a ) even at very high concentration (1 μm) which is about 660-fold higher than its kd indicating absence of nonspecific binding to many membrane-associated human proteins. the igg1 ab1 also did not bind to 5,300 human membrane-associated proteins as measured by a membrane proteome array (fig. 6c ). the high affinity/avidity and specificity of igg1 ab1 along with potent neutralization of virus and good developability properties suggests its potential use for prophylaxis and therapy of sars-cov-2 infection. because it strongly competes with hace2 indicating a certain degree of mimicry, one can speculate that mutations in the rbd may also lead to inefficient entry into cells and infection. in the unlikely case of mutations that decrease the ab1 binding to rbd but do not affect binding to ace2 it can be used in combination with other mabs including those we identified or in bi(multi)specific formats to prevent infection of such sars-cov-2 isolates. ab1 could also be used to select appropriate epitopes for vaccine immunogens and for diagnosis of cov-specific igg1 m336 antibody was expressed in human mammalian cell as described previously (5). the ace2 gene was ordered from origene (rockville, md). the rbd domain (residues 330-532) and s1 domain (residues 14-675) and ace2 (residues 18-740) genes were cloned into plasmid which carries a cmv promotor with an intron, human igg1 fc region and woodchuck posttranscriptional regulatory element (wpre) to generate the rbd-fc, s1-fc and ace2-fc expression plasmids. the rbd-avi-his protein with an avi tag followed by a 6×his tag at c-terminal was subcloned similarly. these proteins were expressed with expi293 expression system (thermo fisher scientific) and purified with protein a resin (genscript) and by ni-nta resin (thermo fisher scientific). the fab cr3022 antibody gene with a his tag was cloned into pcat2 plasmid (developed in house) for expression in hb2151 bacteria and purified with ni-nta resin. protein purity was estimated as >95% by sds-page and protein concentration was measured spectrophotometrically (nanovue, ge healthcare). blitz. antibody affinities and avidities were analyzed by the biolayer interferometry blitz binding ec50 was obtained by using the non-linear mode in graphpad prism 7. igg1 ab1 showed higher binding avidity to 293t-s cells than hace2-fc (0.25 nm v.s. 0.52 nm for igg1 ab1 and hace2-fc to achieve 50% binding, respectively). followed by pbst washing. for detection, an hrp conjugated anti mouse fc antibody was used. competition of ab1 with hace2 tested by blitz. 100 nm hace2-fc was monitored to bind ab1 saturated sensors (red line), which is compared to its independent binding signal to rbd sensor in the absence of ab1(green line were defined as the sample concentration at which a 50% reduction in rlu was observed relative to the average of the virus control wells. a pneumonia outbreak associated with a new coronavirus of probable bat origin convalescent plasma as a potential therapy for covid-19. the lancet infectious diseases sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies potent neutralization of hendra and nipah viruses by human monoclonal antibodies exceptionally potent cross-reactive neutralization of nipah and hendra viruses by a human monoclonal antibody potent cross-reactive neutralization of sars coronavirus isolates by human monoclonal antibodies passive transfer of a germline-like neutralizing human monoclonal antibody protects transgenic mice against lethal middle east respiratory syndrome coronavirus infection a neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection a neutralizing human monoclonal antibody protects african green monkeys from hendra virus challenge safety, tolerability, pharmacokinetics, and immunogenicity of a human monoclonal antibody targeting the g glycoprotein of henipaviruses in healthy adults: a first-in-human, randomised, controlled, phase 1 study construction of a large naive human phage-displayed fab library through one-step cloning construction of a large phagedisplayed human antibody domain library with a scaffold based on a newly identified highly soluble, stable heavy chain variable domain structural basis for the recognition of sars-cov-2 by full-length human ace2 an emerging coronavirus causing pneumonia outbreak in wuhan, china: calling for developing therapeutic and prophylactic strategies the sars-cov s glycoprotein: expression and functional characterization biotinylated hace2-fc (10 nm) was incubated with rbd-fc in the presence of different concentrations of vh ab5. after washing, bound hace2-fc was detected by using hrp conjugated streptavidin after washing, bound cr3022 was detected by using hrp conjugated anti human fc antibody. ab5 showed weak competition with cr3022 for binding to sars-cov-2 rbd. all the elisa experiments were performed in duplicate and the error bars denote ± sd key: cord-326337-s0fp5z1q authors: chan, kui k.; tan, timothy j.c.; narayanan, krishna k.; procko, erik title: an engineered decoy receptor for sars-cov-2 broadly binds protein s sequence variants date: 2020-10-19 journal: biorxiv doi: 10.1101/2020.10.18.344622 sha: doc_id: 326337 cord_uid: s0fp5z1q the spike s of sars-cov-2 recognizes ace2 on the host cell membrane to initiate entry. soluble decoy receptors, in which the ace2 ectodomain is engineered to block s with high affinity, potently neutralize infection and, due to close similarity with the natural receptor, hold out the promise of being broadly active against virus variants without opportunity for escape. here, we directly test this hypothesis. we find an engineered decoy receptor, sace22.v2.4, tightly binds s of sars-associated viruses from humans and bats, despite the ace2-binding surface being a region of high diversity. saturation mutagenesis of the receptor-binding domain followed by in vitro selection, with wild type ace2 and the engineered decoy competing for binding sites, failed to find s mutants that discriminate in favor of the wild type receptor. we conclude that resistance to engineered decoys will be rare and that decoys may be active against future outbreaks of sars-associated betacoronaviruses. zoonotic coronaviruses have crossed over from animal reservoirs multiple times in the past two 19 decades, and it is almost certain that wild animals will continue to be a source of devastating outbreaks. 20 unlike ubiquitous human coronaviruses responsible for common respiratory illnesses, these zoonotic 21 coronaviruses with pandemic potential cause serious and complex diseases, in part due to their tissue 22 tropisms driven by receptor usage. severe acute respiratory syndrome coronaviruses 1 (sars-cov-1) 23 and 2 (sars-cov-2) engage angiotensin-converting enzyme 2 (ace2) for cell attachment and entry (1-24 7). ace2 is a protease responsible for regulating blood volume and pressure that is expressed on the 25 surface of cells in the lung, heart and gastrointestinal tract, among other tissues (8, 9) . the ongoing spread 26 of sars-cov-2 and the disease it causes, covid-19, has had a crippling toll on global healthcare 27 systems and economies, and effective treatments and vaccines are urgently needed. 28 as sars-cov-2 becomes endemic in the human population, it has the potential to mutate and 29 undergo genetic drift and recombination. to what extent this will occur as increasing numbers of people 30 are infected and mount counter immune responses is unknown, but already a variant in the viral spike 31 protein s (d614g) has rapidly emerged from multiple independent events and effects s protein stability 32 and dynamics (10, 11). another s variant (d839y) became prevalent in portugal, possibly due to a 33 founder effect (12). coronaviruses have moderate to high mutation rates. for example, 10 −4 substitutions 34 per year per site occur in hcov-nl63 (13), an alphacoronavirus that also binds ace2, albeit via a 35 smaller interface that is only partially shared with the rbds of sars-associated betacoronaviruses (14). 36 additionally, large changes in coronavirus genomes have frequently occurred in nature from 37 recombination events, especially in bats where co-infection levels can be high (15, 16) . recombination of 38 mers-covs has also been documented in camels (17). this will all have profound implications for the 39 current pandemic's trajectory, the potential for future coronavirus pandemics, and whether drug resistance 40 in sars-cov-2 becomes prevalent. 41 the viral spike is a vulnerable target for neutralizing monoclonal antibodies that are progressing 42 through clinical trials, yet in tissue culture escape mutations in the spike rapidly emerge to all antibodies 43 tested (18). deep mutagenesis of the isolated receptor-binding domain (rbd) by yeast surface display 44 has easily identified mutations in s that retain high expression and ace2 affinity, yet are no longer bound 45 by monoclonal antibodies and confer resistance (19) . this has motivated the development of cocktails of 46 non-competing monoclonals (18, 20) , inspired by lessons learned from the treatment of hiv-1 and ebola, 47 to limit the possibilities for the virus to escape. notably, drug maker eli lilly has a monoclonal 48 monotherapy (ly-cov555) in advanced trials (nct04427501) where the emergence of resistant virus 49 variants has occurred; the trial has been updated to include an arm with a second monoclonal (ly-50 cov016). however, even the use of monoclonal cocktails does not address future coronavirus spill overs 51 from wild animals that may be antigenically distinct. indeed, large screening efforts were required to find 52 antibodies from recovered sars-cov-1 patients that cross-react with sars-cov-2 (21), indicating 53 antibodies have confined capacity for interacting with variable epitopes on the spike surface, and are 54 unlikely to be broad and pan-specific for all sars-related viruses. 55 an alternative protein-based antiviral to monoclonal antibodies is to use soluble ace2 (sace2) as a 56 decoy to compete for receptor-binding sites on the viral spike (6, (22) (23) (24) (25) of diverse sars-associated betacoronaviruses that use ace2 for entry. we further fail to find mutations 78 within the rbd, which directly contacts ace2 and is where possible escape mutations will most likely 79 reside, that redirect specificity towards the wild type receptor. we conclude that resistance to an 80 engineered decoy receptor will be rare, and sace2 2 .v2.4 targets common attributes for affinity to s in 81 sars-associated viruses. 82 the affinities of the decoy receptor sace2 2 .v2.4 were determined for purified rbds from the s 85 proteins of five coronaviruses from rhinolophus bat species (isolates lyra11, rs4231, rs7327, rs4084 86 and rsshc014) and two human coronaviruses, sars-cov-1 and sars-cov-2. these viruses fall 87 within a common clade of betacoronaviruses that use ace2 as an entry receptor (7). they share close 88 sequence identity within the rbd core while variation is highest within the functional ace2 binding site 89 (figures 1 and s1) , possibly due to a co-evolutionary 'arms race' with polymorphic ace2 sequences in 90 ecologically diverse bat species (28). affinity was measured by biolayer interferometry (bli), with 91 sace2 2 (a.a. s19-g732) fused at the c-terminus with the fc moiety of human igg1 immobilized to the 92 sensor surface and monomeric 8his-tagged rbd ( figure s2 ) used as the soluble analyte. this 93 arrangement excludes avidity effects, which otherwise cause artificially tight (picomolar) apparent 94 affinities whenever dimeric sace2 2 in solution is bound to immobilized rbd decorating an interaction 95 surface. wild type sace2 2 bound all the rbds with affinities ranging from 16 nm for sars-cov-2 to 96 91 nm for lyra11, with median affinity 60 nm ( sars-cov-2 to 3.5 nm for isolate rs4231, with median affinity less than 2 nm ( table 1 ). the 100 approximate 35-fold affinity increase of the engineered decoy applies universally to coronaviruses in the 101 test panel and the molecular basis for affinity enhancement must therefore be grounded in common 102 attributes of rbd/ace2 recognition. 103 the rbd of sars-cov-2 (pdb 6m17) is colored by diversity between 7 sars-associated cov strains 105 (blue, conserved; red, variable). a deep mutational scan of the rbd in the context of full-length s reveals residues in the ace2 110 binding site are mutationally tolerant 111 to explore potential sequence diversity in s of sars-cov-2 that may act as a 'reservoir' for drug 112 resistance, the mutational tolerance of the rbd was evaluated by deep mutagenesis (32). saturation 113 mutagenesis was focused to the rbd (a.a. c336-l517) of full-length s tagged at the extracellular n-114 terminus with a c-myc epitope for detection of surface expression. the spike library, encompassing 3,640 115 single amino acid substitutions, was transfected in human expi293f cells under conditions where cells 116 typically acquire no more than a single sequence variant (33, 34). the culture was incubated with wild 117 type, 8his-tagged, dimeric sace2 2 at a sub-saturating concentration (2.5 nm). bound sace2 2 -8h and 118 surface-expressed s were stained with fluorescent antibodies for flow cytometry analysis ( figure 2a ). compared to cells expressing wild type s, the library was poorly expressed, indicating many mutations 120 are deleterious for folding and expression. a cell population was clearly discernable expressing s 121 variants that bind ace2 with decreased affinity ( figure 2b ). after gating for c-myc-positive cells 122 expressing s, cells with high and low levels of bound sace2 2 were collected by fluorescence-activated 123 cell sorting (facs), called the ace2-high and ace2-low populations, respectively ( figure 2c ). both 124 the expression and sace2 2 binding signals decreased over minutes to hours during sorting, possibly due 125 to shedding of the s1 subunit. cells were therefore collected and pooled from three separate facs 126 experiments for a combined 8 hours sort time. 127 averaging the log 2 enrichment ratios for each of the possible amino acids at a residue position. by adding 143 conservation scores for both the ace2-high and ace2-low sorts we derive a score for surface 144 expression, which shows that the hydrophobic rbd core is tightly conserved for folding and trafficking 145 of the viral spike ( figure 3a ). by comparison, residues on the exposed rbd surface are mutationally 146 permissive for s surface expression. this matches the mutational tolerance of proteins generally. 147 for tight ace2 binding (i.e. s variants in the ace2-high population), conservation increases for 161 rbd residues at the ace2 interface, yet mutational tolerance remains high ( figure 3c ). the sequence 162 diversity observed among natural betacoronaviruses, which display high diversity at the ace2 binding 163 site, is therefore replicated in the deep mutational scan, which predicts the sars-cov-2 spike tolerates 164 substantial genetic diversity at the receptor-binding site for function. from this accessible sequence 165 diversity sars-cov-2 might feasibly mutate to acquire resistance to monoclonal antibodies or 166 engineered decoy receptors targeting the ace2-binding site. binding site (e.g. v362, y365 and c391) is free to mutate for yeast surface display, but its sequence is 174 constrained in our experiments; this region of the rbd is buried by connecting structural elements to the 175 global fold of an s subunit in the closed-down conformation (this is the dominant conformation for s 176 subunits and is inaccessible to receptor binding) (2, 4, 38, 39). we used targeted mutagenesis to 177 individually test alanine substitutions to all the cysteines in the rbd ( figure s4 ). we found all cysteine-178 to-alanine mutations severely diminish s surface expression in expi293f cells, including c391a and 179 c525a on the rbd 'backside' that were neutral in the yeast display scan (36). these differences 180 demonstrate that there are tighter sequence constraints on the rbd in the context of a full spike expressed 181 at a human cell membrane, yet overall we consider the two data sets to closely agree. 182 for binding to dimeric sace2 2 , we note that interface residues were more tightly conserved in the 183 starr et al data set (figure 3d ), possibly a consequence of three differences between the deep mutagenesis 184 experiments. first, our selections for ace2 binding of s variants at the plasma membrane appears to 185 primarily reflect mutational effects on surface expression, which is almost certainly more stringent in 186 human cells. yeast permit many poorly folded proteins to leak to the cell surface (40). second, the yeast 187 selections were conducted at multiple sace2 concentrations from which apparent k d changes were 188 computed (36); the starr et al data in this regard is very comprehensive. due to the long sort times 189 required for our human cell libraries where only a small fraction of cells express spike, we sorted at a 190 single sace2 2 concentration that cannot accurately capture a range of different binding affinities 191 quantitatively. third, dimeric sace2 2 may geometrically complement trimeric s densely packed on a 192 human cell membrane, such that avidity masks the effects of affinity-reducing mutations. nonetheless, 193 there is overall agreement that ace2 binding often persists following mutations to the rbd surface, and 194 our data simply suggests mutational tolerance may be even greater than that already observed by starr et 195 al. 196 having shown that the ace2-binding site of sars-cov-2 protein s tolerates many mutations, we 198 asked whether mutations might therefore be found that confer resistance to the engineered decoy 199 sace2 2 .v2.4. resistance mutations are anticipated to lose affinity for sace2 2 .v2.4 while maintaining 200 binding to the wild type receptor, and are most likely to reside in the rbd where physical contacts are 201 made. similar reasoning formed the foundation of a deep mutagenesis-based selection of the isolated 202 rbd by yeast surface display to find escape mutations to monoclonal antibodies, and the results were 203 predictive of escape mutations in pseudovirus growth selections (19). 204 to address whether escape mutations from the engineered decoy might be found in the rbd, we 205 repurposed the s protein library for a specificity selection. cells expressing the library, encoding all 206 possible substitutions in the rbd, were co-incubated with wild type sace2 2 fused to the fc region of 207 igg1 and 8his-tagged sace2 2 .v2.4 at concentrations where both proteins bind competitively (25). it was 208 immediately apparent from flow cytometry of the expi293f culture expressing the s library that there 209 were cells expressing s variants shifted towards preferential binding to sace2 2 .v2.4, but no significant 210 population with preferential binding to the wild type receptor (figures 4a and 4b ). cells expressing s 211 variants that might preferentially bind sace2 2 (wt)-igg1 or sace2 2 .v2.4 were gated and collected by 212 facs (figure 4c ), followed by deep sequencing of s transcripts to determine enrichment ratios. there 213 was close agreement between two independent replicate experiments ( figures 4d-4g ). most rbd 214 mutations were depleted following sorting, consistent with deleterious effects on s folding and 215 expression. 216 soluble ace2 2 .v2.4 has three mutations from wild type ace2: t27y buried within the rbd 233 interface, and l79t and n330y at the interface periphery ( figure 5a) . a substantial number of mutations 234 in the rbd of s were selectively enriched for preferential binding to sace2 2 .v2.4 ( figure 5b , upper-left 235 quadrant). while sace2 2 .v2.4-specificity mutations could be found immediately adjacent to the sites of 236 engineered mutations in ace2 (in particular mutations to s-f486 adjacent to ace2-l79 and s-t500 237 adjacent to ace2-n330), major hot spots for sace2 2 .v2.4-specificity mutations were also mapped to 238 rbd loop 498-506, contacting the region where the ace2-α1 helix packs against a β-hairpin motif 239 ( figure 5a ). by comparison, there were no hot spots in the rbd for sace2 2 (wt)-specificity mutations. 240 indeed, only a small number of mutations were selectively enriched for preferential binding to wild type 241 receptor ( figure 5b ), and the abundance of these putative wild type-specific mutations barely rose above 242 the expected level of noise in the deep mutagenesis data. in this competition assay, s binding to wild type 243 sace2 2 is therefore more sensitive to rbd mutations than s binding to engineered sace2 2 .v2.4. 244 to determine whether the potential wild type ace2-specific mutations found by deep mutagenesis 260 are real as opposed to false predictions due to data noise, we tested 24 mutants of s selectively enriched in 261 the wild type-specific gate by targeted mutagenesis (blue data points in figure 5b ). only minor shifts 262 towards binding wild type sace2 2 were observed ( figure s5 ). two s mutants were investigated further 263 in sace2 2 titration experiments, n501w and n501y, which both retained high receptor binding and 264 displayed small shifts towards wild type sace2 2 in the competition experiment. n501 of s is located in 265 the 498-506 loop and its substitution to large aromatic side chains might alter the loop conformation to 266 cause steric strain with nearby ace2 mutation n330y in sace2 2 .v2.4. after titrating the concentrations 267 of 8his-tagged sace2 2 (wt) and sace2 2 .v2.4 and measuring bound protein to s-expressing cells by flow 268 cytometry, it was found s-n501w and s-n501y do show enhanced specificity for wild type sace2 2 , but 269 the effect is small and sace2 2 .v2.4 remains the stronger binder ( figure 5c ); these mutations therefore 270 will not confer resistance in the virus to the engineered decoy. by comparison, multiple independent 271 escape mutations are readily found in s of sars-cov-2 that diminish the efficacy of monoclonal 272 antibodies by many orders of magnitude (18, 19) . 273 finally, 8 representative mutations to s predicted from the deep mutational scan to increase 274 specificity towards sace2 2 .v2.4 (purple data points in figure 5b ) were cloned and 7 were found to have 275 large shifts towards preferential sace2 2 .v2.4 binding in the competition assay ( figure s6 ). these s 276 mutations were y449k/q/s, l455g/r/y and g504k. none of the mutated sites is in direct contact with 277 an engineered residue on sace2 2 .v2.4 and the molecular bases for specificity changes are therefore 278 ambiguous, but we speculate may involve local conformational perturbations. validation by targeted 279 mutagenesis therefore confirms that the selection can successfully find mutations in s with altered 280 specificity. the inability to find mutations in the rbd that impart high specificity for the wild type 281 receptor means such mutations are rare or may not even exist, at least within the receptor-binding domain 282 where direct physical contacts with receptors occur. we cannot exclude mutations elsewhere having 283 long-range conformational effects. engineered, soluble decoy receptors therefore live up to their promise 284 as broad therapeutic candidates against which a virus cannot easily escape. 285 the allure of soluble decoy receptors is that the virus cannot easily mutate to escape neutralization. 287 mutations that reduce affinity of the soluble decoy will likely also decrease affinity for the wild type 288 receptor on host cells, thereby coming at the cost of diminished infectivity and virulence. however, this 289 hypothesis has not been rigorously tested, and since engineered decoy receptors differ from their wild 290 type counterparts, even if by just a small number of mutations, it is possible a virus may evolve to 291 discriminate between the two. here, we show that an engineered decoy receptor for sars-cov-2 292 broadly binds with low nanomolar k d to the spikes of sars-associated betacoronaviruses that use ace2 293 for entry, despite high sequence diversity within the ace2-binding site. mutations in s of sars-cov-2 294 that confer high specificity for wild type ace2 were not found in a comprehensive screen of all 295 substitutions within the rbd. the engineered decoy receptor is therefore broad against zoonotic ace2-296 utilizing coronaviruses that may spill over from animal reservoirs in the future and against variants of 297 sars-cov-2 that may arise as the current covid-19 pandemic rages on. we argue it is unlikely that 298 decoy receptors will need to be combined in cocktail formulations, as is required for monoclonal 299 antibodies or designed miniprotein binders to prevent the rapid emergence of resistance (18, 41), 300 facilitating manufacture and distribution. our findings give insight into how a potential therapeutic can 301 achieve breath with a low chance of virus resistance for a family of highly infectious and deadly viruses. physiology to exert unacceptable toxicity. for example, the entry receptor for human cytomegalovirus is 312 a growth factor receptor, and growth factor interactions had to be knocked out to make a virus-specific 313 decoy suitable for in vivo administration (43). however, ace2 in this regard is different and its 314 endogenous activity -the catalytic conversion of vasoconstrictive and inflammatory peptides of the renin-315 angiotensin system -may be of direct benefit for addressing covid-19 symptoms. during infection, 316 ace2 activity is dysregulated and the renin-angiotensin system becomes imbalanced, possibly driving 317 aspects of acute-respiratory distress syndrome (ards) (44-46). administration of recombinant sace2 318 converts angiotensin (ang) i and ii to the protective peptides ang-(1-9) and ang-(1-7), respectively, with 319 potential benefits for the pulmonary and cardiovascular systems that include decreased lung elastance, 320 increased blood oxygenation, reduced hypertension and diminished inflammation (44, 45, (47) (48) (49) (50) provide no more than a single coding variant per cell (33, 34). expi293f cells at 2 × 10 6 / ml were 373 transfected with a mixture of 1 ng coding plasmid (i.e. library dna) with 1.5 µg pcep4-δcmv carrier 374 plasmid (described in (34) fitc fluorescence for bound sace2 2 (wt)-8h were collected ( figure 2c ). collection tubes were coated 386 overnight with fetal bovine serum prior to sorting and contained expi293 expression medium. collected 387 cell pellets were frozen at -80°c and were pooled across separate sort experiments prior to extraction of 388 total rna. 389 the competition selection was performed similarly, with the exception that cells expressing the s library 390 were incubated for 30 minutes in a mixture of 20 nm sace2 2 .v2.4-8h and 25 nm sace2 2 (wt)-igg1. 391 after washing twice, bound proteins were stained for 30 minutes with anti-human igg-apc (clone 392 hp6017, 1/250 dilution; biolegend) and anti-his-fitc (chicken polyclonal, 1/100 dilution; 393 immunology consultants laboratory). cells were washed twice and sorted. after gating for the main 394 population of viable cells as described above, the 20 % of cells with the highest fitc-relative-to-apc 395 and highest apc-relative-to-fitc signals were collected ( figure 4c ). 396 total rna was extracted from the collected cells using a genejet rna purification kit (thermo 397 scientific). first strand cdna was synthesized with accuscript (agilent) primed with a gene-specific 398 oligonucleotide. the region of s scanned by saturation mutagenesis was pcr amplified as 3 overlapping 399 fragments that together span the full rbd sequence. following a second round of pcr, primers added 400 adapters for annealing to the illumina flow cell and sequencing primers, together with barcodes for 401 experiment identification. the pcr products were sequenced on an illumina novaseq 6000 using a 402 2×250 nt paired end protocol. data were analyzed using enrich (35), where the frequencies of s variants 403 in the transcripts of the sorted populations were compared to their frequencies in the naive plasmid 404 library. log 2 enrichment ratios for all the individual mutations were calculated and normalized by 405 subtracting the log 2 enrichment ratio for the wild type sequence across the same pcr-amplified fragment. a pneumonia outbreak associated with a new coronavirus of probable bat origin. 435 nature structure, function, and antigenicity of the sars-cov-2 spike glycoprotein receptor recognition by novel coronavirus from 439 wuhan: an analysis based on decade-long structural studies of sars cryo-em structure of the 2019-ncov spike in the prefusion conformation sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked 444 by a clinically proven protease inhibitor angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus. 446 nature functional assessment of cell entry and receptor usage for 448 sars-cov-2 and other lineage b betacoronaviruses much more than just a receptor for sars-cov-2. front angiotensin-converting enzyme 2 and angiotensin 1-7: novel therapeutic targets. 452 the d614g mutation in the sars-cov-2 spike protein reduces s1 shedding and 454 increases infectivity tracking changes in sars-cov-2 spike: evidence that d614g increases 456 infectivity of the covid-19 virus on the track of the d839y mutation in the sars-cov-2 spike fusion peptide: 458 emergence and geotemporal spread of a highly prevalent variant in portugal mosaic structure of human coronavirus nl63, one thousand years of evolution crystal structure of nl63 respiratory coronavirus receptor-binding 463 domain complexed with its human receptor genetic recombination, and pathogenesis of coronaviruses evolutionary origins of the sars-cov-2 sarbecovirus lineage responsible for 468 the covid-19 pandemic co-circulation of three camel coronavirus species and recombination of 470 mers-covs in saudi arabia antibody cocktail to sars-cov-2 spike protein prevents rapid mutational escape 472 seen with individual antibodies complete mapping of mutations to the sars-cov-2 spike receptor-binding 474 domain that escape antibody recognition ultrapotent human antibodies protect against sars-cov-2 challenge via 476 multiple mechanisms cross-neutralization of sars-cov-2 by a human monoclonal sars-cov 478 antibody susceptibility to sars coronavirus s protein-driven infection correlates with 480 expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor neutralization of sars-cov-2 spike pseudotyped virus by recombinant ace2-ig inhibition of sars-cov-2 infections in engineered human tissues using 485 clinical-grade soluble human ace2 engineering human ace2 to optimize binding to the spike protein of sars 487 coronavirus 2 engineered ace2 receptor traps potently neutralize sars-cov-2. biorxiv high affinity modified ace2 receptors prevent sars-cov-2 infection. biorxiv exceptional diversity and selection pressure on sars-cov 493 and sars-cov-2 host receptor in bats compared to other mammals structural basis of receptor recognition by sars-cov-2 stabilized coronavirus spikes are resistant to conformational changes 497 induced by receptor recognition or proteolysis receptor and viral determinants of sars-coronavirus adaptation to human ace2 deep mutational scanning: a new style of protein science mapping interaction sites on human chemokine receptors by deep 503 mutational scanning structural architecture of a dimeric class c gpcr based on co-trafficking of sweet 505 taste receptor subunits enrich: software for analysis of protein function 507 by enrichment and depletion of variants deep mutational scanning of sars-cov-2 receptor binding domain reveals 509 constraints on folding and ace2 binding de novo design of ace2 protein decoys to neutralize sars-cov-2. biorxiv distinct conformational states of sars-cov-2 spike protein molecular architecture of the sars-cov-2 virus global analysis of protein folding using massively parallel design, synthesis, 516 and testing de novo design of picomolar sars-cov-2 miniprotein inhibitors thpdb: database of fda-approved peptide and protein therapeutics engineered receptors for human cytomegalovirus that are orthogonal to normal 522 human biology angiotensin-converting enzyme 2 protects from severe acute lung failure recombinant angiotensin-converting enzyme 2 improves pulmonary blood flow 526 and oxygenation in lipopolysaccharide-induced lung injury in piglets the pivotal link between ace2 deficiency 529 and sars-cov-2 infection renin-angiotensin-system, a potential pharmacological candidate, in acute 531 respiratory distress syndrome during mechanical ventilation sars-cov-2 and ace2: the biology and clinical data settling the arb and 534 acei controversy ace2 improves right ventricular function 536 in a pressure overload model novel ace2-fc chimeric fusion provides long-lasting hypertension control and 538 organ protection in mouse models of systemic renin angiotensin system activation pharmacokinetics and pharmacodynamics of recombinant human angiotensin-541 converting enzyme 2 in healthy human subjects a pilot clinical trial of recombinant human angiotensin-converting enzyme 2 in 543 acute respiratory distress syndrome novel ace2-igg1 fusions with improved activity against sars-cov2. 545 biorxiv computational design of a protein-based enzyme inhibitor cytometer (bd biosciences) and data were processed with fcs express (de novo software). quantification of myc-s surface expression is detailed in figure s4 . part supported by nih award r01ai129719 to e.p. the university of illinois has filed a provisional 432 patent for engineered decoy receptors and e.p. and k.k.c. are co-founders of orthogonal biologics, inc. 433 key: cord-296319-fwn97wds authors: juno, j. a.; tan, h.-x.; lee, w. s.; reynaldi, a.; kelly, h. g.; wragg, k.; esterbauer, r.; kent, h. e.; batten, c. j.; mordant, f. l.; gherardin, n. a.; pymm, p.; dietrich, m. h.; scott, n. e.; tham, w.-h.; godfrey, d. i.; subbarao, k.; davenport, m. p.; kent, s. j.; wheatley, a. k. title: immunogenic profile of sars-cov-2 spike in individuals recovered from covid-19 date: 2020-05-21 journal: nan doi: 10.1101/2020.05.17.20104869 sha: doc_id: 296319 cord_uid: fwn97wds the rapid global spread of sars-cov-2 and resultant mortality and social disruption have highlighted the need to better understand coronavirus immunity to expedite vaccine development efforts. multiple candidate vaccines, designed to elicit protective neutralising antibodies targeting the viral spike glycoprotein, are rapidly advancing to clinical trial. however, the immunogenic properties of the spike protein in humans are unresolved. to address this, we undertook an in-depth characterisation of humoral and cellular immunity against sars-cov-2 spike in humans following mild to moderate sars-cov-2 infection. we find serological antibody responses against spike are routinely elicited by infection and correlate with plasma neutralising activity and capacity to block ace2/rbd interaction. expanded populations of spike-specific memory b cells and circulating t follicular helper cells (ctfh) were detected within convalescent donors, while responses to the receptor binding domain (rbd) constitute a minor fraction. using regression analysis, we find high plasma neutralisation activity was associated with increased spike-specific antibody, but notably also with the relative distribution of spike-specific ctfh subsets. thus both qualitative and quantitative features of b and t cell immunity to spike constitute informative biomarkers of the protective potential of novel sars-cov-2 vaccines. the rapid global spread of sars-cov-2 has highlighted the intrinsic vulnerability of 54 humans to emerging zoonotic infections and spurred frantic efforts to expedite 55 vaccine and antiviral drug development, manufacture and deployment. in contrast to 56 historical pandemics, such as the 1918 "spanish" h1n1 influenza, modern 57 recombinant technology enables a rapid scientific response, with multiple vaccines 58 under development, almost exclusively aimed at eliciting antibodies to the viral 59 "spike" protein. the spike (s) protein of beta-coronaviruses is expressed as a single protein, with 62 proteolytic cleavage yielding s1 and s2 subunits 1 . s localises on the virion surface 63 and mediates both recognition of cellular receptors and membrane fusion. in the case 64 of sars-cov-2, a receptor binding domain (rbd) within s1 directly interacts with 65 high affinity with the peptidase domain of angiotensin-converting enzyme 2 (ace2) 66 2-4 . the s2 subunit of s mediates membrane fusion. the s/ace2 interaction mediates 67 viral entry and provides an attractive target for vaccine-elicited humoral immunity 5 , 68 with antibodies potentially capable of either (i) directly blocking binding of ace2 by 69 s, (ii) blocking conformational changes in s critical for membrane fusion, (iii) 70 eliminating infected cells through antibody effector mechanisms such as antibody-71 dependent cellular cytotoxicity (adcc), or (iv) driving accelerated clearance of free 72 virus. 73 the dominant targets for human antibody against the sars-cov-2 s are unclear. 75 some human mabs originally characterised against sars-cov s cross-react with 76 sars-cov-2. for example cr3022 which binds a cryptic epitope on the rbd 6,7 , 77 while s309, derived from the memory b cells of a sars-cov recovered subject, 78 blocks ace2 engagement by sars-cov-2 s 8 . a recent report of monoclonal 79 antibodies recovered from sars-cov-2 convalescent donors revealed multiple non-80 overlapping epitopes on the rbd, with different capacities for mediating 81 neutralisation 9 . few neutralising epitopes localised outside the rbd have been 82 characterised to date, with preliminary reports of neutralising epitopes within the nimmunogens in humans are poorly resolved. here we undertook an in-depth 94 characterisation of humoral and cellular immunity against spike in humans who 95 recovered from mild to moderate sars-cov-2 infection. we find antibody responses 96 to both s and the rbd are consistently elicited following sars-cov-2 infection, the 97 magnitude of which correlates with both plasma neutralising activity and inhibition of 98 rbd/ace2 binding. s-specific b cells comprise a significant proportion of the 99 circulating memory b cell pool following infection, with rbd-specific b cells 100 constituting a minor subpopulation in most subjects. assessment of the circulating t 101 follicular helper (ctfh) population reveals that s-specific ctfh cells are also readily 102 detected in convalescent subjects, while t cell responses toward the rbd are 103 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 21, 2020 . . https://doi.org/10.1101 /2020 significantly lower in frequency. finally, we find the development of comparatively 104 high plasma neutralisation activity is associated not only with the magnitude of anti-s 105 immune responses, but also with the phenotype of circulating tfh populations, 106 suggesting these features may serve as attractive biomarkers for candidate s-based 107 vaccines entering the clinic. 108 serological responses to spike antigens following sars-cov-2 infection 110 we recruited a cross-sectional cohort (n=41) of australian adults recovered from 111 mild-moderate sars-cov-2 infection and isolated plasma and pbmc samples at a 112 median of 32 (iqr: 28-35) days post-positive pcr test. the cohort had a median age 113 of 59 (iqr: 54-65) and was 43% female (17 of 41). subjects reported mild to 114 moderate upper and lower respiratory tract symptoms with only 5 (12%) requiring 115 hospitalisation, and none requiring mechanical ventilation (table s1) . a control 116 cohort of 27 healthy adults was recruited prior to widespread infection in australia 117 (table s2) . as we had an interest in the degree to which baseline cross-reactive 118 coronavirus immunity affected sars-cov-2 responses, we pre-screened the 27 119 uninfected subjects for serological reactivity against the beta coronavirus hcov-120 hku1 (hku1) ( figure s1 ), selecting individuals with the 5 highest and 5 lowest 121 plasma titres as controls for the study. 122 123 serological profiles are presented stratified across the cohort based on neutralisation 124 activity for each subject. antibodies binding the sars-cov-2 spike ( figure 1a ) or 125 the rbd ( figure 1b) were consistently observed in all 41 infected individuals by 126 elisa, with minimal reactivity in the controls. titres of s-and rbd-specific 127 antibody were highly correlated ( figure s2 ). consistent with previous reports 18 , low-128 level antibody responses cross-recognising the sars-cov rbd were observed in 129 most of our sars-cov-2 infected cohort ( figure 1c ). antibody responses to the 130 human coronavirus strain hku were prevalent at moderate to high levels across the 131 cohort, in line with previous reports of widespread seropositivity to s proteins of 132 human coronaviruses in adults 19,20 ( figure 1d ). the capacity of immune plasma to 133 block interaction between recombinant ace2 and rbd was assessed by elisa, with 134 modest levels of inhibition detected in most subjects, and selected subjects exhibiting 135 potent inhibitory activity ( figure 1e ). virus neutralising activity in the plasma was 136 similarly assessed using a microneutralisation assay with live sars-cov-2 infection 137 of vero cells as previously described for 22 . 138 neutralising antibody titres ranged from 40 to 508 (iqr:113-254) ( figure 1f ). in 139 summary, antibody responses against both s and the rbd are consistently elicited in 140 sars-cov-2 infected individuals, the endpoints titres of which correlate significantly 141 with neutralising activity (r=0.55 and r=0.61 respectively) and ace2 binding 142 inhibition (r=0.72 and r=0.72 respectively) in the plasma ( figure s2) . 143 144 b cell responses to s antigens following sars-cov-2 infection 145 we next examined the frequency and specificity of class-switched b cells in 146 convalescent subjects using sars-cov-2 spike or rbd proteins as flow cytometric 147 probes. clear antigen-specific populations of cd19 + igd -b cells (gating in figure s3 ) 148 binding spike, spike and rbd or rbd alone could be resolved in our cohort of 149 recovered from sars-cov-2 subjects, with minimal background staining in 150 uninfected controls (figure 2a ; figure s4 ). frequencies of spike + rbd -, spike + rbd + 151 and spike -rbd + b cells as a proportion of the cd19 + igdpopulation were a median 152 0.38% (iqr 0.24-0.52), 0.047% (iqr 0.023-0.084) and 0.033% (iqr 0.015-0.045), 153 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 21, 2020 . . https://doi.org/10.1101 /2020 respectively ( figure 2b ). the very low frequencies of spike -rbd + b cells likely 154 constitute a mix of background staining and b cells that recognise rbd epitopes 155 occluded in recombinant s or intact virus. immunoglobulin isotypes were determined 156 for spike + rbdand spike + rbd + populations using igm and igg surface staining, 157 with igm -iggclass-switched b cells previously established to be almost exclusively 158 iga + 23 . in our cohort sampled a median of 36 days after symptom onset, the majority 159 of spike + rbdclass-switched b cells were igg + (median 57.5%; iqr 46.8-64.8), with 160 smaller proportions displaying igm+ (20.9%; iqr 17.4-29.1) and iga + (igm -igg -) 161 (17.4%; iqr 13.2-25.9) ( figure 2c ). isotype distribution of spike + rbd + b cells was 162 more variable due to low event counts, with median frequencies of 45.5% 70.7) igg, 13.6% (iqr 0-30.3) igm and 20.0% iga ). the activation 164 phenotype of antigen-specific b cells was assessed using cd21 and cd27 surface 165 staining 24 ( figure s5 ). most spike + rbd -(median 58.5%; iqr 52.2-66.6) or 166 spike + rbd + (68.7%; iqr 54.4-80) class-switched b cells displayed a resting memory 167 phenotype (cd21 + cd27 + ), also consistent with the median duration of infection. 168 however, a significant proportion of activated memory b cells (cd21 -cd27 + ) was 169 still evident for both spike + rbd -(18.9%; iqr 13.2-25.7) or spike + rbd + b cell 170 populations (13.2%; , with only low proportions of cd21 -cd27and 171 cd21 + cd27phenotypes observed. overall, sars-cov-2 infection efficiently elicits 172 both s-and rbd-specific b cells in most subjects after recovery, which constitute a 173 significant proportion of the memory b cell pool, which are mostly igg + and of a 174 resting memory phenotype. 175 the rbd of sars-cov and sars-cov-2 share significant homology, but with 177 marked diversity within the ace2 binding motif despite shared recognition of this 178 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 21, 2020. . https://doi.org/10.1101/2020.05.17.20104869 doi: medrxiv preprint cellular receptor 2,25 . we examined whether differential staining with sars-cov and 179 sars-cov-2 probes would allow more precise identification of b cells recognising 180 the unique ace2 binding site of sars-cov-2, to understand why some individuals 181 had notable rbd-specific antibody titres but with limited neutralisation activity or 182 rbd/ace2 binding inhibition. pbmcs from a subset of covid+ subjects (n=15) 183 were stained with sars-cov-2 spike, sars-cov-2 rbd and sars-cov rbd 184 probes as before ( figure 2d ). both sars-cov-2 rbd-specific and sars-185 cov/sars-cov-2 cross-reactive igg+ b cells could be resolved in most subjects 186 across sars-cov-2 convalescent and uninfected donors ( figure s8 ). antigen 197 specificity of the ctfh population was determined using an activation induced 198 marker (aim) assay 29 in response to stimulation with sars-cov-2 spike or rbd 199 proteins ( figure 3a ). overall, recovered subjects exhibited robust ctfh responses to 200 the sars-cov-2 spike protein, with a median of 0.92% spike-specific ctfh cells 201 (iqr 0.42 -1.52; figure 3b ). in contrast to the full spike, rbd-specific ctfh 202 responses were significantly lower (p<0.0001), with a median of only 0.12% of ctfh 203 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 21, 2020. . https://doi.org/10.1101/2020.05.17.20104869 doi: medrxiv preprint cells exhibiting rbd specificity (iqr 0.04 -0.38). consistent with the high 204 frequency of hku1 seropositivity among the convalescent cohort ( figure 1d ), ctfh 205 responses to hku1 spike were detected among 97.5% of donors (median 0.52% of 206 ctfh cells, iqr 0.32 -0.99). the frequency of hku1-specific ctfh was generally 207 higher among the convalescent cohort than the uninfected controls ( figure 3b comparison to seb-stimulated cells from a subset of donors confirmed that in vitro 220 tcr stimulation does not preferentially activate or upregulate expression of ccr6 221 among the ctfh population ( figure 3d ). 222 223 analysis of spike-specific non-ctfh cd4 memory (cd3 + cd4 + cd45ra -cxcr5 -) 224 cells revealed similar patterns of antigen reactivity to the ctfh compartment; namely, 225 strong recognition of sars-cov-2 and hku1 spike proteins (median 0.53% and 226 0.54% of cd4 memory cells, respectively) and lower frequencies of rbd-specific t 227 cells (median 0.24% of cd4 memory cells) ( figure s8 ). 228 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 21, 2020 . . https://doi.org/10.1101 /2020 predictors of plasma neutralisation activity the development of serological neutralisation activity will be a critical endpoint for 230 upcoming sars-cov-2 vaccine trials. a co-correlation matrix of subject 231 characteristics and immunological parameters was generated ( figure 4a ). this 232 analysis highlighted broad co-correlation of many immune parameters related to s 233 immunogenicity, namely antibody titres and the circulating frequencies of s-specific 234 b and t cell populations. principal component analysis (pca) on immunological 235 variables revealed clustering of the cohort into subjects with stronger and weaker 236 plasma neutralisation activity ( figure 4b ). using a multiple regression approach, we 237 identified titres of s-specific antibody and the proportion of s-specific ctfh with a 238 th17-like phenotype (ccr6 + cxcr3 -) as the two most significant predictive factors 239 related to neutralisation activity ( figure 4c ). 240 efficient elicitation of potent antibodies capable of neutralising viral entry is likely to 242 be a critical feature of effective vaccines against sars-cov-2. in the current study, 243 we observed that neutralisation activity in the plasma of convalescent subjects ranged 244 from potent to negligible, despite near universal detection of antibodies binding s 245 and/or rbd, suggesting that qualitative aspects of the humoral immune response may 246 be a critical consideration for vaccine development. direct assessment of key 247 immunological events within the respiratory tract and draining lymphoid tissues is 248 challenging in humans, however assessing b and t cell immunity in more readily 249 sampled blood can be informative. 250 251 spike-specific class-switched b cells were expanded in nearly all infected subjects, 252 with a predominantly igg + and resting memory phenotype consistent with the 253 sampling time several weeks after the resolution of infection. b cells binding the 254 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 21, 2020. . https://doi.org/10.1101/2020.05.17.20104869 doi: medrxiv preprint rbd, which contains the ace2 interaction site, were markedly less frequent than s-255 specific b cells, and not detected at all in many subjects. combinatorial b cell 256 staining with both sars-cov and sars-cov-2 probes enabled focused assessment 257 of the uniquely variant epitope on the sars-cov-2 rbd that facilitates high affinity 258 recognition of ace2. a minority of sars-cov-2 rbd-specific b cells also 259 recognise the sars-cov rbd, a finding consistent with the relative infrequency of 260 sars-cov or mers-cov cross-reactive antibodies recovered from convalescent 261 patients to date 9,32 . we find the frequency of igd -igg + b cells that bound s and 262 sars-cov-2 rbd, but not cells binding sars-cov rbd, tracked with serological 263 rbd/ace2 binding inhibition but not with overall neutralising activity. overall, our 264 data suggest that in some subjects, precise antibody recognition and blockade of the 265 rbd ace2-binding site is the principal pathway to generating neutralising antibody. 266 however, the disconnect seen in many subjects between plasma neutralising titres and 267 rbd-specific antibody, b and t cell responses, strongly suggests sufficient non-rbd 268 epitope targets exist to constitute an alternative pathway to comparable virus 269 neutralisation outcomes. to sars-cov-2 rbd were observed, which may reflect limited cd4 t cell epitopes 278 given the small size of rbd. this has implications for rbd-based vaccine strategies, 279 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (ctfh17), consistent with responses to other neo-antigens 289 such as ebola glycoprotein vaccines 34 . however despite this predominance, the 290 relative proportion of s-specific ctfh17 (ccr6 + cxcr3 -) was negatively correlated 291 with virus neutralisation activity. in contrast, increased frequencies of both ctfh1 292 (ccr6 -cxcr3 + ) and ctfh2 (ccr6 -cxcr3 -) were observed in subjects with the 293 highest plasma neutralising activity. expansion of ctfh1 is well characterized 294 following seasonal influenza immunisation, where peak frequencies in the blood 295 correlate with both plasmablast expansion and subsequent serum neutralising 296 antibody titres 23, 30, 35 . similarly, bias toward cxcr3 + phenotypes is reported for 297 antigen-specific ctfh in many chronic infections 36,37 . the functional significance of 298 cxcr3 + ctfh during sars-cov-2 infection is currently unclear, however may 299 reflect differences in lymph node tfh activity or egress from the gc. 300 the impact of widespread pre-existing immunity to human coronaviruses (229e, 302 nl63, hku1, oc43) upon the responses to sars-cov-2 infection is an open 303 question. here we found serum antibody against hku1 was widely prevalent, 304 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 21, 2020. . https://doi.org/10.1101/2020.05.17.20104869 doi: medrxiv preprint consistent with the high seroprevalence rates in adults reported previously 19, 20 . 305 however, we see no evidence of hku-specific immunity modulating binding or 306 neutralising titres against sars-cov-2 antigens. our data suggest cd4 t cell 307 responses to hku1 may be boosted following sars-cov-2 infection, possibly via 308 recognition of conserved epitopes within the s2 domain 38 . the predominantly ccr6 + 309 phenotype of sars-cov-2 and hku-1-specific ctfh may reflect a coronavirus-310 specific tfh response, but further epitope mapping is required to deconvolute the 311 contribution of hku1 memory responses or recently boosted sars-cov-2 cross-312 reactivity. 313 there is understandably considerable scientific interest in predicting the biogenesis of 315 protective immunity against sars-cov-2, of which neutralising antibodies against s 316 are likely to be consequential. although the current study is limited by cohort size, we 317 find that concomitant factors demarking robust humoral immunity, namely increased the study protocols were approved by the university of melbourne human research 328 ethics committee (#2056689) and all associated procedures were carried out in 329 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. subjects who had recovered from covid19 and healthy controls were recruited 333 through contacts with the investigators and invited to provide a blood sample. subject 334 characteristics of sars-cov-2 convalescent subjects are collated in table s1 a set of proteins was generated for serological and flow cytometric assays. the 343 ectodomain of sars-cov-2 (isolate whu1;residues 1 -1208) or hcov-hku1 s 344 protein (isolate n5;residues 1 -1290) were synthesised with furin cleavage site 345 removed and p986/987 stabilisation mutations 39 , a c-terminal t4 trimerisation 346 domain, avitag and his-tag, expressed in expi293 cells and purified by ni-nta 347 affinity and size-exclusion chromatography using a superose 6 16/70 column (ge 348 healthcare) ( figure s9 ). sars-cov s was biotinylated using bir-a (avidity). the 349 sars-cov-2 rbd 40 with a c-terminal his-tag (residues 319-541; kindly provided by 350 florian krammer) was similarly expressed and purified. sars-cov rbd (residues 351 n321-p513) with a c-terminal avitag and his-tag, was expressed in expi293 cells 352 and purified by ni-nta, biotinylated using bir-a (avidity) and purified by ize-353 exclusion chromatography using a s-75 superdex (ge healthcare). the human 354 (residues 19-613) and mouse (residues 19-615) ace2 ectodomain with c-terminal 355 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 21, 2020. . https://doi.org/10. 1101 /2020 his-tag (kindly provided by merlin thomas) were expressed in expi293 cells and 356 purified using ni-nta and size-exclusion chromatography ( figure s10 ). antigenicity 357 of coronaviral proteins was assessed by binding to immune sera, anti-rbd mabs 358 cr3022 and 4b, or human and mouse ace2 ( figure s11 ). the glycosylation profile 359 of recombinant s proteins ( figure s11 ) was assessed using mass spectrometry as 360 previously described 41 by sp3 protein clean up 42 and trypsin in-solution digestion. 361 purified peptides were desalted then separated using a two-column chromatography 362 set up comprising a pepmap100 c18 20 mm ã� 75 î¼m trap and a pepmap c18 500 363 mm ã� 75 î¼m analytical column on dionex ultimate 3000 uplc (thermofisher). 364 samples were concentrated onto the trap column at 5 î¼l/min with buffer a (2% 365 acetonitrile, 0.1% formic acid) for 6 min and infused into a q-exactiveâ�¢ plus mass 366 spectrometry (thermofisher) at 300 nl/min via the analytical column. 125 min 367 gradients were used altering the buffer composition from 2% buffer b (80% 368 acetonitrile, 0.1% formic acid) to 28% b over 95 min, then from 28% b to 40% b 369 over 10 min, then from 40% b to 100% b over 2 min, the composition was held at 370 100% b for 3 min, and then dropped to 3% b over 5 min and held at 3% b for another 371 10 min. the q-exactiveâ�¢ plus mass spectrometer was operated in a data-dependent 372 mode automatically switching between the acquisition of a single orbitrap ms1 scan 373 (70,000 resolution, agc of 3 ã� 10 6 ) followed by 15 data-dependent hcd ms2 events 374 tolerance of â±20 ppm was allowed for hcd ms2 scans. searches were performed 380 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. washed and developed using tmb substrate (sigma), stopped using sulphuric acid 398 and read at 450nm. endpoint titres were calculated as the reciprocal serum dilution 399 giving signal 2x background using a fitted curve (4 parameter log regression). 400 an elisa was performed to measure the ability of plasma antibodies to block 402 interaction between recombinant human ace2 and rbd proteins. 96-well maxisorp 403 plates (thermo fisher) were coated overnight at 4 o c with 8 âµg/ml of recombinant 404 rbd protein in carbonate-bicarbonate coating buffer (sigma). after blocking with 405 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. sars-cov-2 isolate cov/australia/vic01/2020 45 was passaged in vero cells and 414 stored at -80c. plasma was heat-inactivated at 56â°c for 30 min. plasma was serially-415 diluted 1:20 to 1:10240 before addition of 100 tcid 50 of sars-cov-2 in 416 mem/0.5% bsa and incubation at room temperature for 1 hour. residual virus 417 infectivity in the plasma/virus mixtures was assessed in quadruplicate wells of vero 418 cells incubated in serum-free media containing 1 âµg/ml tpck trypsin at 37â°c/5% 419 co2; viral cytopathic effect was read on day 5. the neutralising antibody titre is 420 calculated using the reed/muench method as previously described 21, 22 . 421 422 probes for delineating sars-cov-2 s-specific b cells within cryopreserved human 424 pbmc were generated by sequential addition of streptavidin-pe (thermofisher) to 425 trimeric s protein biotinylated using recombinant bir-a (avidity). biotinylated 426 sars-cov rbd was similarly conjugated to streptavidin-bv421 (bd). sars-cov-427 2 rbd protein was directly labelled to apc using an apc conjugation lightning-428 link kit (abcam). cells were stained with aqua viability dye (thermofisher). 429 monoclonal antibodies for surface staining included: cd19-ecd (j3-119) (beckman 430 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 21, 2020. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. acknowledgements 478 we thank the generous participation of the trial subjects for providing samples. the 479 sars-cov-2 rbd expression plasmids were kindly provided by florian krammer, 480 mt sinai school of medicine, ny, usa. the human and mouse ace2 expression 481 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. blood are clonally convergent but divergent from non-tfh cd4(+) cells. 566 cell reports 30, 137-152.e135 (2020). 567 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 21, 2020. glycoproteomics. journal of proteome research 15, 3904-3915 (2016) . 615 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 21, 2020 . . https://doi.org/10.1101 /2020 caly, l., et al. isolation and rapid sharing of the 2019 novel coronavirus 616 (sars-cov-2) from the first patient diagnosed with covid-19 in australia. 617 med j aust (2020). 618 619 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 21, 2020 . . https://doi.org/10.1101 /2020 figure 3 . specificity of ctfh responses to coronavirus spike proteins (a) representative staining of cd25 and cd134 co-expression on ctfh (cd3 + cd4 + cd45ra -cxcr5 + ) cells following stimulation with 5âµg/ml bsa (negative control), sars-cov-2 spike, sars-cov-2 rbd or hcov hku1 protein, or seb (positive control). (b) antigen-specific ctfh (n=10 sars-cov-2 negative, n=41 sars-cov-2 positive donors) frequencies were calculated as the proportion of cd25 + cd134 + ctfh cells in each stimulation condition after background subtraction using the negative control. (c) representative expression of ccr6 and cxcr3 on bulk ctfh, sars-cov-2 spikespecific, hcov hku1 spike-specific or seb-responsive (cd25 + ox-40 + ) ctfh. (d) quantification of ccr6 + cxcr3 -, ccr6 + cxcr3 + , ccr6 -cxcr3 + or ccr6 -cxcr3 -ctfh populations among sars-cov-2 positive donors (n=41). lymphocytes were identified by fsc-a vs ssc-a gating, followed by doublet exclusion (fsc-a vs fsc-h), and gating on live cd19 + b cells. class-switched b cells were identified as igd -, and surface istoype resolved by staining for igm or igg, with the double negative population (igm -/igg -) previously established as predominantly iga. binding to sars-cov-2 spike (s) and/or sars-cov-2 rbd probes was assessed for each population. figure s4 . representative staining of s-and rbd-specific igd -igg + b cells 3 uninfected subjects (left panels) and 6 subjects after recovery from sars-cov-2 infection (middle and right panels). cd19 + igd -igg + b cells cells were identified using gating strategy shown in figure s5 . binding to sars-cov-2 spike (s) and/or sars-cov-2 rbd probes was assessed. figure s5 . memory b cell phenotypes in subjects after sars-cov-2 infection (a) representative memory b cell phenotypes identified by cd21 and cd27 co-stain of probe + cd19 + igdcells (blue) overlaid on cd19 + igdcells (grey) and (b) the corresponding frequencies of the four populations in subjects previously infected with sars-cov-2 (resting memory -cd21 + cd27 + ; activated memory -cd21 -cd27 + ; naã¯ve/cd27 lo memory -cd21 + cd27 -; atypical b cells -cd21 -cd27 -); n.d -not detected due to absent probe + cells. figure s6 . gating strategy for resolving spike + cd19 + igd -igg + b cells specific for sars-cov-2 and sars-cov rbd (a) lymphocytes were identified by fsc-a vs ssc-a gating, followed by doublet exclusion (fsc-a vs fsc-h), and gating on live cd19 + b cells. igd -igg + b cells were gated and assessed for binding to sars-cov-2 spike. cross-reactive specificities versus those unique to sars-cov-2 were discriminated by co-staining with sars-cov-2 and sars-cov rbd probes. (b) representative staining shown for 4 subjects with prior sars-cov-2 infection. figure s7 . gating strategy for ctfh and memory cd4 + t cell subsets lymphocytes were identified by fsc-a vs ssc-a gating, followed by doublet exclusion (fsc-a vs fsc-h gate), and exclusion of dead or cd14 + cells. t cells were identified as cd3 + cd20 -. following exclusion of gamma delta t cells by vd1/vd2 tcr staining, cd4 + cd8 -t cells were identified. memory cd4 + t cells were defined as cd45ra -cxcr5 -, while ctfh cells were defined as cd45ra -cxcr5 + . ctfh cells were further characterized by pd-1 and ccr6/cxcr3 expression. 10 (24.4%) severe -no. (%) 5 (12.2%) * 3 subjects had a compatible illness and history of exposure but did not have a positive nasal swab ** illness severity was classified as: mild: prominent upper respiratory tract symptoms and not hospitalised. moderate: prominent lower respiratory tract symptoms and not hospitalised. severe: prominent lower respiratory tract symptoms and requiring hospital care. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 21, 2020. . https://doi.org/10. 1101 /2020 sars-cov-2 rbd sars-cov-2 spike igg igd igg igm igd -igg + igd -igm + igd -igm -igg -) sars-cov-2 rbd sars-cov-2 spike sars-cov-2 rbd sars-cov-2 spike immune plasma (n=4) control plasma (n=2) mab cr3022 mab 4b control mab key: cord-309411-2dfiwo65 authors: paris, kristina a.; santiago, ulises; camacho, carlos j. title: loss of ph switch unique to sars-cov2 supports unfamiliar virus pathology date: 2020-06-23 journal: biorxiv doi: 10.1101/2020.06.16.155457 sha: doc_id: 309411 cord_uid: 2dfiwo65 cell surface receptor engagement is a critical aspect of viral infection. this paper compares the dynamics of virus-receptor interactions for sars-cov (cov1) and cov2. at low (endosomal) ph, the binding free energy landscape of cov1 and cov2 interactions with the angiotensin-converting enzyme 2 (ace2) receptor is almost the same. however, at neutral ph the landscape is different due to the loss of a ph-switch (his445lys) in the receptor binding domain (rbd) of cov2 relative to cov1. namely, cov1 stabilizes a transition state above the bound state. in situations where small external strains are applied by, say, shear flow in the respiratory system, the off rate of the viral particle is enhanced. as a result, cov1 virions are expected to detach from cell surfaces in time scales that are much faster than the time needed for other receptors to reach out and stabilize virus attachment. on the other hand, the loss of this ph-switch, which sequence alignments show is unique to cov2, eliminates the transition state and allows the virus to stay bound to the ace2 receptor for time scales compatible with the recruitment of additional ace2 receptors diffusing in the cell membrane. this has important implications for viral infection and its pathology. cov1 does not trigger high infectivity in the nasal area because it either rapidly drifts down the respiratory tract or is exhaled. by contrast, this novel mutation in cov2 should not only retain the infection in the nasal cavity until ace2-rich cells are sufficiently depleted, but also require fewer particles for infection. this mechanism explains observed longer incubation times, extended period of viral shedding, and higher rate of transmission. these considerations governing viral entry suggest that number of ace2-rich cells in human nasal mucosa, which should be significantly smaller for children (and females relative to males), should also correlate with onset of viral load that could be a determinant of higher virus susceptibility. critical implications for the development of new vaccines to combat current and future pandemics that, like sars-cov2, export evolutionarily successful strains via higher transmission rates by viral retention in nasal epithelium are also discussed. although accurate assessments are still evolving, reports from the world health organization indicate that infection with sars-cov-2 (cov2) is significantly different relative to infection with previous respiratory viruses. the biggest distinctions are the longer incubation period and increased viral shedding (both likely responsible for higher rates of transmission), strong correlation of infected-fatality rates (ifr) with age and comorbidities, higher ifr for males relative to females, and minimal impact in children. as much as 40% of deaths from cov2 relate to cardiovascular complications (akhmerov and marban, 2020) , while cellular and animal models have also revealed inappropriate inflammatory responses along with high chemokine production (blanco-melo et al., 2020) . using thermodynamic, kinetic and molecular modeling, we explored potential reasons for these cov2-unique aspects and aimed to identify determinants of its complex pathology. while it stands to reason that some of the answers may be found in the complex genomic changes triggered by the virus in cells, tissues, and organs, longer incubation and infectivity time scales also suggest that differences could have a biophysical origin. work on sars-cov (cov1) has already determined that the virus enters cells via receptormediated endocytosis in a ph-dependent manner (wang et al., 2008) that is characterized by cotranslocation of the viral spike glycoprotein and its specific functional receptor, the angiotensinconverting enzyme 2 (ace2), from the cell surface to early endosomes. key steps that control the fate of the virus in the early and late endosome are driven in part by lowering the ph from 6.5-to-6.0 and from 6.0-to-5.0 (bui et al., 1996) , respectively; exposure to low ph triggers a spike catalyzed fusion between the viral and endosomal membranes followed by viral genome release. the cell machinery is then hijacked to replicate and assemble new virus particles that eventually exit the cell, primarily through budding. broadly speaking, this is the same ph-dependent endocytic path followed by the influenza virus (qin et al., 2019; yamauchi, 2019) , and likely all other coronaviruses (including mers-cov). while infections by cov1 and cov2 are mediated by the ace2 receptor, mers-cov (mers) gains entry to cells through dpp4 (raj et al., 2013) . figure 1 shows structures for the receptorbinding domains (rbd) in complex with their receptors for all three viruses. surprisingly and likely significantly, while the rbd of cov1 (li et al., 2005) and mers (wang et al., 2013) have one histidine on opposite ends of their binding interface, cov2 (wang et al., 2020) does not have his residues in this domain. as we have been able to determine to date, all known strains of cov2 have mutated away their last his residue that is still present in the rbd of cov1/mers and related zoonotic viruses (see below). because cell surface receptor engagement is a critical aspect of viral infection and life cycle, and sensing ph is relevant for both viral replication and regulation of histidine protonation, we set to decipher the mechanistic role of the remaining his residue that distinguishes the rbds of cov1 and cov2. results ph-switch. at low (endosomal) ph, the binding free energy landscape of cov1 and cov2 interactions with their ace2 receptor is almost the same. this is important because low ph is critical for the activation of the spike fusion in late endosomes (martin and helenius, 1991) . in particular, his34 in ace2 (fig. 1) , located at the core of the rbd binding interface, should play a key role in this process. indeed, we predict based on the co-crystal structures that his34 + should readily form a hydrogen bond network that stabilizes the rbd/ace2 complex in both cov1 and cov2 (fig. 2) . thus, loss of the ph-switch in cov2 has no impact on the low ph bound conformation with ace2. on the other hand, at neutral ph the landscape is different due to the loss of the ph-switch (his445lys) in the rbd of the spike protein of cov2 relative to cov1. we studied this loss by performing three independent unconstrained molecular dynamics simulations (mds) of cov1 pdb 2dd8 (prabakaran et al., 2006) and cov2 from the receptor (apo) in pdb 6lzg at both physiological and low ph ~ 5.0 conditions (see methods in supplementary information). in practice, lowering the ph protonates the his residue from neutral to positively charged (his + ) by the addition of an extra hydrogen. of note, although not studied here, the ph-switch in the rbd of mers has previously been observed in ph-dependent crystal structures (zhang et al., 2018) . mds revealed two distinct regions in the binding interface of cov1: (a) a loop motif (f 460 spdgkpctppalncy 475 ) that for his and his + shows mostly not-bound-and bound-like conformations, respectively; and, (b) the rest of the binding interface that consistently adopts bound-like conformations that are independent of ph ( fig. 3a-c) . figure 3a -b shows the dominant clusters observed in the his and his + simulation of cov1, as well as the ph-independent simulation of cov2. figure 3c -d shows detailed analyses of the corresponding root-mean-squaredeviations (rmsds) of these two regions relative to the co-crystals as a function of time (additional mds are shown in fig. s1 ). the plots include the equilibration time (between 100-200 ns) in order to emphasize that the distinct trajectories were not biased by different initial conformations. remarkably, deletion of the ph switch in cov2 that mutates the last histidine, his445, in cov1 by lys458 in cov2 generates a motif, which includes t 470 eiyqag 476 , that yields almost exclusively bound-like conformations (fig. 3d) ¾i.e., cov2 is always ready to bind ace2. it is interesting to note that the effect of both lys458 in cov2 and his + in cov1 ( fig. 3c -d) is to stabilize the bound-like state. implications of these findings in the binding free energy landscape are sketched in fig. 4 . namely, at low (late endosomal) ph, the landscape of cov1 and cov2 interactions with the ace2 receptor are very similar. however, at neutral ph the landscape is different due to the loss of the ph switch in the rbd of cov2 relative to cov1. specifically, the not-bound-like conformations of the phdependent loop in cov1 stabilizes a higher free energy transition state, whereas the persistent bound-like behavior of cov2 yields a much tighter bond. dynamics of virus-receptor interactions for cov1 and cov2. what are the implications of this transition state found in cov1 but not in cov2 at neutral ph? to answer this question, we need to consider that viral particles in the respiratory system are under small external strains from, e.g., shear flow in the respiratory airways, when engaging cell surface receptors. the cell mechanics of these interactions can be described by applying the reaction-limited kinetics of membrane-to-surface adhesion and detachment first envisioned by dembo et al (dembo et al., 1988) . specifically, one can write the free energy of the bound-state (bs) ∆ #$ under a tensile force as a function of the cell-cell gap width l, a constant binding free energy ∆ #$ & and a "spring" energy such that where #$ is the equilibrium length for the bonded state. a similar equation holds for the free energy of the transition state (ts) ∆ .$ at the same cell-cell gap, this treatment assumes equilibrium between bonded and de-bonded states, so there must be a very slow "ramp" rate for the force of pulling or pushing. if these conditions apply, the equilibrium constant for bond formation can be written as where # is the boltzmann factor. note that 12 (0) = 4∆5 67 8 /9 6 : = 12 cd / 12 cee . according to arrhenius theory, the de-bonding rate constant at a given gap-width can then be written as the mechanical or structural basis of the rbd/receptor interactions in fig. s2 can be characterized as a "door-knob" type junction across the gap, as opposed to a gripping or fish-hook bound state. the knob interactions with the receptor entailed two characteristic patches, a large bound-like domain and the smaller switching loop (fig. 3) , which one could model with spring constants k #i and k iccj , respectively (see red and yellow surfaces in fig. s2 ). then, the elastic constant of the bound-and transition-state can be written as k #$ ≈ k #i + k iccj and k .$ ≈ k #i , respectively, with the equilibrium rest lengths being essentially the same, i.e., l #$ = .$ = l 0 . thus, virus detachment to the transition state corresponds to an ideal case of the theory, where the only allowed change between the bonded and transition state is in the spring constants, such that it is clear that the spring approximation should only apply for small deformations. in general, the springs across the gap undergo a "twisting" motion around its long-axis to reach the transition state from the bonded state. the motion will increase "tightness" of the spring if (k #$ − k .$ ) < 0, which defines a catch-bond. here, however, cov1 always loosens tightness, (k #$ − k .$ ) = k iccj > 0, corresponding to a typical slip-bond whose lifetime is shortened by tensile forces acting in the bond. free energy landscapes and estimates of bond detachment for cov1/cov2 and ace2. we use the fastcontact server (champ and camacho, 2007) to compute the electrostatic ∆ 1i1r & and desolvation ∆ s1$ci & binding free energies of the bound and transition states using co-crystal structures and chimeras that incorporate changes triggered by low ph structures. entropies coupled to the unbound state could be somewhat higher for cov1 relative to cov2 due to the larger conformational entropy associated with the switching loop in fig. 3 . other error bars are correlated since interactions are very similar such that ∆∆ ′ have an error bar of ± 1 / . absolute free energies need to account for size-dependent configurational and vibrational entropy changes upon binding, which for high affinity protein-protein complexes have been estimated to be anywhere between 5-to-15 kcal/mol. however, for flat and rather superficial complexes such as those here (fig. s2) , the entropy loss could be much lower. finally, the pre-exponential factor cee 0 (eq. 5) in the absence of a transition state and at equilibrium is exactly 12 cd at 1 m concentration, which for diffusion-limited association can be approximated by 12 cd~1 0 \ s -1 (camacho et al., 2000) . (fig. 3a-c) . experimental equilibrium binding free energy is from (walls et al., 2020) . free energy estimates of bound complexes are fully consistent with experimental data (walls et al., 2020) ; alternative measurements have suggested a 10 fold weaker binding (shang et al., 2020) . these differences will only re-scale cee & by a factor of 10 but will not be significant to our conclusions. the key observation is that cov1 stabilizes a transition state by about 2.6 / above the bound state. as a result, small external strains applied by, say, shear flow in the respiratory system enhance the off rate of the viral particle as shown in eq. 6. thus, cov1 virions are expected to detach from cell surfaces in faster time scales. these binding free energy estimates are depicted in the landscapes in fig. 4 . only at physiological ph should the landscape of cov1 display a ph-dependent transition state. other bonds are expected to break in an all-or-none type of transition. optimal dwelling times and endocytosis. in principle, the ph-switch in cov1 could provide a natural mechanism to optimize virus internalization. namely, cov1 is expected to "bounce around" cell surfaces many times before cell entry. if the density of receptors is high enough, a "stick-and-slip" approach could be an efficient mechanism to find clusters of receptors randomly distributed on the cell surface. on the other hand, if only a small number of cell surface receptors are available, then receptor diffusion will be the limiting step to accrue the critical number of receptors needed for endocytosis, and longer rbd/ace2 dwelling times will be required. of note, tighter binding to ace2 would also make it easier for a smaller number of cov2 particles to establish an initial foothold in the respiratory system compared to weak binding where particles could be exhaled out. broad estimates of "high" concentration, e.g., in the range of 1000-to-10,000 receptors, yield an average separation between receptors ~0.6 − 0.2 µ (see fig. 4b ) that is larger than the diameter of the virus ~0.12 µ . thus, after attachment of the first spike to its receptor, recruitment of a second receptor to stabilized virus attachment will be limited by other receptors circulating in the cell membrane. lateral protein diffusion in cell membranes is length-scale dependent, varying between ~0.01 (kusumi et al., 2005) for 40-100 nm and >100 nm, respectively. thus, diffusion time scales to bring two receptors into close proximity for the above length scales are ~200 − 5 . it is noteworthy that the number of surface receptors in cells have an upper limit of about 20,000, which in the respiratory airways could limit infectivity to dwelling times of about cee &~1 4v (or 12~1 0 \ 4v ) based on ~0.15 µ (fig. 4) . viral infection and its pathology. the lifetime of cov1 rbd/ace2 bonds at physiological ph (~3 s) is marginally short-lived for efficiently triggering endocytosis, even at high ace2 receptor concentrations. as a result, cov1 virions are expected to detach from cell surfaces in time scales that are much faster than the time needed for other receptors to reach out and stabilize virus attachment. and for human nasal goblet cells, it will be significantly worse since, after each bounce, particles will be biased by gravity to either diffuse down the respiratory tract or be exhaled, where they will not find significant amounts of ace2 receptors until reaching lung alveolar epithelial cells (hamming et al., 2004) . on the other hand, deletion of the ph switch allows cov2 to have rbd/ace2 bonds with dwelling times of about ~ 300 s, commensurate with the diffusion time scales needed to recruit enough ace2 receptors to trigger endocytosis. this mechanism implies that, for the most part, cov1 will not co-localize in the nasal cavity. this prediction is consistent with cov1 being mainly a lower respiratory tract disease, causing complications that include acute respiratory distress (ding et al., 2003; hamming et al., 2004) . viral replication in human mucous gland cells will release viruses back into the same area where they can infect new cells until the supply of ace2 receptors is depleted below the critical threshold needed for binding and internalization. this process will trap viral particles in the upper respiratory tract, naturally leading to longer incubation times. similarly, accumulation of viral particles in the nasal mucosa will lead to extended periods of viral shedding. of note, since viral transit to the lower respiratory tract will be significantly slower for cov2 relative to cov1, this period of higher infectivity rates could be for the most part mediated by asymptomatic individuals. based on our findings, incubation times should correlate with the number of ace2-rich cells in the nasal area. it is important to note that children do not have well developed sinuses until adolescence (henson et al., 2020) . thus, large areas for viral replication will not be available in children, resulting in shorter incubation times due to the faster diffusion down to the lower respiratory tract. something similar could apply to females who have smaller nasal cavities relative to males (samolinski et al., 2007) . shorter times in the nasal cavity would lead to a lower viral load in the upper airways and could explain the lower transmission and milder symptoms that are observed in children, as well as the lower ifr in adult women relative to men. our proposed mechanism is also consistent with reported loss of sense of smell (anosmia) that may occur by day 3 of a cov2 infection (speth et al., 2020) , as cells in the nasal cavities support olfactory mucous membranes needed for the perception of smell. proximity to the brain also suggest that cov2 infections could impact the brain in ways that other sars viruses cannot. moreover, cardiovascular and immunological complications triggered by cov2 could also be explained on the basis of long-term insult of endothelial cells by viral sequestration of the ace2 receptor (gurley and coffman, 2008) . ph-switch across species. further supporting the observation that cov2 is unique among other coronaviruses is shown in table 2 that compares sequence alignments of ph domains in rbds of both cov1, cov2, mers, as well as other closely related zoonotic viruses. cov2, and related coronaviruses in one species of pangolin and some bats do not share the ph-switch present in cov1, instead they share the lys458 stabilization motif. however, these zoonotic viruses still have ph-switches that co-localize next to the ph-switch in the rbd of mers structure (fig. 1) . interestingly, different bat-infecting strains show putative ph-switches that are closer in both sequence and structure to either mers or pangolin-associated coronavirus. while we have not yet found the species or strain where the loss of the ph-switch first occurred, these relationships point at the possible zoonotic origin of cov2 as well as evolutionary pressures to preserve the phswitch. it is noteworthy that dpp4, the receptor of the mers rbd, is not found in nasal epithelial cells (meyerholz et al., 2016) . outlook. this newly discovered difference in protein sequence in the receptor binding domain of the spike glycoprotein and its impact on receptor binding reveals a mechanism that allows sars-cov2 internalization to take advantage of the high expression of ace2 in the nasal epithelium¾resulting in increased retention times in the upper respiratory tract and augmented infectivity. this mechanism reconciles observed epidemiological traits and pathologies specific to sars-cov2 and explains differences with those associated with sars-cov, which due to its stick-and-slip ph-switch is unable to efficiently undergo endocytosis in the nasal cavity. sars-cov also has a higher infected-fatality rate than sars-cov2. while the evolutionary advantage of higher infectivity by sars-cov2 in the nasal area is clear, this property comes at the expense of an important regulatory mechanism that would have allowed this virus to more readily move in other organs and tissues. in fact, the life-cycle of sars-cov2 is significantly slower than that of sars-cov because cov2 is essentially immobilized at its initial cell receptor contact. thus, it seems unlikely that the diffusion limited recruitment of ace2 receptors affecting the virus in the respiratory airways would also be the limiting step in tissues. after internalization, the virus is encapsulated in a vesicle supported by rbd/ace2 complexes. the actual final number of complexes in each vesicle should vary above a given threshold, though not much is known about the details of this process. contrary to sars-cov, cov2 complexes would be expected to have greater difficulty slipping and breaking. it is not difficult to imagine that for vesicles compressed by an excess of receptors the fusion with the early endosome might be hindered, hosting a population of viruses that could stay latent or activate at much later times. this simple mechanism could underlie the still anecdotal evidence for infection recurrence (chen et al., 2020) , as well as extremely long-term of viral shedding. collectively, our studies provide insight pertinent to the molecular basis of viral infectivity and, at the same time, validate this form of thermodynamic and molecular modeling as an approach to probe the evolution of the next sars-mediated pandemic. from a therapeutic perspective, our findings linking viral pathology with long-term viral infection/retention in nasal epithelium of the upper respiratory tract suggest that vaccine development should not just concentrate on fighting systemic infection through induction of igg responses, but should instead aim to elicit high titers of secretory iga antibodies capable of neutralizing the virus in the nasal mucosa. therefore, intranasal delivery of a vaccine with strong iga producing potential is a logical approach to consider as the next step in countering the current and future pandemics that, like sars-cov2, export evolutionarily successful strains via higher transmission rates. surface representation of the co-crystals reveal two characteristic lobes with flat and mostly superficial contacts. yellow surface corresponds to ph-switch loop and red surface indicates remaining of binding interface. covid-19 and the heart imbalanced host response to sars-cov-2 drives development of covid-19 effect of m1 protein and low ph on nuclear transport of influenza virus ribonucleoproteins kinetics of desolvation-mediated protein-protein binding fastcontact: a free energy scoring tool for protein-protein complex structures recurrence of positive sars-cov-2 rna in covid-19: a case report the reaction-limited kinetics of membrane-to-surface adhesion and detachment the clinical pathology of severe acute respiratory syndrome (sars): a report from china angiotensin-converting enzyme 2 gene targeting studies in mice: mixed messages tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis anatomy, head and neck, nose sinuses paradigm shift of the plasma membrane concept from the twodimensional continuum fluid to the partitioned fluid: high-speed single-molecule tracking of membrane molecules structure of sars coronavirus spike receptor-binding domain complexed with receptor transport of incoming influenza virus nucleocapsids into the nucleus dipeptidyl peptidase 4 distribution in the human respiratory tract: implications for the middle east respiratory syndrome structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody real-time dissection of dynamic uncoating of individual influenza viruses dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc changes in nasal cavity dimensions in children and adults by gender and age structural basis of receptor recognition by sars-cov-2 single-particle tracking of immunoglobulin e receptors (fcepsilonri) in micron-sized clusters and receptor patches otolaryngol head neck surg function, and antigenicity of the sars-cov-2 spike glycoprotein sars coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic pathway structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 structural and functional basis of sars-cov-2 entry by using human ace2 quantum dots crack the influenza uncoating puzzle structural definition of a unique neutralization epitope on the receptor-binding domain of mers-cov spike glycoprotein the protein data bank the pymol molecular graphics system, version 1.2r3pre, schrödinger, llc routine microsecond molecular dynamics simulations with amber on gpus. 1. generalized born routine microsecond molecular dynamics simulations with amber on gpus. 2. explicit solvent particle mesh ewald simmerling. ff14sb: improving the accuracy of protein side chain and backbone parameters from ff99sb development and testing of a general amber force field software for processing and analysis of molecular dynamics trajectory data vmd -visual molecular dynamics acknowledgements. this work was supported by nih gm097082, ns043277 and protonated state of h34 + is predicted to form stable h-bond network with d30 in ace2, and y440 and y453 in (a) cov1 and (b) cov2, respectively. h-bond network is based on rotamers already observed in co-crystals of the unprotonated forms: (c) pdb 2ajf for cov1, and, (d) pdb 6lzg for cov2 (see also pdb 6m0j). unprotonated co-crystal structures of cov2 assigned h34 with a ndh making a bond with backbone oxygen of d30, which is already making a bond in the a-helix.our mds indicate that even in the unprotonated form the rotamer should be rotated 180 o having nd and ne more readily interacting with d30 and y453 (as shown in panel d). figure 3 . role of ph switch in cov1 relative to cov2. (a) and (b) show the same co-crystals as in fig. 1 superimposed with centroid of largest 1.8 å rmsd cluster of conformations of phswitching loop from mds shown in (c) for cov1 and (d) for cov2. also indicated is the size of the corresponding cluster relative to simulation time. (c) root-mean-squared-deviation (rmsd) of 16 amino acid loop as a function of time that switches between not-bound-like (~ 5.8 å) to bound-like (~ 1.9 å) relative to co-crystal (pdb 2ajf), for his and his + , respectively; (d) same analysis for cov2 homologous loop shows most conformations under 2 å rmsd relative to pdb 6lzg. binding interface, not including loop, stays in a bound-like conformation for 100% of the simulation time for both (e) cov1 and (f) cov2. atomic coordinates for starting structures were acquired from the protein data bank [1] : 2dd8 was used for cov1 rbd and 6lzg was used for the cov2 rbd. the rbd from 2dd8 (bound to neutralizing antibody) was chosen instead of that in pdb id 2ajf (bound to ace2) as a starting structure as it includes otherwise missing portions of the domain. modification of his to different tautomeric or protonation states was done with pymol's mutagenesis wizard [2] . molecular dynamics simulations (mds) were carried out with pmemd.cuda from amber18 [3] [4] [5] using amber ff14sb force field [6] and generalized amber force field (gaff) [7] . we used tleap binary (part of amber18) for solvating the structures in a cubed tip3p water box with a 10 å distance from structure surface to the box edges, and closeness parameter of 0.75 å. the system was neutralized and solvated. simulations were carried out after minimizing the system, gradually heating the system from 0 k to 3 00k over 50 ps, and equilibrating the system for 1 ns at npt. 500 ns of production was then carried out using npt at 300 k with the langevin thermostat, a non-bonded interaction cut off of 8 å, time step of 2 fs, and the shake algorithm to constrain all bonds involving hydrogens. clustering was completed using cpptraj [8] and h-bond and rmsd calculations were done with vmd [9] . all figures were drawn using pymol [2] and gnuplot. key: cord-284102-rovyvv45 authors: wagner, teresa r.; kaiser, philipp d.; gramlich, marius; becker, matthias; traenkle, bjoern; junker, daniel; haering, julia; dulovic, alex; schweizer, helen; nueske, stefan; scholz, armin; zeck, anne; schenke-layland, katja; nelde, annika; strengert, monika; walz, juliane s.; ruetalo, natalia; schindler, michael; schneiderhan-marra, nicole; rothbauer, ulrich title: neutrobodyplex nanobodies to monitor a sars-cov-2 neutralizing immune response date: 2020-09-28 journal: biorxiv doi: 10.1101/2020.09.22.308338 sha: doc_id: 284102 cord_uid: rovyvv45 as the covid-19 pandemic escalates, the need for effective vaccination programs, diagnosis tools and therapeutic intervention ever increases. neutralizing binding molecules have become important tools for acute treatment of covid-19 and also provide a unique possibility to monitor the emergence and presence of a neutralizing immune response in infected or vaccinated individuals. here we identified 11 unique nanobodies (nbs) with high binding affinities to the sars-cov-2 spike receptor domain (rbd). of these, 8 effectively block the rbd:ace2 interface. via competitive binding analysis and detailed epitope mapping, we grouped all nbs into 3 sets and demonstrated their neutralizing effect. combinations from different sets showed a profound synergistic effect by simultaneously targeting different epitopes within the rbd. finally, we established a competitive multiplex binding assay (“neutrobodyplex”) enabling the detection of neutralizing antibodies in serum of infected patients. overall, our nbs have high potential for prophylactic and therapeutic options and provide a novel approach to screen for a neutralizing immune response in infected or vaccinated individuals, helping to monitor immune status or guide vaccine design. 7 phycoerythrin (pe)-labeled streptavidin after stringent washing. additionally, a non-specific nb 135 (gfp-nb, negative control) and two inhibiting mouse antibodies (positive controls) were 136 analyzed 21 . data obtained by this multiplex binding assay showed that 8 of the 10 analyzed 137 nbs inhibit ace2 binding to isolated rbd, s1 domain and homotrimeric spike. ic50 values 138 calculated for inhibition of ace2:rbd interaction ranges between 0.5 nm for nm1228 and 38 139 nm for nm1229 (figure 3) . notably, ic50 values obtained for the most potent inhibitory nbs 140 nm1228 (0.5 nm), nm1226 (0.85 nm) and nm1230 (2.12 nm) are highly comparable to ic50 141 values measured for the mouse iggs (mm43: 0.38 nm; mm57: 3.22 nm). additionally, the 142 assay revealed that all nbs except nm1224, show a similarly strong inhibitory effect of ace2 143 binding to all tested antigens. nm1224 seems to exclusively inhibit rbd:ace2 interaction and 144 does not prevent binding of ace2 to either the homotrimeric spike or the s1 domain. 145 146 after identifying rbd-specific nbs which have an inhibitory effect on ace2 binding, we 148 investigated the relative location of their epitopes within the rbd. firstly, we first performed 149 epitope binning experiments of nb combinations using biolayer interferometry. after coating 150 sensors with biotinylated rbd, a nb was loaded until binding saturation was reached, followed 151 by a short dissociation step to remove excess nb. a second nb from a different family was 152 then exposed to the rbd-nb-complex. using this approach, we identified nbs which recognize 153 overlapping and non-overlapping epitopes on rbd (figure 4, supplementary figure 2) . as 154 expected nbs with only minor differences in their cdr3 (nm1221, nm1222 and nm1230, nb-155 set 2) were suggested to recognize an identical or highly similar epitope as they cannot bind 156 simultaneously to rbd. our analysis revealed that nbs with highly diverse cdr3s such as 157 nm1228, nm1226, nm1227 and nm1229 could not bind simultaneously, suggesting that these 158 nbs recognize similar or at least overlapping epitopes. as a result, we clustered these diverse 159 nbs in nb-set 1. overall, we identified five distinct nbs-sets, comprising at least one candidate 160 targeting a different epitope within the rbd compared to any member of a different nb-set 161 (figure 4) . 162 8 next, we performed hydrogen-deuterium exchange mass spectrometry (hdx-ms) with the 164 most potent inhibitory nbs selected from the different nb-sets. this allowed us to more 165 precisely locate their binding sites at the surface of rbd and compare with the rbd:ace2 166 interface. both members of nb-set1, nm1226 and nm1228, interacted with the rbd at the 167 back/ lower right site (back view, figure 5) . notably, the binding site of nm1226 does not 168 encompass amino acid residues involved in the rbd:ace2 interface. in contrast, nm1228 169 (nb-set1) as well as nm1230 (nb-set2) contacted the rbd at amino acid residues overlapping 170 with the rbd:ace2 binding interface, whereas nm1230 additionally covers parts of the spike-171 like loop region on one edge of the ace2 interface at the top front/ lower left side (front view, 172 which did not contact any amino acid residues involved in the rbd:ace2 interface but rather 179 binds to the opposite site (front view, figure 5 ). comparing the data from epitope binning 180 with the hdx-ms results, provides structural insights into the mechanism by which non-181 competing pairs of nbs can simultaneously bind the rbd. interestingly, the combination of 182 nm1228 (nb-set1) with nm1230 (nb-set2) shows near complete coverage of the ace2 183 interface (figure 5) whereas the observed inhibitory effect of nm1226 might be due to steric 184 hindrance. from these findings, we proposed that the combination of nb-set1 with nb-set2 185 might act synergistically on the inhibition of the interaction between rbd and ace2. 186 after identification of nbs which inhibit the rbd:ace2 interaction biochemically, we employed 189 a cell-based viral infection assay to test for their neutralization potency. to this end, human 190 9 caco-2 cells were co-incubated with the icsars-cov-2-mng strain and serial dilutions of the 191 inhibitory nbs nm1224, nm1226, nm1228 and nm1230. 48 h post-infection neutralization 192 potency was determined via automated fluorescence-microscopy of fixed and nuclear-stained 193 cells (supplementary figure 4) . percentage of the infection rate following nb treatment 194 normalized to a non-treated control was plotted and ic50 values were determined via sigmoidal 195 inhibition curve fits. overall, data obtained from the multiplex binding assay and the viral 196 infection assay were broadly consistent. representatives of nb-set1, nm1226 and nm1228, 197 showed the highest neutralization potency with ic50 values of ~15 nm and ~7 nm followed by 198 nm1230 (~37 nm) and nm1224 (~256 nm). as expected, nm1223 (nb-set3) was not found to 199 considering that nbs targeting diverse epitopes within the rbd:ace2 interface are beneficial 201 in both reducing viral infectivity and preventing mutational escape, we next combined the most 202 potent inhibitory and neutralizing candidates derived from nb-set1 (nm1226, nm1228) and 203 nb-set2 (nm1230) and examined their response in both the multiplex binding assay and viral 204 infection assay. in the multiplex binding assay the combination of nm1226 and nm1230 205 showed an increased effect in competing with ace2 binding to rbd illustrated by a ic50 of 206 0.42 nm which is 2-or 5-fold lower compared to treatment with individual nm1226 or nm1230, 207 respectively (figure 7 a) . notably, the ic50 measured for the combination of nm1228 and 208 nm1230 did not exceed the ic50 identified for nm1228 alone indicating that nm1228 by its own 209 has a very high inhibiting effect (figure 7 a). when we tested both combinations in the viral 210 infection assay, we observed significantly improved effects in both as illustrated by an ic50 of 211 ~4 nm for the combination nm1226 and nm1230 and ~3.5 nm for nm1228 and nm1230 212 (figure 7 b, supplementary figure 5 ). from these findings we conclude, that a combinatorial 213 treatment with two nbs targeting different epitopes within the rbd:ace2 interaction site is 214 beneficial for viral neutralization. context, multiple studies have convincingly shown that neutralizing antibodies preferable bind 226 to the rbd domain and sterically inhibit viral entry via ace2 1,3 . from this, we can assume that 227 our rbd nbs covering large parts of the rbd:ace2 interface might be suitable to monitor the 228 emergence and presence of neutralizing antibodies in patients. to test this hypothesis, we set 229 up a high-throughput competitive binding assay, termed neutrobodyplex, by combining our 230 most potent neutralizing nb combinations with a recently developed, automatable multiplex 231 immunoassay (figure 8 a) 20 . we incubated our previously generated color-coded beads 232 comprising rbd, s1 domain or homotrimeric spike with serum samples from patients or non-233 infected individuals, in addition to dilution series of the combinations nm1226/ nm1230 or 234 nm1228/ nm1230 and used this to detect patient-derived iggs bound to the respective 235 antigens. depending on the nb concentration, neutralizing antibodies targeting the rbd:ace2 236 interaction site within the serum samples are displaced resulting in a reduction of the 237 detectable signal (figure 8 a) . 238 when analyzing rbd specific iggs from serum samples, we detected a distinct signal 239 reduction in the presence of increasing nb concentrations for all tested samples (figure 8 to further demonstrate that our approach is able to determine the presence of iggs targeting 246 the rbd:ace2 interaction site in detailed resolution, we highlight here the effect of competing 247 iggs could be observed when measuring binding to rbd, however using the s1 domain as 249 target antigen distinct differences between both serum samples became visible. while #289 250 comprise a substantial fraction of iggs addressing the rbd:ace2 interface also presented by 251 the s1 domain, in sample #265 iggs binding to additional epitopes of the s1 domain cover the 252 detectable signal reduction derived from displaced iggs (figure 8 for functional analysis we employed a recently developed in vitro multiplex binding assay 20 to 291 monitor the replacement of ace2 as the natural ligand from binding to rbd, s1 domain or 292 homotrimeric spike upon addition of rbd-specific nbs. with this assay, we were able to identify 293 8 inhibiting nbs targeting those spike-derived antigens. interestingly, ic50 values obtained for 294 inhibitory nbs on rbd and homotrimeric spike show a higher correlation compared to ic50 295 values obtained for the s1 domain. based upon detailed epitope mapping, we grouped our 296 nbs in 5 different nb-sets. 3 of those nb-sets, comprise inhibitory nbs which were shown to 297 target different epitopes within the rbd:ace2 interaction site. we confirmed the neutralizing 298 potency of those nbs in a cell-based viral infection assay using fully intact sars-cov-2. 299 through this, we noted that the measurable viral neutralization effect of the individual nbs 300 strongly correlates to the data obtained from the biochemical screen, which demonstrates that 301 13 the multiplex binding assay as presented is highly relevant and suitable to identify virus 302 neutralizing binders. as a result, we modified our previously described multiplex immunoassay 303 (multicov-ab, 20 ) and developed a novel diagnostic test called neutrobodyplex to monitor 304 the presence and the emergence of neutralizing antibodies in serum samples of sars-cov-2 305 infected individuals. using combinations of high affinity nbs covering the rbd:ace2 interface, 306 we were able to directly and specifically displace iggs present in serum samples from these 307 particular rbd epitopes. according to previous studies, human iggs addressing those 308 epitopes were classified as neutralizing antibodies 1,17,18 . in our neutrobodyplex, we further 309 demonstrated that such neutralizing antibodies can be detected best using the rbd. larger expression constructs for bacterial expression of nbs, sequences were cloned into the 331 phen6 vector 28 , thereby adding a c-terminal 6xhis-tag for imac purification as described 332 previously 29,30 . the pcaggs plasmids encoding the stabilized homotrimeric spike protein and 333 the receptor binding domain (rbd) of sars-cov-2 were kindly provided by f. krammer 19 . 334 the cdna encoding the s1 domain (aa 1 -681) of the sars-cov-2 spike protein was obtained 335 by pcr amplification using the forward primer s1_cov2-for 5´-ctt ctg gcg tgt gac 336 cgg -3´ and reverse primer s1_cov2-rev 5´ -gtt gcg gcc gct tag tgg tgg tgg with high-confidence identification (q-value ≤ 0.01) were included to the list. peptides with 499 overlapping mass, retention time and charge in nb and antigen digest, were manually 500 removed. the deuterated samples were recorded in ms mode only and the generated peptide 501 list was imported into hdexaminer v2.5.0 (sierra analytics, modesto, ca, usa). deuterium 502 uptake was calculated using the increase of the centroid mass of the deuterated peptides. 503 hdx could be followed for 79% of the rbd amino acid sequence. the calculated percentage 504 deuterium uptake of each peptide between rbd-nb and rbd-only were compared. any 505 peptide with uptake reduction of 5% or greater upon nb binding was considered as protected. 506 507 cell culture caco-2 (human colorectal adenocarcinoma) cells were cultured at 37°c with 5% 508 co2 in dmem containing 10% fcs, 2 mm l-glutamine, 100 μg/ml penicillin-streptomycin and 509 1% neaa. results from bead-based multiplex ace2 competition assay are shown for the three sars-582 cov-2 spike-derived antigens, rbd, s1 and homotrimeric spike. ace2 bound to the respective 583 antigen was detected. for each nb, a dilution series from 2.106 µm to 0.123 nm is shown in 584 the presence of 80 ng/ml ace2. mfi signals were normalized to the maximal signal per 585 antigen as given by the ace2-only control. ic50 values were calculated from a four-parametric 586 sigmoidal model and are displayed for each nb and antigen. data is presented as mean +/-587 sd of three technical replicates (n =3). 588 isolation of potent sars-cov-2 neutralizing antibodies and 667 protection from disease in a small animal model structural basis for the recognition of sars-cov-2 by full-length human human neutralizing antibodies elicited by sars-cov-2 infection characterization of the receptor-binding domain (rbd) of 2019 novel 674 coronavirus: implication for development of rbd protein as a viral attachment inhibitor 675 and vaccine nanobodies: natural single-domain antibodies structural basis for potent neutralization of betacoronaviruses by 679 neutralizing nanobodies bind sars-cov-2 spike rbd and block 681 interaction with ace2 an ultra-high affinity synthetic nanobody blocks sars-cov-2 infection 683 by locking spike into an inactive conformation. biorxiv humanized single domain antibodies neutralize sars-cov-2 by 686 targeting spike receptor binding domain. biorxiv an alpaca nanobody neutralizes sars-cov-2 by blocking receptor 689 interaction. biorxiv affinity nanobodies block sars-cov-2 spike receptor binding domain interaction 692 with human angiotensin converting enzyme. biorxiv, 2020 fast isolation of sub-nanomolar affinity alpaca nanobody against the 695 spike rbd of sars-cov-2 by combining bacterial display and a simple single-step 696 density gradient selection. biorxiv multivalent nanobody cocktails for highly efficient sars-699 a potent neutralizing nanobody against sars-cov-2 with inhaled delivery 702 potential. biorxiv spike mutation pipeline reveals the emergence of a more transmissible 704 form of sars-cov-2. biorxiv tracking changes in sars-cov-2 spike: evidence that d614g increases infectivity of the covid-19 virus potent neutralizing antibodies against sars-cov-2 identified by high-710 throughput single-cell sequencing of convalescent patients' b cells a neutralizing human antibody binds to the n-terminal domain of the spike 713 protein of sars-cov-2 a serological assay to detect sars-cov-2 seroconversion in humans going beyond clinical routine in sars-cov-2 antibody testing -a 717 multiplex corona virus antibody test for the evaluation of cross-reactivity to endemic 718 coronavirus antigens. medrxiv quantum dot-conjugated sars-cov-2 spike pseudo-virions 721 enable tracking of angiotensin converting enzyme 2 binding and endocytosis evaluation of nine commercial sars-cov-2 immunoassays convergent antibody responses to sars-cov-2 in convalescent 726 individuals a translational multiplex serology approach to profile the prevalence 728 of anti-sars-cov-2 antibodies in home-sampled blood. medrxiv a high-throughput neutralizing antibody assay for covid-19 731 diagnosis and vaccine evaluation speed up to find the right ones: rapid discovery of functional nanobodies a sars-cov-2 surrogate virus neutralization test based on antibody-736 mediated blockage of ace2-spike protein-protein interaction selection and identification of single domain antibody fragments from camel heavy-740 chain antibodies modulation of protein properties in living cells using nanobodies a versatile nanotrap for biochemical and functional studies with 745 fluorescent fusion proteins targeting and tracing antigens in live cells with fluorescent 748 nanobodies sars-cov-2 seroconversion in humans: a detailed protocol for 750 antigen production, and test setup deuterium exchange mass spectrometry to study protein complexes optimization of feasibility stage for hydrogen/deuterium 756 structure of the sars-cov-2 spike receptor-binding domain bound to the 759 ace2 receptor s1 domain or homotrimeric spike of sars-cov-2 was incubated with nb combinations 442 (concentrations ranging from 1.26 µm to 0.08 nm for each nb) and serum samples of 443 convalescent sars-cov-2 patients and healthy donors at a 1:400 dilution. as positive control 444 and maximal signal detection per sample, serum only was included and as negative control for 445nb binding a sars-cov-2-unspecific gfp nanobody (1.26 µm) was used. to compare nb 446 performance, the inhibiting mouse antibody (40591-mm43) was added in concentrations of 447 0.17 µm to 0.08 nm. bound serum iggs were detected via anti-human-igg-pe as previously 448 and fragments mass tolerance were set to 6 ppm and 0.05 da, respectively. no enzyme 495 selectivity was applied, however, identified peptides were manually evaluated to exclude 496 peptides originated through cleavage after arginine, histidine, lysine, proline and the residue key: cord-285758-c18arb6s authors: jiang, shibo; he, yuxian; liu, shuwen title: sars vaccine development date: 2005-07-17 journal: emerg infect dis doi: 10.3201/eid1107.050219 sha: doc_id: 285758 cord_uid: c18arb6s developing effective and safe vaccines is urgently needed to prevent infection by severe acute respiratory syndrome (sars)–associated coronavirus (sars-cov). the inactivated sars-cov vaccine may be the first one available for clinical use because it is easy to generate; however, safety is the main concern. the spike (s) protein of sars-cov is the major inducer of neutralizing antibodies, and the receptor-binding domain (rbd) in the s1 subunit of s protein contains multiple conformational neutralizing epitopes. this suggests that recombinant proteins containing rbd and vectors encoding the rbd sequence can be used to develop safe and effective sars vaccines. safe vaccines is urgently needed to prevent a new sars epidemic and for biodefense preparedness. currently, 3 major classes of sars vaccines are under development: 1) inactivated sars-cov (figure 1 ), 2) full-length s protein (figure 2a) , and 3) those based on fragments containing neutralizing epitopes ( figure 2b ). sars-cov expresses several structural proteins, including nucleocapsid, membrane, envelope, and spike (s) proteins (1) . all may serve as antigens to induce neutralizing antibodies and protective responses. in general, prior to identification of the protein that contains the major neutralizing epitopes, the inactivated virus may be used as the first-generation vaccine because it is easy to generate whole killed virus particles. however, once the neutralizing epitopes are identified, the inactivated virus vaccine should be replaced by vaccines based on fragments containing neutralizing epitopes since they are safer and more effective. several reports have showed that sars-cov inactivated with formaldehyde, uv light, and β-propiolactone can induce virus-neutralizing antibodies in immunized animals (8) (9) (10) (11) , and the first inactivated sars-cov vaccine is being tested in the clinical trials in china. however, safety of the inactivated vaccine is a serious concern; production workers are at risk for infection during handling of concentrated live sars-cov, incomplete virus inactivation may cause sars outbreaks among the vaccinated populations, and some viral proteins may induce harmful immune or inflammatory responses, even causing sars-like diseases (12, 13) . the s protein of sars-cov, a type i transmembrane glycoprotein, is responsible for virus binding, fusion, and entry and is a major inducer of neutralizing antibodies (1, 14) . s protein consists of a signal peptide (sp: amino acids [aa] 1-12) and 3 domains: an extracellular domain (aa 13-1193), a transmembrane domain (aa 11194-1215), and an intracellular domain (aa 1216-1255). its extracellular domain consists of 2 subunits, s1 and s2 (14) , although the cleavage site between these subunits has not been clearly defined. the s1 subunit is responsible for virus binding to the receptor, angiotensin-converting enzyme 2 (ace2) (15, 16) . a fragment located in the middle region of the s1 subunit (aa 318-510) is the receptor-binding domain (rbd) for ace2 (17) (18) (19) . sars-cov may also bind to cells through the alternative receptors dc-sign or l-sign (20, 21) , but the binding sites for these alternative receptors have not been defined. the s2 subunit, which contains a putative fusion peptide and 2 heptad repeats (hr1 and hr2), is responsible for fusion between the viral and target cell membranes. infection by sars-cov is initiated by binding of rbd in the viral s protein s1 subunit to ace2 on target cells. this forms a fusogenic core between the hr1 and hr2 regions in the s2 domain that brings the viral and target cell membranes into close proximity, which results in virus fusion and entry (22) (23) (24) . this scenario indicates that the s protein may be used as a vaccine to induce antibodies for blocking virus binding and fusion. several recombinant vector-based vaccines expressing sars-cov s protein have been assessed in preclinical studies. yang et al. (25) reported that a candidate dna vaccine encoding the full-length s protein induced neutralizing antibodies (neutralizing titers ranging from 1:50 to 1:150) and protected mice from sars-cov challenge. using dna vaccines encoding the full-length and segments of s protein to immunize rabbits, wang et al. have produced higher titers of neutralizing antibodies and demonstrated that major and minor neutralizing epitopes are located in the s1 and s2 subunits, respectively (26) . other groups also found neutralizing epitopes in the s2 subunit (27, 28) . bisht et al. (29) have shown that intranasal or intramuscular inoculations of mice with highly attenuated modified vaccinia virus ankara (mva) vaccines encoding full-length sars-cov s protein also produce neutralizing antibodies with mean neutralizing titers of 1:284. bukreyev et al. (30) reported that mucosal immunization of african green monkeys with an attenuated parainfluenza virus expressing s protein resulted in production of neutralizing antibodies and protected animals from infection by challenge with sars-cov. these data suggest that the s protein can induce neutralizing antibodies and protective responses in immunized animals. using convalescent-phase sera from sars patients and a set of peptides spanning the entire sequence of the sars-cov s protein, we have identified 5 linear immunodominant sites (ids) in the s protein ( figure 2a ). ids i, ii, iii, and v reacted with >50% of the convalescent-phase sera from sars patients, while ids iv was reactive with >80% of sars sera, suggesting that ids iv is the major immunodominant epitope on the s protein (31) . synthetic peptides corresponding to ids could induce high titers of s protein-specific antibodies, but none of these antibodies possesses neutralizing activity. these findings suggest that the ids in s protein may not induce neutralizing antibodies. whether these antibodies enhance infection by heterologous sars-cov strains or mediate harmful immune responses is unclear. the s protein of fipv expressed by recombinant vaccinia can cause antibody-dependent enhancement of disease if vaccinated animals are subsequently infected with wild-type virus (32) . our previous studies on hiv-1 showed that antibodies against some immunodominant epitopes in the hiv-1 envelope glyco rbd, a fragment (≈193 aa residues) in the middle of s1 subunit of s protein ( figure 2b ), is responsible for virus binding to the receptor on target cells. we have demonstrated that the antisera from sars patients and from animals immunized with inactivated sars-cov reacted strongly with rbd (9, 35) . absorption of antibodies by rbd from these antisera results in the removal of most of the neutralizing antibodies, and rbd-specific antibodies isolated from these antisera have potent neutralizing activity (35, 36) . we have also shown that rabbits and mice immunized with rbd produced high titers of neutralizing antibodies against sars-cov with 50% neutralizing titers at a >1:10,000 serum dilution (37) . the immunized mice were protected from sars-cov challenge (unpub. data). the antibodies purified from the antisera against sars-cov significantly inhibited rbd binding to ace2 (9, (36) (37) (38) . using spleen cells from mice immunized with rbd, we have generated a panel of 25 monoclonal antibodies (mabs) that recognize different conformational epitopes on rbd and possess potent neutralizing activity (38) . our result is in agreement with the report by van den brink et al. (39) , who identified 3 human neutralizing anti-s mabs from antibody phage display libraries by using inactivated sars-cov as the target. these researchers also found that all of these mabs specifically bound to rbd and blocked interaction between rbd and ace2. these findings suggest that rbd contains the major neutralizing epitopes in the s protein and is an ideal sars vaccine candidate because rbd contains the receptor-binding site, which is critical for virus attachment to the target cell for infection (15, (17) (18) (19) . antibodies specific for rbd are expected to block binding of virus to the target cell. rbd induces higher titers of neutralizing antibodies than those vaccines expressing the full-length s protein (25, 26, 29, 30, 37, 38) . rbd sequences among the late sars-cov strains are highly conserved. when the early and late sars-cov strains are compared, only 3 to 5 aa residues are variable among the 193 residues in rbd and most of the isolates vary by only 1 residue (4). van den brink et al. (39) showed that 1 human mab (cr3014) specific for rbd of sars-cov strain fm1 can effectively bind to most rbds of the early and late sars-cov strains. these data suggest that antibodies directed against rbd of a sars-cov isolate may neutralize infection by a broad spectrum of sars-cov strains. therefore, recombinant proteins containing rbd or vectors encoding rbd may be used as vaccines for preventing infection by sars-cov with distinct genotypes. an ideal sars vaccine should 1) elicit highly potent neutralizing antibody responses against a broad spectrum of viral strains; 2) induce protection against infection and transmission; and 3) be safe by not inducing any infectionenhancing antibodies or harmful immune or inflammatory responses. currently, an inactivated sars-cov vaccine is in clinical trials in china. safety is the major concern for this type of vaccine (12) . the s protein is the major inducer of neutralizing antibodies. recombinant vector-based vaccines expressing full-length s protein of the late sars-cov are under development. these vaccines can induce potent neutralizing and protective responses in immunized animals but may induce antibodies that enhance infection by early human sars-cov and animal sars-cov-like viruses (6) . recent studies have demonstrated that recombinant rbd consists of multiple conformational neutralizing epitopes that induce highly potent neutralizing antibodies against sars-cov (9, 26, (35) (36) (37) (38) . unlike fulllength s protein, rbd does not contain immunodominant sites that induce nonneutralizing antibodies. rbd sequences are relatively conserved. thus, recombinant rbd or vectors encoding rbd may be used as safe and efficacious vaccines for preventing infection by sars-cov with distinct genotypes. dr. jiang is associate member and head of the viral immunology laboratory, lindsley f. kimball research institute, new york blood center. his primary research interests include development of vaccines and therapeutic agents against sars-cov and hiv. severe acute respiratory syndrome unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage isolation and characterization of viruses related to the sars coronavirus from animals in southern china molecular evolution of the sars coronavirus during the course of the sars epidemic in china molecular epidemiology of the novel coronavirus that causes severe acute respiratory syndrome evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) immunogenicity of sars inactivated vaccine in balb/c mice inactivated sars-cov vaccine elicits high titers of spike protein-specific antibodies that block receptor binding and virus entry epitope mapping and biological function analysis of antibodies produced by immunization of mice with an inactivated chinese isolate of severe acute respiratory syndrome-associated coronavirus (sars-cov) intranasal immunization with inactivated sars-cov (sars-associated coronavirus) induced local and serum antibodies in mice caution urged on sars vaccines glycan arrays lead to the discovery of autoimmunogenic activity of sars-cov sars-associated coronavirus angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus a model of the ace2 structure and function as a sars-cov receptor a 193-amino-acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme 2 the sars-cov s glycoprotein: expression and functional characterization the secret life of ace2 as a receptor for the sars virus ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign cd209l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus interaction between the heptad repeat 1 and 2 regions in spike protein of sarsassociated coronavirus: implication for virus fusogenic mechanism and identification of fusion inhibitors structural characterization of the sars-coronavirus spike s fusion protein core crystal structure of sars-cov spike protein fusion core a dna vaccine induces sars coronavirus neutralization and protective immunity in mice identification of two neutralizing regions on the severe acute respiratory syndrome coronavirus spike glycoprotein produced from the mammalian expression system amino acids 1055 to 1192 in the s2 region of severe acute respiratory syndrome coronavirus s protein induce neutralizing antibodies: implications for the development of vaccines and antiviral agents b-cell responses in patients who have recovered from severe acute respiratory syndrome target a dominant site in the s2 domain of the surface spike glycoprotein severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice mucosal immunisation of african green monkeys (cercopithecus aethiops) with an attenuated parainfluenza virus expressing the sars coronavirus spike protein for the prevention of sars identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (sars) coronavirus: implication for developing sars diagnostics and vaccines identification of antigenic sites mediating antibody-dependent enhancement of feline infectious peritonitis virus infectivity enhancement of human immunodeficiency virus type-1 (hiv-1) infection by antisera to peptides from the envelope glycoproteins gp120/gp41 immunization with modified vaccinia virus ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets identification of a critical neutralization determinant of severe acute respiratory syndrome (sars)-associated coronavirus: importance for designing sars vaccines recombinant modified vaccinia virus ankara expressing the spike glycoprotein of severe acute respiratory syndrome coronavirus induces protective neutralizing antibodies primarily targeting the receptor binding region receptor-binding domain of sars-cov spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine receptor-binding domain of sars coronavirus spike protein contains multiple conformationdependent epitopes that induce highly potent neutralizing antibodies molecular and biological characterization of human monoclonal antibodies binding to the spike and nucleocapsid proteins of severe acute respiratory syndrome coronavirus key: cord-262145-i29e3fge authors: huang, kuan-ying a.; tan, tiong kit; chen, ting-hua; huang, chung-guei; harvey, ruth; hussain, saira; chen, cheng-pin; harding, adam; gilbert-jaramillo, javier; liu, xu; knight, michael; schimanski, lisa; shih, shin-ru; lin, yi-chun; cheng, chien-yu; cheng, shu-hsing; huang, yhu-chering; lin, tzou-yien; jan, jia-tsrong; ma, che; james, william; daniels, rodney s.; mccauley, john w.; rijal, pramila; townsend, alain r. title: breadth and function of antibody response to acute sars-cov-2 infection in humans date: 2020-10-19 journal: biorxiv doi: 10.1101/2020.08.28.267526 sha: doc_id: 262145 cord_uid: i29e3fge serological and plasmablast responses and plasmablast-derived igg monoclonal antibodies (mabs) have been analysed in three covid-19 patients with different clinical severities. potent humoral responses were detected within 3 weeks of onset of illness in all patients and the serological titre was elicited soon after or concomitantly with peripheral plasmablast response. an average of 13.7% and 13.0% of plasmablast-derived mabs were reactive with virus spike glycoprotein or nucleocapsid, respectively. a subset of anti-spike (10 of 32) and over half of anti-nucleocapsid (19 of 35) antibodies cross-reacted with other betacoronaviruses tested and harboured extensive somatic mutations, indicative of an expansion of memory b cells upon sars-cov-2 infection. fourteen of 32 anti-spike mabs, including five anti-rbd, three anti-non-rbd s1 and six anti-s2, neutralised wild-type sars-cov-2 in independent assays. anti-rbd mabs were further grouped into four cross-inhibiting clusters, of which six antibodies from three separate clusters blocked the binding of rbd to ace2 and five were neutralising. all ace2-blocking anti-rbd antibodies were isolated from two patients with prolonged fever, which is compatible with substantial ace2-blocking response in their sera. at last, the identification of non-competing pairs of neutralising antibodies would offer potential templates for the development of prophylactic and therapeutic agents against sars-cov-2. in late 2019, a novel coronavirus emerged and was identified as the cause of a cluster 54 of respiratory infection cases in wuhan, china. it spread quickly around the world. in 55 march of 2020 a pandemic was declared by the world health organization, the virus 56 was formally named as severe acute respiratory syndrome coronavirus 2 (sars-57 cov-2) and the resulting disease was named covid-19. as of 1 october 2020, there (table 1) , suggesting the presence of conserved 137 epitopes on the spike glycoproteins of betacoronaviruses. 138 each of 32 anti-spike glycoprotein mabs was encoded by a unique set of heavy chain 139 vdj and light chain vj rearrangements in the variable domain (supplemental table 140 2). oc43 virus and three of these also cross-reacted on mers (table 1 ). all five cross-146 reactive anti-s2 antibodies had high rates of somatic mutation (25±5), indicating a 147 memory phenotype, and three of the five were neutralising to a moderate level (half 148 maximal effective concentration, ec 50 , 36-133.33 nm, table 1 ). 149 the cdr3 length varied among anti-spike glycoprotein antibodies (supplemental 150 table 2 ). no significant differences were found between anti-s2 and anti-s1 or anti-151 rbd subsets. among anti-s2 mabs, a significantly longer heavy chain cdr3 length 152 was found in the cross-reactive group compared to the specific group (cross-reactive 153 20±2 versus specific 12±4, p= 0.02, two-tailed mann-whitney test; figure 2c) , 154 indicating that a long cdr3 may play a role in antigen binding, which is also found in 155 several broadly reactive human mabs against human immunodeficiency virus and 156 influenza virus (9, 10). the binding activities of 10 anti-rbd mabs were further characterised in detail. 158 using mdck-siat1 cells transduced to express the rbd and flow cytometry, 159 binding activities of the anti-rbd mabs were shown to vary with 50% binding 160 concentration from 0.10 to 1.83 µg/ml (supplemental figure 2 ). the mabs with 161 strong anti-rbd binding have a relatively long heavy chain cdr3 length (50% 162 binding concentration <0.5 µg/ml versus >0.5 µg/ml, p=0.03, two-tailed mann-163 whitney test; supplemental figure 3 the 32 anti-spike glycoprotein mabs were systematically examined by plaque 173 reduction neutralisation (prnt) assay for neutralisation of wild type sars-cov-2 174 virus (see methods; summarised in table 1 ). a total of 14 neutralising antibodies 175 distributed between different regions of the spike glycoprotein were identified: 5 of 10 176 to rbd, 3 of 13 to s1 (non-rbd), 6 of 9 to s2. the ec 50 concentrations, as a 177 measure of potency, ranged from 0.05 to ~133 nm (8 ng/ml -~20 µg/ml). 178 (see methods): inhibition of virus replication was measured by quantitative pcr in the 182 supernatant bathing the infected cells. this results corroborated that anti-rbd fd 183 11a, anti-rbd fi 3a, anti-rbd fd 5d, anti-rbd ey 6a and anti-s2 ew 9c, as 184 crude culture supernatants, reduced the virus signal from ~56-to ~10,085-fold 185 (supplemental figure 5) . 186 potent neutralising antibodies to the rbd of sars-cov-2 spike glycoprotein were 188 identified and we thus analyse the blockade of the ace2-rbd interaction by anti-189 rbd antibodies in two assays ( figure 3 , table 1 the structure of vhh72-fc bound to rbd is known (17) and its footprint on the 198 rbd does not overlap that of ace2, so inhibition is thought to occur by steric 199 hindrance. 200 in the second assay, we employed mdck-siat1 cells overexpressing full-length 201 human ace2 as a transmembrane protein. unlabelled antibodies or ace2-fc were 202 mixed in excess with biotinylated rbd, and binding of rbd was detected with 203 streptavidin-hrp in elisa (figure 3b ). the results of this assay mostly mirrored 204 those of the first assay and confirmed that in this orientation anti-rbd neutralising 205 antibodies fd 11a and fd 5d competed in excess with soluble rbd for binding to 206 ace2 ( figure 3b ). in addition, anti-rbd neutralising antibody ey 6a competed with 207 rbd for ace2 binding. the binding pattern of ey 6a is analogous to a previously 208 described antibody cr3022 (table 1 ) (11). these two antibodies are known to bind to 209 the same region of rbd away from the ace2 binding site, but they influence the 210 binding kinetics of rbd to ace2, presumably through steric effects (15). 211 the ten anti-rbd mabs were then divided into cross-inhibiting groups as described 213 for human mabs to ebola (18) by assessing competition of unlabelled antibodies at 214 10-fold (or greater) excess over a biotin labelled target antibody by elisa. included 215 as controls were the vhh72-fc (17) and h11-h4-fc (14) the ten antibodies formed four cross-inhibiting clusters (table 2) , represented by 222 antibodies ey 6a (cluster 1, which included cr3022), fi 3a (cluster 2, which 223 included h11-h4), fd 11a (cluster 3, which included s309) and fj 10b (cluster 4). 224 the strongest inhibitors of ace2-fc binding were in clusters 2 and 3 (tables 1 and 2) . 225 neutralising antibodies were detected in clusters 1, 2 and 3, with the strongest 226 antibodies fi 3a and fd 11a being in clusters 2 and 3 (tables 1 and 2) . table 2 ) and did not cross-react strongly with other betacoronaviruses 259 (table 1) . fd 10a exhibits the most potent neutralising activity in the prnt assay 260 and also completely inhibits sars-cov-2-induced cytopathic effect (see methods) at 261 8.33 nm. 262 thirteen mabs were defined that bound the s1 region and three, close to germline in 264 sequence, were neutralising. fj 1c showed strong neutralisation (ec 50 55.5 nm), 265 whilst fd 11e (ec 50 70 nm) and fd 1e (ec 50 110 nm) were moderately neutralising 266 (table 1) the 35 mabs were evolved from 33 clonal groups defined by their heavy chain vdj 279 and light chain vj rearrangements (supplemental table 3 the presence of pre-existing immune memory to betacoronavirus that cross-react with 322 sars-cov-2 is supported by the accumulation of somatic mutations in the genes 323 encoding cross-reactive antibodies isolated from covid-19 patients (figures 2c and 324 2d, supplemental tables 2 and 3 ). this situation is reminiscent of re-exposure to 325 immunogenic epitopes shared by closely related viruses leading to induction of 326 broadly cross-reactive antibodies in patients infected with influenza, dengue or zika 327 viruses (29-31). 328 the 32 mabs that bound to the spike glycoprotein were systematically tested for 329 neutralisation (summarised in table 1 ). results established that neutralising epitopes 330 were present on the rbd, s1-ntd, s1-non ntd/rbd, and s2 regions of the spike cd3 neg cd19 pos cd20 neg cd27 hi cd38 hi igg pos plasmablasts were gated and isolated in 425 chamber as single cells as previously described (53) . sorted single cells were used to produce human igg mabs as previously described 428 (53) confluent monolayers of vero e6 cells in 96-well plates were incubated with ~14 501 plaque forming units (pfu) of sars cov-2 (hcov-19/england/02/2020, 502 epi_isl_407073) and antibodies in a 2-fold dilution series (triplicates) for 3 hours at 503 room temperature. inoculum was then removed, and cells were overlaid with plaque 504 assay overlay. cells were incubated at 37°c, 5% co 2 for 24 hours prior to fixation 505 with 4% paraformaldehyde at 4°c for 30 minutes. fixed cells were then 506 permeabilised with 0.2% triton-x-100 and stained with a horseradish peroxidase 507 conjugated-antibody against virus protein for 1 hour at room temperature. tmb 508 substrate was then added to visualise virus plaques as described previously for 509 influenza virus (54). convalescent serum from covid-19 patients was used as a 510 control. 511 in brief, this rapid, high-throughput assay determines the concentration of antibody 513 that produces a 50% reduction in infectious focus-forming units of authentic sarseagle's medium containing 2% fbs), two-fold serially diluted mabs in vgm 541 starting at 100 µg/ml were added to each duplicated well. the plates were 542 immediately transferred to a bsl-3 laboratory and 100 tcid 50 sars-cov-2 (hcov-543 19/taiwan/4/2020, epi_isl_411927) in vgm was added. the plates were further 544 incubated at 37°c with 5% co 2 for three days and the cytopathic morphology of the 545 cells was recorded using an imagexpress nano automated cellular imaging system. competitive binding assays were performed as described previously (18) two assays were used to determine the blocking of binding of ace2 to rbd by 567 mabs. rbd was anchored on the plate in the first assay whereas ace2 was anchored 568 for the second assay. the second ace2 blocking assay was performed as described previously (14, 15) . b non-ntd s1 pos 1.45 0.11 0.13 0.12 0.12 0.14 110.00 ew 8b b non-ntd s1 -ve 1.61 0.11 0.13 0.11 0.11 0.12 -ve fd 11d b ntd pos 1.42 0.18 0.22 0.42 0.25 0.29 -ve fd 11c b non-ntd s1 pos 1.20 0.14 0.20 0.11 0.11 0.12 -ve fd 7d b non-ntd s1 -ve 1.44 0.12 0.14 0.11 0.11 0.12 -ve fd 8b b non-ntd s1 -ve 1.10 0.14 0.12 0.08 0.10 0.09 -ve fd 7c b ntd pos 1.90 0.15 0.15 0.13 0.12 0.13 -ve fg 12c a non-ntd s1 pos 1.74 0.14 0.11 0.08 0.08 0.10 -ve fn 8c c non-ntd s1 -ve 0.54 0.16 0.16 0.11 0.09 0.12 -ve fd 5e b non-ntd s1 pos 0.34 0.16 0.16 0.13 0.11 0.11 -ve ew 9b b non-ntd s1 -ve 0.22 0.17 0.11 0.09 0.10 0.09 -ve deployment of convalescent plasma for the prevention and 650 treatment of covid-19 effect of convalescent plasma therapy on time to clinical 652 improvement in patients with severe and life-threatening use of convalescent plasma therapy in sars patients in hong kong structure, function, and antigenicity of the sars-cov-2 spike breadth of concomitant immune responses prior to patient 659 recovery: a case report of non-severe covid-19 neutralizing antibodies in patients with severe acute respiratory 661 syndrome-associated coronavirus infection antibody responses to sars-cov-2 in patients with covid-19 serology characteristics of sars-cov-2 infection since exposure and 665 post symptom onset cross-neutralization of influenza a viruses mediated by a single 667 antibody loop structural insights on the role of antibodies in hiv-1 vaccine and 669 therapy human monoclonal antibody combination against sars 12. huo, j. et al. neutralization of sars-cov-2 by destruction of the prefusion spike a highly conserved cryptic epitope in the receptor binding domains of neutralizing nanobodies bind sars-cov-2 spike rbd and block 677 interaction with ace2 structural basis for the neutralization of sars-cov-2 by an antibody 679 from a convalescent patient rugged nanoscaffold to enhance plug-and-display vaccination structural basis for potent neutralization of betacoronaviruses by single-domain camelid antibodies therapeutic monoclonal antibodies for ebola virus infection derived 687 from vaccinated humans cross-neutralization of sars-cov-2 by a human monoclonal sars cov antibody protective humoral immunity in sars-cov-2 infected pediatric 691 patients antibody responses to sars-cov-2 in patients of novel coronavirus 693 disease 2019 serologic cross-reactivity of sars-cov-2 with endemic and seasonal an outbreak of human coronavirus oc43 infection and 697 serological cross-reactivity with sars coronavirus recovery in tracheal organ cultures of novel viruses from patients with respiratory 701 disease epidemiology of seasonal coronaviruses: establishing the context for the emergence of coronavirus disease human coronavirus oc43 associated with fatal development of a nucleocapsid-based human coronavirus 708 immunoassay and estimates of individuals exposed to coronavirus in a u.s. 709 metropolitan population the dominance of human coronavirus oc43 and nl63 infections 711 in infants the human immune response to dengue virus is dominated by 713 highly cross-reactive antibodies endowed with neutralizing and enhancing activity zika virus activates de novo and cross-reactive memory b cell 716 responses in dengue-experienced donors broadly cross-reactive antibodies dominate the human b cell 718 response against 2009 pandemic h1n1 influenza virus infection receptor-binding domain of severe convergent antibody responses to sars-cov-2 in 725 convalescent individuals potent neutralizing antibodies against sars-cov-2 identified by single-cell sequencing of convalescent patients' b cells potent neutralizing antibodies against multiple epitopes on sars-cov-730 2 spike a noncompeting pair of human neutralizing antibodies block covid-732 19 virus binding to its receptor ace2 potent neutralizing antibodies from covid-19 patients 734 define multiple targets of vulnerability studies in humanized mice and convalescent humans yield a sars cov-2 antibody cocktail isolation of potent sars-cov-2 neutralizing antibodies and 738 protection from disease in a small animal model human neutralizing antibodies elicited by sars-cov-2 infection a human monoclonal antibody blocking sars-cov-2 infection human monoclonal antibodies block the binding of sars-cov-2 spike protein to angiotensin converting enzyme 2 receptor receptor-binding domain as a target for 747 developing sars vaccines the sars-cov-2 receptor-binding domain elicits a potent 749 neutralizing response without antibody-dependent enhancement a vaccine targeting the rbd of the s protein of sars-cov-2 induces 752 protective immunity structural basis of receptor recognition by sars-cov-2 structure of the sars-cov-2 spike receptor-binding domain bound to 756 the ace2 receptor structural and functional basis of sars-cov-2 entry by using 758 sars-cov-2 neutralizing antibody structures inform therapeutic 760 strategies complete mapping of mutations to the sars-cov-2 spike 762 receptor-binding domain that escape antibody recognition antibody cocktail to sars-cov-2 spike protein prevents rapid 765 mutational escape seen with individual antibodies a neutralizing human antibody binds to the n-terminal domain of the structure-function analysis of neutralizing antibodies to h7n9 769 influenza from naturally infected humans optimisation of a micro-neutralisation assay and its application in 771 antigenic characterisation of influenza viruses. influenza other respir viruses 9 isolation and rapid sharing of the 2019 novel coronavirus from the first patient diagnosed with covid-19 in australia the data are presented 795 as specificity, number of antibodies, and the percentage of total antibodies isolated 796 from each patient. (b) the binding activity of anti-sars-cov-2 mabs with spike 797 glycoprotein, rbd and the s2 subunit in elisa. anti-influenza h3 mab bs-1a and 798 anti-sars rbd cr3022 were included as controls. each experiment was repeated 799 twice. the od 450 values are presented as mean ± standard error of the mean. panels 800 (c) and (d) show numbers of variable domain mutations in mab genes and variation antibodies that 802 strongly cross-react with at least one betacoronavirus (sars or mers or oc43) 803 were defined as cross-reactive mabs. cdr3 length and mutation numbers are 804 presented as mean ± standard error of the mean (anti-s2, specific reactive, n=5; anti-n, specific, n=16 versus cross-reactive, n=19). the two-tailed test was performed to compare the mutations between two groups d, =day ; ns, non-significant hinge and fc region of human igg1 and ace2-fc were included as controls the rbd was colored in 816 green. the epitopes recognized by ey 6a, cr3022 and vhh72 (cluster 1 mab) (11, 817 15, 17) were colored in magenta. the epitopes recognized by ace2 and h11-h4 818 (cluster 2 mab) (14) were overlapping and colored in blue and light blue. the 819 epitopes recognized by s309 convalescent sera were analysed in the ace2-blocking (ace2 anchored) assay anti-rbd antibody fd 11a and anti-822 influenza h3 antibody bs 1a were included as controls. data are presented as mean ± 823 standard error of the mean ace2-blocking activity of anti-rbd antibody compared to ace2-fc (see methods): +, partial; ++ abbreviations: ifa, immunofluorescence; rbd, receptor-binding domain; prnt, plaque reduction 835 neutralisation assay key: cord-301347-22lt6h40 authors: jarvis, matthew c.; lam, ham ching; zhang, yan; wang, leyi; hesse, richard a.; hause, ben m.; vlasova, anastasia; wang, qiuhong; zhang, jianqiang; nelson, martha i.; murtaugh, michael p.; marthaler, douglas title: genomic and evolutionary inferences between american and global strains of porcine epidemic diarrhea virus date: 2016-01-01 journal: prev vet med doi: 10.1016/j.prevetmed.2015.10.020 sha: doc_id: 301347 cord_uid: 22lt6h40 porcine epidemic diarrhea virus (pedv) has caused severe economic losses both recently in the united states (us) and historically throughout europe and asia. traditionally, analysis of the spike gene has been used to determine phylogenetic relationships between pedv strains. we determined the complete genomes of 93 pedv field samples from us swine and analyzed the data in conjunction with complete genome sequences available from genbank (n = 126) to determine the most variable genomic areas. our results indicate high levels of variation within the orf1 and spike regions while the c-terminal domains of structural genes were highly conserved. analysis of the receptor binding domains in the spike gene revealed a limited number of amino acid substitutions in us strains compared to asian strains. phylogenetic analysis of the complete genome sequence data revealed high rates of recombination, resulting in differing evolutionary patterns in phylogenies inferred for the spike region versus whole genomes. these finding suggest that significant genetic events outside of the spike region have contributed to the evolution of pedv. porcine epidemic diarrhea virus (pedv) causes diarrhea, vomiting, and dehydration, leading to high mortality (up to 100%) in suckling piglets. pedv was first discovered in the united kingdom in 1971, and later was found in belgium, hungary, france, italy, and the czech republic (chasey and cartwright, 1978; fan et al., 2012) . in 1986, pedv was first reported in china, and proceeded to spread throughout asia (cui, 1990; song and park, 2012) . in late 2010, a "variant" pedv strain with increased pathogenesis compared to the pedv is a single-stranded, positive sense rna virus belonging to the family coronaviridae, genus alphacoronavirus. the pedv genome is approximately 28 kb in length and roughly two-thirds of the genome consists of open reading frame (orf) 1, which encodes 16 non-structural proteins (nsps) (lai et al., 2013) . these nsps play important roles in viral replication, post-translational processing, and immune evasion (lai et al., 2013) . the virus produces various structural proteins, including spike, membrane, and nucleocapsid (lai et al., 2013) . the spike protein is crucial to cell attachment and infection, and the envelope is an integral membrane protein, aiding in membrane fusion while the nucleocapsid protein is necessary for genomic packaging (hagemeijer and de haan, 2015) . in addition, the pedv genome includes orf3, located between the spike and membrane genes, that encodes an ion channel protein possibly associated with pedv pathogenesis (park et al., 2008; wang et al., 2012) . researchers have explored various regions of the coronavirus (cov) genome to link specific areas with virulence and host cell attachment. for example, the spike gene codes for a viral attachment protein that can be divided into the s1 (1-789 aa) and s2 (790-1383 aa) regions (song and park, 2012) . comparative analysis of transmissible gastroenteritis virus (tgev), porcine respiratory coronavirus (prcv), and murine hepatitis virus (mhv) revealed two main antigenic sites in the s1 region: the n-terminal domain (ntd) and the c-terminal receptor binding domain (rbd) (li et al., 2007) . while both domains can influence virus infectivity, such as in tgev, one domain tends to be central to a cov's tropism: the ntd is important for mhv tropism, and the rbd is central to pedv infectivity and virulence (reguera et al., 2012) . the ntd can bind to various sialic acids on the host cell surface (reguera et al., 2012) . the rbd contains residues that bind to the porcine aminopeptidase-n (papn), the host receptor utilized by tgev and pedv (delmas et al., 1992) . since the last large-scale north american pedv outbreak ended in the spring of 2014, the complete genomes of 93 pedv strains from the us were sequenced and analyzed to further understand the origin and phylogenetic relationships among the american and global pedv strains. in-depth nucleotide and amino acid analysis was conducted to identify genes of high diversity. bayesian analysis was performed to understand the evolution of pedv and the emergence of different clades within us strains. in addition, the rbd was modeled to visualize the differences between american and asian strains to better understand how changes in the rbd might affect vaccine efficacy and development. samples were routinely submitted to the university of minnesota veterinary diagnostic laboratory (umvdl) for pathogen detection. between january 2014 and december 2014 samples were screened for pedv by real time rt-pcr . samples for complete genome sequencing were selected based on the criteria of a high viral concentration from the rt-pcr results and geographical diversity within the us. a total of 83 samples, including fecal (n = 38), intestinal homogenate (n = 21), fecal swab (n = 10), oral fluid (n = 5), feedback (n = 4), and environmental (n = 5) samples were selected for complete genome sequencing using next generation sequencing (ngs) techniques as previously described (genbank numbers kr265759-kr265834, kr265840-kr265846) (marthaler et al., 2013; marthaler et al., 2014) . whole genomic pedv sequences obtained using ngs techniques were also generously supplied from iowa state university (n = 7, genbank numbers km975735-km975741) and the ohio department of agriculture (n = 3, genbank numbers kp641661-kp641663), using previously described methods chen et al., 2014) . using the complete pedv genome sequences from this study (n = 93) and the available pedv sequences from genbank (n = 126), two nucleotide alignments were created and analyzed to determine the phylogenetic relationships between american and global pedv sequences: the concatenation of all orfs (orf1, s, orf3, envelope, membrane, and nucleocapsid), and a s1 alignment. vaccine and cell-passaged strains were excluded from the analysis (table s1 ). nucleotide and amino acid entropy analyses were performed using the matlab software (matlab v8.0 and statistics toolbox v8.1, the mathworks, inc., natick, ma, usa). threshold values were determined using previously published methods (shannon, 1948; litwin and jores, 1992) . recombination analysis was performed using the recombination detection program (rdp) v4, which uses multiple detection algorithms, including the rdp method, genecov, and maxchi, to check for the presence of recombinant sequences in the sequence dataset (martin et al., 2015) . window size was set to 100 bp. breakpoints, the presence of major/minor donor sequences, and confidence intervals were used to determine regions that required excision from the alignment, or if entire sequences needed to be removed from the analysis due to multiple recombination events within the sequence. recombinant sequences were removed only prior to the bayesian analysis, but remained in the alignments for all entropy analysis and molecular modeling. bayesian markov chain monte carlo (mcmc) approach using beast v1.8.1, with a relaxed molecular clock and bayesian skyline population (bsp) prior, with a general-time reversible nucleotide substitution and gamma distributed among-site rate variation was used to infer time-scaled phylogeny (drummond et al., 2002 , 2006 , 2012drummond and rambaut, 2007 minin et al., 2008; drummond and suchard, 2010) . the mcmc chain was run for 800 million generations, with sub-sampling every 80,000 iterations. a maximum clade credibility (mcc) tree was created by discarding the initial 10% of the chains and summarized in treeannotator (v.1.8.0). key nodes were identified using figtree (v.1.4 .2) to determine time to most recent common ancestor (tmrca). the putative papn receptor-binding residues were analyzed to determine residue trends between classical and pandemic strains (reguera et al., 2012) . the c-terminal rbd within the s1 region of the spike gene was modeled using the open-source modeling server swiss-model provided by the swiss institute of bioinformatics (biasini et al., 2014) . predicted tertiary structure of the pedv papn rbd was modeled using prcv as a template since a pedv template was not available. spike monomer and trimer models were developed using a theoretical sars-cov model as a template (bernini et al., 2004) . illustrations were created using the open-source javabased molecular viewer jmol (herraez, 2006) and the python-based molecular viewer pymol (the pymol molecular graphics system, version 1.7.4 schrödinger, llc.). 1932, 1951, 1976, 2467, 2517, 2652, 2658, 2664, 2667, 2679 2720, 2809, 2856, 2883, 2940, 3003, 3021, 3025, 3078, 3105, 3110, 3171, 3516, 3708, 3876 the pedv nucleotide sequences ranged from 27,529 to 28,061 bases in length. two pedv genomes from our study had insertions or deletions. ohio249 had a 3-nt insertion between positions 22,039 and 22,041 in the spike gene while minnesota309 with a 4-nt deletion from positions 27,768 to 27,771 in the 3 utr compared to the original us strain, usa/colorado/2013. entropy analysis was conducted with 219 whole nucleotide and amino acid sequences, containing the concatenated orfs excluding 5 and 3 utrs. entropy values greater than 0.8 and 0.6 were considered highly variable for the nucleotide and amino acid alignments, respectively, based on the level of diversity in the dataset and previously determined entropy values (litwin and jores, 1992) . within the nucleotide alignment, 15 of the 20 pedv regions lacked positions with entropy levels above 0.8 (nsp1, nsp5-nsp10, nsp13, nsp15, nsp16, s2, orf3, envelope, membrane, and nucleocapsid) while 5 regions had entropy levels above 0.8 (nsp2, nsp3, nsp12, nsp14, and s1) (fig. 1) . the nsp2 and 3 were the most divergent regions containing 10 and 16 diverse nucleotide positions, respectively (table 1) . interestingly, the nsp12 gene contained 4 diverse nucleotide positions, which were absent in the amino acid sequence (fig. 1a) . inversely, high amino acid diversity was observed in the nsp4, nsp13, nsp16, and s2 genes, which were absent in the nucleotide alignment (fig. 1b) . higher entropy levels were present in the nsp2, nsp3, and s1 regions in both the nucleotide and amino acid alignments. overall, the orf1a entropy levels were higher compared to orf1b in the amino acid analysis. of the structural genes, the s gene had the highest entropy levels compared to the envelope, membrane, and nucleocapsid genes. recombination was detected in 7 main areas of the concatenated full genome, including the nsp2, nsp3, nsp14-16, s1 domain, and nucleocapsid gene ( fig. 2a) . in these areas, recombination was present in the majority of the sequences, so the entire region was excised from the alignment prior to bayesian analysis. in addition, 35 sequences (23 from asia, 12 from the americas) were omitted from the bayesian analysis due to evidence of widespread recombination throughout the genome (table s1 ). for example, the pandemic sequence minnesota211 contained a recombinant region with the characteristic s-indel deletions and insertions in the s1 domain, indicating a recombinant event occurred between an s-indel strain and a non s-indel pathogenic strain in the us (fig. 2b) . a maximum clade credibility (mcc) phylogeny was inferred for both the concatenated genomic sequences excluding the recombinant regions (12,999 nt) and the spike s1 gene (2142 nt). the analysis was run independently twice until convergence was reached, with high agreement between the two runs. in the concatenated alignment tree, the classical and pandemic asian strains were positioned as basal to the us strains, consistent with an asian origin for the us outbreak (fig. 3) . importantly, the concatenated alignment tree suggests that the us epidemic may have resulted from two independent pedv introductions into the us, including minor and major clade of viruses. the minor clade contained the american and european s-indels, and a small subclade of non s-indel sequences from the ohio, including ohio249/2014, pc21a/2013, and oh15962/2013. the major clade of us pedv strains was supported by high posterior probability (100%) and appears to have diverged further into two highly supported sublineages (99% and 100% posterior probability). the phylogeny is consistent with multiple incursions of the major clade of us pedv viruses into mexico, canada, and south korea. the minor clade includes sequences from late 2013 to early 2014 that are localized to the midwestern and eastern us regions. the estimated tmrca of the minor clade of us pedv strains is july 2009-2011, and the estimated tmrca of the major clade of us pedv strains is september 2010-august 2012. the estimated evolutionary rate for the complete genome (excluding recombinant regions and sequences) is 6.2 × 10 −4 substitutions/site/year (4.8 × 10 −4 -7.6 × 10 −4 , 95% highest posterior density (hpd)). the rate estimate for the us strains is slightly higher, but not significantly: 5.5 × 10 −3 substitutions/site/year (4.6 × 10 −3 -6.5 × 10 −3 , 95% hpd). the spike tree illustrates the evolutionary relationship between the classical strains and the s-indels, which suggests a classical origin for the s-indel genotype (fig. 4) . the pathogenic strains form a highly diverged major clade (fig. 4b) , which braches into 2 large american clades. in addition, the bayesian analysis of the spike gene might suggest 2 separate introductions of pedv into the us. the evolutionary rate for the s1 gene is 1.5 × 10 −3 substitutions/site/year (1.1 × 10 −3 -1.9 × 10 −3 , 95% hpd). considering the high entropy levels in the spike gene and the evolutionary rate determined from the s1 bayesian spike tree, the rbd within the s1 was further examined. the s-indel and classical pedv strains shared similar amino acid substitutions, specifically in the ntd of the s1 region (fig. s1) . furthermore, the pandemic pedv strains from china had an increased number of substitutions within the s1 domain when compared to the american strains due to the longer circulation time in china. compared to the attenuated vaccine strain dr13, 29 of 185 (16%) american strains and 19 of 34 (56%) asian strains had at least one amino acid substitution in the papn rbd ( table 2 ). the majority of the american strains (n = 156) did not represent any amino acid differences in the papn rbd. in this region, 8 positions in the american strains had amino acid differences compared to the vaccine strain dr13 (fig. 5a) . the most common substitutions were in the fourth region of the papn rbd at positions e594d (n = 10) and g598d (n = 9), which were substituted with aspartic acid. more substitutions (n = 13) occurred in the papn rbd of the asian strains (table 2) , with the most common substitutions at position h515l (n = 5). the rbd regions were three-dimensionally modeled to illustrate the 8 and 13 amino acid positions at which substitutions occurred in the north american and asian strains, respectively. the modeling of the spike protein suggests that the papn rbd residues cluster around the inner pore created by the trimer molecule, while the ntd is oriented around the outer surface of the s1 domain ( fig. 5b through 5f). genomic analysis depends critically on complete sequence data to conduct accurate research on phylogeny, evolution, and gene regulation. in the past, it was more economically and time effective to sequence smaller pieces of a genome and develop evolutionary conclusions from these relatively small genomic pieces. however, without full genomic sequences, it is impossible to compare variations within a genome to determine selective pressures on specific genes or regions. because of ngs technology, tools like site-specific entropy analysis can be used to examine variability throughout the genome of many pedv sequences. sun and collaborators reported four regions of diversity within the pedv genome, including v1 in the nsp2 and nsp3, v2 in the s1, v3 in the s2 and orf3, and v4 in the nucleocapsid (sun et al., 2015) . while our results support the nucleotide variance in the nsp2, nsp3, and spike genes, high levels of diversity were not present in the s2, orf3, and the nucleocapsid. this could be due to the omission of pedv isolates in our analysis, as well as the comparatively large number of new us sequences in our dataset. our variance results may more accurately represent variance within american pedv strains, and underrepresent variance within chinese strains. the diversity in the s1 region is comprehensible since it is under strong immunological pressure while the s2 region is more conserved throughout covs (aydin et al., 2014) . the functions of nsp2 and nsp3 remain relatively ambiguous. despite being involved in viral growth and propagation, nsp2 is dispensable for viral replication because cov strains can replicate in absence of the nsp2 (graham et al., 2006) . the function of nsp3 may be related to innate immune evasion since it encodes proteases that facilitate proteasome degradation, changes in intracellular destination, signaling, protein interactivity, and host type i interferon (ifn) antagonist activities (xing et al., 2013) . due to the multifaceted nature of nsp3, other nsp regions could produce proteins with novel effects not yet understood that mediate the virulence of cov species. acquired nucleotide differences throughout the nsp2 and nsp3 regions could contribute to the evasion of host immunity. thus, future research should focus on the functionality and importance of all pedv genes to further understand cov pathogenesis. recombination plays a pivotal role in the evolution of covs by creating new strains with altered virulence. the minnesota211 strain originated from a recombination even between an s-indel and a us pandemic strain, which has been associated with altered pathogenesis. while recombination may occur more often during an epidemic, recombination events occurred in most of the asian strains. recombination events can affect the phylogenetic analy-sis because different regions of the genome may have different evolutionary histories (spade et al., 2015) . our recombination analysis resulted in a significant portion of the complete genome being removed prior to more detailed phylogenetic analysis. at this time, the beast program cannot accommodate genetic data that includes recombined regions. our analysis supported an asian origin for the us outbreak while the inference is biased by the lack of background sequences from other regions. although over 100 genomes were from the us, interpretation of the evolutionary and spatial history of this data is limited by the lack of genomic pedv data from other regions, including europe and asia. us strains had a higher evolutionary rate compared to the global strains, but the bayesian skyline plot did not show any significant increase in evolutionary rate, possibly due to the lack of temporal sampling in the us dataset. the evolutionary rate for the spike gene was higher compared to the rest of the genome, reflecting greater selective pressure. the overall evolutionary rate of pedv (6.2 × 10 −4 substitutions/site/year) is similar to that of tgev and wild animal covs (6.08 × 10 −3 substitutions/site/year, 1.3 × 10 −5 -1.4 × 10 −2 95% hpd), but lower than that of sars-cov (2.3 × 10 −3 nucleotide substitutions/site/year), except during the time of the us pedv 2013 epidemic (5.5 × 10 −3 sub-stitutions/site/year) (song et al., 2005; vijaykrishna et al., 2007) . surprisingly, 3 pandemic strains were positioned within the s-indel clade. possibly, a pandemic and s-indel strain were introduced into the americas, and a recombination event occurred in the ntd that removed the characteristic insertions and deletions of an s-indel strain, as indicated in the minnesota211 sequence. the relationship between the us minor clade and the recent pedv strains from europe is less clear. while these viral populations are closely related (posterior probability of 100%), the direction of transmission is unclear at this time. additional sequences from europe might help to resolve the origins of these recent european pedv cases. (e) a monomer model of the pedv spike protein, with the c-terminal rbd represented in green, dark blue represents the s2 region, light blue represents the s1 region, and yellow represents the n-terminal rbd. (f) a theoretical tertiary structure model of the pedv spike protein. blue represents the s1 region, with the specific n-and c-terminal rbds highlighted in yellow and green, respectively. the papn-rbd is shown in violet.(for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) examining the spike gene can reveal interesting conclusions about the papn rbd. while the ntd spans a larger region of the s1 domain, it has not been directly linked to pedv tropism and functionality, as in tgev and mhv (reguera et al., 2012) . the korean attenuated vaccine strain and the us strains share similar residues in the papn rbd, with numerous differences compared to the older classical strains, suggesting this vaccine may protect against the american strains. however, developing a consistently and longitudinally efficacious vaccine may prove challenging, considering the high evolutionary rate of the s1 region. failures in the development of an efficacious vaccine have been reported, further supporting the difficulty in generating vaccines for pedv . despite some uncertainties in vaccine efficacy, a recent study demonstrated that prior exposure of sows to the s-indel strain provided a level of protective immunity when their piglets were challenged with the more virulent original us pedv strain, which is probably due to conservation within the c-terminal region of the viral genome (goede et al., 2015) . while the exact functionality of all the genes of pedv and other covs is unknown, adding the complete genomes of diverse strains to the global database promotes better understanding of evolutionary and phylogenetic relationships. multiple regions within the genome are variable, and recombination is common between pedv strains. despite excising a large portion of the genome prior to analysis, the bayesian trees illustrate two distinct entries of pedv into the us and characterize the evolution of pedv compared to other covs. modeling of the papn rbd region has revealed that asian strains have increasing diversity compared to previously developed vaccines, and the variability in both the american and asian strains needs to be considered for future vaccine development. as the us swine industry recovers from the pedv epidemic of 2013-2014, research is maturing to understand the regions of diversity, evolution, and the rbd of pedv to prevent future outbreaks and foster vaccine development. influence of hydrophobic and electrostatic residues on sars-coronavirus s2 protein stability: insights into mechanisms of general viral fusion and inhibitor design prediction of quaternary assembly of sars coronavirus peplomer swiss-model: modelling protein tertiary and quaternary structure using evolutionary information virus-like particles associated with porcine epidemic diarrhoea isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the 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diarrhea in suckling piglets evolutionary insights into the ecology of coronaviruses distinct characteristics and complex evolution of pedv strains new variant of porcine epidemic diarrhea virus the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase this study was supported partially by the rapid agricultural response fund, established by the minnesota legislature and administered by the university of minnesota agricultural experiment station, and by boehringer ingelheim vetmedica, inc.the authors thank the faculty and personal at the umvdl for their technical services. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.prevetmed.2015. 10.020. key: cord-285039-9piio754 authors: zhou, haixia; zhang, shuyuan; wang, xinquan title: crystallization and structural determination of the receptor-binding domain of mers-cov spike glycoprotein date: 2019-09-14 journal: mers coronavirus doi: 10.1007/978-1-0716-0211-9_4 sha: doc_id: 285039 cord_uid: 9piio754 three-dimensional structures of the receptor-binding domain (rbd) of mers-cov spike glycoprotein bound to cellular receptor and monoclonal antibodies (mabs) have been determined by x-ray crystallography, providing structural information about receptor recognition and neutralizing mechanisms of mabs at the atomic level. in this chapter, we describe the purification, crystallization, and structure determination of the mers-cov rbd. the first three-dimensional structure of the mers-cov spike glycoprotein receptor-binding domain (rbd), providing the molecular basis of viral attachment to host cells, was determined in the complex with it cellular receptor dipeptidyl peptidase 4 (dpp4, also called cd26) by x-ray crystallography [1] . because of the significance in receptor recognition and specific pathogenesis, rbd became a hot spot in the study of mers-cov. a number of structures of rbd bound by monoclonal antibodies (mabs) have also been determined and deposited in the protein data bank (pdb, http://www.rcsb.org/pdb/) [2] [3] [4] [5] [6] [7] [8] . our group determined the rbd structures in complex with dpp4 and the mabs mers-27, mers-4 and mers-gd27, respectively [2] [3] [4] 9] . all the three-dimensional structures of mers-cov rbd have been determined by x-ray crystallography, which is a powerful method for determining molecular structures at atomic resolution. briefly, the ordered and repeated atoms in a single protein crystal can diffract the incident x-ray beam into many specific directions. the angles and intensities of these diffracted x-rays can be collected and measured in an x-ray diffraction experiment. after obtaining the phases of these diffracted x-rays by heavy-atom derivative, anomalous scattering or molecular replacement methods, a protein crystallographer then calculates the density of electrons with the protein crystal and builds a structural model based on the density map. for details on the principles and methodology of protein crystallography, please refer to the range of other excellent textbooks. in this chapter, an overview of the standard method of protein crystallography is briefly introduced, focusing on crystallization and structural determination of mers-cov rbd using the molecular replacement method. prepare all solutions using ultrapure water (prepared by purifying deionized water, to attain a resistivity of 18 mω cm at 25 c) and analytical grade reagents. when dealing with waste, we strictly follow all waste disposal regulations. 1. pfastbac vector containing the mers-cov rbd gene. 3. lb liquid medium: 10 g tryptone, 5 g yeast extract, 10 g nacl, and 1 l ultrapure water; sterilize by high-pressure steam. 4. liquid lb selection medium: lb liquid medium, 50 μg/ml kanamycin, 7 μg/ml gentamicin, and 10 μg/ml tetracycline. 5. bacmid selection lb agar plate:10 g tryptone, 5 g yeast extract, 10 g agar powder, 10 g nacl, and 1 l ultrapure water. sterilization at high-pressure steam. 50 μg/ml kanamycin, 7 μg/ml gentamicin, 10 μg/ml tetracycline, 100 μg/ml x-gal, 40 μg/ ml iptg mix and pout into sterile plates (see note 1). mer-cov rbd can be expressed using the bac-to-bac baculovirus expression system (fig. 1 ), collected and captured using nta sepharose (ge healthcare) and then further purified by gel filtration chromatography using a superdex 200 high performance column (ge healthcare). crystallization trials are set up using the hanging-drop or sitting-drop vapor diffusion method in conjunction with the sparse-matrix crystal screening kits. the structure of mers-cov rbd in complex with mers-4scfv was determined using the molecular replacement method. when the total volume remaining is reduced to 50 ml, add 100 ml hbs buffer to collect all the liquid in the system in a beaker. then dispense into high-speed centrifuge tubes, centrifuge at 3000 â g for 1 h at 4 c. 4. the supernatant after centrifugation is loaded onto the nickel-nta beads equilibrated with 30 ml of hbs buffer. mers-cov rbd with a his tag could be captured by nickel-nta beads. repeat loading the sample once more. 5. add wash buffer to the beads to remove the nonspecifically bound proteins until the flow-through is not able to discolor the coomassie brilliant blue g250 solution. 6. after adding elution buffer, the target protein will dissociate from the beads; collect it in a 10 kda millipore concentrating tube. similarly, detect protein with coomassie brilliant blue g250. the concentrating tube containing the protein sample is centrifuged at 3000 â g to concentrate the sample to less than 7. mers-covrbd is further purified by gel filtration chromatography. the sample is loaded onto the superdex 200 column pre-equilibrated with hbs buffer. fractions containing rbd are collected and the protein's purity is confirmed by sds-page (fig. 2) . 8. dilute the protein to 1 mg/ml, and digest with endoglycosidase f1 and f3 (f1/f3: rbd at the ratio of 1:100) at 18 c overnight. the digested protein is concentrated and purified by gel filtration chromatography same as above (optional). 9. after preparing the mers-cov rbd protein and mers-4scfv protein (see note 11) detect the absorption of the protein sample at 280 nm (a280). according to the molecular weight and extinction coefficient, the molar concentration can be calculated. the two proteins were mixed at molar ratio of 1:1, incubated on ice for 1 h, and purified using a superdex 200 column. collect the fractions containing the complex and confirm the protein purity by sds-page. sartorius centrifugal concentrator to concentrate to 10-15 mg/ml. after mixing and aspirating, centrifuge at 10,000 â g for 10 min at 4 c. 3. use ttp labtech's mosquito crystallization setup for automated crystallography. absorb 3 μl protein on the 8-well 5 μl micro-reservoir strip (fig. 3a) . then the needles aspirate the protein from the strip onto the swissci plate with 200 nl of protein per well (fig. 3b) , using the sitting-drop vapor diffusion method by mixing 200 nl reservoir and 200 nl reservoir (fig. 3c,d) . 4. seal the plate with tape and gently place it in an 18 c room. 5. check the sample drops under a microscope at 20-100â magnifications after 3 and 7 days (and if necessary, after 1 and 2 weeks, and 1, 3, and 6 months). 6 . a week later, we found crystal growth in the peg/ion, pegrx and jcsg+ kits. specific conditions were as follows (fig. 4a,b) 2. the diffraction images should be collected on the bl17u beamline (fig. 4c) . rotate the mounted crystal and the x-ray diffraction patterns should be recorded at 1 per image, and collected for 360 . 3. the diffraction images in a dataset should be processed with hkl2000 [10] including auto-index, refinement, integration, and scaling steps. after data processing, the crystal unit cell parameters, crystal space group, miller indexes of reflections, intensities, and error estimates of reflections should be determined and stored in a * .sca file, which provides the dataset applicable to structure determination. 4. using ccp4 suite solve the structure as follows: export the * . sca file to a * .mtz file using the program scalpack2mtz. use the * .mtz file to calculate the solvent via matthews_-coef. run with phaser mr (see note 12) with the mers-cov rbd structure (pdb id: 4l72) and the structures of the variable domain of the heavy and light chains available in the pdb with the highest sequence identities as search models (see note 12) [11] . when the phases are determined, the electron density map can be calculated, from which the molecular model can be constructed. 5. subsequent model building and refinement were performed using coot and phenix, respectively (see note 13) [12, 13] . 6. many validation programs are used to check the structure, until the investigator is satisfied, and then the structure can be deposited in the pdb. 1. weigh 10 g tryptone, 5 g yeast extract, 10 g agar powder, and 10 g nacl, and add ultrapure water to 1 l. after sterilization by high-pressure steam, wait until the temperature of the medium drops to about 60 c, add the required antibiotics, inducers etc. (50 μg/ml kanamycin,7 μg/ml gentamicin, 10 μg/ml tetracycline, 100 μg/ml x-gal, 40 μg/ml iptg). mix evenly and then pour into sterile plates. when the culture medium has cooled and solidified, store the bacmid selection lb agar plate at 4 c. 6. endoglycosidase f1 and f3 are expressed and purified from e. coli by our laboratory. the endoglycosidase was added into the reaction system according to the mass ratio of 1:100. 7. the coding sequence of the mers-cov rbd (emc strain, spike residues 367-588) was ligated into the pfastbac-dual vector (invitrogen) with a n-terminal gp67 signal peptide to enable the protein secreting outside the cell and a c-terminal his-tag to facilitate further purification processes. 8. allow the pellet to dissolve for at least 10 min at room temperature. to avoid shearing the dna, pipet only 1-2 times to resuspend. store the bacmid at 4 c and use it as soon as possible, usually within 1 week. aliquot the bacmid dna into separate tubes and store at à20 c (not in a frost-free fridge). avoid multiple freeze/thaw cycles as this decreases the transfection efficiency. 9. characteristics of infected cells: a 25-50% increase in cell diameter can be seen and the size of cell nuclei increases at the early stage. cells release from the plate and appear lysed, showing signs of clearing in the monolayer. 10 . p0 virus can be stored for years, adding 2% (v/v) fbs at 4 c, protected from light. 11. the expression of mers-4scfv protein was conducted in 293f cells transiently transfected with plasmid dna. after 72 h, the supernatant was collected and concentrated. the purified mers-4scfv protein was obtained by ni-nta affinity chromatography and superdex 200 size-exclusion chromatography. the purification method is the same as that of rbd protein. 12. if the crystal structure of the same protein or a similar protein has been solved, the molecular replacement method can be applied. after obtaining the solutions of the rotation and translation functions, initial phases can be calculated from the reference model, after which the electron density can be calculated. 13. the accuracy of the constructed model is confirmed by the crystallographic r-factor and r-free, which indicate the discrepancy between the calculated and observed amplitudes. the stereo-chemical parameters of the model can also be checked using programs such as molprobity, procheck, or rampage. molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 structural basis for the neutralization of mers-cov by a human monoclonal antibody mers-27 structural definition of a unique neutralization epitope on the receptorbinding domain of mers-cov spike glycoprotein ultrapotent human neutralizing antibody repertoires against middle east respiratory syndrome coronavirus from a recovered patient evaluation of candidate vaccine approaches for mers-cov junctional and allelespecific residues are critical for mers-cov neutralization by an exceptionally potent germline-like antibody introduction of neutralizing immunogenicity index to the rational design of mers coronavirus subunit vaccines importance of neutralizing monoclonal antibodies targeting multiple antigenic sites on mers-cov spike to avoid neutralization escape structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 processing of x-ray diffraction data collected in oscillation mode phaser crystallographic software coot: modelbuilding tools for molecular graphics phenix: building new software for automated crystallographic structure determination key: cord-308310-wtmjt3hf authors: zha, lisha; zhao, hongxin; mohsen, mona o.; hong, liang; zhou, yuhang; li, zehua; yao, chuankai; guo, lijie; chen, hongquan; liu, xuelan; chang, xinyue; zhang, jie; li, dong; wu, ke; vogel, monique; bachmann, martin f; wang, junfeng title: development of a covid-19 vaccine based on the receptor binding domain displayed on virus-like particles date: 2020-05-14 journal: biorxiv doi: 10.1101/2020.05.06.079830 sha: doc_id: 308310 cord_uid: wtmjt3hf the recently ermerging disease covid-19 is caused by the new sars-cov-2 virus first detected in the city of wuhan, china. from there it has been rapidly spreading inside and outside china. with initial death rates around 4%, covid-19 patients at longer distances from wuhan showed reduced mortality as was previously observed for the sars coronavirus. however, the new coronavirus spreads more strongly, as it sheds long before onset of symptoms or may be transmitted by people without symptoms. rapid development of a protective vaccine against covid-19 is therefore of paramount importance. here we demonstrate that recombinantly expressed receptor binding domain (rbd) of the spike protein homologous to sars binds to ace2, the viral receptor. higly repetitive display of rbd on immunologically optimized virus-like particles derived from cucumber mosaic virus resulted in a vaccine candidate (rbd-cumvtt) that induced high levels of specific antibodies in mice which were able to block binding of spike protein to ace2 and potently neutralized the sars-cov-2 virus in vitro. onset of symptoms or may be transmitted by people without symptoms. rapid development of a protective vaccine against covid-19 is therefore of paramount importance. here we demonstrate that recombinantly expressed receptor binding domain (rbd) of the spike protein homologous to sars binds to ace2, the viral receptor. higly repetitive display of rbd on immunologically optimized virus-like particles derived from cucumber mosaic virus resulted in a vaccine candidate (rbd-cumvtt) that induced high levels of specific antibodies in mice which were able to block binding of spike protein to ace2 and potently neutralized the sars-cov-2 virus in vitro. covid-19 is caused by a novel coronavirus closely related to viruses causing sars and mers. as the disease caused by the other two viruses, covid-19 mainly manifests symptoms in the lung and causes cough and fever 1 . the disease covid-19 is less severe than sars and mers, which is beneficial per se but leads to easier and wider spread of the virus, in particular due to infected individuals with very little symptoms ("spreaders") and a long incubation time (up to 3 weeks) combined with viral shedding long before disease onset 2 . a vaccine with rapid onset of protection is therefore in high demand for the control of the pandemic that is currently taking its course. the spike protein of covid-19 is highly homologous to the spike protein of sars and both viruses share the same receptor, which is angiotensin converting enzyme 2 (ace2) 3, 4 . the receptor binding domain (rbd) of the sars spike protein binds to ace2 and is an important target for neutralizing antibodies [5] [6] [7] . by analogy, the rbd of covid-19 spike protein may be expected to similarly be the target of neutralizing antibodies, blocking the interaction of the virus with its receptor. we have previously shown that antigens displayed on virus-like particles (vlp) induce high levels of antibodies in all species tested, including humans 8 . more recently, we have developed an immunologically optimized vlp platform based on cucumber mosaic virus. these cumvtt vlps (hereafter cumvtt) incorporate a universal t cell epitope derived from tetanus toxin providing pre-existing t cell help. in addition, during the production process these vlps package bacterial rna which is a ligand for toll-like receptor 7/8 and serves as potent adjuvants 9 . using antigens displayed on these vlps, it 4 was possible to induce high levels of specific antibodies in mice, rats, cats, dogs and horses and treat diseases such as atopic dermatitis in dogs or insect bite hypersensitivity in horses [9] [10] [11] . to generate a covid-19 vaccine candidate, we therefore attempted to display the rbd domain on cumvtt (fig. 1a) . to this end we gene-synthesized the covid-19 rbd domain and fused it to an fc molecule for better expression. as expected, the protein bound efficiently to the viral receptor ace2 as determined by sandwich elisa (fig. 1b) . in a next step, the protein was chemically coupled to the surface of cumvtt using the well established chemical cross-linkers sata and smph (ref. 9). sds-page and western blotting confirmed efficient coupling of the rbd-fusion molecule to cumvtt, resulting in the vaccine candidate rbd-cumvtt (fig. 1c,d) . to test immunogenicity of the vaccine candidate, mice were immunized three times (weekly schedule) with the rbd-fusion molecule alone or conjugated to the surface of cumvtt formulated in montanide adjuvants. as shown in fig. 2a -c, coupling to vlps dramatically increased the immunogenicity of the rbd. as shown by elisa on recombinant rbd, rbd-cumvtt showed strongly increased immunogenicity at all time-points tested (one week after the vaccine injection time-points). to assess the potential for anti-viral activity, we assessed whether the induced antibodies were able to block binding of the rbd protein to the viral receptor ace2. as shown in fig. 3 , immune sera obtained after two boosts (day 21) were able to strongly inhibit rbd binding to ace2. the best correlate of protection is viral neutralization. to this end, we generated pseudotyped retroviruses 12 expressing the sars-cov-2 spike protein and luciferase for quantification of infection (fig. 4a ). using these viruses, the neutralizing capacity of the sera from immunized mice was assessed on ace2-transfected cells (fig. 4b) , directly demonstrating high anti-viral neutralizing activity of the induced antibodies. hence, the rbd-cumvtt vaccine candidate is able to induce high levels of sars-cov-2 neutralizing antibodies. furthermore, the cumvtt based vaccine is based on highly efficient expression systems and chemical conjugation technologies, rendering it an attractive candidate for large scale production under cgmp. previous studies with a similar vlp-based conjugate vaccine has demonstrated that high levels of specific antibodies can be mounted within a week 13, 14 (see also fig. 2a) , offering the additional possibilities to rapidly immunize individuals that have been exposed to infected humans or those that are kept in quarantine. thus, vaccines based on the sars-cov-2 rbd domain displayed on vlps may have the potential to critically interfere with global spread of the virus. 5 the sars-cov-2 receptor-binding domain (rbd) and the n-terminal peptidase domain of human ace2 were expressed using 293f cells (invitrogen). the sars-cov-2 rbd (residues arg319-phe541) with an n-terminal il-2 signal peptide for secretion and a cterminal fc tag for purification was inserted into pfuse-migg1-fc2 vector (invitrogen). the construct was transformed into bacterial dh5α competent cells, and the extracted plasmid was then transfected into 293f cells at a density of 3×10 6 cells/ml using pei (invitrogen). the cell culture supernatant containing the secreted rbd was harvested 96 h after infection, concentrated and buffer-exchanged to hbs (10 mm hepes, ph 7.2, 150 mm nacl). rbd was captured by protein a resin (ge healthcare) and eluted with gly-hcl buffer ph 2.2. fractions containing rbd were collected and neutralized to ph 7.0 with 1m tris. for elisa coating, ace2 was cleaved from the fc part using thrombin as described in the manufacturer's manual. the human ace2 (residues ser19-ser741) with an n-terminal il-2 signal peptide for secretion and a c-terminal 6×his tag for purification was inserted into pfuse-vector (invitrogen). the human ace2 was expressed by essentially the same protocol used for the sars-cov-2 rbd. ace2 was captured by ni-nta resin (ge healthcare) and eluted with 500 mm imidazole in hbs buffer. rbd was then purified by gel filtration chromatography using the superdex 200 column (ge healthcare) pre-equilibrated with hbs buffer. fractions containing ace2 were collected. the antibody competitive binding activities of the serum were assayed by elisa. ace2 (1ug/ml) was incubated in 96-well plate overnight at 4°c. after incubation, the plate was blocked with 2% bsa for 2h at 37°c and then washed five times with pbs containing 0.05% tween 20. bsa was used as negative control followed by the addition of a mixture of 40-fold diluted serum and rbd-mfc (0.15ug/ml) followed by incubation for 30 min with gentle shaking at 37°c. plates were washed five times with pbs containing 0.05% tween 20 (pbt) followed by 100 µl of horseradish peroxidase/anti-mfc antibody conjugate (diluted 1:5000 in pbt buffer), incubated 30 min with gentle shaking. plates were washed five times pbt buffer and developed with 100 µl of freshly prepared 3,3',5,5'-tetramethylbenzidine (tmb) substrate. reaction was stopped with 100 µl of 1.0 m h3po4 and read spectrophotometrically at 450 nm in a microtiter plate reader. 6 the production of cumvtt was described in detail in zeltins et al. 9 briefly, e coli c2566 cells the rbd was conjugated to cumvtt using the cross-linker succinimidyl 6-(betamaleimidopropionamido) hexanoate (smph) (thermo fisher scientific, 10-molar excess, 60 minutes, 23°c). the coupling reactions were performed with 0.3x molar excess of rbd, 0.3x rbd, or equal molar amount of rbd regarding the cumvtt (shaking at 23°c for 3 hours at 1200 rpm on dsg titertek; flow laboratories, irvine, united kingdom). unreacted smph and rbd proteins were removed using amicon-ultra 0.5, 100k (merck-millipore, burlington, mass). vlp samples were centrifuged for 2 minutes at 14,000 rpm for measurement on nd-1000. coupling efficiency was calculated by densitometry (as previously described for il17a-cumvtt vaccine 9 ), with a result of approximately 20% to 30%. pseudovirus expressing the sars-cov-2 spike protein was produced by lentivirus second the 293t-ace2 cells which stably express ace2 receptors on the cell membrane were prepared by transfection of ace2 gene into 293t cells using lentivirus system. pseudoviruses prepared above were added to the 293t-ace2 cells (3 × 10 4 cells/well) with 100 μl polybrene (16μg/ml). after 48 h, the infection was monitored using the luciferase assay system (promega). titer was calculated based on serial dilutions of pseudovirus. the mouse serum samples (2 μl) were diluted to 1:10, 1:40, 1:160, 1:640 and 1:2560 respectively, and then mixed with an equal volume of pseudovirus stock. after incubation at 37°c for 1 h, the mixture was inoculated on the 293t-ace2 cells (3 x 10 4 cells/well). at the same time, pseudovirus+dmem medium was set as a positive control and dmem medium only was set as a negative control. after the cells were incubated for 72 hours, serum neutralization was measured by luciferase activity of infected pseudovirus. a cut-off of >80% was used as to determine neutralizing titer. clinical characteristics of coronavirus disease 2019 in china characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a cases from the chinese center for disease control and prevention angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus composition and divergence of coronavirus spike proteins and host ace2 receptors predict potential intermediate hosts of sars-cov-2 a 193-amino acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme 2 an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus receptor-binding domain of sars-cov spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine therapeutic vaccines for chronic diseases: successes and technical challenges incorporation of tetanus-epitope into virus-like particles achieves vaccine responses even in older recipients in models of psoriasis, alzheimer's and cat allergy vaccination against il-31 for the treatment of atopic dermatitis in dogs treating insect-bite hypersensitivity in horses with active vaccination against il-5 pseudotyped lentiviral vectors: one vector, many guises. hum interaction of viral capsidderived virus-like particles (vlps) with the innate immune system vaccine against peanut allergy based on engineered virus-like particles displaying single major peanut allergens generation of high-titer pseudotyped lentiviral vectors key: cord-260412-yjr83ef6 authors: hotez, peter j.; bottazzi, maria elena title: developing a low-cost and accessible covid-19 vaccine for global health date: 2020-07-29 journal: plos negl trop dis doi: 10.1371/journal.pntd.0008548 sha: doc_id: 260412 cord_uid: yjr83ef6 nan there is an urgent need to advance safe and affordable covid-19 vaccines for low-and middle-income countries of asia, africa, and latin america. such vaccines rely on proven technologies such as recombinant protein-based vaccines to facilitate its transfer for emerging market vaccine manufacturers. our group is developing a two-pronged approach to advance recombinant protein-based vaccines to prevent covid-19 caused by sars-cov-2 and other coronavirus infections. one vaccine is based on a yeast-derived (pichia pastoris) recombinant protein comprised of the receptor-binding domain (rbd) of the sars-cov formulated on alum and referred to as the cov rbd219-n1 vaccine. potentially, this vaccine could be used as a heterologous vaccine against covid-19. a second vaccine specific for covid-19 is also being advanced using the corresponding rbd of sars-cov-2. the first antigen has already undergone current good manufacturing practices (cgmp) manufacture and is therefore "shovel ready" for advancing into clinical trials, following vialing and required good laboratory practice (glp) toxicology testing. evidence for its potential efficacy to cross-protect against sars-cov-2 includes cross-neutralization and binding studies using polyclonal and monoclonal antibodies. evidence in support of its safety profile include our internal assessments in a mouse challenge model using a lethal mouse-adapted sars strain, which shows that sars-cov rbd219-n1 (when adsorbed to aluminum hydroxide) does not elicit eosinophilic lung pathology. together, these findings suggest that recombinant protein-based vaccines based on the rbd warrant further development to prevent sars, covid-19, or other coronaviruses of pandemic potential. "the thing we have to think about now that's different is, how do we produce vaccines specifically for the developing world if this is a truly global epidemic."-seth berkley, ceo, gavi as of june 2020, covid-19 caused by the sars-cov-2 coronavirus has infected more than 7 million people globally (confirmed cases) and caused almost 400,000 deaths [1] . although the epidemic began in china, europe, and the united states, there are significant concerns about the risks of disease emergence in low-and middle-income nations. there are now more almost 750,000 cases in brazil, 300,000 cases in india, and 50,000 cases in south africa, such that covid-19 will become widespread among the poor living in the group of 20 nations [1, 2] . moreover, sars-cov-2 infection is expected to emerge in the global south [3] . in the african region of the world health organization (who), covid-19 is now spreading in the populated areas of ghana, nigeria, and democratic republic of congo, and presumably across the region [1] . in nations such as india, for example, the feasibility of enforcing social distancing in large and crowded urban centers will be particularly daunting [3] , so that ensuring access to a safe and affordable covid-19 vaccine will become a global priority. dr. seth berkley, the ceo of gavi, the vaccine alliance, has highlighted the importance of prioritizing a covid-19 vaccine specifically for these countries [4] . at least a dozen covid-19 candidate vaccines are under development using different technology platforms [5] , with an emphasis on speed, maximizing safety, and avoiding vaccineinduced immunopathology [6] . many of these will enlist cutting-edge nucleic acid delivery technologies and other innovative approaches. in the meantime, there is urgency to address and rapidly respond to gavi's charge and pursue safe, low-cost, easily administered, and rapidly scalable approaches. for instance, texas children's center for vaccine development (cvd) at baylor college of medicine, in collaboration with its nonprofit product development partners-seattle-based path and infectious disease research institute (idri)-have been spearheading a coronavirus vaccine program focusing on recombinant subunit protein vaccines produced in a globally available microbial fermentation platform, and optimized to maximize yield following expression and protein purification [7, 8] . towards this goal, we are now also developing the sars-cov-2 rbd recombinant protein as a potential vaccine candidate, in parallel with the existing cov rbd219-n1 candidate vaccine, which was previously developed and manufactured under cgmp in 2016 [7] [8] [9] [10] . the bulk drug substance has been stored frozen (−70˚c to 80˚c) and remains stable through ongoing testing. furthermore, an independent quality assessment confirmed the suitability of the material through phase 2 clinical trials. both rbd vaccine candidates have potential as vaccine antigens to prevent sars-cov-2 infection and/or covid-19. overall, our initial approach relies on advancing the already manufactured cov rbd219-n1 as a heterologous recombinant subunit vaccine to protect against both sars and covid-19 [9] , and in parallel accelerate the advancement of the sars-cov-2 rbd candidate as a homologous covid-19 vaccine (fig 1) . our preliminary studies now show that the sars-cov-2 rbd candidate, which is specific for the sequence of the sars-cov-2, can also be highly produced in the yeast p. pastoris. both approaches reinforce each other, as the processes developed for the cov rbd219-n1 candidate also apply to the sars-cov-2 candidate, and both antigens downstream could be further developed as potentially a bivalent or a universal coronavirus vaccine. the sars-cov protein known as cov rbd219-n1 was selected on its ability to elicit high titers of neutralizing antibodies against both sars-cov pseudotype virus and live sars-cov virus [7, 8] , prior to confirmatory testing against sars-cov challenge in animal models. it also induced high-level neutralizing antibodies and protective immunity with minimal immunopathology in mice after a homologous virus challenge with sars-cov (ma15 strain) [9, 10] . there are several advantages of the cov rbd candidate antigens and vaccines for purposes of global health: 1. high yield and low cost. the antigens are expressed in p. pastoris, a low-cost expression platform, which can be produced and scaled at high yields [7, 8] . by deleting an n-linked glycosylated asparagine at the n-1 position of rbd219, both the yield and antigenicity improved. at a 10-liter scale production process, the cov rbd219-n1 antigen was produced through fermentation at 400 mg/l fermentation supernatant (fs) with purification recovery >50% [7, 8] . a panel of characterization tests indicates that the process is reproducible and robust and that the purified, tag-free rbd219-n1 protein has high purity and a well-defined structure. it is therefore suitable for both pilot scale manufacturing and for transition into process improvements leading to industrial scale manufacturing. 2. technology transfer. the process is suitable for technology transfer to emerging market vaccine manufacturers (aka dcvms, developing country vaccine manufacturers) having expertise in fermentation technology (https://www.dcvmn.org/) [11] . the p. pastorisderived recombinant protein is currently produced by several dcvms, including those in bangladesh, brazil, cuba, india, and indonesia. 3. shovel ready. the cov rbd219-n1 antigen was manufactured under cgmp and can be vialed to produce between 20,000 and 200,000 doses, with the possibility of transferring production processes and cell banks to dcvms for large-scale production sufficient to meet global needs. beyond low cost and ease of potential technology transfer to dcvms, an advantage of employing a recombinant protein subunit vaccine is the long-standing safety record of this class of vaccines, and the fact that this technology has been used for the licensure of two other antiviral vaccines-hepatitis b and human papillomavirus, as well as biologics (e.g., insulin) [11] . in addition to their low cost and suitability for use in public immunization programs in lowand middle-income countries, we pursued rbd recombinant protein-based vaccines as a technology to maximize safety relative to other platforms, such as virus vectors that have previously been found to induce immune enhancement. for instance, immune enhancement in children following a formalin-inactivated respiratory syncytial virus (rsv) vaccine was first reported in the 1960s and later shown to occur in laboratory animals with early prototype sars-cov vaccines using virus-vectored platforms or inactivated virus constructs [12] . we have recently summarized the major safety concerns of some prototype coronavirus vaccines based on studies conducted in laboratory animals (rodents, ferrets, and nonhuman primates) [12] . they include the following points. some of the earliest sars-cov vaccine candidates used vectored-based platforms, and these were associated with immune enhancement or activation. in 2004-2005, scientists at the public health agency of canada's national microbiology laboratory in winnipeg, manitoba (who helped to develop the first successful ebola vaccine), found that a recombinant modified vaccinia ankara (rmva) expressing the s-spike protein resulted in severe liver pathology upon sars-cov virus challenge. similarly, rmva expressing the s-spike also resulted in lung immunopathology in rhesus macaques, as did other virus-vectored constructs. lung immunopathology is also linked to whole inactivated viral vaccines. however, it was determined that in many cases eosinophilic pathology is driven by the sars nucleocapsid (n) protein, although a recent trial in nonhuman primates found that an alum-adjuvanted inactivated sars-cov-2 vaccine did not induce immunopathology [13] . among the major conclusions of these studies was that they may be driven by t helper-17 (th17) responses linked to interleukin-6 [12, 14] , and that aluminum formulations exhibit greatly reduced immunopathology [15] . given the history of virus-vector platforms and inactivated vaccines in eliciting eosinophilic immunopathology, our emphasis has been on the evaluation of inexpensive recombinant proteins produced in microbial systems. these are comprised of the cov rbd219-n1 antigen, encoding amino acids 319-536 (219 aa) of the sars-cov s-spike protein [7] [8] [9] [10] , and now a second, cov2 rbd antigen, which is also expressed without the n-terminal amino acid. the rationale for selecting the rbd domain of the s protein includes focusing on the key component that binds to the human angiotensin converting enzyme 2 (ace2) receptor, and removing the known elements of the s protein involved in immune enhancement. supporting studies summarized elsewhere emphasize how s protein peptides outside of the rbd can induce immune enhancement in non-human primates [12] . moreover, cov rbd219-n1 induce high titers of neutralizing antibodies in mice and 100% infection against sars cov virus challenge [10] . alum formulations of cov rbd 219-n1 do not induce immunopathology [10] , a finding consistent with other published studies [13] [14] [15] . there is evidence to justify advancing the cov rbd219-n1 antigen as either a homologous vaccine against sars [7] [8] [9] [10] or as a heterologous vaccine against covid-19 [9] . in parallel, a cov2 rbd protein candidate is being advanced. regarding the former, against sars cov homologous virus challenge the vaccine formulated on alum exhibits high levels of protective immunity and with evidence of minimal or no immune enhancement [10] . with regards to cross-protection against sars cov2, the rbd of the sars-cov-2 and cov rbd219-n1 share significant similarity of amino acid sequence (> 75% identity, >80% similarity) and there is evidence that both viruses use the human ace2 receptor for cell entry [9] . further published studies indicate strong antigenic similarities between the sars-cov and sars-cov-2 rbds, and the potential for cross protection. for example, serum from a convalescent sars-cov patient was shown to neutralize sars-cov-2 driven entry [16] . moreover, new studies by tai and colleagues find that using pseudotyped sars-cov-2, the sars-cov rbd blocks the entry of both sars-cov and sars-cov-2 pseudovirus into human ace2-expressing 293t cells [17] . through pseudovirus neutralization activity, it was found that sars-cov rbd-specific antisera could neutralize sars-cov-2 pseudovirus infection, suggesting that sars-cov rbd-specific antibodies can cross-react with sars-cov-2 rbd and cross neutralize sars-cov-2 pseudovirus infection [17] . additional studies find that multiple (but not all) neutralizing monoclonal antibodies bind to both rbds [9, 18, 19 ]. an international priority is the scale-up and global access of an affordable and safe recombinant vaccine to prevent emerging coronavirus infections, including covid-19. our aspirational goal is to protect global populations at risk for this important emerging virus infection. a low-cost recombinant protein antigen expressed in p. pastoris and formulated on aluminum or other accessible adjuvants represents a highly accessible technology to transfer to low-and middle-income countries. it represents one of several key mechanisms for ensuring that populations across the major affected nations of africa, asia, and the americas will benefit from covid-19 vaccinations. world health organization. covid-19 situation reports poverty and the impact of covid-19: the blue-marble health approach millions in india under coronavirus lockdown as major cities restrict daily life will vaccines reach low-income countries during a global pandemic the outbreak of sars-cov-2 pneumonia calls for viral vaccines don't rush to deploy covid-19 vaccines and drugs without sufficient safety guarantees optimization of the production process and characterization of the yeast-expressed sars-cov recombinant receptor-binding domain (rbd219-n1), a sars vaccine candidate yeast-expressed recombinant protein of the receptor-binding domain in sars-cov spike protein with deglycosylated forms as a sars vaccine candidate potential for developing a sars-cov receptor-binding domain (rbd) recombinant protein as a heterologous human vaccine against coronavirus infectious disease (covid)-19 yeast-expressed sars-cov recombinant receptor-binding domain (rbd219-n1) formulated with alum induces protective immunity and reduces immune enhancement the yeast stands alone: the future of protein biologic production covid-19 vaccine design: the janus face of immune enhancement rapid development of an inactivated vaccine candidate for sars-cov-2 the potential role of th17 immune responses in coronavirus immunopathology and vaccine-induced immune enhancement. microbes and infection covid-19 vaccines: neutralizing antibodies and the alum advantage pö hlmann s. the novel coronavirus 2019 (2019-ncov) uses the sars coronavirus receptor ace2 and the cellular protease tmprss2 for entry into target cells characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine cryo-em structure of the 2019-ncov spike in the prefusion conformation identification of sars-cov rbd-targeting monoclonal antibodies with cross-reactive or neutralizing activity against sars-cov-2 key: cord-268144-maa8c4a4 authors: zhang, yuan; zheng, nan; zhong, yang title: computational characterization and design of sars coronavirus receptor recognition and antibody neutralization date: 2007-02-17 journal: comput biol chem doi: 10.1016/j.compbiolchem.2007.02.005 sha: doc_id: 268144 cord_uid: maa8c4a4 the sequential determination of crystal structures of the sars coronavirus spike receptor-binding domain (rbd) in complex with its cellular receptor or neutralizing antibody opened a door for the design and development of antiviral competitive inhibitors. based on those complex structures, we conduct computational characterization and design of rbd-mediated receptor recognition and antibody neutralization. the comparisons between computational predictions and experimental evidences validate our structural bioinformatics protocols. and the calculations predict a number of single substitutions on rbd, receptor or antibody that could remarkably elevate the binding affinities of those complexes. it is reasonable to anticipate our structure-based computation-derived hypotheses could be informative to the future biochemical and immunological tests. as an envelope glycoprotein, the spike protein of severe acute respiratory syndrome coronavirus (sars-cov) plays a key role in the viral entry and neutralization (bartlam et al., 2005; denison, 2004; lau and peiris, 2005; xu and gao, 2004; zhu, 2004) . this structural protein consists of two functional regions: the outer globular s1 region responsible for the initial attachment to cellular receptor and the inner stalk s2 region contributing to the subsequent fusion between viral envelope and cellular membrane (beniac et al., 2006; hofmann and pohlmann, 2004; lin et al., 2005; xiao and dimitrov, 2004) . a membrane-associated zinc metallopeptidase, angiotensinconverting enzyme 2 (ace2), has been identified as the functional receptor for sars-cov (li et al., 2003) . and a soluble form of ace2 could block the association of s1 region with the permissive vero e6 cells (li et al., 2003; moore et al., 2004) . in addition, a 193-amino acid fragment (residues 318-510), located within the s1 region, was demonstrated as an independently folded receptor-binding domain (rbd) capable of attaching ace2 more efficiently (ic 50 < 10 nm) compared with the full s1 region (ic 50 ≈ 50 nm) . besides, this rbd was able to elicit highly potent neutralizing antibodies in the immunized animals, which conferred those animals significant protection from the challenge of pathogenic sars-cov (du et al., 2006; he et al., 2004 he et al., , 2005a he et al., ,b, 2006a zakhartchouk et al., 2006; zhao et al., 2006) . moreover, a human monoclonal antibody 80r, isolated from a nonimmune human antibody library, was shown to potently neutralize sars-cov through targeting the rbd and blocking receptor recognition (sui et al., 2004) . the epitope mapping illustrated a 180-amino acid conformationally sensitive fragment (residues 324-503) within the rbd was the neutralizing epitope of 80r (sui et al., 2005) . furthermore, another human monoclonal antibody m396 also exhibited potent neutralization of sars-cov by competition with ace2 for binding to rbd (prabakaran et al., 2006) . together those data suggest the receptor association process of sars-cov is an attractive opportunity for therapeutic intervention (de clercq, 2006; he and jiang, 2005; hofmann and pohlmann, 2004; jiang et al., 2005; kuhn et al., 2004; yeung et al., 2006) . the peptide or peptidomimetic antagonist leads, including the sars-cov spike rbd, the soluble form of ace2 and the neutralizing antibodies 80r plus m396, should be able to potently abolish viral attachment to host cells. in this study, we conducted structural bioinformatics analyses on the crystal structures of the sars-cov rbd complexed with functional receptor or neutralizing antibody (hwang et al., 2006; li et al., 2005a li et al., ,2006 prabakaran et al., 2006) to predict single substitutions on spike rbd, receptor or antibodies possibly causing remarkable elevation in the binding affinities of complexes for the design and development of anti-sars agents. three coordinates files were retrieved from the protein data bank (pdb) (berman et al., 2000) . one file is the ace2-bound rbd (pdb code: 2ajf) (li et al., 2005a) , while the others are the rbd complexed with 80r (pdb code: 2ghw) (hwang et al., 2006) or m396 (pdb code: 2dd8) (prabakaran et al., 2006) . both the first and second files harbor a pair of sister complexes. and in the third file, either the heavy chain or the light chain of m396 makes its own contacts with the rbd. thus, a total of six complex structures (ae/bf for ace2-rbd, ab/cd for 80r-rbd and hs/ls for m396-rbd) are subject to computational simulations, respectively. firstly, the program foldx , based on an empirical effective energy function, was employed for calculation of the binding free energy values of wild type complexes. then, a computational alanine scanning on the protein-protein interfaces was performed for evaluation of energetical contribution from single binding sites to the complex formation. those positions yielding a calculated increase in association energy of more than 1 kcal/mol on alanine substitution were defined as energetic hot (important) spots according to previous criteria (guerois et al., 2002; guerois and serrano, 2000; kiel and serrano, 2006; kiel et al., 2004 kiel et al., , 2005 . the next step was to redesign the interactions between rbd and its binding partners through the software deepview (arnold et al., 2006; guex and peitsch, 1997) . each of the binding sites on the rbd, receptor or antibody was saturated with virtual substitutions, i.e., replaced with all the 20 natural amino acid residues except the original one. finally, the reconstructed models were feed to the program foldx to compute their binding energies. here, only the variants rewarded a value of at least 1 kcal/mol lower than that of the wild type were taken into consideration. the calculated binding energy values and hot spots of the wild type complexes are shown in table 1 . the complexes ace2-rbd (ae/bf) and 80r-rbd (ab/cd) show a close correlation between their interaction energies (−15.78/−14.82 kcal/mol versus −18.36/−16.53 kcal/mol) and buried surface area (1700å 2 versus 2200å 2 ), gap volume (7000å 3 versus 4000å 3 ), or binding affinity (1.70 nm versus 1.59 nm) (hwang et al., 2006) . those obvious associations indicate that the higher geometric complementarity, corresponding to the larger buried surface area and the smaller gap volume, offers the complex 80r-rbd rather than the complex ace2-rbd the lower interaction energy and consequently the stronger binding affinity. similarly, the correlation of binding energy with buried surface area is also found for the complex m396-rbd in which the heavy chain and the light chain contribute 66% and 34% to the total buried surface (prabakaran et al., 2006) . and the rbd association energy of the former chain (−8.64 kcal/mol) is remarkably lower than that of the latter (−5.42 kcal/mol). the perfect agreements of computational predictions with structural observations or biochemical evidences strongly suggest the reliability of our protocols. as to the hot spots of complexes, the consistency between computational predictions and experimental evidences is clearly detected for rbd and receptor. in ace2-rbd complexes, three receptor residues (glu37, asp38 and tyr41 on the chain a of complex ae or glu37, tyr41 and lys353 on the chain b of complex bf) form one hot spot cluster interacting with another hot cluster formed by five or six rbd residues (arg426, tyr436, tyr475, tyr484 and tyr491 on the chain e of complex ae and the chain f of complex bf, whereas asn473 only on the chain f). the interactions between the two hot clusters make the major contribution to the binding free energy of ace2-rbd complexes. notably, our predictions are in agreement with previous experimental alanine mutagenesis, which identified two hot spots on rbd (arg426 and asn473) (chakraborti et al., 2005) and another two on receptor (tyr41 and lys353) . in addition, computational alanine scanning on the sister complexes ab and cd successfully identify a rbd hot spot (asp480) revealed in mutational binding analyses (sui et al., 2005) . in sharp contrast to ace2, the antibody 80r possesses four hot residues (tyr102, asn164, arg223 and trp226) being scattered on the binding surface rather than centralized into a cluster. the difference in the number and distribution of hot spots might account for the large gap between the interaction energies of 80r-rbd (−18.3627 and −16.5309 kcal/mol) and those of ace2-rbd (−15.7765 and the complexes ae and bf with the chains a, b for ace2 and the chains e, f for rbd; the complexes ab and cd with the chains a, c for rbd and the chains b, d for 80r; the complexes hs and ls with the chains h, l and s for heavy and light chains of m396 plus rbd. for association with rbd, or the higher spike-binding affinity of 80r compared to that of receptor. finally, only two neighboring hot spots (trp91 and asp92) are found on the light chain of m396 while none on the heavy chain. thus, an interesting discovery is the fact that among the five or six ace2-binding hot spots of rbd, three (tyr436, tyr484 and tyr491) are simultaneously 80r-neutralizing hot spots whereas only one (tyr491) is important for m396 neutralization. this finding indicates that 80r might have the greater potential than m396 for inhibition of spike-mediated infection. in summary, the consistency of calculations with experiments mentioned above further validates our approaches to characterize protein-protein interactions. the predicted replacements on spike rbd, cellular receptor or neutralizing antibody with significant increase in binding affinity are listed in table 2 . the comparisons between virtual mutants derived from sister complexes of ace2-rbd or 80r-rbd consistently identify a number of substitutions worth of biochemical and immunological experimental tests. for instance, recent experimental evidences revealed the great potential of ace2 in the protection of several animal models from sars-cov-induced lung injury or severe acute lung failure kuba et al., 2005 kuba et al., , 2006 . simultaneously, the crystal structures of the native and inhibitor-bound forms of ace2 towler et al., 2004; turner et al., 2004) successfully laid a solid foundation for the discovery of novel small-molecule inhibitors of its enzymatic activity or spikemediated virus entry by chemical genetics (huentelman et al., 2004; kao et al., 2004) and the identification of its crucial activesite residues by site-directed mutagenesis (guy et al., 2005a,b) . very recently, a modest anti-sars activity (ic 50 ≈ 0.1 mm) was observed for an ace2-derived peptide containing two segments of receptor (residues 22-44 and 351-357) linked by glycine (han et al., 2006) . it should be pointed out that both the experimentally confirmed hot spots (tyr41 and lys353) and the predicted sites for replacements (thr27, lys31 and his34) are nested in those two segments. similarly, a small peptide derived from spike protein (residues 483-493) also block viral receptor recognition with ic 50 of 6.99 nm (ho et al., 2006) . and our calculated two hot spots (tyr484 and tyr491) in combination with two target positions (tyr484 and gln492) are located in this short fragment, too. consequently, it is reasonable to anticipate that our blueprint could effectively increase the binding affinity of the two novel peptides to disrupt sars-cov infection. the swiss-model workspace: a web-based environment for protein structure homology modelling structural insights into sars coronavirus proteins architecture of the sars coronavirus prefusion spike the protein data bank the sars coronavirus s glycoprotein receptor binding domain: fine mapping and functional characterization potential antivirals and antiviral strategies against sars coronavirus infections severe acute respiratory syndrome coronavirus pathogenesis, disease and vaccines: an update receptorbinding domain of sars-cov spike protein induces long-term protective immunity in an animal model the sh3-fold family: experimental evidence and prediction of variations in the folding pathways predicting changes in the stability of proteins and protein complexes: a study of 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ace2: from vasopeptidase to sars virus receptor a 193-amino acid fragment of the sars coronavirus s protein efficiently binds angiotensinconverting enzyme 2 immunological responses against sars-coronavirus infection in humans severe acute respiratory syndrome coronavirus entry into host cells: opportunities for therapeutic intervention immunogenicity of a receptor-binding domain of sars coronavirus spike protein in mice: implications for a subunit vaccine a study on antigenicity and receptor-binding ability of fragment 450-650 of the spike protein of sars coronavirus key: cord-288761-fyvr0tc1 authors: santiago, césar; mudgal, gaurav; reguera, juan; recacha, rosario; albrecht, sébastien; enjuanes, luis; casasnovas, josé m. title: allosteric inhibition of aminopeptidase n functions related to tumor growth and virus infection date: 2017-04-10 journal: sci rep doi: 10.1038/srep46045 sha: doc_id: 288761 cord_uid: fyvr0tc1 cell surface aminopeptidase n (apn) is a membrane-bound ectoenzyme that hydrolyzes proteins and peptides and regulates numerous cell functions. apn participates in tumor cell expansion and motility, and is a target for cancer therapies. small drugs that bind to the apn active site inhibit catalysis and suppress tumor growth. apn is also a major cell entry receptor for coronavirus, which binds to a region distant from the active site. three crystal structures that we determined of human and pig apn ectodomains defined the dynamic conformation of the protein. these structures offered snapshots of closed, intermediate and open apn, which represent distinct functional states. coronavirus envelope proteins specifically recognized the open apn form, prevented ectodomain progression to the closed form and substrate hydrolysis. in addition, drugs that bind the active site inhibited both coronavirus binding to cell surface apn and infection; the drugs probably hindered apn transition to the virus-specific open form. we conclude that allosteric inhibition of apn functions occurs by ligand suppression of ectodomain motions necessary for catalysis and virus cell entry, as validated by locking apn with disulfides. blocking apn dynamics can thus be a valuable approach to development of drugs that target this ectoenzyme. the apn ectodomain structure. as the apn protein is a type ii membrane protein, ectodomain expression required deletion of the n-terminal cytoplasmic and transmembrane domains, and introduction of a secretion signal sequence, as well as a hemagglutinin (ha) tag to allow protein detection and purification ( supplementary fig. s1a ). as the n-terminal and middle portions of the hapn and papn ectodomains are heavily glycosylated, we produced them in cho cells (see methods). the purified proteins generated distinct crystal forms under different crystallization conditions (table 1 and methods). in the past we reported a papn ectodomain crystal structure in complex with a cov spike (s) fragment (pdb code 4f5c) 16 , here we show three new structures for apn (table 1 ). in the four structures, the n-terminal ha tag and ~30 ectodomain residues were very disordered, indicating a large degree of flexibility in the membrane proximal polypeptide. the ectodomains have a hook-like conformation formed by domain i to iv and contained a zinc ion at the active site in domain ii (fig. 1 ). the exposed convex side of domain iv mediates similar protein dimerization in the distinct crystals. approximately 950 å 2 of each monomer is buried at the dimer interface ( table 2) , indicative of a stable protein-protein interaction. domain iv is the largest apn domain and the most divergent in the m1 aminopeptidase family. in apn, domain iv has seven helix-turn-helix heat repeats and a single arm repeat formed by three alpha helices (α 25-α 27) . the arm repeat is the most variable domain iv region in the hapn and papn structures, and can contact the peptide substrate bound to the active site (see below). although the dimeric assembly of human and pig apn ectodomains was preserved in various crystals, the conformation of each monomer differed among crystal forms, such that the distance between the n-terminal region of the ectodomains that formed the dimer varied from 95 to 131 å in the structures ( table 2 and supplementary fig. s1b) . each crystal captured a single apn conformation, with all the monomers in the same form. these structures identified three distinct apn conformations, based on active site accessibility, which we termed closed, intermediate and open forms (fig. 1a) . as reported for other m1 family members 23, 24 , the observed apn structural diversity indicated ectodomain dynamics in solution and on the cell surface. the active site accessibility at domain ii differed among crystal forms because of interdomain adjustments in the apn. the contacts between domain iv and other domains in the monomers varied among the structures, whereas the domain iv-iv buried surfaces in each monomer at the dimerization interfaces were preserved ( table 2) . domain iv contacts with domain i or iii changed markedly less (~100-200 å 2 buried surface) than with domain ii (1000 å 2 ); domain ii-iv interaction thus mainly stabilized the closed apn conformation. there were no notable differences in the other interdomain contacts in the distinct apn forms (table 2) . domains i-ii are distant from domain iv in the open conformation of the apn monomer (fig. 1a,b) , where the zinc ion at the catalytic site is more accessible to the solvent (fig. 1c) . the domain i to iii module swings 15° toward domain iv, closing the active site (fig. 1b,c) . the hapn structure adopts an intermediate conformation in the crystals (fig. 1a) ; the distance between the n terminus of each monomer in the dimer is 116 å, and the angle difference of fig. 1a and table 2 ). on the cell surface, the domain i to iii module must swing over domain iv, which is fixed by dimerization (fig. 1b, supplementary video s1). the module movement must be facilitated by the flexible ~30-residue polypeptide that links domain i to the transmembrane domain ( supplementary fig. s1a ), although polypeptide length probably limits the interdomain movement shown here with apn (15°), which is less pronounced than that reported for erap-1 (22°, determined as in table 2 ). the type of interdomain movement also differs between erap-1 and the apn. the domain iii-iv module moves together relative to domain i-ii in erap-1, whereas domain i to iii swings over domain iv in the apn. in addition, the erap-1 hinge region is at the domain iii n terminus, whereas that of apn is in the domain iv n terminus. domains i, ii and iii can pivot at the beginning of the first (α 13) or third (α 15) domain iv helix, which are perpendicular to the swing angle (fig. 1b) . these differences in apn motion compared to other aminopeptidases are probably related to dimer formation, which is not observed in other m1 family members. the domain ii buried surface increases due to its interaction with domain iv when the conformation changes from open to closed (table 2) , thus reducing accessibility of the active site cavity (fig. 1c) . m1 aminopeptidase dynamics is thought necessary for catalysis, and the closed the met residues of the papneh were replaced by seleno-met (see methods). highest-resolution shell is in parentheses. favored, allowed and outlier residues (%) in the ramachandran plot, as well as number of ectodomains in the asymmetric unit (asu) are shown. statistics for the papn-rbd crystal structure discussed here have been reported earlier (pdb code 4f5c) 16 . structure representations in supplementary fig. 1b . (fig. 2a) . in the closed papn, the side chain of a phenylalanine (phe893) at domain iv was placed at about 4.5 å from the hydrolyzable peptide bond, whose carbonyl group is coordinated to the zinc ion. the phenylalanine was located in the loop that connects α 26 and α 27 in the single domain iv arm repeat of human and pig apn (fig. 2a) ; it penetrated the active site groove in the closed conformation and locked the peptide, ready for hydrolysis. domain iv residues that precede phe893 in the α 26-α 27 loop contacted domain ii in the closed papn. a similar loop conformation is seen in a closed hapn structure (pdb code 4fys) 19 . the phenylalanine side chain in closed apn probably hinders peptide release or translocation for further processing after p1 hydrolysis. it is likely that binding of the p1′ residue to the zinc ion required removal of the phenylalanine plug by domain ii displacement away from domain iv. the phenylalanine adopted a distinct conformation in the intermediate and open apn conformations (fig. 2a) . domain ii movement was accompanied by a conformational change of the α 26-α 27 loop in domain iv (supplementary video s1), which became more solvent-exposed; the phenylalanine side chain faced into domain iv in the intermediate and open conformations and the peptide plug was removed from the active site. these changes would facilitate release of the n-terminal residue after hydrolysis. the small interdomain movement of the intermediate apn structure would be sufficient for peptide processing (fig. 2a) . we previously described in detail the cov spike rbd-papn binding interface 16 . the porcine cov spike rbd binds to a papn region that is distant from the catalytic site ( supplementary fig. s1b) . a critical cov receptor-binding motif, which bears an exposed tryptophan, penetrates a narrow cavity formed by domain ii and iv (fig. 2b) 16 . the tryptophan aromatic side chain stacks onto papn domain iv residues his786-pro787, and is trapped by domain iv residues asn783-pro787 on one side and domain ii residues gln367-ser368 on the other (fig. 2b) . the main chain of domain ii residues is in close contact (3.9 å) with the tryptophan side chain, and its imino nitrogen forms a hydrogen bond with the domain iv asn783 main chain carbonyl. domain iv-based superposition of the open papn with bound rbd and that of closed papn showed a shift in the domain ii main chain region that contacts the rbd; this region collides (< 3.0 å) with the cov tryptophan (fig. 2b) . closing of the ectodomain would hinder penetration of the viral tryptophan between the papn domain ii and iv. cov binding to apn would lock the protein in its open conformation (fig. 2b) , preventing the ectodomain movement probably necessary for peptide hydrolysis (fig. 2a) . we analyzed the catalytic activity of soluble human and pig apn ectodomains in the presence of porcine cov s fragments bearing the rbd (fig. 3) . the soluble s proteins specifically inhibited papn-mediated catalysis, measured as the hydrolysis of the l-pna substrate, and had no effect on hapn activity. the tgev (transmissible gastroenteritis coronavirus) spike does not bind hapn because it lacks the n-linked glycan recognized by porcine cov in papn 15, 16 . the isolated rbd was sufficient to inhibit papn catalysis (fig. 3a) ; inhibition was dependent on rbd concentration. a high rbd:papn ratio was needed to achieve maximum inhibition (50-60%; fig. 3b ), which decreased slowly after 30 min (fig. 3c) drugs that bind the catalytic site inhibit cov binding to apn. non-hydrolyzable drugs that bind the apn catalytic site inhibit catalysis and prevent angiogenesis and tumor growth 7, 10, 26, 27 . they appear to restrict ectodomain conformational changes, as shown by reduction in the number of some apn conformation-specific fig. s1b) , with residues that contact the rbd in sticks with carbons in yellow (domain ii) and green (domain iv). the same residues are shown for the superposed closed structure (carbons in grey). the rbd motif that penetrates the papn cavity is shown with a grey surface and with residues as sticks (carbons in cyan or in magenta for trp). scientific reports | 7:46045 | doi: 10.1038/srep46045 mab epitopes 25 . on the cell surface, active site epitopes recognized by the my7 mab decrease in the presence of actinonin, which indicates apn closure. crystal structures of m1 aminopeptidases in complex with these drugs show preferential adoption of a closed state 19, 20, 24, 28 . drug binding would thus not only compete with substrates for active site binding, but might also restrict the aminopeptidase dynamics needed for peptide processing. the structure of the papn-rbd complex indicates that porcine cov would be specific for the open conformation (fig. 2b) . restriction of apn ectodomain opening by active site-binding drugs would thus have an allosteric effect on cov binding. to test this hypothesis, we studied tgev rbd binding to cell surface papn in the presence of drugs that bind to the active site (fig. 4) . in flow cytometry, we determined the binding of an rbd-fc fusion protein to cells that expressed papn or an active site mutant (papn-hh/aa), alone or with various drugs (fig. 4a,b) . we analyzed the effect of the natural apn inhibitors actinonin and bestatin 29 ; both reduced rbd-fc binding to cell surface papn (fig. 4a, left) . we then evaluated four synthetic amino-benzosuberone (abs) derivatives that bind with high affinity and selectivity to apn (supplementary fig. s3a) 30 ; all four abs molecules prevented rbd binding to papn and its effectiveness increased with apn-binding affinity (fig. 4b, left) . the bulkier abs2 and abs4 compounds, which contain a phenyl group and bind with the highest affinity to apn, more efficiently blocked binding of the tgev rbd to papn on the cell surface. the inhibitory molecules bind to the apn active site 19, 21 , which is distant from the apn region recognized by cov (supplementary fig. s1b) . to further determine whether the inhibitory effect was linked to drug binding to the papn active site, we analyzed rbd binding to the papn-hh/aa mutant, which lacks the two histidines (h383 and h387) that coordinate the zinc ion ( supplementary fig. s2) . staining for the rbd-fc protein was similar in cells expressing the mutant, alone or with the drugs (fig. 4a right and b right) , which showed that compound binding to the papn active site was necessary to prevent rbd binding to a distant site. in addition, inhibition of rbd binding to papn was drug concentration-dependent (fig. 4c) , and the amount of compound needed to reach 50% inhibition (ic 50 ) decreased with compound affinity for apn (~30 μ m for bestatin (ki ~4 μ m), ~1 μ m for actinonin (ki ~1 μ m), ~0.1 μ m for abs4 (ki ~0.06 nm) ). these results show that drugs that bind to the cov cell entry and infection 31 . we nonetheless found that active site-binding molecules hindered cov s protein binding and might inhibit virus infection. studies with low affinity binding drugs such as bestatin show no reduction in tgev infection 31 . virus particles have high receptor-binding avidity, and these drugs might not have sufficient affinity to maintain most apn molecules closed. the selective compounds abs1-4 have high affinity for apn and, at 1-10 μ m concentrations, inhibit capillary tube formation in cell cultures, with no cytotoxicity 27 . in our cultures, we observed no toxicity at abs concentrations < 100 μ m (not shown). we therefore analyzed the tgev-mediated cytopathic effect for each of the four abs molecules and actinonin at a 50 μ m concentration and monitored inhibition of virus infection with abs4 (2 log) and abs2 (1 log) (fig. 5a) ; at the same concentration, the lower-affinity abs1 and abs3 compounds or actinonin did not inhibit. abs4 has a bromo substituent that is predicted to interact with the phenylalanine that plugs the substrate in the closed conformation 32 ; this interaction likely helped maintain the closed ectodomain and efficiently prevented virus binding. tgev is a representative, extensively studied animal cov that use papn for cell entry 4, 14 . to further determine whether abs4 inhibition of virus infection was linked to cell entry, we analyzed tgev replication at 6 h post-infection and found that virus entry decreased with the abs4 concentration (fig. 5b ). abs4 addition after virus absorption at 4 °c did not inhibit virus growth (not shown), which indicates that it prevented virus binding to cells. in addition, we studied the effect of abs4 concentration on tgev cell infection, and we observed that the tgev cytopathic effect was reduced and cell survival increased at higher abs4 concentrations (fig. 5c) . abs compounds are selective for apn molecules and designed to inhibit apn catalytic activity and tumor growth; here we show that they also prevent cov cell infections. to further analyze the importance of apn dynamics, we engineered disulfide bonds to bridge domains ii and iv and restrict ectodomain motion. we replaced the papn domain ii ser464 and domain iv ser892 and/or ser929 with cysteine to lock the ectodomain in the closed form with interdomain disulfide bridges. the ser464 main chain cα is ~5 and ~6 å, respectively, from ser892 and ser929 in the closed form, but ser464 moves ~12 å away in the open form ( supplementary fig. s4 ). disulfide bond formation between papn cys464 and cys892 or cys929 should thus prevent ectodomain motion. we expressed the papn-cysteine mutants (c2-c4) on the 293t cell surface and compared their catalytic and cov binding activity with that of the wild type papn (fig. 6) . the papn cysteine mutants showed reduced catalytic activity (fig. 6a) and tgev rbd binding (fig. 6b ) relative to the wild type protein in 293t transfectants that express similar protein amounts. the higher activity of the papn c2 than the c3 or c4 mutants suggested that the cys464-cys892 disulfide bond was more labile than the cys464-cys929 bond, probably because cys892 is in a flexible loop (supplementary fig. s4 ). treatment with a reducing agent restored catalysis and rbd binding in the cysteine mutants and did not affect wild type papn binding activity (fig. 6) . reducing the disulfide bonds fully restored rbd binding, but catalysis was partially recovered in the papn c3 and c4 mutants. substrate hydrolysis is proposed to close the ectodomain (see above), which would facilitate rebuilding the disulfides. locking the closed form and the phenylalanine in the domain iv arm repeat inside the active site probably impeded substrate processing (fig. 2a) . the papn cysteine mutants bound markedly less rbd than the wild type protein, which confirmed that cov s protein binding to the closed papn was sterically hindered (fig. 2b) , and that cov recognized the open form. overall, these results validate the functional relevance of the apn ectodomain conformations and its motion. structural dynamics is an intrinsic property of aminopeptidases. the apn crystal structures reported here indicate the dynamic conformation of its ectodomain, and functional studies show its relevance in catalysis and virus infection. distinct ectodomain regions mediate these functions, but agents that bind to one region prevent activities linked to the other. these allosteric effects with ligands are probably caused by restrictions in apn conformational dynamics, as confirmed with disulfide bond mutants. they demonstrated that preventing ectodomain motion and locking apn forms inhibits its functions. apn ectodomain movement is less pronounced and differs from that reported for other m1 aminopeptidases. these differences could be due to the apn dimeric conformation and its linkage to the cell surface. dimerization only engages the domain iv region, and we found that the dimer is conserved in all apn structures, closed, intermediate and open. apn domain iv thus does not move as described for erap-1 or f3 23, 24 , proteins that do not form dimers. the fixed conformation of the apn dimer determines that the domain i to iii module swings over domain iv (supplementary video s1), with the hinge at the domain iv n-terminal region. the length of this movement is less marked in apn (15°) than in erap-1 (22°), although the two proteins have very similar closed conformations. displacement of the apn domains i, ii and iii must be limited by the length of the flexible polypeptide that links domain i to the transmembrane region, whose movement is restricted by membrane fluidity. the extent of apn movement nonetheless appears to be sufficient for release of the hydrolyzed peptide n-terminal residue, which is not plugged by domain iv in the open or in the intermediate apn conformations (fig. 2a) . it is not clear how each monomer in the dimer moves, whether their movement is random or synchronized in the same or inverse directions. experiments with hapn antibodies 25 and those shown here with the tgev rbd (fig. 3) suggest that ~50% of the molecules adopt different forms; these data imply that each apn monomer maintains a distinct conformation (supplementary video s1). the crystal structures reported here provide snapshots of apn dynamic conformation, and also guided experiments that demonstrate its role in virus entry into cells and catalysis. the switch between a proteolytic active (closed) and an inactive (open) conformation has been proposed for several m1 aminopeptidases 20, 23, 24, 28 . this dynamics is thought to be important for peptide hydrolysis and release from the aminopeptidase active site. the region that joins α 26 and α 27 in the domain iv arm repeat penetrates the active site groove in closed pig and human apn structures reported here and elsewhere (fig. 2a) 19 , and a conserved phenylalanine in this region locks the substrate coordinated to the zinc ion, permitting hydrolysis. further peptide processing likely requires removal of the phenylalanine lock by opening the apn ectodomain, which facilitates n-terminal residue release and peptide translocation, both sterically hindered in the closed conformation (fig. 2a) . the inherent flexibility in the domain iv arm repeat that we demonstrate here is linked to interdomain arrangements might also enable substrate processing, and indicate how ectodomain movements participate in peptide hydrolysis. local changes in a conserved tyrosine (tyr472 in papn) at the active site of m1 aminopeptidases are also suggested to be important 20, 24, 28 . among apn forms, the absence of conformational switches in active site residues at domain ii ( supplementary fig. s2 ) nonetheless indicates that tyrosine movement is not linked to interdomain motion. disulfide bonds that lock the apn closed conformation or drugs that prevent opening of the ectodomain inhibited cov protein binding and cell infection, whereas porcine cov s proteins probably hinder apn transition to the closed form and peptide hydrolysis. our results verify the critical role of apn dynamics in cov infection and catalysis, and demonstrate that the open apn structure is inactive in peptide hydrolysis. anti-apn antibodies that inhibit apn activity and reduce tumor growth 25, 33 likely block ectodomain movements. the allosteric inhibition of apn functions shown here using viral proteins and drugs is likely to be due to suppression of apn transient conformational states, as shown for other enzymes 34 . blocking apn movement prevents its functions, and suggests a new approach for the development of drugs that target this protein. small molecules or conformation-specific antibody inhibitors of ectodomain motions can bind to the active site or interact with distant sites, as shown here with cov spike fragments. high affinity drugs designed to inhibit catalysis and tumor growth prevent cov infections, which indicates that targeting apn ectodomain dynamics can be a valuable approach to block apn functions related to cancer progression and virus infections. . catalysis was determined at 60 min as absorbance at od405 nm (see fig. 3 and methods). relative rbd-fc binding to transfected cells determined from mean fluorescence intensity computed by flow cytometry as in fig. 4c . domain ii and iv residues replaced by cysteine are indicated at bottom (see supplementary fig. s4 ). mean ± sd (n ≥ 5). 35 . a papn protein with met residues replaced by seleno-met (se-met papn) was produced using methionine-and glutamine-free dmem (invitrogen) supplemented with 10% dialyzed fetal calf serum (fcs) and l-seleno-methionine (both from sigma). apn proteins secreted to culture supernatants were purified by affinity chromatography with anti-ha 12ac5 mab (roche), followed by size exclusion chromatography in hepes-saline buffer (20 mm hepes, 150 mm nacl) ph 7.5. preparation of most soluble cov s proteins used here has been described 16, 35 . s1h and s3h proteins were derived from the hol87 porcine cov s glycoprotein and bear the papn-binding domain. soluble tgev rbd was derived from the s glycoprotein of the tgev sc11 strain (genbank acc. n° aj271965). it contains s residues 505 to 657, an n-terminal ha peptide, and a flag sequence (monovalent rbd variant) or human igg1 fc (bivalent rbd-fc variant) at the c-terminal end. cov s proteins were produced in cho-lec or 293 t cells and purified as described 35 . analysis of apn catalysis. apn catalytic activity was determined using leucine p-nitroanilide (l-pna) (sigma) in a standard spectrophotometric assay in 96-well plates with soluble proteins or transfected cells. to study cov protein inhibition of apn catalysis, soluble apn ectodomains (5 μ g/ml; ~40 nm) were added to duplicate wells, alone or with soluble cov s protein variants, followed by the l-pna substrate (1 mm) in 100 μ l final volume (4 °c). plates were incubated at room temperature and od at 405 nm was measured at different times. background od of wells without apn was subtracted to determine specific catalytic activity. similar procedure was used with 293t cells (3 × 10 4 ) expressing papn 36 hr after transfection. od of well with mock-transfected cells were taken as background. cell samples expressing various amounts of papn at the membrane were used to normalize the activity of the papn cysteine mutants. relative activity of the mutant to wild type was determined as the ratio of the papn mutant to the wild type od from samples with the same protein expression, as monitored by flow cytometry (see below). cov protein binding to apn. stably transfected bhk21-papn, cho-papn and cho-papn mutant cells or transiently transfected 293t cells were used. the papn contained the ha peptide at the c terminus to monitor cell surface expression in cho and 293t cells. in the papn-hh/aa mutant, the two active site histidines were replaced with alanines, whereas in the papn cysteine mutants, the domain ii ser464 and the domain iv ser892 and/or ser929 were substituted by cysteine. we analyzed the effect on rbd binding to papn of two natural inhibitors of apn enzyme activity, bestatin and actinonin (sigma) 29 , as well as four synthetic abs compounds 30 . bestatin and actinonin were dissolved at 25 mm in pbs, whereas abs compounds were used at 20 mm in dmso. in wild type or mutant papn-expressing cells, we used flow cytometry to monitor tgev rbd binding to cell surface papn, essentially as reported 16, 35 . cells were washed three times with cold pbs and resuspended (10 6 cells/ml) in pbs supplemented with 0.3% heat-inactivated fcs and 0.125% bovine serum albumin (bsa; binding buffer); 200 μ l of cell suspension were added to 96-well plates (nunc), cells were sedimented and resuspended in 20 μ l of 1-2 μ g/ml rbd-fc solution alone or with inhibitors at indicated concentrations (30 min, 4 °c). an unrelated fc fusion protein was used as control. cells were washed and incubated with anti-human igg fluorescein isothiocyanate (fitc)-labeled secondary antibody (30 min, 4 °c). the mean fluorescent intensity was determined in a beckman coulter epics xl1; background cell staining with the fc protein was subtracted to determine the specific rbd-fc binding to cell surface papn. in parallel, the amount of cell surface papn expression was determined by flow cytometry with the anti-ha 12ac5 mab (roche) and an anti-mouse fitc-labeled secondary antibody (invitrogen). analysis of the papn cysteine mutants binding activity was normalized by the cell surface protein amounts as explained above for the catalytic activity. by qrt-pcr (quantitative reverse transcription polymerase chain reaction). stable transfected bhk21-papn or bhk21 cells (5 × 10 4 cells/well) in dmem (dulbecco's modified eagle's medium) with 10% fcs were plated in 24-well plates (18 h). plates were transferred to 4 °c, medium was removed and 200 μ l binding buffer alone or with apn-binding drugs or rdb protein were added to wells; after 10 min, the solution was replaced with 200 μ l virus inoculum at a multiplicity of infection (m.o.i) of 1, alone or with inhibitors in binding buffer. after virus adsorption at 4 °c, cells were washed three times with binding buffer, and incubated in dmem with 5% fcs (6 h, 37 °c, 5% co 2 ). cells were detached and lysed with 100 μ l tri reagent (sigma) for rna extraction, and cdna was generated from 1 μ g rna using the high capacity cdna reverse transcription kit (applied biosystems). real-time pcr reactions (10 μ l) were performed in triplicate using 5 μ l cdna sample, 2 μ l of 5x hot firepol evagreen qpcr mix plus (rox) (solis biodyne) and 0.3 μ l of specific primers for mouse β -actin or for the tgev s gene, in a 7500 real time pcr system (applied biosystems) using a standard protocol. data were analyzed with 7500 software using the comparative ct method (δ δ ct). tgev s expression relative to β -actin was determined and the ratio of values alone and with inhibitor used as relative cell entry. infection or cytopathic effect of tgev was inhibited in porcine st cells. one day after seeding (2.5 × 10 4 cells/well) in 96-well plates, cells were transferred to 4 °c and pre-incubated with 50 μ l binding buffer alone or with inhibitors, in duplicate. solutions were replaced with 50 μ l of serial 10-fold dilutions of virus inoculum with inhibitors or with dmso (≤ 0.25%) as control. after incubation (1 h, 4 °c), cells were washed three times with dmem with 5% fcs and incubated alone or with inhibitors for two days at 37 °c. to determine cell survival after infection, medium was removed, cells were formalin-fixed, stained with crystal violet and viability determined by optical density (od) at 590 nm. ratios from wells with and without virus were determined to calculate cell survival (see supplementary fig. s3b ). crystallization and diffraction data collection. the endoglycosidase h-treated (16 h, 30 °c) papn (papneh) ectodomain was crystallized by the sitting drop technique with a crystallization solution of 5% polyethylene glycol (peg)-1000 and 10% peg-8000 (ph ~6) and a 15 mg/ml protein sample. alternatively, native glycosylated papn ectodomain crystals were prepared with a crystallization solution of 20% peg-3350 and 100 mm sodium acetate ph 5.6. the hapn ectodomain (15 mg/ml) was crystallized with a solution of 20% peg-6000, 50 mm imidazole-hcl ph 8.0. crystals were frozen in crystallization solutions containing 20% ethylene glycol for diffraction data collection at the european synchrotron radiation facility (esrf; id14 and id23) and swiss light source (sls; pxii) beamlines. diffraction data were processed with xds 36 and scaled with scala programs 37 . for statistical data, see table 1 . structure determination. the structure of se-met papneh protein was solved by a combination of molecular replacement (mr) and single-wavelength anomalous dispersion (sad) methods. the crystals contained two molecules in the asymmetric unit (table 1) . a partial structure was obtained by mr using the phaser program 38 and domains i to iii of the tricorn-interacting factor f3 (pdb code 1z1w), which share ~30% residue identity with papn. the phaser llg value for the best mr solution was 161, whereas rfz values were 6.5 and 5.0, and tfz values of 3.0 and 9.9. we then used the mrsad protocol in the auto-rickshaw server 39 to determine the complete papneh structure, starting from the partial mr structure and using se-met papneh crystal diffraction data collected at the selenium peak wavelength. the final structure included the two papn molecules of the asymmetric unit, which were adjusted manually and refined with phenix.refine 40 using data extending to 2.5 å resolution (for statistics, see table 1 ). the papneh structure comprises residues 60 to 963 and the zinc atoms coordinated in the enzyme active site. the other apn ectodomain structures (table 1) were determined by the mr method using the papneh structure as search model. two ensembles including domain i, ii and iii or isolated domain iv were used for mr structure determination with phaser. structures were refined with phenix.refine (statistics in table 1 ). in all structures, the engineered tags and 25-30 residues of the n-terminal ectodomains were very disordered and are not included in the final models. electron density maps of active site residues and of n-linked glycans are shown in supplementary figs s2 and s5 , respectively. structure representations prepared with pymol (pymol.org). aminopeptidases: structure and function families of zinc metalloproteases the moonlighting enzyme cd13: old and new functions to target aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev human aminopeptidase n is a receptor for human coronavirus 229e human melanoma invasion and metastasis enhancement by high expression of aminopeptidase n/cd13 aminopeptidase n is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis role of aminopeptidase in angiogenesis aminopeptidase n in arterial hypertension aminopeptidase n (cd13) as a target for cancer chemotherapy impaired angiogenesis in aminopeptidase n-null mice the neovasculature homing motif ngr: more than meets the eye novel aminopeptidase n (apn/cd13) inhibitor 24f can suppress invasion of hepatocellular carcinoma cells as well as angiogenesis the molecular biology of coronaviruses mutational analysis of aminopeptidase n, a receptor for several group 1 coronaviruses, identifies key determinants of viral host range structural bases of coronavirus attachment to host aminopeptidase n and its inhibition by neutralizing antibodies biosynthesis of intestinal microvillar proteins. dimerization of aminopeptidase n and lactase-phlorizin hydrolase structure and function of aminopeptidase n the x-ray crystal structure of human aminopeptidase n reveals a novel dimer and the basis for peptide processing structure of aminopeptidase n from escherichia coli suggests a compartmentalized, gated active site structural basis for multifunctional roles of mammalian aminopeptidase n a structural view of coronavirus-receptor interactions crystal structures of the tricorn interacting factor f3 from thermoplasma acidophilum, a zinc aminopeptidase in three different conformations crystal structures of the endoplasmic reticulum aminopeptidase-1 (erap1) reveal the molecular basis for n-terminal peptide trimming cryptic and regulatory epitopes in cd13/aminopeptidase n development of synthetic aminopeptidase n/cd13 inhibitors to overcome cancer metastasis and angiogenesis selective aminopeptidase-n (cd13) inhibitors with relevance to cancer chemotherapy structural basis for antigenic peptide precursor processing by the endoplasmic reticulum aminopeptidase erap1 aminopeptidase-n/cd13 (ec 3.4.11.2) inhibitors: chemistry, biological evaluations, and therapeutic prospects a novel amino-benzosuberone derivative is a picomolar inhibitor of mammalian aminopeptidase n/cd13 determinants essential for the transmissible gastroenteritis virus-receptor interaction reside within a domain of aminopeptidase-n that is distinct from the enzymatic site exploring s1 plasticity and probing s1′ subsite of mammalian aminopeptidase n/cd13 with highly potent and selective aminobenzosuberone inhibitors mt95-4, a fully humanized antibody raised against aminopeptidase n, reduces tumor progression in a mouse model allosteric inhibition through suppression of transient conformational states antigenic modules in the n-terminal s1 region of the transmissible gastroenteritis virus spike protein collaborative computational project, n. the ccp4 suite: programs for protein crystallography pushing the boundaries of molecular replacement with maximum likelihood on the combination of molecular replacement and singlewavelength anomalous diffraction phasing for automated structure determination phenix: a comprehensive python-based system for macromolecular structure solution coot: model-building tools for molecular graphics we thank the esrf for provision of synchrotron radiation facilities through bag-madrid projects, as well as the swiss-sls facility, s. rodríguez for technical support, and c. mark for editorial assistance. gm was a recipient of a la caixa fellowship. jr was supported by the juan de la cierva program and rr by nih grant 2p01ai054456-06a1. the work was supported by grants from the spanish ministry of science (bfu2011-23940 and bio2014-52683-r to jmc). key: cord-300707-k9uk14b3 authors: bouwman, kim m.; tomris, ilhan; turner, hannah l.; van der woude, roosmarijn; bosman, gerlof p.; rockx, barry; herfst, sander; haagmans, bart l.; ward, andrew b.; boons, geert-jan; de vries, robert p. title: multimerizationand glycosylation-dependent receptor binding of sars-cov-2 spike proteins date: 2020-09-04 journal: biorxiv doi: 10.1101/2020.09.04.282558 sha: doc_id: 300707 cord_uid: k9uk14b3 receptor binding studies using recombinant sars-cov proteins have been hampered due to challenges in approaches creating spike protein or domains thereof, that recapitulate receptor binding properties of native viruses. we hypothesized that trimeric rbd proteins would be suitable candidates to study receptor binding properties of sars-cov-1 and -2. here we created monomeric and trimeric fluorescent rbd proteins, derived from adherent hek293t, as well as in gnti mutant cells, to analyze the effect of complex vs high mannose glycosylation on receptor binding. the results demonstrate that trimeric fully glycosylated proteins are superior in receptor binding compared to monomeric and immaturely glycosylated variants. although differences in binding to commonly used cell lines were minimal between the different rbd preparations, substantial differences were observed when respiratory tissues of experimental animals were stained. the rbd trimers demonstrated distinct ace2 expression profiles in bronchiolar ducts and confirmed the higher binding affinity of sars-cov-2 over sars-cov-1. our results show that fully glycosylated trimeric rbd proteins are attractive to analyze receptor binding and explore ace2 expression profiles in tissues. afford additional means for fluorescent-based experiments [13] , and thus are 101 attractive to be fused to rbd proteins. the resulting proteins were analyzed for 102 binding to cell culture cells and paraffin-embedded tissues of various hosts 103 including susceptible and non-susceptible animals. the results demonstrate 104 that fully glycosylated trimeric sars-cov-2 rbd proteins reveal the 105 differences in ace2 expression between cell cultures and tissue sections. 106 these trimeric rbd proteins bind ace2 efficiently in a species-dependent 107 manner and can be used to profile ace2 tissue expression. finally, we 108 without the gcn4 trimerization domain, fused to either sfgfp or morange (fig 116 1a ). the monomeric and trimeric rbds were efficiently expressed in both hek 117 293t as well as gnti cells (data not shown), with an increased yield up to 2-to 118 5-fold when fused to a c-terminal sfgfp ( fig 1b) . expression yields of the 119 morange2 fusions were comparable to that of sfgfp fusions (data not shown). 120 to illustrate the expression yields of sars spike proteins or domains thereof 121 we measured the fluorescence in the cell culture supernatant (fig 1c) . the 122 wild-type full-length ectodomains were difficult to express even with the addition 123 of sfgfp or morange2 fusion (fig 1b) . to increase yields for the full-length 124 ectodomain we introduced the 2p and additional hexapro mutations [14] , and 125 analyzed the fluorescence in cell culture supernatants five days post-126 transfection after incubation at 33 or 37°c. although we did not observe a large 127 increase in yields, we were able to purify sufficient protein to compare full-length 128 ectodomain trimers vs monomeric and trimeric rbd and ntd proteins. 129 130 spike rbd domains in frame with a c-terminal gcn4 and fluorescent reporter 131 protein display multimeric features on gel and maintain antigenicity 132 after purification, all rbd proteins were analyzed on gel under non-and 133 reducing conditions (fig 2a) . without reducing agent, monomeric rbd proteins 134 revealed dimeric fractions which could be reduced to a single monomeric form. 135 the ntd trimers were reduced under non-reducing conditions, thus solely by 136 sds. the trimeric rbd variants, on the other hand, revealed dimers and trimers 137 that could be reduced. besides, the ntd of prototypical γ-coronavirus ibv-m41 138 and influenza a virus ha pr8 as control proteins were included. finally, we 139 determined the extent of n-glycosylation maturation on purified proteins 140 expressed in either gnti or 293t by subjecting the monomeric and trimeric 141 proteins to pngasef and endoh treatment ( fig s1) next, we examined the antigenicity of the sars-cov-1 and -2 proteins using 149 serum collected from macaques 21 days post-infection with sars-cov-2 [15] . 150 both sars-cov-2 rbd monomers and trimers derived from 293t cells were 151 efficiently recognized, indicating proper folding ( fig 2b) . as expected, sars-152 cov-1 rbd proteins were poorly recognized, and the negative controls m41 153 ntd and pr8 ha displayed baseline binding identical to pre-infection serum. 154 the ntd trimers were likewise not recognized by the serum (not shown), 155 indicating that the majority of antibodies in naïve animals after infection are 156 directed against the sars-cov rbd [16] . similar results were obtained using 157 gnti-derived proteins, with the rbd trimer being less efficiently recognized by 158 the macaque serum than its monomeric counterpart. this is in line with recent 159 observations that insect cell-derived proteins are less well bound by serum to determine whether the fluorescent rbd trimers are indeed structured in a 165 trimeric manner we subjected these proteins to negative stain single-particle 166 em. the em data revealed that the rbd proteins form stable trimers that 167 resemble known spike structures (fig. 2c) . initially, 58,018 individual particles 168 were picked, placed into a stack, and submitted to reference-free two-169 dimensional (2c) classification. from the initial 2d classes, particles that did 170 not resemble rbd were removed, resulting are final particle stacks of 32,152 171 particles, which were then subject to relion 2d classification. all resultant 172 classes demonstrated evident and distinct trimeric rbd, gcn4, and three 173 sfgfp protein structures that could be identified in the em images. from the 174 em images, we generated a model in which we took the crystal structures of 175 sfgfp, the gcn4 trimerization domain (pdb:2o7h), and the sars-cov-2 176 rbd (pdb: 6xm4) to demonstrate the likely structure of our rbd trimer (fig 177 2d) . to determine the biological activity of our rbd proteins we stained a549 and 182 vero cells that are reported to support sars-cov replication, with the latter 183 being more susceptible [17] . however, a549 cells were bound by all our rbd 184 proteins with a slight increase in intensity from monomeric gnti derived rbd 185 proteins to trimeric 293t derived rbds (fig 3) . trimeric 293t rbd binding was 186 efficiently blocked using 4μm recombinant ace2 whereas 400nm ace2 pre-187 incubation was not sufficient to prevent binding of fully glycosylated trimeric 188 rbd proteins to cells completely. sars-cov-2 rbd proteins bound slightly 189 more intensely to a549 cells compared to the same sars-cov-1 rbd 190 proteins. a similar pattern was observed for vero-e6 cells, however, the fully 191 glycosylated sars-cov-2 rbd trimer bound markedly stronger compared to 192 the other rbd preparations ( fig s2a) . importantly, the full-length ectodomain 193 also bound efficiently to a549 cells ( fig s2b) . we did not observe any binding 194 of the trimeric ntd domains to a549 cells ( fig s2b) . mdck cells, derived from 195 canine kidney, served as negative controls, to which we indeed did not observe 196 any binding with any of the indicated proteins ( fig s2c) . applied. in all cases, sars-cov-2 displayed a higher avidity compared to 213 sars-cov-1. a similar trend of binding intensities was observed for 214 monomeric, trimeric, and different n-glycosylated sars-cov-rbd proteins 215 fused to morange2 ( fig s3a) . again specific binding was seen to the epithelium 216 of terminal bronchioles and, to a much lower extent, to alveoli and endothelium. 217 the results were confirmed using horseradish peroxidase readout with a 218 hematoxylin counterstain (fig s3b) , which output is enzyme driven and purely 219 qualitative, however, we did observe similar differences in staining intensities. 220 here, very minimal staining using the sars-cov ntd domains was observed 221 (fig s3b) , which we did not detect using a fluorescent readout (fig s3c) . to 222 determine if the binding was ace2 dependent we pre-incubated trimeric rbd 223 proteins with recombinant ace2. while 4 μm was sufficient to block binding to 224 cell culture cells (fig 4c) , 8μm was needed to prevent all detectable binding to 225 ferret lung tissue. 226 227 to confirm our observations of different binding on tissues, we quantified the 228 intensities of the ace2 antibody and sars-cov-1 and -2 rbd proteins, except 229 for the monomeric gnti derived proteins as these were almost at the 230 background ( fig 4d) . as expected a noteworthy trend was observed of 231 increasing binding strength from sars-cov1 gnti derived monomers to 232 sars-cov-2 fully glycosylated rbd trimers. interestingly multimerization 233 appears to be more important for strong ace2 interaction to tissue compared 234 to the glycosylation status. viroscience, erasmus university, the netherlands, respectively. tissue 374 sections were rehydrated in a series of alcohol from 100%, 96% to 70%, and 375 lastly in distilled water. tissues slides were boiled in citrate buffer ph 6.0 for 10 376 min at 900 kw in a microwave for antigen retrieval and washed in pbs-t three 377 times. endogenous peroxidase activity was blocked with 1% hydrogen peroxide 378 for 30 min. tissues were subsequently incubated with 3% bsa in pbs-t 379 overnight at 4 °c. the next day, the purified viral spike proteins (50μg/ml) were human monoclonal 416 antibodies block the binding of sars-cov-2 spike protein to angiotensin 417 converting enzyme 2 receptor structural and 420 functional basis of sars-cov-2 entry by using human ace2 functional assessment of cell entry and 424 receptor usage for sars-cov-2 and other lineage b betacoronaviruses structure, function, and antigenicity of the sars-cov-2 spike glycoprotein epub 2020/03/11 a serological assay to detect sars-cov-2 seroconversion 433 in humans a highly 435 conserved cryptic epitope in the receptor-binding domains of sars-cov-2 436 and sars-cov potent neutralizing antibodies from covid-19 patients 441 define multiple targets of vulnerability conformational 445 dynamics of sars-cov-2 trimeric spike glycoprotein in complex with receptor 446 ace2 revealed by cryo-em closing coronavirus spike 449 glycoproteins by structure-guided design structures, 452 conformations and distributions of sars-cov-2 spike protein trimers on intact 453 virions stabilizing the closed sars-cov-2 spike trimer structure-based design of prefusion-stabilized sars-459 fluorescent trimeric hemagglutinins reveal multivalent 463 receptor binding properties structure-based design of prefusion cov-2 spikes. science. 2020. epub 2020/07/25 comparative pathogenesis of covid-19, mers, and 472 sars in a nonhuman primate model structural 476 basis of a shared antibody response to sars-cov-2 severe acute respiratory syndrome coronavirus 2 from patient with infection 483 and rapid transmission of sars-cov-2 in ferrets okba 486 nma, et al. sars-cov-2 is transmitted via contact and via the air between 487 ferrets sars-cov-2 infection in farmed minks, the 492 netherlands syrian hamsters as a small animal model for sars-cov-2 497 infection and countermeasure development severe acute respiratory syndrome coronavirus infection of golden syrian 502 pathogenesis and transmission of sars-cov-2 in golden hamsters deducing the n-and 508 o-glycosylation profile of the spike protein of novel coronavirus sars-cov-2 site-specific 512 glycan analysis of the sars-cov-2 spike epub 2020/05/06 site-516 specific n-glycosylation characterization of recombinant sars-cov-2 spike 517 glycans on the sars-cov-2 spike control the receptor binding 520 domain conformation sars-cov-2 receptor ace2 and tmprss2 are primarily expressed in 525 bronchial transient secretory cells the protein expression profile 528 of ace2 in human tissues the sars-cov-2 spike protein has a broad tropism for mammalian ace2 532 proteins cell 534 entry of sars-cov-2 conferred by angiotensin-converting enzyme 2 (ace2) 535 of different species the 538 protein expression profile of ace2 in human tissues tissue distribution of ace2 protein, the functional receptor for sars 542 coronavirus. a first step in understanding sars pathogenesis de haan 546 ca. the influenza a virus hemagglutinin glycosylation state affects receptor-547 binding specificity improving the photostability of bright monomeric orange 551 and red fluorescent proteins three amino acid changes in avian coronavirus spike protein 556 560 leginon: a system for fully automated acquisition of 1000 electron 561 micrographs a day appion: an integrated, database-driven pipeline to facilitate em image 565 processing dog 568 picker and tiltpicker: software tools to facilitate particle selection in single 569 particle electron microscopy 5μg of protein was subjected without or with pngase f or endoh for 1hr and 726 subjected to sds-page and western blot analyzes supplemental figure 2. binding of rbd proteins to cell lines 733 (a) protein binding of rbd proteins observed on vero e6 cells proteins were applied 50μg/ml and where indicated pre-incubated with 735 spike proteins were detected using anti-strep and 736 goat-anti-mouse antibodies binding of full-length sars-cov-2 ectodomain, ibv-m41, antibodies 738 only, and ntd spike proteins to a549 cells. proteins were applied 50μg/ml 739 and detected using anti-strep and goat-anti-mouse antibodies c) non-binding of rbd trimers to mdck cells binding of rbd proteins to tissues 747 (a) binding of rbd proteins fused to morange2 to ferret lung tissues 748 proteins were applied 50μg/ml and detected using anti-strep and goat-anti-749 mouse antibodies binding of rbd proteins fused to sfgfp proteins to ferret lung tissues, 751 using hrp as a readout identical experiment to (a) but using an hrp readout using anti-strep and goat-753 anti-mouse antibodies control staining on ferret lung tissues using hrp as readout proteins were 756 applied 50μg/ml and detected using anti-strep and goat-anti-mouse antibodies scalebar is 100μm lack of ntd binding to ferret lung tissue using fluorescence 759 proteins were applied 50μg/ml and detected using anti-strep and goat-anti-760 mouse antibodies control stainings to syrian hamster tissues, antibodies only and m41 proteins were applied 50μg/ml and where indicated pre-incubated with 763 recombinant ace2 protein key: cord-312560-onfabcfv authors: klingler, j.; weiss, s.; itri, v.; liu, x.; oguntuyo, k. y.; stevens, c.; ikegame, s.; hung, c.-t.; enyindah-asonye, g.; amanat, f.; baine, i.; arinsburg, s.; bandres, j. c.; kojic, e. m.; stoever, j.; jurczyszak, d.; bermudez-gonzalez, m.; simon, v.; liu, s.; lee, b.; krammer, f.; zolla-pazner, s.; hioe, c. e. title: role of igm and iga antibodies to the neutralization of sars-cov-2 date: 2020-08-21 journal: medrxiv : the preprint server for health sciences doi: 10.1101/2020.08.18.20177303 sha: doc_id: 312560 cord_uid: onfabcfv sars-cov-2 has infected millions of people and is on a trajectory to kill more than one million globally. virus entry depends on the receptor-binding domain (rbd) of the spike protein. although previous studies demonstrated anti-spike and -rbd antibodies as essential for protection and convalescent plasma as a promising therapeutic option, little is known about the immunoglobulin (ig) isotypes capable of blocking virus entry. here, we studied spikeand rbd-specific ig isotypes in plasma/sera from two acutely infected and 29 convalescent individuals. spikeand rbd-specific igm, igg1, and iga1 antibodies were produced by all or nearly all subjects at varying levels and detected at 7-8 days post-disease onset. igg2, igg3, igg4, and iga2 were also present but at much lower levels. all samples also displayed neutralizing activity. igm, igg, and iga were capable of mediating neutralization, but neutralization titers correlated better with binding levels of igm and iga1 than igg. in december 2019, the first patients with coronavirus disease 2019 , caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) were identified in the city of wuhan, hubei province, china 1 . since then, the epidemic has rapidly spread to most regions of the world, infecting millions of people 2 . effective therapeutics and vaccines against sars-cov-2 are urgently needed. to this end, more information about the ig isotypes present in the plasma of covid-19 convalescent individuals and their antiviral activities are needed, as convalescent plasma transfusion showed promising results in patients with severe to life-threatening covid-19 3-5 . the data would also inform vaccine development, as more than 100 vaccine candidates are in different stages of preclinical development, and many are now in phase 2 and 3 clinical trials 6 . although using different strategies 7 , many vaccines are based on one of the three membrane-anchored proteins present on the virus envelope surface: the sars-cov-2 spike protein 8, 9 , which contains the receptor-binding domain (rbd) required for binding to and entry into the cells [10] [11] [12] [13] [14] [15] [16] [17] . these vaccines aim to protect by inducing neutralizing antibodies (abs) capable of blocking the viral infection. however, which of the immunoglobulin (ig) isotypes are protective is not yet clear. monomeric igg constitutes approximately 75% of the abs found in serum and exists as four subtypes: igg1 (~66% of igg), igg2 (~23% of igg), igg3 (~7% of igg) and igg4 (~4% of igg) 18, 19 . igm abs represent 10% of total serum abs and are the first to arise in response to new antigens 19, 20 . although igm abs do not undergone extensive somatic hypermutation to increase their affinity as do igg and iga abs, their higher valency due to the oligomerization of igm enhances their avidity and potency against pathogens 19, 21, 22 . iga abs exist as two subtypes: iga1 and iga2, and represent 15% of total serum abs 19 . they are dimeric in the mucosa, but in the circulation, these two iga subtypes are monomeric. sars-cov-2 spike-, rbd-and nucleocapsid-specific serum/plasma abs of igm, igg, and iga . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 21, 2020. . https://doi.org/10.1101/2020.08.18.20177303 doi: medrxiv preprint isotypes are found in most covid-19 patients 23-30 , with ab neutralizing activities reported among the convalescent patients 26,28,31,32 . however, the neutralizing titers appear to vary greatly 26,28,31,32 and they correlate with ab binding levels against rbd, spike, and/or nucleocapsid, and also with age, duration of symptoms, and symptom severity 26,28,32 . several rbd-specific monoclonal abs of igg isotype with potent antiviral activities has been generated from individuals with high neutralization titers and these confer protection in animal models 26,31,33,34 . moreover, a monoclonal ab of iga isotype capable of recognizing both the sars-cov-1 and sars-cov-2 spike proteins, and blocking ace2 receptor binding was recently described 35 . however, no data are available regarding the neutralizing capacity of plasma igm and iga abs from covid-19 patients. studies on other respiratory viruses such as influenza show that, in addition to igg, iga could also mediate virus neutralization, and their relative contribution depends on the physiologic compartment in which they are found, with iga contributing to the protection of mostly the upper respiratory tract while igg was protecting the lower respiratory tract 36-39 . of note, an anti-hemagglutinin monoclonal iga has been demonstrated to mediate more potent antiviral activities against influenza when compared to a monoclonal igg against the same epitope 40 . interestingly, an igm ab with potent antiviral activities targeting the receptor binding site of influenza b has also been described 41 . in addition, mucosal respiratory syncytial virus (rsv)-specific iga neutralizing abs is a better correlate of protection than serum rsv-specific igg neutralizing abs 42 . in the case of sars-cov-1, high titers of mucosal iga in the lungs are correlated with reduced pathology upon viral challenge in animal models 43 . whether iga in the blood and the respiratory tract mucosa offers protection against sars-cov-2 infection remains an open question. moreover, few data are available concerning the contribution of igm to neutralization and protection against viruses, including sars-cov-2. we have recently published a luminex assay detecting ig total against spike and rbd 44 . based on this assay, we studied here the ig isotype profiles against spike and rbd in the plasma and serum . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 21, 2020. . https://doi.org/10.1101/2020.08.18.20177303 doi: medrxiv preprint from acutely infected or convalescent individuals using a luminex assay that detects antigen-specific igm, igg1-4, iga1 and iga2. using a pseudovirus assay 45 , we also measured neutralizing activities in plasma and serum samples and in ig isotype fractions to determine the neutralizing capacity of igm, iga, and igg. the data demonstrate high prevalence of spike-and rbd-specific igm and iga, similar to that of igg1, in plasma/serum from covid-19 patients and their significant contributions to virusneutralizing activities. this is the first evidence that purified plasma igg, igm, and iga contributes to sars-cov-2 neutralization. a total of 29 serum (p#5-8) and plasma (tf#1-25) specimens from covid-19-convalescent individuals was tested. sera from three uninfected individuals banked as part of an ongoing longitudinal study prior to the covid-19 outbreak (n#1-3), and an additional ten plasma from covid-negative contemporaneous blood bank donors (n#4-13) were included for comparison. the specimens were initially titrated for total ig against spike and rbd (fig. 1) . all 29 covid-19 positive specimens exhibited titration curves of total ig abs against spike, while none of the negative controls did. similar results were observed with rbd, except that one covid-19-negative sample had low titrating levels of rbd-specific ig. overall the background mfi values were higher for rbd than spike. the areas under curves (aucs) highly correlated with the 1:200 dilution mfi (p < 0.0001; supplementary fig. 1) , consequently all samples were tested for isotyping at this dilution. to assess the reproducibility of the assay, the samples were tested in at least two separate experiments run on different days, and a strong correlation was observed between the mfis from these independent experiments (supplementary fig. 2 ). to evaluate for the presence of spike-specific and rbd-specific total ig, igm, igg1, igg2, igg3, igg4, iga1 and iga2, the specificity of the secondary abs used to detect the different isotypes were first validated with luminex beads coated with myeloma proteins of known ig isotypes (igg1, igg2, igg3, . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted august 21, 2020. . https://doi.org/10.1101/2020.08.18.20177303 doi: medrxiv preprint igg4, iga1, iga2, and igm) (supplementary fig. 3) . while all 29 convalescent individuals had anti-spike and anti-rbd total ig (fig. 2) , the ig levels were highly variable, with mfi values ranging from 36,083 to 190,150. all 29 convalescent individuals displayed igm abs against spike at varying levels, although only 55% (16/29) were positive for anti-rbd igm, when evaluated using cut-off values calculated as mean + 3 standard deviation (sd) of the 13 covid-negative samples. the lower percentage of igm abs specific for rbd might be due to the high background observed for igm against rbd with covid-negative specimens (fig. 2b,c) . an igg1 response was also detected against both spike and rbd in 97% of the convalescent subjects, with mfi values that ranged from 1012.5 to 59,880, and the responses against spike and rbd were highly correlated for every isotype (supplementary fig. 4) . in contrast igg2, igg3, and igg4 abs against spike and rbd were detected in only a small fraction of the subjects, and the levels were very low (mfi values < 1300) (fig. 2) . surprisingly, almost all the individuals produced iga1 abs against spike (90%) and rbd (86%) while 21% exhibited iga2 against spike, and 48% exhibited iga2 against rbd (fig. 2) . overall, these data demonstrate that igm, igg1, and iga1 abs were all strongly induced against spike and rbd in all or almost all covid-19 convalescent individuals (fig. 2) . the levels, however, were highly variable among individuals. while not reaching statistical significance, a general trend was observed toward higher levels of total ig, igm, igg1, and iga1 in women compared to men (supplementary fig. 5 ). in fig. 3 , comparing levels of total ig with the different ig isotypes showed a highly significant correlation with igg1 for both abs specific for spike and rbd indicating that igg1 is the major isotype induced by sars-cov-2 infection. there was no other isotype which showed a significant correlation with total ig abs for anti-spike abs, although there was a significant correlation between total ig and iga1 for anti-rbd abs. moreover, none of the igm, igg1, or iga1 isotypes correlated with one another (fig. 3c) . iga1 is induced early after disease onset in covid-19 patients. since almost all convalescent . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted august 21, 2020. . https://doi.org/10.1101/2020.08.18.20177303 doi: medrxiv preprint 7 covid-19 patients displayed iga ab responses to spike and rbd antigens, we sought to evaluate the kinetics of iga versus other ig isotype induction at early time points. we tested longitudinal samples collected from two patients (p#1 and p#2) from days 7 to 15 post-onset of symptoms. the earliest samples from both patients were positive for iga1 abs against spike (fig. 4a) and rbd (fig. 4b) , and the levels increased over time. like iga1 ab, total ig, igm, and igg1 abs were also detectable as early as 7-8 days post symptom onset, and their levels increased over time. on the contrary, iga2 ab levels were near or below background on days 7-8 and remained unchanged over two weeks post-onset. igg4 abs also remained low or near background, whereas igg2 and igg3 abs increased slightly to above background after 10-15 days. neutralizing activities is detected in all convalescent covid-19 individuals. we subsequently tested the ability of samples from convalescent subjects to neutralize a vsv∆g pseudovirus bearing the sars-cov-2 spike protein (cov2pp). the results, shown in all specimens from covid-19-convalescent individuals were able to neutralize the virus at levels superior to 50% (fig. 5a) . for 26 of 28 specimens, neutralization reached more than 90% (fig. 5a) . interestingly, one sample demonstrated highly potent neutralization with a reciprocal ic50 titer > 40,960, and neutralization was still 75% at the highest dilution tested. in contrast, one sample had a low neutralization titer (reciprocal ic50 titer = 37) and reached a neutralization plateau of only ~60%. in comparison, none of the samples from covid-19-negative individuals reached 50% neutralization (fig. 5b) , while the srbd positive control demonstrated potent neutralization with an ic50 of 0.06 µg/ml ( fig. 5c) , similar to that recently reported 45 . . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted august 21, 2020. . https://doi.org/10.1101/2020.08.18.20177303 doi: medrxiv preprint 8 isotype levels and neutralization titers varied tremendously among convalescent covid-19 individuals ( fig. 6) , we investigated if neutralizing activities correlated with a particular ab isotype. igm abs specific for spike and rbd displayed the strongest correlation with neutralizing abs (p < 0.0001 and p = 0.0002, respectively), while igg1 did not show a significant correlation (fig. 7a) . neutralization reciprocal ic90 titers weakly correlated with rbd-specific total ig levels but did not correlate with spike-specific total ig levels (fig. 7a) . correlation with ic50 titers yielded similar results (data not shown). iga1 levels also correlated with reciprocal ic90 titers (p = 0.013 and p = 0.006 for spike and rbd, respectively; fig. 7a ). although there were significant correlations between anti-spike and -rbd abs of the igg2 and igg3s isotypes, most of the neutralization values were below the cut-off and for the few igg3 and igg2 responders, the levels were near background (fig. 7b) . neutralizing activities are mediated by igm, igg, and iga fractions. the data above show the strongest correlation of neutralizing activity with igm. to ask directly to what extent abs of different isotypes mediate neutralization, we evaluated the neutralization activities of igm, igg, and iga fractions purified from plasma from five covid-19-convalescent individuals (rp#1-5). the enrichment of igm, igg1, and iga1 abs reactive with spike and rbd was validated using the isotyping method used above (supplementary fig. 6 and not shown). these igm, igg and iga fractions were then evaluated for neutralizing activity along with the original plasma (fig. 8) . the rp#1-5 plasma neutralizing reciprocal ic50 titers ranged from 35 to 690 (fig. 8a,b) . the purified igg, iga and igm fractions all displayed neutralization of more than 50%, while the negative control ig fractions (rn#1) did not (fig. 8c,d) . the ic50 values for igm and igg were similar, and both were significantly better than iga (fig 8c,d) . our study demonstrates the presence of igg1, iga1 and igm within 7-8 days after the onset of symptom, and the correlation of neutralizing activity is strongest with igm, followed by iga1, but does not correlate with igg1 or other igg isotypes. while correlations are important, direct testing of . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted august 21, 2020. . https://doi.org/10.1101/2020.08.18.20177303 doi: medrxiv preprint neutralizing activities of different isotypes was performed, and these experiments showed that neutralizing activity was displayed most potently by igm and igg and less strongly by iga. these data indicate the protective potential of all three major ig isotypes and suggest that induction of each of these isotypes by vaccination may offer optimal protection against infection. these data also carry important implications for the use of hyperimmune globulin as treatment and prophylactic modalities. several sars-cov-2 vaccine candidates tested in animal models and humans were shown to induce igg responses against spike and rbd as well as virus neutralizing activities, but in many of these studies, the induction of other ig isotypes was not evaluated 46-49 . dna vaccines expressing full-length and truncated spike proteins were able to curtail virus infection in the respiratory tract by varying degrees. virus reduction correlated with levels of neutralization and also fc-mediated effector functions such as antibody-dependent complement deposition (adcd) 9 . interestingly, these dna vaccines elicited spike-and rbd-specific igg1, igg2, igg3, iga, and igm abs, and similar to our findings, neutralization correlated most strongly with igm. adenovirus serotype 26 vaccine vectors encoding seven different sars-cov-2 spike variants showed high protection, and virus reduction correlated best with neutralizing ab titers together with igm binding levels, fcγrii-binding, and adcd responses 50 . we noted that while all covid-19 convalescent individuals exhibited plasma/serum neutralization activities, reaching 50% neutralization, and 26 of 28 specimens attained 90% neutralization, neutralization levels were highly variable with reciprocal ic50 and ic90 titers ranging over three orders of magnitude. similarly, the levels of spike-and rbd-binding total ig and ig isotypes also varied greatly. a trend to higher levels of total ig and each ig isotype was seen in female compared to male subjects, as reported in another study 51 . moreover, except for tf#11 (a male elite neutralizer), the median neutralizing reciprocal ic90 titer was higher in females than males, although the difference did not reach significance (data not shown). gender differences in ab induction have been observed following vaccination against influenza in humans and mice and were shown to result from the impact . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 21, 2020. . https://doi.org/10.1101/2020.08.18.20177303 doi: medrxiv preprint of sex steroids 52,53 . whether and to what extent this contributes to gender differences seen in clinical outcomes of covid-19 54 remains to be investigated. other studies have shown that the ab levels were associated with multiple factors, including time from disease onset 55 and disease severity 25 . however, clinical data are not available for the subjects studied here, limiting our analysis only to neutralization and ig isotypes. one remarkable finding from our study is that neutralization levels correlated with binding levels of igm and iga1, but not igg1. this is consistent with our data showing the neutralization activities mediated by purified igm and iga fractions from covid-19 patients. nonetheless, purified igg fractions from convalescent plasma also exhibited potent neutralization, but since the fractions contained all four igg subtypes, it remains unknown which igg subtypes contribute to neutralization, although igg1, the most abundant igg isotypes in the blood, did not correlate with neutralization titers, and igg2 and igg3 present at low levels displayed weak correlations. the lack of correlation between igg1 binding titers and neutralization activities may be explained by the fact that the dominant igg1 responses may target sites not critical for blocking virus entry. indeed, among mabs isolated from six covid-19 convalescent patients, > 40% of rbd-specific mabs did not display neutralizing activities 26 and another study demonstrated that mabs that bind to non-rbd epitopes on the spike protein had poor neutralization potencies 33 . nonetheless, the absence of a correlation between igg1 binding titers and neutralizing activities reported above requires more study given that data remain controversial since other studies have demonstrated correlations between neutralization titers and sars-cov-2 specific igg levels 26,28,32 . in addition to neutralization, non-neutralizing ab activities have been implicated in protection from virus infection through potent fc-mediated functions such as antibody-dependent cellular cytotoxicity (adcc), antibody-dependent cellular phagocytosis (adcp), and complement-mediated lysis; this is true for hiv, influenza, marburg, and ebola viruses 38,56-59 . these fc activities were not evaluated in our . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 21, 2020. . https://doi.org/10.1101/2020.08.18.20177303 doi: medrxiv preprint study, and their contribution to protection against infection and disease progression in humans is yet unclear 9,60,61 . interestingly, a recent study demonstrated enrichment of spike-specific igm and iga1 abs and spike-specific phagocytic and adcd activity in plasma of individuals who recovered from infection, while nucleocapsid-specific igm and iga2 responses and nucleocapsid-specific adcd activity were features enriched in deceased patients 62 . defining the full potential of abs against sars-cov-2 that includes neutralizing, non-neutralizing and enhancing activities is vital for developing the most effective vaccines and determining the optimal convalescent ab treatment against covid-19. when we examined plasma specimens collected within 7-8 days after covid-19 onset, we detected igg and iga against spike and rbd, as well as igm. this is consistent with published reports showing that 100% of covid-19-infected individuals developed igg within 19 days after symptom onset and that seroconversion for igg and igm occurred simultaneously or sequentially 25 . iga was also found early after infection (4-6 days after symptom onset) in another study 23 . these studies suggest that measuring total ig, rather than igg, would provide a better outcome for diagnosis of early disease. indeed, we found no correlation between the levels of different isotypes examined in our study. this lack of correlation may result from their non-synchronous, sequential induction (igm first, then igg, and finally iga), but the presence of iga early during acute infection also suggests the potential contribution of natural iga, which, similar to natural igm, arise spontaneously from innate b1 cells to provide the initial humoral responses before the induction, maturation, and class-switching of adaptive classical b cells 63,64 . in summary, this study demonstrates that spike-and rbd-specific igm, igg1, and iga1 abs were present in serum or plasma of all or almost all analyzed covid-19 convalescent subjects and were detected at extremely early stages of infection. the plasma of convalescent individuals also displayed neutralization activities that were mediated by igm, igg, and iga1, although neutralization titers correlated more strongly with levels of igm and iga1 than igg. the contribution of igm and iga abs to . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 21, 2020. . https://doi.org/10.1101/2020.08.18.20177303 doi: medrxiv preprint the neutralizing activities against sars-cov-2 demonstrates their importance of protective immunity against this virus. recombinant proteins. the recombinant spike and rbd proteins were produced as previously described 65 in expi293f cells (thermofisher) by transfections of purified d.n.a. using an expifectamine transfection kit (thermofisher). the soluble version of the spike protein included the protein ectodomain (amino acids 1-1213), a c-terminal thrombin cleavage site, a t4 foldon trimerization domain, and a hexahistidine tag. the protein sequence was also modified to remove the polybasic cleavage site (rrar to a) and two stabilizing mutations (k986p and v987p, wild type numbering). the rbd (amino acids 319-541) included the signal peptide (amino acids 1-14) and a hexahistidine tag. supernatants from transfected cells were harvested on day three post-transfection by centrifugation of the culture at 4000 g for 20 min. the supernatant was then incubated with 6 ml ni-nta agarose (qiagen) for one to two hours at room temperature. next, gravity-flow columns were used to collect the ni-nta agarose, and the protein was eluted. each protein was concentrated in amicon centrifugal units is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 21, 2020. . https://doi.org/10.1101/2020.08.18.20177303 doi: medrxiv preprint 13 obtained from study participants enrolled in irb-approved protocols at icahn school of medicine at mount sinai and the james j. peter va medical center. study participants provided written consent at enrollment and agreed to sample banking and future research use of their banked biospecimen. samples from these protocols included sera from seven participants with documented sars-cov-2 infection: specimens from p#1 (d8, d11, and d15 after symptom onset), p#2 (d7, and d10 after symptom onset), and rp#1-5. in addition, sera were used, which had been collected from healthy donors prior to the spread of sars-cov-2 in the u.s.a. ig fractionation. iga was first isolated from plasma by mixing 1:2 diluted plasma with peptide m agarose beads (600 µl/28 ml plasma, invivogen #gel-pdm) for 1.5 hours at room temperature. beads were then collected on a column and washed with pbs until protein reading (280 nm) by nanodrop reached background. iga was eluted from beads with a ph 2.8 elution buffer (thermo scientific #21004) and neutralized with ph 9 tris buffer. the pass-through plasma sample was collected for igg enrichment using protein g agarose beads (invivogen #gel-agg) using the same protocol as above and subsequently for igm isolation using a hitrap igm column (g.e. healthcare #17-5110-01) according to the manufacturer's instruction. an additional purification step was performed using protein a plus mini-spin columns to separate igg from igm. protein concentrations were determined with nanodrop prior to use in luminex and neutralization experiments. luminex binding ab assay. the sars-cov-2 antigens used in this assay were a soluble recombinant trimerized form of the spike protein and a recombinant rbd protein 66 . antigens were coupled as previously described, with minor changes 44 . each antigen was covalently coupled individually to a uniquely labeled fluorochrome carboxylated xmap bead set at 2.0 μg protein/million beads using a twostep carbodiimide reaction with the xmap ab coupling (abc) kit following to the manufacturers' instructions (luminex, austin, tx). the coupled beads were pelleted, resuspended at 5x10 6 beads/ml in storage buffer (pbs, 0.1% bovine serum albumin (b.s.a.), 0.02% tween-20, and 0.05% sodium azide, . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 21, 2020. . https://doi.org/10.1101/2020.08.18.20177303 doi: medrxiv preprint ph 7.4), and stored at -80°c. three to five million beads per batch were prepared in a 1.5 ml conical tube. before each experiment, the beads needed for a single run (2,500 beads/well x number of wells) were pelleted and resuspended in assay buffer (pbs, 0.1% b.s.a., 0.02% tween-20) to deliver 2,500 beads in a volume of 50 μl/well. sera/plasma samples were serially titrated (1:50 to 1:6400 final dilution) or diluted in assay buffer to 1:100 (for a final dilution of 1:200). the samples were then added as 50 μl/well to the wells containing the beads and incubated at room temperature for 1 hour at 600 rpm. after two washes in assay buffer, 100 μl/well of biotinylated antibodies specific for total ig, igg1, igg2, igg3, igg4, iga1, iga2, or igm was added and incubated for 30 min at room temperature on a plate shaker; these antibodies were rabbit biotinylated-anti-human total ig (abcam, catalog #ab97158) at 2 μg/ml, mouse biotinylated-anti-human igg1 fc (invitrogen #mh1515) at 4 μg/ml, mouse biotinylated-anti-human igg2 fc (southern biotech #9060-08) at 1 μg/ml, mouse biotinylated-antihuman igg3 hinge (southern biotech #9210-08) at 3 μg/ml, mouse biotinylated-anti-human igg4 fc (southern biotech #9200-08) at 4 μg/ml, mouse biotinylated-anti-human iga1 fc (southern biotech #9130-08) at 4 μg/ml, mouse biotinylated-anti-human iga2 fc (southern biotech #9140-08) at 4 μg/ml or goat biotinylated-anti-human igm (southern biotech #2020-08) at 3 μg/ml. after two washes, 100 μl/well of streptavidin-phycoerythrin (p.e.) at 1 μg/ml was added (biolegend #405204) followed by a 30 min incubation at room temperature on a plate shaker. after two additional washes, 100 μl of assay buffer/well was added and put on a shaker to resuspend the beads. the plate was read with a luminex flexmap 3d instrument. specimens were tested in duplicate, and the results were recorded as mean fluorescent intensity (mfi). cov2pp production and titration. the sars-cov-2 pseudovirus (cov2pp) was produced as previously described 45 . briefly, 293t cells were transfected to overexpress sars-cov-2 glycoproteins. for background entry with particles lacking a viral surface glycoprotein, pcagg empty vector was . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 21, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 21, 2020. . https://doi.org/10.1101/2020.08.18.20177303 doi: medrxiv preprint r.l.u.) / virus control r.l.u.) *100). the inhibitory concentration 50% (ic50) and 90% (ic90) were respectively defined as the reciprocal sample dilution or purified ig fraction concentration achieving 50% and 90% neutralization. statistical analysis. the statistical significance was determined by a two-tailed mann-whitney test and correlations analyzed with a spearman rank-order correlation test using graphpad prism 8. the raw data that support the findings of this study are available from the corresponding author upon request. zhu . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 21, 2020. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 21, 2020. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 21, 2020. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 21, 2020. immunol. 20, 392-394 (2020). . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 21, 2020. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 21, 2020. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 21, 2020. . https://doi.org/10.1101/2020.08.18.20177303 doi: medrxiv preprint cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 21, 2020. . https://doi.org/10.1101/2020.08.18.20177303 doi: medrxiv preprint of cov2pp by samples from (a) 28 covid-19-convalescent individuals and (b) 11 covid-19-negative individuals, compared to (c) a recombinant soluble rbd (srbd) control. each plasma or sera specimen was tested at 4-fold dilutions from 1:10 to 1:40,960, and srbd was tested at 4-fold dilutions from 100 to 0.02 µg/ml. the data are shown as mean percentage of neutralization + sd of triplicate. the extrapolated titration curves were generated using a nonlinear regression model in graphpad prism (inhibitor versus response -variable slope (four parameters), least squares regression). the dotted horizontal lines highlight 50% neutralization. tf#1 tf#2 tf#3 tf#4 tf#5 tf#6 tf#7 tf#8 tf#9 tf#10 tf#11 tf#12 tf#13 tf#14 tf#15 tf#16 tf#17 tf#18 tf#19 tf#20 tf#21 tf#22 tf#23 tf#24 tf#25 covid-19-positive subjects tf#1 tf#2 tf#3 tf#4 tf#5 tf#6 tf#7 tf#8 tf#9 tf#10 tf#11 tf#12 tf#13 tf#14 tf#15 tf#16 tf#17 tf#18 tf#19 tf#20 tf#21 tf#22 tf#23 tf#24 tf#25 covid-19-positive subjects tf#1 tf#2 tf#3 tf#4 tf#5 tf#6 tf#7 tf#8 tf#9 tf#10 tf#11 tf#12 tf#13 tf#14 tf#15 tf#16 tf#17 tf#18 tf#19 tf#20 tf#21 tf#22 tf#23 tf#24 tf#25 covid-19-positive subjects control. each plasma or sera specimen was tested at 4-fold dilutions from 1:10 to 1:40,960, and srbd was tested at 4-fold dilutions from 100 to 0.02 µg/ml. the data are shown as mean percentage of neutralization + sd of triplicate. the extrapolated titration curves were generated using a nonlinear regression model in graphpad prism (inhibitor versus responsevariable slope (four parameters), least squares regression). the dotted horizontal lines highlight 50% neutralization. phase 1/2 study to describe the safety and immunogenicity of a covid rna vaccine candidate (bnt162b1) in adults 18 to 55 years of age: interim report dna vaccine protection against sars-cov-2 in rhesus macaques structural basis for the recognition of sars-cov-2 by full-length human ace2 the novel coronavirus 2019 (2019-ncov) uses the sars-coronavirus receptor ace2 and the cellular protease tmprss2 for entry into target cells sars-cov-2 invades host cells via a novel route: cd147-spike protein neuropilin-1 facilitates sars-cov-2 cell entry and provides a possible pathway into the central nervous system a pneumonia outbreak associated with a new coronavirus of probable bat origin genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor plasma were tested at 4-fold dilutions from 1:10 to 1:40,960 or 1:20 to 1:81,920. data are shown as the mean percentage of neutralization. the dotted horizontal lines highlight 50% neutralization. (b) neutralization reciprocal ic 50 and ic 90 titers of rp#1-5 plasma. (c) neutralization of cov2pp by purified igm, igg, and iga fractions from 5 covid-19-infected individual plasma (rp#1-5) compared to a control ig fraction. the fractions were tested at 4-fold dilutions from 500 to 0.02 µg/ml. data are shown as the mean percentage of neutralization. the dotted horizontal lines highlight 50% neutralization. (d) ic 50 of purified igm, igg, and iga fractions from rp#1-5 plasma. the statistical significance was determined by a two we thank all the donors for their contribution to research and dr. arthur nadas (nyu school of medicine) for statistical consultation. the authors declare no competing interests.. cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. fig. 6 . summary of relative ig isotype levels and neutralization titers. key: cord-252919-647zcjgu authors: chen, yun; guo, yao; pan, yihang; zhao, zhizhuang joe title: structure analysis of the receptor binding of 2019-ncov date: 2020-02-17 journal: biochem biophys res commun doi: 10.1016/j.bbrc.2020.02.071 sha: doc_id: 252919 cord_uid: 647zcjgu abstract 2019-ncov is a newly identified coronavirus with high similarity to sars-cov. we performed a structural analysis of the receptor binding domain (rbd) of spike glycoprotein responsible for entry of coronaviruses into host cells. the rbds from the two viruses share 72% identity in amino acid sequences, and molecular simulation reveals highly similar ternary structures. however, 2019-ncov has a distinct loop with flexible glycyl residues replacing rigid prolyl residues in sars-cov. molecular modeling revealed that 2019-ncov rbd has a stronger interaction with angiotensin converting enzyme 2 (ace2). a unique phenylalanine f486 in the flexible loop likely plays a major role because its penetration into a deep hydrophobic pocket in ace2. ace2 is widely expressed with conserved primary structures throughout the animal kingdom from fish, amphibians, reptiles, birds, to mammals. structural analysis suggests that ace2 from these animals can potentially bind rbd of 2019-ncov, making them all possible natural hosts for the virus. 2019-ncov is thought to be transmitted through respiratory droplets. however, since ace2 is predominantly expressed in intestines, testis, and kidney, fecal-oral and other routes of transmission are also possible. finally, antibodies and small molecular inhibitors that can block the interaction of ace2 with rbd should be developed to combat the virus. a mysterious pneumonia illness was first reported in late december 2019 in wuhan, china, and has rapidly spread to a dozen of countries including the united states with thousands of infected individuals and hundreds of deaths within a month [1] . scientists in china have isolated the virus from patients and determined its genetic code. the pathogen responsible for this epidemic is a new coronavirus designated 2019-ncov by the world health organization. 2019-ncov belongs to the same family of viruses as the well-known severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov), which have killed hundreds of people in the past 17 years. coronaviruses consist of a large diverse family of viruses. they can be classified into four genera: alpha-, beta-, gamma-, and delta coronavirus [2, 3] . representative alphacoronaviruses include human coronavirus nl63 (hcov-nl63), while the betacoronaviruses include the best-known sars-cov and mers-cov. based on nucleic acid sequence similarity, the newly identified 2019-ncov is a betacoronavirus. the entry of all coronaviruses into host cells is mediated by spike glycoprotein that gives coronaviruses a crownlike appearance by forming spikes on their surface. the amino acid sequence of spike glycoprotein consists of a large ectodomain, a single-pass transmembrane anchor, and a short c-terminal intracellular tail [3] . the ectodomain contains a receptor-binding unit s1 and a membrane-fusion unit s2. electron microscopic imaging illustrated that spike glycoprotein forms a clove-shaped spike with three s1 heads and a trimeric s2 stalk. for a virus to enter a host cell, s1 binds to a specific cell surface receptor via its receptorbinding domain (rbd), and s2 fuses the host cell and viral membranes, enabling the entry of viral genomes into host cells. specific rbd-receptor binding determines if a cell or animal can be infected and also serves as a target for therapeutic inventions to treat diseases caused by coronaviruses. previous studies have identified angiotensin converting enzyme 2 (ace2) as a functional receptor for sars-cov [4, 5] . in this study, we analyzed the structure of spike glycoprotein rbd of 2019-ncov and identified a unique feature that potentially allows a high affinity binding to ace2 in human cells. we further discussed potential candidates for natural hosts of 2019-ncov, routes of transmission, and strategies to inhibit virus entry for therapeutic applications. the genomic sequence of 2019-ncov as deposited by wang et al. was downloaded from the genbank database (mn908947.3). dna and protein sequences were compared by using the blast program. multiple sequence alignment was performed by using the clustal omega program. three-dimensional structure was analyzed by using the cn3d program from the ncbi. protein structure simulation was performed by using swiss-model based on the cocrystal structure of human ace2 with the sars-cov spike glycoprotein rbd (6, pdb id 2ajf). ace2 and rdb interaction was analyzed by molecular docking using the patchdock and firedock programs. by using the initially reported sequence mn908947.3, a blast search of the ncbi database revealed 6 inputs for the virus with essentially identical sequences (accession nc_045512.2, mn908947.3, mn975262.1, mn985325.1, mn988713.1, and mn938384.1). the closest homolog of 2019-ncov is a sars-like coronavirus isolated from bat (mg772933.1) with a sequence identity of 87.99% at 99% coverage (fig. 1a) . it also shows 80% sequence identity with sars coronavirus isolated from human patients or civet with 98% coverage. throughout the entire 29,903bp genome of 2019-ncov, the least conserved region encodes for the spike glycoprotein with sequence identity of 74e83%. spike glycoprotein forms spikes on the surface of coronaviruses and is responsible for entrance of the viruses into the host cells. the rbd in the spike glycoprotein molecule directly binds receptors on the surface of host cells [3] . in the case of sars-cov and bat/civet sars-like cov, the receptor is ace2, an exopeptidase that catalyzes the conversion of angiotensin i to the nonapeptide angiotensin1-9 or the conversion of angiotensin ii to angiotensin1-7 [3e7]. at the protein level, the whole spike glycoprotein and its rbd share 76% and 72% sequence identity with sars-cov, respectively. sars-cov spike glycoprotein is known to be glycosylated. a total of 22 predicated n-glycosylation sites is found in spike glycoprotein of 2019-ncov, which are shared by sars-cov except that the latter contains an extra glycosylation site at n370. a detailed sequence alignment of the rbd of sars-cov spike glycoprotein with those from closely related coronaviruses at the protein level is shown in fig. 1b . the crystal structure of sars-cov rbd in complex with its receptor, human ace2, has been solved [6] . by performing molecular simulation, we obtained a ternary structure for rbd of 2019-ncov that is essentially superimposable with that of sars-cov ( fig. 2aec) , except for a noted structural variation in a loop (loop 2). the backbone of the deduced rbd structure consists of 7 beta sheets. peptide segments involved in the formation of this secondary structure are all highly conserved without the presence of secondary structure breakers. four cysteinyl residues that form disulfide bonds (corresponding to c366/c418 and c467/c474 of sars-cov) are also conserved (see also fig. 1b) . we furthermore performed molecular docking to examine the binding of rbd with ace2. the deduced complex structure reveals similar mode of extensive interaction as seen with sars-cov with a more favorable binding energy (à21.82 21 vs. à13.38 kcal/mol) (fig. 2def) . the contact between ace2 and rbd involves two b-sheets and three loops (see figs. 1b and 2a). there are 16 amino acid residues in sars-cov rbd that are directly in contact with ace2, of which 8 are conserved in 2019-ncov (see fig. 1b ). presumably, the substituted amino acids can either reduce or enhance the interaction. to define the contribution of the variant amino acids to the rbd/ ace2 interaction, we compare the sequences of rbd from three other sars-cov-associated viruses (fig. 1b) . these include coronaviruses isolated from patients during a short, weak sars outbreak in 2003e2004 (denoted sarsv here) and from palm civets and bats, possible sources of sars-cov found in humans [8e11]. recombinant proteins containing rbd of these 3 viruses are all known to bind to human ace2 [11, 12] . in comparison with sars-cov (responsible for the major sras outbreak during the 2002e2003), binding with sarsv and civet rbds is substantially weaker [12] , while quantitative binding affinity with bat rbd has been not been determined [11] . amino acid residues in the rbd/ ace2 binding interface plays a crucial role in determining the binding affinity. among the 16 amino acid residues in rbd of sars that are in contact with ace2, 14, 14, 7, and 8 are shared by sarsv, civet, bat, and 2019-ncov, respectively (fig. 1b) . n479 found in both sars viruses isolated from human patients is changed to k and r in civet and bat, respectively. an earlier study demonstrated that an n479 to k substitution resulted in significantly lower affinity (30fold increase in kd values) [12] . interestingly, this amino acid is substituted by a similar amino acid glutamine (q493) in 2019-ncov, which also contains an amide group but at an extended position, which can potentially carry out similar functions. in comparison with sars-cov, t487 is changed to asparagine (n501) in 2019-ncov but alanine or serine in the other viruses. it has been shown that a t487 to s substitution increased kd by 20-fold, suggesting the methyl group rather than the hydroxyl group in this threonine residue is more important for the interaction [12] . it is hard to predict if n501 with an amide group can confer a better interaction. hydrophobic amino acid l472 is also important for interaction between rbd and ace2. interestingly, it is substituted by proline in sarsv and phenylalanine in 2019-ncov (corresponding to f486). l472 is located in a loop formed by disulfide bond c467/c474. interestingly, this loop with ctppalnc in sars-cov is replaced by cngvegfnc in 2019-ncov containing one extra amino acid residue and totally different amino acid compositions. the replacement of two proline residues by two flexible glycine residues converts a rigid structure to a very flexible one. further examination of the deduced rbd/ace2 complex structure reveals that this unique phenylalanine f486 in the flexible loop can penetrate deep into a hydrophobic pocket in ace2 formed by f28, l79, y83, and l97 (fig. 2f) . the presence of two aromatic amino acids in the pocket may provide additional binding force via pstacking interactions [13] . taken together, 2019-ncov likely has a stronger binding to ace2 via its spike glycoprotein. glycosylation may also affect the interaction of rbd with ace2. among the 23 glycosylation sites on spike glycoprotein, two are in rbd (fig. 1b) . glycosylation has been detected on one of these residues, asn330 [6] . n330 corresponds to n343 in the spike glycoprotein of 2019-ncov and is a conserved glycosylation site. since it is well separated from the rbd/ace2 interaction interphase, glycosylation at this site is unlikely to interfere with the interaction [6] . it should be noted that another potential glycosylation site corresponding to n357 in sars-cov is not conserved in 2019-ncov because of substitution of t by a in the þ2 position. lack of this glycosylation is not expected to affect the receptor binding. the 2019-ncov outbreak is thought to be initiated from a seafood market that also carried many other wild live animals including snakes, birds, and various mammals. interestingly, a study by ji et al. suggests that snakes might serve as a likely reservoir for the novel ncov-2019 based on the observation that the codon usage of ncov-2019 was more similar to snakes than other potential hosts they investigated [14] . while the data and premise are being debated, we sought to address the problem by analyzing the structure of ace2 in different animals. ace2 is widely expressed in the animal kingdom from fish, amphibians, reptiles, birds, to mammals. remarkably, its structure is highly conserved. comparison of human ace2 with that of a civet (paguma larvata, aax63775.1), a bat (rhinolophus sinicus, adn93475.1), a bird (nipponia nippon, kfq92425.1), a snake (protobothrops mucrosquamatus, xp_029140508.1), a frog (xenopus laevis, xp_018104311.1), and a fish (callorhinchus milii, xp_007889845.1) revealed amino acid sequence identity of 83%, 81%, 83%, 61%, 60%, and 59%, respectively. fig. 3 aligns parts of ace2 sequences that contain all the interaction sites in contact with sars-cov rbd according to the published co-crystal structure [6] . the interaction involved mainly two a-helices of ace2. out of 20 amino acid residues involved in the direct interaction, 4 of them are shared by all seven species of animals analyzed in the study, including f28 that supposedly interacts with f486 of spike glycoprotein from 2019-ncov (fig. 2f) . many of the remaining resides in the contact are conserved or replaced by amino acids of similar chemical properties. it is interesting to note that bird ace2 shares as many conserved contacting amino acid residues as bat and civet ace2. ace2 molecules from any of these has the potential to interact with rbd of 2019-ncov with high affinity. therefore, it would not be a surprise if any of these wild animals is found to be a primary or secondary host of 2019-ncov. sars-cov-like coronaviruses have been found in many bats that are considered as natural reservoirs for the viruses. they may well be the host for 2019-ncov. however, the possibility that cold-blooded animals like snakes can serve as a host cannot be ruled out. the flexible interacting loop identified in our study may allow the virus to adapt to both the cold-blooded and warm-blooded hosts. by performing immunostaining, earlies studies have demonstrated the expression of ace2 in lung alveolar epithelial cells as well as arterial and venous endothelial cells, arterial smooth muscle cells, renal tubular epithelium, and epithelia of the small intestine [15, 16] . the lung expression provides strong support for infection of sars-cov and 2019-ncov through the airways of the lung. however, by searching the human protein atlas database, we found that ace2 mrna is mainly detected in small intestine, colon, duodenum, kidney, testis, and gallbladder. its expression level in the lung is minimal (fig. 4) . furthermore, by examining data from two single-cell rna-seq studies [17, 18] , we only identified 2 out of 4599 and 13 out of 540 lung epithelial cells expressed a detectable level of ace2 (www.ebi.ac.uk/gxa/sc). this confirms that the overall expression of ace2 in the lung is low and may also suggest the presence of selected cells with upregulated ace2 expression under certain conditions. the tissue expression pattern of ace2 suggests other modes of virus transmission that may involve the functions of intestine, kidney, testis, and other tissues. particular attention should be paid to the intestines which expressed the highest level of ace2. earlier studies have demonstrated that diarrhea was present in up to 70% of patients infected with sars-cov [19] . more importantly, a recent case report demonstrated the presence of 2019-ncov in feces of a patient with an initial diarrhea episode [20] . while this finding has been noted in other reports, tests of feces and urine samples for the presence of 2019-ncov is warranted, which may help to reveal alternative routes of virus transmission. since its initial outbreak, the 2019-ncov infection is much more contagious than it was originally thought. we know that the virus is capable of spreading quickly from human to human and that people can spread the virus even before they become symptomatic [1] . this makes it harder to contain the virus, and many are concerned about the possibility of a new pandemic. our study suggests unique structural features of the spike glycoprotein rbd of 2019-ncov that confers potentially higher affinity binding for its receptor than found with sars-cov. with a higher affinity binding capability, the number of viruses required to infect a cell is much reduced. this partly explains why 2019-ncov appears to be more aggressive than sars-cov. this also reminds us of a lesser-known coronavirus hcov-nl63 that also uses ace2 also as a receptor. hcov-nl63 was initially isolated from a child with bronchiolitis in the netherlands [21] . it belongs to the alphacoronavirus subfamily. the rbd of sars-cov shares no structural homology with that of sars-cov but recognizes the same region in ace2. however, cocrystal structure reveals that rbd of nl63-cov has a narrower contact with ace2, involving fewer amino acids [22] . this presumably results in a weaker interaction. evidently, nl63-cov does not spread aggressively and only causes mild to moderate respiratory infections [23] . the exact mode of transmission for 2019-ncov has not been firmly established. sars-cov is thought to be transmitted by respiratory droplets produced when an infected person coughs or sneezes [19] . the respiratory droplets spread can occur only through direct person-to-person contact or at a close distance. presumably, 2019-ncov can be transmitted through respiratory droplets. it may also be transmitted more effectively through the air over a long distance (airborne spread) or by other ways. considering the predominant expression of ace2 in intestines and kidney, 2019-ncov may infect cells in these tissues and find its way into feces and urine. this makes transmission through the fecal-oral route and bodyfluids (urine) possible. the presence of 2019-ncov in feces supports such a notion [20] . specific rbd-receptor binding determines if a cell or animal can be infected and also serves as a target for therapeutic inventions to treat diseases caused by coronaviruses. by binding directly to ace2 on the surface of host cells, spike glycoprotein plays an essential role in virus infection. an obvious way to stop the virus infection is to block the rbd and ace2 interaction. this can be achieved by using antibodies or small molecular inhibitors. naturally, antibodies and inhibitors that can disrupt the interaction of rbd with ace2 is of therapeutic importance. by using a molecular docking approach, an earlier study identified n-(2-aminoethyl)-1 aziridineethanamine as a novel ace2 inhibitor that effectively blocks the sars-cov rbd-mediated cell fusion [24] . this has provided a potential candidate and lead compound for further therapeutic drug development. meanwhile, biochemical and cell-based assays can be established to screen chemical compound libraries to identify novel inhibitors. on the other hand, many ace inhibitors are currently used to treat hypertension and other cardiovascular diseases [25] . among them are captopril, perindopril, ramipril, lisinopril, benazepril, and moexipril. although these drugs primarily target ace, a homolog of ace2 with 42% sequence identity and 61% sequence similarity in the catalytic domain, they may be effective toward ace2 as well [26] . it should be noted that ace inhibitors bind to the catalytic center rather than rbd binding site. nonetheless, these enzymatic inhibitors may indirectly alter conformation of the rbd binding site and thereby affect the interaction of ace2 with rbd. it is certainly worthwhile to test these drugs for their ability to block the rbd/ace2 interaction. a novel coronavirus outbreak of global health concern coronaviruses: an overview of their replication and pathogenesis structure, function, and evolution of coronavirus spike proteins angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus the sars-cov s glycoprotein: expression and functional characterization structure of sars coronavirus spike receptor-binding domain complexed with receptor ace2 of the heart: from angiotensin i to angiotensin (1-7) analysis of multimerization of the sars coronavirus nucleocapsid protein cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human isolation and characterization of viruses related to the sars coronavirus from animals in southern china isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor receptor and viral determinants of sarscoronavirus adaptation to human ace2 rapp e, pi-stacking interactions. alive and well in proteins homologous recombination within the spike glycoprotein of the newly identified coronavirus may boost cross-species transmission from snake to human tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis severe acute respiratory syndrome coronavirus infection of human ciliated airway epithelia: role of ciliated cells in viral spread in the conducting airways of the lungs a cellular census of human lungs identifies novel cell states in health and in asthma single-cell rna sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis severe acute respiratory syndrome: historical, epidemiologic, and clinical features first case of 2019 novel coronavirus in the united states identification of a new human coronavirus crystal structure of nl63 respiratory coronavirus receptor-binding domain complexed with its human receptor, proc. natl human coronavirus nl63: a clinically important virus? structure-based discovery of a novel angiotensin-converting enzyme 2 inhibitor intrarenal angiotensin-converting enzyme: the old and the new angiotensin-converting enzyme-2 (ace2): comparative modeling of the active site, specificity requirements, and chloride dependence this study is supported in part by the 100 top talents program of sun yat-sen university, national natural science foundation of china (nsfc, grant no. 31871400), and the sanming project of medicine in shenzhen (no. szsm201911004). the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. key: cord-297747-kifqgskc authors: lupala, cecylia s.; li, xuanxuan; lei, jian; chen, hong; qi, jianxun; liu, haiguang; su, xiao-dong title: computational simulations reveal the binding dynamics between human ace2 and the receptor binding domain of sars-cov-2 spike protein date: 2020-03-27 journal: biorxiv doi: 10.1101/2020.03.24.005561 sha: doc_id: 297747 cord_uid: kifqgskc a novel coronavirus (the sars-cov-2) has been identified in january 2020 as the causal pathogen for covid-19 pneumonia, an outbreak started near the end of 2019 in wuhan, china. the sars-cov-2 was found to be closely related to the sars-cov, based on the genomic analysis. the angiotensin converting enzyme 2 protein (ace2) utilized by the sars-cov as a receptor was found to facilitate the infection of sars-cov-2 as well, initiated by the binding of the spike protein to the human ace2. using homology modeling and molecular dynamics (md) simulation methods, we report here the detailed structure of the ace2 in complex with the receptor binding domain (rbd) of the sars-cov-2 spike protein. the predicted model is highly consistent with the experimentally determined complex structures. plausible binding modes between human ace2 and the rbd were revealed from all-atom md simulations. the simulation data further revealed critical residues at the complex interface and provided more details about the interactions between the sars-cov-2 rbd and human ace2. two mutants mimicking rat ace2 were modeled to study the mutation effects on rbd binding to ace2. the simulations showed that the n-terminal helix and the k353 of the human ace2 alter the binding modes of the cov2-rbd to the ace2. the outbreak of a new type of severe pneumonia covid-19 started in december 2019 1 has been going on world-wide, and caused over 15,000 fatalities, infected more than 350,000 individuals globally. although the earlier infected cases were mainly found in china before march, 2020, particularly in hubei province, the confirmed covid-19 cases have been reported in more than 160 countries or territories by the end of march, 2020, and still increasing rapidly. one urgent desire in coping with this global crisis is to develop or discover drugs that can treat the diseases caused by the novel coronavirus, the sars-cov-2 (also known as 2019-ncov)2. according to the genome comparative studies, the sars-cov-2 belongs to the genus beta-coronavirus, with nucleotide sequence identity of about 96% compared to the closest bat coronavirus ratg13, about 89% compared to two other bat sars-like viruses (bat-sl-covzc45 & bat-sl-covzxc21), and 79% compared to the sars-cov 3,4. furthermore, the sars-cov-2 spike protein has a protein sequence identity of 73% for the receptor binding domain (rbd) with the sars-cov rbd (denoted as sars-rbd in the following). the sars-cov and sars-cov-2 both utilize the human angiotensin converting enzyme 2 protein (ace2) to initiate the spike protein binding and facilitate the fusion to host cells 5-9. the 193-residue rbd of the sars-cov spike protein has been found to be sufficient to bind the human ace2 6. based on this fact, the rbd of sars-cov-2 becomes a critical protein target for drug development to treat the covid-19. when this study was started, neither the crystal structure of the sars-cov-2 spike protein nor the rbd segment were determined, so the homology modeling approach was applied to construct the model of the sars-cov-2 spike rbd in complex with the human ace2 binding domain (denoted as cov2-rbd/ace2 in the following). similar approach has been applied to predict the complex structure and estimate the binding energies 3. because of the high sequence similarity between cov2-rbd and sars-rbd, the predicted structure was found to be highly consistent with the resolved crystal structures 10 (see another crystal structure at http://nmdc.cn/ncov entry:nmdcs0000001, pdbid: 6lzg). these structures laid the foundation for the dynamics investigation of the cov2-rbd/ace2 complex using computational simulation method. the predicted cov2-rbd/ace2 model was subjected to all-atom molecular dynamics (md) simulations to study the binding interactions. although the crystal structure and the predicted model of the cov2-rbd/ace2 complex provide important information about the binding interactions at the molecular interfaces, md simulations can extend the knowledge to a dynamics regime in a fully solvated environment. the importance of the ace2 residues was investigated by simulating the complexes with ace2 mutants, in which partial dissociation from the ace2 was observed within 500 ns simulations. the control simulations of the sars-rbd/ace2 complexes allowed the detailed comparison in receptor binding for the two different types of viruses. the results showed that the wild type cov2-rbd/ace2 complex is stable in 500 ns simulations, especially in the well-defined binding interface. on the other hand, the mutations on the helix-1 or k353 of the ace2 can alter the binding, revealing new binding poses with reduced contacts compared to those in the crystal structures. the analysis of the interaction energy showed that the binding is enhanced by adjusting conformations to form more favorable interactions as the simulation progressed, consistent with the increased hydrogen bonding patterns. furthermore, the analysis also showed that sars-rbd and cov2-rbd have comparable binding affinities to the ace2, with the former slightly stronger than the latter. the dynamic information obtained by this study shall be useful in understanding sars-cov-2 host interaction and for designing inhibitors to block cov2-rbd binding. the computer model of the sars-cov-2 spike rbd in complex with human ace2 the spike rbd of sars-cov-2 (genbank: mn908947 2) comprises cys336-gly526 residues according to the sequence homology analysis with sars-cov spike rbd. the predicted three-dimensional structure model of these residues was obtained with the swiss-model server 11. this predicted sars-cov-2 rbd model was subsequently superimposed into the x-ray structure of sars-cov rbd in complex with human ace2 (pdb code:2ajf, chain d 7). finally, the computer model of sars-cov-2 rbd with human ace2 (cov2-rbd/ace2) was obtained for further simulations and analysis. based on the analysis of the predicted model, sequence alignment, and literature survey, two other systems containing mutations in the human ace2 were prepared and subject to md simulation studies. the mutant construct is based on the fact that rat ace2 markedly diminishes interactions with sars spike protein 12, and it was proposed that the rat ace2 likely has reduced binding affinity to the cov2-rbd 13. to investigate the roles of critical residues on the ace2, we created two mutants of the human ace2 (see table 1 ): (1) mutant mut_h1, with the ace2 n-terminal (residue 19-40) mutated to the residues of rat ace2; and (2) mutant k353h, in which the highly conserved k353 was mutated to histidine (the corresponding amino acid in rat and mouse ace2 proteins). to focus on the impact of these two binding sites, the rest of the ace2 were kept to be the same as human ace2. the predicted model of cov2-rbd/ace2 complex was used as the starting models for md simulations. the spike protein rbd domain is composed of 180 residues (323-502), while the ace2 protein contains 597 residues from the n-terminal domain. the simulation parameterization and equilibration were prepared for complex structures including the mutant systems, using the charmm-gui webserver 14. each system was solvated in tip3p water and sodium chloride ions to neutralize the systems to a salt concentration of 150 mm. approximately, each system was composed of about 220,000 atoms that were parametrized with the charmm36 force field 15. after energy minimization using the steepest descent algorithm, each system was equilibrated at human body temperature 310.15 k, which was maintained by nose-hoover scheme with three independent trajectories starting from random velocities based on maxwell distributions were simulated for both cov2-rbd/ace2 and sars-rbd/ace2 complex systems in their wild types. in all simulations, a time step of 2.0 fs was used and the pme (particle mesh ewald) 20 was applied for long-range electrostatic interactions. the van der waals interactions were evaluated within the distance cutoff of 12.0 å. hydrogen atoms were constrained using the lincs algorithm 21. the human ace2 mutants in complex with cov2-rbd were constructed as described previously. each mutant complex model was simulated in two independent trajectories. furthermore, as the crystal structure of the cov2-rbd/ace2 complex became available, two additional simulations were carried out using the crystal structure as the starting model to cross-validate the simulation results based on the homology model. each trajectory was propagated to 500 ns by following the same protocol as the wild type cov2-rbd/ace2 complex simulations. analyses were carried out with tools in gromacs (such as rmsd, rmsf, energy, and pairdist) to examine the system properties, such as the overall stability, local residue and general structure fluctuations through the simulations. the g_mmpbsa program 22 was applied to extract the molecular mechanics energy emm (lennard jones and electrostatic interactions) between ace2 and the rbd of spike proteins. vmd and chimera were applied to analyze the hydrogen bonds, molecular binding interface, water distributions, visualization and rending model images 23, 24 . the homology structure of the cov2-rbd/ace2 was compared to the sars-rbd/ace2 crystal structure and the newly resolved crystal structure of the cov2-rbd/ace2. the results indicated that the homology model is accurate, especially at the binding interface. the md simulations further refined the side chain orientations to improve the model quality. the simulation data revealed the stable binding between the cov2-rbd and the ace2, in spite of the conformational changes of the ace2. the relative movement between the cov2-rbd and the ace2 mainly exhibited as a swinging motion pivoted at the binding interface. simulations also revealed the roles of water molecules in the binding of the rbd to the ace2 receptor. the md simulation of complex with ace2 mutants suggested that mutation to the ace2 helix-1 and the k353 can alter the binding modes and binding affinity. the predicted cov2-rbd/ace2 complex structure is highly similar to the sars-rbd/ace2, as shown in figure 1 . the rbd domain has an rmsd of 0.99 å for the aligned residues (1.53 å for all 174 residue pairs), indicating that the homology model of the cov2-rbd is in good agreement with the sars-rbd. for ace2 residues near the binding interface (within 4.0 å of the rbd), the rmsd is smaller than 0.43 å compared to the sars-rbd/ace2 complex. the superposed structures revealed that the rbd/ace2 interfaces are almost identical in two complexes (figure 1c ). in a retrospective comparison, the predicted complex structure was superposed to the newly resolved crystal models (see figure 2d for a detailed comparison at the interface). the results indicated that the homology model is very accurate, especially for the binding interface. the residues near the cov2-rbd/ace2 interface (defined as the combined set of ace2 residues within 4.0 å of rbd and the rbd residues within 4.0 å of the ace2) exhibited a difference of 0.43 å rmsd, which is comparable to the difference between the two independently reported crystal models (an rmsd of 0.25 å for the same comparison). the rmsd is about 0.77 å for residues in an extended region within 10.0 å of the binding interface. the rbd domain of the spike protein showed an overall rmsd values less than 1.5 å, and the ace2 domain with an rmsd about 2.0 å between the predicted model and the crystal structures. in three simulations of the cov2-rbd/ace2 systems, the binding interface was highly stable, exhibiting very small conformational changes, especially for the interfacing residues of the ace2 protein. the rmsd for the residues at the rbd binding interface is 0.85å (+/-0.13å) on average. side chain atom positions were refined to form more favorable interactions (figure 2d) . one outstanding example is the k31 side-chain, which pointed in the wrong orientation in the predicted structure, was quickly refined to the correct orientation, consistent with the crystal structure (right panel of figure 2d ). in terms of collective conformational changes, the cov2-rbd/ace2 complex showed two interesting movements: (1) the loop (l67) between β6 and β7 (residues between s477 and g485 in particular) of cov2-rbd was found to expand its interactions with the nterminal helix (the helix-1) of the ace2 (figure 2a) , while it pointed away from the helix-1 in the predicted and the crystal structures (figure 2d, left) ; (2) a tilting movement of the rbd relative to the ace2 was observed, which can be depicted as a swinging motion with the binding interface as the pivot (see figure 4 for an illustration). in both predicted and the crystal structures of the cov2-rbd/ace2 complex, the l67 does not form close contacts with the ace2. the analysis of the crystal packing revealed that this loop participated in the interaction with another asymmetric unit (see supplemental information). interestingly, the simulation data suggested that the l67 could move towards the ace2 and form contacts with the helix-1. this can potentially enhance the binding, as reflected in the change of interaction energies. in the crystal structure, the c480 and c488 of the rbd are cross-linked via a disulfide bond that reduces the flexibility of the l67 region, limiting its access to the ace2. on the other hand, it has been reported that the binding of sars-rbd to ace2 is insensitive to the redox states of the cysteines to a high extend 26. based on the simulation results, we hypothesize that the reduced form of c480 and c488 can also exist during the virus invasion to host cells, and the reduced cysteines can potentially enhance the binding to ace2. in the other two simulation trajectories, we found that the l67 remained in conformations similar to that in the crystal structure and the cysteines (c480 and c488) were close enough for disulfide bond formation. by examining the binding interface of cov2-rbd and the ace2, we found the polar and charged residues account for a large fraction, therefore the electrostatic interactions play critical roles for the complex formation. based on the distances between the two proteins, the key residues at the binding interface were identified and summarized in table 2 for the three representative models (see figure 2 ). majority of these residues are conserved for these three models, except that the model#1 (figure 2a) has additional contacts to the ace2 from residues in the l67 region (highlighted with green color in table 2 ). as shown in figure 2 , the l67 remained in the starting position for the other two representative models (figure 2b,c) . the same simulations were carried out for the sars-rbd/ace2 complex, serving as a comparative system. interestingly, the sars-rbd counterpart of the l67 in cov2-rbd did not form close contacts with the ace2 in three simulations. it is worthwhile to mention that the sequence identity between cov2-rbd and sars-rbd is low in this loop region, suggesting the loop region might be partially responsible for the difference in the receptor binding. the hydrogen bonds between the cov2-rbd and ace2 were extracted using vmd based on the statistics of three simulation trajectories, the cov2-rbd/ace2 complex has 2.7 hydrogen bonds between the subunits on average with stringent criteria. in comparison, the sars-rbd/ace2 has 3.2 hydrogen bonds on average (see supplementary information ). the statistics of hydrogen bonds suggest a slightly weaker binding between the cov2-rbd and the ace2, relative to the sars-rbd/ace2 complex. it is also noteworthy to point out the important roles of water molecules at the complex interface for cov2-rbd/ace2 complex. at any instant time, there are approximately 15 water molecules at the binding interface, simultaneously located within 2.5 å of both the cov2-rbd and the ace2 (figure 6 ). these water molecules can function as bridges by forming hydrogen bonds with the residues from the rbd or the ace2. the dwelling time of water molecules at the interface can be a few nanoseconds, as revealed by the md simulations. this results is also consistent with the crystal structure, which has 12 water molecules at the interface (figure 6c ). these discoveries emphasize the role of the water molecules, which desires detailed quantification to understand the interactions between the rbd and the ace2. it has been demonstrated that the ace2 from several mammalian species possess high sequence similarities, yet their binding to the sars-rbd differs significantly. in particular, the binding of sars-rbd to the rat ace2 is much weaker as discovered in experiments 12. inspired by these information, two mutants of the cov2-rbd/ace2 were constructed: (1) ace2-mut-h1 by mutating n-terminal helix-1 to that of the rat ace2; (2) ace2-k353h by mutating k353 to histidine (the amino acid in wild type rat ace2). two 500ns md simulations were carried out for each mutant system. the simulation showed that the mutations in ace2-mut-h1 reduced the interaction between the cov2-rbd and the helix-1, and the ace2-k353h showed weaker binding between the cov2-rbd and the β-hairpin centered at the h353. using the clustering analysis, the representative structures were identified from each simulation trajectory (figure 7) . although the overall topology is very similar to the wild type complex structure, there are pronounced differences. for the ace2-mut-h1 system, the cov2-rbd tilted further away from the ace12 helix-1 in one simulation (figure 7a) ; and the cov2-rbd lost its contact with helix-13 (g326 to n330) in another simulation for the ace2-k353h (figure 7c ). in the wild type ace2, the k353 is a hydrogen donor, and its mutant h353 cannot form the hydrogen bond with the cov2-rbd as in the wild type cov2-rbd/ace2 complex. the number of contacting residue pairs was significantly reduced in the ace2-k353h mutant system. this is in line with the report that k353 is more critical than the other residues, as its hydrophobic neighborhood enables this positively charged residue high selectivity to the rbd 27,28. the physical interactions between the rbd and the ace2 were quantified for the simulated structures. we considered the molecular mechanics energy emm , which is composed of the van der waals and the electrostatic interactions. furthermore, the number of residue contacts (nc) between (rbd and ace2) was extracted from simulated structures. both the emm and nc indicate that the rbd interactions with the ace2 are comparable for cov2 and sars spike proteins (figure 8) . from the simulations, the we would like to point out that the energy emm is the physical interaction between the rbd and the ace2, rather than the binding energy, which requires accurately incorporating solvation energy and entropy. furthermore, the standard deviations of emm are 70.2 kj/mol and 65.5 kj/mol for the two complexes. therefore, we infer that the binding affinities are comparable for cov2-rbd/ace2 and sars-rbd/ace2. the simulations started from the predicted and crystal models yielded very similar results (purple triangles). this is in line with a recent study, in which the authors showed similar binding affinity to human ace2 for both sars-cov-2 and sars-cov spike proteins 29. they found the association rate constants kon to be the same at 1.4x105 m-1s-1, while the sars-cov spike protein showed a faster dissociation, with the rate constant koff to be 7.1x10-4 s-1, about 4.4 times larger than the sars-cov-2 spike protein koff =1.6x10-4 s-1. similar kon values and the equilibrium dissociation constants kd in nanomolar range were reported in other studies for sars-cov-2 spike protein (or rbd) binding to human ace210,30. more interestingly, the mutation impacts were reflected in the emm and nc analysis: the ace2-mut_h1 is likely to reduce the binding to the ace2 due to the tilting movement of cov2-rbd, making it further from the ace2 helix-1 (the blue triangle symbol at lower right, see figure 7a for the representative structure). in the other simulation trajectory for the cov2-rbd/ace2-mut_h1 complex (blue triangle at the left upper corner), the largest nc was observed among all simulations. for simulations of the complex with ace2-k353h mutants (green diamonds), the number of contacts were both reduced compared to the wild type system. in one simulation, the contacts between the cov2-rbd and the helix-13 of the ace2 were completely lost (see figure 7c) , consistent with the less favorable interactions reflected on an increase of emm. for the sars-rbd interaction with the ace2-mut_h1, both simulations revealed fewer contacts compared to the wild type sars-rbd/ace2 complex (purple stars in figure 8 ). the homology modeling of the cov2-rbd/ace2 complex yielded highly consistent models compared to the crystal structures. all-atom molecular dynamics simulations were carried out to study the dynamic interactions of cov2-rbd with human ace2, the results were compared to the sars-rbd/ace2 system. the human ace2 mutants were also constructed to mimic the rat ace2 to investigate the roles of critical residues, and possible binding modes in other mammals. it is observed that md simulations improved the structure at the binding interface and strengthened the interactions between the subunits. the structure of the complex interface is highly stable for all simulations of cov2-rbd/ace2 complex in the wild type. the loop region between β6 and β7 can potentially form more contacts with the ace2 as observed in one simulation trajectory. the simulations results also reveal that the interactions between cov2-rbd and the ace2 are mediated by water molecules at the interfaces, stressing the necessity of accounting for the explicit water molecules when quantifying the binding affinity. the interactions between the rbd and the ace2 were quantified by physical interaction energies (molecular mechanics energy) and the number of contacting residues. the detailed comparison results suggest that the cov2-rbd and the sars-rbd bind to human ace2 with comparable affinity. the comparison between the sars-rbd/ace2 and the cov2-rbd/ace2 complexes, with the former forms fewer contacts than the latter (figure 8 ), yet exhibiting stronger interactions. the decomposition of the emm to the van der waals and the electrostatic interactions revealed that the major difference is attributed to the electrostatic interactions. furthermore, we compared the major contacting residues and found that the sars-rbd has two charged residues (r426 and d463) and the cov2-rbd has only one charged residue (k417) at the complex interface. the polar and hydrophobic residues are comparable in the two rbds. this is consistent with the statistics of hydrogen bonds at the complex interfaces. this study was started with a structure predicted using homology modeling method, which later found to be highly consistent with the crystal structure, demonstrating the potentiality of structure prediction and dynamics simulation in revealing molecular details before the availability of high resolution experimental information. furthermore, the interactions between cov2-rbd and the ace2 mutants mimicking rat ace2 protein were investigated. the results provide valuable information at the atomic level for the reduced binding affinity. the recent report on the sars-cov-2 infection to a dog 31 remark mut_h1 t20l, q24k, k26e, t27s, d30n, h34q, f40s l20, n30, q34, and s40 are conserved between rat and mouse. k353h k353h h353 is conserved between rat and mouse table 2 . contact residues between the cov2-rbd and the ace2. green color denotes new interaction not observed in crystal structure. model#2 t27 f28 d30 k31 h34 e35 e37 d38 y41 q42 m82 y83 n330 k353 g354 d355 r357 k417 y453 l455 f456 q474 a475 g476 s477 t478 g485 f486 n487 y489 q493 y495 g496 q498 t500 n501 g502 y505 s19 q24 t27 f28 d30 k31 h34 e35 d38 y41 q42 l45 m82 y83 n330 k353 g354 d355 r357 k417 g446 y449 y453 l455 f456 a475 f486 n487 y489 q493 y495 g496 q498 t500 n501 g502 y505 q24 t27 f28 d30 k31 h34 e35 e37 d38 y41 q42 m82 y83 n330 k353 g354 d355 r357 k417 g446 y449 y453 l455 f456 a475 f486 n487 y489 q493 y495 g496 q498 t500 n501 g502 y505 table s1. contact residues at the sars-cov-rbd/ace2 interface traj 1 traj2 traj 3 ace2 cov ace2 cov ace2 cov s19 q24 t27 k31 h34 e37 d38 y41 q42 l45l l79l m82 y83 q325 e329 n330 k353 g354 d355 r357 r426 y436 y440 y442 l443 d463 l472 n473 y475 n479 g482 y484 t486 t487 g488 i489 y491 q24 t27 d30 k31 h34 e37 d38 y41 q42 l45l l79l m82 y83 q325 e329 n330 k353 g354 d355 r357 r426 y436 y440 y442 l443 p462 d463 g464 l472 n473 y475 n479 g482 y484 t486 t487 g488 i489 y491 q24 t27 k31 h34 e37 d38 y41 q42 l45l l79l m82 y83 q325 e329 n330 k353 g354 d355 r357 r426 y440 y442 l443 p462 d463 g464 p470 l472 n473 y475 l478 n479 g482 y484 t486 t487 g488 i489 y491 m82 t27 k31 k353 y41 e329 m82 t27 k31 k353 y41 e329 fig.1 cov2-rbd/ace2 sars-rbd/ace2 superposed a. b. c. fig.2 a. b. c. a novel coronavirus from patients with pneumonia in china a new coronavirus associated with human respiratory disease in china evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus a 193-amino acid fragment of the sars coronavirus s protein efficiently binds angiotensinconverting enzyme 2 structure of sars coronavirus spike receptor-binding domain complexed with receptor trilogy of ace2: a peptidase in the renin-angiotensin system, a sars receptor, and a partner for amino acid transporters the pathogenicity of 2019 novel coronavirus in hace2 transgenic mice crystal structure of the 2019-ncov spike receptor-binding domain bound with the ace2 receptor swiss-model: homology modelling of protein structures and complexes efficient replication of severe acute respiratory syndrome coronavirus in mouse cells is limited by murine angiotensin-converting enzyme 2 receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars simulations using the charmm36 additive force field optimization of the additive charmm all-atom protein force field targeting improved sampling of the backbone φ, ψ and side-chain χ1 and χ2 dihedral angles a unified formulation of the constant temperature molecular dynamics methods canonical dynamics: equilibrium phase-space distributions polymorphic transitions in single crystals: a new molecular dynamics method gromacs: high performance molecular simulations through multi-level parallelism from laptops to supercomputers particle mesh ewald: an n·log(n) method for ewald sums in large systems lincs: a linear constraint solver for molecular simulations g-mmpbsa -a gromacs tool for highthroughput mm-pbsa calculations vmd: visual molecular dynamics ucsf chimera -a visualization system for exploratory research and analysis receptor and viral determinants of sars-coronavirus adaptation to human ace2 significant redox insensitivity of the functions of the sars-cov spike glycoprotein: comparison with hiv envelope structural analysis of major species barriers between humans and palm civets for severe acute respiratory syndrome coronavirus infections receptor recognition mechanisms of coronaviruses: a decade of structural studies structure, function, and antigenicity of the sars-cov-2 spike glycoprotein cryo-em structure of the 2019-ncov spike in the prefusion conformation. science (80-. ) coronavirus: hong kong confirms a second dog is infected the authors declare no competing interests. key: cord-256572-sqz8yc7b authors: huo, jiandong; zhao, yuguang; ren, jingshan; zhou, daming; duyvesteyn, helen me; ginn, helen m; carrique, loic; malinauskas, tomas; ruza, reinis r; shah, pranav nm; tan, tiong kit; rijal, pramila; coombes, naomi; bewley, kevin; radecke, julika; paterson, neil g; supasa, piyasa; mongkolsapaya, juthathip; screaton, gavin r; carroll, miles; townsend, alain; fry, elizabeth e; owens, raymond j; stuart, david i title: neutralization of sars-cov-2 by destruction of the prefusion spike date: 2020-05-06 journal: biorxiv doi: 10.1101/2020.05.05.079202 sha: doc_id: 256572 cord_uid: sqz8yc7b there are as yet no licenced therapeutics for the covid-19 pandemic. the causal coronavirus (sars-cov-2) binds host cells via a trimeric spike whose receptor binding domain (rbd) recognizes angiotensin-converting enzyme 2 (ace2), initiating conformational changes that drive membrane fusion. we find that monoclonal antibody cr3022 binds the rbd tightly, neutralising sars-cov-2 and report the crystal structure at 2.4 å of the fab/rbd complex. some crystals are suitable for screening for entry-blocking inhibitors. the highly conserved, structure-stabilising, cr3022 epitope is inaccessible in the prefusion spike, suggesting that cr3022 binding would facilitate conversion to the fusion-incompetent post-fusion state. cryo-em analysis confirms that incubation of spike with cr3022 fab leads to destruction of the prefusion trimer. presentation of this cryptic epitope in an rbd-based vaccine might advantageously focus immune responses. binders at this epitope may be useful therapeutically, possibly in synergy with an antibody blocking receptor attachment. highlights cr3022 neutralises sars-cov-2 neutralisation is by destroying the prefusion spike conformation this antibody may have therapeutic potential alone or with one blocking receptor attachment incursion of animal (usually bat)-derived coronaviruses into the human population has caused several outbreaks of severe disease, starting with severe acute respiratory syndrome (sars) 3 in 2002 (menachery et al., 2015) . in late 2019 a highly infectious illness, with cold-like symptoms progressing to pneumonia and acute respiratory failure, resulting in an estimated 6% overall death rate (baud et al., 2020) , with higher mortality among the elderly and immunocompromised populations, was identified and confirmed as a pandemic by the who on 11 th march 2020. the etiological agent is a novel coronavirus (sars-cov-2) belonging to lineage b betacoronavirus and sharing 88% sequence identity with bat coronaviruses (lu et al., 2020a) . the heavily glycosylated trimeric surface spike protein mediates viral entry into the host cell. it is a large type i transmembrane glycoprotein (the ectodomain alone comprises over 1200 residues) (wrapp et al., 2020) . it is made as a single polypeptide and then cleaved by host proteases to yield an n-terminal s1 region and the c-terminal s2 region. spike exists initially in a pre-fusion state where the domains of s1 cloak the upper portion of the spike with the relatively small (~22 kda) s1 rbd nestled at the tip. the rbd is predominantly in a 'down' state where the receptor binding site is inaccessible, however it appears that it stochastically flips up with a hinge-like motion transiently presenting the ace2 receptor binding site (roy, 2020; song et al., 2018; walls et al., 2020; wrapp et al., 2020) . ace2 acts as a functional receptor for both sars-cov and sars-cov-2, binding to the latter with a 10 to 20-fold higher affinity (k d of ~15 nm), possibly contributing to its ease of transmission (song et al., 2018; wrapp et al., 2020) . there is 73% sequence identity between the rbds of sars-cov and sars-cov-2 ( figure s1 ). when ace2 locks on it holds the rbd 'up', destabilising the s1 cloak and possibly favouring conversion to a postfusion form where the s2 subunit, through massive conformational changes, propels its fusion domain upwards to engage with the host membrane, casting off s1 in the process (song et al., 2018; wrapp et al., 2020) . structural studies of the rbd in complex with ace2 (lan et al., 2020; wang et al., 2020b; yan et al., 2020) how that it is recognized by the extracellular peptidase domain (pd) of ace2 through mainly polar interactions. the s 4 protein is an attractive candidate for both vaccine development and immunotherapy. potent nanomolar affinity neutralising human monoclonal antibodies against the sars-cov rbd have been identified that attach at the ace2 receptor binding site (including m396, cr3014 and 80r (ter meulen et al., 2006; sui et al., 2004; zhu et al., 2007) ). for example 80r binds with nanomolar affinity, prevents binding to ace2 and the formation of syncytia in vitro, and inhibits viral replication in vivo (sui et al., 2004) . however, despite the two viruses sharing the same ace2 receptor these ace2 blocking antibodies do not bind sars-cov-2 rbd (wrapp et al., 2020) . in contrast cr3022, a sars-cov-specific monoclonal selected from a single chain fv phage display library constructed from lymphocytes of a convalescent sars patient and reconstructed into igg1 format (ter meulen et al., 2006) , has been reported to cross-react strongly, binding to the rbd of sars-cov-2 with a k d of 6.3 nm (tian et al., 2020) , whilst not competing with the binding of ace2 (ter meulen et al., 2006) . furthermore, although sars-cov escape mutations could be readily generated for ace2blocking cr3014, no escape mutations could be generated for cr3022, preventing mapping of its epitope (ter meulen et al., 2006) . furthermore a natural mutation of sars-cov-2 has now been detected at residue 495 (y n) (gisaid (shu and mccauley, 2017) : accession id: epi_isl_429783 wienecke-baldacchino et al., 2020), which forms part of the ace2 binding epitope. finally, cr3022 and cr3014 act synergistically to neutralise sars-cov with extreme potency (ter meulen et al., 2006) . whilst this work was being prepared for publication a paper reporting that cr3022 does not neutralise sars-cov-2 and describing the structure of the complex with the rbd at 3.1 å resolution was published (yuan et al., 2020) . here we extend the structure analysis to significantly higher resolution and, using a different neutralisation assay, show that cr3022 does neutralise sars-cov-2, but via a mechanism that would not be detected by the method of yuan et al (yuan et al., 2020) . we use cryo-em analysis of the interaction of cr3022 with the full spike ectodomain to confirm 5 this mechanism. taken together these observations suggest that the cr3022 epitope should be a major target for therapeutic antibodies. to understand how cr3022 works we first investigated the interaction of cr3022 fab with isolated recombinant sars-cov-2 rbd, both alone and in the presence of ace2. surface plasmon resonance (spr) measurements (methods and figure s2 ) confirmed that cr3022 binding to rbd is strong (although weaker than the binding reported to sars-cov (ter meulen et al., 2006) ), with a slight variation according to whether cr3022 or rbd is used as the analyte (k d = 30 nm and 15 nm respectively, derived from the kinetic data in table s1 ). an independent measure using bio-layer interferometry (bli) with rbd as analyte gave a k d of 19 nm (methods and figure s2 ). these values are quite similar to those reported by tian et al. (tian et al., 2020 ) (6.6 nm), whereas weaker binding (k d ~ 115 nm) was reported recently by yuan et al. (yuan et al., 2020) . using spr to perform a competition assay revealed that the binding of ace2 to the rbd is perturbed by the presence of cr3022 ( figure s3 ). the presence of ace2 slows the binding of cr3022 to rbd and accelerates the dissociation. similarly, the release of ace2 from rbd is accelerated by the presence of cr3022. these observations are suggestive of an allosteric effect between ace2 and cr3022. a plaque reduction neutralisation test using sars-cov-2 virus and cr3022 showed an nd50 of 1:201 for a starting concentration of 2mg/ml (calculated according to grist (grist, 1966) ), superior to that of mers convalescent serum (nd50 of 1:149) used as a nibsc 6 international standard positive control (see methods and table s2 ). this corresponds to 50% neutralisation at ~70 nm (~10.5 ug/ml). this is similar to the neutralising concentration (50% neutralisation at 11 ug/ml) reported by ter meulen et al. (ter meulen et al., 2006) for sars-cov, however, as discussed below, it is in apparent disagreement with the result reported recently by yuan et al. (yuan et al., 2020) . we determined the crystal structure of the sars-cov-2 rbd-cr3022 fab complex (see methods and table s3 ) to investigate the relationship between the binding epitopes of ace2 and cr3022. crystals grew rapidly and consistently. two crystal forms grew in the same drop. the solvent content of the crystal form solved first was unusually high (ca 87%) with the ace2 binding site exposed to large continuous solvent channels within the crystal lattice ( figure s4 ). these crystals therefore offer a promising vehicle for crystallographic screening to identify potential therapeutics that could act to block virus attachment. the current analysis of this crystal form is at 4.4 å resolution and so, to avoid overfitting, refinement used a novel real-space refinement algorithm to optimise the phases (vagabond, hmg unpublished, see methods). this, together with the favourable observation to parameter ratio resulting from the exceptionally high solvent content, meant that the map was of very high quality, allowing reliable structural interpretation ( figure s5 , methods). full interpretation of the detailed interactions between cr3022 and the rbd was enabled by the second crystal form which diffracted to high resolution, 2.4 å, and the structure of which was refined to give an r-work/r-free of 0.213/0.239 and good stereochemistry (methods, table s3, figure s5 ). the high-resolution structure is shown in figure 1a . there are two complexes in the crystal asymmetric unit with residues 331-529 in one rbd, 332-445 and 448-532 in the other rbd well defined, whilst residues133-136 of the cr3022 heavy chains are disordered. the rbd has a very similar structure to that seen in the complex of sars-cov-2 rbd with ace2, rmsd for 194 ca atoms of 0.6 å 2 (pdb, 6m0j (lan et al., 2020) ), and an rmsd of 1.1 å 2 compared to the sars cov rbd (pdb, 2ajf (li et al., 2005) ). only minor conformational changes are introduced by binding to cr3022, at residues 381-390. the rbd was deglycosylated (methods) to leave a single saccharide unit at each of the n-linked glycosylation sites clearly seen at n331 and n343 ( figure s5 ). cr3022 attaches to the rbd surface orthogonal to the ace2 receptor binding site. there is no overlap between the epitopes and indeed both the fab and ace2 ectodomain can bind without clashing ( figure 1d ) (tian et al., 2020) . such independence of the ace2 binding site has been reported recently for another sars-cov-2 neutralising antibody, 47d11 . the fab complex interface buries 990 å 2 of surface area (600 and 390 å 2 by the heavy and light chains respectively, figure 2a and figure s6 ), somewhat more than the rbd-ace2 interface which covers 850 å 2 (pdb 6m0j (lan et al., 2020) ). typical of a fab complex, the interaction is mediated by the antibody cdr loops, which fit well into the rather sculpted surface of the rbd (figure 1b , c). the heavy chain cdr1, 2 and 3 make contacts to residues from α 2, β 2 and α 3 (residues 369-386), while two of the light chain cdrs (1 and 2) interact mainly with residues from the β 2-α3 loop, α 3 (380-392) and the α 5-β4 loop (427-430) (figures 1, s1, s7). a total of 16 residues from the heavy chain and 14 from the light chain cement the interaction with 26 residues from the rbd. for the heavy chain these potentially form 7 h-bonds and 3 salt bridges, the latter from d55 and e57 (cdr2) to k378 of the rbd. whilst the light chain interface comprises 6 h-bonds and a single salt bridge between e61 (cdr2) and k386 of the rbd. the binding is consolidated by a number of hydrophobic 8 interactions ( figure s7b ). of the 26 residues involved in the interaction 23 are conserved between sars-cov and sars-cov-2 ( figure 2b and figure s1 ). the cr30222 epitope is much more conserved than that of the receptor blocking anti-sars-cov antibody 80r for which only 13 of the 29 interacting residues are conserved (hwang et al., 2006) , in-line with the lack of cross reactivity observed for the latter. the reason for the conservation of the cr3022 epitope becomes clear in the context of the complete pre-fusion s structure (pdb ids: 6vsb (wrapp et al., 2020) , 6vxx, 6vyb (walls et al., 2020) ) where the epitope is inaccessible ( figure 3 ). when the rbd is in the 'down' configuration the cr3022 epitope is packed tightly against another rbd of the trimer and the n-terminal domain (ntd) of the neighbouring protomer. in the structure of the pre-fusion form of trimeric spike the majority of rbds are 'down', although presumably stochastically one may be 'up' (walls et al., 2020; wrapp et al., 2020) . the structure of a sars-cov complex with ace2 ectodomain shows that this 'up' configuration is competent to bind receptor, and that there are a family of 'up' orientations with significantly different hinge angles (song et al., 2018) . however, the cr3022 epitope remains largely inaccessible even in the 'up' configuration. modelling the rotation of the rbd required to enable fab interaction in the context of the spike trimer, showed a rotation corresponding to a > 60° further declination from the central vertical axis was required, beyond that observed previously (walls et al., 2020; wrapp et al., 2020) (figure 3i ), although this might be partly mitigated by more complex movements of the rbd and if more than one rbd is in the 'up' configuration this requirement would be relaxed somewhat. since locking the up state by receptor blocking antibodies is thought to destabilise the pre-fusion state (walls et al., 2019) binding of cr3022 presumably introduces further destabilisation, leading to a premature conversion to the post-fusion state, inactivating the virus. cr3022 and ace2 blocking antibodies can bind 9 independently but both induce an 'up' conformation, presumably explaining the observed synergy between binding at the two sites (ter meulen et al., 2006) . to test if cr3022 binding destabilises the prefusion state of spike, the ectodomain construct described previously (wrapp et al., 2020) was used to produce glycosylated protein in hek cells (methods). cryo-em screening showed that the protein was in the trimeric prefusion conformation. spike was then mixed with an excess of cr3022 fab and incubated at room temperature, with aliquots being taken at 50 minutes and 3 hours. aliquots were immediately applied to cryo-em grids and frozen (methods). for the 50 minutes incubation, collection of a substantial amount of data allowed unbiased particle picking and 2d classification which revealed two major structural classes with a similar number in each, (i) the prefusion conformation, and (ii) a radically different conformation (methods , table s4 and figure s8 ). detailed analysis of the prefusion conformation led to a structure at a nominal resolution of 3.4 å (fsc = 0.143), based on a broad distribution of orientations, that revealed the same predominant rbd pattern (one 'up' and two 'down') previously seen (wrapp et al., 2020) with no evidence of cr3022 binding (figure 4a , figure s9 ). analysis of the other major particle class revealed strong preferential orientation of the particles on the grid ( figure s10a ). despite this a reconstruction with a nominal resolution of 3.9 å within the plane of the grid, and perhaps 7 å resolution in the perpendicular direction ( figure s10b ), could be produced which allowed the unambiguous fitting of the cr3022-rbd complex (figure 4b ). note that in addition there is less well defined density attached to the rbd, in a suitable position to correspond to the spike n-terminal domain (wrapp et al., 2020) . these structures are no longer trimeric, rather two complexes associate to form an approximately symmetric dimer (however, application of this symmetry in the reconstruction process did not improve 1 0 the resolution). the interactions responsible for dimerisation involve the ace2 binding site on the rbd and the elbow of the fab, however the interaction does not occur in our lowresolution crystal form and is therefore probably extremely weak and not biologically significant. since conversion to the post-fusion conformation leads to dissociation of s1 (which includes the n-terminal domain and rbd) these results confirm that cr3022 destabilises the prefusion spike conformation. further evidence of this is provided by analysis of data collected after 3 h incubation. by this point there were no intact trimers remaining and a heterogeneous range of oligomeric assemblies had appeared, which we were not able to interpret in detail but which are consistent with the lateral assembly of fab/rbd complexes ( figure s11 ). note that the relatively slow kinetics will not be representative of events in vivo, where the conversion might be accelerated by the elevated temperature and the absence of the mutations which were added to this construct to stabilise the prefusion state (kirchdoerfer et al., 2018; pallesen et al., 2017; wrapp et al., 2020) . until now the only documented mechanism of neutralisation of coronaviruses has been through blocking receptor attachment. in the case of sars-cov this is achieved by presentation of the rbd of the spike in an 'up' conformation. although not yet confirmed for sars-cov-2 it is very likely that a similar mechanism can apply. here we define a second class of neutralisers, that bind a highly conserved epitope ( figure s1 ) and can therefore act against both sars-cov and sars-cov-2 (cr3022 was first identified as a neutralising antibody against sars-cov (ter meulen et al., 2006) ). we find that binding of cr3022 to the isolated rbd is tight (~20 nm) and the crystal structure of the complex reveals the atomic detail of the interaction. despite the spatial separation of the cr3022 and ace2 epitopes we find an allosteric effect between the two binding events. the role of the 1 1 cr3022 epitope in stabilising the prefusion spike trimer explains why it has, to date, proved impossible to generate mutations that escape binding of the antibody (ter meulen et al., 2006) . whilst in our assay cr3022 neutralises sars-cov-2, a recent paper (yuan et al., 2020) reported an alternative assay that did not detect neutralisation. the difference is likely due to their removal of the antibody/virus mix after adsorption to the indicator cells, before incubating to allow cytopathic effect (cpe) to develop. this would be in-line with the distinction previously seen between neutralisation tests for influenza virus by antibodies which bind the stem of hemagglutinin and therefore do not block receptor binding (thomson et al., 2012) . these antibodies did not appear to be neutralising when tested with the standard who neutralisation assay, in which a similar protocol is used to that adopted by yuan et al, in which the inoculum of virus/antibody is washed out before development of cpe. neutralisation was observed, however, when the antibodies were left in the assay during incubation to produce cpe. by analogy we would expect antibodies to the rbd that block attachment to ace2 to behave in a similar way to antibodies against the globular head of ha, whilst antibodies such as cr3022, that neutralise by an alternative mechanism to blocking receptor attachment, may need to be present throughout the incubation period with the indicator cells to reveal neutralisation. this agrees with our observation that, in the absence of ace2, the cr3022 fab destroys the prefusion-stabilised trimer (t 1/2 ~1h at room temperature as measured by cryo-em). with monoclonal antibodies now recognised as potential antivirals (lu et al., 2020b; salazar et al., 2017) our results suggest that cr3022 may be of immediate utility, since the mechanism of neutralisation will be unusually resistant to virus escape. in contrast antibodies which compete with ace2 (whose epitope on sars-cov-2 is reported to have already 1 2 shown mutation at residue 495 (gisaid: accession id: epi_isl_429783 wienecke-baldacchino et al., 2020 (shu and mccauley, 2017) ), are likely to be susceptible to escape. furthermore, with knowledge of the detailed structure of the epitope presented here a higher affinity version of cr3022 might be engineered. alternatively, since the same mechanism of neutralisation is likely to be used by other antibodies, a more potent monoclonal antibody targeting the same epitope might be found (for instance by screening for competition with cr3022). additionally, since this epitope is sterically and functionally independent of the well-established receptor-blocking neutralising antibody epitope there is considerable scope for therapeutic synergy between antibodies targeting the two epitopes (indeed this type of to further validate the spr results the k d of fab cr3022 for rbd was also measured by bio-layer interferometry. kinetic assays were performed on an octet red 96e (fortebio) at 30 ℃ with a shake speed of 1000 rpm. fab cr3022 was immobilized onto amine reactive 2nd generation (ar2g) biosensors (fortebio) and serially diluted rbd (80，40，20，10 and 5 nm) was used as analyte. pbs (ph 7.4) was used as the assay buffer. recorded data were analysed using the data analysis software ht v11.1 (fortebio), with a global 1:1 fitting model. neutralising virus titres were measured in serum samples that had been heat-inactivated at 56 °c for 30 minutes. sars-cov-2 (strain victoria/1/2020 at cell passage 3 (caly et al., 2020) ) was diluted to a concentration of 1.4e+03 pfu/ml (70 pfu/50 µl) and mixed 50:50 in 1% fcs/mem containing 25 mm hepes buffer with doubling serum dilutions from 1:10 to 1:320 in a 96-well v-bottomed plate. the plate was incubated at 37 °c in a humidified box for 1 hour to allow the antibody in the serum samples to neutralise the virus. cr3022 (ph7.2) at a starting concentration of 2 mg/ml was diluted 1 in 10. the dilutions were then made 2-fold up to 320. the neutralised virus was transferred into the wells of a twice dpbs-washed plaque assay 24-well plate that had been seeded with vero/hslam the previous day at 1.5e+05 cells per well in 10% fcs/mem. neutralised virus was allowed to adsorb at 37 °c for a further hour, and overlaid with plaque assay overlay media (1x mem/1.5% cmc/4% fcs final). after 5 days incubation at 37 °c in a humified box, the plates were fixed, stained and plaques counted. dilutions and controls were performed in duplicate. median neutralising titres (nd50) were determined using the spearman-karber formula (kärber, 1931) relative to virus only control wells. purified and deglycosylated rbd and cr3022 fab were concentrated to 8.3 mg/ml and 11 mg/ml respectively, and then mixed in an approximate molar ratio of 1:1. crystallization screen experiments were carried out using the nanolitre sitting-drop vapour diffusion method in 96-well plates as previously described (walter et al., 2003 (walter et al., , 2005 transmission). data were indexed, integrated and scaled with the automated data processing program xia2-dials (winter, 2010; winter et al., 2018) . the data set of 720° was collected from a single frozen crystal to 4.4 å resolution with 52-fold redundancy. the crystal belongs to space group p4 1 2 1 2 with unit cell dimensions a = b = 150.5 å and c = 241.6 å. the structure was determined by molecular replacement with phaser (mccoy et al., 2007) using search models of human germline antibody fabs 5-51/o12 (pdb id, 4kmt (teplyakov et al., 2014) ) heavy chain and ighv3-23/igk4-1 (pdb id, 5i1d (teplyakov et al., 2016) ) light chain, and rbd of sars-cov-2 rbd/ace2 complex (pdb id, 6m0j (lan et al., 2020) ). there is one rbd/cr3022 complex in the crystal asymmetric unit, resulting in a crystal solvent content of ~87%. during optimization of the crystallization conditions, a second crystal form was found to grow in the same condition with similar morphology. a data set of 720° rotation with data extending to 2.4 å was collected on beamline i03 of diamond from one of these crystals (exposure time 0.004 s per 0.1° frame, beam size 80×20 μ m and 100% beam transmission). the crystal also belongs to space group p4 1 2 1 2 but with significantly different unit cell dimensions (a = b = 163.1 å and c = 189.1 å). there were two rbd/cr3022 complexes in the asymmetric unit and a solvent content of ~74%. the initial structure was determined using the lower resolution data from the first crystal form. data were excluded at a resolution below 35 å as these fell under the beamstop shadow. one cycle of refmac5 (murshudov et al., 2011) was used to refine atomic coordinates after manual correction in coot (emsley and cowtan, 2004) figure s5 ). the final refined structure had an r work of 0.331 (r free , 0.315) for all data to 4.36 å resolution. this structure was later used to determine the structure of the second crystal form, which has been refined with phenix (liebschner et al., 2019) to r work = 0.213 and r free = 0.239 for all data to 2.42 å resolution. this refined model revealed the presence of one extra residue at each heavy chain n-terminus and 3 extra residues at the n-terminus of one rbd from the signal peptide. there is well ordered density for a single glycan at each of the glycosylation sites at n331 and n343 in one rbd, and only one at n343 in the second rbd. data collection and structure refinement statistics are given in table s3 . structural comparisons used shp (stuart et al., 1979) , residues forming the rbd/fab interface were identified with pisa (krissinel and henrick, 2007) , figures were prepared with pymol (the pymol molecular graphics system, version 1.2r3pre, schrödinger, llc). purified spike protein was buffer exchanged into 2 mm tris ph 8.0, 200 mm nacl, 0.02 % nan3 buffer using a desalting column (zeba, thermo fisher μ m and at a nominal magnification of x105,000, corresponding to a calibrated pixel size of 0.83 å/pixel, see table s4 . cryo-em data processing 2 2 for both the 50 minute and 3 h incubation datasets, motion correction and alignment of 2x binned super-resolution movies was performed using relion3.1. ctf-estimation with gctf (v1.06) (zhang, 2016) and non-template-driven particle picking was then performed within cryosparc v2.14.1-live followed by multiple rounds of 2d classification (punjani et al., 2017) . for the 50 minutes dataset. 2d class averages for structure-a and structure-b were then used separately for template-driven classification before further rounds of 2d and 3d classification with c1 symmetry. both structures were then sharpened in cryosparc. data processing and refinement statistics are given in table s4 . an initial model for the spike (structure-a) was generated using pdb id, 6vyb (walls et al., 2020) and rigid body fitted into the final map using coot (emsley and cowtan, 2004) . the model was further refined in real space with phenix (liebschner et al., 2019) which resulted in a correlation coefficient of 0.84. two copies of rbd-cr3022 were fitted into structure-b in the same manner. because of the strongly anisotropic resolution the overall correlation coefficient vs the model was lower (0.47). for the 3 h incubation dataset, particles were extracted with a larger box size (686 pixels as compared to 540 pixels), and, following multiple rounds of 2d classification, 2d class averages from 'blob-picked' particles showing signs of complete 'flower-like' structures were selected for ab initio reconstruction. for the 3 h data no detailed fitting was attempted. t/e (red, negative; blue, positive). real estimates of mortality following covid-19 infection isolation and rapid sharing of the 2019 novel coronavirus (sar-cov-2) from the first patient diagnosed with covid-19 in australia coot: model-building tools for molecular graphics diagnostic methods in clinical virology. x structural basis of neutralization by a human anti-severe acute respiratory syndrome spike protein antibody, 80r beitrag zur kollektiven behandlung pharmakologischer reihenversuche stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis inference of macromolecular assemblies from crystalline state structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 structural biology: structure of sars coronavirus spike receptor-binding domain complexed with receptor macromolecular structure determination using x-rays, neutrons and electrons: recent developments in phenix genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding development of therapeutic antibodies for the treatment of diseases phaser crystallographic software a sars-like cluster of circulating bat coronaviruses shows potential for human emergence human monoclonal antibody combination against sars coronavirus: synergy and coverage of escape mutants refmac5 for the refinement of macromolecular crystal structures a pipeline for the production of antibody fragments for structural studies using transient expression in hek 293t cells the production of glycoproteins by transient expression in mammalian cells hek 293 cells: an alternative to e. coli for the production of secreted and intracellular mammalian proteins immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen immunopathogenesis of coronavirus infections: implications for sars cryosparc: algorithms for rapid unsupervised cryo-em structure determination dynamical asymmetry exposes 2019-ncov prefusion spike antibody therapies for the prevention and treatment of viral infections gisaid: global initiative on sharing all influenza datafrom vision to reality cryo-em structure of the sars coronavirus spike glycoprotein in complex with its host cell receptor ace2 crystal structure of cat muscle pyruvate kinase at a resolution of 2.6 å potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association antibody modeling assessment ii structural diversity in a human antibody germline library pandemic h1n1 influenza infection and vaccination in humans induces cross-protective antibodies that potent binding of 2019 novel coronavirus spike protein by a sars coronavirusspecific human monoclonal antibody immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus unexpected receptor functional mimicry elucidates activation of coronavirus fusion function, and antigenicity of the sars-cov-2 spike glycoprotein a procedure for setting up high-throughput nanolitre crystallization experiments. i. protocol design and validation a procedure for setting up highthroughput nanolitre crystallization experiments. crystallization workflow for initial screening, automated storage, imaging and optimization molecular mechanism for antibody-dependent enhancement of coronavirus entry a human monoclonal antibody blocking sars-cov-2 infection structural and functional basis of sars-cov-2 entry by using human ace2 xia2: an expert system for macromolecular crystallography data reduction dials: implementation and evaluation of a new integration package cryo-em structure of the 2019-ncov spike in the prefusion conformation structural basis for the recognition of the sars-cov-2 by full-length human ace2 a highly conserved cryptic epitope in the receptor-binding domains of sars-cov-2 and sars-cov gctf: real-time ctf determination and correction potent cross-reactive neutralization of sars coronavirus isolates by human monoclonal antibodies rbds in the down conformation (generated by superposing our rbd structure on the prefusion trimer of ref (wrapp et al., 2020) ). the viral membrane would be at the bottom of the picture. all of s1 and s2 are shown in yellow, apart from the rbd, which is shown in grey, with the cr3022 epitope coloured green. a, a cut-way of the trimer showing, in red, the dipeptide (residues 986-987) which has been mutated to pp to confer stability on the pre-fusion state. note the proximity to the cr3022 epitope. c, showing a top view of the molecule (also used for panels d-f). one of the rbds has been drawn in light grey in the down configuration 2 8 and hinged up in dark grey, using the motion about the hinge axis observed for several coronavirus spikes, but extending the motion sufficiently to allow cr3022 to bind. the pp motif is shown in red and the glycosylated residue n343 in magenta. panels d-f show the trimer viewed from above d -all rbds down, e -one rbd up f -one rbd rotated (as in c)to allow access to cr3022. panels g-i are equivalent structures to d-f, but are viewed from the side. in e bound ace2 is shown and in f cr3022. key: cord-318018-ybdkp398 authors: bruni, margherita; cecatiello, valentina; diaz-basabe, angelica; lattanzi, georgia; mileti, erika; monzani, silvia; pirovano, laura; rizzelli, francesca; visintin, clara; bonizzi, giuseppina; giani, marco; lavitrano, marialuisa; faravelli, silvia; forneris, federico; caprioli, flavio; pelicci, pier giuseppe; natoli, gioacchino; pasqualato, sebastiano; mapelli, marina; facciotti, federica title: persistence of anti-sars-cov-2 antibodies in non-hospitalized covid-19 convalescent health care workers date: 2020-10-01 journal: j clin med doi: 10.3390/jcm9103188 sha: doc_id: 318018 cord_uid: ybdkp398 although antibody response to sars-cov-2 can be detected early during the infection, several outstanding questions remain to be addressed regarding the magnitude and persistence of antibody titer against different viral proteins and their correlation with the strength of the immune response. an elisa assay has been developed by expressing and purifying the recombinant sars-cov-2 spike receptor binding domain (rbd), soluble ectodomain (spike), and full length nucleocapsid protein (n). sera from healthcare workers affected by non-severe covid-19 were longitudinally collected over four weeks, and compared to sera from patients hospitalized in intensive care units (icu) and sars-cov-2-negative subjects for the presence of igm, igg and iga antibodies as well as soluble pro-inflammatory mediators in the sera. non-hospitalized subjects showed lower antibody titers and blood pro-inflammatory cytokine profiles as compared to patients in intensive care units (icu), irrespective of the antibodies tested. noteworthy, in non-severe covid-19 infections, antibody titers against rbd and spike, but not against the n protein, as well as pro-inflammatory cytokines decreased within a month after viral clearance. thus, rapid decline in antibody titers and in pro-inflammatory cytokines may be a common feature of non-severe sars-cov-2 infection, suggesting that antibody-mediated protection against re-infection with sars-cov-2 is of short duration. these results suggest caution in using serological testing to estimate the prevalence of sars-cov-2 infection in the general population. the coronavirus disease-19 (covid-19) is a respiratory illness caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), a novel beta-coronavirus firstly described in wuhan city, china, on december 2019 [1] . sars-cov-2 spreading has been declared pandemic in mid-march 2020 by who [2] . at present, the virus has infected more than 33 million people worldwide with an associated case mortality rate of 1% to 15%, depending on the country [3] . covid-19 is associated with a broad range of mild-to-severe symptoms, potentially leading to hospitalization in intensive care units (icu) for the most severe cases. the respiratory tract is initially involved with possible development of severe interstitial pneumonia [4, 5] , albeit the gastrointestinal tract can also significantly participate in disease pathogenesis as a consequence of the expression of the ace2 receptor, that mediates sars-cov-2 viral entry [6] , on both alveolar and enteric epithelial cells [7] . infected subjects manifest a complex clinical pattern appearing as early as two days post exposure and lasting several weeks [1] . infection with sars-cov-2 induces a prompt activation of the immune system, finalized to the clearance of infected cells [8] . innate and adaptive immune cells accumulate at the site of infection, where production of cytokines and inflammatory mediators may result in patient recovery or, in case of ineffective viral clearance, in hyperactivation of the immune system and development of severe complications, such as acute respiratory distress syndrome ards [4, 9] . overexpression of pro-inflammatory cytokines (i.e., il-1 beta, il-2, il-6, il-17, tnfα etc.) and impairment of humoral immunity have been described in patients with the most severe form of the disease [5] . antibodies against sars-cov-2 proteins are produced as a consequence of the activation of the humoral arm of the immune system. virus-specific igm antibodies are secreted as first class of immunoglobulins, followed by the more specific igg [10] . among the latter, those specific for the viral spike receptor binding domain (rbd) when expressed at higher titer manifest direct neutralizing activity towards the viral entry into cells, as they prevent effective engagement of surface ace2 receptors by the spike protein [11, 12] . the iga response against sars-cov-2 has been shown to be rapid and persistent [13, 14] and is associated with mucosal responses, including respiratory and gastrointestinal responses. serological testing is a valuable tool to monitor viral spreading throughout the population [15] . furthermore, serological assays allow the identification of past infection in individuals with viral rna levels undetectable by rt-pcr for epidemiological purposes [16] . various commercial and in-house assays that utilize distinct viral antigens and detect different antibody classes are currently available. however, sars-cov-2 serological tests available on the market do not always allow systematic simultaneous detection of a wide antibody spectrum for several antigens in a reliable manner, and this may hamper a proper population testing for clinical or epidemiological purposes [17] . conversely, serological enzyme-linked immunosorbent assays (elisa) to detect immunoglobulins raised against the viral spike soluble ectodomain (spike) and its highly immunogenic receptor binding domain (rbd), or against the nucleocapsid protein (n), provide promising results in terms of accuracy and reproducibility [11] . recently, these elisa assays have been used to show that neutralizing antibodies (nab) against different viral antigens may decline after 20-30 days post symptoms onset, and that the magnitude of nab response may be associated with disease severity in covid-19 patients [18] . in order to measure the presence and variation of antibody responses against different viral proteins, we set up and validated an in-house direct elisa assay based on three distinct sars-cov-2 viral antigens, i.e., eukaryotically-expressed rbd and spike and bacterially-expressed nucleocapsid protein. using this assay, we simultaneously measured igm, igg and iga anti-viral antibodies titers in the sera of covid-19 patients, as well as levels of pro-inflammatory cytokines. in addition, we longitudinally collected the sera of 16 convalescent healthcare workers who tested positive for sars-cov-2 by nasopharyngeal (nf) swabs, and were symptomatic but not hospitalized. our data show that humoral immune responses against sars-cov-2 correlated with disease severity in terms of both antibody titers, persistence over time and serum levels of pro-inflammatory cytokines. notably, 90% of covid-19 mildly symptomatic patients halved their anti-rbd igg titers after 4 weeks from viral negativization, thus confirming the short lifespan of humoral immune responses against sars-cov-2. health care workers of two different covid hospitals in milan (n = 16) with documented covid-19 infection (by nf swab), not hospitalized but with manifested covid-19 symptoms (supplementary table s2 ) were monitored for seroconversion by igm, igg and iga serum levels at two time points after viral clearance between april and june 2020. the study has been conducted in accordance with the standards of good clinical practice, with the ethical principles deriving from the helsinki declaration and the current legislation on observational studies. clearance from the ethical committee has been obtained (ieo1271). additional study populations were icu hospitalized severe covid-19 patients (n = 24) and (n = 58) covid-19 negative subjects whose sera were collected between april and june 2020. in total, 436 pre-covid subjects enrolled in ieo studies between 2009 and 2015 were used to calculate the roc curves for the assays. the exclusion criterion was, for all subjects involved in the study, the inability to provide informed consent. the inclusion criteria were, for those not hospitalized with covid-19, (i) being health care workers (medical doctors, practitioners, post-graduate students, nurses), potentially exposed to sars-cov-2 between february and june 2020, (ii) documented sars-cov-2 infection by nf swab, (iii) not being hospitalized for covid-19; for those hospitalized with covid-19: (i) documented sars-cov-2 infection by nf swab, (ii) being admitted in the icu between february and june 2020 for covid-19; for negative controls: (i) sera being collected before 2019. the recombinant spike sars-cov-2 glycoprotein receptor binding domain (rbd) and the soluble full-length trimeric ectodomain have been produced in mammalian hek293f cells as glycosylated proteins by transient transfection with pcaggs vectors generated in prof. krammer's laboratory [11] . the constructs were synthesized using the genomic sequence of the isolated virus, wuhan-hi-1 released in january 2020, and contain codons optimized for expression in mammalian cells. briefly, hek293f cells were seeded at a final concentration of 0.5 million/ml in freestyle medium (thermo fisher scientific, milano, italy), incubated at 37 • c, 5% co 2 at 120 rpm o/n in an eppendorf new brunswick s41i incubator. the day after hek293f cells were transfected using 1 µg of dna per 1 × 10 6 cells and a dna: pei max ratio of 1:5 in optimem medium. four hours post-transfection, the medium was supplemented with peptone primatone rl (merck) to a final concentration of 0.6% w/v. cells were then incubated for 6 days, checking cell viability daily if needed (a mortality higher than 30% is indicative of a toxic protein). for protein purification, the culture supernatant was transferred to conical centrifuge tubes, cleared by centrifugation at 1000×·g for 15 min and filtered with 0.22 µm stericup filters. the filtered medium addition with 1:10 volume of 570 mm nah 2 po 4 ph 8.0, 300 mm nacl and loaded on a hisprep fast flow 16/60 column (ge-healthcare) equilibrated in 57 mm nah 2 po 4 , 300 mm nacl. his-tagged protein was eluted with step gradients of 50-100-150-235 mm imidazole. peak fractions were pooled, dialyzed overnight against pbs and concentrated to 0.4 mg/ml (spike soluble) or 1.0 mg/ml (rbd) in 10 kda-mwco amicon filter units. retrieved proteins were quantified, flash frozen in liquid nitrogen in aliquots and stored at −80 • c. his-tagged sars-cov-2 full length n-protein plasmid (kind gift of david d. ho, md, columbia university, new york, ny, usa) was transformed in e.coli bl21 plyss cells. protein expression was induced with 0.5 mm iptg and carried on at 18 • c overnight. cells were harvested by centrifugation in lysis buffer (25 mm tris-hcl ph 8.0, 500 mm nacl, 1 mm dtt, 5% glycerol, 10 mm imidazole, with calbiochem protease inhibitor cocktail iii). all following steps were carried out at 4 • c or using ice-cold buffers. cells were lysed by sonication; lysate was cleared by centrifugation at 20,000× g for 40 min, then pei (ph 7.5-final concentration 0.02%) was dropwise added, under stirring, and lysate was then further cleared by centrifugation at 20,000× g for 40 min. next, 5 ml ni-nta beads per liter of culture, pre-equilibrated in lysis buffer, were added to the cleared lysate and protein binding was continued for 1 h in gentle agitation at 4 • c. beads were washed with at least 20 column volumes of 25 mm tris-hcl ph 7.4, 500 mm nacl, 1 mm dtt, 5% glycerol, 50 mm imidazole and his-tagged protein was eluted with 4 column volumes of 25 mm tris-hcl ph 7.4, 200 mm nacl, 1 mm dtt, 5% glycerol, 250 mm imidazole. the eluted fractions containing protein were diluted with heparin buffer (25 mm tris-hcl ph 7.4, 1 mm dtt) to reach final a nacl concentration of 0.1 m and were subsequently loaded onto a hi-trap heparin hp column (ge healthcare) equilibrated in 25 mm tris-hcl ph, 50 mm nacl, 1 mm dtt (buffer a). a linear gradient reaching 100% buffer b (25 mm tris-hcl ph, 1 m nacl, 1 mm dtt) in 30 column volumes was applied and fractions containing his-tagged n-protein were pooled, concentrated and loaded onto a superdex 200 16/60 size exclusion chromatography. fractions containing n-protein were pooled. a 4 l culture yielded 12.5 ml of 1.12 mg/ml pure n-protein, which was flash frozen in liquid nitrogen in aliquots and stored at stored at −80 • c. the elisa assay to detect immunoglobulins (ig) uses fragments of the sars-cov-2 spike glycoprotein (s-protein) and the nucleocapsid (n) as antigens based on the protocol published in [11, 19] . after binding of the proteins to a nunc maxisorp elisa plate, and blocking aspecific bindings with pbs-bsa 3%, patients' sera to be analyzed were applied to the plate to allow antibody binding at a final dilution of 1:200, revealed with secondary anti-human-igg (bd, clone g18-145), igm (merck, polyclonal code a6907), iga (biolegend, poly24110) antibody conjugated to hrp. samples are read on a glomax reader at 450 nm. this elisa test is not intended for commercial use and is currently under evaluation at the italy's ministry of health (aut.min.rich. 15.05.2020) for emergency use approval. the assay has been validated with a cohort of n = 56 covid-19 subjects (severe, moderate and mild disease) and n = 463 (subjects collected in pre-covid era (between 2012 and 2015)). roc curves have been implemented to determine the sensitivity and specificity of the assay (supplementary figure s1 ). quantification of soluble biomarkers was performed in sera of patients collected immediately after virus clearance (2 consecutive negative nf swabs) and one month post virus clearance using a luminex immunoassay (human cytokine/chemokine/gf procartaplex 45plex, thermo fisher) with map technology according to manufacturer's protocol. samples were acquired on a luminex 200sd and analyzed with xponent software 4.2. the sera of healthy subjects (n = 20) collected between april and june 2020 as well as icu covid-19 patients (n = 24) were used as control groups. the categorical variables were described as absolute frequency and percentage. the continuous variables with normal distribution were described as median ± standard deviation (sd), whereas the continuous variables without normal distribution were given as median and range. normality of continuous variables was checked with d'agostino-pearson omnibus normality test. the mann-whitney test or student's t-test for continuous variables, and the chi-square or fisher's exact tests for categorical variables, were used to associate clinical variables with the result of sars-cov-2 serological test (positive or negative). the p values lower than 0.05, two-tailed, will be considered statistically significant. graphpad prism software was used for all statistical analyses. to evaluate the antibody response of individuals infected by sars-cov-2, elisa assays were developed in-house by producing and purifying recombinant rbd, spike and nucleocapsid proteins of the sars-cov-2 virus following the protocols published in [19] ( figure 1a) . the performances of these elisa assays were assessed for the different viral antigens and classes of antibodies by determining roc curves using (i) a cohort of 56 sera from covid-19 patients collected between april and june 2020 and tested positive for nasopharyngeal swabs, and (ii) 436 pre-covid-19 sera, collected between 2012 and 2015 (supplementary table s1 and figure s1 ). anti-sars-cov-2 igg showed the highest specificity and sensitivity, irrespective of the antigen used (supplementary figure s1a,b) . anti-rbd igg showed a specificity and sensitivity of 97% and 95%, respectively, while the assay performed with the spike ectodomain reached values of 98.5% and 77% and the one with the n protein values of 91% and 95% (supplementary table s1 and figure s1 ). these performances are in line with those published for both in-house and commercial assays approved for emergency use by the fda [20, 21] . the performance of iga detection was high for the rbd assay (91.5% specificity and 95% sensitivity), while it was slightly lower for the n protein (85% and 69%) and for the spike (73% and 71%). the performance of the igm assay was comparatively lower for all the viral proteins tested (supplementary figure s1a,b) . the validated elisa assays were then used to systematically test the antibody titers of different classes of sars-cov-2 specific antibodies in sera from the following groups of patients: (i) 24 severe covid-19 patients admitted to icus; (ii) 16 health care workers from two hospitals in milan, exposed to the virus between february and march 2020 and confirmed positive to sars-cov-2 rna by rt-qpcr on nasopharyngeal swabs. fifty-eight sars-cov-2-negative subjects collected between april and june 2020 were used as negative controls (supplementary table s1 ). sera of the 16 health care workers were collected in the convalescence phase of the disease after two consecutively negative nasopharyngeal swab tests. time between the first detection of the virus and the first negative swab ranged from 14 to 35 days from onset of symptoms to disappearance of viral rna (supplementary table s2 ). these subjects all manifested clinical symptoms strongly related to sars-cov-2 infection, including fever, ageusia, anosmia, fatigue, myalgia, diarrhea, coryza and cough [5] . two of them manifested a more severe disease course with episodes of dyspnea. none of the patients required hospitalization and they all recovered from the disease (supplementary table s2 ). non-hospitalized covid-19 subjects manifested a lower antibody titer as compared to severe icu patients for all the tested antibody classes and viral antigens ( figure 1b-d) . this finding is in accordance to what published for asymptomatic [22] and paucisymptomatic [14] patients whose antibody titers were detected using commercial elisa or chemiluminescence assays against either the spike or the n-protein. when comparing the presence of the different classes of antibodies, all the covid-19 positive subjects resulted positive for the presence of igg antibodies against all the viral antigens tested ( figure 1e ). interestingly, a few of them were igm negative or with an antibody concentration close to the detection limit of the spike and rbd assay, as compared to the n protein. the observation that all of them instead showed n-specific igm antibodies may be a genuine persistence of anti-n protein igm or the consequence of a lower specificity of the n assay, possibly reflecting the high conservation of the n proteins among beta-coronaviruses other than sars-cov-2 [23] . interestingly, 25% of the non-hospitalized covid-19 patients did not develop rbd-specific iga, and only 1 out of 4 developed n-specific iga antibodies, a percentage that was instead above 80% for the hospitalized ones ( figure 1e ). since severe covid-19 is associated with a strong release of pro-inflammatory cytokines [8] , the sera from covid-19 patients were analyzed for the presence of pro-and anti-inflammatory cytokines, chemokines and growth factors by multidimensional analysis (figure 2, supplementary figures s2 and s3 and supplementary table s3 ). icu patients, whose sera were collected in the acute phase of the disease, showed a sustained production of pro-inflammatory mediators, among which il-6, il-17a, il-12p70, il-1beta, il-4, il-5 and il-13, all associated with the "cytokine storm" observed in very severe covid-19 patients, were the most abundantly detected (figure 2a ). on the contrary, even in the early convalescent phase, those cytokines were undetectable in the sera of non-hospitalized covid-19 patients (figure 2a) . interestingly, pro-inflammatory cytokines-such as ifn-gamma, tnf, il-23, il-15, il-21 and ip-10/cxcl-10-were detected both in the sera of severe icu hospitalized and of non-hospitalized covid-19 patients ( figure 2b ). to note, chemokines involved in the recruitment in inflamed tissues of both monocytes and t cells like mcp1/ccl2, rantes/ccl5, mip1alpha/ccl3 and eotaxin/ccl11 ( figure 2c ) were present at comparable concentrations in severe icu hospitalized and in non-hospitalized patients, indicating active recruitment of immune cell populations also in milder forms of covid-19. in order to evaluate the kinetics of antibody titers in convalescent non-hospitalized covid-19 patients, serum ig levels were measured at different time points, i.e., two days (t1) and one month (t2) after the first negative nf swab ( figure 3a) . interestingly, within a month after negativization of the viral rna, rbd-and spike-specific antibody titers halved in the sera of the vast majority of convalescent covid-19 patients ( figure 3b ,c). when tested against the rbd, 14/16, 13/16 and 14/16 patients showed a decrease in the antibody title ranging from 30% to 90% in their viral-specific igm, igg and iga antibodies classes ( figure 3b,e) . similarly, 8/16, 11/16 and 13/16 patients showed a decrease of at least 50% of their spike igm, igg and iga antibody titers ( figure 3c ,e). in both cases antibodies titers were still above the od detection threshold. on the contrary, antibodies against the viral nucleocapsid protein did not show a significant decrease at the second time point of evaluation ( figure 3d,e) . interestingly, similarly to the antibody titers, the presence of proinflammatory mediators in the sera of convalescent patients also decreased over time and became almost undetectable one month after a negative pcr for viral rna, a finding that mirrors the successful control of the infection and the consequent switch off of the immune response ( figure 3f, supplementary figure s4 ). during the last months many key aspects of the immune response to sars-cov-2 have been elucidated. however, given the complexity and diversity of the clinical manifestation of covid-19 disease, several outstanding questions remain still to be addressed. here we show that humoral immune responses against sars-cov-2 correlated with disease severity in terms of both antibody titers, persistence over time and serum levels of pro-inflammatory mediators. moreover, we showed that the vast majority of covid-19 mildly symptomatic patients analyzed in the study halved their anti-rbd antibody titers after 4 weeks from viral negativization, thus confirming the short lifespan of humoral immune responses against sars-cov-2. humoral immune response against sars-cov-2 proteins leads the production of antibodies against the portions of the viral proteins [10] [11] [12] . in this sense, serological tests, based on the search of specific anti-sars-cov-2 antibodies, represent a useful tool aimed at identifying patients who contracted the infection and, consequently, comparing the clinical course and eventual complications between the general population and population at risk, such as health care workers [15] . importantly, measurable variations in the humoral response might account for a re-activation of the immune system as a consequence of viral re-exposure, both in healthcare workers and in the general population. serological monitoring of antibody levels can thus provide information on the actual circulation of the virus, which can be used by decision makers to adapt safety and restriction measures according to the real presence of the virus within the population. nonetheless, the specificity and sensitivity of the different assays greatly vary among kits taking into consideration the different techniques implemented (elisa, clia, lateral flow) and the antigens used (spike ectodomain, s1-s2 of the spike, spike rbd, nucleocapsid). thus, only highly sensitive tests can detect with high accuracy whether people, including mildly symptomatic or asymptomatic subjects, have specific anti-sars-cov-2 antibodies present in their blood. the test utilized in this study is a robust elisa assay imported from the laboratory of prof. krammer at mount sinai, that has been approved for emergency use by the fda [11, 19] . we reproduced its excellent performance in our lab, that allowed us to detect a broad range of antibody levels, spanning form those measured in the blood of severe hospitalized patients and not hospitalized mild covid-19+ individuals. the elisa assay has been validated with a cohort of more than 500 positive and negative subjects, giving rise to extremely high performance values. specificity and sensitivity of the elisa assays were high for anti-rbd igg and iga (92-97%) and slightly lower for igm and the spike and n proteins (70-85%). these performances are in line with those published for both in-house and commercial assays [20, 21] . for this reason, this test is also being currently evaluated by the italy's istituto superiore di sanita' (iss) for its emergency use approval. one additional key strength of this assay as compared to other types of serological assays is its flexibility, i.e., the possibility to simultaneously assess different classes of antibodies against a broad panel of sars-cov-2 antigens within the same assay. thus, this elisa assay gave us a comprehensive understanding of the magnitude and persistence of antibody titer against different viral proteins and their correlation with the strength of the immune response, as measured by the serum levels of pro-inflammatory mediators. the presence of few false positives among the covid-negative population tested with the viral nucleocapsid protein as compared to the rbd might be a consequence of a mistakenly detection of anti-n antibodies previously raised against common cold coronaviruses which cross-react with the sars-cov-2 nucleocapsid [23] . the nucleocapsid protein is the more conserved protein among different coronaviruses. it is possible to speculate that antibodies produced against previous common cold coronaviruses (and cross-reacting with the sars-cov-2 antigens) might still be present in the sera at high levels, and therefore be detectable. as a consequence, when analyzed longitudinally, we observed that only the antibodies specific to sars-cov-2 decline while those aspecific and possibly reacting to previous coronaviruses remain detectable at the same levels over time. a similar observation was recently published by a large longitudinal study [24] . moreover, a recent paper evaluated the persistence of anti-n specific antibodies raised against four different common cold coronaviruses in a cohort of hiv+ individuals followed longitudinally for more than 10 years [25] . the study confirmed that n-specific antibodies undergo fluctuations in their detection levels as a consequence of seasonal re-infections with a kinetic of 6-12 months. interestingly, the authors reported that 2 out of 10 patients (20% of the individuals enrolled in the study) showed cross-reactive antibodies against the viral n-proteins of the four viruses, and in one of them these cross-reactive antibodies persisted over the years. the duration of circulating igg antibodies is still unclear and might depend on several factors, including the type and extent of immune response elicited upon the encounter with the virus [17] . in this study, non-hospitalized subjects showed lower antibody titers and blood pro-inflammatory cytokine profiles compared to patients in intensive care units (icu), irrespective of the antibodies tested. this finding is in accordance to what published for asymptomatic [22] and paucisymptomatic [14] patients whose antibody titers were detected using commercial elisa or chemiluminescence assays against either the spike or the n-protein. anti-rbd iga antibodies manifested a similar kinetic compared to that of igg. iga response against sars-cov-2 has been reported to be rapid and persistent [18, 19] and possibly associated with mucosal immune response in the gut and lungs. notably, iga production has been associated with disease severity, suggesting that iga production might occur locally at the mucosal sites, possibly correlating with the viral load, the duration of the viral exposure and the virus entry route [13, 26] . consistently, a recent communication [14] confirmed that the highest levels of igg and iga antibodies against the spike s1 domain, encompassing the n-terminal half of the protein with the rbd, were associated with severe disease [13, 14] . severe hospitalized covid-19 patients overexpressed pro-inflammatory cytokines (i.e., il-1 beta, il-2, il-6, il-17, tnfα). in one of the very first reports of the clinical course of covid-19 patients, as early as march 2020, serum increase in interleukin (il)-2, il-7, gmcsf, ip-10, mcp 1, mip1-α, and tnf-α was associated to disease severity [5] . elevated il-6 levels were detected in hospitalized patients and have been associated with icu admission, respiratory failure, and poor prognosis in several studies [5, 27, 28] . presently, conflicting results regarding il-1b and il-4 have been reported [29] [30] [31] . the elevation of pro-inflammatory cytokines, albeit being widely described in covid-19 patients, does not seem presently to have prognostic value, because they do not always differentiate moderate cases from severe cases [32] . levels of il-6 at first assessment might predict respiratory failure [33] , other publications with longitudinal analyses demonstrated that il-6 increases fairly late during the disease's course, consequently compromising its prognostic value at earlier stages [34] . moreover, serum concentrations of kl-6, a molecule elevated in serum of patients with interstitial lung diseases (ilds), such as idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, was recently proposed to be capable of differentiating between severe and mild covid-19 patients, being mainly produced by damaged or regenerating alveolar type ii pneumocytes [35, 36] . conversely, ip-10, mcp-3, and il-1ra were capable of differentiating between severe and mild covid-19 patients [32] . interestingly, mip 1 alpha, il8 and eotaxin, similarly to the results published by long et al. [22] , were expressed to a greater extent by healthy subjects compared to covid-19 patients. human mip 1 alpha and eotaxin were reported to be potent inhibitors of m-tropic hiv-1 infection, and were therefore considered as potential hiv-1 inhibitors [37] . a similar protective mechanism of action might be envisaged in sars-cov-2 infection. we also observed that during non-severe covid-19 infections, pro-inflammatory cytokines are produced and correlate with the severity of the disease. similarly to anti-sars-cov-2 antibodies, pro-inflammatory mediators also decreased within a month after viral clearance, as expected upon the resolution of the disease. overall, we suggest that the decline in antibody titer and pro-inflammatory cytokines is a common characteristic of sars-cov-2 infection. this study therefore has important implications for the use of serological testing for the monitoring of infection outbreaks against re-infection with sars-cov-2. our results indicate that the detection of antibodies with serological assays for epidemiological and monitoring purposes in non-hospitalized seroconverted covid-19+ subjects, who most likely represent the majority of people who encountered the virus, is only highly reliable within a limited window of time after viral clearance. supplementary materials: the following are available online at http://www.mdpi.com/2077-0383/9/10/3188/s1, figure s1 : roc curves, figure s2 : cytokine levels in sera of covid-19 patients, figure s3 : sera growth factors concentration, figure s4 : not significant longitudinal variation of serum cytokines and chemokines in non-hospitalized covid-19 patients table s1 : patients' clinical characteristics table s2: covid-19 non-hospitalized patients clinical symptoms table s3 . luminex analytes. funding: this research was funded by a generous contribution from giuseppe caprotti and the fondazione guido venosta and partially supported by the italian ministry of health with ricerca corrente and 5 × 1000 funds; we thank the enthusiastic support of francesco niutta and nicolo' fontana-rava. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. a new coronavirus associated with human respiratory disease in china johns hopkins coronavirus resource center clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in clinical features of patients infected with 2019 novel coronavirus in angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target evidence for gastrointestinal infection of sars-cov-2 the trinity of covid-19: immunity, inflammation and intervention risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease antibody responses to sars-cov-2 in patients with covid-19 a serological assay to detect 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(accessed on covid-19 in vitro diagnostic devices and test methods database clinical and immunological assessment of asymptomatic sars-cov-2 infections crystal structure of sars-cov-2 nucleocapsid protein rna binding domain reveals potential unique drug targeting sites humoral immune response to sars-cov-2 in iceland seasonal coronavirus protective immunity is short-lasting distinct early iga profile may determine severity of covid-19 symptoms: an immunological case series detectable serum sars-cov-2 viral load (rnaaemia) is closely correlated with drastically elevated interleukin 6 (il-6) level in critically ill covid-19 patients the role of interleukin-6 in monitoring severe case of coronavirus disease 2019 virologic and clinical characteristics for prognosis of severe covid-19: a retrospective observational study in wuhan correlation analysis between disease severity and inflammation-related parameters in patients with covid-19 pneumonia immune cell profiling of covid-19 patients in the recovery stage by single-cell sequencing plasma ip-10 and mcp-3 levels are highly associated with disease severity and predict the progression of covid-19 elevated levels of interleukin-6 and crp predict the need for mechanical ventilation in covid-19 clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study serum kl-6 concentrations as a novel biomarker of severe covid-19 peripheral lymphocyte subset monitoring in covid19 patients: a prospective italian real-life case series identification of rantes, mip-1α, and mip-1β as the major hiv-suppressive factors produced by cd8+t cells the authors declare no conflict of interest. key: cord-263481-w5ytp1q7 authors: lokman, syed mohammad; rasheduzzaman, m.d.; salauddin, asma; barua, rocktim; tanzina, afsana yeasmin; rumi, meheadi hasan; hossain, m.d. imran; siddiki, a.m.a.m. zonaed; mannan, adnan; hasan, m.d. mahbub title: exploring the genomic and proteomic variations of sars-cov-2 spike glycoprotein: a computational biology approach date: 2020-06-02 journal: infect genet evol doi: 10.1016/j.meegid.2020.104389 sha: doc_id: 263481 cord_uid: w5ytp1q7 the newly identified sars-cov-2 has now been reported from around 185 countries with more than a million confirmed human cases including more than 120,000 deaths. the genomes of sars-cov-2 strains isolated from different parts of the world are now available and the unique features of constituent genes and proteins need to be explored to understand the biology of the virus. spike glycoprotein is one of the major targets to be explored because of its role during the entry of coronaviruses into host cells. we analyzed 320 whole-genome sequences and 320 spike protein sequences of sars-cov-2 using multiple sequence alignment. in this study, 483 unique variations have been identified among the genomes of sars-cov-2 including 25 nonsynonymous mutations and one deletion in the spike (s) protein. among the 26 variations detected, 12 variations were located at the n-terminal domain and 6 variations at the receptor-binding domain (rbd) which might alter the interaction of s protein with the host receptor angiotensin converting enzyme-2 (ace2). besides, 22 amino acid insertions were identified in the spike protein of sars-cov-2 in comparison with that of sars-cov. phylogenetic analyses of spike protein revealed that bat coronavirus have a close evolutionary relationship with circulating sars-cov-2. the genetic variation analysis data presented in this study can help a better understanding of sars-cov-2 pathogenesis. based on results reported herein, potential inhibitors against s protein can be designed by considering these variations and their impact on protein structure. wuhan, hubei province of china in december 2019. the death toll rose to more than 68,000 among 1,250,000 confirmed cases around the globe (until april 4, 2020) [1] . the virus causing covid-19 is named as severe acute respiratory syndrome coronavirus 2 (sars-cov-2). based on the phylogenetic studies, the sars-cov-2 is categorized as a member of the genus betacoronavirus, the same lineage that includes sars coronavirus (sars-cov) [2] that caused sars (severe acute respiratory syndrome) in china during 2002 [3] . recent studies showed that sars-cov-2 has a close relationship with bat sars-like covs [4, 5] [7] ]. interestingly, s glycoprotein is characterized as the critical determinant for viral entry into host cells which consists of two functional subunits namely s1 and s2. the s1 subunit recognizes and binds to the host receptor through the receptor-binding domain (rbd) whereas s2 is responsible for fusion with the host cell membrane [ [8] , [9] , [10] ]. mers-cov uses dipeptidyl peptidase-4 (dpp4) as entry receptor [11] whereas sars-cov and sars-cov-2 utilize ace-2 (angiotensin converting enzyme-2) [12] , abundantly available in lung alveolar epithelial cells and enterocytes, suggesting s glycoprotein as a potential drug target to halt the entry of sars-with remarkable properties like glutamine-rich 42 aa long exclusive molecular signature (dsqqtvgqqdgsednqtttiqtivevqpqlemeltpvvqtie) in position 983-1024 of polyprotein 1ab (pp1ab) [16] , diversified receptor-binding domain (rbd), unique furin cleavage site (prrar↓sv) at s1/s2 boundary in s glycoprotein which could play roles in viral pathogenesis, diagnosis and treatment [17] . to date, few genomic variations of sars-cov-2 are reported [ [18] , [19] ]. there is growing evidence that spike protein, a 1273 amino acid long glycoprotein having multiple domains, possibly plays a major role in sars-cov-2 pathogenesis. viral entry to the host cell is initiated by the receptor-binding domain (rbd) of s1 head. upon receptor-binding, proteolytic cleavage occurs at s1/s2 cleavage site and two heptad repeats (hr) of s2 stalk form a six-helix bundle structure triggering the release of the fusion peptide. as it comes into close proximity to the transmembrane anchor (tm), the tm domain facilitates membrane destabilization required for fusion between virus-host membranes [ [20] , [21] ]. insights into the sequence variations of s glycoprotein among available genomes are key to understanding the biology of sars-cov-2 infection, developing antiviral treatments and vaccines. in this study, we have analyzed 320 genomic sequences of sars-cov-2 to identify mutations between the available genomes followed by the amino acid variations in the glycoprotein s to foresee their impact on the viral entry to host cell from structural biology viewpoint. analysis. the ncbi reference sequence of sars-cov-2 s glycoprotein, accession number yp_009724390 was used as the canonical sequence for the analyses of spike protein variants. variant analyses of sars-cov-2 genomes were performed in the genome detective coronavirus typing tool version 1.13 which is specially designed for this virus the dataset was then aligned with muscle [24] . entropy (h(x)) plot of nucleotide variations in sars-cov-2 genome was constructed using bioedit [25] . mega x (version 10.1.7) was used to construct the msas and the phylogenetic tree using pairwise alignment and neighborjoining methods in clustalw [26, 27] . tree structure was validated by running the analysis on 1000 bootstraps [28] replications dataset and the evolutionary distances were calculated using the poisson correction method [29] . variant sequences of sars-cov-2 were modeled in swiss-model [30] using the cryo-em spike protein structure of sars-cov-2 (pdb id 6vsb; [8] ) as a template. the overall quality of models was assessed in rampage server [31] by generating ramachandran plots (supplementary table 1 ). pymol and biovia discovery studio were used for structure visualization and superpose [32, 33] . j o u r n a l p r e -p r o o f 3. results multiple sequence alignment of the available 320 genomes of sars-cov-2 were performed and 483 variations were found throughout the 29,903 bp long sars-cov-2 genome with in total 115 variations in utr region, 130 synonymous variations that cause no amino acid alteration, 228 non-synonymous variations causing change in amino acid residue, 16 indels, and 2 variations in non-coding region (supplementary table 2 ). among the 483 variations, 40 variations (14 synonymous, 25 non-synonymous mutations and one deletion) were observed in the region of orf s that encodes s glycoprotein which is responsible for viral fusion and entry into the host cell [34] . notable that, most of the sars-cov-2 genome sequences were deposited from the usa (250) and china (50) (supplementary fig. 1 ). positional variability of the sars-cov-2 genome was calculated from the msa of 320 sars-cov-2 whole genomes as a measure of entropy value (h(x)) [35] . excluding 5′ and 3′ utr, ten hotspot of hypervariable position were identified, of which seven were located at orf1ab (1059c>t, 3037c>t, 8782c>t, 14408c>t, 17747c>t, 17858a>g, 18060c>t) and one at orf s (23403a>g), orf3a (25563g>t), and orf8 (28144t>c) respectively. the variability at position 8782 and 28144 were found to be the highest among the other hotspots ( fig. 1 ). the phylogenetic analysis of a total of 66 sequences (26 unique sars-cov-2 and 40 different coronavirus s glycoprotein sequences) was performed. the evolutionary distances showed that all the sars-cov-2 spike proteins cluster in the same node of the phylogenetic tree confirming the sequences are similar to refseq yp_009724390 (fig. 2) . bat coronaviruses has a close evolutionary relationship as different strains were found in the nearest outgroups and clades (bat coronavirus bm48-31, bat hp-beta coronavirus, bat coronavirus hku9) conferring that j o u r n a l p r e -p r o o f journal pre-proof coronavirus has vast geographical spread and bat is the most prevalent host (fig. 2) . in other clades, the clusters were speculated through different hosts which may describe the evolutionary changes of surface glycoprotein due to cross species transmission. viral hosts reported from different spots at different times is indicative of possible recombination. the s glycoprotein sequences of sars-cov-2 were retrieved from the ncbi virus variation resource repository and aligned using clustalw. the position of sars-cov-2 spike protein domains was measured by aligning with the sars-cov spike protein (fig. 3 ) [36, 37] . from the sequence identity matrix, 26 unique variants among unique sars-cov-2 spike glycoprotein sequences were identified to have 25 substitutions and a deletion ( fig. 4a and supplementary table 3 ). 215 sequences were found identical with sars-cov-2 s protein reference sequence (yp_009724390) while 64 sequences were identical with the same variation of d614g (supplementary table 4 respectively due to substitution of amino acid that differs in charge. the remaining 15 variants were mutated with the amino acids that are similar in charge (fig. 4 a) . the sars-cov-2 spike protein variants were superposed with the cryo-electron microscopic structure of sars-cov-2 spike protein [8] . fig. 3) . the s2 subunit of spike protein, especially the heptad repeat region 2, fusion peptide domain, transmembrane domain, and cytoplasmic tail, were found to be highly conserved in the sars-cov and the sars-cov-2 variants while the s1 subunit was more diverse, specifically the n-terminal domain (ntd) and receptor-binding domain (rbd). the spatial distribution of s protein sequences having different variation over time reveals that most of the variants (17 out of 240 s glycoprotein sequences) were reported from the us j o u r n a l p r e -p r o o f journal pre-proof followed by 3 out of 2 sequences (including y145 deletion) and 2 out of 50 sequences from india and china, respectively (fig. 5) . only one variant was found out of only one available sequence in the repository from sweden, australia, south korea and peru. interestingly, all sequences are unique among countries from the sequence reported except d614g, which was found in the us and peru (fig. 5) . moreover, we have also analyzed sequences from brazil, italy, nepal, pakistan, spain, taiwan and vietnam but there is no variation in the s glycoprotein sequence was found when compared to refseq yp_009724390. covid 19 is one of the most contagious pandemics the world has ever had with 1,250,000 confirmed cases to date (april 4, 2020) and the cases have increased as high as 5 times in less than a month [1] . phylogenetic analysis showed that the sars-cov-2 is a unique coronavirus presumably related to bat coronavirus (bm48-31, hp-betacoronavirus). during this study, we [38] , [39] , [40] ]. likewise, a number of studies targeting sars-cov-2 spike protein have been undertaken for the therapeutic measures [41] , but the unique structural and functional details of sars-cov-2 spike protein are still under scrutiny. we also found a variant (r408i) at receptor binding domain (rbd) that mutated from positively charged arginine residue to neutral and smaller sized isoleucine residue (fig. 4 i) . this change might alter the interaction of viral rbd with the host receptor because the r408 residue of sars-cov-2 is known to interact with the ace2 receptor for viral entry [42] . similarly, alterations of rbd (g476s, v483a, h519q, and a520s) also could affect the interaction of sars-cov-2 spike protein with other molecules j o u r n a l p r e -p r o o f which require further investigations. qia98583 and qis30615 variants were found to have an alteration of alanine to valine (a930v), and aspartic acid to tyrosine (d936y) respectively in the alpha helix of the hr1 domain. previous reports have indicated that hr1 domain plays a significant role in viral fusion and entry by forming helical bundles with hr2, and mutations including alanine substitution by valine (a1168v) in hr1 region are predominantly responsible for conferring resistance to mouse hepatitis coronaviruses against hr2 derived peptide entry inhibitors [43] . this study hypothesizes the mutation (a930v) found in that of sars-cov-2 might also have a role in the emergence of drug-resistance virus strains. also, the mutation (d1168h) found in the heptad repeat 2 (hr) sars-cov-2 could play a vital role in viral pathogenesis. moreover, we found that 20 variants including one deletion out of 26 were located within s1 especially within ntd and rbd region of glycoprotein s (fig. 4a) which region is responsible for the preliminary interaction with the host cell receptor ace2. this indicates that the ntd and rbd are very prone to mutations. however, the ntd and rbd portions harbour potential epitopes that might serve as potential peptide vaccine candidates against sars-cov-2 as reported in different studies [44] [45] [46] . the reason behind choosing the sequences from s protein domain ntd and rbd is they are situated in the outer surface of the virus that could be more accessible for the immune system (fig. 3c ). so the variations reported herein within the outer domains of s glycoprotein could help to design effective epitope-based vaccines or antivirals. the sars-cov-2 s protein contains additional furin protease cleavage site, prrars, in s1/s2 domain which is conserved among all 320 sequences as revealed during this study ( supplementary fig. 3 ). this unique signature is thought to make the sars-cov-2 more virulent than sars-cov and regarded as novel features of the viral pathogenesis [9] . according to previous reports the more the host cell protease can process the coronavirus s can accelerate viral tropism accordingly in influenza virus [[9] , [47] , [48] , [49] ]. apart from that, this could also promote viruses to escape antiviral therapies targeting transmembrane protease j o u r n a l p r e -p r o o f tmprss2 (clinicaltrials.gov, nct04321096) which is well reported protease to cleave at s1/s2 of s glycoprotein [50] . comparative analyses between sars-cov and sars-cov-2 spike glycoprotein showed 77% similarity between them where the most diverse region was coronavirus disease (covid-2019) situation reports severe acute respiratory syndrome-related coronavirus--the species and its viruses, a statement of the coronavirus study group lim, others, a novel coronavirus associated with severe acute respiratory syndrome bats are natural reservoirs of sars-like coronaviruses, science (80-. ) huang, others, a pneumonia outbreak associated with a new coronavirus of probable bat origin pei, others, a new coronavirus associated with human respiratory disease in china genome composition and divergence of the novel coronavirus (2019-ncov) originating in china cryo-em structure of the 2019-ncov spike in the prefusion conformation, science (80-. ) structure, function, and antigenicity of the sars-cov-2 spike glycoprotein structure analysis of the receptor binding of 2019-ncov fouchier, others, dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc greenough, others, angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses a. nitsche, others, sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor an exclusive 42 amino acid signature in pp1ab protein provides insights into the evolutive history of the 2019 novel human-pathogenic coronavirus (sars-cov2) the spike glycoprotein of the new coronavirus 2019-ncov contains a furin-like cleavage site absent in cov of the same clade genomic variance of the 2019-ncov coronavirus genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex interaction between heptad repeat 1 and 2 regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors muscle: multiple sequence alignment with improved accuracy and speed bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt mega x: molecular evolutionary genetics analysis across computing platforms the neighbor-joining method: a new method for reconstructing phylogenetic trees bootstrap confidence levels for phylogenetic trees evolutionary divergence and convergence in proteins swiss-model: homology modelling of protein structures and complexes structure validation by calpha geometry: phi, psi and cbeta deviation pymol: an open-source molecular graphics tool receptor recognition mechanisms of coronaviruses: a decade of structural studies a parvovirus b19 synthetic genome: sequence features and functional competence cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding long-term protection from sars coronavirus infection conferred by a single immunization with an attenuated vsv-based vaccine human monoclonal antibodies against highly conserved hr1 and hr2 domains of the sars-cov spike protein are more broadly neutralizing a truncated receptor-binding domain of mers-cov spike protein potently inhibits mers-cov infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines fusion mechanism of 2019-ncov and fusion inhibitors targeting hr1 domain in spike protein role of changes in sars-cov-2 in the interaction with the human ace2 receptor: an in silico analysis coronavirus escape from heptad repeat 2 (hr2)-derived peptide entry inhibition as a result of mutations in the hr1 domain of the spike fusion protein development of epitope-based peptide vaccine against novel coronavirus 2019 (sars-cov-2): immunoinformatics approach in silico identification of novel b cell and t cell epitopes of wuhan coronavirus (2019-ncov) for effective multi epitope-based peptide vaccine production epitope-based chimeric peptide vaccine design against s, m and e proteins of sars-cov-2 etiologic agent of global pandemic covid-19: an in silico approach host cell proteases controlling virus pathogenicity role of hemagglutinin cleavage for the pathogenicity of influenza virus host cell proteases: critical determinants of coronavirus tropism and pathogenesis coronaviruses: an overview of their replication and pathogenesis receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins laude, others, aminopeptidase n is a major receptor for the enteropathogenic coronavirus tgev positional organization of major structural protein-encoding genes in orange color (s = spike protein, e = envelope protein, m = membrane protein, n = nucleocapsid protein) and accessory protein orfs in blue colors. b. variability within 320 sars-cov-2 genomic sequences represented by entropy (h(x)) value across genomic location key: cord-299783-8ti6r0eh authors: bruni, m.; cecatiello, v.; diaz-basabe, a.; lattanzi, g.; mileti, e.; monzani, s.; pirovano, l.; rizzelli, f.; visintin, c.; bonizzi, g.; giani, m.; lavitrano, m.; faravelli, s.; forneris, f.; caprioli, f.; pelicci, p. g.; natoli, g.; pasqualato, s.; mapelli, m.; facciotti, f. title: persistence of anti-sars-cov-2 antibodies in non-hospitalized covid-19 convalescent health care workers date: 2020-08-01 journal: nan doi: 10.1101/2020.07.30.20164368 sha: doc_id: 299783 cord_uid: 8ti6r0eh background. coronavirus disease-19 (covid-19) is a respiratory illness caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), a novel beta-coronavirus. although antibody response to sars-cov-2 can be detected early during the infection, several outstanding questions remain to be addressed regarding magnitude and persistence of antibody titer against different viral proteins and their correlation with the strength of the immune response, as measured by serum levels of pro-inflammatory mediators. methods. an elisa assay has been developed by expressing and purifying the recombinant sars-cov-2 spike receptor binding domain (rbd), soluble ectodomain (spike), and full length nucleocapsid protein (n protein). sera from healthcare workers affected by non-severe covid-19 were longitudinally collected over four weeks, and compared to sera from patients hospitalized in intensive care units (icu) and sars-cov-2-negative subjects for the presence of igm, igg and iga antibodies as well as soluble pro-inflammatory mediators in the sera. results. specificity and sensitivity of the elisa assays were high for anti-rbd igg and iga (92-97%) and slightly lower for igm and the spike and n proteins (70-85%). the elisa allowed quantification of igm, igg and iga antibody responses against all the viral antigens tested and showed a correlation between magnitude of the antibody response and disease severity. non-hospitalized subjects showed lower antibody titers and blood pro-inflammatory cytokine profiles as compared to patients in intensive care units (icu), irrespective of the antibodies tested. noteworthy, in non-severe covid-19 infections, antibody titers against rbd and spike, but not against the n protein, as well as pro-inflammatory cytokines decreased within a month after viral clearance. conclusions. rapid decline in antibody titers and in pro-inflammatory cytokines may be a common feature of non-severe sars-cov-2 infection, suggesting that antibody-mediated protection against re-infection with sars-cov-2 is of short duration. these results suggest caution in use serological testing to estimate the prevalence of sars-cov-2 infection in the general population. syndrome coronavirus 2 (sars-cov-2), a novel beta-coronavirus firstly described in wuhan city, china, on december 2019 [1] . sars-cov-2 spreading has been declared pandemic in mid-march 2020 by who [2] . at present the virus has infected more than 12 million people worldwide with an associated case fatality rate of 1 to 15%, depending on the country [3] . covid-19 is associated with a broad range of mild-to-severe symptoms, potentially leading to hospitalization in intensive care units (icu) for the most severe cases. the respiratory tract is initially involved with possible development of severe interstitial pneumonia [4, 5] , albeit the gastrointestinal tract can also significantly participate in disease pathogenesis as a consequence of the expression of the ace2 receptor, that mediates sars-cov-2 viral entry [6] , on both alveolar and enteric epithelial cells [7] . infected subjects manifest a complex clinical pattern appearing as early as two days post exposure and lasting several weeks [1] . infection with sars-cov-2 induces a prompt activation of the immune system, finalized to the clearance of infected cells [8] . innate and adaptive immune cells accumulate at the site of infection, where production of cytokines and inflammatory mediators may result in patient recovery or, in case of ineffective viral clearance, in hyperactivation of the immune system and development of severe complications, such as acute respiratory distress syndrome ards [4, 9] . overexpression of pro-inflammatory cytokines (i.e. il-1 beta, il-2, il-6, il-17, tnfα etc.) and impairment of humoral immunity have been described in patients with the most severe form of disease [5] . antibodies against sars-cov-2 proteins are produced as a consequence of the activation of the humoral arm of the immune system. virus-specific igm antibodies are secreted as first class of immunoglobulins, followed by the more specific igg [10] . among the latter, those specific for the viral spike receptor binding domain (rbd) when expressed at higher titer manifest direct neutralizing activity towards the viral entry into cells, as they prevent effective engagement of surface ace2 receptors by the spike protein [11, 12] . the iga response against sars-cov-2 has been shown to be rapid and persistent [13, 14] and is associated with mucosal responses, including the respiratory and gastrointestinal ones. serological testing is a valuable tool to monitor viral spreading throughout the population [15] . furthermore, serological assays allow the identification of past infection in individuals with viral rna levels undetectable by rt-pcr for epidemiological purposes [16] . various commercial and inhouse assays that utilize distinct viral antigens and detect different antibody classes are currently available. however, sars-cov-2 serological tests available on the market do not always allow systematic simultaneous detection of a wide antibody spectrum for several antigens in a reliable manner, and this may hamper a proper population testing for clinical or epidemiological purposes [17] . conversely, serological enzyme-linked immunosorbent assay (elisa) to detect immunoglobulins raised against the viral spike soluble ectodomain (spike) and its highly immunogenic receptor binding domain (rbd), or against the nucleocapsid protein (n), are providing promising results in terms of accuracy and reproducibility [11] . recently, these elisa assays have been used to show that neutralizing antibodies (nab) against different viral antigens may decline after 20-30 days post symptoms onset, and that the magnitude of nab response may be associated with disease severity in covid-19 patients [18] . in order to measure the presence and evolution of antibody responses against different viral proteins, we set up and validated an in-house direct elisa assay based on three distinct sars-cov-2 viral antigens, i.e. eukaryotically-expressed rbd and spike and bacterially-expressed nucleocapsid protein. using this assay, we simultaneously measured igm, igg and iga anti-viral antibodies titers in the sera of covid-19 patients, as well as levels of pro-inflammatory cytokines. in addition, we longitudinally collected the sera of 16 convalescent healthcare workers who tested positive for sars-cov-2 by nasopharyngeal (nf) swabs, and were symptomatic but not hospitalized. our data show that humoral immune responses against sars-cov-2 correlated with disease severity in terms of both antibody titers, persistence over time and serum levels of pro-inflammatory cytokines. notably, 90% of covid-19 mildly symptomatic patients halved their anti-rbd igg titers after 4 weeks from viral negativization, thus confirming the short lifespan of humoral immune responses against sars-cov-2. to evaluate the antibody response of individuals infected by sars-cov-2, elisa assays were developed in-house by producing and purifying recombinant rbd, spike and nucleocapsid proteins of the sars-cov-2 virus following the protocols published in [19] ( figure 1a ). the performances of these elisa assays were assessed for the different viral antigens and classes of antibodies by determining roc curves using i) a cohort of 56 sera from covid-19 patients collected between april and june 2020 and tested positive for nasopharyngeal swabs, and ii) 436 pre-covid-19 sera, collected between 2012 and 2015 (supplementary table 1 and figure s1 ). anti-sars-cov2 igg showed the highest specificity and sensitivity irrespective of the antigen used (supplementary figures 1a,b) . anti-rbd igg showed specificity and sensitivity of 97% and 95%, respectively, while the assay performed with the spike ectodomain reached values of 98.5% and 77% and the one with the n protein values of 91% and 95% (supplementary table 1 and figure s1 ). these performances are in line with those published for both in-house and commercial assays approved for emergency use by the fda [20, 21]. performance of iga detection was high for the rbd assay (91.5% specificity and 95% sensitivity), while it was slightly lower for the n protein (85% and 69%) and for the spike (73% and 71%). the performance of the igm assay was comparatively lower for all the viral proteins tested (supplementary figures 1a,b) . the validated elisa assays were then used to systematically test the antibody titers of different classes of sars-cov-2 specific antibodies in sera from the following groups of patients: i) 24 severe covid-19 patients admitted to icus; ii) 16 health care workers from two hospitals in milan, exposed to the virus between february and march 2020 and confirmed positive to sars-cov-2 rna by rt-qpcr on nasopharyngeal swabs. 58 sars-cov-2-negative subjects collected between april and june 2020 were used as negative controls (supplementary table 1 ). sera of the 16 health care workers were collected in the convalescence phase of the disease after two consecutively negative nasopharyngeal swab tests. time between the first detection of the virus and the first negative swab ranged from 14 to 35 days from onset of symptoms to disappearance of viral rna (supplementary table 2 ). these subjects all manifested clinical symptoms strongly related to sars-cov-2 infection, including fever, ageusia, anosmia, fatigue, myalgia, diarrhea, coryza and cough [5] . two of them manifested a more severe disease course with episodes of dyspnea. none of the patients required hospitalization and they all recovered from the disease (supplementary table 2 ). non-hospitalized covid-19 subjects manifested a lower antibody titer as compared to severe icu patients for all the tested antibody classes and viral antigens ( figure 1b-d) . this finding is in accordance to what published for asymptomatic [22] and paucisymptomatic [14] patients whose antibody titers were detected using commercial elisa or chemiluminescence assays against either the spike or the n-protein. when comparing the presence of the different classes of antibodies, all the covid-19 positive subjects resulted positive for the presence of igg antibodies against all the viral antigens tested ( figure 1e ). interestingly, a few of them were igm negative or with an antibody concentration close to the detection limit of the spike and rbd assay, as compared to the n protein. the observation that all of them instead showed n-specific igm antibodies may be a genuine persistence of anti-n protein igm or the consequence of a lower specificity of the n assay, possibly reflecting the high conservation of the n proteins among beta-coronaviruses other than sars-cov2 [23] . interestingly, 25% of the non-hospitalized covid-19 patients did not develop rbd-specific iga, and only 1 out of 4 developed n-specific iga antibodies, a percentage that was instead above 80% for the hospitalized ones ( figure 1e) . notably, iga production has been associated with disease severity, suggesting that iga production might occur locally at the mucosal sites, possibly correlating with the viral load, the duration of the viral exposure and the virus entry route [13, 24] . consistently, a recent communication [14] confirmed that the highest levels of igg and iga antibodies against the spike s1 domain, encompassing the n-terminal half of the protein with the rbd, were associated with severe disease [13, 14] since severe covid-19 is associated with a strong release of pro-inflammatory cytokines [8, 25] table 3 ). icu patients, whose sera were collected in the acute phase of the disease, showed a sustained production of pro-inflammatory mediators, among which il-6, il-17a, il-12p70, il-1b, il-4, il-5 and il-13, all associated with the "cytokine storm" observed in very severe covid-19 patients, were the most abundantly detected (figure 2a ). on the contrary, even in the early convalescent phase, those cytokines were undetectable in the sera of non-hospitalized it is tempting to speculate that as a consequence of the higher conservation of the n protein compared to the spike protein across different coronavirus species [23] , antibodies produced against previous common cold coronaviruses (and cross-reacting with the sars-cov2 antigens) might still be present in the sera at high levels, and therefore be detectable at the same titers, while the antibodies specific to sars-cov-2 decline. interestingly, similarly to the antibody titers, the presence of proinflammatory mediators in the sera of convalescent patients also decreased over time and became almost undetectable one month after a negative pcr for viral rna, a finding that mirrors the successful control of the infection and the consequent switch off of the immune response ( figure 3f, supplementary figure 4) . overall, we suggest that the decline in antibody titer and pro-inflammatory cytokines is a common characteristic of sars-cov-2 infection. this study has therefore important implications for the use of serological testing for the monitoring of infection outbreaks against re-infection with sars-cov-2. our results indicate that the detection of antibodies with serological assays for epidemiological and monitoring purposes in non-hospitalized seroconverted covid19+ subjects, who most likely represent the majority of people who encountered the virus, is highly reliable only within a limited window of time after viral clearance. human subjects. infection (by nf swab), not hospitalized but with manifested covid-19 symptoms (supplementary table 2 ) were monitored for seroconversion by igm, igg and iga serum levels at two time points after viral clearance between april and june 2020. the study has been conducted in accordance with the standards of good clinical practice, with the ethical principles deriving from the helsinki declaration and the current legislation on observational studies. clearance from the ethical committee has been obtained (ieo1271). additional study populations were icu hospitalized severe covid-19 patients (n=24) and (n=58) covid-19 negative subjects whose sera were collected between april and june 2020. 436 pre-covid subjects enrolled in ieo studies between 2009 and 2015 were used to calculate the roc curves for the assays. the recombinant spike sars-cov2 glycoprotein receptor binding domain (rbd) and the soluble fulllength trimeric ectodomain have been produced in mammalian hek293f cells as glycosylated proteins by transient transfection with pcaggs vectors generated in prof. krammer's laboratory [11] . the constructs were synthesized using the genomic sequence of the isolated virus, wuhan-hi-1 released in january 2020, and contain codons optimized for expression in mammalian cells. briefly, hek293f cells were seeded at a final concentration of 0.5 million/ml in freestyle medium (thermo fisher scientific), incubated at 37 °c, 5% co2 at 120 rpm o/n in an eppendorf new brunswick s41i incubator. the day after hek293f cells were transfected using 1 µg of dna per 1x10 6 cells and a dna:pei max ratio of 1:5 in optimem medium. 4h post-transfection, the medium was supplemented with peptone primatone rl (merck) to a final concentration of 0.6% w/v. cells were then incubated for 6 days, checking cell viability daily if needed (a mortality higher than 30% is indicative of a toxic protein). the elisa assay to detect immunoglobulins (ig) uses fragment of the sars-cov2 spike glycoprotein (s-protein) and the nucleocapsid (n protein) as antigens based on the protocol published in [11, 19] . after binding of the proteins (rbd and n proteins) to a nunc maxisorp elisa plate, and blocking aspecific bindings with pbs-bsa 3%, patients' sera to be analyzed were applied to the plate to allow for emergency use approval. the assay has been validated with a cohort of n=56 covid-19 + subjects (severe, moderate and mild disease) and n=463 (subjects collected in pre-covid era (between 2012 and 2015)). roc curves have been implemented to determine sensitivity and specificity of the assay (supplementary figure 1) multiplexing analysis of sera cytokines. quantification of soluble biomarkers was performed in sera of patients collected immediately after virus clearance (2 consecutive negative nf swabs) and one month post virus clearance using a luminex immunoassay (human cytokine/chemokine/gf procartaplex 45plex, thermo fisher) with map technology according to manufacturer's protocol. samples were acquired on a luminex 200sd and analyzed with xponent software 4.2. the sera of healthy subjects (n=20) collected between april and june 2020 as well as icu covid-19+ patients (n=24) were used as control groups. the categorical variables were described as absolute frequency and percentage. the continuous variables with normal distribution were described as median ± standard deviation (sd), whereas the continuous variables without normal distribution were given as median and range. normality of continuous variables was checked with d'agostino-pearson omnibus normality test. the mann-whitney test or student's t-test for continuous variables, and the chi-square or fisher's exact tests for categorical variables, were used to associate clinical variables with the result of sars-cov-2 serological test (positive or negative). the p values lower than 0.05, two-tailed, will be considered statistically significant. prism software was used for all statistical analyses. cytokines not significantly different between icu (dark blue symbols) and non-hospitalized (blue symbols) covid+ patients (c) chemokines levels in sera of patients (icu, dark blue, not hospitalized blue symbols, healthy subjects light blue). p < 0.05 (*), p < 0.01 (**) p < 0.001 (***), p < 0.0001 (****) were regarded as statistically significant. ns, not significant (e) cumulative fold decrease between t1 and t2 antibody titers in elisa assays against the rbd (squares), the spike ectodomain (circles) and the n (triangles) sars-cov2 viral proteins. (f) longitudinal variation of serum cytokines and chemokines in non-hospitalized covid-19+ patients. statistical significance was calculated using kruskal-wallis nonparametric test for multiple comparisons. p < 0.05 (*), p < 0.01 (**) p < 0.001 (***) were regarded as statistically significant. ns, not significant. figure 2 : cytokine levels in sera of covid19+ patients. cytokines not significantly different between hospitalized (dark blue) and non-hospitalized (blue) covid+ patients. statistical significance was calculated using kruskal-wallis nonparametric test for multiple comparisons. p < 0.05 (*) were regarded as statistically significant. supplementary figure 3 : (a) growth factors present in icu hospitalized (dark blue) but not in non-hospitalized (blue) covid+ patients (b) growth factors not significantly different between icu hospitalized (dark blue) and non-hospitalized (blue) covid+ patients. statistical significance was calculated using kruskal-wallis nonparametric test for multiple comparisons. p < 0.05 (*), p < 0.01 (**) were regarded as statistically significant. a new coronavirus associated with human respiratory disease in china clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in clinical features of patients infected with 2019 novel coronavirus in wuhan angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target evidence for gastrointestinal infection of sars-cov-2 the trinity of covid-19: immunity, inflammation and intervention risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease antibody responses to sars-cov-2 in patients with covid-19 a serological assay to detect sars-cov-2 seroconversion in humans analysis of a sars-cov-2-infected individual reveals development of potent neutralizing antibodies with limited somatic mutation iga-ab response to spike glycoprotein of sars-cov-2 in patients with covid-19: a longitudinal study piano mortari e, terreri s, spectrum of innate and adaptive immune response to sars-cov-2 infection across asymptomatic, mild and severe cases-a longitudinal study meta-analysis of diagnostic performance of serological tests for sars-cov-2 antibodies up to 25 april 2020 and public health implications temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study antibody tests in detecting sars-cov-2 infection: a meta-analysis. diagnostics (basel) longitudinal evaluation and decline of antibody responses in sars-cov2 infection sars-cov-2 seroconversion in humans: a detailed protocol for a serological assay, antigen production, and test setup clinical and immunological assessment of asymptomatic sars-cov-2 infections crystal structure of sars-cov-2 nucleocapsid protein rna binding domain reveals potential unique drug targeting sites distinct early iga profile may dteremine severity of covid-19 symptoms: an immunological case series. medrxive phenotype and kinetics of sars-cov-2-specific t cells in covid-19 patients with acute respiratory distress syndrome key: cord-321166-nvphu1fm authors: thomson, emma c.; rosen, laura e.; shepherd, james g.; spreafico, roberto; da silva filipe, ana; wojcechowskyj, jason a.; davis, chris; piccoli, luca; pascall, david j.; dillen, josh; lytras, spyros; czudnochowski, nadine; shah, rajiv; meury, marcel; jesudason, natasha; de marco, anna; li, kathy; bassi, jessica; o’toole, aine; pinto, dora; colquhoun, rachel m.; culap, katja; jackson, ben; zatta, fabrizia; rambaut, andrew; jaconi, stefano; sreenu, vattipally b.; nix, jay; jarrett, ruth f.; beltramello, martina; nomikou, kyriaki; pizzuto, matteo; tong, lily; cameroni, elisabetta; johnson, natasha; wickenhagen, arthur; ceschi, alessandro; mair, daniel; ferrari, paolo; smollett, katherine; sallusto, federica; carmichael, stephen; garzoni, christian; nichols, jenna; galli, massimo; hughes, joseph; riva, agostino; ho, antonia; semple, malcolm g.; openshaw, peter j.m.; baillie, j. kenneth; rihn, suzannah j.; lycett, samantha j.; virgin, herbert w.; telenti, amalio; corti, davide; robertson, david l.; snell, gyorgy title: the circulating sars-cov-2 spike variant n439k maintains fitness while evading antibody-mediated immunity date: 2020-11-05 journal: biorxiv doi: 10.1101/2020.11.04.355842 sha: doc_id: 321166 cord_uid: nvphu1fm sars-cov-2 can mutate to evade immunity, with consequences for the efficacy of emerging vaccines and antibody therapeutics. herein we demonstrate that the immunodominant sars-cov-2 spike (s) receptor binding motif (rbm) is the most divergent region of s, and provide epidemiological, clinical, and molecular characterization of a prevalent rbm variant, n439k. we demonstrate that n439k s protein has enhanced binding affinity to the hace2 receptor, and that n439k virus has similar clinical outcomes and in vitro replication fitness as compared to wildtype. we observed that the n439k mutation resulted in immune escape from a panel of neutralizing monoclonal antibodies, including one in clinical trials, as well as from polyclonal sera from a sizeable fraction of persons recovered from infection. immune evasion mutations that maintain virulence and fitness such as n439k can emerge within sars-cov-2 s, highlighting the need for ongoing molecular surveillance to guide development and usage of vaccines and therapeutics. sars-cov-2, the cause of covid-19, emerged in late 2019 and expanded globally, resulting in over 41 million confirmed cases as of october 2020. molecular epidemiology studies across the world have generated over 135,000 viral genomic sequences and have been shared with unprecedented speed via the gisaid initiative (https://www.gisaid.org/). these data are essential for monitoring virus spread (meredith et al., 2020) and evolution. of particular interest is the evolution of the sars-cov-2 surface protein, spike (s), which is responsible for viral entry via its interaction with the human angiotensin-converting enzyme 2 (hace2) receptor on host cells. the s protein is the target of neutralizing antibodies generated by infection or vaccination (folegatti et al., 2020; jackson et al., 2020; keech et al., 2020) as well as monoclonal antibody (mab) drugs currently in clinical trials (hansen et al., 2020; jones et al., 2020; pinto et al., 2020) . a sars-cov-2 s variant, d614g, is now dominant in most places around the globe (callaway, 2020) . studies in vitro indicate that this variant may have greater infectivity while molecular epidemiology indicates that it spreads efficiently and likely maintains virulence (hu et al., 2020; korber et al., 2020; volz et al., 2020; . amino acid 614 is outside the receptor binding domain (rbd) of s, the domain targeted by 90% of neutralizing antibody activity in serum of sars-cov-2 survivors (piccoli et al., 2020) . initial studies suggest that d614g actually exhibits increased sensitivity to neutralizing antibodies, likely due to its effects on the molecular dynamics of the spike protein (hou et al., 2020; yurkovetskiy et al., 2020) . therefore, this dominant variant is unlikely to escape antibody-mediated immunity. the low numbers of novel mutations reaching high frequency in sequenced sars-cov-2 isolates may relate to the moderate intrinsic error rate of the replication machinery of sars-cov-2 (li et al., 2020c; robson et al., 2020) and to this new human coronavirus requiring no significant adaption to humans (maclean et al., 2020) . nevertheless, the increasing number of infected individuals and the large reservoir of individuals susceptible to infection increases the likelihood that novel variants that impact vaccine and therapeutic development will emerge and spread. moreover, the full impact of immune selection, which can drive variant selection, likely has not yet had a dominant influence on the pandemic, since herd immunity has not yet been attained. as population immunity increases and vaccines are deployed at scale this might change. the potential for circulating viral variants to derail promising vaccine or antibody-based prophylactics or treatments, even in the absence of selective pressure from the drug or vaccine, is demonstrated by the failure of a phase iii clinical trial of a mab targeting the respiratory syncytial virus (simoes et al., 2020) , and the need for new influenza vaccines on a yearly basis. it is therefore critical to understand whether and how sars-cov-2 may evolve to evade antibody-dependent immunity. here, we examined the immunodominant sars-cov-2 receptor binding motif (rbm), the primary target of the neutralizing ab response within the rbd (piccoli et al., 2020) and found it to be less conserved than the rbd or the entire spike protein in circulating viruses. to understand the implications of this structural plasticity for immune evasion, we defined the clinical and epidemiological impact, the molecular features, and the immune response to an rbm variant, n439k. this variant has arisen independently twice, in both cases forming lineages of more than 500 sequences. as of october 2020, it has been observed in 12 countries and is the second most commonly observed rbd variant worldwide. we find that the n439k mutation is associated with a similar clinical spectrum of disease and slightly higher viral loads in vivo compared with isolates with the wild-type n439 residue, and that it results in immune escape from polyclonal sera from a proportion of recovered individuals and a panel of neutralizing mabs. n439k provides a sentinel example of immune escape, indicating that rbm variants must be evaluated when considering vaccines and the therapeutic or prophylactic use of mabs. long term control of the pandemic will require systematic monitoring of immune escape variants and selection of strategies that address the variants circulating in targeted populations. competing pressures influence the evolution of the spike rbm. first, the rbm mediates viral entry (shang et al., 2020; walls et al., 2020; wrapp et al., 2020b) and therefore it must maintain sufficient affinity to engage the entry receptor hace2. second, it is a major target of neutralizing antibodies (robbiani et al., 2020; rogers et al., 2020; wec et al., 2020) and could be a primary location for the emergence of immune escape mutations. we set out to understand these competing pressures by evaluating the landscape of rbm sequence divergence observed in circulating sars-cov-2 variants and in other viruses of the sarbecovirus lineage. we used published x-ray structures of sars-cov and sars-cov-2 rbd:hace2 complexes (lan et al., 2020; li et al., 2005) to define the rbm residues using a 6 å distance cutoff (figures 1a-c) . we evaluated ~130,000 sars-cov-2 genomic sequences deposited in gisaid as of october 7, 2020 and observed a high number of variants occurring in the rbm (figure 1a) . to understand how the divergence of the rbm compares to the divergence of the entire rbd and the whole spike protein, we divided the spike protein into three non-overlapping regions: the rbm, the rbd outside of the rbm, and the full s protein outside of the rbd. we counted individual variants occurring at least ten times, and quantified substitutions of different amino acids at the same position as separate variants. we found that the rbm is the least conserved region of s ( figure 1b) . to understand this result further, we evaluated a published deep mutational scanning (dms) data set of the rbd and compared it to sequences of circulating viruses. the dms data defines the effect of each possible single amino acid change on both expression of the rbd and its capacity to bind hace2. for each position in the rbm, we compared the dms results for all amino acid substitutions at that position versus only substitutions that have been observed in circulating sars-cov-2 isolates ( figure 1c) . a subset of residues shows the largest loss of hace2 binding upon mutation (top ~1/3 of rbm residues in figure 1c ) and, as would be expected, few natural variants of these residues have been observed to be circulating to date. surprisingly, these conserved residues each contribute weakly to the rbd:hace2 total interaction energy (the sum of pair-wise interaction energies for all residues at the binding interface in the x-ray structure; "binding energy" in figure 1c ). for the majority of the rbm (bottom ~2/3 of rbm residues in figure 1c ), variation in circulating virus sequences confirms the tolerance to mutation predicted by the dms data. notably, several rbm residues forming the strongest interactions with the receptor, e.g. k417 and e484, are not highly conserved despite their predicted importance. these results suggest that the rbm has a degree of structural plasticity whereby it is able to accommodate mutations without disrupting hace2 binding. evolutionary analysis of sarbecoviruses provides further support for rbm plasticity li et al., 2020b; rambaut et al., 2020) . the sars-cov rbm is highly divergent from the sars-cov-2 rbm (figure s1a-b) while maintaining hace2 binding affinity. additionally, there are many sequence changes in the rbm across a panel of related coronaviruses from animal isolates (figure s1a-b, table s1 ). to determine the ability of members of the sarbecovirus lineage to bind hace2, we produced nine recombinant rbd proteins corresponding to seven animal isolates, sars-cov-2, and sars-cov and evaluated their binding to recombinant hace2 ( figure s1c ). we found that three of the rbds from animal isolates showed strong affinity for hace2: gd pangolin, which has a highly similar rbm to sars-cov-2, and gx pangolin and bat cov wiv1, which have highly divergent rbms (figure s1a-b) . this further supports the conclusion that the rbm is structurally plastic, while retaining binding with hace2 as a receptor. given this plasticity, we next considered whether an rbm variant can lead to immune evasion while retaining virulence. the two most commonly observed circulating rbd variants as of october 2020 contain mutations in the rbm (s477n and n439k). we first identified the n439k variant in march 2020, circulating in scotland from lineage b.1 on the background of d614g (da silva filipe et al., 2020) . using phylogenetic analysis, we determined this variant represented a single lineage (figure 2a ) that increased in frequency to 553 sequences by june 20, 2020 (~10% of the available scottish viral genome sequences for this time period). numbers of n439k and all other isolates decreased in scotland concurrent with control of the pandemic by initiation of stringent public health measures and this lineage has not been detected in scotland after june. however, the n439k variant has been observed in >659 sequences in a second lineage in europe, first sampled in romania on may 13, 2020, then norway on june 23, 2020 and is now circulating in 12 countries, as well as arising independently in the u.s. (figure 2a-c) . as of oct 6, 2020, all 1201 n439k variants arose from a c-to-a transversion in the third codon position, though these counts are heavily influenced by sampling frequency which varies widely between countries. as scotland has a high sampling frequency for its population size (~5.5m), it is possible to calculate a growth rate (voltz and frost, 2017) based on a comparison of the scottish lineages. we find that the growth rate is similar to what has already been shown for the d614g background with no evidence for a faster rate of growth than n439 lineages ( figure s2a ). in addition to its frequency and spread, the n439k variant stood out from other circulating rbm variants as having a plausible mechanism for maintenance of viral fitness. the equivalent position to n439k in the sars-cov rbm is also a positively-charged amino acid (r426), which forms a salt bridge with hace2 (li et al., 2005) . we therefore hypothesized that the n439k sars-cov-2 variant may form this additional salt bridge at the rbd-hace2 interface (rbd n439k:hace2 e329). structural modeling supported that this salt bridge could form without disrupting the binding interface, including the two original salt bridges (rbd k417:hace2 d30 and rbd e484:hace2 k31) (figure 3a-c) . a salt bridge is the strongest type of non-covalent bond and the n439k mutation could plausibly increase the number of salt bridges at the binding interface from two to three, presenting the hypothesis that the n439k variant may have enhanced binding for hace2. to test this hypothesis, we used surface plasmon resonance (spr) to evaluate binding of recombinant n439k s or rbd protein to recombinant hace2. we also evaluated the n439r and k417v variants, each of which is found in sars-cov at these positions. across multiple assay formats, we found that the n439k and n439r variants exhibited a ~2-fold enhanced binding affinity for hace2 as compared to the original n439 variant (termed herein wt) ( figure 3d ). the magnitude of this enhancement was paralleled by a ~2-fold loss of binding affinity for the k417v variant relative to wt. lastly, we also tested the effect of the n439k/r and k417v mutations in combination. these double variants form the same number of salt bridges at the hace2 binding interface as compared to wt, but one is at rbd position 439 rather than 417; we found they had an hace2 affinity similar to the wt ( figure 3d ). these data indicate that acquisition of the n439k mutation enhances binding affinity, which could have implications in vivo in the context of natural infection. also, the enhanced affinity could plausibly compensate for other mutations that would otherwise be detrimental (e.g. k417v), further highlighting the plasticity of the rbm. the enhanced hace2 affinity of the n439k variant, its geographical emergence as independent lineages as well as its prevalence among circulating viral isolates is consistent with maintained viral fitness. we set out to directly examine fitness by evaluating clinical data and outcomes of virus carrying the n439k mutation versus wt n439, as well as by direct in vitro viral growth and competition. we used qpcr to evaluate viral load (as measured by cycle threshold, ct) in 1,918 scottish patients whose viral isolates had been sequenced (figures 4a-b ). viral isolates were either n439k/d614g (n=406), n439/d614g (n=978) or ancestral (n439/d614) (n=534). our analysis found strong evidence that the n439k/d614g genotype was associated with marginally lower cycle threshold (ct) than the n439/d614g genotype (mean ct value difference between n439k/d614g and n439/d614g: -0.65, 95% ci: -1.22, -0.07) ( figure 4b ). as ct measurements were carried out in multiple sites, a sub-analysis of viral load using rna standards was carried out with available samples and showed a near-complete correlation with ct ( figure 4b ). d614g has previously been associated with higher viral loads/lower ct values than d614 (korber et al., 2020) but we did not detect this difference in this statistical analysis due to the intercept of the model being imprecisely estimated (table s2) . clinical outcomes were also obtained for a subset of these patients (n=1,591), who were scored for severity of disease based on oxygen requirement: 1. no respiratory support, 2: supplemental oxygen, 3: invasive or non-invasive ventilation or high flow nasal cannulae, 4: death (figures 4c and s2b ). genotype counts for this analysis were n439k/d614g (n=399), d614g (with n439) (n=735) or ancestral (n439/d614) (n=457). analysis based on our ordinal scale indicated that the n439k/d614g viral genotype was associated with similar clinical outcomes compared to d614g or ancestral genotypes (posterior mean: 0.06, 95% ci: -1.21, 1.33) ( table s3 ). all other results from the severity analysis were qualitatively similar to a previous analysis of the d614g mutation (volz et al., 2020) . these clinical data indicate that the n439k virus is not attenuated. we next tested growth of two representative sars-cov-2 isolates, gla1 (wt n439) and gla2 (n439k), both with the d614g background (table s4) . culture was carried out for 72 hours in vero e6-ace2 cells either with or without tmprss2 expression. there was no significant difference between the growth of these strains after inoculation at multiplicities of infection (mois) of 0.005 and 0.01. the n439k strain replicated slightly faster early after inoculation ( figure 4d ). these data indicate that the n439k mutation does not exhibit dominant negative effects on viral growth, and most likely supports normal replication. to further assess fitness for replication in cultured cells, we carried out a cross-competition assay using inoculation of cells at a matched moi followed by quantitation of n439 and n439k by metagenomic ngs over time (figure 4e ). the n439k strain demonstrated similar fitness as the wt n439 strain, with a possible fitness advantage for n439k in cells expressing tmprss2. taken together with the clinical outcomes, these results indicate that the n439k mutation results in viral fitness that is similar or possibly slightly improved compared to the wild-type n439. having established that virus carrying the n439k mutation is fit, we sought to understand whether this mutation evades antibody-mediated immunity by evaluating recognition of the n439k variant by monoclonal antibodies and by polyclonal immune serum from 445 recovered individuals, including 6 donors who were infected by the sars-cov-2 n439k variant. 7.4% of the tested sera showed a greater than 2-fold reduction in binding to n439k rbd as compared to wt rbd (figures 5a-b and s3) . in some individuals the rbd response was diminished to low titers of <1:30 by the n439k mutation. thus, the response to the rbd is significantly influenced by the n439k mutant within the immunodominant rbm domain (piccoli et al., 2020) in a significant portion of persons potentially immune to wt sars-cov-2. the majority of sera demonstrating loss of binding were those that had overall lower responses to wt rbd, indicating lower ab titers. the sera from the six individuals known to have recovered from infection with sars-cov-2 n439k virus showed no change in binding levels to wt rbd as compared to n439k rbd (figures 5a-b and s3) . this may reflect a true variant-specific response or that differential binding could not be measured due to the limited number of samples analyzed. to understand our results at the level of individual antibodies, we evaluated a panel of 144 mabs isolated from individuals recovered from sars-cov-2 infection early in the pandemic (likely with n439 wt virus) (piccoli et al., 2020; tortorici et al., 2020) , as well as clinical-stage mabs regn10933, regn10987, ly-cov555, and s309 (the parent of vir-7831) hansen et al., 2020; chen et al., 2020; pinto et al., 2020) . 15.5% of these mabs demonstrated a >2-fold reduction of rbd binding in response to the n439k mutation ( figures 5c-d and s4 ). for comparison, we also evaluated the k417v mutation which eliminates one salt bridge at the rbm:hace2 interface and the n439k/k417v double mutation. a similar percentage (12.8% for k417v vs 15.5% for n439k) of mabs lost >2-fold binding to these variants, including several (13.5%) which were not sensitive to either single mutant but were sensitive to the double mutant ( figures 5c-d) . the reduced binding of mabs to these rbd mutants were also confirmed by bio-layer interferometry analysis (bli) (figures 5e and s5a) . to define the potential biological importance of these mutations for evasion of antibody-mediated neutralization, we tested mabs against pseudoviruses expressing s variants n439k, k417v or n439k/k417v (figures 5f-h and s5b ). neutralization of pseudoviruses containing these mutations was significantly diminished for certain mabs, including some that are in clinical development. as predicted by its non-rbm epitope , s309 was capable of neutralizing each of these variants. sensitivity of some neutralizing mabs to mutations at these positions have also been reported in other studies greaney et al., 2020; li et al., 2020a; weisblum et al., 2020) but combinations of mutations have not typically been evaluated. overall, our results demonstrate that mutations compatible with viral fitness can result in immune evasion from both monoclonal and polyclonal antibody responses. the evolution of the sars-cov-2 rbm, a critical epitope for vaccine response and therapeutic mabs, will depend on the fitness of rbm variants. the findings herein describe an example of a naturally-occurring rbm variant which can evade antibody-mediated immunity while maintaining fitness. fitness of this variant, n439k, was demonstrated by repeated emergence by convergent evolution, spread to multiple countries and significant representation in the sars-cov-2 sequence databases, the fact that the n439k rbd retains a high affinity interaction with the hace2 receptor, efficient viral replication in cultured cells, and no disease attenuation in a large cohort of infected individuals. the fitness of n439k is consistent with our findings that the rbm is the most divergent region of s. this divergence indicates an ability of sars-cov-2 to accommodate mutations at the rbm while retaining the functional requirement of hace2 binding, and is likely to be linked to immune pressure from neutralizing ab responses. there is precedent for the most immunogenic region of a viral surface protein to be the fastest mutating despite harboring the receptor binding site; for example, the immunogenic globular head domain of the influenza virus hemagglutinin surface protein, which contains the sialic acid receptor binding site, evolves faster than the stalk region (doud et al., 2018; kirkpatrick et al., 2018) . the ability to accommodate mutations in the rbm indicates a high likelihood that immune-evading sars-cov-2 variants compatible with fitness will continue to emerge, with implications for reinfection, vaccines, and both monoclonal and polyclonal antibody therapeutics. in our profile of immune escape from the n439k variant, we observed resistance to a mab currently being evaluated in clinical trials as part of a two-mab cocktail. the promise of using cocktails of mabs is that they should significantly lower the likelihood of drug-induced selection of resistant viruses . however, if circulating viral strains already carry resistant mutations to one antibody in the cocktail, this could reduce the cocktail to a monotherapy. additionally, considering the high level of plasticity of the rbm demonstrated in the present study, there could be many combinations of rbm mutations compatible with viral fitness while leading to immune escape. this is supported by our result that n439k can compensate for a mutation (k417v) that otherwise decreases receptor binding affinity ( figure 3d ). this particular combination of mutations is plausibly compatible with fitness as it parallels sars-cov rbm:hace2 interactions (salt bridge at sars-cov rbd position r426 and no salt bridge at v404, figure 3a) . notably, several mabs which were not sensitive to these mutations individually were sensitive to them in combination, including the two-mab cocktail ( figure 5c-h) . we propose two approaches that will be critical for minimizing the impact of mab escape mutations. one is to develop mabs with epitopes that are highly resistant to viral escape. this may include epitopes outside of the rbm and/or epitopes that are crossreactive across sars-cov and sars-cov-2, indicating conserved epitopes with a low tolerance for mutation wec et al., 2020; wrapp et al., 2020a) . a comparison of epitopes of rbm-targeting mabs with the most conserved regions of the rbm ( figure 1c ) may also identify rbm mabs with a higher barrier to escape. the second approach is to screen patients, likely at the population level, for the presence of potential resistance variants prior to drug administration. the availability of multiple different mab therapeutics in the clinic could provide the opportunity to tailor the choice of therapeutic to local circulating variants. in general, given that access to therapeutic monoclonal antibodies via clinical trials and emergency use authorization is expanding, and as more people develop immune responses to the wildtype virus, monitoring the evolution of sars-cov-2 will be increasingly critical. although sars-cov-2 is evolving slowly and at present should be controllable by a single vaccine (dearlove et al., 2020) , variation accumulating in the rbm could put this at risk, especially for individuals with a moderate ab response to vaccination or infection. while we only report on evasion of antibody-mediated immunity here, it would be surprising to us if similar changes are not observed to evade t cell immunity and innate immunity. wec, a.z., wrapp, d., herbert, a.s., maurer, d.p., haslwanter, d., sakharkar, m., jangra, r.k., dieterle, m.e., lilov, a., huang, d., et al. (2020) . broad neutralization of sars-related viruses by human monoclonal antibodies. science 369, 731-736. weisblum, y., schmidt, f., zhang, f., dasilva, j., poston, d., lorenzi, j.c.c., muecksch, f., rutkowska, m., hoffmann, h.-h., michailidis, e., et al. (2020) . escape from neutralizing antibodies by sars-cov-2 spike protein variants. (2020) . a new coronavirus associated with human respiratory disease in china. nature 579, 265-269. yurkovetskiy, l., wang, x., pascal, k.e., tomkins-tinch, c., nyalile, t.p., wang, y., baum, a., diehl, w.e., dauphin, a., carbone, c., et al. (2020) . structural and functional analysis of the d614g sars-cov-2 spike protein variant. cell. zhang, l., jackson, c.b., mou, h., ojha, a., rangarajan, e.s., izard, t., farzan, m., and choe, h. (2020) . the d614g mutation in the sars-cov-2 spike protein reduces s1 shedding and increases infectivity. https://wwwbiorxivorg/content/101101/20200612148726v1. samples from 439 sars-cov-2 infected individuals were obtained from the ticino healthcare workers cohort (switzerland), described previously (piccoli et al., 2020) , and under study protocols approved by the local institutional review board (canton ticino ethics committee, switzerland). all donors provided written informed consent for the use of blood and blood components (such as pbmcs, sera or plasma). in the ticino region of switzerland and during the time period of collection (february-march 2020) no n439k sars-cov-2 isolates were reported. samples from six n439k variant infected individuals were obtained from the isaric4c consortium (https://isaric4c.net/). ethical approval was given by the south central-oxford c research ethics committee in england (reference 13/sc/0149), and by the scotland a research ethics committee (reference 20/ss/0028). the study was registered at https://www.isrctn.com/isrctn66726260. residual nucleic acid extracts derived from the nose-throat swabs of 1918 sars-cov-2 positive individuals whose diagnostic samples were submitted to the west of scotland specialist virology centre between 3 rd march and 30 th june 2020 were sequenced as part of the cog-uk consortium under study protocols approved by the relevant national biorepositories (16/ws/0207nhs and 10/s1402/33) (consortiumcontact@cogconsortium.uk, 2020) . rbm residues were determined based on the rbd:ace2 complex crystal structures 2ajf for sars-cov (li et al., 2005) and 6m0j for sars-cov-2 (lan et al., 2020) . the 2ajf structure was obtained from the pdb-redo server (pdb-redo.eu) and was subsequently prepared in the molecular modeling software moe (v2019.0102, https://www.chemcomp.com) using the structure preparation, protonation and energy minimization steps with default settings. rbd residues within 6.0a distance of any ace2 atoms (determined using moe) were determined for each of the two copies of the complex in the asymmetric unit, and then were combined to obtain the rbm. 6m0j was obtained from the coronavirus structural task force server (https://github.com/thornlab/coronavirus_structural_task_force) and was further refined (using refmac5 v5.8.0258), manually fitted (using coot v0.9) and prepared (using moe, as described above) in multiple iterative cycles. the final structure was analyzed for rbd-ace2 contact residues with a 6.0a cutoff to obtain the rbm (using moe). the final list of rbm residues (figure 1c ) was arrived at by combining the sars-cov and sars-cov-2 results. using moe, the pairwise binding energy between each residue in sars-cov-2 rbd and each residue in ace2, and the total binding energy for all interactions, was determined at cutoff distances 3.0a, 3.5a, 4.0a, 4.5a, 5.0a, 5.5a, 6.0a, 6.5a and 7.0a. the percentage of the total binding energy for each interacting rbd residue was calculated for each distance cutoff and was then averaged over all cutoffs. the resulting values are shown in green in figure 1c . differential accumulation of amino acid variants in the rbm, rbd or spike protein was computed taking into account only the presence or absence of a variant at any residue. each variant called present counts one. a variant is called present if there are at least x number of supporting sequences deposited in gisaid, where x varies from 2 to 20. the number of variants is then normalized to the size of the domain (number of residues). dms data was retrieved from . variant-level dms scores were aggregated to residue-level by taking the minimum (most disruptive variant) or the average score across all variants of a residue, except for the reference residue and the stop codon. alternatively, minimum and average scores are computed only across variants that have been observed as naturally occurring. data were represented as a heatmap annotated with: frequency of non-reference amino acids in deposited gisaid sequences (n ≈ 130,000, at least 4 sequences were required to call a variant as present), in log10 scale; number of countries in which a variant was observed; and percentage of total binding energy computed from an x-ray crystal structure (cf. structural analysis methods section). prefusion-stabilized sars-cov-2 spike protein variants (residues 14-1211, containing the 2p and furin cleavage site mutations with a muphosphatase signal sequence and a c-terminal avi-8xhis-epea-tag in a pd2610-v5 vector (atum bio) were expressed in expi293f cells at 37°c and 8% co2 according to manufacturer's instructions (thermo fisher scientific). cell culture supernatant was collected after four days and purified over a 5 ml c-tag affinity matrix (thermo fisher scientific). elution fractions were concentrated and injected on a superose 6 increase 10/300 gl column with 1x pbs ph 7.4 as running buffer. sars-cov-2 rbd variants (residues 328-531 with a c-terminal thrombin-cleavage site-twinstrep-8xhis-tag, and n-terminal signal sequence) were expressed in expi293f cells at 37°c and 8% co2 in a humidified incubator. transfection was performed using expifectamine 293 reagent (thermo fisher scientific). cell culture supernatant was collected three days after transfection and supplemented with 10x pbs to a final concentration of 2.5x pbs (342.5 mm nacl, 6.75 mm kcl and 29.75 mm phosphates), or 3.2x for rbd n439r. sars-cov-2 rbds were purified using 1 or 5 ml histalon superflow cartridges (takara bio) and subsequently buffer exchanged into cytiva 1x hbs-n buffer or pbs. rbds from other sarbecoviruses were expressed in expi293f cells at 37°c and 8% co2. cells were transfected using pei max. cell culture supernatant was collected seven days after transfection. proteins were purified using a 5 ml strep-tactin xt superflow high capacity cartridge followed by buffer exchange to pbs using hiprep 26/10 desalting columns. for s binding measurements, recombinant ace2 (residues 19-615 from uniprot q9byf1 with a c-terminal thrombin cleavage site-twinstrep-10xhis-ggg-tag, and nterminal signal sequence) was expressed in expi293 cells at 37°c and 8% co2 in a humified incubator. transfection was performed using expifectamine 293 reagent (thermo fisher scientific). cell culture supernatant was collected seven days after transfection, supplemented with buffer to a final concentration of 80 mm tris-hcl ph 8.0, 100 mm nacl, and then incubated with biolock solution for one hour. after filtration through a 0.22 µm filter, ace2 was purified using a 1 ml streptrap hp column (cytiva) followed by isolation of the monomeric ace2 by size exclusion chromatography using a superdex 200 increase 10/300 gl column pre-equilibrated in pbs (gibco 10010-023). for binding measurements with surface-captured rbd, recombinant ace2 (residues 19-615 from uniprot q9byf1 with a c-terminal avitag-10xhis-ggg-tag, and nterminal signal sequence) was expressed in hek293.sus using standard methods (atum bio). protein was purified via ni sepharose resin followed by isolation of the monomeric ace2 by size exclusion chromatography using a superdex 200 increase 10/300 gl column pre-equilibrated with pbs. for binding measurements with surface-captured ace2, recombinant ace2 (residues 18-615 with a c-terminal gs-igg2a-mm-fc tag, and n-terminal signal sequence) was stably transfected in cho-k1 gs knock-down cell line (atum bio). protein was purified via protein a and buffer exchanged into pbs. spr binding measurements were performed using a biacore t200 instrument. s protein was surface captured via anti-avitag pab covalently immobilized on a cm5 chip, rbd protein was surface captured via streptactin xt covalently immobilized on a cm5 chip, and ace2-mfc was surface captured via covalent immobilization of the cytiva mouse antibody capture kit on a c1 chip. running buffer was cytiva hbs-ep+ (ph 7.4) and all measurements were performed at 25 °c. all experiments were performed as singlecycle kinetics, with a 3-fold dilution series of monomeric ace2 starting from 300 nm, each concentration injected for 180 sec, or a 3-fold dilution series of rbd starting from 50 nm, each concentration injected for 240 sec. all data were double reference-subtracted and fit to a binding model using biacore evaluation software. for one representative replicate, capture levels were normalized to wt for visualization. binding data with ace2 as analyte were fit to a 1:1 binding model. binding data with rbd as analyte were fit to a heterogeneous ligand binding model, due to an artifactual kinetic phase with very slow dissociation that arises when rbd is an analyte; the lower affinity of the two kds reported by the fit is reported as the kd of the rbd-ace2 interaction (the two reported kds are separated by at least two orders of magnitude for all fits). the measured kd for ace2 binding to s is likely influenced by conformational dynamics of the rbds in the context of the prefusion s trimer. reported kds are an average of 3-4 replicates measured on at least two separate days, with error given as sem. a national sequencing collaboration formed at the start of the epidemic in the uk, cog-uk consortium (consortiumcontact@cogconsortium.uk, 2020), has facilitated the tracking of sars-cov-2 sequences across scotland since the start of the outbreak in february 2020 (6,825 sequences by oct 6, 2020) and real-time monitoring of genetic changes in the spike gene that might be associated with changes in virulence or transmissibility. sequencing was carried out using an amplicon-based protocol in real-time at a rate of up to 300 genomes per week. 50% of samples were selected as surveillance samples, representing scottish health boards proportionately based on population size, while 50% were selected to allow intervention with local issues such as nosocomial infection in hospitals and nursing homes. a gradual increase in the prevalence of the n439k polymorphism was noted to become increasingly prevalent during april 2020. this was noted to be particularly common in the greater glasgow & clyde nhs health board region but spread to adjacent scottish health boards also. sequencing libraries were prepared according to the artic ncov-2019 described in detail at https://artic.network/ncov-2019. briefly, pcr amplicons were generated using the ncov-2019 primalseq sequencing primers using 25-35 cycles of amplification. generated amplicons were used to prepare either oxford nanopore or illumina sequencing libraries. oxford nanopore libraries were prepared as described in the link above and sequenced in a flow cell r9.4.1 (oxford nanopore technologies, part number flo-min106d), using minknow version 19.12.6. raw fast5 files were basecalled using guppy version 3.2.10 in high accuracy mode with a minimum quality score of 7. reads were size filtered, demultiplexed and trimmed with porechop (https://github.com/rrwick/porechop), and mapped against reference strain wuhan-hu-1 (mn908947). variants were called using nanopolish 0.11.3 and accepted if they had a loglikelihood score of greater than 200 and minimum read coverage of 20. for illumina sequencing, amplicons were used to prepare libraries using the kapa hyperprep kit (kapa biosystems, part number kk8504) and further processed as described in the competition assay sequencing method. sequencing was carried out on illumina's miseq system (illumina, part number sy-410-1003) using a miseq reagent v2 500 cycle kit (illumina, part number ms-102-2003) . reads were trimmed with trim_galore (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and mapped with bwa (li and durbin, 2009) ) to the wuhan-hu-1 (mn908947) reference sequence, followed by primer trimming and consensus calling with ivar (grubaugh et al., 2019 ) and a minimum read coverage of 10. uk sequences were obtained from the cog-uk consortium, https://www.cogconsortium.uk. global sequences were obtained from the gisaid initiative, https://www.gisaid.org on oct 19 2020. the sequences were mapped using minimap2 and padded against the wuhan/wh04/2020 reference. the sequences were downsampled with weights that normalise sequence count per epiweek, maximise the number of countries and lineages represented, and enriching for sequences with the n439k mutation. a maximum-likelihood phylogenetic tree was constructed using iq-tree with the the following parameters: -czb -blmin 0.0000000001 -m hky --runs 5 and all other parameters set to default. the tree was visualised with custom python code using the baltic library, https://github.com/evogytis/baltic. for the phylodynamic analysis, scottish "introduction" lineages were identified (lycett et al., 2020, in prep and see http://sars2.cvr.gla.ac.uk/risefallscotcovid), and the skygrowth package in r was used to estimate the effective population size over time, and the growth rate of the lineage within scotland (volz and frost, 2017) . clinical samples submitted to the west of scotland specialist virology centre for sars-cov-2 diagnostic rt-pcr testing were selected for sequencing as part of the covid-19 uk genomics uk consortium (cog-uk) project, resulting in 1918 whole genome sequences originating from the nhs greater glasgow and clyde health board region. sequences were linked to electronic patient records and basic metadata including sample date, age, sex, admission to hospital and mortality at 28 days post diagnosis extracted. the electronic patient records of a subset of 1591 patients underwent full casenote review and clinical severity was recorded based on a 4-level ordinal scale: 1. no requirement for respiratory support, 2. treatment with supplemental oxygen via facemask or low-flow nasal cannulae, 3. intubation and ventilation, non-invasive ventilation or oxygen delivery by high flow nasal cannulae devices, 4. death within the 28 days following diagnosis. we modified the who ordinal scale to these 4 points as described previously (volz et al., 2020) to avoid using hospitalisation as a criterion of severity because 1) many patients in nursing homes had severe infection but were not admitted to hospital, and 2) early in the outbreak, all cases were hospitalised irrespective of the severity of their infection. these data had previously been analysed to test for an effect of the d614g mutation on the severity of disease (volz et al., 2020) ; we extend that analysis here using the same methodology to test for an effect of the n439k mutation. additionally, we perform a new analysis using a model with the same structure to test for an effect of both the d614g mutation and the d614g/n439k mutation combination on the viral load of infected patients, as measured by cycle threshold value. in both cases we cannot estimate the marginal effect of the n439k mutation, as we only have the mutation on the 614g genetic background, so the individual effect of n439k cannot be separated from any potential epistatic interactions between the mutations. briefly, the structure of the model used previously (volz et al., 2020) and in the present study is a phylogenetic generalised additive model with mutation being the primary predictor of interest. the model controls for biological sex, age and the number of days since the first reported case in the dataset, with the latter two being included as penalised splines with a maximum of 30 knots. if the patient was part of a cluster of cases, this was included as a random effect, with individuals not part of clusters being assigned their own levels. correlations driven by the rest of the genome are controlled for by a phylogenetic random effect using a correlation matrix generated under a brownian motion assumption from a phylogeny estimated in iq-tree 2 v. 2.0.6 (minh et al., 2020) using a hky + γ model, masking the positions recommended by de maio et al. as of 22/7/2020 (https://virological.org/t/issues-with-sars-cov-2-sequencing-data/473/13), rooted on the first sequenced sars-cov-2 genome (wu et al., 2020) . the priors for the severity model were those used in the previous analysis of this data. the priors for the model of the viral load were a student-t (mean = 20, scale = 10, degrees of freedom = 3) prior on the model intercept, a gaussian (mean = 0, standard deviation = 10) prior over the fixed effects, and an exponential (lambda = 0.1) prior over the random effect, penalised spline and residual standard deviations. there are two key structural differences between the model used previously (volz et al., 2020) and the model used here. firstly, mutation is a three level rather than two level factor (d614/n439, d614g/n439 and d614g/n439k) with the ancestral d614/n439 being the reference level. secondly, as we are now interested in two mutations, we estimated the phylogeny used to control for the effect of the rest of the genome excluding both the nucleotide position underlying the d614g mutation and the nucleotide position underlying the n439k mutation (in addition to the sites from de maio et al mentioned above). the severity model used a cumulative error structure while the model on the ct values used a gaussian error structure. in both cases, the models were estimated in brms v. 2.13.5 (bürkner, 2018) . the presented models had no divergent transitions, rhat values less than 1.01, and appropriate bulk and tail effective sample sizes for all parameters. shortest probability intervals were calculated using the r package spin v. 1.1 (liu et al., 2015) . analysis code is available at https://github.com/dpascall/sars-cov-2-mutationanalysis. all samples were tested in duplicate using the 2019-ncov_n1 assay rt-qpcr assay (https://www.fda.gov/media/134922/download). ready-mixed primers and probe were obtained from idt (leuven, belgium). pcr was carried out using neb luna universal probe one-step reaction mix and enzyme mix (new england biolabs, herts, uk), primers and probe at 500 nm and 127.5 nm, respectively, and 5 µl of rna sample in a final volume of 20 µl. no template negative controls were included after every seventh sample. six ten-fold dilutions of sars-cov-2 rna standards were tested in duplicate in each assay; standards were calibrated using a plasmid containing the n sequence that had been quantified using droplet digital pcr. thermal cycling was performed on an applied biosystems™ 7500 fast pcr instrument running sds software v2.3 (thermofisher scientific) under the following conditions: 55 °c for 10 minutes and 95 °c for 1 minute followed by 45 cycles of 95 °c for 10 s and 58 °c for 1 minute. assays were repeated if the reaction efficiency was <90% or the r2 value of the standard curve was ≤0.998. where possible, testing of samples was repeated if the %cv of the duplicates was <10%. veroe6-ace2 cells (veroe6 cells induced to overexpress ace2) either with or without tmprss2 overexpression (rhin et al., 2020 under review) were seeded in a 12well plate and inoculated with an moi of 0.01 with either the gla1 (n439/d614g) or gla2 (n439k/d614g) virus isolates for 1hr before washing the cells three times in pbs and replacing with 2% dmem. 100ul of media was removed at each timepoint, rna was extracted, and the presence of sars-cov-2 determined using 2019-ncov-n1 assays (idt) with an neb luna universal probe one-step rt-qpcr kit. a standard curve was used to determine the copy number present per ml of cell culture media. 100ul of the fresh media was also tested for the presence of virus, which was undetectable in all wells. three t25 flasks were seeded with veroe6-ace2 or veroe6-ace2-tmprss2 and inoculated with either single viruses or both gla1 and gla2 virus strains at an moi of 0.01 for 1 hr. the flasks were washed three times with pbs, with 100 ul of the final wash being retained to determine the presence of free virus, before adding 5 ml of fresh 2% dmem. at 24, 48, and 72 hrs, 500 ul of media was removed, which was replaced with 500 ul fresh media. 300 ul was used for rna extraction and ngs analysis of the frequencies of the specific positions within the spike protein. the single virus inoculations showed no alternations in the frequency of the amino acid positions and the final wash showing no free virus in the supernatant. we used an unbiased metagenomic ngs sequencing pipeline to quantify variation across the whole viral genome on the illumina ngs next seq platform. briefly, extracted nucleic acid was incubated with dnasei (thermo fisher, part number am2222) followed by cdna synthesis using superscript iii (thermo scientific, part number 18080044) and nebnext ultra ii non-directional rna second strand synthesis module (new england biolabs, part number e6111l). samples were further processed using the kapa ltp library preparation kit for illumina platforms (kapa biosystems, part number kk8232) and indexed with the nebnext multiplex oligos for illumina 96 unique dual index primer pairs (new england biolabs, part number e6442s). libraries were sequenced on illumina's nextseq 550 system (illumina, part number sy-415-1002), generating 10 million pairs of reads per sample. human mabs were isolated from plasma cells or memory b cells of sars-cov or sars-cov-2 immune donors, as previously described (corti et al., 2011; pinto et al., 2020; tortorici et al., 2020) . ly-cov555 mab was obtained from eli lilly and company. regn10933 and regn10987 mabs were produced recombinantly based on published sequences (hansen et al., 2020) . a total of 148 human monoclonal antibodies or 445 human sera were tested for binding to rbd wt and mutants. spectraplate-384 plates with high protein binding treatment (custom made from perkin elmer) were coated overnight at 4 °c with 0.5 µg/ml (for mabs) or 5 ug/ml (for sera) sars-cov-2 rbd wt, n439k, k417v or n439k/k417v in phosphate-buffered saline (pbs), ph 7.2. plates were subsequently blocked with blocker casein 1% supplemented with 0.05% tween 20 (sigma-aldrich) for 1 h at room temperature. the coated plates were incubated with serial dilutions of the monoclonal antibodies or of the sera for 1 h at room temperature. the plates were then washed with pbs containing 0.1% tween-20 (pbs-t), and alkaline phosphatase-goat anti-human igg (southern biotech) was added and incubated for 1 h at room temperature. after 3 washing steps with pbs-t, p-nitrophenyl phosphate (pnpp, sigma-aldrich) substrate was added and incubated for 30 min at room temperature. the absorbance of 405 nm was measured by a microplate reader (biotek). fitting was performed using a 4-parameter logistic (4pl) model, yielding dose-response curves from which the area under the curve (auc) between 5 and 500 ng/ml was computed. the auc allows to capture, in a single metric, shifts of interest in two parameters of the 4pl model: ec50 and upper asymptote. bli binding measurement was performed on a selection of human monoclonal antibodies tested by elisa. antibodies were diluted to 2.7 µg/ml in kinetic buffer (pbs supplemented with 0.05% bsa) and immobilized on protein a biosensors of an octet red96 system (fortébio). antibody-coated biosensors were incubated for 5 min with a solution containing 5 µg /ml of sars-cov2 rbd wt, n439k, k417v or n439/k417v in kinetic buffer. a dissociation step was then performed by incubating the biosensors for 5 min in kinetic buffer. change in molecules bound to the biosensors caused a shift in the interference pattern that was recorded in real time and plotted using graphpad prism 8 software. replication defective vsv pseudovirus (takada et al., 1997) expressing sars-cov-2 spike protein were generated as previously described (riblett et al., 2016) with some modifications. plasmids encoding sars-cov-2 spike variants were generated by site-directed mutagenesis of the wild-type plasmid, pcdna3.1(+)-spike-d19 (giroglou et al., 2004) . lenti-x™ 293t cells (takara, 632180) were seeded in 10-cm dishes at a density of 1e5 cells/cm 2 and the following day transfected with 5 µg of spike expression plasmid with transit-lenti (mirus, 6600) according to the manufacturer's instructions. one day post-transfection, cells were infected with vsv-luc (vsv-g) (kerafast, eh1020-pm) for 1 h, rinsed three times with pbs, then incubated for an additional 24 h in complete media at 37°c. the cell supernatant was clarified by centrifugation, filtered (0.45 µm), aliquoted, and frozen at -80°c. vero e6 cells (atcc crl-1586) were seeded into clear bottom white 96 well plates (costar, 3903) at a density of 2e4 cells per well. the next day, mabs were serially diluted in pre-warmed complete media, mixed at a 1:1 ratio with pseudovirus and incubated for 1 h at 37°c in round bottom polypropylene plates. media from cells was aspirated and 50 µl of virus-mab complexes were added to cells and then incubated for 1 h at 37°c. an additional 100 µl of prewarmed complete media was then added on top of complexes and cells incubated for an additional 16-24 h. conditions were tested in duplicate wells on each plate and at least six wells per plate contained uninfected, untreated cells (mock) and infected, untreated cells ('no mab control'). virus-mab-containing media was then aspirated from cells and 100 ml of a 1:4 dilution of bio-glo (promega, g7940) in pbs was added to cells. plates were incubated for 10 mins at room temperature and then were analyzed on the envision plate reader (perkinelmer). relative light units (rlus) for infected wells were subtracted by the average of rlu values for the mock wells (background subtraction) and then normalized to the average of background subtracted "no mab control" rlu values within each plate. percent neutralization was calculated by subtracting from 1 the normalized mab infection condition. data were analyzed and visualized with prism (version 8.4.3). ic50 curves were calculated from the interpolated value from the log(inhibitor) vs. response -variable slope (four parameters) nonlinear regression with an upper constraint of <100. each neutralization infection was conducted on three independent days. . dms score is the binding or expression fold change over wt on a log10 scale. aggregated dms data is shown for each residue by taking the minimum (most disruptive variant) or the average score across all possible variants of a residue, except for the reference residue and the stop codon ('mutagenesis' columns). alternatively, minimum and average scores are computed only across variants that have naturally occurred ('observed variants' columns). when no natural variants have been observed, cells are grey. the heatmap is annotated with frequency of non-reference amino acids in deposited sequences (at least 4 sequences were required to call a variant), in log10 scale; number of countries in which a variant was observed; and percentage of total binding energy between rbd and hace2 computed from an x-ray crystal structure. data were sorted on the leftmost dms column. legend on next page (h) correlation of elisa-binding fold change and neutralization fold change for each variant relative to wt (where a smaller elisa auc and therefore a smaller ratio represents loss of binding, and a larger ic50 and therefore a larger ratio represents loss of neutralization) a rbm rbd table s1 . details of the sarbecovirus sequences used for figure s1 . the top 8 sequences shaded in gray were used for the similarity plot and all 69 sequences were used for the entropy plot. parameter estimates on the link scale from the model estimating the impact of the n439k mutation on the ct value of patients infected with sars-cov-2 in scotland. credible intervals represent 95% the shortest posterior density intervals. the difference between d614g/n349 and d614g/n349k was estimated by direct subtraction of the hamiltonian monte carlo samples of the d614g/n349k estimate from the d614g/n349 estimate. ct value did not appear strongly correlated with biological sex or age after controlling for the other factors. patients infected with related viral genomes had correlated ct values at testing potentially implying that there are other undescribed mutations in the genome that are affecting the viral load. parameter estimates on the link scale from the model estimating the impact of the n439k mutation on the severity of infection of patients infected with sars-cov-2 in scotland. credible intervals represent 95% the shortest posterior density intervals. thresholds correspond to the positions of the boundaries between the different severity classes. amino acid change gene mutation gla1 c3037t nsp12 p323l c14408t s d614g a23403g e v5a a24388t t26258c gla2 c3037t nsp12 p323l c14408t nsp15 v35a t19724c s n439k c22879a s d614g a23403g orf 10 v6f g29573t table s4 nucleotide differences between gla1 and gla2. snps determined by cov-glue on consensus sequences relative to wuhan-hu-1 (nc_045512.2). antibody cocktail to sars-cov-2 spike protein prevents rapid mutational escape seen with individual antibodies evolutionary origins of the sars-cov-2 sarbecovirus lineage responsible for the covid-19 pandemic advanced bayesian multilevel modeling with the r package brms an integrated national scale sars-cov-2 genomic surveillance network sars-cov-2 neutralizing antibody ly-cov555 in outpatients with covid-19 a neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza a hemagglutinins genomic epidemiology of sars-cov-2 spread in scotland highlights the role of european travel in covid-19 emergence a sars-cov-2 vaccine candidate would likely match all currently circulating variants how single mutations affect viral escape from broad and narrow antibodies to h1 influenza hemagglutinin safety and immunogenicity of the chadox1 ncov-19 vaccine against sars-cov-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus s protein complete mapping of mutations to the sars-cov-2 spike receptor-binding domain that escape antibody recognition an amplicon-based sequencing framework for accurately measuring intrahost virus diversity using primalseq and ivar sars-cov-2 d614g variant exhibits enhanced replication ex vivo and earlier transmission in vivo d614g mutation of sars-cov-2 spike protein enhances viral infectivity an mrna vaccine against sars-cov-2 -preliminary report neutralizing antibodies against sars-cov-2 and other human coronaviruses ly-cov555, a rapidly isolated potent neutralizing antibody, provides protection in a non-human primate model of sars-cov-2 infection phase 1-2 trial of a sars-cov-2 recombinant spike protein nanoparticle vaccine the influenza virus hemagglutinin head evolves faster than the stalk domain tracking changes in sars-cov-2 spike: evidence that d614g increases infectivity of the covid-19 virus structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor structure of sars coronavirus spike receptorbinding domain complexed with receptor fast and accurate short read alignment with burrows-wheeler transform the impact of mutations in sars-cov-2 spike on viral infectivity and antigenicity emergence of sars-cov-2 through recombination and strong purifying selection transmission dynamics and evolutionary history of 2019-ncov simulation-efficient shortest probability intervals natural selection in the evolution of sars-cov-2 in bats, not humans rapid implementation of sars-cov-2 sequencing to investigate cases of health-care associated covid-19: a prospective genomic surveillance study iq-tree 2: new models and efficient methods for phylogenetic inference in the genomic era refmac5 for the refinement of macromolecular crystal structures mapping neutralizing and immunodominant sites on the sars-cov-2 spike receptor-binding domain by structure-guided high-resolution serology cross-neutralization of sars-cov-2 by a human monoclonal sars-cov antibody a dynamic nomenclature proposal for sars-cov-2 lineages to assist genomic epidemiology a haploid genetic screen identifies heparan sulfate proteoglycans supporting rift valley fever virus infection convergent antibody responses to sars-cov-2 in convalescent individuals coronavirus rna proofreading: molecular basis and therapeutic targeting isolation of potent sars-cov-2 neutralizing antibodies and protection from disease in a small animal model structural basis of receptor recognition by sars-cov-2 suptavumab for the prevention of medically attended respiratory syncytial virus infection in preterm infants deep mutational scanning of sars-cov-2 receptor binding domain reveals constraints on folding and ace2 binding a system for functional analysis of ebola virus glycoprotein ultrapotent human antibodies protect against sars-cov-2 challenge via multiple mechanisms scalable relaxed clock phylogenetic dating evaluating the effects of sars-cov-2 spike mutation d614g on transmissibility structure, function, and antigenicity of the sars-cov-2 spike glycoprotein top -pairwise similarity to sars-cov-2 (sliding window size of 30 amino acids) for seven related sarbecoviruses (see figure key) across the rbd region of the spike protein. bottom -site-specific entropy plot across the rbd protein alignment of sars-cov-2 and 68 related viruses (data s1). entropy for each position l (h(l)) was calculated using shannon's entropy formula with a natural log as sites constituting the rbm are annotated in blue the x-axis refers to absolute positions in the sars-cov-2 spike protein sequence. rightbox plot of site-specific entropy values for the rbm sites (blue) and remaining non-rbm rbd sites (gray) sequence alignment (left) and identity for rbm and rbd (right) to sars-cov-2 of the rbd sequences showing binding to hace2. rbm residues indicated by blue boxes. (c) binding of hace2 to human, pangolin and bat sarbecovirus rbds by bli. bat cov ratg13 we thank all scottish nhs virology laboratories who provided samples for sequencing and scott arkison for hpc maintenance. we thank chiara silacci-fregni from humabs biomed, sandra jovic, blanca fernandez rodriguez, federico mele, from the institute for research in biomedicine in bellinzona and tatiana terrot from ente ospedaliero cantonale in lugano for the help in collecting sera samples. we thank cindy ng for help with protein production. we thank julia di iulio for help with analyzing gisaid sequences. we gratefully acknowledge the authors, originating and submitting laboratories of the sequences from gisaid, https://www.gisaid.org, on which much of this research is based.the isaric who ccp-uk study protocol is available at https://isaric4c.net/protocols; study registry https://www.isrctn.com/isrctn66726260. this work uses data provided by patients and collected by the nhs as part of their care and support #datasaveslives. we are grateful to the 2648 frontline nhs clinical and research staff and volunteer medical students who collected the data in challenging circumstances; and the generosity of the participants and their families for their individual contributions in these difficult times. we also acknowledge the support of jeremy j farrar, nahoko shindo, devika dixit, nipunie rajapakse, lyndsey key: cord-263090-29n9tsk9 authors: roy, susmita title: dynamical asymmetry exposes 2019-ncov prefusion spike date: 2020-04-21 journal: biorxiv doi: 10.1101/2020.04.20.052290 sha: doc_id: 263090 cord_uid: 29n9tsk9 the novel coronavirus (2019-ncov) spike protein is a smart molecular machine that instigates the entry of coronavirus to the host cell causing the covid-19 pandemic. in this study, a structural-topology based model hamiltonian of c3 symmetric trimeric spike is developed to explore its complete conformational energy landscape using molecular dynamic simulations. the study finds 2019-ncov to adopt a unique strategy by undertaking a dynamic conformational asymmetry induced by a few unique inter-chain interactions. this results in two prevalent asymmetric structures of spike where one or two spike heads lifted up undergoing a dynamic transition likely to enhance rapid recognition of the host-cell receptor turning on its high-infectivity. the crucial interactions identified in this study are anticipated to potentially affect the efficacy of therapeutic targets. one sentence summary inter-chain-interaction driven rapid symmetry breaking strategy adopted by the prefusion trimeric spike protein likely to make 2019-ncov highly infective. movement that generates the 'up' and 'down' conformations (7, 8, 10) . other betacoronaviruses, like sars-cov, mers-cov and distantly related alphacoronavirus porcine epidemic diarrhea virus (pedv) also have this apparently stochastic rbd movement (11, 12) . the combination of rbd up-down rearrangement may lead each s1-head of the trimeric prefusion spike protein of coronavirus to adopt different possible conformations: (i) 3down, (ii) 1up-2down, (iii) 2up-1down, and (iv) 3up (fig. 1c) . among them 3down, 3up are symmetric conformers and 1up-2down, 2up-1down are asymmetric conformers. single-particle cryo-electron microscopy (cryo-em) determined few such symmetric and asymmetric structures referred to as the receptorbinding inactive state and receptor-binding active state, respectively (8) . the asymmetric structure where one of the rbds rotates up was thought to be less stable for sars-cov s (10) . in comparison, the recent cryo-em study found three rbds in 1up-2down conformation as a predominant arrangement in the prefusion state of 2019-ncov s trimer (7) . this arrangement apparently appears legitimate for sars-cov-2 s in order to explain the higher affinity of 1up-2down for ace2 receptor than that of sars-cov s. however, we cannot rule out the possibility of 2up-1down conformation as a functional state, which may provide even stronger binding with ace2 considering the fact that ace2 is a dimeric receptor (9, 13) . this hypothesis is consistent with a recent crystallographic study demonstrating that cr3022, a neutralizing antibody isolated from convalescent sars patients targets the rbd when at least two rbd on the trimeric spike protein are in the up conformation (14) . assembling all these experimental results it is high time to understand the molecular mechanism of s1-head coordination of trimeric sar-cov-2 s and to identify important interaction in regulating spike up-down conformations. a schematic of receptor-bound spike protein including the receptor-binding subunit s1, the membrane-fusion subunit s2 of a coronavirus is shown. b. side and top views of the homo-trimeric structure of sars-cov-2 spike protein with one rbd of the s1 subunit head rotated in the up conformation. c. rbd up-down movement expected to lead s1 heads of the trimeric spike protein to attain the following possible conformers: (i) 3 down (ii) 1up-2down (iii) 2up-1down, and (iv) 3up. these are an analogue demonstration of the spike protein top-view where ntds are represented by colored ovals, rbds are represented by flexible sticks and s2 domains are represented by filled circles. a major challenge was simulating the gigantic structure of the full-length trimeric spike, as it is associated with the largescale conformational transition. it is indeed a daunting task to explore the full conformational landscape at an atomic length-scale. to overcome this, a structure-based coarse-grained molecular dynamic simulation approach has been adopted (15) . the simulation started with a full-length homo-trimeric spike protein structure generated from homology modeling which involves the alignment of a target sequence and a template structure (pdb: 6vsb) (7, 16) . this also helped to build the missing loops. the domain-specific residuerange for the full-length, trimeric sars-cov-2 s is given in fig. 2a . the s1 head coordination of the trimeric spike is programmed by developing a super-symmetric topology-based modeling framework ( fig. 2b ) (described in the method pipeline in the supplementary material). with this, the molecular machine is ready to swing each of its s1 head between its 'up' and 'down' conformations (movie s1, s2). a number of cryo-em structures captured the 'up' and 'down' conformations of the rbd domain of spike proteins of other coronaviruses including sars-cov-2 where the s1 subunit undergoes a hinge-like conformational movement prerequisite for receptor binding (fig. 2c) (7, 8, 10, 17) . apart from the hinge-responsive rbd-cleft interaction, in this study, a few inter-chain interactions are found to assist the 'rbd-up' and the 'rbd-down' conformations (shown in fig. 2d and 2e, movie s3). these few interactions are identified to impact the breathing of rbd of sars-cov-2 s. this makes the early referred 'rbd-up/down' conformations slightly different from the 's1-head-up/down' conformation for trimeric sars-cov-2 s as the former is regulated only by intra-chain interactions while the latter is regulated by both intra and inter-chain interactions (fig. s1 ). after identifying all these unique intra and inter-chain contacts (18, 19) extracted from the corresponding 's1-head-up' and 's1-head-down' conformations, a super-symmetric contact map is generated. this follows the development of a structure-based model hamiltonian (materials and methods in supplementary) which is based on the energy landscape theory of protein folding (20) (21) (22) (23) (24) . this approach not only potentiates the trimeric spike to adopt c3 symmetric '3up' and '3down' states but also to break the symmetry in a thermodynamically governed way ( fig. s1-s4 ) (25, 26) . residue-residue native contact map identifying unique intra and inter-chain contact-pairs formed by any single monomer in its s1-head up and s1-head down states. c. within intra-chain contacts, the unique contacts that drive hinge motion leading to rbd-up and rbd-down states are highlighted in the structure, as well as in the contact map. d. inter-chain unique contacts between rbd and ntd domains upholding the s1-head-up state. e. inter-chain unique contacts are responsible for connecting the rbd of chaina with the s2-stalk of chainb and the s2 stalk of chainc. to monitor the transition between the 's1-head-up' and the 's1-head-down' states for each monomer with the trimeric interactions, a large pool of unbiased longtime trajectories generated where multiple occurrences of up and down states for each monomer have been sampled. we employ a reaction coordinate, q, the fraction of the native contact (19, 27) corresponding to the inter-chain contacts associated with the 's1-head-up' and the 's1-head-down' states. a typical trajectory plot of q extracted from the equilibrium simulation of the trimeric prefusion spike clearly shows the hopping between different conformational states as hypothesized earlier (fig. 3a) . furthermore, the dynamic transitions between the two major asymmetric states (1up-2down: q s1-head-down ≈0.45 and 2up-down: q s1-head-down ≈0.15) are evident in the q-trajectory. analysis of all the simulations yields the 2-d free energy landscape of the trimeric spike protein of sars-cov-2 ( fig 3b) with its all possible conformations. the conformations corresponding to the minima of the free energy landscape are shown in fig.3c . the temperature dependence of conformational transition indicates that the configurational entropy and enthalpy compensation results in the enhanced population of the asymmetric 1up-2down to 2up-1down conformations ( fig s4) . while the predominant population of the 1up-2down state is consistent with the recent cryo-em data, (7) (movie s1, s2) the other asymmetric structure (2up-1down) emerges as a best binding epitope for cr3022 (an antibody collected from convalescent sars patients) according to a recent antibody recognition study of sars-cov-2 s (14). .conformational transition of sars-cov-2 spike protein in its prefused state. a. the fraction of native contact (q) dynamics counting inter-chains contact-pairs formed in the s1head-up state and the s1-head-down state. b. a two-dimensional free energy landscape of conformational transition as a function of inter-chain contacts supporting s1-head-down (x-axis) and s1-head-up state (y-axis) explores all possible conformations. c. the representative structure corresponding to each minimum of the free energy landscape is designated as follows: (i) 3up, (ii) 2up-1down, (iii) 1up-2down, and (iv) 3down state (as shown in the one-dimension population distribution plot). a. unique inter-chain interactions formed by rbd of one chain with ntd of the adjacent chain stabilizing the s1-head-up conformation in sars-cov-2 s (pdb:6vsb). interchain domain closure is analyzed by inter-chain proline-proline distance measurement. the same distance measured for the following spikes: b. sars-cov spike (pdb:5x5b) and c. mers-cov spike (pdb: 5x5f). d. rbd up-down hinge dynamics triggered by inter-chain rbd-ntd domain interaction. e. in the absence of rbd-ntd inter-chain interaction, the hinge motion of rbd is hindered by populating more 'rbd-down' conformations and allows to sample 'rbd-up' conformation only rarely in a stochastic manner. in this study, sequence and interaction level (fig. s5, fig. s1 ) comparison has been made over the cryo-em structure of sars-cov-2 s (pdb: 6vsb), sars-cov s (pdb:5x5b) and mers-cov s (pdb: 5x5f) (7, 12) . this comparison results that sars-cov-2 s has ntd-rbd domain association where a proline residue of chaina forms ch-п type interaction with the tyrosine residue (28) and hydrophobic interaction with another proline of chainb (fig. 4a, fig. s1 ). inter-chain proline-proline distance measurement shows that the corresponding rbd-ntd domains are far away in the case of sars-cov s (fig. 4b ) and further away in the case of mers-covs (fig. 4c ). this measurement involves their respective cryo-em structures. despite the relatively high degree of sequence similarity between the sars-cov-2 s and the sars-cov s and also with the spike protein from the bat coronavirus ratg13, a single histidine residue at the relevant rbd-ntd domain interface is found unique in the vase of sars-cov-2 s (fig. s5) (29) . the imidazole ring of histidine is pointing towards the hydrophobic assembly of aforesaid proline-tyrosine in the juxtaposition of the rbd-s1 hinge region. such inter-chain rbd-ntd connection is thus found to impact the rbd hinge interaction by upregulating more rbd-up conformation (fig. 4d ). in the absence of such interchain interaction, the rbd mostly stays in the down conformation allowing rbd to break the symmetry rarely in a stochastic manner (fig. 4e) . the absence of inter-chain rbd-ntd connection also appears to impact the sars-cov rbd hinge interaction. here, the opening of rbd-s1 cleft is significantly less than that of sars-cov-2 s in their respective s1-head-up state (fig. s6 ). the assistance from the inter-chain rbd-s2-stalk related interfacial contacts are also found to modulate the population dynamics of rbd-down conformation (fig. s7 ). the influence of this inter-chain rbd-s2-stalk interaction has also been observed in an early cryo-em analyses where two proline mutations at the top of s2 stalk (inferring rbd-s2 inter-chain connection) helped to stabilize the 'up' conformers of sars-cov s (30) . the synergy between internal rbd-hinge interactions and inter-chain interactions allows trimeric sars-cov-2 s to adopt a unique dynamical feature than other corona-virus spikes. it appears that the inter-chain interactions driven rapid symmetry breaking strategy potentiates this spike machine to turn on its high-infectivity. the energy landscape framework used in this study indeed helps to unify and compare different spike protein interactions present in other coronaviruses. while in the current situation to develop diagnostics and antiviral therapies are of utmost priority, the present structure-based model derived information at the microscopic interaction level might provide deep insight to design effective decoys or antibodies to fight against 2019-ncov infection. movies s1 to s3 method pipeline of building a super-symmetric contact map of sars-cov-2 prefusion spike protein. coarse-grained structure-based simulations have been performed for full-length trimetric sars-cov-2 spike protein. the structure-based hamiltonians for different simulations were derived after processing the recent cryo-em structure (pdb:6vsb) thorough the swiss model to complete missing loops present in the structure (7, 16) . this generates a homo-trimeric sars-cov-2 spike where this initial structure has important components in terms of intra and inter-chain contacts (interaction) leading to an 's1-head-up' and an 's1-head-down' conformation for each protomer. in this prevalent trimeric variant, only one monomer adopts 's1-head-up' and the same of the other two adopts the 's1-head-down' conformation. few characteristic intra-chain contacts cause the receptor-binding domain to perform a hinge-motion resulting 'rbd-up' and 'rbdconformations driven by intra c as defined in the pipeline method. contact calculation is performed using the shadow criterion (19) . interesting components are inter-chain contacts residing at the interface of the dimer. now, two categories of interactive dimeric interfaces are there: asymmetric-dimer interface and symmetricdimer interface. chaina (s1-head-up) and the adjacent chainb (s1-head-down) represent an asymmetric dimer unit. similarly, chainb (s1-head-down) and the adjacent chainc (s1-headdown) represent a symmetric dimer unit. at the asymmetric-dimer interface, the rbd-domain of chaina forms a few unique contacts with the ntd domain of the adjacent chainb as shown in has been cycled over all the interfaces making each of interfaces dynamically capable of inducing s1-head movement. developing a structure-based hamiltonian of trimeric spike protein simulation: a structure-based hamiltonian of trimeric spike protein for sar-cov2 is derived using the super-symmetric contact map. in the current structure-based model amino acids are represented by single beads at the location of the c-α atom (15, 25, 31, 32) . the coarse-grained structurebased model, a well-established model, comprehends a novel way to investigate the mechanisms associated with protein folding and function (20-22, 24, 33-38) . in the current context of decoding virus entry mechanism, this model successfully characterized class-i viral fusion protein dynamics including conformational rearrangement of a viral surface glycoprotein, influenza hemagglutinin (ha) during its prefusion and postfusion states (26, 39) . as described in the pipeline method, the complete hamiltonian comprises of two terms: and, intra up down shared the first non-local term of the hamiltonian used in a/b/c intra h represents non-bonded interaction potential in the form of 10-12 lennard-jones potential that is used to describe the interactions that stabilize the native contacts (15) . a native contact is defined for a pair of residues (i and j) present in the native state using shadow criteria and when (i−j)>3. δ ij is defined in such a way that if any i and j residues belong to intra c , δ ij = 1 turning on 10-12 lennard-jones potential; otherwise δ ij = 0. for all non-native pairs for which δ ij = 0, a repulsive potential with σ = 4å is used. all the interaction coefficients used in this potential are given in table s1 . as described in the method pipeline, inter h will include only the non-local inter-chain contacts residing at the interface of the dimer which comprises of accounting for asymmetric-dimer similar to our early approach, δ ij is such defined that if any i and j residues belong to inter c , δ ij = 1, turning on 10-12 lennard-jones potential; otherwise δ ij = 0. here, inter to begin every simulation an initial structure is energetically minimized under the structure-based hamiltonian using the steepest descent algorithm. atomic coordinates of the energy minimized structure have been evolved using langevin dynamics with a time step of 0.0005 r  . we used an underdamped condition for rapid sampling (40) . for explicit particles, reduced mass of 1 r  and a drag coefficient all temperatures mentioned here are in reduced units. temperature dependence of the conformational transition has been performed over several temperatures. three representative reduced temperature-dependent (t*=0.50t r , t*=0.58t r, and t*=0.83t r ) analyses are shown for clarity in fig. s4 . population distribution as a function of the fraction of native inter-chain contacts formed in the s1-head-down state is monitored over these temperatures. four states emerge as indicated in fig. 3b and fig. s4 . as the temperature increases the population shifts more towards the s1-head-up state. at t*=0.58t r the population of 1up-2down state appears as a predominant population in the conformational landscape which correlates well with the recent cryo-em data (7) . we have performed all our simulations being consistent with this selected temperature. the rmsd analyses ensure the correctness of the simulation progress and the emergence of the correct structure (fig. s3) . the population shifts more towards the s1-head-up state conformations as the temperature increases. it suggests that the s1-head-up states are more dynamic and entropically stable. note that the dynamical transition between 1up-2down and 2up-1down states may tolerate a wide range of temperatures by a population shift mechanism. so far, we have examined that it tolerates the temperature range from t*=0.50t r to t*=1.67t r . temperature dependence of rbd hinge motion has also been studied (fig. s4) . population distribution as a function of the fraction of native intra-chain hinge-region contacts formed by the rbd at different temperatures has been monitored. a bimodal distribution reflects the population of the 'rbd-up' and 'rbddown' states for any individual chain being in trimeric spike. as temperature increases, the rbd-up states start to enhance their populations. free energy calculation: in a system, if a state "a" described by its reaction coordinate, x a (which in our case is the fraction of native contact) is separated from another state "b" described by its reaction coordinate, x b , by a finite barrier, the free energy of transition from a to b can be expressed as, where, ( ) b p x is the probability to find the system in state b at the reaction coordinate, q b . the same holds for ( ) a p x . from a finite set of unbiased simulations of trimeric spike protein, a complete thermodynamic description is obtained. probability distributions are obtained by sampling the configurational space running 50 molecular dynamics simulation sets. fig. s1 : inter-chain interaction from the 's1-head-up' and the 's1-head-down' states of sars-cov-2 spike. a. inter-chain rbd-ntd domain closure in the s1-head-up state. the domain closure is mediated by double hydrogen bonds connecting arg of chaina with asn and cys residue of chainb. b. inter-chain rbd-s2 domain closure in the s1-head-down state. the s2 stalk connection with rbd is mediated by a proline residue of chaina with the formation of a ch-п type interaction with tyrosine and hydrophobic interaction with another proline of chainb. fig. s2 : the structural alignment of two chains in the s1-head-down state. chainb (orange) and chainc (green) in the s1-head-down state extracted from the cryo-em structure (pdb:6vsb) of trimeric spike. low rmsd between these two chains suggests that contact information extracted from any of these chains will be equivalent. this supports our contact map generation shown in the method pipeline. fig. s3 . rms deviation of each chain from their initial state during a typical simulation progress. a. the initial state of chain a in the trimeric spike was in 's1-head-up' state and chain b/c was in 's1-head-down' state. b. the lower rmsd for chain a corresponds to chain a's head-up state. c. the lower rmsd for chain b corresponds to chain b 's head-down state. d. the lower rmsd for chain c corresponds to chain c's head-down state. the rmsd analyses ensure the correctness of the simulation progress and the emergence of the correct structure. fig. s4 . temperature dependence of s1-head up-down transition and rbd open-close breathing transition. a. population distribution as a function of the of native inter-chain contacts formed in the s1-head-down state as shown in fig. s1 . four states emerge as shown in fig. 3b . as temperature increases the population shifts more towards the s1-head-up state conformations indicating that s1-head-up states are more dynamic and entropically stable.note that the dynamical transition between 1up-2down and 2up-1down states may tolerate a wide range of temperatures by a population shift mechanism. b. population distribution as a function of the fraction of native intra-chain hinge-region contacts formed by the rbd. a bimodal distribution reflects the 'rbd-up' and the 'rbd-down' states for any individual chain being in trimeric spike. as the temperature increases, rbd-up started populating more. temperature analysis helps to choose an intermediate temperature to obtain correct population distribution. fig. s5 . sequence alignment of sars-cov-2 spike (pdb: 6vsb) with that of sars-cov spike(pdb:5x5b), mers-cov spike (pdb: 5x5f) and ratg13 spike. only the rbd is highlighted in green. the unique histidine residue (highlighted in yellow) of the rbd of sars-cov-2 is noted. identical residues are denoted by an "*" beneath the consensus position. the multiple sequence alignment is continued over the next page. fig. s6 .the opening of rbd-s1 cleft in the 's1-head-up' state of sars-cov-2 s differs from that of sars-cov s. the opening is measured by a characteristic distance between a serine and proline residues at two edges of the cleft. for sars-cov-2 s the distance is 2.43 nm while for sars-cov-s, it is 1.65nm. it appears that inter-chain rbd-ntd connection influences the sars-cov s rbd hinge motion significantly where the cleft opening is supported by those inter-chain interactions. fig. s7 . the free energy landscape in the presence and absence of inter-chain rbd-s1 contacts. a. in the presence of inter-chain rbd-s1 contacts, the enhanced population of the 1up-2down compared to 2up-1down. b. in the absence of inter-chain rbd-s1 contacts, the population shifts from 1up-2down state 2up-1down state. movie s1: conformational dynamics of full-length trimeric sars-cov-2 spike protein showing rapid symmetry breaking. movie s2:conformational dynamics of full-length trimeric sars-cov-2 spike protein showing rapid symmetry breaking. in this movie the ntd domains are not shown for better demonstration of the rbd movement. movie s3: conformational dynamics of a monomer of the full-length sars-cov-2 showing rbd hinge motion. and notes: 1. in microbial evolution and co-adaptation: a tribute to the life and scientific legacies of joshua lederberg: workshop summary plagues and peoples sars-cov-2: an emerging coronavirus that causes a global threat evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission identifying sars-cov-2 related coronaviruses in malayan pangolins structure, function, and evolution of coronavirus spike proteins cryo-em structure of the 2019-ncov spike in the prefusion conformation structure, function, and antigenicity of the sars-cov-2 spike glycoprotein structural basis for the recognition of sars-cov-2 by full-length human ace2 cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding the 3.1-angstrom cryo-electron microscopy structure of the porcine epidemic diarrhea virus spike protein in the prefusion conformation cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor a highly conserved cryptic epitope in the receptor-binding domains of sars-cov-2 and sars-cov topological and energetic factors: what determines the structural details of the transition state ensemble and "en-route" intermediates for protein folding? an investigation for small globular proteins swiss-model: homology modelling of protein structures and complexes the human coronavirus hcov-229e s-protein structure and receptor binding smog 2: a versatile software package for generating structure-based models the shadow map: a general contact definition for capturing the dynamics of biomolecular folding and function. the journal of physical chemistry protein folding funnels: a kinetic approach to the sequence-structure relationship symmetry and the energy landscapes of biomolecules chemical physics of protein folding levinthal's paradox funnels, pathways, and the energy landscape of protein folding: a synthesis microbial evolution and co-adaptation: a tribute to the life and scientific legacies of joshua lederberg: workshop summary plagues and peoples sars-cov-2: an emerging coronavirus that causes a global threat evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission identifying sars-cov-2 related coronaviruses in malayan pangolins structure, function, and evolution of coronavirus spike proteins cryo-em structure of the 2019-ncov spike in the prefusion conformation structure, function, and antigenicity of the sars-cov-2 spike glycoprotein structural basis for the recognition of sars-cov-2 by full-length human ace2 cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding the 3.1-angstrom cryo-electron microscopy structure of the porcine epidemic diarrhea virus spike protein in the prefusion conformation cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor a highly conserved cryptic epitope in the receptor-binding domains of sars-cov-2 and sars-cov topological and energetic factors: what determines the structural details of the transition state ensemble and "en-route" intermediates for protein folding? an investigation for small globular proteins swiss-model: homology modelling of protein structures and complexes the human coronavirus hcov-229e s-protein structure and receptor binding smog 2: a versatile software package for generating structure-based models the shadow map: a general contact definition for capturing the dynamics of biomolecular folding and function. the journal of physical chemistry protein folding funnels: a kinetic approach to the sequence-structure relationship symmetry and the energy landscapes of biomolecules chemical physics of protein folding levinthal's paradox funnels, pathways, and the energy landscape of protein folding: a synthesis the origin of minus-end directionality and mechanochemistry of ncd motors order and disorder control the functional rearrangement of influenza hemagglutinin landscape approaches for determining the ensemble of folding transition states: success and failure hinge on the degree of frustration pi-interactions in proteins the embl-ebi search and sequence analysis tools apis in 2019 stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis role of aaa3 domain in allosteric communication of dynein motor proteins intersubunit assisted folding of dna binding domains in dimeric catabolite activator protein. the journal of physical chemistry online service), computational modeling of biological systems : from molecules to pathways chemical physics of protein folding from levinthal to pathways to funnels from structure to function: the convergence of structure based models and co-evolutionary information protein folding mechanisms and the multidimensional folding funnel navigating the folding routes rotation-activated and cooperative zipping characterize class i viral fusion protein dynamics the nature of folded states of globular proteins strain mediated adaptation is key for myosin mechanochemistry: discovering general rules for motor activity key: cord-274480-aywdmj6o authors: song, wenfei; wang, ying; wang, nianshuang; wang, dongli; guo, jianying; fu, lili; shi, xuanling title: identification of residues on human receptor dpp4 critical for mers-cov binding and entry date: 2014-10-21 journal: virology doi: 10.1016/j.virol.2014.10.006 sha: doc_id: 274480 cord_uid: aywdmj6o middle east respiratory syndrome coronavirus (mers-cov) infects host cells through binding the receptor binding domain (rbd) on its spike glycoprotein to human receptor dipeptidyl peptidase 4 (hdpp4). here, we report identification of critical residues on hdpp4 for rbd binding and virus entry through analysis of a panel of hdpp4 mutants. based on the rbd–hdpp4 crystal structure we reported, the mutated residues were located at the interface between rbd and hdpp4, which potentially changed the polarity, hydrophobic or hydrophilic properties of hdpp4, thereby interfering or disrupting their interaction with rbd. using surface plasmon resonance (spr) binding analysis and pseudovirus infection assay, we showed that several residues in hdpp4–rbd binding interface were important on hdpp4–rbd binding and viral entry. these results provide atomic insights into the features of interactions between hdpp4 and mers-cov rbd, and also provide potential explanation for cellular and species tropism of mers-cov infection. middle east respiratory syndrome (mers), a novel coronavirus which causes severe respiratory illness, was first reported in a patient from saudi arabia in 2012 (de groot et al., 2013) . to date, individual cases as well as small clusters and large outbreaks have been reported in several countries and the mortality rate is estimated at 30% among laboratory-confirmed cases (organization, 2014) . phylogenetic analysis demonstrates that the mers coronavirus (mers-cov) is genetically closest to clade 2c betacoronavirus found in camels and insectivorous bats (ithete et al., 2013) although the true viral reservoir remains uncertain. the clinical symptoms caused by mers-cov are similar to those caused by severe acute respiratory syndrome coronavirus (sars-cov) although the two viruses use two distinct receptors; mers-cov uses dipeptidyl peptidase 4 (dpp4) while sars-cov uses angiotensin-converting enzyme 2 (ace2). other coronaviruses use other receptors and perhaps this provides partial explanation for their cellular and species tropism. mers-cov can replicate in a range of cell lines derived from human, non-human primate, porcine, and bat (de wit et al., 2013) . traditional small laboratory animals, such as mice (coleman et al., 2014) , hamsters (de wit et al., 2013) , and ferrets (raj et al., 2014) , were shown to resist mers-cov infection. the finite host range of mers-cov has seriously restricted the development of appropriate animal models to study the pathogenesis of this virus and to assess the efficacy of potential therapeutic strategies. raj et al. (2014) demonstrated that human receptor dpp4 (hdpp4) domain (residues 246 to 505) could confer the susceptibility of ferret dpp4 to mers-cov infection. zhao et al. (2014) are the first to describe a method of developing a small-animal model for mers-cov in which an adenovirus expressing hdpp4 was utilized to transiently transduce mouse airway cells and make mice susceptible to mers-cov infection. recently van doremalen et al. (2014) showed that dpp4 played an important role in the observed species tropism of mers-cov infection and identified residues in dpp4 responsible for this restriction. these results indicate that the insusceptibility to infection is primarily determined by the inability of mers-cov binding to dpp4 of a non-permissive cell line. previous findings have shown that hdpp4 extracellular domain consists of a variable n-terminal eight-blade β-propeller domain and a conserved c-terminal α/β-hydrolase domain (engel et al., 2003; rasmussen et al., 2003) . however, our understanding of critical residues of hdpp4 on mers-cov interaction and entry is quite limited. we and others have previously characterized rbd-hdpp4 crystal structure lu et al., 2013; wang et al., 2013) . the rbd-hdpp4 crystal structure showed that the viral rbd recognized blades iv and v of the dpp4 β-propeller domain. the atomic interaction details of the binding interface revealed that the rbd receptor recognition was predominantly mediated by several amino-acid residue interactions, including rbd residue d539 with dpp4 residue k267, rbd y499 with dpp4 r336, rbd residues d510 and e513 with dpp4 residues r317 and q344, rbd l506, w553 and v555 with dpp4 l294 and i295. previously, we have generated a panel of mers-cov mutant rbd proteins at the residues d539, y499, d510, e513, l506, w553 and v555 to characterize their impacts on binding activity to hdpp4 and the entry efficiency into target cells. however, the impacts of the corresponding residues on hdpp4 have not been well characterized. here, through structure-guided mutagenesis, we identified several key residues in hdpp4 that were critical for rbd binding measured by both real-time surface plasmon resonance (spr) and pseudovirus entry. these residues included k267 and r336 on binding patch 1, and l294, i295, r317 and q344 on binding patch 2. the mutations of three positively charged residues k267, r336 and r317 perhaps interfere with the interaction of the negatively charged residues on the surface of rbd; the mutations of l284, i295 and q344 may lead to the change of hydrophobic or hydrophilic properties of hdpp4 at the interface with rbd. our previous findings have shown that the binding interface between hdpp4 and mers-cov rbd is mainly composed of two binding patches, patch 1 and patch 2 (fig. 1a) . the patch 1 interface is characterized by interactions between c-terminal end of the long linker connecting the rbd β6/β7 strands and the hdpp4 blade 4. the contact in patch 1 is critically determined by the polar interactions among a group of hydrophilic amino-acid residues, including rbd e536, d537, d539 and y499 and hdpp4 k267 and r336. in this patch, dpp4 residue k267 interacts with rbd d539 by salt bridge (fig. 1b) , while dpp4 residue r336 forms hydrogen bond with rbd residue y499 (fig. 1c) . patch 2 has a hydrophobic core surrounded by a hydrophilic periphery. in the hydrophobic core, rbd and hdpp4 contacts are critically dependent on a few 'hot spot' residues including rbd l506, w553 and v555, and dpp4 l294 and i295. however, the surrounding hydrophilic surface consists of rbd residues d510, e513 and y540, and dpp4 residues h298, r317 and q344. among these hydrophilic residues, the salt bridge and hydrogen bond between d510 and r317, e513 and q344 contribute to the maintenance of rbd-receptor contact (fig. 1d) . to study the impacts of the substitutions of the critical residues on hdpp4 described above on the interaction between mers-cov rbd and hddp4, we determined the binding efficiency between these two proteins by employing spr technique. first, we constructed a series of hdpp4 mutants guided by the rbd-hdpp4 complex crystal structure information . the wide-type and mutant hdpp4 were introduced into baculovirus expression system. all wide-type and mutant forms of hdpp4 were expressed efficiently (data not shown). second, the binding efficiency was measured by spr. as shown in fig. 2 and table 1 , mutations at several hdpp4 residues, in individual or combination, resulted in a significant attenuation in binding to mers-cov rbd. in patch 1, residue k267 mutation (k267a and k267e) presumably damaged the salt-bridge interaction, completely abrogated the binding between hdpp4 and rbd, while r336a reduced rbd and hdpp4 binding about 100 fold. in patch 2, double mutations at l294 and i295 (l294a þ i295a and l294d þi295d) completely eliminated the binding between rbd and hdpp4, presumably by disrupting hydrophobic interactions with rbd l506, w553 and v555. in contrast, the single-residue substitution of r317a and q344a in the hydrophilic surface of patch 2 had negligible effect on binding efficiency. to further study the importance of the critical residues on hdpp4 on viral entry, we measured the entry efficiency of pseudovirus into cos7 cells expressing the wide-type and mutant forms of hdpp4. the expression levels of the wide-type and mutant hdpp4 were analyzed by fluorescence-activated cell sorting (facs) using goat anti-hdpp4 polyclonal antibody. all of the wide-type and mutant hdpp4 proteins could be expressed on the surface of cos7 cells with the similar expression efficiency (fig. 3a) . forty-eight hours later, these cells were exposed to pseudovirus infection and their entry efficiency was measured by luciferase activity 48 h later. as showed in fig. 3b , the residue mutations located at patch 1 (k267a, k267e and r336a) and hydrophobic region of patch 2 (l294a þi295a and l294aþ i295d) fig. 1 . the amino-acid residue interaction details at the binding interface. (a) two patches of the binding interface. patch 1 interface is characterized by interactions between the c-terminal end of the long linker connecting the rbd β6/β7 strands (light magenta) and the hdpp4 blade 4 (cyan). in patch 2, a gently concaved outer surface in rbd (light magenta) accommodates a linker containing a short α helix between hdpp4 blades 4 and 5 (cyan). (b) and (c) hydrophilic residues of rbd and hdpp4 interact through polar contacts in patch 1. rbd d539 has salt-bridge interaction with hdpp4 residue k267 (b). dpp4 residue r336 forms hydrogen bond with rbd residue y499 (c). the polar contacts (salt-bridge and hydrogen bond) are drawn as black dashed sticks. (d) hot spot residues in the hydrophobic core and hydrophilic periphery of patch 2. resulted in significantly reduction in viral entry. this is consistent with the binding results described previously. in the hydrophilic region of patch 2, residue substitution r317 led to partial loss of viral infection (41.4%), while the mutation q344 modestly increased viral infection (22.8%). in summary, we have identified several key residues in hdpp4 critical for viral binding and entry into target cells. these residues include positively charged residues of patch 1 (k267 and r336) and hydrophobic zone of patch 2 (l294 and i295). in contrast, the mutations at hydrophilic zone of patch 2 (r317 and q344) had little influence on binding and virus entry efficiency. these results showed that the positively charged residues at the outer surface of blade 4 and the hydrophobic regions of blade 5 may play an important role in mediating viral binding and entry into the target cells, while the impact of mutations at hydrophilic region of patch 2 was barely detectable. this is consistent with our earlier findings where residue mutations at the corresponding negatively charged and hydrophobic core positions on rbd of mers-cov could significantly reduce both binding and viral entry efficiency. sequence analysis of dpp4 from multiple animal species (fig. 4 ) showed that mers-cov susceptible animals, such as macaque, camel and bat, shared the same sequence with hdpp4 at blades iv and v. in contrast, those mers-cov resistant animals, such as mouse, rat and ferret, have residues at l294, i295 and r366 that are all different from hdpp4. raj et al. (2014) reported that when these sites of hdpp4 were changed to the residues of ferret, the binding and viral infection efficiency could also be decreased. van doremalen et al. (2014) found 5 residues involved in the hdpp4-rbd interaction which were important to determine the susceptibility to mers-cov infection, in which i295 and r336 were included. these results are consistent with our findings and suggest these residues play an important role in rbd binding and viral entry, and determining the tropism to mers-cov infection. mers-cov rbd (residues 367-606) and the extracellular domain of hdpp4 (residues 39-766) were expressed using a bacto-bacs baculovirus expression system (invitrogen). in brief, the dna encoding rbd and hdpp4 were respectively cloned into the pfastbac™ dual vector (invitrogen) incorporating an n-terminal gp67 signal peptide to facilitate secretion and a c-terminal hexa histidine-tag for purification. the constructed dna was then transformed into the bacterial dh10bac competent cells and the recombined bacmid dna was extracted and transfected into sf9 cells using cellfectin ii reagent (invitrogen). after 5-7 days of incubation at 300 k, the low-titer viruses were harvested and then amplified. the amplified high-titer viruses were then used to infect sf9 cells and the cell culture supernatant containing target protein was harvested 60 h after infection, concentrated, loaded to nickel (ni)-charged resin (ge healthcare), and eluted with 0.5 m imidazole and further purified using the superdex™ 200 highperformance column (ge healthcare) pre-equilibrated with tris buffer (50 mm tris, ph 8.8, 40 mm nacl). fractions containing the purified protein were collected and applied directly to a preequilibrated resource™ q column (ge healthcare) and then eluted with a 0.05-1 m nacl gradient in 40 mm tris buffer (ph 8.8). fractions containing protein were finally purified using super-dex™ 200 column pre-equilibrated with hbs (10 mm hepes, ph 7.2, 150 mm nacl) and centrifuged to 1 mg/ml. mutants of the extracellular domain of hdpp4 were constructed using a standard pcr-based cloning strategy. and the mutant proteins were expressed and purified in the same way. the spr analyses were carried out using a biacore t200 instrument (ge healthcare) equipped with a research-grade cm5 sensor chip. to measure the affinity binding between rbd and wide-type or mutant hdpp4, the rbd was immobilized on the sensor chip by standard amine coupling procedure. the flow cell 1 was left blank to serve as a reference. purified rbd at a concentration of 5 μg/ml in sodium acetate buffer (10 mm, ph 5.0) was immobilized to a density of 300-400 response units on the flow cell 2. for the collection of binding data, hdpp4 or its mutants in a buffer of 10 mm hepes, ph 7.2, 150 mm nacl, and 0.005% (v/v) tween-20 were injected over the two flow cells at a series of concentration at a 30 μl/min flow rate and 298 k. the rbd-hdpp4 complex was allowed to associate for 60 s and dissociated for 60 s. the surfaces were regenerated with an injection of 5 mm naoh between each cycle if needed. the data was analyzed with the biacore t200 evaluation software by fitting to a 1:1 langmuir binding model. mers-cov pseudovirus was generated by co-transfection of human immunodeficiency virus (hiv) backbone expressing firefly luciferase (pnl43r-e-luciferase) and mers-cov spike glycoprotein expression vector (pcdna3.1 þ , invitrogen) into the 293 t cells. viral supernatants were harvested 48 h later, normalized by p24 elisa kit (beijing quantobio biotechnology co., ltd, china) before infecting the target cos7 cells transiently expressing wide-type or mutant hdpp4. the wide-type and mutant hdpp4 expressing cos7 cells were incubated with goat anti-hdpp4 polyclonal antibody (r&d) followed by incubation with fluorescein phycoerythrin (pe)labeled rabbit anti-goat igg antibody (santa cruz). the expression levels of wide-type and mutant hdpp4 were measured by flow cytometer (bd aria ii) and the mean fluorescence intensity (mfi) was analyzed. the cos7 cells infected by mers-cov pseudovirus were lysed at 48 h post infection and viral entry efficiency was quantified by comparing the luciferase activity between pseudoviruses-infected cos7 cells expressing wide-type and those infected cos7 cells expressing mutant hdpp4. the entry efficiency (%) of pseudovirus was calculated on the basis of luciferase activity. and the percentages of pseudovirus entry efficiency shown for mutant hdpp4 were luciferase activity values versus that of the wide-type hdpp4, as the entry efficiency for wide-type hdpp4 was defined as 100%. data shown were corrected for the expression of different hdpp4 constructs by the parameter of mfi. error bars represent standard errors of the means of three independent experiments. student's t-test; n po 0.05; nn po 0.01. crystal structure of the receptor-binding domain from newly emerged middle east respiratory syndrome coronavirus wild-wide-type and innate immune-deficient mice are not susceptible to the middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters the crystal structure of dipeptidyl peptidase iv (cd26) reveals its functional regulation and enzymatic mechanism close relative of human middle east respiratory syndrome coronavirus in bat molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 middle east respiratory syndrome coronavirus (mers-cov) -update. world health organization adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase 4-mediated entry of the middle east respiratory syndrome coronavirus crystal structure of human dipeptidyl peptidase iv/cd26 in complex with a substrate analog host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 rapid generation of a mouse model for middle east respiratory syndrome we thank drs linqi zhang and xinquan wang for their kind support and helpful suggestions. this work was supported by national natural science fund 81101236, 81471929, ministry of science and technology of china (2014cb542500), the national science and technology major projects (2012zx10001-004). key: cord-296657-mymndjvd authors: higuchi, yusuke; suzuki, tatsuya; arimori, takao; ikemura, nariko; kirita, yuhei; ohgitani, eriko; mazda, osam; motooka, daisuke; nakamura, shota; matsuura, yoshiharu; matoba, satoaki; okamoto, toru; takagi, junichi; hoshino, atsushi title: high affinity modified ace2 receptors prevent sars-cov-2 infection date: 2020-09-16 journal: biorxiv doi: 10.1101/2020.09.16.299891 sha: doc_id: 296657 cord_uid: mymndjvd the sars-cov-2 spike protein binds to the human angiotensin-converting enzyme 2 (ace2) receptor via receptor binding domain (rbd) to enter into the cell. inhibiting this interaction is a main approach to block sars-cov-2 infection and it is required to have high affinity to rbd independently of viral mutation for effective protection. to this end, we engineered ace2 to enhance the affinity with directed evolution in human cells. three cycles of random mutation and cell sorting achieved more than 100-fold higher affinity to rbd than wild-type ace2. the extracellular domain of modified ace2 fused to the fc region of the human immunoglobulin igg1 had stable structure and neutralized sars-cov-2 pseudotyped lentivirus and authentic virus with more than 100-fold lower concentration than wild-type. engineering ace2 decoy receptors with directed evolution is a promising approach to develop a sars-cov-2 neutralizing drug that has affinity comparable to monoclonal antibodies yet displaying resistance to escape mutations of virus. coronavirus disease 2019 has spread across the world as a tremendous pandemic and presented an unprecedented challenge to human society. the causative agent of covid-19, sars-cov-2 is a single-stranded positive-strand rna virus that belongs to lineage b, clade 1 of the betacoronavirus genus 1-3 . the virus binds to host cells through its trimeric spike glycoprotein composed of two subunits; s1 is responsible for receptor binding and s2 for membrane fusion 4 . angiotensin-converting enzyme 2 (ace2) is lineage b clade 1 specific receptor including sars-cov-2 3 . the receptor binding domain (rbd) of s1 subunit directly binds ace2 with high affinity, therefore, it is the most important targeting site to inhibit viral infection. in fact, the rbd is the common binding site of effective neutralizing antibodies identified from convalescent patients [5] [6] [7] . rna viruses such as sars-cov-2 have high mutation rates 8 , which are correlated with high evolvability including the acquisition of anti-viral drug resistance. neutralizing antibodies are one of the promising approaches to combat covid-19. accumulating evidence demonstrated that monoclonal antibodies isolated from convalescent covid-19 patients have high potency in neutralizing viruses. however, mutations in the spike gene can lead to the sars-cov-2 adaptation to such neutralizing antibodies. in the replicating sars-cov-2 pseudovirus culture experiment, escape mutation was observed against monoclonal antibody as early as in the first passage 9 and evasion was seen even against the polyclonal convalescent plasma 10 . notably, some mutations identified in in vitro replicating culture experiment are present in natural population according to the database 10 similarly to the anti-rbd antibodies, extracellular domain of ace2, soluble ace2 (sace2), can also be used to neutralize sars-cov-2 as a decoy receptor. the therapeutic potency was confirmed using human organoid 10 , and now apeiron biologics conducts european phase ii clinical trial of recombinant sace2 against covid-19. in addition, fusing sace2 to the fc region of the human igg1 has been shown to enhance neutralization capacity 11 as well as to improve the pharmacokinetics to the level of igg in mice 12 . most importantly, sace2 has a great advantage over antibodies due to the resistance to the escape mutation. the virus with escape mutation from sace2 should have limited binding affinity to cell surface native ace2 receptors, leading to a diminished or eliminated virulence. unfortunately, many reports, including our current study, have revealed that the binding affinity of wild-type sace2 to the sars-cov-2 spike rbd is much weaker (kd ~50 nm) than that of clinical grade antibodies 4, 11, [13] [14] [15] . thus, the therapeutic potential of the wild-type sace2 as a neutralizing agent against sars-cov-2 is uncertain. here we conducted protein engineering with human cell-based directed evolution to improve the binding affinity of ace2 to the spike rbd. random mutations were introduced in the protease domain containing the interface to the rbd, then full length ace2 mutant library was expressed in 293t cells and incubated with fluorescence-labelled rbd. high binding population was sorted and underwent dna extraction, the bulk of which was further induced with random mutations for the next cycle of selection. three cycles of screening resulted in an identification of mutant ace2 clones with more than 100-fold higher binding affinity to the rbd and lower half-maximal inhibitory concentration (ic50) for sars-cov-2 pseudotyped lentivirus as well as authentic virus. the present protein engineering system generates a virus-neutralizing drug that has high affinity comparable with antibodies and can resolve the issue of drug resistance caused by escape mutation. we engineered ace2 to bind the rbd of the sars-cov-2 spike protein with the combination of surface display of mutagenized library and fluorescence-activated cell sorting (facs) to perform the evolution in 293t human cells. the protease domain (pd) of ace2 is known to harbor the interface to viral spike protein, located in the top-middle part of ace2 ectodomain. in this study, ace2 residues 18-102 and 272-409, referred to as pd1 and pd2, respectively, were mutagenized independently. synthetic signal sequence and ha tag were appended and restriction sites were introduced in both sides of pd1 and pd2 by optimizing codon (fig. 1a) . we used error-prone pcr to mutagenize the protease domain of ace2 with an average of about one amino acid mutation per 100 bp, then inserted the fragment into the introduced restriction site by homologous recombination. the reaction sample was transformed to competent cell, generating a library of ~10 5 mutants. mutant plasmid library was packaged into lentivirus, followed by expression in human 293t cells in less than 0.3 moi (multiplicity of infection) to yield no more than one mutant ace2 per cell. cells were incubated with recombinant rbd of sars-cov-2 spike protein fused to superfolder gfp (sfgfp; fig. 1b) . we confirmed the level of bound rbd-sfgfp and surface expression levels of ha-tagged ace2 with alexa fluor 647 in twodimensional display of flow cytometry. top 0.05 % cells showing higher binding relative to expression level were harvested from ~5 x 10 7 cells by facs. to exclude the structurally unstable mutants, cells with preserved signal of surface ace2 were gated. genomic dna was extracted from collected cells and mutagenized again to proceed to the next cycle of screening (fig. 1c) . random mutagenesis screening for pd1 was performed 3 times and mutated sequences from top 0.05% population were reconstructed into the backbone plasmid, and expressed in 293t cells individually. one to three hundred clones were validated for the binding capacity to the rbd-sfgfp. as the selection cycle advances, the two-dimensional distribution of library cells in flowcytometry became broader and higher in rbd-binding signal, and individual clone validation identified several mutants with higher binding capacity (fig.2a) . to evaluate the neutralization activity in the form of sace2, we first generated fusion protein of the soluble extracellular domain of mutagenized ace2 residues 18-614 and sfgfp (sace2-sfgfp) and used them to compete with the cell-surface wt ace2 for the rbd binding. to this end, concentration of each mutant sace2-sfgfp in the cultured medium from transfected cells was quantitatively standardized with sfgfp signal, serially diluted, preincubated with rbd-sfgfp for 30 min, and then transferred to wild-type ace2 expressing 293t cells. after 30 min, the rbd-bound cells were analyzed in flowcytometry. higher neutralization activity against the rbd was confirmed for the mutants that have accumulated mutations (fig. 2b , table. s1). second mutagenesis based on the top hit of first screening, 1-19 mutant, was also performed, but the distribution of the library cells did not expand so much, and we could not isolate clones with significantly higher affinity than the bulk of top 0.05% (fig. s1 ). we next performed pd2 mutagenesis in both the bulk of top 0.05% and one of the highest mutants of the 3 rd library, the clone 3n39. again, the binding distribution of the pd2 library cells was similar to the basal cells, suggesting the inability of this strategy to further increase the affinity to rbd. a recent study reported, via deep mutational scanning, that several specific mutations in pd2 were enriched in high rbd-binding clones 13 . when we added these mutations in 3n39, it did not improve the capacity of the rbd neutralization further ( fig. s2 ). to identify essential mutation(s) in the high affinity ace2 mutants, each mutation was altered to wild-type in mutant 3n39, 3j113 and 3j320. mutant 3n39 contains 7 mutations of a25v, k26e, k31n, e35k, n64i, l79f, and n90h. among them, individual back-mutation of v25, n31, k35, and f79 to wild-type residues resulted in modest to severe reduction of the rbd-neutralization capacity (fig. 3a) , while multiple back mutations of e26, i64, and h90 in various combination did not alter the high activity of the original 3n39 ( fig. 3b) , indicating that a25v, k31n, e35k, and l79f was necessary and sufficient components. in the case of mutant 3j113 that is composed of k31m, e35k, q60r, s70f, l79f, and n90d, similar back mutation experiment revealed that k31m, e35k, and q60r was essential (fig. 3c) . simultaneous back mutations of two (f70/f79 and f70/d90) but not three (f70/f79/d90) nonessential residues were tolerated ( fig. 3d) , suggesting that l79f and n90d may exert their positive effect on the activity only when they coexist. the third mutant 3j320 has t20i, a25v, h34a, t78r, t92q, and q101h. single and multiple back mutation experiments showed that h34a, t92q, and q101h were essential in securing the high inhibitory activity of 3j320 (figs. 3e, f). these top 3 high affinity mutants exhibited higher rbd-neutralization activity when compared with the same sace2 scaffold carrying the two high affinity mutation sets reported recently 11, 13 (fig. 3g ). first mutant library was also sorted in the manner of high and low rbd-sfgfp binding signal (fig. s2a ). the affinity value of each mutant was defined as the ratio of high and low read counts. then, the impact of each amino acid mutation on rbd binding was analyzed as a semi-deep mutational scanning (dms) (fig. s2b ). this analysis revealed that some mutations such as k31n, e35k, and q60r in top 3 high affinity mutants had very mild impact in itself. simple combination of high value mutations, a25v, k26t, q42l, l79v and t92k, referred to here as the dms mutant, and its derivatives showed less neutralization activity than top three high affinity mutants, indicating that each mutation works coordinately in high affinity mutants (fig. 3h ). next, we characterized the binding affinities of the mutant sace2s for spike rbd using surface plasmon resonance, where igg1-fc fused rbd was immobilized as a ligand and the association and dissociation kinetics of his-tagged sace2 were determined. the kd value of wild type sace2 was 41.4 nm, whereas those of mutants 3j38 and 3n39 were determined to be 6.5 nm and 0.37 nm (fig. 4a) . analytical size exclusion chromatography (sec) showed no signs of protein aggregation in the ace2 mutant samples, confirming that the apparent high affinity was not caused by the avidity effect and the observed kd values represent genuine 1:1 affinity toward rbd (fig. s4a ). recombinant soluble ace2 (rsace2) was reported to have a fast clearance rate in human blood with a half-life of hours 16, 17 . recently, it was demonstrated that a rsace2 fused with a fc fragment show high stability in mice 12 as well as higher neutralization activity toward both pseudotyped and authentic sars-cov-2 in cultured cells 11 . we formulated our high affinity mutant sace2s as fc fusion (sace2-fc) and found that the purified proteins were folded well and devoid of aggregation, showing solution behavior indistinguishable from wild type protein (fig. s4b) . to evaluate their efficacy in neutralizing sars-cov-2 infections, affinity-enhanced sace2-fc mutants were assayed for viral neutralization against pseudotyped lentivirus and authentic sars-cov-2. the ic50 values of wild-type, 3j38, and 3n39 for pseudovirus neutralization in ace2-expressing 293t cells were 65.2, 8.9, and 0.43 μg/ml, respectively. in the same way, 3n39 neutralized pseudovirus very efficiently with an ic50 value more than 100 times lower than the wild-type in tmprss2-expressing veroe6 cells, (fig. 4b) . most importantly, when the neutralization potential against the authentic sars-cov-2 in tmprss2expressing veroe6 cells was evaluated, wild-type sace2-fc showed no efficiency even at 100μg/ml, whereas 3n39 sace2-fc demonstrated significant neutralizing effect in 6.3μg/ml (fig. 4c) . sars-cov-2 neutralization is one of the preventative or therapeutic approaches against covid-19. monoclonal antibodies have become one of the common drug modalities, especially as therapeutics against autoimmune diseases and cancer. as virus-neutralizing antibodies, palivizumab is clinically used to prevent hospitalization from respiratory syncytial virus infection in high-risk infants 18 , and cocktail of monoclonal antibodies has been shown to reduce mortality from ebola virus disease 19 . engineered recombinant decoy receptor drugs are also developed to neutralize various cytokines including vascular endothelial growth factor, tumor necrosis factor alpha, and ctla-4 and approved for orbital vascular diseases and rheumatoid arthritis. recombinant sace2 or sace2-fc fusion protein has potency to neutralize sars-cov-2 12,20 , however its modest binding affinity requires higher dose than monoclonal antibody. we developed the screening system based on the cycle of random mutation and sorting of high affinity population in 293t cells followed by validation of neutralizing activity in a soluble form. in this screening, an additional random mutation was induced in the bulk of sorted mutants, which worked better than mutagenesis in the top mutant. engineering of decoy receptors with improved affinity was previously reported for cancer-related molecules and ace2 11, 21, 22 . they used yeast display system to perform directed evolution. large scale library (~10 7 mutants) was prepared and high affinity mutants were identified by repeating sorting from initial library. fast growth rate of yeast is suitable for library screening involving repeated sorting and propagation. we on the other hand employed human cells for the display purpose. since post-translational modification can modulate protein binding affinity, human cell-based screening is better to understand the impact of ace2 variants on viral affinity and also to proceed biologics development. repeating mutagenesis after cell sorting without propagation enabled us to conduct screening with relatively small library (~10 5 mutants) and human cells. during the validation, we noticed that high affinity pattern in the flow cytometry assay of full length ace2 binding rbd-sfgfp did not always correlate with its neutralization activity. thus, it is evident that experimental validation of each mutation at the level of sace2 protein was important for efficient identification of high affinity mutants. our mutant ace2s have affinity comparable to typical anti-spike monoclonal antibodies, but they also offer some advantages over antibodies when considered as a drug candidate. interface of ace2 to the rbd is larger than that of antibodies, which potentially increases efficacy. escape mutation to modified ace2 is likely to result in lower affinity to the native receptor, making such virus much less virulent. sars-cov-2 also enters into host cell via endocytosis. sars-cov-2 infection is mediated not only by tmprss family proteases but by cathepsin l that is catalytically active at ph 3.0-6.5 23 . some antibodies are susceptible to impaired affinity at lower ph, leading to lower viral neutralization. high affinity modified ace2 fused with fc is the promising strategy to neutralize sars-cov-2. the time frame for running one cycle of mutagenesis and sorting was just one week in our system, and we succeeded in developing optimized mutants in a couple of months without depending on patientsderived cells or tissues. thus, our system can rapidly generate therapeutic candidates against various viral diseases and may be well suited for fighting against future viral pandemics. lenti-x 293t cells were purchased from clontech and cultured at 37 °c with 5% co2 in dulbecco's modified eagle's medium (dmem, wako) containing 10% fetal bovine serum (gibco) and penicillin/streptomycin (100 u/ml, invitrogen). veroe6/tmprss2 cells were a gift from national institutes of biomedical innovation, health and nutrition (japan) and cultured at 37 °c with 5% co2 in dmem (wako) containing 5% fetal bovine serum (gibco) and penicillin/streptomycin (100 u/ml, invitrogen). all the cell lines were routinely tested negative for mycoplasma contamination. for a semi-deep mutational scanning of ace2 residues 18-102, 30 % of ha positive cells with the highest and 30% with the lowest gfp fluorescence were also collected in 1st mutated library (fig. s3a) and their genomic dna was extracted by nucleospin tissue (takara). ace2 residues 18-102 plasmid sequence was amplified with primers containing adaptor and barcode sequence to perform deep sequencing on the illumina miseq platform using 300nt paired-end protocol. data were analyzed as follows; high and low gating read count of each mutant was normalized with total counts and log10 ratio of high/low was defined as affinity value. then, each amino acid mutation-containing mutant affinity values were aggregated. . the pcdna4to ha-ace plasmid was transfected into 293t cells (500 ng dna per ml of culture kinetic binding measurement using biacore (spr) the binding kinetics of sace2 (wild-type or mutants) to rbd were analyzed by spr using a biacore pseudotyped reporter virus assays were conducted as previously described 25 . a plasmid coding sars-cov-2 spike was obtained from addgene #145032 14 , and deletion mutant cδ19 (with 19 amino acids deleted from the c terminus) was cloned into pcdna4to (invitrogen) to enhance virus titer 26 . spike-pseudovirus with a luciferase reporter gene was prepared by transfecting plasmids (cδ19, pspax2, and plenti firefly) into lentix-293t cells with lipofectamine 3000 (invitrogen). after 48 hours, supernatants were harvested, filtered with a 0.45 μm low protein-binding filter (sfca), and frozen at -80 °c. ace2-expressing 293t cells were seeded at 10,000 cells per well in 96-well plate. pseudovirus and three-fold dilution series of sace2-fc protein were incubated for 1 hour, then this mixture was administered to ace2-expressing 293t cells. after 1 hour pre-incubation, medium was vero-tmprss2 were seeded on 24 well plates (80,000 cells/well) and incubated for overnight. the culture supernatants serially diluted by medium were inoculated and incubated for 2 hours. culture medium was removed, fresh medium containing 1% methylcellulose (1.5ml) was added, and the culture was further incubated for 3 days. the cells were fixed with 4% paraformaldehyde phosphate buffer solution (nacalai tesque) and plaques were visualized by using a crystal violet. table. s1 amino acid sequence and rbd neutralization activity value of validated mutants. the value of rbd neutralization activity was calculated as -log2 concentration of 50% rbd-sfgfp bound competing relative to 3n39. a pneumonia outbreak associated with a new coronavirus of probable bat origin a new coronavirus associated with human respiratory disease in china functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses cryo-em structure of the 2019-ncov spike in the prefusion conformation a human neutralizing antibody targets the receptor-binding site of sars-cov-2 human neutralizing antibodies elicited by sars-cov-2 infection potent neutralizing antibodies against sars-cov-2 identified by high-throughput single-cell sequencing of convalescent patients' b cells why are rna virus mutation rates so damn high? antibody cocktail to sars-cov-2 spike protein prevents rapid mutational escape seen with individual antibodies escape from neutralizing antibodies by sars-cov-2 spike protein variants engineered ace2 receptor traps potently neutralize sars-cov-2. biorxiv neutralization of sars-cov-2 spike pseudotyped virus by recombinant ace2-ig engineering human ace2 to optimize binding to the spike protein of sars coronavirus 2 structural basis of receptor recognition by sars-cov-2 structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor targeting the degradation of angiotensin ii with recombinant angiotensinconverting enzyme 2: prevention of angiotensin ii-dependent hypertension pharmacokinetics and pharmacodynamics of recombinant human angiotensinconverting enzyme 2 in healthy human subjects humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants. the impact-rsv study group controlled trial of ebola virus disease therapeutics inhibition of sars-cov-2 infections in engineered human tissues using clinical-grade soluble human ace2 an engineered axl 'decoy receptor' effectively silences the gas6-axl signaling axis antitumor activity of an engineered decoy receptor targeting clcf1-cntfr signaling in lung adenocarcinoma genome-wide crispr screen reveals host genes that regulate sars-cov-2 infection human secretory signal peptide description by hidden markov model and generation of a strong artificial signal peptide for secreted protein expression protocol and reagents for pseudotyping lentiviral particles with sars-cov-2 spike protein for neutralization assays retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus s protein we would like to thank sho hashimoto, toshiyuki nishiji, tomohiro hino, and keiko tamura key: cord-281536-8y7yxcp4 authors: lim, hocheol; baek, ayoung; kim, jongwan; kim, min sung; liu, jiaxin; nam, ky-youb; yoon, jeonghyeok; no, kyoung tai title: hot spot profiles of sars-cov-2 and human ace2 receptor protein protein interaction obtained by density functional tight binding fragment molecular orbital method date: 2020-10-08 journal: sci rep doi: 10.1038/s41598-020-73820-8 sha: doc_id: 281536 cord_uid: 8y7yxcp4 the prevalence of a novel β-coronavirus (sars-cov-2) was declared as a public health emergency of international concern on 30 january 2020 and a global pandemic on 11 march 2020 by who. the spike glycoprotein of sars-cov-2 is regarded as a key target for the development of vaccines and therapeutic antibodies. in order to develop anti-viral therapeutics for sars-cov-2, it is crucial to find amino acid pairs that strongly attract each other at the interface of the spike glycoprotein and the human angiotensin-converting enzyme 2 (hace2) complex. in order to find hot spot residues, the strongly attracting amino acid pairs at the protein–protein interaction (ppi) interface, we introduce a reliable inter-residue interaction energy calculation method, fmo-dftb3/d/pcm/3d-spies. in addition to the sars-cov-2 spike glycoprotein/hace2 complex, the hot spot residues of sars-cov-1 spike glycoprotein/hace2 complex, sars-cov-1 spike glycoprotein/antibody complex, and hcov-nl63 spike glycoprotein/hace2 complex were obtained using the same fmo method. following this, a 3d-spies-based interaction map was constructed with hot spot residues for the hace2/sars-cov-1 spike glycoprotein, hace2/hcov-nl63 spike glycoprotein, and hace2/sars-cov-2 spike glycoprotein complexes. finally, the three 3d-spies-based interaction maps were combined and analyzed to find the consensus hot spots among the three complexes. as a result of the analysis, two hot spots were identified between hace2 and the three spike proteins. in particular, e37, k353, g354, and d355 of the hace2 receptor strongly interact with the spike proteins of coronaviruses. the 3d-spies-based map would provide valuable information to develop anti-viral therapeutics that inhibit ppis between the spike protein of sars-cov-2 and hace2. domain (rbd) of s1 undergoes hinge-like conformational changes that transiently conceal or reveal the determinants of receptor binding 3 . sars-cov-2 could possibly use angiotensin-converting enzyme 2 (hace2), the same receptor as sars-cov 4 and hcov-nl63. since the essential function of the s protein is to penetrate host cells, it is considered as the optimal target for the prevention of cell infection. for this reason, s protein-targeted antibody-mediated neutralization has been considered as a suitable treatment for sars-cov diseases. therefore, the hot spot analysis on the interface between the rbd domain of the s1 subunit and the hace2 receptor would provide crucial information for antibody engineering and for small-molecular drug development. to investigate protein-protein interactions (ppis) between hace2 and the rbd domain of s1 subunit at the molecular level, an ab initio quantum mechanical (qm) method was introduced. this method was used to obtain the most accurate information on the ppis through analysis of the wave function obtained from the qm calculation, especially of the fragment molecular orbital (fmo) approximation method. even with the fmo method, the calculations in a biomolecular system need a huge amount of computer resources. in order to obtain results within a reasonable computation time while maintaining a certain degree of accuracy of ab initio mo, we introduced the density functional tight-binding (dftb) method, which is an efficient parameterized qm method and is expected to exhibit reasonable accuracy at a remarkably reduced computational cost 7 . the fmo method is one of various linear-scaling methods to reduce the huge computational cost of qm calculations by the fragmentation of target molecules. the energies of fragment and their pairs are computed in the embedding electrostatic potential 8 . recently, the fmo method has been combined with dftb, and the polarizable continuum model (pcm) was introduced to consider the effect of a solvent on a model system 9 . pair interaction energies (pies) among the fragments of the model system from the fmo-dftb/pcm method correlate well with pies from ab initio dft fmo/pcm and with an ignition møller-plesset perturbation theory (mp2) fmo/pcm 9 . in our earlier work, we investigated ppis between programmed cell death 1 and its ligand pd-l1 using fmo-mp2/ pcm and the results efficiently explained the experimental site-directed mutagenesis data 10 . in this work, to find common hot spot amino acids on the interfaces between the rbd domain and hace2 of the three complexes, rbd-sars-cov-2/hace2 (twelve experimental structural data), rbd-sars-cov-1/ hace2 (four experimental structural data), and rbd-hcov-nl63/hace2 (one experimental structural data), we performed fmo-dftb3/d/pcm calculations. to visualize the interaction energy and the distance of the interacting amino acid pairs, the fmo/3d-spies analysis tool was introduced. to narrow down the hot spot region, we also performed the same calculation with rbd-sars-cov-1/antibody complexes (five experimental structural data). based on the fmo/3d-spies results, we constructed 3d-spies-based interaction maps of the hace2 and rbd domains from sars-cov-1, hcov-nl63, and sars-cov-2. in order to validate the ppi predictability of the fmo-dftb3/d/pcm results, we compared them with the site-directed mutagenesis results. consequently, we summarized the fmo-dftb3/d/pcm/3d-spies results as interaction maps and found the hot spot regions in rbd-sars-cov-2 and hace2 at a qm level. all experimental structures calculated in this work are summarized in table 1 . all missing side chains were filled using prime implemented in maestro program 11 . hydrogen atoms were added to the crystal structures at ph 7.0 and their positions were optimized with the propka function implemented in maestro program 12 . water molecules in the crystal structures were included in the fmo calculations to explore their roles in ppis. in the fmo calculations, all n-acetylglucosamine (glcnac) residues in the rbd domains and hace2 were included, only rbd domains of three coronaviruses were included, and only fv domains of antibodies that bind to rbd domains were included. all fmo calculations were performed with the version feb 14, 2018 gamess 13 . the two-body fmo method was applied to all calculations in this work for the fmo2/dftb3 method; this is a recent extension of the selfconsistent-charge density functional tight-binding method and derived via a third-order expansion of the dft method 9 . dftb3 calculations were performed using the 3ob parameter set 14, 15 , and the uff-type dispersion correction (dftb3/d) 16, 17 . due to the exchange-repulsion term is not computed in dftb, the ex terms are all zero (see supplementary table s2-s25) . the polarizable continuum model (pcm), an implicit solvation model, was employed with the explicitly expressed water molecules present in the x-ray crystal and cryo-em structures. all input files were prepared in compliance with the hybrid orbital projection (hop) scheme fragmentation 18 . each residue and water molecule was defined as one fragment. two cysteine residues forming the disulfide bridge were defined as one fragment, and glcnac, with which the asparagine residue formed covalent bonds, was defined as one fragment. all 3d-spies results were generated with the reported protocol 10 . in the protocol, we selected only pies within a specific distance (5.0 å) between two fragments, which reflected the distance used for the approximate of electrostatic potential in fmo method 19 . we considered the interaction with an pie more stable than − 3.0 kcal/mol to be significant on the basis of previous reports 10, 20, 21 . to investigate ppis between hace2 and rbd domains of the three coronaviruses, we collected 23 experimental structures summarized in table 1 and performed fmo-dftb3/d/pcm calculations for all the experimental structures. due to structural arrangements from mutations summarized in table s1 , we collected all available structures to consider them together. subsequently, we performed hot spot analysis using the fmo/3d-spies tool. hot spot region between hace2 and rbd-sars-cov-1. in order to investigate the hot spot region in the rbd of the sar-cov-1 and hace2 receptor complex, we performed fmo calculations on 12 rbd-sars-cov-1/hace2 complexes (supplementary table s2 -s13). we summarized the fmo results in fig 23 . when comparing the 69 amino acid pairs of this study with the mutagenesis experimental results from two papers, it was confirmed that 32 of the 69 amino acid pairs correlated with the experimental results. the changes in the binding affinity between the proteins that form a complex by mutation can be explained by comparing the structural changes (i.e. changes in the amino acid pairs that contribute to the increase or decrease of the binding affinity) of the mutated proteins with those of the wild-type proteins. qu et al. reported that the n479k/t487s mutation on rbd-sars-cov-1 lowers the binding affinity 24 . one complex (pdb id: 3d0h) has the t487s mutation in rbd-sars-cov-1. t487 in wt rbd-sars-cov-1 attractively interacts with 6 amino acids, y41, g326, n330, g354, f356, and r357, whereas s487 in the mutated complex attractively interacts with only 3 amino acids, n330, g354, and r357. wu et al. reported that the k31t mutation on hace2 increases the binding affinity 25 , because the k31 in wt hace2 (pdb id: 2ajf) interacts only with y442 of rbd-sars-cov-1, whereas t31 in the mutated hace2 (pdb id: 3d0g) interacts with two amino acids, y442 and y475. the common hot spot region in rbd-sars-cov-1 against hace2 and sars-cov-1 antibodies. in order to narrow down the hot spot regions between hace2 and rbd-sars-cov-1, we performed fmo calculations on four rbd-sars-cov-1/antibody complexes (supplementary table s14-s19). we summarized the fmo results in fig. 1 . 26 when comparing the 30 amino acid pairs of this study with the previously reported results, it was confirmed that 17 of the 30 amino acid pairs are correlated: r426/y53, t433/w226, n437/r162, y440/d182, p470/d202, n479/d182, d480/r162, d480/s163, d480/n164, d480/r223, y481/r223, y484/y102, t486/y53, t487/y53, t487/d99, g488/a33, and y491/d99. in rbd-sars-cov-1/m395 (pdb id: 2dd8) complex, the fmo results detected 18 amino acid pairs, which are summarized in supplementary table s15 . the amino acid pairs that contributed to the stability of the complexes are well correlated with the published sitedirected mutagenesis study, in which the t487 mutation does not significantly affect the neutralizing activity of the antibody 27 . the fmo results supported that t487s mutation would change only minor van der waals interactions between t487 and hc y32. in the rbd-sars-cov-1/s230 (pdb id: 6nb6, 6nb7) complex, the fmo results detected 25 amino acid pairs, which are summarized in supplementary table s16-s18. the s230 binds to rbd-sars-cov-1 in different two states. the fmo results of state 1 are detailed in supplementary table s16, and those of the state 2 are mentioned in supplementary table s17-s18. in the rbd-sars-cov-1/ the interactions between four antibodies (80r, m395, s320, and f26g13) and rbd domain from sar-cov-1 are shown in the right-hand with color bars. the main hot spot region is colored in light red, and the secondary hot spot region in hace2 is colored in light blue, and all interactions shown in this map have attractive pie value more stable than − 3.0 kcal/mol, whose magnitudes are ignored. in order to find common hot spot amino acids in rbd-sars-cov-1 against hace2 and sars-cov-1 antibodies, we illustrated the fmo results with a 3d-spies-based map. (see fig. 1 ). all four antibodies (80r, m395, s230, and f26g19) and hace2 have two common amino acids, r426 and t487, in rbd-sars-cov-1. three of the four antibodies and hace2 have four common amino acids, t486, g488, i489, and y491, in rbd-sars-cov-1. two of the four antibodies and hace2 have two common amino acids, f483 and q492, in rbd-sars-cov-1. only s230 and hace2 share four common amino acids, d463, n473, y475, and y442, in rbd-sars-cov-1. only 80r and hace2 share two common amino acids, q479 and y484, in rbd-sars-cov-1. other interactions between antibodies and rbd-sars-cov-1 do not share interactions between hace2 and rbd-sars-cov-1. considering the possibility of mutation prediction in viruses by the fmo methods 28, 29 , the evolutionary process of sars-cov-1 can be performed to elude neutralization of antibody by switching the unshared interactions between the antibody and hace2 receptor. according to the map, there are two hot spot regions between hace2 and rbd-sars-cov-1 (see fig. 1 ). the main hot spot region on hace2 consists of d38, y41, k353, d355, and several residues. the counter part of that on rbd-sars-cov-1 comprises r426, t486, t487, i489, y491, and so on. the secondary hot spot region on hace2 receptor consists of d30, k31, and several residues. the counter part of that on rbd-sars-cov-1 comprises y442, d463, n473, n479, and so on. we found that sars-cov-1 antibodies focus on the main hot spot to block the formation of amino acid pairs between hace2 and rbd-sars-cov-1. although the rbd of hcov-nl63 does not share structural homology with the rbds of sars-cov-1 and sars-cov-2, the three viruses recognize the same hace2 receptor to invade host cells. in order to investigate the hot spot region between hcov-nl63 and hace2, we performed fmo calculations on the hcov-nl63/hace2 complex (pdb id: 3kbh). the fmo results in which 23 amino acid pairs were detected are summarized in supplementary table s20 . the fmo results were in agreement with the six amino acid pairs (hace2/rbd-hcov-nl63) previously reported by wu et al. 30 : d30/s496, h34/g495, h34/s496, e37/y498, m323/h586, and g354/g537. in order to find amino acids in hot spot regions in the ppi interface between sars-cov-2 and hace2, we performed fmo calculations on four sars-cov-2/hace2 complexes (supplementary table s21 to investigate the common hot spot region on hace2 against rbds from the three viruses, and vice versa, we illustrated the fmo results in fig. 1 . in the three viruses, all rbds have common interactions with d30, k31, e37, k353, g354, and d355 in hace2. sars-cov-1 and sars-cov-2 have common interactions with the s19, q24, f28, e35, a36, d38, y41, q42, y83, e329, n330, and r357 in hace2. only sars-cov-1 and hace2 share interactions with e23, a25, f32, t324, q325, g326, and f356, whereas only nl63-cov and hace2 share interactions with n33, m323, and f327. the common interactions between sars-cov1 and nl63-cov were h34 and r393 in hace2. we created a 3d-spies based interaction map to find the hot spot regions from the ppi information between hace2 and rbd-sars-cov-2 (see figs. 1 and 2) . when comparing the interacting residues between hace2 and rbd of the three viruses, there are two hot spot regions consisting of shallow grooves on the hace2 receptor. the main hot spot is formed by e37, k353, g354 and d355. the secondary hot spot consists of d30 and k31. according to the map, the main hot spot is expected to be the most important hot spot between hace2 and rbd-sars-cov-2. we observed that the secondary hot spot on hace2 has interactions with k417, l455, e484, p491, and q493 in rbd-sars-cov-2, whereas the main hot spot has interactions with r403, f497, q498, t500, n501, g502, y505, and q506 in rbd-sars-cov-2. the results from the common hot spot region in sars-cov-1 antibodies supported the results that the main hot spot region was important for the ppi between rbd-sars-cov-1 and its antibodies. in the results of sars-cov-2 and its antibody (b38) summarized in supplementary table s25 , the antibody had interactions with r403, q498, n501, g502, and y505 of rbd-sars-cov-2, which are the counterpart of the main hot spot. it can be used to develop antibodies and antiviral agents by using the information of the hot spot regions suggested in this work. even though the fmo method was successfully applied to evaluate ppis, analysis of biomolecular systems still requires huge computational costs. here, we combined parameterized quantum chemical approaches (fmo-dftb3/d/pcm) and the 3d-scattered pair interaction energies (3d-spies) protocol to analyze ppis between sars-cov-2 and hace2 complex. the fmo-dftb3/d/pcm/3d-spies results also showed a qualitative scientific reports | (2020) 10:16862 | https://doi.org/10.1038/s41598-020-73820-8 www.nature.com/scientificreports/ www.nature.com/scientificreports/ correlation with site-directed mutagenesis results, such as the fmo-mp2/pcm/3d-spies results in our earlier work 10 . the reliable inter-residue interaction energy calculation method, fmo-dftb3/d/pcm/3d-spies, would be a powerful tool for drug discovery and protein engineering in the future. furthermore, the quantum-mechanical-level hot spot analysis results will provide new directions for antibody engineering and small-molecule development. the 3d-spies-based map would provide valuable information for the discovery of anti-viral therapeutics that inhibit ppis between the spike protein of sars-cov-2 and hace2. a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster clinical features of patients infected with 2019 novel coronavirus in wuhan cryo-em structure of the 2019-ncov spike in the prefusion conformation the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak-an update on the status structure, function, and evolution of coronavirus spike proteins the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex dftb3: extension of the self-consistent-charge density-functional tight-binding method (scc-dftb) fragment molecular orbital method: an approximate computational method for large molecules the fragment molecular orbital method combined with density-functional tight-binding and the polarizable continuum model investigation of protein-protein interactions and hot spot region between pd-1 and pd-l1 by fragment molecular orbital method on the role of the crystal environment in determining protein side-chain conformations propka3: consistent treatment of internal and surface residues in empirical p k a predictions gamess as a free quantum-mechanical platform for drug research parametrization and benchmark of dftb3 for organic molecules parameterization of dftb3/3ob for sulfur and phosphorus for chemical and biological applications an efficient a posteriori treatment for dispersion interaction in density-functional-based tight binding uff, a full periodic table force field for molecular mechanics and molecular dynamics simulations fragment molecular orbital method: application to polypeptides fragment molecular orbital method: use of approximate electrostatic potential the fragment molecular orbital method reveals new insight into the chemical nature of gpcr-ligand interactions exploring chemistry with the fragment molecular orbital method a virus-binding hot spot on human angiotensin-converting enzyme 2 is critical for binding of two different coronaviruses receptor and viral determinants of sars-coronavirus adaptation to human ace2 identification of two critical amino acid residues of the severe acute respiratory syndrome coronavirus spike protein for its variation in zoonotic tropism transition via a double substitution strategy mechanisms of host receptor adaptation by severe acute respiratory syndrome coronavirus structural basis of neutralization by a human anti-severe acute respiratory syndrome spike protein antibody, 80r structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody possibility of mutation prediction of influenza hemagglutinin by combination of hemadsorption experiment and quantum chemical calculation for antibody binding prediction of probable mutations in influenza virus hemagglutinin protein based on large-scale ab initio fragment molecular orbital calculations crystal structure of nl63 respiratory coronavirus receptor-binding domain complexed with its human receptor h.l. a.b., and j.k. contributed equally to this work. m.k. and j.l. supported this work by collecting mutation data. all authors contributed to writing the manuscript and approved the final version of the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/10.1038/s4159 8-020-73820 -8.correspondence and requests for materials should be addressed to k.t.n.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. key: cord-267001-csgmc155 authors: george, parakkal jovvian; tai, wanbo; du, lanying; lustigman, sara title: the potency of an anti-mers coronavirus subunit vaccine depends on a unique combinatorial adjuvant formulation date: 2020-05-27 journal: vaccines (basel) doi: 10.3390/vaccines8020251 sha: doc_id: 267001 cord_uid: csgmc155 vaccination is one of the most successful strategies to prevent human infectious diseases. combinatorial adjuvants have gained increasing interest as they can stimulate multiple immune pathways and enhance the vaccine efficacy of subunit vaccines. we investigated the adjuvanticity of aluminum (alum) in combination with rasp-1, a protein adjuvant, using the middle east respiratory syndrome coronavirus mers-cov receptor-binding-domain (rbd) vaccine antigen. a highly enhanced anti-mers-cov neutralizing antibody response was induced when mice were immunized with rasp-1 and the alum-adjuvanted rbd vaccine in two separate injection sites as compared to mice immunized with rbd + rasp-1 + alum formulated into a single inoculum. the antibodies produced also significantly inhibited the binding of rbd to its cell-associated receptor. moreover, immunization with rasp-1 co-administered with the alum-adjuvanted rbd vaccine in separate sites resulted in an enhanced frequency of tfh and gc b cells within the draining lymph nodes, both of which were positively associated with the titers of the neutralizing antibody response related to anti-mers-cov protective immunity. our findings not only indicate that this unique combinatorial adjuvanted rbd vaccine regimen improved the immunogenicity of rbd, but also point to the importance of utilizing combinatorial adjuvants for the induction of synergistic protective immune responses. vaccination is one of the most successful strategy to prevent infectious diseases in the human population, including those caused by emerging viruses [1] . among various vaccine types, such as inactivated virus, live attenuated virus, and viral vector-based vaccines, subunit vaccines using proteins or peptides are believed to be much safer as they do not contain any live virus components and/or cause undesirable severe side effects [2] . however, unlike attenuated vaccines (composed of a virus or bacterium that replicates within the host) or inactivated vaccines (composed of either heat or chemically-inactivated parts of the pathogen), subunit vaccines (that are derived from known pathogen target antigens) are generally much less immunogenic, which can be improved with the addition of appropriate adjuvant(s) to the vaccine [1] . adjuvants play an important role in enhancing the potency of subunit vaccines by improving humoral and/or cellular immune responses to the various subunit protein vaccines, decreasing the antigen dosages, and/or reducing immunization regimens [3, 4] . aluminum salts (hereinafter alum) c57bl/6 mice were immunized intramuscularly (i.m.) twice, three weeks apart, with mers-rbd-fd (5 µg; hereinafter rbd) formulated with or without alum (alhydrogel ® 250 µg) (invivogen, san diego, ca, usa), rasp-1 (25 µg), or with distinct adjuvant combinations. inoculums were prepared in a final volume of 100 µl per mouse and 50 µl was injected in the caudal thigh muscle of each hind leg in the appropriate site as outlined in table 1 . control mice were injected with pbs in 0.1% sds, the buffer solution of rasp-1 and is referred to as naive mice hereafter. complete adsorption of the rbd and rasp-1 proteins by alum was confirmed by sds-gel electrophoresis of the unbound protein samples after absorption with alum for 30 min on a rotator at rt. sera samples were collected 7 days post-2nd immunization for analyses of anti-mers-cov neutralizing antibody titers, inhibition of mers-cov rbd-dpp4 receptor binding, and mers-cov rbd-specific antibody responses using mers-cov s1 as a target. the draining lymph nodes from each hind leg per mouse were also recovered at day 7 post-2nd immunization for analyses of the various cell profiles within. the neutralizing activity of sera from the immunized mice against mers-cov infection in vitro was carried out using our established pseudovirus neutralization assay [17, 20] . briefly, 293t cells were co-transfected with a plasmid encoding the s protein of mers-cov (strain emc2012) and a plasmid encoding env-defective, luciferase-expressing hiv-1 genome (pnl4-3.luc.re). the supernatants containing mers-cov s expressing pseudovirus collected 72 h post-transfection were incubated with serially diluted mouse sera at 37 • c for 1 h. the virus-serum mixtures were then added into huh-7 cells expressing the mers-cov receptor dpp4. the cells were refed with fresh medium 24 h later, and after 72 h, lysed using cell lysis buffer (promega, madison, wi, usa) before the supernatants were transferred into 96-well luminometer plates. after addition of luciferase substrate (promega, madison, wi, usa), the plates were measured for relative luciferase activity using infinite 200 pro luminator (tecan, männedorf, switzerland). neutralizing activity was calculated using the calcusyn computer program [25] and is expressed as 50% pseudovirus neutralizing antibody titers (nt 50 ). sera from immunized mice were tested for their ability to inhibit the binding of the recombinant mers-rbd-fc protein to the cell-associated hdpp4 receptor in huh-7 cells using flow cytometry analysis [26] . briefly, cells were incubated with the mers-rbd-fc protein (5 µg/ml) in the presence or absence of diluted mouse sera (1:50) for 30 min at room temperature. after three washes and staining with fitc-labeled goat anti-human igg fc secondary antibody (1:500, thermo fisher scientific, waltham, ma, usa) for 30 min at room temperature, the cells were measured for fluorescence in a flow cytometer (bd lsrfortessa 4 system). mean fluorescence intensity (mfi) values of the fitc channel from cells incubated with mers-rbd-fc protein in the absence of diluted sera were treated as 100 percentage binding. inhibition of binding was calculated as the percentage of reduced binding to hdpp4 receptor in the presence of diluted sera from the different immunization groups versus the maximum binding observed in the absence of sera. elisa to measure mers-cov rbd-specific antibody responses in immunized mouse sera was performed with mers-cov s1 as the target antigen and as previously described with some modifications [17, 20] . the mers-cov s1 subunit of the mers-cov s protein contains the rbd region of the virus. briefly, 96-well elisa plates were coated with mers-cov s1 (1 µg/ml) overnight at 4 • c, blocked with 2% fat-free milk in pbs containing tween-20 (pbst) at 37 • c for 2 h, and then washed with pbst 3 times. the plates were subsequently incubated at 37 • c for 1 h with serially diluted mouse sera, and horseradish peroxidase (hrp)-conjugated anti-mouse igg (1:5000), igg1 (1:5000), or igg2c (1:2000) antibodies (thermo fisher scientific, waltham, ma, usa). the substrate 3,3 ,5,5 -tetramethylbenzidine (sigma-aldrich, st. louis, mo, usa) was added to the plates after additional washes, and the reaction was stopped by the addition of 1 n h 2 so 4 . absorbance at 450 nm was measured using an elisa plate reader (tecan, männedorf, switzerland). endpoint titers were calculated as the reciprocal of the highest dilution of sera giving an optical density greater than the mean ± 3 times the standard deviation of sera from naïve mice. draining lymph nodes from each hind leg per mouse were harvested at day 7 post-2nd immunization. the lymph nodes were dissociated into single cell suspensions using a syringe plunger, then passed through a 70 µm cell strainer and resuspended in complete rpmi 1640 media containing 10% fetal bovine serum (r10). subsequently, 2.5 × 10 6 cells were washed and resuspended in fresh r10 media in a 96-well cell culture plate for flow cytometry staining. 1.5 × 10 6 cells were stained with a labeled antibodies: cd45-af700, cd11b-pe-cy5, cd11c-bv711, ly6c-percp, cd40-apc, ccr7-pe, cd80-bv650, cd86-bv421, ly6g-pe-cy7, and b220-bv605. while 1 × 10 6 cells were stained with a cocktail of the following fluorescently labeled antibodies: cd45-af700, cd4-pe-cy7, cxcr5-bv605, pd-1-bv421, b220-bv650 and gl-7-af647 (all from biolegend, san diego, ca, usa) , and cd95-bv510 (bd biosciences, dublin, ireland), in a brilliant violet cell stain buffer (bd biosciences, dublin, ireland) for 20 min in the dark at room temperature. cells were then washed, resuspended in cell stain buffer (biolegend, san diego, ca, usa), and the number of stained cells was acquired using bd lsrfortessa cell analyzer (bd biosciences, dublin, ireland). the data were analyzed using flowjo software (tree star, ashland, or, usa). cd45 + cd11c -ly6c + cells were identified as monocytes, cd45 + cd11c -ly6c + cd40 + cells were identified as activated monocytes, cd45 + cd11c -ly6c + ccr7 + cells were identified as migratory monocytes, cd45 + cd4 + cells were identified as cd4 + t cells, cd45 + cd4 + cxcr5 + pd-1 + cells were identified as tfh cells, cd45 + b220 + cells were identified as b cells and cd45 + b220 + cd95 + gl-7 + cells were identified as gc b cells. one-way anova test with tukey's multiple comparison was used for statistical analysis using graphpad prism v6 (graphpad, san diego, ca, usa). spearman correlation was performed to determine the association of the fold increase in the frequency of tfh and gc b cells with neutralizing antibody titers using graphpad prism v6 (graphpad, san diego, ca, usa). p < 0.05: *, p < 0.01: **, p < 0.001: ***, p < 0.0001: ****. nd: not detectable. to investigate whether rasp-1 in combination with alum enhances the humoral immune responses induced by the mres-rbd-fd (herein after rbd) vaccine, we immunized c57bl/6 mice twice. three weeks apart, using a formulation where rasp-1 and the rbd vaccine proteins were completely adsorbed to alum and then administered as a single inoculum (table 1 ; group 5). this adjuvanted vaccine was compared to that in which rasp-1 was co-administered with the alum-adjuvanted rbd vaccine as two inoculums and in two separate sites of the caudal thigh muscle (table 1 ; group 6). rbd formulated with either rasp-1 or alum alone, rbd alone, and pbs alone were included as controls. table 1 . immunization of mice using different combinations and/or formulations of the vaccines and the site of injection: c57bl/6 mice were immunized intramuscularly (i.m.) with mers-rbd-fd (rbd) formulated with or without alum and/or rasp-1 alone or together in different combinations and/or formulations. mice were immunized twice, 3 weeks apart according to the various g1-g6 experimental groups either at the front (a: 50 µl of inoculum) and/or the back (b: 50 µl of inoculum) of the caudal thigh muscle in each hind leg. injection immunization of mice with rasp-1 and the alum-adjuvanted rbd vaccine in separate sites (g6, figure 1 ) significantly resulted in the highest neutralizing antibody titers against mers-cov infection in vitro, nt 50 = 17,657. it was approximately four-fold higher than in mice that were vaccinated with rbd + rasp-1 + alum in a single inoculum (g5 − nt 50 = 4453; figure 1 ),~10-,~3-, and 85-fold higher when compared to rasp-1-adjuvanted rbd vaccine, alum-adjuvanted rbd vaccine, and rbd only, respectively (g3 − nt 50 = 1839, g4 − nt 50 = 6528, g2 − nt 50 = 207, respectively; figure 1 ). it appears that this unique rasp-1 and alum combinatorial adjuvants promoted synergy in the functional humoral response produced vs. the vaccines that utilized the other formulations and/or regimens, including the rasp-1-adjuvanted rbd vaccine. ). it appears that this unique rasp-1 and alum combinatorial adjuvants promoted synergy in the functional humoral response produced vs. the vaccines that utilized the other formulations and/or regimens, including the rasp-1-adjuvanted rbd vaccine. (table 1 and x-axis legend). sera samples were collected 7 days post-2nd immunization and analyzed for neutralization of the pseudotyped mers-cov. the data represents the mean and standard error (sem) of nt50 titers from at least two independent experiments with 3 to 5 mice per group. "+" indicates the presence and "−" indicates the absence of the protein or adjuvants in the formulation. statistics was performed using one-way anova with tukey's multiple comparison. p < 0.001: ***, p < 0.0001: ****, nd: not detectable. when the total igg response to the mers-rbd antigen was studied using the mers-cov s1 protein as the target protein, we found that although the rbd-specific total igg antibody titers were ~8 times higher in mice that were vaccinated by co-administrating rasp-1 and the alum-adjuvanted rbd vaccine in separate sites (g6-142,525 end point titer), they were not significantly different from those elicited by immunization with rbd + rasp-1 + alum administered in a single inoculum (g5-18,149 end point titer), or with the alum-adjuvanted rbd vaccine (g4-53,104 end point titer; figure s1a ). nevertheless, the co-administration of rasp-1 and the alum-adjuvanted rbd vaccine in separate sites significantly increased the rbd-specific total igg antibody titers by ~20-fold compared to rasp-1-adjuvanted mers-rbd vaccine or ~160-fold compared to the rbd vaccine alone (g6-142,525, g3-6700 and g2-872 end point titers respectively; figure s1a ); clearly showing that immunization using the unique rasp-1 and alum combinational adjuvants had a beneficiary effect in comparison to the rasp-1 (~20 fold) or the alum (~3 fold) adjuvanted vaccines. to elucidate the igg subtypes induced in the different immunization groups, we also analyzed the rbd-specific igg1 and igg2c antibody titers. we observed that the highest titer of igg1 antibodies was induced when the rbd vaccine was formulated with alum alone or with rasp-1 + alum in one inoculum (g4-250,951 and g5-369,000 end point titers respectively; figure s1b ), with the titers being (table 1 and x-axis legend). sera samples were collected 7 days post-2nd immunization and analyzed for neutralization of the pseudotyped mers-cov. the data represents the mean and standard error (sem) of nt 50 titers from at least two independent experiments with 3 to 5 mice per group. "+" indicates the presence and "−" indicates the absence of the protein or adjuvants in the formulation. statistics was performed using one-way anova with tukey's multiple comparison. p < 0.001: ***, p < 0.0001: ****, nd: not detectable. when the total igg response to the mers-rbd antigen was studied using the mers-cov s1 protein as the target protein, we found that although the rbd-specific total igg antibody titers werẽ 8 times higher in mice that were vaccinated by co-administrating rasp-1 and the alum-adjuvanted rbd vaccine in separate sites (g6-142,525 end point titer), they were not significantly different from those elicited by immunization with rbd + rasp-1 + alum administered in a single inoculum (g5-18,149 end point titer), or with the alum-adjuvanted rbd vaccine (g4-53,104 end point titer; figure s1a ). nevertheless, the co-administration of rasp-1 and the alum-adjuvanted rbd vaccine in separate sites significantly increased the rbd-specific total igg antibody titers by~20-fold compared to rasp-1-adjuvanted mers-rbd vaccine or~160-fold compared to the rbd vaccine alone (g6-142,525, g3-6700 and g2-872 end point titers respectively; figure s1a ); clearly showing that immunization using the unique rasp-1 and alum combinational adjuvants had a beneficiary effect in comparison to the rasp-1 (~20 fold) or the alum (~3 fold) adjuvanted vaccines. to elucidate the igg subtypes induced in the different immunization groups, we also analyzed the rbd-specific igg1 and igg2c antibody titers. we observed that the highest titer of igg1 antibodies was induced when the rbd vaccine was formulated with alum alone or with rasp-1 + alum in one inoculum (g4-250,951 and g5-369,000 end point titers respectively; figure s1b ), with the titers being significantly higher when the vaccine was with both adjuvants and in one inoculum. the rbd-specific igg1 titers were significantly decreased when rasp-1 was used as an adjuvant in aqueous formulations; when the rasp-1 and the alum-adjuvanted rbd were co-administered in separate sites (~7-fold decrease; g6-55,945 end point titers) or with the rasp-1-adjuvanted rbd vaccine (~40-fold decrease; g3-9165 end point titers) when it was compared to the administration of rbd + rasp-1 + alum in a single inoculum (g5-369,000 end point titers; figure s1b ). rasp-1 is known as an igg2 (th1)-biased adjuvant [8] . notably, igg2c responses were only elevated when rasp-1 was also administered as an adjuvant (g3-254, g5-1064, and g6-726 end point titers), with the combinatorial adjuvanted vaccines performing similarly and best (g5 and g6; figure s1c ). these data suggest that when rasp-1 is adsorbed to alum in a vaccine formulation (g5), the two adjuvants work in synergy not only to elicit a stronger igg1 response than the alum-adjuvanted vaccine formulation, but also for inducing the igg2c antibody response as compared to alum-adjuvanted vaccine formulation alone (~14-fold increase), suggesting that the combination of rasp-1 and alum in a vaccine with the rbd antigen works in synergy to elicit a more balanced igg1-igg2c antibody response (igg1/igg2c ratio of 1626 in g5 vs. 4403 in g4; figure s1d ). the reduced igg1/igg2c ratio is more pronounced in a vaccine formulation where rasp-1 is not adsorbed to alum but co-administered separately (igg1/igg2c ratio of 601 in g6; figure s1d ). sera samples from day 7 post-2nd immunization were also tested for their ability to inhibit the binding of mers-rbd-fc protein to the hdpp4 receptor-expressing huh-7 cells by flow cytometry. similarly to the enhanced induction of neutralizing antibodies, immunization with rasp-1 and the alum-adjuvanted rbd vaccine in separate injection sites also increased the ability of the generated antibodies to inhibit the binding of the rbd protein to its receptor by~2-fold (g6-50%; figure 2 ) as compared to rbd + rasp-1 + alum adjuvanted vaccine in a single inoculum or with alum-adjuvanted rbd vaccine (g5-25% and g4-35%; figure 2 ), and by >3-fold as compared to the rasp-1-adjuvanted rbd vaccine (g3-16%; figure 2 ). when group g6 was compared to groups g3 and g4, a synergy of the combinatorial adjuvants, though when rasp-1 is not adsorbed to alum, was evident. however, when rasp-1 was adsorbed to alum (g5), the functionality of the antibodies elicited by the vaccine is much reduced. the inhibitory activity in group g5 was reduced by~30%, albeit not significantly, as compared to g4. (table 1 and x-axis legend). sera samples were collected on day 7 post-2nd immunization and assayed for inhibition of the binding of mers-cov rbd-fc to huh-7 cells expressing mers-cov receptor dpp4. the data represents the mean and standard error (sem) of percentage inhibition of binding from at least two independent experiments with 3 to 5 mice per group. "+" indicates the presence and "−" indicates the absence of the protein or adjuvants in the formulation. statistics was performed using one-way anova with tukey's multiple comparison. p < 0.05: *, p < 0.0001: ****. nd: not detectable. the draining lymph nodes (ln) were harvested from each leg 7 days post-2nd immunization and analyzed for the number of monocytes as well as their activation and migratory status. migration of innate cells from the site of injection to the lns are required to initiate an effective adaptive immune response [27] . the analyses demonstrated that there was no significant difference in the total number of monocytes (cd45 + cd11c -ly6c + ; figures s2a and s3a) recruited into the ln per mouse between the various immunization groups. however, the number of activated monocytes (c45 + cd11c -ly6c + cd40 + ) in the ln were significantly higher in mice where rasp-1 and the alumadjuvanted rbd vaccine were co-administered in separate sites than in mice immunized with rbd + rasp-1 + alum in a single inoculum, or with alum-adjuvanted rbd vaccine (g6 vs. g5 and g4, respectively; figure 3b ). notably, the number of activated monocytes in the ln of mice that received only the rasp-1-adjuvanted rbd vaccine (g3) were similar to those present in mice where rasp-1 was administered without being adsorbed to alum (g6). it appears that alum does not add to the activation of the monocytes in any of the formulations tested (g4, g5 or g6; figure 3b ). (table 1 and x-axis legend). sera samples were collected on day 7 post-2nd immunization and assayed for inhibition of the binding of mers-cov rbd-fc to huh-7 cells expressing mers-cov receptor dpp4. the data represents the mean and standard error (sem) of percentage inhibition of binding from at least two independent experiments with 3 to 5 mice per group. "+" indicates the presence and "−" indicates the absence of the protein or adjuvants in the formulation. statistics was performed using one-way anova with tukey's multiple comparison. p < 0.05: *, p < 0.0001: ****. nd: not detectable. the draining lymph nodes (ln) were harvested from each leg 7 days post-2nd immunization and analyzed for the number of monocytes as well as their activation and migratory status. migration of innate cells from the site of injection to the lns are required to initiate an effective adaptive immune response [27] . the analyses demonstrated that there was no significant difference in the total number of monocytes (cd45 + cd11c -ly6c + ; figures s2a and s3a ) recruited into the ln per mouse between the various immunization groups. however, the number of activated monocytes (c45 + cd11c -ly6c + cd40 + ) in the ln were significantly higher in mice where rasp-1 and the alum-adjuvanted rbd vaccine were co-administered in separate sites than in mice immunized with rbd + rasp-1 + alum in a single inoculum, or with alum-adjuvanted rbd vaccine (g6 vs. g5 and g4, respectively; figure 3b) . notably, the number of activated monocytes in the ln of mice that received only the rasp-1-adjuvanted rbd vaccine (g3) were similar to those present in mice where rasp-1 was administered without being adsorbed to alum (g6). it appears that alum does not add to the activation of the monocytes in any of the formulations tested (g4, g5 or g6; figure 3b ). nevertheless, a synergistic effect was observed when rasp-1 was added to the rbd vaccine with or without alum with respect to ccr7 + (migratory) monocyte subset. the number of migratory monocytes (cd45 + cd11c -ly6c + ccr7 + ) in the ln were significantly higher in mice where rasp-1 and the alum-adjuvanted rbd vaccine were co-administered in separate sites, rbd + rasp-1 + alum was administered in a single inoculum or the rasp-1-adjuvanted rbd vaccine vs. the alum-adjuvanted rbd vaccine (g6, g5 and g3 vs. g4; figure 3c ). the number of migratory monocytes in the ln of mice that received the alum-adjuvanted rbd vaccine were actually similar to those in the control group that received only rbd (g4 vs. g2). as observed with the number of activated monocytes, migratory monocytes in the lns of mice that received rasp-1 and the alum-adjuvanted rbd vaccine co-administered in separate sites were significantly higher in mice than in mice received rbd + rasp-1 + alum administered in a single inoculum (g6 vs g5; figure 3c ). nevertheless, a synergistic effect was observed when rasp-1 was added to the rbd vaccine with or without alum with respect to ccr7 + (migratory) monocyte subset. the number of migratory monocytes (cd45 + cd11c -ly6c + ccr7 + ) in the ln were significantly higher in mice where rasp-1 and the alum-adjuvanted rbd vaccine were co-administered in separate sites, rbd + rasp-1 + alum was administered in a single inoculum or the rasp-1-adjuvanted rbd vaccine vs. the alum-adjuvanted rbd vaccine (g6, g5 and g3 vs. g4; figure 3c ). the number of migratory monocytes in the ln of mice that received the alum-adjuvanted rbd vaccine were actually similar to those in the control group that received only rbd (g4 vs. g2). as observed with the number of activated monocytes, migratory monocytes in the lns of mice that received rasp-1 and the alum-adjuvanted rbd vaccine co-administered in separate sites were significantly higher in mice than in mice received rbd + rasp-1 + alum administered in a single inoculum (g6 vs g5; figure 3c ). to investigate the possible contribution of tfh and gc b cells to the robust functional antibody responses induced in mice administered with rasp-1 and the alum-adjuvanted rbd vaccine in separate injection sites, ln from 7 days post-2nd immunization were analyzed for the tfh ( figure 4a ) and gc b ( figure 5a ) cell frequencies. although the frequency of total cd4 + t cells within the ln was not significantly different between the various immunization groups (figure s4b ), the frequency of the tfh (cd4 + cxcr5 + pd-1 + ) cells within the ln of mice administered with rasp-1 and the alum-adjuvanted rbd vaccine in separate sites (g6) was 2.3-fold higher than in mice administered with the rbd + rasp-1 + alum vaccine in a single inoculum, and 5.5-and 2.1-fold higher versus rasp-1-and alum-adjuvanted rbd vaccines (g6 vs. g3 and g4, respectively; figure 4b) . importantly, the fold increase in the tfh cells was positively and significantly associated with the neutralizing antibody titers against the pseudotyped mers-cov in two immunization groups, namely mice that were immunized with rasp-1 and the alum-adjuvanted rbd vaccine in separate sites (g6; r = 0.535, p = 0.0150) and mice immunized with rbd + rasp-1 + alum vaccine in a single inoculum (g5; r = 0.593, p = 0.0058) ( figure 4c) . notably, the frequency of b cells (b220 + ) in the ln was also significantly higher in mice administered with rasp-1 and the alum-adjuvanted rbd vaccine in separate sites than in mice administered with rbd + rasp-1 + alum in a single inoculum, or with the alum-adjuvanted rbd vaccine or with the rbd vaccine alone (g6-33% vs. g5-25%, g4-25%, and g2-24% respectively; figure 5b ). additionally, the frequency of b cells in mice immunized with rasp-1-adjuvanted rbd was also significantly higher as compared to mice immunized with rbd + rasp-1 + alum in a single inoculum (g3-29% vs. g5-25% respectively; figure 5b ). formulating the vaccine with rasp-1 in an aqueous formulation (not adsorbed to alum) might have been critical for the increased number of b cells in these two vaccine formulations (g6 and g3). when the frequency of gc b cells (b220 + cd95 + gl-7 + ) in the draining ln ( figure 5c ) were analyzed, it appeared that immunization of mice with rasp-1 and the alum-adjuvanted rbd vaccine in separate sites (g6) induced~2-fold increase in the frequency of the gc b cells versus immunization with rbd + rasp-1 + alum in a single inoculum and the alum-adjuvanted rbd vaccine (g5 and g4), and~4-fold increase in gc b cells was induced versus immunization with the rasp-1-adjuvanted rbd vaccine (g3; figure 5c ). interestingly, the alum-adjuvanted vaccines (g5 and g4) had >2-fold increase in gc b cells in the ln compared to rasp-1-adjuvanted rbd vaccine (g3; figure 5c ). importantly, only when rasp-1 and the alum-adjuvanted rbd vaccine were co-administered in separate sites (g6) was the fold increase in the gc b cells positively and significantly associated with the neutralizing antibody titers against the pseudotyped mers-cov (r = 0.550, p = 0.029). no significant association was observed in mice when the rbd + rasp-1 + alum vaccine was administered (g5) as a single inoculum (r = −0.051, p = 0.829; figure 5d ). "−" indicates the absence of the protein or adjuvants in the formulation. statistics was performed using one-way anova with tukey's multiple comparison. p < 0.05: *, p < 0.001: ***. spearman correlation was performed to determine the association of tfh cells with neutralizing antibody titers. adjuvants are essential components in both prophylactic and therapeutic vaccines since they ameliorate antigen-specific protective immune responses [28] . however, choosing the appropriate adjuvant that can be employed safely and that enhances vaccine efficacy is still elusive and needs to be optimized experimentally first [29] . besides a handful of adjuvants such as cpg, poly i:c, mpla and mf59, aluminum-based (alum) adjuvants are being used in most of the adjuvanted vaccines for humans globally even today since its inception 85 years ago [30] . although beneficial effects of alum as an adjuvant were observed with the dtap, hepb, and hepa vaccines, a biased th2-type immune response, absence of strong cellular responses, and the induction of adverse reactions were some of the limitations found with the various alum-adjuvanted vaccines [30] . therefore, the utilization of a combination of adjuvants in vaccines that can improve the safety and efficacy of vaccines against emerging pathogens is being actively pursued by the research community [31] . the use of combinatorial adjuvant system is beneficial since they can be tailored to target varied pattern-recognition receptors (prrs) with each being able to enhance antigen-specific responses (cellular and humoral) in a complementary or synergistic outcome [32] . for instance, intranasal vaccination with emulsified fine particles like pelc in combination with ld-indolicidin enhanced protective influenza-specific serological immunity in mice [33] . mpl and cpg combination adjuvants promoted homologous and heterosubtypic cross protection when used with the inactivated split influenza virus vaccine [34] . the co-administration of alum and a tlr-7 adjuvant enhanced memory b cell response to lymphocytic choriomeningitis virus (lcmv) antigen [35] . alum in combination with mpla-ha-adjuvanted hbsag increased both the magnitude and the persistence of hbsag-specific immune responses against hepatitis b virus infection [36] . the aim of the present study was to explore the synergistic potential of combining the o. volvulus-derived protein adjuvant, rasp-1 with alum as a novel combinatorial adjuvant system using mers-rbd-fd as the model vaccine antigen. we have previously shown that rasp-1 enhances the immune response when co-administered in an aqueous formulation with several bystander vaccine antigens [9] [10] [11] . moreover, we have also reported that rasp-1-adjuvanted trivalent influenza vaccine (iiv3) elicits a balanced igg1/igg2c response to iiv3 and protects mice following h1n1 virus challenge, potentially via myd88-independent tlr4 signaling [8, 12] . in this study, we have shown that mice immunized with rbd + rasp-1 + alum in a single inoculum elicited neutralizing antibody titers against pseudotyped mers-cov that were not significantly different from mice that received either rasp-1-adjuvanted rbd vaccine or alum-adjuvanted rbd vaccine alone ( figure 1) . notably, when rasp-1 and the alum-adjuvanted rbd vaccine were co-administered in separate sites the vaccine ameliorated the production of neutralizing antibody titers by~4-fold as compared to the combinatorial adjuvant system administered in a single inoculum (figure 1) . we also observed that mice that received two immunizations at three-week intervals of the combinatorial adjuvant system where rasp-1 and the alum-adjuvanted rbd vaccine were co-administered separately elicited neutralizing antibody titers against pseudotyped mers-cov infection that were similar to, or slightly lower than, those elicited in mice that received three immunizations of the montanide isa51-adjuvanted, or two immunizations of the mf59-adjuvanted mers cov-rbd-fc or mers-rbd-fd vaccines [20] . another noteworthy observation is that the heightened neutralizing antibody titers induced by the unique experimental combinatorial adjuvant system was achieved using 5 µg of the mers-rbd-fd vaccine protein, compared to 10 µg of mers-rbd-fc or mers-rbd-fd proteins used in previous studies [20] . this suggests that the rasp-1 in this unique combinatorial adjuvant system enabled also rbd dose sparing and with two immunizations. we have previously reported that rasp-1 also facilitates iiv3 antigen dose sparing up to a 10-or 40-fold decrease, and with a single immunization of the rasp-1-adjuvanted iiv3, mice were still protected from a lethal h1n1 influenza virus challenge [12] . importantly, the antibodies elicited by the different combinatorial rbd-adjuvanted vaccines were also functional in their ability to inhibit the binding of mers-rbd to the human dpp4 receptor. mice that received rasp-1 and the alum-adjuvanted rbd vaccine separately inhibited the binding bỹ 2-fold more as compared to mice administered with rbd rasp-1 + alum in a single inoculum (figure 2 ). although mice that received alum-adjuvanted rbd vaccine significantly inhibited binding (35% ± 2.3) compared to rasp-1-adjuvanted rbd vaccine, they were not significantly different compared to the combinatorial adjuvant system administered in a single inoculum (figure 2 ). the inhibition of binding, however, was only enhanced in the combinatorial adjuvant system where rasp-1 and alum-adjuvanted rbd vaccine are co-administered separately. these data collectively suggest that adsorption of rasp-1 to alum in a combinatorial adjuvant system does not enhance the functional antibody responses elicited by alum-adjuvanted rbd vaccine. while rasp-1 when not adsorbed to alum in a combinatorial adjuvant system was able to ameliorate the functional antibody responses. an important concern raised when anti-viral vaccine are developed, especially with the ongoing covid-19 crisis, is that some vaccine approaches may induce unwantedly adverse side effects due to antibody dependent enhancement (ade) and thus more severe pathology [37] . since ade is generally correlated to the neutralizing antibody titers, studies have also shown that high neutralizing antibody titer may eliminate the potential induction of ade [38, 39] . [18] [19] [20] 40] . in our study, we show immunization of mice with rasp-1 and the alum-adjuvanted mers-cov rbd vaccine in separate sites have induced nt 50 neutralizing antibody titers greater than 1:15,000 against pseudotyped mers-cov infection. we expect that such high-titer neutralizing may prevent mers-cov infection in vivo without causing any adverse effects. however, this will have to be proven experimentally in the future. in this study, we also observed that immunization with the alum-adjuvanted rbd vaccine elicits an rbd-specific igg1-biased response, while the rasp-1-adjuvanted rbd vaccine elicits a balanced rbd specific igg1-igg2c response ( figure s1b,c) . notably, in the combinatorial adjuvant system where rasp-1 and the alum-adjuvanted rbd vaccine are co-administered separately the balanced igg1/igg2c response ( figure s1d ) was preserved, while mice that received the combinatorial adjuvant system in a single inoculum elicited an igg1-biased response ( figure s1b,d) . these results suggest that the presence of rasp-1 in an aqueous formulation shifts the dominant igg1 response elicited by the alum-adjuvanted rbd vaccine to a balanced igg1-igg2c response and this was more pronounced when rasp-1 was not adsorbed to alum. interestingly, the administration of the combinatorial adjuvant system showed differences in the ly6c + activated monocyte but not in cd11c + activated dc subsets ( figure s3 ). this may likely be due to the presence of rasp-1 in the vaccine, since we have previously shown that intra-muscular injection rasp-1 alone or the rasp-1-adjuvanted trivalent influenza (iiv3) vaccine elicited an increased recruitment of monocytes than dcs at the site of injection (24 h after injection) as compared to pbs control group or iiv3 alone [12] . in the present study, the administration of the combinatorial adjuvant system where rasp-1 was completely adsorbed to alum (single inoculum immunization group) significantly reduced the number of cd40 + (activated) monocytes and ccr7 + (migratory) monocytes to the draining ln compared to the administration of the combinatorial adjuvant system where rasp-1 was not adsorbed to alum ( figure 3b,c) . interestingly, the number of cd80 + monocytes in the ln was similar whether rasp-1 + alum + rbd were administered as a single inoculum or as a co-administered vaccine in two separate sites. however, both of these vaccine formulations as well as the rasp-1 adjuvanted-mers-rbd vaccine resulted in significantly higher number of recruited cd80 + monocytes than the alum-adjuvanted rbd vaccine, suggesting that the presence of alum did not significantly alter the number of cd80 + monocytes recruited by the combinatorial rasp-1 and alum adjuvanted-mers-rbd vaccines ( figure s3b ). moreover, there was a 2-fold increase in the number of the activated monocytes and migratory monocytes recruited to the draining ln in mice that received the rasp-1-adjuvanted rbd vaccine when compared to the alum-adjuvanted rbd vaccine ( figure 3b ,c). the number of migratory monocytes doubled in the draining ln of mice immunized with the combinatorial adjuvant system where rbd + rasp-1 + alum was administered in a single inoculum as compared to the alum-adjuvanted rbd vaccine alone. the number of migratory monocytes further increased in mice that received the combinatorial adjuvant system where rasp-1 and the alum-adjuvanted rbd vaccine were co-administered separately ( figure 3c ). there were no significant differences observed in the number of activated and migratory dcs across all the immunization groups. collectively, these data suggest that the rasp-1 in the combinatorial adjuvant system may play a significant role in the enhanced recruitment of monocyte subsets. this is supported with the data where the administration of the rasp-1-adjuvanted rbd vaccine also significantly increased the number of activated (cd40 + ) and migratory (ccr7 + ) monocytes in the draining ln compared to alum-adjuvanted rbd vaccine ( figure 3b,c) . also, rasp-1 and alum may work in synergy to improve the number of migratory monocytes in the draining ln compared to what alum could do alone. one of the important events in the generation of an adaptive cellular response is the effective migration of innate cells to the lymph nodes to encounter naïve t cells, a process in which ccr7, a chemokine receptor, is known to play a dominant role [41] . in addition, the absence of ccr7 has been shown to affect the magnitude of protective responses against viral infections in mouse models [42, 43] . therefore, we suggest that rasp-1, when not adsorbed to alum in a vaccine formulation, may improve the effective recruitment of innate cells that lead to the induction of effector adaptive cellular responses. tfh cells can determine humoral immunity that is also derived from gc b cells, and therefore both of these cell types have become an important aspect for rational designs of more effective vaccines, in particular those depending on functional antibodies for their efficacy [44, 45] . to better understand what contributed to the improved elicitation of functional anti-mers-cov neutralizing antibodies, the frequencies of tfh (cd4 + cxcr5 + pd-1 + ) cells and gc b (b220 + cd95 + gl-7 + ) cells in the draining ln of immunized mice were analyzed. a two-fold increase in both tfh and gc b frequencies were induced when rasp-1 and the alum-adjuvanted rbd vaccine were co-administered in separate sites as compared to the combinatorial adjuvant system where rbd + rasp-1 + alum were administered in a single inoculum (figures 4b and 5c ). while no significant difference was observed in the fold increase of the frequency of gc b cells in the ln of mice that were immunized with rasp-1 and the alum-adjuvanted rbd vaccine co-administered separately as compared to the alum-adjuvanted rbd vaccine, a six-fold increase was observed when this was compared to rasp-1-adjuvanted rbd vaccine alone ( figure 5c ). these data suggest that the complete adsorption of rasp-1 to alum diminished not only the ability to induce migratory monocyte, but also the development of cells that are important for mounting an effective humoral response. importantly, we found a significant and positive correlation between the neutralizing antibody titers in sera of mice vaccinated with rasp-1 and the alum adjuvanted rbd vaccine separately and the fold increase in the frequency of tfh and gc b cells recruited in the draining ln ( figures 4c and 5d) . interestingly, the fold increase in the frequency of tfh cells was also significantly and positively associated with the titers of neutralizing antibodies in mice that were immunized with the combinatorial adjuvant system administered in a single inoculum (rbd + rasp-1 + alum; figure 4b ), suggesting that the rasp-1 and alum may work in synergy. our study demonstrates that a unique combination of rasp-1 (a helminth-derived protein) protein adjuvant with alum and the mers-rbd-fd using the model vaccine antigen enhanced the protective immune responses to mers-cov, despite the fact that adjuvants have to be co-administered separately (where rasp-1 was not adsorbed to alum). also, for the first time, we were able to determine that the tfh and gc b cells in the lns in mice immunized with combinatorial adjuvanted-mers-rbd vaccine were significantly and positively associated with the essential functional protective immune responses to mers-cov neutralizing antibodies. further studies will be necessary, however, to elucidate the precise underlining mechanisms of this unique adjuvant combination of rasp-1 and alum. in our study, it appeared that the adsorption of rasp-1 to alum reduced the immunopotentiating activities of either rasp-1 or alum. as the potency of rasp-1 is highest when it is in an aqueous formulation, a better understanding of the targets of multiple immune pathways that are induced may also help us utilize the rasp-1 protein adjuvant in combination with other prr agonists that can be used in aqueous formulations as adjuvants in novel combinatorial formulations. such combinatorial adjuvants may be more advantageous with subunit vaccine models that generally are known to induce suboptimal protective immune responses alone and/or induce vaccine enhanced disease (ved) when used with the alum adjuvant [46] . the following are available online at http://www.mdpi.com/2076-393x/8/2/251/s1, figure s1 : induction of mers-cov-rbd specific igg subtypes in sera of immunized mice, figure s2 : representative flow cytometry plot determining the gating strategy of the immune cells recruited to the draining lymph nodes (lns) of immunized mice, figure s3 : number of monocyte and dc subsets recruited into the lymph nodes (lns) of immunized mice, figure s4 : frequency of cd4 + t cells in the lymph nodes (lns) of immunized mice. funding: this research was funded by nih grants u01ai124260 and r01ai139092. recent advances of vaccine adjuvants for infectious diseases the latest advancements in zika virus vaccine development augmentation of vaccine-induced humoral and cellular immunity by a physical radiofrequency adjuvant from 1920 to 2015 and beyond. vaccines (basel) aluminium adjuvants-in retrospect and prospect optimizing the utilization of aluminum adjuvants in vaccines: you might just get what you want old and new adjuvants -1, a th1-biased protein adjuvant derived from the helminth onchocerca volvulus, can directly bind and activate antigen-presenting cells the adjuvanticity of an o. volvulus-derived rov-asp-1 protein in mice using sequential vaccinations and in non-human primates asp-1, a recombinant secreted protein of the helminth onchocercavolvulus, is a potent adjuvant for inducing antibodies to ovalbumin, hiv-1 polypeptide and sars-cov peptide antigens enhanced humoral response to influenza vaccine in aged mice with a novel adjuvant, rov-asp-1. vaccine the parasite-derived rov-asp-1 is an effective antigen-sparing cd4(+) t cell-dependent adjuvant for the trivalent inactivated influenza vaccine, and functions in the absence of myd88 pathway isolation of a novel coronavirus from a man with pneumonia in saudi arabia crystal structure of the receptor-binding domain from newly emerged middle east respiratory syndrome coronavirus dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc advances in mers-cov vaccines and therapeutics based on the receptor-binding domain recombinant receptor-binding domains of multiple middle east respiratory syndrome coronaviruses (mers-covs) induce cross-neutralizing antibodies against divergent human and camel mers-covs and antibody escape mutants receptor-binding domain of mers-cov with optimal immunogen dosage and immunization interval protects human transgenic mice from mers-cov infection introduction of neutralizing immunogenicity index to the rational design of mers coronavirus subunit vaccines a recombinant 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peripheral lymph nodes vaccine safety evaluation: practical aspects in assessing benefits and risks efficacy and safety of immunological adjuvants. where is the cut-off? advances in aluminum hydroxide-based adjuvant research and its mechanism from discovery to licensure, the adjuvant system story triggering intracellular receptors for vaccine adjuvantation mucosal delivery of a combination adjuvant comprising emulsified fine particles and ld-indolicidin enhances serological immunity to inactivated influenza virus mpl and cpg combination adjuvants promote homologous and heterosubtypic cross protection of inactivated split influenza virus vaccine alum/toll-like receptor 7 adjuvant enhances the expansion of memory b cell compartment within the draining lymph node evaluation of hyaluronic acid-based combination adjuvant containing monophosphoryl lipid a and aluminum salt for hepatitis b vaccine the potential danger of suboptimal antibody responses in covid-19 cross-reactivity, and function of antibodies elicited by zika virus infection engineering a stable cho cell line for the expression of a mers-coronavirus vaccine antigen rot, a. ccr7 and its ligands: balancing immunity and tolerance impact of ccr7 on priming and distribution of antiviral effector and memory ctl antiviral immune responses in the absence of organized lymphoid t cell zones in plt/plt mice the adjuvant gla-se promotes human tfh cell expansion and emergence of public tcrbeta clonotypes can follicular helper t cells be targeted to improve vaccine efficacy? a unique combination adjuvant modulates immune responses preventing vaccine-enhanced pulmonary histopathology after a single dose vaccination with fusion protein and challenge with respiratory syncytial virus this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we gratefully acknowledge kathy tang, head of the lars facility at nybc for providing animal and veterinary care. the authors also thank mihaela barbu-stevanovic, head of the flowcytometry core facility at nybc. the authors also acknowledge maria elena bottazzi and bin zhan from baylor college of medicine, texas children's hospital center for vaccine development, houston, texas for the production of the recombinant ov-asp-1 protein. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord-263042-qdmunb9l authors: zhao, yongkun; wang, chong; qiu, boning; li, chufang; wang, hualei; jin, hongli; gai, weiwei; zheng, xuexing; wang, tiecheng; sun, weiyang; yan, feihu; gao, yuwei; wang, qian; yan, jinghua; chen, ling; perlman, stanley; zhong, nanshan; zhao, jincun; yang, songtao; xia, xianzhu title: passive immunotherapy for middle east respiratory syndrome coronavirus infection with equine immunoglobulin or immunoglobulin fragments in a mouse model date: 2016-11-24 journal: antiviral res doi: 10.1016/j.antiviral.2016.11.016 sha: doc_id: 263042 cord_uid: qdmunb9l middle east respiratory syndrome (mers) is a highly lethal pulmonary infection caused by a coronavirus (cov), mers-cov. with the continuing spread of mers-cov, prophylactic and therapeutic treatments are urgently needed. in this study, we prepared purified equine f(ab’)(2) from horses immunized with mers-cov virus-like particles (vlps) expressing mers-cov s, m and e proteins. both igg and f(ab’)(2) efficiently neutralized mers-cov replication in tissue culture. passive transfer of equine immune antibodies significantly reduced virus titers and accelerated virus clearance from the lungs of mers-cov infected mice. our data show that horses immunized with mers-cov vlps can serve as a primary source of protective f(ab’)(2) for potential use in the prophylactic or therapeutic treatment of exposed or infected patients. middle east respiratory syndrome (mers)-cov is an emerging pathogen that causes severe pneumonia in humans in the arabian peninsula and in travelers from this region (assiri et al., 2013a; zaki et al., 2012b; zumla et al., 2015) . human-to-human spread has been documented (assiri et al., 2013b) . while infections of immunocompetent patients generally present with only mild symptoms, the elderly and patients with pre-existing illnesses such as diabetes or renal failure are likely to develop more severe disease (assiri et al., 2013a) . as of september 21, 2016, 1806 cases with 643 deaths (35.6% mortality) had been reported to the world health organization, although the actual number of infections could be much larger since mild, asymptomatic or undiagnosed cases are likely to be common (drosten et al., 2014) . as yet there are neither licensed vaccines nor any prophylactic or therapeutic treatments effective against mers-cov. given the ability of coronaviruses to rapidly adapt to new hosts, a major public health concern is that mers-cov will further adapt to replication in humans, triggering a global severe acute respiratory syndrome (sars)-like pandemic (peiris et al., 2004; zaki et al., 2012a) . as of now, the most promising treatment is the passive administration of anti-mers-cov neutralizing antibodies. several research groups have developed and produced anti-mers patientderived or humanized monoclonal neutralizing antibodies in vitro that were able to protect mers-cov infected mice (corti et al., 2015; li et al., 2015; zhao et al., 2014) . however, since these antibodies react with a single epitope on the mers-cov spike (s) protein and since coronaviruses are prone to mutate, this approach has raised concerns about possible antibody escape (corti et al., 2015; sabir et al., 2016) . recently, we showed that sera from middle east dromedary camels contained high levels of anti-mers-cov neutralizing antibodies. passive immunotherapy with sera from these animals significantly reduced virus loads and accelerated virus clearance from the lungs of mers-cov infected mice . this provides proof of concept that immune animal sera are potentially useful in the treatment of patients with mers (hayden et al., 2014) . passive immunotherapy with animal sera or antibodies has been successfully used to prevent rabies and to neutralize snake venom (both et al., 2012; gutierrez et al., 2014) . convalescent plasma used to treat patients with sars has been found safe and has demonstrated some efficacy in a study with a small number of patients (mair-jenkins et al., 2015) . however, neutralizing antibody titers in mers patients are generally low and the limited number of mers survivors makes this approach impractical (drosten et al., 2013) . here, we show that immunization of healthy horses with mers-cov virus-like particles (vlps) expressing mers-cov s, m and e proteins induces strong polyclonal neutralizing antibodies against mers-cov. since administration of whole antibodies can induce allergic responses in some humans, we further tested f(ab') 2 fragments prepared by digestion of antibody with pepsin. prophylactic or therapeutic treatment of mers-cov infected mice with either igg or f(ab') 2 significantly decreased the virus load in their lungs. mers-cov vlps were produced and purified as previously described . in brief, army worm sf9 cells were infected with a single recombinant baculoviruses co-expressing mers-cov structural protein genes s, m, and e, at a multiplicity of infection (moi) of 0.5. culture supernatants were harvested at 96 h post-infection and centrifuged at 2000 g for 30 min to remove cell debris. following centrifugation of the clarified supernatants at 100,000 g for 1 h at 4 c the resulting vlp pellets were resuspended in pbs and loaded onto a 30e40e50% discontinuous sucrose gradient. after an additional centrifugation at 100,000 g for 1.5 h at 4 c, bands between 30 and 40% sucrose containing mers-cov vlp were collected. four 4-year-old healthy horses received multi-point intramuscular injections of 0.5, 1.5, 2, 3, and 5 mg mers-cov vlps in 4 ml pbs at weeks 0, 2, 4, 6, and 8, respectively. freund's complete adjuvant (sigma) was included in the first dose, and incomplete adjuvant in the remaining ones. sera were collected from the jugular vein 2 weeks after each injection, and stored at à20 c before further analysis. mers-cov specific antibodies in the sera were measured by an indirect enzyme-linked immunosorbent assay (elisa) using purified mers-cov receptor-binding domain (rbd) protein (i.e., s protein residues 358e662 cloned into the pet-30a expression vector and purified by ni-nta affinity chromatograph column). briefly, 96-well microtitration plates (corning costar, usa) were pre-coated with 100 ml purified rbd antigen diluted in 0.05 mol/l carbonate sodium buffer (ph 9.6) to a final concentration of 1 mg/ ml and incubated at 4 c overnight. after blocking with skimmed milk for 2 h at 37 c, 100 ml twofold serially diluted serum samples were added to the wells, and incubated at 37 c for 1 h. the plates were washed three times with pbs containing 0.05% tween-20 (pbst), before addition of 100 ml hrp-labeled rabbit antibody against horse igg (bioss, china; 1:20,000) and incubation at 37 c for 1 h. after washing with pbst, 100 ml 3, 3 0 , 3, 5'-tetramethylbenzidine (tmb) (sigma, usa) as substrate was added to each well and incubated for 30 min. the reaction was stopped with 50 ml 2 m h 2 so 4 . optical densities at 450 nm were measured in an elisa plate reader (bio-rad, usa). horse antiserum was diluted with 2 vol of normal saline (0.9% nacl) and a half volume of saturated ammonium sulfate was then added and mixed gently at room temperature for 30 min before centrifugation at 5000 g for 20 min. the resulting sediment was redissolved in saline and mixed with a one-third volume of saturated ammonium sulfate. after incubation at ambient temperature for 30 min and centrifugation at 5000 g for 20 min, the second sediments were dissolved in normal saline and dialyzed against normal saline to remove any remaining ammonium salt. immunoaffinity resins were prepared by coupling 10 mg rbd protein to 0.02 m sodium periodate-activated sepharose 4b (4 g), and then incubating with 150 ml sodium borohydride for 30 min. after reaction with 1 m tris (ph 7.5) for 30 min, a purified igg sample was diluted 9-fold with pbs and incubated with the rbd resin overnight at 4 c with constant rotation. the flowthroughs (anti-rbd depleted) were collected, and then the flowthroughs were tested against the rbd protein by elisa to ensure rbdspecific igg all bound with the rbd sepharose 4b. after washing with pbs, the bound antibodies (anti-rbd) were eluted in 0.2 m glycine-hcl buffer (ph 2.7). the eluates were neutralized with 1 m tris buffer (ph 9.0), and then dialyzed against pbs. all samples were adjusted to the same protein concentration and sterilized by passage through microspin filters (0.2 mm pore size; millipore). neutralizing activity of the igg, rbd-specific igg, and flowthroughs were tested. the ph of the horse antiserum was adjusted to 3.3 with 1 mol/l hcl. following incubation with pepsin (10000 iu/ml) at 30 c for 2.5 h, the reaction was stopped by adjusting the ph to 7.2 with 1 mol/l naoh. the solution was then applied to protein-a and protein-g columns sequentially to remove whole immunoglobulins. the purity of the resulting f(ab') 2 protein was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) followed by coomassie blue staining and the target fraction in the gel was analyzed in a thin layer chromatography scanner (transmission, zigzag scan, dual wavelength, swing width:8 mm, delta y: 0.1 mm) (cs-9301, shimadzu). specific pathogen-free 6 week old balb/c mice were purchased from charles river laboratories international and maintained in the animal care facility, university of iowa. briefly, all mice were housed in thoren individually ventilated cages. caging and bedding were autoclaved. irradiated diet was fed. filtered water (0.2 mm filter) was provided with edstrom automatic watering system. hepa-filtered cage changing stations were used. all persons entering animal rooms worn autoclaved gowns, gloves, hair bonnets, face masks, and shoe covers. serum samples, purified igg or f(ab') 2 were serially diluted in dmem and mixed with an equal volume of mers-cov containing 80 pfu. following incubation at 37 c for 1 h, aliquots were added to cultures of vero 81 cells in 48 well plates and incubated at 37 c in 5% co 2 for 1 h with gentle rocking every 15 min. plates were then overlaid with 1.2% agarose/dmem/2% calf serum. after further incubation for 3 days, agarose plugs were removed using a small spatula, and the remaining plaques were visualized by staining with 0.1% crystal violet. six-week-old female balb/c mice were lightly anesthetized with isoflurane and transduced intranasally with 2.5 â 10 8 pfu of ad5-hdpp4 in 75 ml dmem as described elsewhere (zhao et al., 2014) . five days post transduction, mice were infected intranasally with mers-cov (1 â 10 5 pfu) in a total volume of 50 ml dmem. mice were monitored daily for morbidity (weight loss) and mortality. all work with mers-cov was conducted in the university of iowa biosafety level 3 (bsl-3) laboratory. separate groups were injected with 200 ml horse antiserum or 500 mg igg or f(ab') 2 intraperitoneally (ip) 1 day before or after intranasal infection with 1 â 10 5 pfu mers-cov. control mice were given an equal volume of normal horse serum (sigma). to obtain virus titers, lungs were harvested from subgroups of 3 animals at the indicated time points (see results) and homogenized into 3 ml of phosphate buffered saline (pbs), using a manual homogenizer. lung homogenates were aliquoted into micro tubes and kept in à80 c. virus was titered on vero 81 cells. cells were fixed with 10% formaldehyde and stained with crystal violet three days post-infection (p.i.). viral titers are expressed as pfu/g tissue for mers-cov (zhao et al., 2014) . due to the biosafety risk, mers-cov must be handled in a bsl-3 laboratory, whereas vlps can be rapidly generated under bsl-2 conditions as an immunogen inducing high antibody titers. in addition, the horse provides little risk to humans and produces high antibody yields, making these animals an effective source for production of hyperimmune sera (zheng et al., 2016) . rbd-specific igg titers in the sera were all above 1:20,480 after five immunizations (fig. 1) as assessed by elisa. rbd contains the major neutralizing epitopes of the s protein, as shown by the observation that absorption of sars patient convalescent sera with sars-cov rbd removes the majority of neutralizing antibodies (he et al., 2005) . independent research groups have also shown more directly that the mers-cov rbd sequence contains the major antigenic determinants for inducing neutralizing antibodies, and that neutralizing epitopes within mers-cov s1 are also localized primarily in the rbd region (du et al., 2013; mou et al., 2013) . here, we have demonstrated that anti-rbd antibodies function as major components of neutralizing antibodies. we found that rbd-specific igg neutralized mers-cov infection with half maximal inhibitory concentration of 15.74 mg/ml, and 2.612 â 10 3 mg/ml for flowthroughs (fig. 2) , suggesting that the rbd of s protein act as an important neutralization determinant of mers-cov. our results demonstrate that equine antibodies are polyclonal and recognize more antigen determinants in mers-cov s protein than single mabs, which could potentially prevent antibody escape. the integrity of igg and f(ab') 2 fragments was evaluated using an sds-page gel (fig. 3a) . the purity of the f(ab') 2 fragments after protein-a/g chromatography was >91% after gel electrophoresis (fig. 3b ). passive transfer of blood products from other humans poses a safety concern, with possible contamination with agents of blood-borne diseases (e.g., hiv, hepatitis). heterologous antibody carries a potential risk of allergic reaction, but generation of f(ab') 2 fragments, results in antibodies being less immunoreactive and safer for use in humans. while we successfully generated equine antibodies against mers-cov vlps, their protective effect against authentic mersfig. 1 . robust mers-cov rbd-specific antibody in immunized horse sera. horses (n ¼ 4) were injected intramuscularly with mers-cov vlps and boosted every two weeks an additional 4 times. sera were collected 2 weeks after each immunization. rbd-specific antibodies in immunized horse sera were detected using elisa. cov infection remained untested. using a plaque reduction neutralizing assay, we confirmed that immune sera significantly neutralized mers-cov infection in vitro, with a half effective maximal dilution of 1: 20,900 (fig. 4a, b) . further, we found that equine igg and f(ab') 2 also neutralized mers-cov infection with half effective maximal concentrations (ec 50 ) of 2.16 mg/ml and 2.60 mg/ml for igg and f(ab') 2 , respectively (fig. 4c, d) . collectively, these results show that equine antibody products exhibit highly potent neutralizing activity against mers-cov. next we asked if adoptive transfer of equine antibodies could protect mice from mers-cov infection prophylactically and therapeutically. by using a mouse model we previously generated (zhao et al., 2014) , we injected animals with immune serum (fig. 5a, b) , purified igg (fig. 5c, d) or f(ab') 2 (fig. 5e, f) i.p. 1 day before (fig. 5a , c, e) or after (fig. 5b, d, f) mers-cov challenge. in both prophylactic and therapeutic settings, passive transfer of equine immune antibodies resulted in a 2e4 log reduction of virus titers in the lungs of mers-cov infected mice, and accelerated virus clearance in the serum treated group (fig. 5a, b) . we did not observe any difference in body weight loss and pathologic changes on the exterior surface of the lungs in treated and untreated mice after fig. 2 . neutralizing activity of the rbd-specific antibodies in igg. in vitro neutralization tests of total igg, rbd-specific igg, and flowthroughs, were determined in a series of 2-fold dilutions and 50% neutralization was calculated using graphpad prism. infeciton, since in this model, mice only develope mild lung disease. rapid virus replication and inflammatory cell infiltration in the infected lungs are the major parameters to measure (zhao et al., 2014) . since the half-life of f(ab') 2 in vivo is relatively short and mers-cov is cleared within 6 days in this model (zhao et al., 2014) , we did not inject f(ab') 2 antibodies before day à1 or after day 1 p.i. of note, the purified igg seemed to have lower protective potency than that of the immune serum in vivo (fig. 5) . the concentration of igg in serum is > 10 mg/ml. we used 200 ml of immune serum (equal to 2 mg igg) per mouse which is much higher than the immune igg we used (500 mg/mice). the other reason could be we purified immune igg using saturated ammonium sulfate precipitation method, which needed to be performed under room temperature. we speculated that some iggs were degraded or misfolded, and unable to bind to mers-cov spike protein under this circumstance. while, immune sera were properly stored at à20 c and contained high concentration of bsa and other proteins, which made the antiserum more stable. to date, there are several anti-mers-cov antibodies developed from different origins. each antibody contains its own advantages and disadvantages. for monoclonal antibodies, mouse-derived monoclonal antibody needs to be humanized before human use (li et al., 2015) ; a human neutralizing antibody derived from a convalescent mers patient can be produced in large amount from cho cells (corti et al., 2015) . however, the single clone antibody raises the concern of viral escape mutant when applied to human. administration of transchromosomic bovine human immunoglobulins (luke et al., 2016) or dromedary immune serum resulted in rapidly viral clearance in infected mouse lungs. the disadvantage of these antibodies is that these animals are not readily available. compared to the antibodies described above, the administration of equine igg-derived f(ab') 2 fragment proved to be a versatile and feasible method (lu et al., 2006; zhou et al., 2007) . it provides a useful platform to produce therapeutics against emerging infectious diseases. in summary, by immunizing healthy horses with mers-cov vlps, we have successfully developed the first equine igg-derived f(ab') 2 fragment that neutralizes mers-cov in vitro and in vivo. both prophylactic and therapeutic treatments decreased virus loads and accelerated virus clearance in the lungs of mers-cov-infected mice. therefore, horses immunized with mers-cov vlps can serve as a useful initial source for developing protective f(ab') 2 fragments, for the purpose of preparedness and to serve as a strategic reserve for 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saudi arabia mers-cov virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques isolation of a novel coronavirus from a man with pneumonia in saudi arabia isolation of a novel coronavirus from a man with pneumonia in saudi arabia rapid generation of a mouse model for middle east respiratory syndrome passive immunotherapy with dromedary immune serum in an experimental animal model for middle east respiratory syndrome coronavirus infection treatment with hyperimmune equine immunoglobulin or immunoglobulin fragments completely protects rodents from ebola virus infection inhibition of infection caused by severe acute respiratory syndrome-associated coronavirus by equine neutralizing antibody in aged mice middle east respiratory syndrome ad5-hdpp4 transduced balb/c mice (6wks, female) were injected intraperitoneally with 200 ml horse serum key: cord-310230-9wfb43gt authors: ghorbani, mahdi; brooks, bernard r.; klauda, jeffery b. title: critical sequence hot-spots for binding of ncov-2019 to ace2 as evaluated by molecular simulations date: 2020-06-27 journal: biorxiv doi: 10.1101/2020.06.27.175448 sha: doc_id: 310230 cord_uid: 9wfb43gt the novel coronavirus (ncov-2019) outbreak has put the world on edge, causing millions of cases and hundreds of thousands of deaths all around the world, as of june 2020, let alone the societal and economic impacts of the crisis. the spike protein of ncov-2019 resides on the virion’s surface mediating coronavirus entry into host cells by binding its receptor binding domain (rbd) to the host cell surface receptor protein, angiotensin converter enzyme (ace2). our goal is to provide a detailed structural mechanism of how ncov-2019 recognizes and establishes contacts with ace2 and its difference with an earlier coronavirus sars-cov in 2002 via extensive molecular dynamics (md) simulations. numerous mutations have been identified in the rbd of ncov-2019 strains isolated from humans in different parts of the world. in this study, we investigated the effect of these mutations as well as other ala-scanning mutations on the stability of rbd/ace2 complex. it is found that most of the naturally-occurring mutations to the rbd either strengthen or have the same binding affinity to ace2 as the wild-type ncov-2019. this may have implications for high human-to-human transmission of coronavirus in regions where these mutations have been found as well as any vaccine design endeavors since these mutations could act as antibody escape mutants. furthermore, in-silico ala-scanning and long-timescale md simulations, highlight the crucial role of the residues at the interface of rbd and ace2 that may be used as potential pharmacophores for any drug development endeavors. from an evolutional perspective, this study also identifies how the virus has evolved from its predecessor sars-cov and how it could further evolve to become more infectious. the novel coronavirus (ncov-2019) outbreak emerging from china has become a global pandemic and a major threat for human public health. according to world health organization (who) as of june 27 th 2020, there has been about 10 million confirmed cases and approaching 500,000 deaths due to coronavirus in the world. [1] [2] much of the human population including the united states of america were under lockdown or official stay-at-home orders to minimize the continued spread of the virus. coronaviruses are a family of single-stranded enveloped rna viruses. phylogenetic analysis of coronavirus genome has shown that ncov-2019 belongs to the beta-coronavirus family, which also includes mers-cov, sars-cov and bat-sars-related coronaviruses. [3] [4] it is worth mentioning that sars-cov, which was widespread in 2002 caused more than 8,000 cases and about 800 deaths and mers-cov (middle east respiratory syndrome coronavirus) in 2012 also spread in more than 25 countries, causing about 2,500 cases and more than 850 deaths. (www.who.int/health-topics/coronavirus). 5 in all coronaviruses, a homotrimeric spike glycoprotein on the virion's envelope mediates coronavirus entry into host cells through a mechanism of receptor binding followed by fusion of viral and host membranes. 3, 6 coronavirus spike protein contains two functional subunits s1 and s2. the s1 subunit is responsible for binding to host cell receptor, and the s2 subunit is responsible for fusion of viral and host cell membranes. 3, 7 the spike protein in ncov-2019 exists in a meta-stable pre-fusion conformation that undergoes a substantial conformational rearrangement to fuse the viral membrane with the host cell membrane. 7, 8 ncov-2019 is closely related to bat coronavirus ratg13 with about 93.1% sequence similarity in the spike protein gene. the sequence similarity of ncov-2019 and sars-cov is less than 80% in the spike genome. 2 s1 subunit in the spike protein includes a receptor binding domain (rbd) that recognizes and binds to the host cells receptor. the rbd of ncov-2019 shares 72.8% sequence identity to sars-cov rbd and the root mean squared deviation (rmsd) for the structure between the two proteins is 1 . which shows the high structural similarity. 4, 8, 9 experimental binding affinity measurements using surface plasmon resonance (spr) have shown that ncov-3 fold higher affinity than sars-cov binding to ace2. 7 based on the sequence similarity between rbd of ncov-2019 and sars-cov and also the tight binding between rbd of ncov-2019 and ace2, it is most probable that ncov-2019 uses this receptor on human cells to gain entry into the body. 3, 6, 7, 10 the spike protein and specifically the rbd domain in coronaviruses have been a major target for therapeutic antibodies. however, no monoclonal antibodies targeted to rbd have been able to bind efficiently and neutralize ncov-2019. 7, 11 the core of ncov-2019 rbd is a 5 the sequence alignment between sars-cov in human, sars civet, bat ratg13 coronavirus and ncov-2019 in the rbm is shown in figure 2 . there is a 50% sequence similarity between the rbm of ncov-2019 and sars-cov. rbm mutations played an important role in the sars epidemic in 2002. 12, 3 two mutations in the rbm of sars-2002 from sars-civet were observed from strains of these viruses. these two mutations were k479n and s487t. these two residues are close to the virus binding hotspots in ace2 including hotspot-31 and hotspot-353. hotspot-31 centers on the salt-bridge between k31-e35 and hotspot-353 is centered on the salt-bridge between k353-e358 on ace2. residues k479 and s487 in sars-civet are in close proximity with these hotspots and mutations at these residues caused sars to bind ace2 with significantly higher affinity than sars-civet and played a major role in civet-to-human and human-to-human transmission of sars coronavirus in 2002. 3, [13] [14] [15] numerous mutations in the interface of sars-cov rbd and ace2 from different strains of sars isolated from humans in 2002 have been identified and the effect of these mutations on binding ace2 have been investigated by surface plasmon resonance. 16, 17 two identified rbd mutations (y442f and l472f) increased the binding affinity of sars-cov to ace2 and two mutations (n479k, t487s) decreased the binding affinity. it was demonstrated that these mutations were viral adaptations to either human or civet ace2. 16, 17 a pseudotyped viral infection assay of the interaction between different spike proteins and ace2 confirmed the correlation between high affinity mutants and their high infection. 16 further investigation of rbd residues in binding of sars-cov and ace2 was performed through ala-scanning mutagenesis, which resulted in identification of 10 residues that reduce binding affinity to ace2 upon mutation to alanine. these residues are k390, r429, d429, d454, i455, n473, f483, q492, y494 and r495. 18 rbd mutations have also been identified in mers-cov, which affected their affinity to receptor (dpp4) on human cells. 17 it is not known whether these mutations are linked to the severity of coronavirus in these regions. the focus of this article is to elucidate the differences between the interface of sars-cov and ncov-2019 with ace2 to understand with atomic resolution the interaction mechanism and hotspot residues at the rbd/ace2 interface using long-timescale molecular dynamics (md) simulation. an alanine-scanning mutagenesis in the rbm of ncov-2019 helped to identify the key residues in the interaction, which could be used as potential pharmacophores for future drug development. furthermore, we performed molecular simulations on the seven most common mutations found from surveillance of rbd mutations n439k, t478i, v483a, g476s, s494p, v483f and a475v. from an evolutionary perspective this study shows the residues in which the virus might further evolve to be even more dangerous to human health. ncov-2019 shares 76% sequence similarity with sars-2002 spike protein, 73% sequence identity for rbd and 50% for the rbm. 1 bat coronavirus ratg13 seems to be the closest relative of ncov-2019 sharing about 93% sequence identity in the spike protein. 6 the 25 the mutations selected are listed in table s1 along with their location in rbd. the crystal structure of ncov-2019 in complex with hace2 (pdb id:6m0j) 28 as well as sars-cov complex with human ace2 (pdb id: 6acj) 29 were obtained from rcsb (www.rcsb.org). 30 the 5000 steps of energy minimizations were done using the steepest descent algorithm. in all steps the lincs algorithm was used to constraint all bonds containing hydrogen atoms and a time step of 2 fs was used as the integration time step. 33 equilibration of all systems were performed in three steps. in the first step, 100,000 steps of simulation were performed using a velocity-rescaling thermostat to maintain the temperature at 310k with a 0.1 ps coupling constant in nvt ensemble under periodic boundary conditions and harmonic restraints on the backbone and sidechain atoms of the complex. 34 the velocity rescaling thermostat was used in all other steps of simulation. in the next step, we performed 300,000 steps in the isothermalisobaric npt ensemble at temperature of 310k and pressure of 1bar using a berendsen barostat. 35 this was done by decreasing the force constant of the restraint on backbone and side chain atoms of the complex from 1000 to 100 and finally to 10 మ . berendsen barostat was only used for the equilibration step due to usefulness in rapidly correcting density. in the next step the restraints were removed, and the systems were subjected to 1,000,000 steps of md simulation under npt ensemble. in the production run, harmonic restraints were removed and all the systems were simulated using a npt ensemble where the pressure was maintained at 1bar using the parrinello-rahman barostat 36 with a compressibility of and a coupling constant of 0.5ps. it is important to note that all the berendsen barostat was only used for the equilibration step as it was shown that this barostat can cause unrealistic temperature gradients. 37 the production run lasted for 500ns for sars-cov and ncov-2019 complexes and 300ns for all the mutants using with a 2fs timestep and the particle-mesh ewald (pme) 36 for long range electrostatic interactions using gromacs 2018.3 package. 38 all mutant systems were constructed as described before and all complexes ran for 300ns of production run. the principal components were used to calculate and plot the approximate free energy landscape (afel). we refer to the free energy landscape produced by this approach to be approximate in that the ensemble with respect to the first few pc's (lowest frequency quasiharmonic modes) is not close to convergence, but the analysis can still provide valuable information and insight. g_sham, g_covar and g_anaeig functions in gromacs 41 were used to obtain principal components and afel. in each afel the deep valleys represent the most stable conformations separated by some intermediate states. the dynamic cross-correlation maps (dccm) were obtained using md_task package to identify the correlated motions of rbd residues. 42 in dccm the cross-correlation matrix by setting a value of 80 and 2 for solvent and solute dielectric constants. the non-polar free energy is simply estimated from solvent accessible surface area (sasa) of the solute from equation 5. to compute the rmsd of systems, the rotational and translational movements were removed by first fitting the c α atoms of the rbd to the crystal structure and then computing the rmsd with respect to c α atoms of rbd in each system. in most of the variants, the rmsd is stable during the 300ns simulation. however, a few mutations show some rmsd variance. in mutation y489a, the rmsd increases from the first two eigenvectors were used to calculate and plot the afel as a function of first two principal components using the last 200ns of the simulation for mutant systems. afel for a few mutants are shown in figure 6 and the rest of them are shown in figure s4 . the binding energetics between ace2 and the rbd of sars-cov, ncov-2019 and all its mutant complexes were investigated by the mmpbsa method. 44 for ncov-2019. the binding free energy for ncov-2019 and sars-cov was decomposed into a perresidue based binding affinity to find the residues that contribute strongly to the binding and complex formation (figure 9 ). most of the investigated residues in the rbm of ncov-2019 had a favorable contribution to total binding energy. binding free energy decomposition to its individual components for all mutants is represented in contributes the most to this low binding energies for these mutants. the contribution of rbm residues to binding with ace2 for ncov-2019 were mapped to the rbd structure and is shown in figure 11b . natural mutants exhibited similar or higher binding affinities compared to wild-type ncov-2019. importantly, mutation t478i which is one of the most occurred mutations in england based on gisaid database 25 table 1 contribution of interface residues to structure in rbd of ncov-2019. the rbd domain is purple and the ace2 is yellow. the rbd in contact with ac2 is rendered in a surface format with more red being a favorable contribution to binding (more negative) and blue unfavorable contribution (positive). in this work, we preformed md simulations to unveil the detailed molecular mechanism . d460 in sars-cov is located in a region of high negative charge from residues e35, e37 and d38 on ace2. electrostatic repulsion between d480 on sars-cov and the acidic residues on ace2 is the reason for highly negative contribution of this residue to binding of sars-cov to ace2. mutation to s494 in this location removes this highly negative contribution. to our knowledge this is first detailed molecular simulation study on the effect of mutations on binding of ncov-2019 to ace2. previous computational studies have found that ncov-2019 binds to ace2 with a total binding affinity which was about 3 0 stronger than sars-cov and is in fair agreement with the results here. 52 the critical role of interface residues and residues are computationally investigated here and in other articles and the results of all the studies indicate the importance of these residues for the stability of the complex and finding hotspot residues for the interaction with receptor ace2. 47, [52] [53] [54] it is interesting to note the role of shown there is a correlation between higher binding affinity to receptor and higher infection rate by coronavirus. [55] [56] [57] [58] high binding affinity for some mutants such as t478i could be the reason for higher human-to-human transmission rate in regions where these mutations are found. it is also alerting that mutations at other residues such as g446a, g449a, y449a and y489a increase the binding affinity considerably and should be monitored. mutations of ncov-2019 rbd that do not change the binding affinity and complex stability, could have implications for antibody design purposes since they could act antibody escape mutants. escape from monoclonal antibodies are observed for mutations of sars-cov in 2002 and these mutations should be considered for any antibody design endeavors against consider these escape mutations. in conclusion, this study unraveled key molecular traits underlying the higher affinity of ncov-2019 for ace2 compared to sars-cov and unveiled critical residues for the interaction higher affinity than wild-type. other occurring mutations n439k and e484a are found to increase the electrostatic interaction of rbd with ace2. it is also alerting that some of the alanine substitutions at residues g446, g447 and y489 substantially increased the binding affinity that may lead to a strongly rbd attachment to ace2 and influence the infection virulence. on the other hand, most mutations are found not to impact the binding affinity of rbd with ace2 in ncov-2019 which could have implications for vaccine design endeavors as these mutations could act as antibody escape mutants. receptor recognition is the first line of attack for coronavirus and this study gives novel insights to key structural features of interface residues for advancement of effective therapeutic strategies to stop the coronavirus pandemic. the authors would like to dedicate this article to the doctors and nurses who sacrificed their time, health and even their lives to fight covid-19, particularly those in iran and the united states. jbk would also like to dedicate this work to family friend 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title: covid-19 neutralizing antibodies predict disease severity and survival date: 2020-10-20 journal: medrxiv : the preprint server for health sciences doi: 10.1101/2020.10.15.20213512 sha: doc_id: 310636 cord_uid: y7n22ykt covid-19 exhibits variable symptom severity ranging from asymptomatic to life-threatening, yet the relationship between severity and the humoral immune response is poorly understood. we examined antibody responses in 113 covid-19 patients and found that severe cases resulting in intubation or death exhibited increased inflammatory markers, lymphopenia, and high anti-rbd antibody levels. while anti-rbd igg levels generally correlated with neutralization titer, quantitation of neutralization potency revealed that high potency was a predictor of survival. in addition to neutralization of wild-type sars-cov-2, patient sera were also able to neutralize the recently emerged sars-cov-2 mutant d614g, suggesting protection from reinfection by this strain. however, sars-cov-2 sera was unable to cross-neutralize a highly-homologous pre-emergent bat coronavirus, wiv1-cov, that has not yet crossed the species barrier. these results highlight the importance of neutralizing humoral immunity on disease progression and the need to develop broadly protective interventions to prevent future coronavirus pandemics. coronavirus infectious disease of 2019 , caused by infection with severe acute respiratory syndrome coronavirus 2 (sars-cov-2), exhibits significant variability in the severity of presentation. the impact of this variability on the development of protective immune responses and the role of antibodies in disease progression is unclear. there is currently no standard treatment regimen for either mild or severe cases of covid-19, and there is limited understanding of the impact that current investigational therapies have on immune responses against sars-cov-2. non-human primates (nhp) that have been exposed to sars-cov-2 have been found to develop potent antibody responses and are largely immune to reinfection (chandrashekar et al. , 2020; deng et al. , 2020) . similarly, animal models testing candidate vaccine approaches have demonstrated that protection against sars-cov-2 challenge is positively correlated with the development of high titers of neutralizing antibodies (mercado et al. , 2020; yu et al. , 2020) . importantly, passive transfer of convalescent sera has been shown to prevent infection in otherwise naive animals, highlighting the crucial role of antibodies in mediating protection against viral infection (hassan et al. , 2020; rogers et al. , 2020) . in contrast, the role of antibodies on the clearance of established sars-cov-2 infection and clinical outcomes is less clear. ordinarily, infections with viruses require cell-mediated immunity for viral clearance. antibodies mediate functions such as antibody-dependent cellular cytotoxicity (adcc) and phagocytosis (adcp) via innate immune cells such as nk cells and macrophages. yet, the need for antibodies in the clearance of sars-cov-2 infection has been challenged by two recent cases of patients with x-linked agammaglobulinemia who acquired and survived sars-cov-2 infection without requiring oxygen or intensive care (soresina et al. , 2020) . some studies even propose the possibility of a pathogenic role of antibodies in primary infection via antibody dependent enhancement (ade) and augmentation of inflammation (liu et al. , 2019) , although it is believed that this is insufficient to explain the prevalence of severe cases of sars-cov-2 infection (arvin et al. , 2020) . as such, a beneficial, neutral, or harmful role of antibodies in active coronavirus infection remains controversial. despite numerous clinical studies presently in progress, no broadly effective standard-of-care treatment has yet emerged for covid-19. remdesivir, a nucleotide analog active against sars-cov-2, has shown modest benefit in severe covid-19 cases by improving time to recovery (beigel et al. , 2020; . hydroxychloroquine was initially tested in patients based on in vitro studies z. chen et al. , 2020) , but subsequent meta-analyses and randomized controlled trials have demonstrated no benefit in preventing or treating covid-19 (boulware et al. , 2020; tang et al. , 2020; ullah et al. , 2020) . morbidity and mortality due to covid-19 is largely a consequence of adult respir atory distress syndrome (ards) caused by a combination of both hyperinflammatory and hypercoagulable states (domingo et al. , 2020) . among experimental treatments currently being evaluated, dexamethasone and other corticosteroids that result in immunosuppression have been shown to reduce disease severity (siemieniuk et al. , 2020) and improve survival (horby et al. , 2020) . given the involvement of immune dysregulation in the pathology of infection, the consequence of current interventions on the development of humoral immunity is not known. recent studies have demonstrated the emergence of sars-cov-2 variants containing amino acid substitutions in the viral spike protein targeted by antibodies, raising concerns for potential resistance to neutralization. one mutation, d614g, has rapidly become the predominant transmitted variant by outcompeting wildtype infections (korber et al. , 2020) . while it has been suggested that this mutant results in a more fit virus, the serological consequences of this change are unclear. additionally, recent studies in bats have described a novel coronavirus (wiv1-cov) with high homology to sars-cov-2 that uses the same ace2 receptor for cell entry (menachery et al. , 2016) . it has been postulated that this virus may present a similar pandemic risk if it were to spread from bats to humans. however, the consequence of prior sars-cov-2 seroconversion on neutralization of related coronaviruses like wiv1-cov has not been described. in this study, we characterized humoral immune responses and clinical outcomes in 113 sars-cov-2-infected patients of varying severity who received a range of treatments, as well as 1,257 pre-pandemic individuals. our covid-19 patient cohort contained a wide range of outcomes, including non-hospitalized, hospitalized, intubated, and deceased individuals. we assessed inflammatory markers, il-6 levels, lymphocyte counts, and demographic variables such as age and sex. a quantitative elisa that measures igg, igm, and iga antibodies to the receptor binding domain (rbd) of sars-cov-2 and a high-throughput neutralization assay using lentiviral vectors pseudotyped with sars-cov-2 and wiv1-cov were developed to assess neutralization potency and cross-neutralizing responses. remarkably, we find that anti-rbd antibody levels, neutralization titer, and neutralization potency index predicted disease severity and survival, yet lacked cross-neutralizing activity to pre-emergent wiv1-cov. taken together, our results highlight the impact of an effective humoral immune response on covid-19, as quantified by a neutralization potency index, and describe the a cross-sectional cohort of 113 covid-19 cases confirmed by sars-cov-2 nasopharyngeal pcr was studied and followed for at least 3 months. the cohort was divided into the following five groups based on disease severity, outcomes, and pre-existing health status: ( i ) non-hospitalized, which were never admitted to the hospital due to ( ii ) hospitalized, which were admitted for at least one day but were never intubated and were eventually discharged, ( iii ) intubated, which were intubated for at least one day but were subsequently extubated and discharged; ( iv ) deceased, which passed away due to and ( v ) immunosuppressed, which were includes some non-hospitalized, hospitalized, and intubated patients, but none deceased) ( supplementary table 1 ). when compared to non-hospitalized individuals, all cases of covid-19 resulting in hospital admission were significantly older in age (median age 63 versus 28, p < 0.0001) and there was a significant enrichment for males in severe cases resulting in intubation and/or death (74% versus 51% males, p = 0.02) ( figure 1a ), consistent with prior studies (meng et al. , 2020; n. chen et al. , 2020) . laboratory data showed that clinical severity correlated with markers of inflammation, namely, peak serum levels of c-reactive protein ( figure 1b ), ferritin ( figure s1a ), d-dimer ( figure s1b ), lactate dehydrogenase ( figure s1c ), and il-6 ( figure 1c ), as well as lymphopenia ( figure 1d ), as has been previously shown wynants et al. , 2020; x. chen et al. , 2020; zhou et al. , 2020) . interestingly, covid-19 severity was also associated with peak serum levels of troponin-t ( figure s1d ), a marker of myocardial damage and/or ischemia that may reflect cardiac injury, as has been previously described (tersalvi et al. , 2020) . altogether, our cohort contained a wide range of clinical presentations of sars-cov-2 infection with our analyses confirming previously described associations. its specificity to sars-cov-2 as well as its ease of production and stability (stadlbauer et al. , 2020) . full-length spike has more regions of homology to other coronaviruses that may cause greater false positivity, as has been shown between sars-cov, sars-cov-2, mers-cov, and common cold covs (chan et al. , 2005; ju et al. , 2020) . in addition, studies have shown that rbd is the main target of coronavirus neutralizing antibodies (he et al. , 2005) . we determined the sensitivity and specificity of this assay by assessing anti-rbd antibody levels in a cohort of sars-cov-2-infected patient serum samples collected between 14 to 42 days after symptom onset ( n = 85) in order to maximize seropositivity for igg, igm, and iga. we also assessed 1,257 pre-pandemic serum samples composed of a large unbiased cohort ( n = 1,124) and selected cohorts of individuals ( n = 133) with positive serology results for cytomegalovirus, varicella-zoster virus, hepatitis b virus, hepatitis c virus, hiv, syphilis, toxoplasma, and/or rheumatoid factor ( figure 2b ). anti-rbd igg, igm, and iga levels were measured for each sample by interpolation on to the standard curve and a receiver operating curve (roc) analysis was used to determined optimal cut-offs that distinguished sars-cov-2-infected patients from pre-pandemic controls ( figure 2c ). cut-offs of 1.18 u/ml for anti-rbd igg achieved 73% sensitivity, 2.14 u/ml for anti-rbd igm achieved 66% sensitivity, and 0.95 u/ml for anti-rbd iga achieved 48% sensitivity, with >99.0% specificity for all three. to assess the cross-reactivity of anti-rbd igg in sera of sars-cov-2 seropositive individuals, we modified our elisa to detect igg antibodies against the rbd of sars-cov and mers-cov. interestingly, no cross-reactivity was seen to sars-cov rbd despite 73% homology, nor to mers-cov, which has only 17% homology ( figure s2c and s2d ). additional experiments measuring igg antibodies against the rbd of two common cold coronaviruses-nl63, which has 20% homology to sars-cov-2 rbd, and hku1, which has 1.9% homology ( figure s2c )-showed a seroprevalence of >95% ( figure s2e ), as has been shown in previously published studies (gorse et al. , 2010) , with no correlation between the igg antibody levels of nl3 or hku1 with sars-cov-2 ( figure s2e ). these data show that anti-rbd igg antibodies induced during sars-cov-2 infection do not cross-react to recognize the rbd of other pandemic coronaviruses. in addition, anti-rbd igg antibodies to common cold coronaviruses appear to not provide detectable pre-existing reactivity to sars-cov-2 rbd nor do they correlate with anti-rbd igg levels in covid-19 patients. overall, these data suggest that natural infection with coronavirus results in anti-rbd antibodies with limited cross-reactivity. previous studies have demonstrated the potential to pseudotype retroviral vectors with sars-cov spike proteins (moore et al. , 2004) . however, pseudoviruses bearing sars-cov-2 spike produced by these methods yield low titers (nie et al. , 2020) , hampering large-scale testing of neutralization. recently, a forward genetics approach identified an efficiently replicating vesicular stomatitis virus (vsv) variant encoding sars-cov-2 spike containing a truncated form lacking the c-terminal 21 amino acids (case et al. , 2020) . interestingly, previous studies also showed a role of the cytoplasmic tail of sars-cov in altering surface expression and fusogenic potential (corver et al. , 2009) . to determine whether analogous truncations might improve sars-cov-2 pseudovirus production, we examined the cell-surface expression of truncated forms of sars-cov-2 spike and found that removal of 18 amino acids from the c-terminus (δ18) resulted in significantly greater cell-surface expression and higher titers of pseudovirus ( figure s2f -h ). this truncation removed a putative er-retention signal (lontok, corse and machamer, 2004; mcbride, li and machamer, 2007; ujike et al. , 2016) while retaining cysteine-rich domains that are highly conserved among coronaviruses. using these spike modifications, we developed a cov pseudovirus neutralization assay compatible with high-throughput liquid handling instrumentation in 384-well plate format using our previously published lentiviral vector system expressing both luminescent and fluorescent marker transgenes ( figure 2d ) (crawford et al. , 2020) . to validate our assay, the potency of a neutralizing monoclonal antibody, b38, and a non-neutralizing monoclonal antibody, cr3022, both of which target sars-cov-2 rbd with known ic50 values, was determined. this yielded ic50 values of~6 μg/ml for b38 and undetectable (>100 μg/ml) for cr3022, which were in agreement with previous reports (tian et al. , 2020; wu et al. , 2020) ( figure 2e and s2i ). in addition, we found that luciferase activity was directly proportional to the number of infected (i.e. zsgreen+) cells, providing flexibility in assay readout ( figure s2j ). to determine the performance of our assay on human sera, we measured the neutralization potency of human sera from 1,220 pre-pandemic individuals and 118 covid-19 patient samples >15 days after symptoms onset, with a dilution range of 1:12 to 1:8,748. the dilution titer that achieved 50% neutralization (nt50) was calculated for each specimen and roc analysis was performed, revealing that an nt50 threshold of 1:20 achieves a sensitivity of 94% and specificity of >99.0% in identifying covid-19 patients ( figure 2g -h ). overall, we found median titers of 1:664 in covid-19 patients, with potency ranging from <1:12 to >1:8,748. comparatively, titers of 1:5 for yellow fever vaccination, 1:8 for rubella vaccination, . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . and 1:40 for influenza vaccination are considered to indicate protective immunity (hannoun, megas and piercy, 2004; plotkin, 2010) . altogether, we established a highly accurate high-throughput sars-cov-2 pseudovirus neutralization assay that can accurately quantify the neutralization potency of humoral immune responses directed to sars-cov-2 spike protein. as a separate note for investigators using pseudovirus neutralization assays, we excluded pre-pandemic individuals taking antiretroviral therapy for human immunodeficiency virus infection or pre-exposure prophylaxis ( n = 37 in the original cohort of 1,257) after finding that potent inhibition of pseudovirus infection occurred in a majority of these individuals ( figure s2k ). we believe this was due to antiretroviral compounds in the patients' sera inhibiting transduction with our lentivirus-based vector system, thus generating an artifact. of note, non-documented antiretroviral use may explain a proportion of the false positives observed in the remaining specimens ( n = 12 out of 1,220). we proceeded to analyze antibody responses in our cohort of 113 covid-19 patients as well as a negative control cohort of 37 healthy blood donors, and found that in contrast to the typical kinetics of antibody responses in viral infections (i.e. igm before class-switched igg and iga), anti-rbd igg antibodies appear almost simultaneously with anti-rbd igm antibodies after symptom onset, and only a subset of individuals generate anti-iga antibodies concomitantly ( figure 3a -c ). interestingly, the development and quantity of anti-rbd igg antibodies appeared to be increased and sustained in the time frame analyzed (up to 72 days), while anti-rbd igm and iga antibodies waned after~42 days. neutralization titers closely resembled anti-rbd igg levels and were similarly sustained over time ( figure 3d ). overall, seropositivity at >14 days after symptom onset was 74% for anti-rbd igg, 61% for anti-rbd igm, 41% for anti-rbd iga, and 89% overall for any antibody. neutralization was detected in 95% of samples >14 days after symptom onset. to assess the humoral immune response among the pre-defined cohorts of varying disease severity, we focused on patients for which samples were collected between 14 and 42 days after symptom onset ( n = 85). this time frame was chosen to prevent biases resulting from time of sampling post-infection ( figure s3a ), which is known to have a significant impact on the magnitude of antibody responses. we found that severely ill patients that were intubated or passed away due to covid-19 had the highest anti-rbd igg and iga levels, but no significant . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . https://doi.org/10.1101/2020.10.15.20213512 doi: medrxiv preprint differences were seen for igm ( figure 3e -g ). these individuals also had the highest neutralization titers ( figure 3h ). in contrast, individuals that were not hospitalized had the lowest anti-rbd igg and iga levels and neutralization titers. unsurprisingly, immunosuppressed individuals-none of whom passed away-had significantly blunted igg, iga, and neutralizing responses. upon analyzing anti-rbd antibody seropositivity and neutralization titer, we found igg seropositivity was an excellent predictor of neutralization with a sensitivity of 78% and specificity of 100% ( figure 3i ). when seropositivity for any anti-rbd antibody was present, neutralization could be predicted with a sensitivity of 91% and specificity of 94%. anti-rbd igg levels correlated the most with neutralization ( r 2 = 0.76) ( figure 3j although anti-rbd igg levels correlated with neutralization by regression analysis, there was variability that appeared to segregate by our pre-defined severity cohorts ( figure 3j ). to better visualize this, we plotted residuals of each neutralization titer subtracted from its predicted titer based on the regression ( figure 3k ). this revealed that samples from severely ill patients were biased towards lower-than-predicted neutralization titers, suggesting that they harbored higher levels of non-neutralizing anti-rbd igg antibodies that did not contribute to neutralization. consequently, we calculated a neutralization potency index (nt50/igg) for each patient, and found that intubated or subsequently deceased patients had a significantly lower index ( figure 3l ), with all deceased patients having an index <100. accordingly, when patients were classified as having neutralization potency indices that were 'high' (≥100) or 'low' (<100), there was a significant risk of death in the days following sample collection in the 'low' index group (87% 30-day survival, n = 68) and there were no deaths in the 'high' index group (100% 30-day survival, n = 30) (p = 0.03; figure 3m ). of note, this finding was true across our entire cohort of 111 covid-19 patients (including non-hospitalized and immunosuppressed individuals) for which both anti-rbd igg. in addition, neutralization potency index did not correlate with days after symptom onset and remained predictive of survival when using a cox proportional hazards model that accounted for age, sex, hospitalization status, intubation status, . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . https://doi.org/10. 1101 and days between symptom onset and sample collection ( p = 0.03). these results suggest that neutralization potency index may help risk stratify patients irrespective of where they are in their disease course. altogether, severity of sars-cov-2 infection significantly correlates with higher anti-rbd antibody levels but sub-optimal neutralization potency is a significant predictor of mortality. to explore the influence of pre-existing medical conditions and covid-19 therapies on humoral immune responses to sars-cov-2 we performed multivariate analysis of all available demographic, clinical, laboratory, and experimental data ( figure s4 ). with the exception of immunosuppressed individuals, which had significantly decreased antibody and neutralizing responses, our cohort was not large enough to conclusively detect the effects of particular pre-existing medical conditions on the overall humoral immune response. however, a principle components analysis (pca) that included demographic data, pre-existing medical conditions, laboratory data, treatments received, anti-rbd antibody levels and neutralization titers but not clinical outcomes demonstrated clustering of patients by the severity cohorts ( figure 4a ). principal components were mainly influenced by inflammatory markers, anti-rbd antibody levels, and neutralization titers, but a contribution from pre-existing medical conditions such as hypertension and diabetes was observed ( figure 4b ). to assess the effect of different treatments on the humoral immune response, we performed an analysis limited to samples collected from patients that had initiated treatment and were in the hospital for at least 3 days ( n = 69). covid-19-directed treatment regimens included azithromycin (an antibiotic with anti-inflammatory properties), remdesivir, hydroxychloroquine, corticosteroids, and tocilizumab (an anti-il-6 receptor antibody). of note, individuals in the tocilizumab-treated cohort included 3 individuals known to receive tocilizumab for compassionate use and 16 patients enrolled in a blinded clinical trial with 2:1 tocilizumab-to-placebo randomization (i.e. some patients might have received placebo). azithromycin, remdesivir, and hydroxychloroquine-for which there was concern of attenuating antibody responses (de miranda santos and costa, 2020) -did not significantly affect anti-rbd antibody levels or neutralization titers in our cohort ( figure 4c ). however, we found that use of corticosteroids and tocilizumab significantly decreased anti-rbd igg concentration, and in the case of corticosteroids, neutralization titer ( figure 4c ). corticosteroids are a general . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . immunosuppressant known to decrease antibody production, whereas il-6 signaling is important in several aspects of antibody responses (kopf et al. , 1998) . interestingly, tocilizumab-treated patients had a significant increase in the neutralization potency index stemming from the larger effect on anti-rbd igg as compared to neutralization ( figure 4c ). this result raises new questions regarding the role of il-6 signaling in the production of non-neutralizing versus neutralizing antibodies and how these might become de-coupled, although a selection bias cannot be excluded. altogether, immunomodulatory therapies, some of which have shown clinical efficacy or are actively being studied, influence humoral immune responses in sars-cov-2-infected patients. given the importance of humoral immunity in preventing most viral infections, the recent emergence of a mutation in the sars-cov-2 spike protein (d614g) has raised concerns for the potential for convalescent patients to become re-infected. studies have demonstrated that this variant may possess greater replicative fitness and an altered conformation of the spike protein that may render pre-existing immunity less effective (korber et al. , 2020) . to determine the impact of this variant on the neutralization potency of sera from patients previously infected with sars-cov-2, we introduced the d614g mutation into the sars-cov-2 δ18 spike ( figure 5a ). when characterizing this new construct, we found that both surface expression and infectivity were further increased relative to that of the d614 sars-cov-2 δ18 spike ( figure 5b and s5a,c,d,f ), in line with previous studies (korber et al. , 2020) . we tested this new pseudovirus, normalizing for infectious units per well, against the same panel of patient samples and found an increase in neutralizing titers that was very small but statistically significant ( figure 5c -d ), an effect that was seen in a prior study (korber et al. , 2020) . this indicates that individuals that have been infected with either d614 wild-type or g614 mutant sars-cov-2 will have cross-neutralization to the opposite strain, both of which are circulating in boston, massachusetts (lemieux et al. , 2020) and were likely represented in our study cohort. the emergence of sars-cov, mers-cov, and now sars-cov-2 within the last two decades has demonstrated the ability of zoonotic coronaviruses to cross the species barrier and pose pandemic threats. this has prompted microbiologists and epidemiologists to seek out and characterize zoonotic coronaviruses that have the potential to cross into humans. recent studies in bats have identified a novel coronavirus, wuhan institute of virology 1 coronavirus (wiv1-cov), which, like sars-cov-2 and sars-cov, has a spike that uses ace2 receptor for . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . cell entry and bears high sequence homology to both sars-cov (92%) and sars-cov-2 (77%). we generated wiv1-cov pseudovirus using an analogous spike truncation (δ18) ( figure 5a ), which resulted in high expression of wiv1-cov spike on producer cells as well as infectivity and titer ( figure 5e and s5b,c,e,f ). these results suggest that this c-terminal truncation can serve as a general approach for modifying coronavirus spike proteins for efficient pseudovirus production. interestingly, wiv1-cov spike could be detected at the cell surface by the sars-cov and -cov-2-specific monoclonal antibody cr3022 ( figure s5b ), a finding that, to the best of our knowledge, has not been previously described. using wiv1-cov pseudovirus, we found that sera from sars-cov-2-infected individuals showed a lack of cross-neutralization except for relatively low-level neutralization in a few individuals with very high sars-cov-2 neutralization titers ( figure 5f -g ). this indicates that humoral immunity raised against one coronavirus is generally insufficient to generate cross-neutralizing immunity to even highly related coronavirus strains. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . https://doi.org/10.1101/2020.10.15.20213512 doi: medrxiv preprint traditionally, cellular immunity is responsible for clearing an established viral infection, while humoral immune responses play a more critical role in preventing future infection. here we found that severely ill covid-19 patients had the highest levels of anti-rbd antibodies, which other studies have similarly described (shrock et al. , 2020) . to further characterize this antibody response, we measured neutralization titers and developed a neutralization potency index derived from our quantitative readouts (nt50/igg) to assess the quality of anti-rbd antibodies irrespective of the quantity produced. remarkably, neutralization potency was significantly diminished in severely ill patients, and survival analysis demonstrated that an index of ≥100 was predictive of 100% 30-day survival, whereas <100 was associated with 87% 30-day survival in our limited cohort of 113 covid-19 patients. thus, this neutralization potency index may be a useful metric for physicians seeking to risk-stratify covid-19 patients. despite the clear correlation between covid-19 severity and development of humoral immunity, the cause-effect relationship between these two is unclear. one possibility is that severe disease caused by hyperinflammation and/or uncontrolled viral replication induces overproduction of antibodies that serve as a 'biomarker' of severity. this is supported by our finding that the most severely affected patients had the highest levels of inflammatory markers and cytokines, which can drive antibody production. in support of this possibility, a recent study suggests a pathogenic role of immune activation and exuberant antibody production from extrafollicular b cells in critically ill patients (woodruff et al. , 2020) . indeed, of all the covid-19 treatment regimens being used and tested, dampening of the immune response with corticosteroids has proven to have one of the greatest benefits in improving outcomes and survival (siemieniuk et al. , 2020) , and we find that corticosteroids decrease both anti-rbd igg levels and neutralization titers. however, another possibility is that high levels of antibodies with low neutralization potency worsen disease severity, possibly via ade. this is supported by our finding of decreased neutralization potency in severely ill patients, and raises concerns over the use of convalescent plasma as a treatment strategy. one exception, however, may be in immunosuppressed individuals, which generally have sub-optimal antibody levels and neutralization titers. further studies in animal models of covid-19 testing passive transfer of low-potency index sera may help resolve this controversy. a multitude of vaccines are presently being evaluated for sars-cov-2 prevention, including inactivated virus (gao et al. , 2020) , spike antigen (jackson et al. , 2020; keech et al. , 2020) , and rbd antigen (dai et al. , 2020; mulligan et al. , 2020) . each will likely result in . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . https://doi.org/10.1101/2020.10.15.20213512 doi: medrxiv preprint humoral immunity with different ratios of neutralizing and non-neutralizing antibodies. given our results, it will be important to assess the potency index of each candidate to determine those with maximal potential. interestingly, one study showed that vaccination of mice with rbd generated potently neutralizing antibodies without antibody-dependent enhancement. this was postulated to be due to the lack of immunodominant non-neutralizing epitopes present on the remainder of the spike protein (quinlan et al. , 2020) . the diverse and atypical kinetics of antibody production-in particular, early rise of igg and in some cases iga-suggests the possibility of a contribution from class-switched (igg+ or iga+) memory b cells early in the humoral immune response rather than solely from the naive (igm+) b cell pool, as has been recently postulated (song et al. , 2020) . regardless, our results support a role for anti-rbd igm and iga in contributing to sars-cov-2 neutralization, despite their transient nature in serum. anti-rbd igg responses and neutralization, on the other hand, were sustained in the time frame analyzed (~60 days), but several studies have emerged that question the longevity of these responses, which have yet to be determined. it is tempting to speculate that severely afflicted individuals may have more enduring immunity than mild cases. the differences in humoral response induction may stem from a combination of factors, including host permissibility to viral replication and a rapid response from innate immune effector cells and cytotoxic t cells, some of which have been postulated to arise from cross-reactive memory cells to other coronaviruses (grifoni et al. , 2020) . although the mutation rate of coronaviruses is very low when compared to other viruses such as influenza or hiv, certain mutations in the spike protein of sars-cov-2 have emerged in the setting of the rapidly spreading pandemic. we found that one such mutation, d614g, which has now spread and become a dominant strain worldwide, does not affect the neutralizing ability of patient sera, reducing concerns for re-infection. still, prior coronavirus pandemics (e.g. sars-cov, mers-cov, and now sars-cov-2) have occurred due to zoonotic coronaviruses crossing the species barrier, indicating an ongoing threat of future pandemics even in the face of effective vaccines to current viruses. one pre-emergent bat coronavirus, wiv1-cov, is highly homologous to sars-cov and sars-cov-2 and can infect ace2-expressing human cells (menachery et al. , 2016) . our data demonstrate that sera from sars-cov-2 infected patients exhibit very limited cross-neutralization of wiv1-cov, except for rare individuals with relatively low-level neutralization of wiv1-cov, suggesting that generation of broadly neutralizing antibodies is indeed possible, as has been previously described (wec et al. , 2020) . in summary, the development of potently neutralizing humoral immunity against . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . https://doi.org/10.1101/2020.10.15.20213512 doi: medrxiv preprint sars-cov-2 appears to increase survival, and may protect against re-infection with other circulating strains of sars-cov-2. however, it is generally unlikely to provide protection against subsequent coronavirus pandemics. as such, future efforts should focus on the development of broadly active therapies and prevention modalities that generate potently neutralizing antibodies with activity across different coronavirus strains. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . https://doi.org/10.1101/2020.10.15.20213512 doi: medrxiv preprint to quantitatively detect igg, igm, and iga antibodies to sars-cov-2 receptor binding domain (rbd), we developed an indirect elisa using an anti-sars-cov and -cov-2 monoclonal antibody (cr3022) with igg1, igm, and iga1 isotypes (kindly provided by galit tween-20 detergent served as an inactivation agent to render samples non-infectious, as has been previously described for other enveloped viruses (mayo and beckwith, 2002) . a seven-point standard curve was created using each of the standards (i.e. cr3022-igg1, cr3022-igm, cr3022-iga1) starting at 2 μg/ml by performing 1:3 serial dilutions with dilution buffer. samples and standards were added to corresponding wells and incubated for 1 h at 37°c, followed by washing. human antibody isotypes were detected with specific antibodies (bethyl) diluted as indicated: anti-human igg-hrp (1:25,000), anti-human igm-hrp (1:20,000), and anti-human iga-hrp (1:5,000). these were added to each plate and incubated for 30 min at room temperature. after washing, tmb substrate (inova) was added to each well and incubated for 7 min (for igg), 13 min (for igm), and 10 min (for iga), before stopping with 1 m h 2 so 4 . buffer compositions, reagent concentrations and incubation times and temperatures were optimized in separate experiments for each analyte to maximize signal-to-noise ratio. optical density (o.d.) was measured at 450 nm with subtraction of the o.d. at 570 nm as a reference wavelength on a spectramax abs microplate reader. anti-rbd antibody levels were calculated by interpolating onto the standard curve and correcting for sample dilution; one unit per ml (u/ml) was defined as the equivalent reactivity seen by 1 μg/ml of cr3022. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . https://doi.org/10.1101/2020.10.15.20213512 doi: medrxiv preprint to compare the neutralizing activity of patient sera against coronaviruses, we produced lentiviral particles, pseudotyped with different spike proteins, by transient transfection of 293t cells and titered the viral supernatants by flow cytometry on 293t-ace2 cells (moore et al. 2004 ). virus production was also quantified by p24 elisa on viral supernatants using the hiv-1 p24 ca antigen capture assay (leidos biomedical research, inc). to increase throughput and consistency, assays and readouts were performed on a fluent automated workstation (tecan) using 384-well plates (grenier). following an initial 12-fold dilution, the liquid handler performed serial three-fold dilutions (ranging from 1:12 to 1:8,748) of each patient serum and/or purified antibody in 20 μl followed by addition of 20 μl of pseudovirus containing 125 infectious units and incubation for 1 h at room temperature. finally, 10,000 293t-ace2 (moore et al. , 2004) cells in 20 μl cell media containing 15 μg/ml polybrene were added to each well and incubated at 37°c for 60-72 h. cells were lysed using a previously described assay buffer (siebring-van olst et al., 2013) and luciferase expression was quantified using a spectramax l luminometer (molecular devices). percent neutralization was determined by subtracting background luminescence measured in cell control wells (cells only) from sample wells and dividing by virus control wells (virus and cells only). of note, repeated sera neutralization measurements in independent assays using 500, 250, 125 infectious units of pseudovirus per well generated similar results (data not shown), indicating that the nt50 is not significantly influenced by pseudovirus titers. data was analyzed using graphpad prism and nt50 values were calculated by taking the inverse of the 50% inhibitory concentration value for all samples with a neutralization value of 80% or higher at the highest concentration of serum or antibody. to quantify the pseudotyped lentiviral supernatants in terms of infectious units, we plated 400,000 of either 293t or 293t-ace2 cells in 1 ml in a 12-well plate format (corning). 24 h later, ten-fold serial dilutions of lentiviral transfection supernatant were made in 100 μ l, which was then used to replace 100 μ l of media on the plated cells. cells were then incubated with lentivirus supernatant for 48 h at 37°c and then harvested with trypsin-edta (corning), resuspended in pbs supplemented with 2% fbs (pbs+), and measured on a stratedigm s1300exi flow cytometer. samples were gated for zsgreen expression. to compare the relative surface expression of pseudovirus spike protein, we plated 400,000 293t cells per well in 1 ml in a 12-well plate. 24 h later, we transfected each well with . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . https://doi.org/10.1101/2020.10.15.20213512 doi: medrxiv preprint a lentiviral helper vector coding for different spike proteins. the cells were incubated for 48 h at 37°c and harvested into pbs containing 1% fetal bovine serum (sigma) (called pbs+). cells transfected with each vector were divided into 3 aliquots, stained with either pbs+, cr3022 sars-cov antibody (10 μ g/ml in pbs+), or b38 sars-cov-2 antibody (10 μg/ml in pbs+) for 30 minutes at room temperature. cells were then washed with 1 ml pbs+, spun at 1,150 x g, and stained with anti-human igg af647 polyclonal antibody (invitrogen) at 2 μg/ml in pbs+ for 30 minutes at rt. cells were washed with 1 ml of pbs+, spun at 1,150 x g, resuspended in 150 μ l of pbs+ and measured on a stratedigm s1300exi flow cytometer. 60-72 hours after neutralization assay setup, each well in a serum dilution series within a 384-well plate was imaged using a fitc filter to detect cellular zsgreen expression. images were acquired using a 20x air objective on a zeiss lsm510 instrument. acquired images were analyzed using imagej to produce overlays. use of patient samples for the development and validation of sars-cov-2 diagnostic tests was approved by partners institutional review board (protocol 2020p000895). serum samples from 113 patients diagnosed with covid-19 (confirmed by at least one sars-cov-2 pcr-positive nasopharyngeal swab at massachusetts general hospital) were collected over course of several weeks, resulting in partially longitudinal, cross-sectional cohort consisting of 165 serum samples, with a prospective follow-up period of at least 3 months to assess clinical course and outcomes by manual chart review by at least two physicians. for each patient, the following information was obtained: age, sex, sars-cov-2 pcr results, date of symptom onset, hospitalization and discharge dates, intubation and extubation dates, and deceased date. date of symptom onset was defined as the earliest date that at least one of the following covid-19-related symptoms was reported as developing acutely and new from baseline: fever, chills, loss of smell or taste, body aches, rhinorrhea, nasal congestion, sore throat, cough, shortness of breath. if the date of symptom onset could not be determined with confidence, this information was excluded from the analysis. patients were assessed for the presence of absence of the following pre-existing medical conditions: lung disease (e.g. asthma, copd), heart disease (e.g. coronary artery disease, heart failure), vascular disease (e.g. peripheral vascular disease), hypertension, diabetes, obesity (bmi >30), kidney disease, autoimmune . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020 . . https://doi.org/10.1101 disorder, solid organ cancer, chemotherapy for solid organ cancer, hematologic cancer, chemotherapy or immunotherapy for hematologic cancer, history of organ transplant, history of hematopoietic stem cell transplant, and pre-existing use of corticosteroids or other immunosuppressive medications. based on these information, the cohort was divided into the following groups based on severity of disease and underlying health status: ( i ) non-hospitalized, consisting of individuals that were never admitted to the hospital and were sent home to quarantine; ( ii ) hospitalized, which included individuals that were hospitalized for at least one night but were never intubated and were eventually discharged; ( iii ) intubated, comprising hospitalized individuals that were intubated for at least one day but survived and were eventually discharged; ( iv ) deceased, for which we had obtained a specimen before they eventually passed away in the hospital; and ( v ) immunosuppressed, which consisted of people that were on immunosuppressive medication (including high-dose corticosteroid) and/or were afflicted by a clinically significant hematologic malignancy before being diagnosed with covid-19. laboratory data throughout admission were analyzed, and the maximum documented serum levels of ferritin, c-reactive protein, d-dimer, lactate dehydrogenase, troponin-t, and il-6 were recorded for each patient, as well as the lowest absolute lymphocyte count documented (lymphocyte count nadir). in addition, use of the following treatments were documented: corticosteroids, hydroxychloroquine, azithromycin, atorvastatin, remdesivir, lopinavir/ritonavir, tocilizumab (part of treatment versus placebo trial, currently blinded), and anakinra. all information obtained from medical records was verified by at least two physicians. pre-pandemic serum samples ( n = 1,257) were obtained from the clinicals laboratories at massachusetts general hospital. these samples were comprised of an unbiased cohort of individuals being tested for measles, mumps, and rubella titers ( n = 1124), as well as a selected subset of 133 individuals with positive serology results for cytomegalovirus ( n = 10), varicella-zoster virus ( n = 25), hepatitis b virus ( n = 25), hepatitis c virus ( n = 24), hiv ( n = 37), syphilis ( n = 16), toxoplasma ( n = 1), and rheumatoid factor ( n = 1). statistical and data analyses were performed using graphpad prism 8. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020 . . https://doi.org/10.1101 proportional hazards models performed by both jmp pro and r confirmed these findings after accounting for additional variables. when using r, the cox proportional hazards model was performed using the coxph function from the survival package v3.2-7 (https://cran.rproject.org/package=survival) in r v4.0.2 (r core team 2020). in . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020 . . https://doi.org/10.1101 ( c ) roc analyses for each assay were done to assess how seropositivity predicted covid-19 . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . status. area under the curve (auc) was 0.94 for igg, 0.92 for igm, and 0.86 for iga. cut-offs of 1.18 u/ml for igg achieved a sensitivity of 73%, 2.14 u/ml for igm achieved 66%, and 0.95 u/ml for iga achieved 48%, with >99.0% specificity for all three. ( d ) a schematic of the high-throughput sars-cov-2 pseudovirus neutralization assay is shown. ( e ) validation of the neutralization assay using a recently discovered neutralizing monoclonal antibody, b38, was performed and showed an ic50 of 6 μg/ml. ( f ) neutralization titers that achieved 50% neutralization (nt50) were calculated for pre-pandemic samples ( n = 1,220, individuals on antiretroviral therapy excluded) and covid-19 patient samples ( n = 118). ( g ) an roc analysis demonstrated an auc of 0.97, with an nt50 cut-off of 20 achieving sensitivity of 94% and specificity of >99.0%. ( a-c ) anti-rbd igg, igm, and iga levels were plotted over days after symptom onset for confirmed covid-19 cases for which data of symptom onset was known ( n = 98 patients, n = 147 samples total). healthy blood donors ( n = 37) are included as a negative control within the gray region. the dotted lines indicate the cut-offs for anti-rbd igg, igm, and iga seropositivity. ( d ) titers that achieve 50% neutralization (nt50) were plotted over days after symptom onset for each patient sample. ( e-h ) patient samples were selected for collection between 14 and 42 days after symptom onset (earliest time point for each patient), and for each cohort of healthy blood donors, non-hospitalized, hospitalized, intubated, deceased, and immunosuppressed patients, anti-rbd igg, igm, iga, and neutralization (nt50) was plotted. non-parametric multivariate anova was performed for each (excluding healthy blood donors); statistical significance is indicated as follows: **** p < 0.0001, *** p < 0.001, ** p < 0.01, and * p < 0.05. ( i-j ) an roc and log-log regression analyses were performed on igg versus neutralization. for j , the severity cohort is indicated as follows: healthy (white), non-hospitalized (green), hospitalized (yellow), intubated (red), deceased (gray), and immunosuppressed (blue). for j , pearson correlations were performed and r 2 and p values are indicated. ( k ) a residual plot for neutralization titer was generated from the log-log correlation. the gray ellipse indicates a cluster of samples from intubated (red) and deceased (gray) patients. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . ( c ) sub-analyses on covid-19 patients that were in the hospital for at least 3 days to ( n = 69) were performed on the last collected specimen to show the effect of azithromycin ( n = 10 treated), remdesivir ( n = 9 treated), hydroxychloroquine ( n = 8 treated), corticosteroids ( n = 9 treated), and tocilizumab ( n = treated as part of a trial with 2:1 randomization to placebo) on anti-rbd igg levels (upper panel), neutralization titer (middle panel), and neutralization potency index (nt50/igg) (lower panel). a t test was performed for each comparison; * indicates unadjusted p < 0.05. mutant sars-cov-2 spike but not the highly homologous pre-emergent bat coronavirus wiv1-cov. ( a ) a schematic of the sars-cov-2 and wiv1-cov spike proteins, including full-length, truncated (δ18), and mutant (d614g) forms is shown. full-length wiv1-cov spike has 77.5% sequence homology to sars-cov-2 spike and has the same putative er retention signal (errs) as sars-cov-2. ( b ) expression of full-length, δ18, and δ18 d614g sars-cov-2 spike constructs in 293t cells . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . https://doi.org/10. 1101 in comparison to empty vector (neg. ctrl) was measured by flow cytometry (upper panel). infectivity of lentivirus, which was defined as the infectious units divided by the quantity of p24 in lentiviral supernatant, was also measured and compared to vsv-g-pseudotyped lentivirus (lower panel). ( c-d ) cross-neutralization of serum samples from covid-19 patients that were non-hospitalized (green, n = 16), hospitalized (yellow, n = 67), intubated (red, n = 43), deceased (gray, n = 15), or immunosuppressed (blue, n = 21) and healthy blood donors ( n = 35) was measured for wild-type versus d614g mutant sars-cov-2 δ18 spike pseudovirus. for c, pearson correlations were performed and r 2 and p values are indicated; for d , paired, non-parametric t test was performed; *** indicates p < 0.001. ( e ) similar to b , expression and infectivity of full length and δ18 wiv1-cov spike was measured. ( f-g ) similar to c-d , cross-neutralization of serum samples from covid-19 patients was measured for wild-type sars-cov-2 versus wiv1-cov pseudovirus. for f, pearson correlations were performed and r 2 and p values are indicated; for g , paired, non-parametric t test was performed; **** indicates p < 0.0001. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020 . . https://doi.org/10.1101 clinical laboratory-defined cut-offs of the upper limit of normal are indicated with a dotted line. for each parameter, a non-parametric anova was performed; statistical significance is indicated as follows: **** p < 0.0001, *** p < 0.001, ** p < 0.01, and * p < 0.05. ( h ) pseudovirus titers of the indicated spike constructs were quantified. ( i ) lack of neutralizing ability of cr3022 mab was confirmed in pseudovirus neutralization . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . a multi-variate analysis of all available data including age, sex, language, hospital course and events, pre-existing medical conditions, treatments received, clinical laboratory data, and antibody and neutralization data was performed, with pearson coefficients ( r ) ranging from -1 (red) to 0 (white) to +1 (blue). the presence of an 'x' indicates that there were insufficient data to correlate the variables in question. the following abbreviations were used: daso, days after symptom onset; dapp, days after pcr positivity; dpp, days pcr positive (total number of days between first pcr positive results and last pcr positive result that was followed by one . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . negative result); dhos, days hospitalized; hsct, hematopoietic stem cell transplant; crp, c-reactive protein; ldh, lactate dehydrogenase; ck, creatine kinase; anti-rbd, anti-receptor binding domain; anti-nc ab, anti-nucleocapsid antibody (as measured by the commercially available roche sars-cov-2 total antibody chemiluminescent assay); sc2, sars-cov-2. figure s5 . characterization of cov spike expression vectors. ( a ) surface level expression of sars-cov-2 spike protein following transfection of 293t cells. several constructs of spike were tested: codon-optimized full-length spike from sars-cov-2, a truncated version with 18 amino acids deleted from the cytoplasmic tail (δ18), and a truncated version that also includes a d614g mutation. expression was measured via flow cytometry by staining with b38 antibody at a concentration of 10 μg/ml followed by staining with an anti-human igg antibody conjugated to af647 at 2 μg/ml. ( b ) surface level expression of full-length and truncated (δ18) wiv1-cov spike proteins were also measured following transfection of 293t cells via flow cytometry. expression was measured via flow cytometry by staining with cr3022 antibody at a concentration of 10 μg/ml followed by staining with an anti-human igg antibody conjugated to af647 at 2 μg/ml. ( f ) transduction with 10-fold serial dilutions and subsequent assessment of zsgreen expression by flow cytometry was performed to calculate pseudovirus titer (u/ml) for each construct indicated. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . https://doi.org/10.1101/2020.10.15.20213512 doi: medrxiv preprint (a) a cross-sectional cohort of covid-19 patients (n = 113) was divided into groups of varying clinical severity, i.e., non-hospitalized (n = 18), hospitalized (n = 45), intubated (n = 27), deceased (n = 10), and immunosuppressed (n = 13) and analyzed for their age and sex. median age was 28 years in patients who were never hospitalized (n = 20; includes 2 from immunosuppressed group) and 63 years in all patients who were admitted to the hospital (n = 93), with statistical significance of p < 0.0001 with t test. fisher's exact test on the number of males who were intubated or deceased (n = 31 males out of 42 total; includes 5 from immunosuppressed group who were intubated) versus not (n = 36 males out of 71 total) demonstrated a significant enrichment with p = 0.02. (b-d) peak levels of c-reactive protein and il-6 as well as lymphocyte count nadir are presented in violin plots when data was available. in c, none of the non-hospitalized patients had serum il-6 levels measured (n.a., not assessed). for b and c, clinical laboratory-defined cut-offs of the upper limit of normal are indicated with a dotted line; for d, the dotted line represents the lower limit of normal. for each parameter, a non-parametric anova was performed; statistical significance is indicated with the following notations: **** p < 0.0001, *** p < 0.001, ** p < 0.01, and * p < 0.05. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. values from samples and calculate units/ml (u/ml), with 1 u/ml defined as the equivalent reactivity caused by 1 μg/ml of the corresponding cr3022 monoclonal antibody (b) anti-rbd igg, igm, and iga antibodies were measured in both pre-pandemic samples (n = 1,257) and covid-19 patient samples (n = 85). dotted lines indicate the threshold of seropositivity that achieves >99.0% specificity on roc analyses. (c) roc analyses for each assay were done to assess how seropositivity predicted covid-19 status. area under the curve (auc) was 0.94 for igg, 0.92 for igm, and 0.86 for iga. cut-offs of 1.18 u/ml for igg achieved a sensitivity of 73%, 2.14 u/ml for igm achieved 66%, and 0.95 u/ml for iga achieved 48%, with >99.0% specificity for all three. (d) a schematic of the high-throughput sars-cov-2 pseudovirus neutralization assay is shown. (e) validation of the neutralization assay using a recently discovered neutralizing monoclonal antibody, b38, was performed and showed an ic50 of 6 μg/ml. (f) neutralization titers that achieved 50% neutralization (nt50) were calculated for pre-pandemic samples (n = 1,220, individuals on antiretroviral therapy excluded) and covid-19 patient samples (n = 118). (g) an roc analysis demonstrated an auc of 0.97, with an nt50 cut-off of 20 achieving sensitivity of 94% and specificity of >99.0%. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . https://doi.org/10.1101/2020.10.15.20213512 doi: medrxiv preprint (a-c) anti-rbd igg, igm, and iga levels were plotted over days after symptom onset for confirmed covid-19 cases for which data of symptom onset was known (n = 98 patients, n = 147 samples total). healthy blood donors (n = 37) are included as a negative control within the gray region. the dotted lines indicate the cut-offs for anti-rbd igg, igm, and iga seropositivity. (d) titers that achieve 50% neutralization (nt50) were plotted over days after symptom onset for each patient sample. (e-h) patient samples were selected for collection between 14 and 42 days after symptom onset (earliest time point for each patient), and for each cohort of healthy blood donors, non-hospitalized, hospitalized, intubated, deceased, and immunosuppressed patients, anti-rbd igg, igm, iga, and neutralization (nt50) was plotted. non-parametric multivariate anova was performed for each (excluding healthy blood donors); statistical significance is indicated as follows: **** p < 0.0001, *** p < 0.001, ** p < 0.01, and * p < 0.05. (i-j) an roc and log-log regression analyses were performed on igg versus neutralization. for j, the severity cohort is indicated as follows: healthy (white), non-hospitalized (green), hospitalized (yellow), intubated (red), deceased (gray), and immunosuppressed (blue). for j, pearson correlations were performed and r 2 and p values are indicated. (k) a residual plot for neutralization titer was generated from the log-log correlation. the gray ellipse indicates a cluster of samples from intubated (red) and deceased (gray) patients. (l) neutralization potency index (nt50/igg) was calculated for all 113 patients (at earliest time point) and plotted by cohort. a nonparametric multivariate anova was performed without correction for multiple comparisons; unadjusted p values are indicated as follows: ** p < 0.01, * p < 0.05. (m) survival analysis of covid-19 patients classified as having a high (≥100) (n = 35) or low (<100) (n = 76) neutralization potency index (nt50/igg) was performed using kaplan-meier method and revealed significantly decreased risk of death in low neutralization potency individuals (p = 0.03). . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 20, 2020. . https://doi.org/10.1101/2020.10.15.20213512 doi: medrxiv preprint (c) sub-analyses on covid-19 patients that were in the hospital for at least 3 days to (n = 69) were performed on the last collected specimen to show the effect of azithromycin (n = 10 treated), remdesivir (n = 9 treated), hydroxychloroquine (n = 8 treated), corticosteroids (n = 9 treated), and tocilizumab (n = treated as part of a trial with 2:1 randomization to placebo) on anti-rbd igg levels (upper panel), neutralization titer (middle panel), and neutralization potency index (nt50/igg) (lower panel). a t test was performed for each comparison; * indicates unadjusted p < 0.05. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 20, 2020. . https://doi.org/10.1101/2020.10.15.20213512 doi: medrxiv preprint : sars-cov-2-infected patient sera cross-neutralizes both wild-type and d614g mutant sars-cov-2 spike but not the highly homologous pre-emergent bat coronavirus wiv1-cov. (a) a schematic of the sars-cov-2 and wiv1-cov spike proteins, including full-length, truncated (δ18), and mutant (d614g) forms is shown. full-length wiv1-cov spike has 77.5% sequence homology to sars-cov-2 spike and has the same putative er retention signal (errs) as sars-cov-2. (b) expression of full-length, δ18, and δ18 d614g sars-cov-2 spike constructs in 293t cells in comparison to empty vector (neg. ctrl) was measured by flow cytometry (upper panel). infectivity of lentivirus, which was defined as the infectious units divided by the quantity of p24 in lentiviral supernatant, was also measured and compared to vsv-g-pseudotyped lentivirus (lower panel). (c-d) cross-neutralization of serum samples from covid-19 patients that were non-hospitalized (green, n = 16), hospitalized (yellow, n = 67), intubated (red, n = 43), deceased (gray, n = 15), or immunosuppressed (blue, n = 21) and healthy blood donors (n = 35) was measured for wild-type versus d614g mutant sars-cov-2 δ18 spike pseudovirus. for c, pearson correlations were performed and r 2 and p values are indicated; for d, paired, non-parametric t test was performed; *** indicates p < 0.001. (e) similar to b, expression and infectivity of full length and δ18 wiv1-cov spike was measured. (f-g) similar to c-d, cross-neutralization of serum samples from covid-19 patients was measured for wild-type sars-cov-2 versus wiv1-cov pseudovirus. for f, pearson correlations were performed and r 2 and p values are indicated; for g, paired, non-parametric t test was performed; **** indicates p < 0.0001. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. 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efficacy of hydroxychloroquine in covid-19: a systematic review and meta-analysis c-reactive protein levels in the early stage of covid-19 remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro broad neutralization of sars-related viruses by human monoclonal antibodies extrafollicular b cell responses correlate with neutralizing antibodies and morbidity in covid-19' a noncompeting pair of human neutralizing antibodies block covid-19 virus binding to its receptor ace2 dna vaccine protection against sars-cov-2 in rhesus macaques a new predictor of disease severity in patients with covid-19 in wuhan, china', medrxiv key: cord-276833-haci44cy authors: kim, ju; yang, ye lin; jang, yong-suk title: human β-defensin 2 is involved in ccr2-mediated nod2 signal transduction, leading to activation of the innate immune response in macrophages date: 2019-05-18 journal: immunobiology doi: 10.1016/j.imbio.2019.05.004 sha: doc_id: 276833 cord_uid: haci44cy beta-defensins contribute to host innate defense against various pathogens, including viruses, although the details of their roles in innate immune cells are unclear. we previously reported that human β-defensin 2 (hbd 2) activates primary innate immunity against viral infection and suggested that it plays a role in the induction of the adaptive immune response. we analyzed the mechanisms by which hbd 2 primes innate antiviral immunity and polarized activation of macrophage-like thp-1 cells using the receptor-binding domain (rbd) of middle east respiratory syndrome coronavirus (mers-cov) spike protein (s rbd) as a model antigen. the expression of nucleotide-binding oligomerization domain containing 2 (nod2), type i interferons, (ifns), and proinflammatory mediators was enhanced in s rbd-hbd 2-treated thp-1 cells. s rbd-hbd 2 treatment also enhanced phosphorylation and activation of receptor-interacting serine/threonine-protein kinase 2 and ifn regulatory factor 3 compared to s rbd alone. finally, hbd 2-conjugated s rbd interacted with c-c chemokine receptor 2 (ccr2), and nod2 was involved in hbd 2-mediated ccr2 signaling, which was associated with the activation and m1 polarization of thp-1 cells. therefore, hbd 2 promotes ccr2-mediated nod2 signaling, which induces production of type i ifns and an inflammatory response, and enhances primary innate immunity leading to an effective adaptive immune response to hbd 2-conjugated antigen. antimicrobial peptides (amps) are a component of the primary host defense in the mucosa. amps are produced mainly by epithelial cells and the cells involved in innate immunity. the functions of amps in defense against various infections are better characterized than their role as innate immune modulators (boyton and openshaw, 2002) . defensins are small cationic amps and are found in various organisms including mammals, insects, and plants. among them, β-defensins are primarily produced by epithelial cells and leukocytes in vertebrates (stolzenberg et al., 1997) . immature dendritic cells (dcs) can be activated by β-defensins, and β-defensins also inhibit infection by haemophilus influenzae and viruses (zhao et al., 2016; lafferty et al., 2017) . inactivation of human β-defensins (hbds) leads to the recurrent airway infections experienced by patients with cystic fibrosis (smith et al., 1996) . also, a lack of human β-defensin 2 (hbd 2) results in immune dysfunction, such as reduced numbers of b and regulatory t cells, resulting in decreased production of antigen (ag)-specific immunoglobulin a (lugering et al., 2005; mcdonald et al., 2007) . therefore, an understanding of the regulatory mechanism by which amps modulate the immune response during the early stage of viral infection is critically needed to deal with various diseases caused by virus infection. virus infection of host cells triggers innate antiviral responses, which are initiated via pattern recognition receptor (prr) signaling pathways (ishii et al., 2008) . among the prr families, nucleotidebinding oligomerization domain containing 2 (nod2) is an important mediator of the innate immune response to viral infection, and induces expression of type i interferons (ifns) to promote the expression of proinflammatory cytokines and restrict viral replication (wiese et al., 2017) . furthermore, nod2-deficient mice exhibit decreased production of ifns and increased susceptibility to viral infection (sabbah et al., 2009) . presumably, the ability of a virus to counteract innate antiviral immunity during the early stage of infection influences pathogenicity and disease severity (perlman and dandekar, 2005) . type i ifns play a major role in the antiviral innate immune response by upregulating the https://doi.org/10.1016/j.imbio.2019.05.004 received 8 april 2019; received in revised form 13 may 2019; accepted 16 may 2019 production of antiviral proteins and the recruitment of immune cells (haller et al., 2006) . production of type i ifn is initiated by ubiquitously expressed cytoplasmic viral sensors in response to detection of viral pathogen-associated molecular patterns such as double-stranded rna (kato et al., 2011; li and zhong, 2018) . stimulated viral sensors activate downstream signaling pathways, leading to expression of transcription factors including ifn regulatory factor 3 (irf3) and nuclear factor-κb (nf-κb), which drive ifn-β expression (yoneyama et al., 2004) . however, some viruses, including middle east respiratory syndrome coronavirus (mers-cov), inhibit these type i ifn induction pathways . for example, various proteins of mers-cov, including m protein, papain-like protease protein (plpro), and accessory proteins 4a and 4b, are antagonists of ifns (shokri et al., 2019) . accordingly, the virulence of mers-cov is linked to its immune evasion mechanisms, such as suppression of ifn production during the early stage of infection, induction of macrophage apoptosis, and inactivation of t cells with downregulation of ag presentation (niemeyer et al., 2013) . macrophages are professional phagocytes capable of internalizing and degrading pathogens and apoptotic cells. macrophages are present in the respiratory mucosa, such as in the lung and various fluid compartments, where the detection of and the response to infection occur. due to their location, macrophages detect viral ags first and promote an antiviral innate immune response as well as an ag-specific adaptive immune response by presenting ags to t-cells (manicassamy et al., 2010) . we previously reported that hbd 2 promotes an antiviral innate immune response in macrophage-like thp-1 cells and elicits an enhanced ag-specific and virus-neutralizing antibody (ab) response in vivo using the receptor binding domain (rbd) of mers-cov spike protein (s rbd) as a model ag (kim et al., 2018) . moreover, the type i ifn response and the production of primary antiviral molecules such as nod2 were enhanced by s rbd-hdb 2 treatment of thp-1 cells (kim et al., 2018) . consequently, we assumed that modulation of nod2 signaling by hbd 2 would prevent infection by viruses that suppress innate antiviral immunity. in this study, we investigated the mechanism by which hbd 2 enhances the type i ifn immune response in thp-1 cells by modulating nod2 signaling pathways using hbd 2-conjugated s rbd of mers-cov. macrophage-like thp-1 (atcc ® tib-202™) and vero e6 (atcc ® crl-1586™) cells were obtained from the american type culture collection (manassas, va, usa). mers-cov (1-001-mer-is-2015001) was provided by the korean center for disease control and prevention (kcdc). all experiments using mers-cov were performed in accordance with the world health organization recommendations in a biosafety level 3 facility in the korea zoonosis research institute at chonbuk national university (iksan, korea). the chemicals and laboratory wares were obtained from sigma chemical co. (st. louis, mo, usa) and spl life sciences (pocheon, korea), respectively, unless otherwise specified. production of recombinant mers-cov s rbd with or without hbd 2 at the c-terminus (residues 291-725) of the s1 domain were performed as described previously with minor modifications (ma et al., 2014) . briefly, the gene encoding s rbd was synthesized with codon optimization based on the mers-cov s protein sequence (genbank akl59401.1; genscript, piscataway, nj, usa). the s rbd gene with the hbd 2 gene at its 3′ terminus was amplified by polymerase chain reaction (pcr) using forward and reverse primers reported previously (kim et al., 2018) . the amplified genes were cloned into the pcoldii escherichia coli expression vector (takara bio, shiga, japan). recombinant proteins with an n-terminal his tag were purified using ni-nta superflow (qiagen, valencia, ca, usa) according to the manufacturer's instructions. thp-1 cells were cultured in rpmi medium (welgene, gyeongsan, korea) containing 10% fetal bovine serum (fbs; gibco, grand island, ny, usa) at 37°c in a co 2 incubator. the cells were treated with phorbol-12-myristate-13-acetate (1 μg/ml for 1 × 10 6 cells) for 2-3 days to induce differentiation into macrophages (daigneault et al., 2010) . the cultures were replenished with fresh medium, maintained for 3 days, and incubated with recombinant s rbd or s brd-hbd 2 (1 μg/ml per 1 × 10 6 cells) with or without inhibitors (rs 102,895 for ccr2 and gsk 717 for nod2). the cells were harvested after 6 or 24 h and subjected to quantitative real-time reverse transcription polymerase chain reaction (rt-pcr) or western blotting. rna was extracted using trizol ® reagent (thermo-fisher scientific, waltham, ma, usa) following the manufacturer's instructions. rna was used to synthesize cdna with an mmlv reverse transcription kit (promega, fitchburg, wi, usa). gene expression levels were measured by quantitative real-time rt-pcr with the quantitect sybr green pcr kit (qiagen, hilden, germany) and an abi 7500 system (applied biosystems, foster city, ca, usa) using 50 ng of first-strand cdna under the following conditions: 95°c for 5 min followed by 40 cycles at 95°c for 15 s, 55°c for 30 s, and 72°c for 30 s. the expression levels were normalized to that of β-actin (hactb) using 7500 fast software version 2.0.6 (applied biosystems). the primer sets used to amplify target genes are listed in table 1 . cells were washed twice with cold phosphate-buffered saline (pbs), and lysed in a lysis buffer containing 1% triton x-100 supplemented table 1 sequences of the primers used for qrt-pcr. primers used to measure the expression levels of genes associated with antiviral innate immune responses and macrophage differentiation. the β-actin gene (hactb) was used as an endogenous control. with a complete protease inhibitor cocktail (roche applied science, mannheim, germany) and 2 mm dithiothreitol. total cell lysates were prepared by centrifugation at 10,000 rpm for 10 min. equal amounts of lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page), transferred to an immobilon-p polyvinylidene difluoride membrane (merck millipore, burlington, ma, usa), and immunoblotted using specific abs. primary abs against human p-rip2 and p-irf3 were obtained from abcam (cambridge, ma, usa) and abs against nod2 and iκbα were from invitrogen (grand island, ny, usa) and cell signaling (danvers, ma, usa), respectively. the primary ab against human β-actin was purchased from bioss (woburn, ma, usa). target proteins were detected by enhanced chemiluminescence (thermo-fisher scientific). mers-cov was propagated in vero e6 cells, which were cultured in dmem medium (welgene) containing 10% fbs at 37°c in a co 2 incubator. to assess the viral loads and the expression levels of target genes in mers-cov-infected cells, mers-cov was passaged six times in vero e6 cells and transferred to thp-1 cells (2 × 10 6 /well) in a tissue culture plate. after incubation for 24 h, we extracted total rna and performed quantitative real-time rt-pcr using the primers in table 1 to measure the expression levels of mers-cov upe and target genes (kim et al., 2018) . thp-1 cell monolayers in confocal dishes were fixed with 4% paraformaldehyde. the cells were permeabilized using buffer containing triton x-100 (0.1%), blocked, and incubated with the recombinant protein and a specific primary ab. primary abs against human c-c chemokine receptor 2 (ccr2) and nod2 were purchased from invitrogen. a penta-his ab conjugated to alexa fluor ® 488 (qiagen) and anti-rabbit and -mouse igg conjugated to alexa fluor 555 or 568 (invitrogen), respectively, were used as the secondary abs. finally, washed cells were stained with 4′6-diamidino-2-phenylindole (dapi), covered with slowfade gold antifade reagent (invitrogen), and observed using a confocal laser scanning microscope (clsm, lsm 510, carl zeiss, thornwood, ny, usa). statistical analyses were performed using prism 7 (graphpad, san diego, ca, usa). data are means ± standard deviations (sds). the significance of differences was assessed by two-way analysis of variance (anova), and p < 0.05 was considered indicative of statistical significance. 3.1. hbd 2-conjugated ag stimulates the nod2 signaling procedure, which leads to type i ifn production in macrophage-like thp-1 cells mers-cov infection inhibits the production of ifn-α/β and the host antiviral immune response . we evaluated the influence of mers-cov infection of thp-1 cells on the innate immune response (fig. 1) . the expression level of the upe gene was assessed by quantitative real-time rt-pcr at 24 h post-infection of thp-1 cells with 10 3 , 10 4 , and 10 5 plaque-forming units (pfu) of mers-cov (fig. 1a) . importantly, mers-cov infection dose-dependently decreased the expression of factors critical in antiviral innate immunity, such as nod2, ifn-α, and ifn-β ( fig. 1b-d) . to analyze whether hbd 2 affects immune induction, we determined the expression levels of the ifn-β, nod2, and tumor necrosis factor (tnf)-α genes in thp-1 cells treated with s rbd or s rbd-hbd 2 ( fig. 2) . s rbd-hbd 2 significantly (p < 0.01) enhanced the expression of ifn-β and nod2 by 2.18-and 7.35-fold, respectively, compared to s rbd alone-treated thp-1 cells ( fig. 2a and b) . the expression of tnfα, an nf-κb-dependent proinflammatory cytokine, was increased in s rbd-hbd 2-treated cells compared with that in s rbd alone-treated cells, albeit not significantly so (fig. 2c) . we next assessed the levels of the nod2-associated signal transducing mediators, receptor interacting protein-2 (rip2) and irf3, in s rbd-or s rbd-hbd 2-treated thp-1 cells (fig. 3) . as expected, treatment with s rbd-hbd 2 remarkably upregulated the phosphorylation of rip2 and irf3 and the nod2 protein level. in addition, hbd 2-conjugated s rbd reduced iκbα accumulation, degradation of which requires nf-κb activation (fig. 3c) . therefore, hbd 2-conjugated ag treatment induces the production of the type i ifn, ifn-β, and the proinflammatory cytokine, tnf-α, through nod2-associated rip2 response by activating the transcription factors irf3 and nf-κb. 3.2. hbd 2-conjugated ag-mediated induction of a type i ifn response was mediated by ccr2 signaling β-defensin-fused proteins retain their antibacterial and chemotactic activity for c-c chemokine receptor 6 (ccr6)-expressing cells, as do monocytes that do not express ccr6 (röhrl et al., 2010a) . in addition, the β-defensin-conjugated proteins induced ccr2-specific chemotaxis on ccr2-transfected hek293 cells, human peripheral blood monocytes, and mouse peritoneal exudate cells in a dose-dependent manner (röhrl et al., 2010b) . therefore, we determined whether hbd 2 binds to ccr2 rather than ccr6 (fig. 4) by immunofluorescence assay in thp-1 cells (fig. 4a ). ccr2 and s rbd-hbd 2 co-localized, suggesting a direct interaction, but no such co-localization with ccr2 was observed for s rbd alone (data not shown). we next evaluated whether the interaction of hbd 2 with ccr2 mediates intracellular signaling. interestingly, the significantly (p < 0.01) enhanced ifn-β expression by s rbd-hbd 2 was abrogated by the ccr2 antagonist, rs 102,895 (fig. 4b) . moreover, the nod2 expression and nod2-associated rip2 and irf3 expression enhanced significantly (p < 0.05) by s rbd-hbd 2 was abrogated by the ccr2 antagonist ( fig. 4c-e) , as was tnf-α, whose expression is dependent on nf-κb activation (fig. 4f) . thus, hbd 2 interacts directly with ccr2, which promotes signal transduction, activation of type i ifn, and an inflammatory response. we next focused on endosomal and/or cytoplasmic signal transmediators involved in hbd 2-mediated activation of primary antiviral responses. in our previous report, a proinflammatory cytokine response, which requires the activation of nf-κb, was induced by hbd 2 (kim et al., 2018) . additionally, early stimulation of an innate immune response is dependent on toll-like receptor (tlr) 4 and/or nod2-triggered nf-κb signaling (tsai et al., 2011) . we thus determined whether the hbd 2-mediated enhancement is due to nod2-associated signaling because nod2 expression was enhanced by s rbd-hbd 2 (fig. 5) . s rbd-hbd 2 co-localized with nod2, suggesting an interaction (fig. 5a) , but no such co-localization with nod2 occurred for s rbd without hbd 2 conjugation (data not shown). consequently, we speculated that nod2 interacts with s rbd-hbd 2 and that nod2-mediated signaling is involved in hbd 2-mediated activation of nf-κb. we evaluated the influence of nod2 signaling on hbd 2-mediated type i ifn and proinflammatory responses using the inhibitor of nod2 signaling, gsk 717. gsk 717 abrogated the hbd 2-mediated (p < 0.01) enhanced expression of ifn-β, rip2, and irf3 ( fig. 5b-d) . additionally, a nod2 inhibitor abrogated the enhanced expression of tnf-α in hbd 2-treated thp-1 cells (fig. 5e ). these observations suggest that nod2 functions as an intracellular signal transmediator in hbd 2-induced activation of type i ifn production and the inflammatory response. macrophages can differentiate into m1 (classically activated) or m2 (alternatively activated) macrophages. classical m1-type macrophages are key effector cells for the elimination of pathogens, virus-infected cells, and malignant cells, while m2-type macrophages exhibit anti-inflammatory and tissue repair activities (gordon and martinez, 2010) . nf-κb signaling is an intracellular proinflammatory pathway (hoesel and schmid, 2013) and activates m1-type macrophage differentiation (saijo and glass, 2011) . consequently, we analyzed the expression of marker genes of m1 and m2 macrophages in s rbd-hbd 2-treated thp-1 cells by quantitative real-time rt-pcr (fig. 6) . the expression of cd16 and cd80 was significantly (p < 0.05) enhanced by the hbd 2conjugate, suggesting differentiation into m1-type macrophage cells. by contrast, there was no significant difference in the expression of m2 marker genes (cd163 and cd206) between cells treated with s rbd and s rbd-hbd 2 (data not shown). to identify the signaling pathways involved in hbd 2-mediated activation and polarization of macrophages, we evaluated the effect of a ccr2 inhibitor, rs 102,895, and nod2 inhibitor, gsk 717, on cells treated with s rbd with or without hbd 2 conjugation. expression of the m1-type macrophage marker genes, cd16 and cd86, was markedly downregulated by the ccr2 and nod2 inhibitors compared to the control ( fig. 6a and b) . interestingly, expression of cd14, a marker of early activation of macrophages, was stably upregulated in both s rbd alone-and s rbd-hbd 2-treated thp-1 cells, while cd14 expression was markedly downregulated by pretreatment with a ccr2 or nod2 inhibitor, although the ccr2 inhibitor did not completely reverse the hbd 2-mediated enhanced expression of cd14 (fig. 6c) . these results demonstrate that ccr2-mediated activation of nod2 signaling pathway by s rbd-hbd 2 is associated with the activation and m1 polarization of macrophage-like thp-1 cells. innate immunity is the first line of defense against exogenous and endogenous threats, including pathogen infection and tissue damage. innate immunity not only precedes the ag-specific adaptive immune response but also enables a long-lasting memory response by innate agpresenting cells (apcs), which interact with adaptive immune cells. innate immune cells, such as macrophages and mast cells, recruited into infected sites produce a wide range of cytokines that regulate the balance between pro-and anti-inflammatory responses and so maintain immunological homeostasis (gallenga et al., 2019) . cytokines have pleiotropic effects on the functions of immune cells and immune responses that constitute the host defense against infectious agents. for instance, members of the il-1 family, which are produced by macrophages and mast cells, are important regulators of the innate immune response and play a role in inflammatory processes (varvara et al., 2018) . several il-1 family cytokines are pro-inflammatory, while others, including il-37, il-38, il-1ra, and il-36ra, are anti-inflammatory . in addition, il-1 indirectly participates in t-lymphocyte-mediated immunity by inducing helper type 2 t-cell polarization and the formation of abs by plasma cells by producing il-6 (gallenga et al., 2019) . these findings suggest that further studies on modulation of the balance between pro-and anti-inflammatory cytokines and network among innate immune cells, such as m1/m2 macrophage polarization, would facilitate the development of novel therapeutic approaches for immunological disorders. macrophages, dcs, neutrophils, natural killer cells, and innate lymphoid cells play major roles in pathogen recognition through specialized receptors such as prrs (akira et al., 2006; shim and lee, 2015; seo et al., 2013) . viral infection activates danger signals which are transmitted via prrs, including tlrs, nucleotide-binding oligomerization domain-like receptors (nlrs), retinoic acid-inducible gene 1-like receptors (rlrs), c-type lectin receptors, cytosolic dna sensors, and inflammasome signaling cascades. cross-talk between prrs and to that of hactb were analyzed by quantitative real-time rt-pcr in duplicate. the resulting values normalized to that of hactb using the level in the cells treated with 10 3 pfu of mers-cov as a reference basal expression value are shown as means ± sd. *p < 0.05, **p < 0.01, and ***p < 0.001. activation of these signaling cascades induces an antiviral immune response by upregulating the expression of antiviral cytokines, including type i ifns. however, viruses can evade the antiviral function of ifn by inhibiting ifn production and signal transduction (ferran and skuse, 2017) . type i ifns, mainly ifn-α and ifn-β, are major effector cytokines in the innate antiviral response (gonzález-navajas et al., 2012) and their encoding genes are regulated by several transcription factors, including nf-κb and irf3 (seth et al., 2005) . upon virus infection, irf3 is phosphorylated, dimerizes, and enters the nucleus to upregulate the expression of type i ifn, melanoma differentiation-associated protein 5 (mda5), and cytoplasmic retinoic acid-inducible gene i (rig-i), leading to activation of nf-κb and irf3 (kato et al., 2011) . the hosts react to infection by mounting a primary response involving inflammation, followed by a pathogen-specific adaptive response. although inflammation is a double-edged response, it is an important mechanism of protective innate immunity against infection by viruses, bacteria, fungi, prions, and parasites. inflammatory monocyte-derived macrophages and innate immune cells are rapidly recruited to inflamed sites, where they remove harmful stimuli and induce t-cell responses by ifn-dependent mechanisms (ginhoux et al., 2016) . we previously reported that hbd 2 promotes the antiviral innate immune response in thp-1 cells and the ability of an hbd 2-conjugated fig. 2 . hbd 2-conjugated s rbd ag treatment of thp-1 cells stimulates the expression of genes involved in innate immunity and antiviral responses. thp-1 cells were stimulated with 1 μg/ml recombinant s-rbd or hbd 2-conjugated s rbd for 24 h and the expression of the indicated genes was analyzed by quantitative real-time rt-pcr in duplicate, with normalization to the expression of the internal control (hactb). expression levels relative to those of pbstreated control cells are shown as means ± sd. **p < 0.01. fig. 3 . hbd 2-conjugated s rbd ag treatment of thp-1 cells stimulates the expression and activation of genes related to nod2-mediated innate immune signaling. thp-1 cells were stimulated with 1 μg/ml recombinant s rbd or hbd 2-conjugated s rbd for the indicated periods. cell lysates were prepared and immunoblotted with the indicated abs. β-actin was used as the loading control. mers-cov ag to elicit a greater ag-specific and mers-cov neutralizing ab response compared to hbd 2 non-conjugated ag in vivo (kim et al., 2018) . also, immunization with s rbd-hbd 2 prior to viral infection enhanced the humoral and protective immune response to mers-cov infection in human dipeptidyl peptidase 4 (hdpp4)-expressing mice, a model of mers-cov infection (data not shown). in addition, the type i ifn response, the expression of primary antiviral molecules including nod2, a cytoplasmic viral prr that activates irf3, and production of ifn-β were enhanced by hbd 2 treatment of thp-1 cells. these cells are widely used as an in vitro model for studies of human macrophages involved in the inflammatory response and immunological homeostasis (ginhoux et al., 2016) . here, we investigated the immunomodulatory ability of hbd 2 and the mechanism by which hbd 2 induces production of type i ifn and an inflammatory response in thp-1 cells. β-defensins exert regulatory activity in host innate and adaptive immune responses. for example, mouse β-defensin 2 activates immature dcs via tlr4, triggering a th1 response, and human β-defensin 3 activates apcs via tlr1 and tlr2 in an nf-κb-dependent manner (funderburg et al., 2007) . additionally, it was suggested that β-defensin is an endogenous ligand for tlr4 and shares a signal transduction pathway with other tlr4 ligands. we evaluated the possible interaction between hbd 2 and tlr4 as well as nod2 and found that hbd 2-conjugated ag co-localized with nod2 ( fig. 5a) but not with tlr4 (data not shown). moreover, inhibition of nod2 signaling in thp-1 cells abrogated the hbd 2-mediated enhanced ifn-β expression by suppressing rip2 and irf3 signaling as well as tnf-α expression (fig. 5b-e) . human and mouse β-defensins induce ccr2-and ccr6dependent chemotaxis (röhrl et al., 2010b) and ccr2 and ccr6 recruit fig. 4 . hbd 2-conjugated s rbd co-localizes with ccr2 and the interaction between hbd 2-conjugated ag and ccr2 induces nod2 signaling. (a) thp-1 cells treated with 1 μg/ml hbd 2-conjugated s rbd were subjected to immunofluorescence assay using monoclonal abs against s rbd and ccr2 and visualized by clsm. dapi-stained nuclei, blue; s rbd signal, green (alexa fluor 488-coupled secondary ab); ccr2 signal, red (alexa fluor 555-coupled secondary ab). representative fields are shown at ×400 magnification. (b-f) thp-1 cells were stimulated with 1 μg/ml recombinant s rbd protein with or without hbd 2 at 6 h or 24 h after treatment with or without rs 102,895 (ccr2 antagonist) and their expression, together with that of the internal control gene hactb, was analyzed by qrt-pcr in duplicate. expression levels relative to those of the pbs-treated control are shown as means ± sd. *p < 0.05 and **p < 0.01. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). professional apcs to inflamed tissues and initiate an adaptive immune response (osterholzer et al., 2005) . ccr2 is expressed on various types of myeloid cells, including monocytes and neutrophils, which are crucial for innate immunity and phagocytosis (iida et al., 2005) . we found that hbd 2-conjugated ag co-localized with ccr2 and contributed to nod2-mediated signaling, leading to the activation of type i ifn production and an inflammatory response in thp-1 cells. consequently, the mechanism underlying hbd 2-induced ccr2-mediated signaling should be investigated further. macrophages help clear infectious cells by internalizing and degrading pathogens. during infection with influenza a, the phagocytic capacity of mouse peritoneal exudate macrophages is enhanced by coculture with virus-infected epithelial cells (fujimoto et al., 2000) , and decreased phagocytic uptake of opsonized influenza a virus is correlated with decreased cell surface expression of cd16 and cd32, which are highly expressed by classically activated m1 macrophages. also, expression of cd16 and cd32 is decreased in macrophages infected with viruses capable of replicating productively in them (marvin et al., 2017) . m1 macrophages exert a proinflammatory effect, present ag, perform phagocytosis, produce tnf-α and il-1β, and express cd80 and cd86 on their surface. by contrast, m2 macrophages are responsible for tissue repair and wound healing, produce il-10 and tgf-β, and express arginase-1 and cd163 (gordon and martinez, 2010) . we report here that hbd 2 induces the expression of m1 markers by activating ccr2mediated nod2 signaling pathway in thp-1 cells (fig. 6) . although further studies are needed, these results provide insight into the mechanism by which hbd 2 induces antiviral innate and ag-specific adaptive immune responses. there are no competing financial interests in this study. 5 . hbd 2-conjugated s rbd co-localizes with nod2 and the interaction between hbd 2conjugated ag and nod2 induces signaling involved in innate antiviral immune responses. (a) thp-1 cells treated with 1 μg/ml hbd 2conjugated s rbd were subjected to immunofluorescence assay using monoclonal abs against s rbd and nod2 and visualized by clsm. dapi-stained nuclei, blue; s rbd signal, green (alexa fluor 488-coupled secondary ab); nod2 signal, red (alexa fluor 555-coupled secondary ab). representative fields are shown at ×630 magnification. (b-e) thp-1 cells were stimulated with 1 μg/ml recombinant s rbd protein with or without hbd 2 at 6 h or 24 h after treatment with or without gsk 717 (nod2 antagonist), and their expression, together with that of the internal control gene hactb, was analyzed by qrt-pcr in duplicate. expression levels relative to those of the pbstreated control are shown as means ± sd. *p < 0.05 and **p < 0.01. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). the bk21 plus program in the department of bioactive material sciences. confocal laser scanning microscopy was performed using the instruments installed in the center for university-wide research facilities (curf) at chonbuk national university. pathogen recognition and innate immunity pulmonary defences to acute respiratory infection the identification of markers of macrophage differentiation in pma-stimulated thp-1 cells and monocyte-derived macrophages evasion of host innate immunity by emerging viruses: antagonizing host rig-i pathways virus clearance through apoptosis-dependent phagocytosis of influenza a virus-infected cells by macrophages human β-defensin-3 activates professional antigenpresenting cells via toll-like receptors 1 and 2 interleukin-1 family cytokines and mast cells: activation and inhibition new insights into the multidimensional concept of macrophage ontogeny, activation and function immunomodulatory functions of type i interferons alternative activation of macrophages: mechanism and functions the interferon response circuit: induction and suppression by pathogenic viruses the complexity of nf-κb signaling in inflammation and cancer identification of ccr2, flotillin, and gp49b genes as new g-csf targets during neutrophilic differentiation host innate immune receptors and beyond: making sense of microbial infections rig-i-like receptors: cytoplasmic sensors for non-self rna human β-defensin 2 plays a regulatory role in innate antiviral immunity and is capable of potentiating the induction of antigen-specific immunity impact of mold on mast cell-cytokine immune response human beta defensin 2 selectively inhibits hiv-1 in highly permissive ccr6+cd4+ t cells regulation of cellular antiviral signaling by modifications of ubiquitin and ubiquitin-like molecules absence of ccr6 inhibits cd4+ regulatory tcell development and m-cell formation inside peyer's patches searching for an ideal vaccine candidate among different mers coronavirus receptor-binding fragments-the importance of immunofocusing in subunit vaccine design analysis of in vivo dynamics of influenza virus infection in mice using a gfp reporter virus influenza virus overcomes cellular blocks to productively replicate, impacting macrophage function cc chemokine receptor 6 expression by b lymphocytes is essential for the development of isolated lymphoid follicles middle east respiratory syndrome coronavirus accessory protein 4a is a type i interferon antagonist ccr2 and ccr6, but not endothelial selectins, mediate the accumulation of immature dendritic cells within the lungs of mice in response to particulate antigen immunopathogenesis of coronavirus infections: implications for sars thp-1 cells were stimulated with 1 μg/ml recombinant s rbd protein with or without hbd 2 at 24 h after treatment with rs 102,895 (ccr2 antagonist) or gsk 717 (nod2 antagonist), and their expression, together with that of the internal control gene hactb, was analyzed by qrt-pcr in duplicate. expression levels relative to those of the pbs-treated control are shown as means ± sd specific binding and chemotactic activity of mbd4 and its functional orthologue hbd2 to ccr6-expressing cells human beta-defensin 2 and 3 and their mouse orthologs induce chemotaxis through interaction with ccr2 activation of innate immune antiviral responses by nod2 microglial cell origin and phenotypes in health and disease dectin-1 stimulation selectively reinforces lps-driven igg1 production by mouse b cells identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-κb and irf3 caspase-1 independent viral clearance and adaptive immunity against mucosal respiratory syncytial virus infection modulation of the immune response by middle east respiratory syndrome coronavirus cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid epithelial antibiotic induced in states of disease dual roles of nod2 in tlr4-mediated signal transduction and -induced inflammatory gene expression in macrophages stimulated mast cells release inflammatory cytokines: potential suppression and therapeutical aspects protection from influenza a virus infection by modulating nucleotide-binding oligomerization domain containing 2 (nod2) signaling a novel peptide with potent and broad-spectrum antiviral activities against multiple respiratory viruses human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus key: cord-319855-78xmxymu authors: br, bharath; damle, hrishikesh; ganju, shiban; damle, latha title: in silico screening of known small molecules to bind ace2 specific rbd on spike glycoprotein of sars-cov-2 for repurposing against covid-19 date: 2020-07-01 journal: f1000res doi: 10.12688/f1000research.24143.1 sha: doc_id: 319855 cord_uid: 78xmxymu background: human coronavirus (sars-cov-2) is causing a pandemic with significant morbidity and mortality. as no effective novel drugs are available currently, drug repurposing is an alternative intervention strategy. here we present an in silico drug repurposing study that implements successful concepts of computer-aided drug design (cadd) technology for repurposing known drugs to interfere with viral cellular entry via the spike glycoprotein (sars-cov-2-s), which mediates host cell entry via the hace2 receptor. methods: a total of 4015 known and approved small molecules were screened for interaction with sars-cov-2-s through docking studies and 15 lead molecules were shortlisted. additionally, streptomycin, ciprofloxacin, and glycyrrhizic acid (ga) were selected based on their reported anti-viral activity, safety, availability and affordability. the 18 molecules were subjected to molecular dynamics (md) simulation. results: the md simulation results indicate that ga of plant origin may be repurposed for sars-cov-2 intervention, pending further studies. conclusions: repurposing is a beneficial strategy for treating covid-19 with existing drugs. it is aimed at using docking studies to screen molecules for clinical application and investigating their efficacy in inhibiting sars-cov-2-s. sars-cov-2-s is a key pathogenic protein that mediates pathogen-host interaction. hence, the molecules screened for inhibitory properties against sars-cov-2-s can be clinically used to treat covid-19 since the safety profile is already known. the complete genome of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is 82% identical to sars-cov, both viruses share a common clade encompassing the genus betacoronavirus as the root node 1, 2 . currently no novel antivirals exist that are effective against either of the viruses 3-6 . drug repurposing is a commercially viable strategy, as it exploits existing drugs, thus significantly reducing the cost and time involved in developing effective therapeutics 7-9 . experimental approaches, however, at pre-clinical and clinical stages for drug repurposing involve high cost and time 10 . computational approaches can offer quick, considerable, and novel testable hypotheses for systematic drug repositioning 8 . current drugs in different phases of clinical trials are being investigated for inhibitory activity against viral targets that play a significant role in the coronavirus infection lifecycle. the drug targets might be involved in entry into the host (e.g. umifenovir and chloroquine), replication (e.g. lopinavir/ ritonavir), or rna synthesis (e.g. remdesivir/favipiravir). among these, targeting sars-cov-2 cellular entry via the spike glycoprotein (sars-cov-2-s) has emerged as the leading option for repurposing 9 . as sars-cov-2-s is a surface protein involved in adhesion/fusion and entry into host cells, it has been identified as a potential drug target for both biologics and small molecules 10 . the entry of covid-19 pathogen is mediated by the homotrimeric transmembrane protein sars-cov-2-s. it is comprised of two functional subunits, s1 and s2, which are non-covalently bound in the pre-fusion conformation. the s1 subunit interacts with the human ace2 receptor through the receptor binding domain (rbd), while the s2 subunit is one of the components of viral envelope [11] [12] [13] [14] [15] [16] [17] [18] [19] . apart from interacting with the ace2 receptor, the rbd site also contributes to the stabilisation of the prefusion state of the s2 subunit equipped with fusion machinery [18] [19] [20] [21] [22] [23] [24] . in covs, the s-protein is cleaved by host proteases at the s20 site located above the fusion peptide 16, 25 . this activates the protein via extensive irreversible conformational changes 11, 16, 17, 23, 26 . it is well understood that the entry of cov into the susceptible host is a complex process that requires the vigorous actions of receptor binding and proteolytic processing of the s-protein to promote fusion with the pathogen 27 . hence, the current study aims to predict and validate the structure of sars-cov-2-s protein using computer-aided homology modelling tools and screen a library of small molecules for their interaction with the sars-cov-2-s protein. the whole genome of sars-cov-2 (genbank accession number: mt159721.1, length: 29882 bp) was retrieved from ncbi and used as a query to perform a sequence similarity search using ncbi-blast 28 topological analysis of pathogen-host interactome for target validation drug target identification and validation were carried out using a network-based topological analysis method using the webbased application pathogen-host interaction search tool (phisto) by setting pathogen type to virus, family to coronaviridae, species to sars-related coronavirus and strain to sars-cov. the node properties like the degree of connectivity (k) and betweenness centrality (bc) were assessed 29,30 . the statistical significance of k and bc values were assessed by the fligner-killeen (median) test. the similarity between rbd domains of s-protein from sars-cov-2 (accession number qii57328, length:1273aa), sars-cov (accession number: afr58728, length:1255aa) and ratg13 (accession number: qhr63300, length:1269aa) was evaluated by using the multiple sequence alignment (msa) tool clustal omega from embl-ebi. conservation in ace2 receptor interaction was seen among all the three sequences aligned. this conservation aided in the active binding site prediction. the protein-protein interaction between sars-cov-2-s and host ace2 receptor complex was studied using the crystal structure from protein data bank (pdb id: 6cs2). the amino acids involved in the interaction were identified as ligand binding sites for inhibitor molecules. homology modelling of sars-cov-2-s protein homology modelling was performed with swiss-model for the protein sequence of sars-cov-2-s using the crystal structure of sars-cov-s and ace2 complex (pdb id: 6acd) as a template. the modelled protein was validated for quality using a ramachandran plot and prepared for molecular docking studies using the protein preparation wizard feature of the schrodinger small molecule suite 31 . this analysis could also have been performed using open source software such as autodock 32 or swissdock 33 . the modelled receptor was processed for docking studies by deleting crystallographic water molecules with less than three h-bonds. this could also be done manually by editing the .pdb file in a text editor. next, hydrogen atoms corresponding to neutral ph were added in consideration of ionisation states of amino acids. following this, coordinates for any missing side-chain atoms were added using prime v4.0, schrödinger 2019-2 34 . finally, the energy of the modelled structure was minimised using the opls_2005 force field 35 . this analysis could also have been performed using open source software such as autodock 32 . the three-dimensional conformations of the 4015 small molecule drugs already in use to treat various diseases and as nutritional supplements were downloaded from the drugcentral database and subjected to ligand minimisation using ligprip (ligprep, version 2.3, schrödinger, llc, new york, ny, 2009). this analysis could also have been performed using open source software such as autodock 32 . the compounds were minimised by assigning force field opls_2005 and stereoisomers were calculated after retaining specific chiralities. the absorption, distribution, metabolism and excretion (adme) predictions were performed for all ligands using the qikprop package 36 . this analysis could also have been performed using open source software such as swiss-adme 37 . the active site on the prepared receptor was defined around the selected residues (arg426, tyr436, pro462, thr486, gly488, and tyr491) with a 10å radius. this generated a grid box measuring 20x20x20å. the docking of small molecules over sars-cov-2-s was performed using glide v7.8, this analysis could also have been performed using open source software such as autodock 32 . schrödinger 2019-2 38 in different modes sequentially with defined and incremental precision, and computational time differences. the best-docked conformer with minimum glide energy and e model energy was selected and lowest-energy docked complex of three known molecules streptomycin, ciprofloxacin, and glycyrrhizic acid (ga) in complex with sars-cov-2-s were selected for molecular dynamic simulations. the md 39 of shortlisted complexes were studied using the opls_2005 force field 40 in a plane tip3p water model 41 . md simulations were performed using desmond version 4.2 42 . this analysis could also have been performed using open source software such as gromacs 43 . the system was built by dissolving the streptomycin/sars-cov-2-s, ciprofloxacin/sars-cov-2-s, and ga/sars-cov-2-s complexes in an orthorhombic box containing water molecules, allowing a buffer region of 10å between atoms and box peripherals. the system was further minimised using the l-bfgs algorithm for a minimum of 10 steepest descent steps and a maximum of 2000 iterations until a gradient threshold of 25 kcal/mol/å and convergence threshold of 1.0 kcal/mol/å was reached. for short-range electrostatic interactions, the solid-phase microextraction 44 method was employed at 1e-09 tolerance and 9å cut-off radius. the built systems were gradually warmed up to 300k in the npt ensemble with a time step of 2fs. a 100ns md simulation in the npt ensemble was performed using a nose-hoover thermostat 40 . resulting root mean square deviation (rmsd) and root mean square fluctuation (rmsf) values were analysed. multiple sequence alignment of complete genomes the complete genome of coronavirus sars-cov-2, bat coronavirus ratg13, pangolin coronavirus isolate mp789, and sars-cov obtained as blast hits were aligned and a phylogenetic tree was constructed ( figure 1 ). the msa demonstrated the molecular similarities between the organisms. ratg13 has been identified as a neighbour genome for sars-cov-2 and this justifies the hypothesis that the infection may be transmitted from bats. meanwhile, the subsequent neighbours were pangolin mp789 and sars-cov. this preliminary sequence alignment enabled the understanding of sequence similarities and evolutionary information, which is deeply fundamental to the process of drug discovery. a detailed investigation of the pathogen-host interactome can shed clear insights on the mechanism of viral infection and the pathology involved. due to a lack of interaction data on sars-cov-2, the sars-cov proteome was considered and the sars-cov/human interactome was built by screening domain interactions between sars-cov/human protein-protein interactions, and then the network distribution, topological and functional analyses were performed ( figure 2 ). the circular shapes correspond to proteins (nodes) which are labelled by uniprot_ids and details about the nodes are listed in table 1 . among 14 proteins of sars-cov, the majority of sars-cov/human interaction involves five non-structural proteins (ns3b, ns6, ns7a, ns7b and ns8a with four, three, eight, three and one human proteins, respectively), three open reading frame (orf) polyproteins (orf9b, a7j8l3 and a7j8l2 with four, one and one human proteins, respectively), two replicase proteins (r1a and r1ab with two and one human proteins, respectively), the membrane protein (vme1 with human ikkb), envelope membrane protein (vemp with human b2cl1), nucleoprotein (ncap with four human proteins) and spike glycoprotein (spike with human ace2). with these observations, we determine the high specificity of membrane, envelope and spike glycoprotein interactions with the host through specific entry points. hence, these three sars-cov proteins can be a potential target to inhibit the pathogen-host interaction specifically, while other interactions are more versatile. to ensure the impact of inhibition of ikkb, b2cl1, and ace2 mediated interaction, the landscape of the sars-cov/ human interaction was further analysed for degree and betweenness centrality distributions of the host, as shown in table 2 . the degree of connectivity estimates the number of directly connecting neighbours to a particular node, while betweenness centrality estimates the frequency of nodes occurring on the shortest paths in the context of other nodes. in the protein interactomes, a node with a high degree of connectivity is identified as hub protein and a node with maximum betweenness centrality is identified as bottleneck protein. in the current topological analysis, the node with the lowest degree of distribution 1 and betweenness centrality 0.0 was a2a3r6. however, the molecular function of a2a3r6 (uniprot id: a2a3r6) is not well understood in both human physiology or pathology. hence, ace2, with the degree of distribution 4 and betweenness centrality 2271, was the next most significant node, as shown in figure 3 , and it was identified as a key node or key player in the sars-cov/host interaction. hence, the sars-cov-s interaction with host ace2 was identified as a potential drug target. as information about the sars-cov-2/human interaction is not available, the sars-cov/human interaction data was used. we studied the similarity between sars-cov and sars-cov-2 by sequence analysis and rbd prediction. as depicted in figure 4 , the alignment between the s-protein of sars-cov-2 and that of bat coronavirus ratg13 was closer than with the s-protein of sars-cov. the alignment at rbd site residues 317 to 569 was found to be more than 80% similar to sars-cov and ratg13, particularly at major residues including tyr436, thr486, gly488 and tyr491 but excluding arg426 and pro462, as shown in figure 5 . considering the evolution, the available elucidated structure of the sars-cov/ace2 complex (pdb id: 6cs2) was used as a template for homology modelling. the residues involved in the interaction of sars-cov with ace2 were predicted using the prime module available in schrodinger small molecule suite and the major interactions are tabulated in table 3 and shown in figure 6 . a very strong interaction was seen between the smallest amino acid, gly488, with lys353, gly354, and asp355. this interaction is facilitated by two features; one hydrogen bond and 94.9% buried solvent accessible surface area. remaining residues also showed significantly strong interactions with ace2. hence, the same residues were made centric to generate the grid. the modelling of sars-cov-2 was performed using the crystal structure of sars-cov-s as a template, which was 97% identical to the query. the modelled protein shown in figure 7a was validated for quality and preparedness. the ramachandran plot generated using the protein preparation wizard confirmed the quality of modelled structure by plotting >95% residues in the allowed region, as shown in figure 7b . the repurposing of small molecules as therapeutics to treat covid-19 requires knowledge of the interaction of the therapeutic molecule with sars-cov-2-s. initial high-throughput virtual screening suggested 142 molecules that exhibit reasonable interaction with sars-cov-2-s. following this, the shortlisted molecules were docked in sp mode where the accuracy of prediction was improved. the docking in sp mode suggested 15 top table 4 as lead molecules. as hydroxychloroquine has been identified as a possible treatment for covid-19, it was also subjected to subsequent docking in xp mode. all 15 molecules showed better interaction than hydroxychloroquine with sars-cov-2-s. the three molecules streptomycin, ciprofloxacin, and ga had low interaction penalties and displayed better interactions with the ace2 binding site on the rbd of sars-cov-2-s, as shown in figure 8a -c, respectively. the three molecules were selected based on their reported anti-viral activity, safety, availability, and affordability 45-47 . for sars-cov-2-s, the glide generated docking model showed that streptomycin could bind to sars-cov-2-s in a manner highly similar to the sars-cov-2-s and ace2 interaction. the binding pocket of streptomycin was in the rbd site, which has been observed to be an acceptor for ace2. streptomycin was well-fitted with the shape of the pocket, as shown in figure 8a and figure 9a , with an xp score of -6.5, where it formed a total five hydrogen bonds, among which two hydrogen bonds were formed by donating electrons from n31 and n32 atoms to the glu493 side-chain atoms. simultaneously, two other hydrogen bonds were observed between the backbone atoms of leu501 by receiving electrons from hydroxyl groups at the 5th and 6th carbon atoms of the s1 six-carbon ring of streptomycin. the remaining h-bonds were formed between the backbone atom of ser503 and the hydroxyl group at the 6th carbon atom at the g3 group of streptomycin. however, the stability of the interaction cannot be pronounced without molecular dynamic simulations. the docking model of ciprofloxacin illustrated its binding mode on the rbd site, which has been observed to be a key interference site for virus-host interaction. the ciprofloxacin fit with reasonable steric complementarity into the rbd pocket, as shown in figure 8b and figure 9b , with an xp score of -5.31. the interaction of ciprofloxacin with sars-cov-2-s was facilitated by two hydrogen bonds between val492 and phe499; each bond being formed by receiving and donating electrons from hydroxyl and ketone groups, respectively. the docking model of ga illustrated its binding mode on the rbd site, which has been observed to be a key site for interference of the virus-host interaction. the ga fit with steric complementarity in the rbd pocket, as shown in figure 8c and figure 9c , with an xp score of -7.474. the docking of ga with sars-cov-2-s was facilitated by three hydrogen bonds with leu464, val492, and glu493 by receiving electrons from the hydroxyl groups of ga. additionally, the ketone group of ga formed a hydrogen bond, with the backbone atoms of phe499 receiving the electrons. as the sars-cov-2-s receptor has 1273aa, it requires enormous computational time to perform md simulation for the whole range of protein, hence, we confined this study only to the rbd portion, ranging from 317th residue to 569th residue, for md simulation. the rmsd can illustrate the average difference in the displacement of selected atoms in a particular frame compared to its reference frame. the plots in figure 10 illustrate the evolution of a protein (left y-axis) and ligand (right y-axis) rmsd. post simulation, the protein and ligand frames are initially aligned over the backbone atom coordinates of the reference frame, and then the rmsd is extrapolated. the information on protein-ligand rmsd can dissect and demonstrate the conformational differences that occurred throughout the simulation. the rmsd of between 1-3 å is fairly acceptable for small, globular proteins. an rmsd exceeding this indicates a major conformational change during the simulation and pronounces the instability of the complex. the rmsd plot for the streptomycin/sars-cov-2-s complex, shown in figure 10a , attained equilibrium at 5ns and thereafter showed stability with a maximum rmsd of 1 å (peaks of 2.5 å -3.0 å) up to 55ns. after 55ns a change in the equilibrium state was observed. however, the rmsd was within 1.5 å, which is acceptable. similarly, the streptomycin rmsd (right y-axis) was observed to be significantly higher than the rmsd of the receptor at the rbd site. thus, it is likely that streptomycin diffuses from its initial binding site after 48ns. the rmsd plot for the ciprofloxacin/sars-cov-2-s complex, shown in figure 10b , attained equilibrium at 2ns and thereafter showed stability with a maximum rmsd of 1.8 å (peaks of 2.4 å -4.2 å) up to 58ns. after 58ns a sudden change in equilibrium state was observed. however, the rmsd was within 2 å, which is acceptable. on the other hand, the rmsd values for ciprofloxacin were observed to be significantly in alignment with the rmsd of sars-cov-2-s at the rbd site. thus, it is likely that ciprofloxacin can retain its initial binding site up to 100ns. the rmsd plot for the ga/sars-cov-2-s complex, shown in figure 10c , attained equilibrium until 100ns. compared to sars-cov-2-s complexes with streptomycin and ciprofloxacin, it was found that the sars-cov-2-s complex with ga was stable until the end of the simulation without any drift in equilibrium. on the other hand, the rmsd values for ga were observed to be significantly in alignment with the rmsd of the sars-cov-2-s rbd domain in almost all the frames. hence, it is likely that it remains in its initial binding site up to 100ns. it is predicted to inhibit sars-cov-2-s at the rbd domain comparatively better than streptomycin and ciprofloxacin and for a longer duration, but its contact with key ligands has to be confirmed through rmsf and protein-ligand contact analysis. the rmsf helps characterise minute differences in the protein chain during the simulation. in rmsf plots, peaks correspond to the residues on the protein that fluctuate more during the course of a simulation. usually, terminals and loop regions fluctuate more than other secondary structures like alpha-helices and beta-strands. the secondary structure of the rbd of sars-cov-2-s has the same secondary structural elements as the rbd from sars-cov, with 74% homologous residues. these residues are majorly formed of loops and are highly flexible. a unique phe486 residue in the loop plays a key role in ace2 interaction by occupying a deep hydrophobic pocket in ace2. in the trimmed rbd structure this loop starts from 148th residue and ends at 172nd residue. since the ligand-binding site is located in this loop region, a higher rmsd was noticed. in the rmsf plot for the rbd domain of the streptomycin/sars-cov-2-s complex, shown in figure 11a , the rmsf at the loop region was 5.6å with many ligand contacts (green-coloured vertical bars). this was on par with molecular docking interactions. in the rmsf plot for the rbd domain of the ciprofloxacin/sars-cov-2-s complex, shown in figure 11b , the rmsf at the loop region was 5.6å with a few ligand contacts (green-coloured vertical bars). this justifies the interactions seen in molecular docking. further, in the rmsf plot for the rbd domain of the ga/sars-cov-2-s complex, shown in figure 11c , the rmsf at loop region was 6.3å with a high number of ligand contacts (green-coloured vertical bars), justifying the interactions seen in molecular docking. though the ligand contacts are seen in interactions, the simulation time coverage determines their stability. protein-ligand interactions can be traced throughout the simulation and can be categorised into four types: hydrogen bonds, hydrophobic, ionic, and water bridges, as summarised in figure 12a -12c. the stacked bars in the plots are normalised over the course of the trajectory and help us to understand the retention of contact throughout the simulation time. the contacts with a value of more than 0.7 are expected to be retained for over 70% of the total simulation time. in the protein-ligand contact plot for the streptomycin/ sars-cov-2-s complex, shown in figure 12a , residues glu493 and lys544 showed maximum interactions fractions, i.e. 0.20 facilitated by hydrogen bonds and water bridges. this suggests that the specific interaction is maintained for 20% of the simulation time, and such short interactions are not promising. hence, streptomycin cannot be a potential inhibitor of sars-cov-2-s to offer anti-covid-19 activity. in the protein-ligand contact plot for the ciprofloxacin/ sars-cov-2-s complex shown in figure 12b , residues phe465, tyr482, tyr498, and phe499 were seen to have the interactions fractions 0.75, 0.6, 0.35 and 0.39 respectively facilitated by hydrophobic, hydrogen bonds and water bridges. this suggests that for 70%, 60%, 35% and 39% of the simulation time, the specific interaction is maintained by respective residues and such interactions are considered good. hence, ciprofloxacin may be a potential inhibitor of sars-cov-2-s and may offer anti-covid19 activity. in the protein-ligand contact plot for the ga/sars-cov-2-s complex shown in figure 12c , residues val492, glu493, asn496, cys497, and phe499 had the interactions fractions 0.78, 1.12, 0.80, 0.60 and 0.80, respectively, facilitated by hydrophobic, hydrogen bonds and water bridges. this suggests that for 78%,100%, 80%, 60% and 80% of the simulation time, the specific interaction is maintained by respective residues and such interactions are excellent and promising. hence, ga can be a potential inhibitor of sars-cov-2-s and can offer anti-covid19 activity. through our topological analysis, we have determined the degree of distribution for viral proteins, and we show that, due to its low degree of distribution, ace2 is likely to be targeted by viruses like sars-cov. hence, the interaction between the viral protein sars-cov-2-s and the host ace2 receptor is a potential drug target for the repurposing of known drugs. further, sequence alignment and domain analysis suggest that the rbd is the ligand-binding site. molecular docking studies have suggested streptomycin, ciprofloxacin, and ga as possible leads to inhibit sars-cov-2-s. molecular dynamic simulation analysis has indicated that ga is a promising small molecule that could be repurposed as a potential inhibitor of sars-cov-2-s to offer anti-covid19 activity. all data underlying the results are available as part of the article and no additional source data are required. current peer review status: 27 july 2020 reviewer report https://doi.org/10.5256/f1000research.26631.r67868 © 2020 hl r. this is an open access peer review report distributed under the terms of the creative commons attribution , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is license properly cited. college of medical and health sciences, wollega university, nekemte, ethiopia the manuscript entitled " screening of known small molecules to bind ace2 specific rbd on in silico spike glycoprotein of sars-cov-2 for repurposing against covid-19" addresses the repurposability of known drugs against covid-19. spike glycoprotein is the most popular drug target for sars-cov-2 having a significant role in host entry. with this basic understanding, the manuscript is reviewed and the observations are listed below. abstract: conclusion: the statement "hence, the molecules screened for inhibitory properties against sars-cov-2-s can be clinically used to treat covid-19 since the safety profile is already known." can be rewritten as "the ga of plant origin shown superior interaction with sars-cov-2-s compared to rest other molecules, hence, ga can be clinically investigated to confirm its efficacy to treat covid-19." methods: homology modelling of sars-cov-2-s protein the crystal structure of sars-cov-s and ace2 complex (pdb id: 6acd) is used as a template for homology modelling. it is expected to discuss the identity, query coverage between the query and template. discuss the criteria for template selection. in the figure legend for figures 8c, 9c , 10c, 11c, and 12c glycyrrhizic acid can be replaced by ga. the manuscript can be accepted for indexing with above-mentioned changes. are sufficient details of methods and analysis provided to allow replication by others? yes are all the source data underlying the results available to ensure full reproducibility? yes no competing interests were disclosed. reviewer expertise: phytochemical screening, pharmacology, molecular docking i confirm that i have read this submission and believe that i have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. the benefits of publishing with f1000research: your article is published within days, with no editorial bias you can publish traditional articles, null/negative results, case reports, data notes and more the peer review process is transparent and collaborative your article is indexed in pubmed after passing peer review dedicated customer support at every stage for pre-submission enquiries, contact research@f1000.com a pneumonia outbreak associated with a new coronavirus of probable bat origin scalable algorithms for molecular dynamics simulations on commodity clusters gromacs: high performance molecular simulations through multi-level parallelism from laptops to supercomputers publisher full text 45. thomas db, susan ow: streptomycin as an antiviral agent: mode of action possible antiviral effect of ciprofloxacin treatment on polyomavirus bk replication and analysis of non-coding control region sequences in silico screening of known small molecules to bind ace2 specific rbd on spike glycoprotein of sars-cov-2 for repurposing against covid-19 in the present study authors have made an effort to repurpose the known drugs against sars-cov-2. although the study is well organized and presented, a few minor changes are suggested for betterment.the name of the molecules should begin with uppercase.in an abstract, authors have mentioned about the anti-viral activity of streptomycin, ciprofloxacin, and glycyrrhizic acid. but, it requires the literature support. authors can cite the references for the anti-viral/ ant-microbial activity in methods section.in results and discussions, authors could claim the better affinity of glycyrrhizic acid towards sars-cov-2-s in comparison with other two molecules. change suggested: additionally, the ketone group of ga formed a hydrogen bond, with the backbone atoms of phe499 receiving the electrons and possess the better affinity towards sars-cov-2-s when compared to streptomycin and ciprofloxicine.in conclusion section, authors have mentioned that, ace2 is likely to be targeted by viruses like sars-cov. since, authors have tabulated the methods and references for ace2 and sars-cov spike protein interaction in table 1, it is better to rewrite as "ace2 is known to be targeted by viruses like sars-cov". key: cord-292883-7hvq9qaj authors: nguyen-contant, phuong; embong, a. karim; kanagaiah, preshetha; chaves, francisco a.; yang, hongmei; branche, angela r.; topham, david j.; sangster, mark y. title: s protein-reactive igg and memory b cell production after human sars-cov-2 infection includes broad reactivity to the s2 subunit date: 2020-09-25 journal: mbio doi: 10.1128/mbio.01991-20 sha: doc_id: 292883 cord_uid: 7hvq9qaj the high susceptibility of humans to severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection, the cause of coronavirus disease 2019 (covid-19), reflects the novelty of the virus and limited preexisting b cell immunity. igg against the sars-cov-2 spike (s) protein, which carries the novel receptor binding domain (rbd), is absent or at low levels in unexposed individuals. to better understand the b cell response to sars-cov-2 infection, we asked whether virus-reactive memory b cells (mbcs) were present in unexposed subjects and whether mbc generation accompanied virus-specific igg production in infected subjects. we analyzed sera and peripheral blood mononuclear cells (pbmcs) from non-sars-cov-2-exposed healthy donors and covid-19 convalescent subjects. serum igg levels specific for sars-cov-2 proteins (s, including the rbd and s2 subunit, and nucleocapsid [n]) and non-sars-cov-2 proteins were related to measurements of circulating igg mbc levels. anti-rbd igg was absent in unexposed subjects. most unexposed subjects had anti-s2 igg, and a minority had anti-n igg, but igg mbcs with these specificities were not detected, perhaps reflecting low frequencies. convalescent subjects had high levels of igg against the rbd, s2, and n, together with large populations of rbdand s2-reactive igg mbcs. notably, igg titers against the s protein of the human coronavirus oc43 were higher in convalescent subjects than in unexposed subjects and correlated strongly with anti-s2 titers. our findings indicate cross-reactive b cell responses against the s2 subunit that might enhance broad coronavirus protection. importantly, our demonstration of mbc induction by sars-cov-2 infection suggests that a durable form of b cell immunity is maintained even if circulating antibody levels wane. t he betacoronavirus severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the causative agent of a respiratory disease termed coronavirus disease 2019 , emerged in china in late 2019 and rapidly spread worldwide (1) . a pandemic was declared in march 2020, and global deaths from covid-19 now exceed 900,000. the rapid increase in cases in many countries has challenged health care systems, and shutdowns and quarantine measures introduced to slow virus spread have caused major disruptions to society and economies (2) . sars-cov-2 infection produces a wide spectrum of outcomes. a proportion of infections, likely more than 20%, remain asymptomatic. most clinical cases develop mild to moderate respiratory symptoms, but up to 20% progress to a more severe disease with extensive pneumonia (3, 4) . when sars-cov-2 emerged and began to spread, the severity of the threat was primarily attributed to the novelty of the virus to the human immune system and, consequently, a lack of preexisting immune memory to quickly clear virus and limit disease progression. four types of common cold coronavirus are endemic in humans, including the alphacoronaviruses 229e and nl63 and the betacoronaviruses oc43 and hku1. however, limited relatedness between key structural proteins of these human coronaviruses (hcovs) and those of sars-cov-2 suggested that significant crossreactive immunity was unlikely (5, 6) . initial studies of non-sars-cov-2-exposed individuals found negligible levels of igg against the sars-cov-2 spike (s) protein, the viral attachment protein that binds receptor angiotensin converting enzyme 2 (ace2) on host cells to initiate infection (7) . more recently, however, studies have provided evidence of sars-cov-2-reactive b and t cell memory in unexposed subjects that could confer some protection against sars-cov-2 or modulate disease pathogenesis (8) (9) (10) . sera from non-sars-cov-2-exposed individuals have been screened for igg binding to the s1 and s2 subunits of the sars-cov-2 s protein. the membrane-distal s1 subunit contains the receptor binding domain (rbd) for receptor recognition, and the membrane-proximal s2 subunit, which has higher homology among coronaviruses than does s1 (6, 8) , mediates membrane fusion to release viral rna into the host cell. in two large cohorts of unexposed subjects, approximately 10% had igg that bound s2 but not s1 or the rbd. approximately 4% of the subjects had igg against the sars-cov-2 nucleocapsid (n) protein, which is highly conserved among coronaviruses (10, 11) . although n is an internal viral protein and not a target of neutralizing antibodies (abs), coronavirus infections typically elicit strong anti-n ab production (12) . the idea that circulating hcovs elicit igg that cross-reacts with sars-cov-2 is supported by the finding that sars-cov-2 infection increases igg titers against the s proteins of multiple hcovs (13) . in t cell studies, cd4 ϩ t cells in up to 50% of non-sars-cov-2-exposed donors responded to epitopes in s and non-s proteins of sars-cov-2 (8, 9) . notably, s-reactive cd4 ϩ t cells in unexposed subjects were mostly reactive to the conserved s2 subunit, consistent with cross-reactivity to circulating hcovs (8) . sars-cov-2-reactive cd8 ϩ t cells were also detected in unexposed donors, but the response was less marked than for cd4 ϩ t cells (9) . sars-cov-2-reactive memory b cells (mbcs) generated in b cell responses to hcovs are also likely to be present in non-sars-cov-2-exposed individuals. indeed, mbcs might be more important than preexisting cross-reactive abs as a source of protection against sars-cov-2. igg mbcs are more broadly reactive than bulk serum abs generated against the same antigen, they persist after circulating ab levels wane, and they are readily activated to generate strong ab responses or seed germinal centers for additional rounds of affinity maturation (14) . concurrent early production of virusspecific igm and igg in the response to sars-cov-2 infection suggests a response mediated by igg mbcs as well as by naive b cells (10, (15) (16) (17) . this picture is supported by identification of b cell subsets with high and low immunoglobulin v gene mutation frequencies during the response to sars-cov-2 infection (18) . however, little direct analysis of sars-cov-2-reactive mbcs in unexposed subjects has been performed. characterization of populations of mbcs generated and/or expanded by sars-cov-2 infection can also provide insights into cross-reactivity between coronaviruses and participation of preexisting mbcs in the response. wec et al. (19) used cells from a survivor of the 2003 sars-cov outbreak as a source of mbcs that bound the s protein of sars-cov-2; a comprehensive panel of abs expressed by the mbcs were cloned and characterized. notably, most of the highly mutated mabs bound the s2 subunit of multiple hcov s proteins, often with higher affinity than to the s2 of sars-cov-2. a screening of healthy donors identified low frequencies of mbcs reactive to the s proteins of the 2003 sars-cov and sars-cov-2 (19) . findings suggest that s2-reactive mbcs generated by hcovs were activated and expanded by the 2003 sars-cov. rbd-binding mbcs sampled in the convalescent phase of sars-cov-2 infection expressed abs with relatively low numbers of v gene mutations, suggesting that this component of the response largely reflected naive b cell activation by novel epitopes (20) . to extend our understanding of the b cell response to sars-cov-2 infection, the current study compared ab and mbc immunities to sars-cov-2 in unexposed individuals and individuals in the convalescent phase of infection. in particular, we were interested in the presence of sars-cov-2-reactive mbcs in unexposed subjects that could confer some protection against sars-cov-2 and in formation of mbcs by sars-cov-2 infection to provide durable protection against reinfection. most importantly, we demonstrate that sars-cov-2 infection generates both igg and igg mbcs reactive to the novel rbd and the conserved s2 subunit of the s protein. long-lived mbcs are thus likely to be available to mediate rapid protective ab responses if circulating ab levels wane and reinfection occurs. our results also draw attention to preexisting sars-cov-2-cross-reactive b cell memory corresponding to the s2 subunit in sars-cov-2-naive subjects. we speculate that the strong response to s2 after sars-cov-2 infection reflects preexisting s2-reactive mbc activation and strengthens broad coronavirus protection. igg against sars-cov-2 proteins in unexposed subjects primarily targets the s2 subunit of the s protein. to investigate preexisting b cell immunity to sars-cov-2 in unexposed individuals and sars-cov-2-reactive b cell immunity generated by infection, we analyzed sera and peripheral blood mononuclear cells (pbmcs) from (i) 21 healthy donors sampled prior to the emergence of sars-cov-2 and (ii) 26 nonhospitalized covid-19 convalescent subjects sampled 4 to 9 weeks after symptom onset. reactivity was measured against the s protein (including the rbd and s2 subunit) and n protein of sars-cov-2 and the s proteins of the human alphacoronavirus 229e and betacoronavirus oc43. h1 influenza virus hemagglutinin and tetanus toxoid (ttd) were included as control antigens that humans are commonly exposed to through infection and vaccination. serum igg levels were measured by enzyme-linked immunosorbent assay (elisa). approximately one-third of non-sars-cov-2-exposed subjects in the healthy donor cohort had low levels of serum igg against the s and n proteins of sars-cov-2, likely reflecting cross-reactivity with seasonal hcovs (fig. 1a) . notably, 86% of unexposed subjects had igg against the highly conserved s2 subunit of the s protein. it is possible that inherent features of the bulky s reagent used in our analysis reduced binding by anti-s2 abs. igg that bound the highly novel rbd was not detected in unexposed subjects. all non-sars-cov-2-exposed subjects had igg against s proteins of hcovs 229e and oc43, indicating previous infection, and against control proteins h1 and ttd ( fig. 1c to f). s-and n-specific igg production following sars-cov-2 infection includes a strong response to the s2 subunit. levels of igg against s, rbd, s2, and n were markedly higher in convalescent subjects than in unexposed subjects, indicating strong induction of these abs by sars-cov-2 infection (fig. 1a) . in a lower number of convalescent subjects, high anti-s igg titers were associated with low levels of anti-n igg. indeed, more than 40% of convalescent subjects had anti-n igg levels within the range seen in unexposed subjects, questioning the reliability of using anti-n igg measurement to identify previous sars-cov-2 infection in recovered patients (21) . notably, serum igg titers against s2 were consistently higher than against the rbd in convalescent subjects, perhaps reflecting the novelty of the rbd and a response dependent on naive b cell activation (fig. 1b) . interestingly, titers of igg were higher against the s protein of the hcov oc43 in convalescent subjects than in unexposed subjects, but this was not the case for the s protein of hcov 229e (or for the control proteins h1 and ttd) (fig. 1c to f) . the anti-oc43 s igg titers correlated with those against the sars-cov-2 s (r s ϭ 0.49, p ϭ 0.0109), rbd (r s ϭ 0.57, p ϭ 0.0025), and s2 (r s ϭ 0.86, p ͻ 0.0001), indicating a relationship with sars-cov-2 infection (fig. 1g) . the particularly strong correlation between igg titers against oc43 s and the sars-cov-2 s2 suggests a cross-reactive response to the s2 subunit. since the healthy donor samples in our analysis were collected 6 to 10 years before the emergence of sars-cov-2, we considered the possibility that a recently circulating hcov was responsible for the higher anti-oc43 s igg titers in the convalescent subjects. to exclude this possibility, we measured anti-oc43 s igg titers in sera collected from 20 health care workers in 2020. the health care workers had cared for hospitalized sars-cov-2 patients, but all were negative for igg against sars-cov-2 s and rbd, consistent with the effectiveness of personal protective equipment and appropriate work practices. oc43 s-reactive igg levels in health care worker sera were similar to those in non-sars-cov-2-exposed healthy donor sera and significantly lower than those in sera from convalescent subjects (fig. 1c) . taken together, our results indicate that sars-cov-2 infection generates a strong igg response that cross-reacts with the s2 of human betacoronaviruses. strong s-reactive mbc formation following sars-cov-2 infection includes reactivity to the rbd and s2 subunit. pbmcs from non-sars-cov-2-exposed subjects and convalescent subjects were analyzed for the presence of mbcs reactive to sars-cov-2 proteins. circulating antigen-specific igg mbc populations were measured by in vitro stimulation of mbcs to induce differentiation into ab-secreting cells (ascs). poststimulation antigen-specific measurement of levels of mbc-derived ascs (mascs) by enzyme-linked immunosorbent spot (elispot) assay or of mbc-derived polyclonal abs (mpabs) by elisa provided a measure of the levels of precursor mbcs (22) . analysis of mascs by elispot assay was performed against the sars-cov-2 s, rbd, and n proteins and against influenza virus h1 and ttd. mpab levels were measured against those of antigens used in the elispot assay, as well as sars-cov-2 s2 and the s proteins of hcovs oc43 and 229e. antigen-specific igg mpab concentrations correlated strongly with the frequency of igg mascs derived from stimulated mbcs (determined for sars-cov-2 s, sars-cov-2 rbd, influenza virus h1, and ttd, r s ϭ 0.89, 0.67, 0.83, and 0.95, respectively, p յ 0.0002), validating the use of the mpab concentration as a measure of the size of specific mbc populations. the presence of a low level of igg against the sars-cov-2 s, rbd, and n proteins in a proportion of unexposed subjects suggested that igg mbcs with the same specificity had also been formed. however, these mbcs were not detected (fig. 2c) , possibly because of very low frequencies in the circulation. in contrast, igg mbcs reactive to the s proteins of the hcovs oc43 and 229e and the control proteins h1 and ttd were detected in nearly 50% or more of non-sars-cov-2-exposed subjects, consistent with the higher levels of serum igg against these antigens (fig. 2e to h) . as expected, sars-cov-2 rbd-reactive mbcs were not detected in unexposed subjects. in marked contrast to non-sars-cov-2-exposed subjects, the vast majority of convalescent subjects had circulating igg mbcs reactive to sars-cov-2 s, rbd, and s2, indicating strong induction by sars-cov-2 infection of mbcs reactive to novel and conserved regions of the s protein ( fig. 2a and c) . notably, numbers of igg mbcs reactive to the s protein of the hcov oc43 were higher in convalescent subjects than in unexposed subjects (fig. 2e) , but there was no difference between the two subject groups in the levels of igg mbcs reactive to the hcov 229e s protein or influenza virus h1 or ttd (fig. 2b and f to h) . s2-reactive igg mbc numbers correlated well with levels of igg mbcs reactive to sars-cov-2 s (r s ϭ 0.77, p ͻ 0.0001) and rbd (r s ϭ 0.60, p ϭ 0.0012) and to s of hcov oc43 (r s ϭ 0.52, p ϭ 0.0059) but not with those reactive to s of hcov 229e (r s ϭ ϫ0.13, p ϭ 0.53), influenza virus h1 (r s ϭ 0.13, p ϭ 0.54), or ttd (r s ϭ 0.29, p ϭ 0.15). the findings of our mbc analysis are consistent with serum igg measurement and indicate that sars-cov-2 infection generates igg mbcs reactive to the sars-cov-2 s2 that cross-react with the s2 of human betacoronaviruses. interestingly, only a small proportion of the convalescent subjects generated detectable n-reactive igg mbcs, even though most subjects produced high levels of anti-n igg in serum ( fig. 2c and d) . it is unclear whether this reflects a real difference between s-reactive mbc formation and n-reactive mbc formation or an effect of the sampling time. overall, we demonstrate that sars-cov-2 infection induces strong s-reactive mbc formation that would be expected to provide lasting protection against reinfection and, potentially, broad protection against betacoronaviruses. our goals in this study were to investigate sars-cov-2-reactive b cell memory in unexposed subjects that could provide some protection against sars-cov-2 infection and the generation of b cell memory by sars-cov-2 infection that could provide lasting protection against reinfection. in particular, we were interested in igg mbcs, which respond to cognate antigens with rapid, vigorous, and high-affinity ab production. importantly, mbcs are long-lived cells that continue to provide strong protection when circulating ab levels wane. our approach was to analyze circulating igg as well as igg mbcs from the sars-cov-2-naive and sars-cov-2-convalescent subject groups. our key findings are as follows: (i) the presence of igg reactive to the s2 subunit of sars-cov-2 in most unexposed subjects, likely reflecting cross-reactivity to hcovs; (ii) markedly increased levels of igg against the sars-cov-2 s and n proteins, including reactivity to the rbd and s2 subunit of s, in convalescent subjects; (iii) increased igg binding to the s protein of the oc43 hcov, but not the 229e hcov, in convalescent subjects, reflecting greater cross-reactivity between s2 subunits of betacoronaviruses; (iv) strong formation of igg mbcs reactive with the rbd and s2 subunit of the sars-cov-2 s protein in convalescent subjects; and (v) formation of igg mbcs reactive with the s protein of oc43, but not with that of 229e, in convalescent subjects, consistent with s2 subunit cross-reactivity between betacoronaviruses. approximately one-third of our cohort of non-sars-cov-2-exposed subjects had low levels of igg against the sars-cov-2 s and n proteins. the low anti-n igg level likely reflects infection with hcovs, which have low-level (20% to 30%) homology with the sars-cov-2 n protein (11) . however, a protective function for anti-n abs has not been established (23) . notably, 86% of unexposed subjects had igg against the s2 subunit, reflecting homology with hcovs, but none had igg against the highly novel sars-cov-2 rbd (6, 8, 24) . abs that target the s2 subunit have been shown to have virus-neutralizing activity, raising the possibility that the presence of preexisting anti-s2 igg confers some protection against sars-cov-2 (25) . the processes that generate anti-s2 igg are also likely to generate s2-reactive igg mbcs, and these might provide more significant protection than low levels of anti-s2 abs. however, s2-reactive mbcs (or s-reactive and n-reactive mbcs) were not detected in non-sars-cov-2-exposed subjects. taking those findings together with the identification of s-reactive mbcs in unexposed healthy donors (19) , it is likely that the levels of s2-reactive mbcs were below the limit of detection in our assays. on the basis of an estimate of 10 to 20 igg mascs generated per igg mbc after in vitro stimulation (26) , our analysis suggests that the frequency of s2-reactive mbcs, if present in unexposed healthy donors, would be ͻ1/10 6 pbmcs. most mbcs are resident in lymphoid tissues and, except for mbcs against frequently seen immunogenic antigens (for example, the influenza virus h1 or ttd in this study), are at very low frequencies in the circulation in the steady state (27, 28) . anti-rbd, anti-s, and anti-n igg levels were markedly higher in the convalescent subjects than in non-sars-cov-2-exposed subjects, indicating strong induction by sars-cov-2 infection. perhaps notably, the majority of convalescent subjects had higher igg titers against the s2 than against the rbd. this is particularly surprising because of the accessibility of the rbd to b cells and the expected immunodominance over the s2 subunit (29, 30) . our demonstration of strong anti-s2 igg production is consistent with the activation of a preexisting population of igg mbcs against the conserved s2 subunit in the absence of mbcs reactive to the novel rbd. however, we cannot exclude the possibility of inherent differences in the stability or antigenicity of rbd and s2 reagents as an explanation. igg levels against the s protein of hcov oc43 (but not 229e) were significantly higher in convalescent subjects than in non-sars-cov-2-exposed subjects and correlated strongly with anti-s2 igg levels. these findings support the idea of stronger b cell cross-reactivity between the s2 subunits of sars-cov-2 and human betacoronaviruses than alphacoronaviruses (8) . importantly, we demonstrated that sars-cov-2 infection generates rbd-reactive and s2-reactive igg mbcs. recently, long et al. (4) found that levels of sars-cov-2reactive abs, including neutralizing abs, start to decrease within 8 to 12 weeks of infection, especially when the infection is asymptomatic. since mbc populations are maintained for many years, perhaps decades, our findings indicate that mbcs generated by sars-cov-2 infection would be available to rapidly generate protective abs if waning ab levels were to allow reinfection to occur (31) . notably, three convalescent subjects in our analysis had undetectable rbd-reactive igg levels but nevertheless had rbd-reactive igg mbcs. this might reflect mbc production by germinal centers that remained active after recovery from infection (32) . the proportion of subjects with mbcs reactive to the hcovs oc43 and 229e was greater for the convalescent group than for the unexposed group, likely reflecting the increase in levels of s2-reactive mbcs in the convalescent group and cross-reactivity with hcovs. s2-reactive mbc expansion mediated by sars-cov-2 infection could enhance protection against a broad range of coronaviruses (25) . the level of n-reactive mbc formation in convalescent subjects was lower than expected given the large number of subjects with high titers of n-reactive igg, but additional sampling times are required to confirm this observation. the antigen-specific b cell response to infection and vaccination in humans is characterized by entry into the circulation of recently proliferated class-switched b cells, termed activated b cells (abcs), which are phenotypically and transcriptionally distinct from ascs (33) . circulating abc frequencies peak at 2 to 4 weeks after antigen exposure and have substantially decreased by 3 months. frequencies of antigen-specific resting mbcs (negative for markers of recent proliferation) increase together with those of abcs and decrease much more slowly (22, 34) . abcs, like resting mbcs, were activated by the in vitro stimulation conditions used in our study to divide and differentiate into ascs (33) . we therefore cannot exclude the possibility that abc activation contributes, to some degree, to measurement of what we designate mbcs. on the basis of the kinetics of abc and resting mbc formation and maintenance of immunoglobulin gene clonal lineages in the two populations, ellebedy et al. (33) suggested that at least a subset of abcs form resting mbcs. however, the differentiation pathways of abcs are not well established (34) and the proportion that becomes part of long-maintained mbc populations remains uncertain. in conclusion, our analysis investigated ab and mbc immunity to sars-cov-2 in unexposed subjects and individuals soon after recovery from sars-cov-2 infection. the findings emphasized the novelty of the sars-cov-2 s protein rbd in unexposed subjects. however, igg reactive to the s2 was widespread in unexposed subjects and likely resulted from exposure to hcovs. although our approach was unable to directly identify s2-reactive mbcs in the unexposed subjects, we suggest that these cells were present and strongly contributed s2-reactive igg early in the response to sars-cov-2 infection. the igg response in convalescent sars-cov-2 subjects was also strong against the rbd and, less consistently, against the n protein. importantly, the convalescent sars-cov-2 subjects had generated rbd-reactive and s2-reactive igg mbcs. the rbd-reactive mbcs are likely to provide strong long-term protection if rbd-reactive neutralizing ab levels wane and reinfection occurs. additional studies are required to establish the importance of s2-reactive igg in providing broad anticoronavirus activity and the influence of expanded s2-reactive mbc populations on a de novo b cell response to the rbd. study participants and clinical samples. all study participants were recruited at the university of rochester medical center, rochester, ny, and provided written informed consent prior to inclusion in the studies. the studies were approved by the university of rochester human research subjects review board (protocols 16-0064, 07-0090, and 07-0046) and conducted in accordance with the principles of good clinical practice. a prepandemic cohort of 21 healthy donors (median age, 48 years; interquartile range [iqr], 25 to 70 years) were enrolled from 2011 to 2014 (non-sars-cov-2-exposed subjects). a cohort of 20 health care workers (median age, 38 years; iqr, 30 to 52 years) at strong memorial hospital, rochester, ny, were enrolled in may 2020. the health care workers had not been diagnosed with covid-19 prior to enrollment. a cohort of 26 nonhospitalized covid-19 convalescent subjects (9 males and 17 females) (median age, 49 years; iqr, 36 to 63 years) were enrolled in may 2020 and consisted of 22 pcr-confirmed patients and 4 non-pcr-confirmed subjects who were contacts of confirmed cases or displayed covid-19-like symptoms. the convalescent subjects were sampled 4 to 9 weeks after symptom onset. symptoms reported (percentages of subjects) were fever (67%), cough (74%), sore throat (48%), stuffy/runny nose (56%), difficulty breathing (52%), fatigue (85%), headache (67%), body aches (67%), nausea/vomiting (19%), and diarrhea/loose stool (41%). recombinant proteins. rbd and stabilized ectodomain s protein from sars-cov-2 (isolate wuhan-hu-1) were expressed in-house in hek293 cells using pcaggs plasmid constructs kindly provided by florian krammer (icahn school of medicine at mount sinai) (7) . baculovirus-expressed s2 subdomain and hek293 cell-expressed n protein were obtained from sino biological (chesterbrook, pa) and raybiotech (peachtree corners, ga), respectively. baculovirus-expressed s proteins from seasonal hcovs oc43 and 229e were obtained from sino biological. in-house hek293 cell-expressed hemagglutinin from eggderived h1n1 a/california/7/2009 and ttd (milliporesigma, burlington, ma) were used as noncoronavirus control proteins. mbc analysis. measurement of levels of antigen-specific mbcs was essentially performed as described previously (22) . briefly, cryopreserved pbmcs were thawed and rested overnight at 37°c in complete medium. rested pbmcs were stimulated for 6 days at 1 ϫ 10 6 pbmcs/well in 24-well plates to induce mbc expansion and differentiation into ascs. the stimulation cocktail consisted of complete medium supplemented with 1 g/ml r848 (sigma, st. louis, mo), 10 ng/ml interleukin-2 (il-2) (gibco, gaithersburg, md), and 25 ng/ml il-10 (stemcell technologies, vancouver, canada). after stimulation, cells were harvested and pelleted by centrifugation. the undiluted supernatant containing abs secreted by ascs generated from stimulated mbc precursors (mpabs) was collected and stored for analysis by elisa. supernatants from unstimulated cultures of rested pbmcs were collected to control for abs produced by preexisting ascs. antigen-specific ascs in the cell pellet (mascs) were enumerated by elispot assay. for each antigen, 300,000 stimulated pbmcs were analyzed by elispot assay and the limit of masc detection was set at 8 spots (mascs)/10 6 pbmcs. on the basis of elispot assay results, antigen-specific mbcs in peripheral blood were quantified as antigen-specific igg mascs as a proportion of stimulated pbmcs. antigen-specific igg concentrations in mpab samples (after subtraction of ab concentrations in supernatants from the levels seen in unstimulated pbmc control cultures) were also used as a measure of the relative sizes of reactive mbc populations. enzyme-linked immunosorbent assay (elisa). concentrations of ag-specific serum abs and mpabs were measured by elisa as previously described (22) . briefly, nunc maxisorp 96-well plates (thermo fisher, waltham, ma) were coated overnight with optimized concentrations of antigens. serially diluted samples were added to blocked plates and incubated for 2 h at room temperature. alkaline phosphatase-conjugated anti-human igg (clone mt78; mabtech, stockholm, sweden) and p-nitrophenyl phosphate substrate (thermo fisher) were subsequently added to detect bound antigen-specific abs. absorbance was read at 405 nm after color development. a weight-based concentration method was used to quantify antigen-specific ab levels in test samples as described previously (22, 35) . sera from healthy donors and convalescent subjects with high titers for test antigens were used to establish human serum standards. the cutoff for assay positivity was set at approximately 2ϫ the mean optical density (od) value for negative wells. statistical analyses. the medians (with q1 and q3) were summarized by subject group and compared by the wilcoxon rank sum test. spearman correlation analysis was used together with corresponding robust regression models to assess monotonic associations among ab responses. multiple-test adjustment was not applied for this explorative study; thus, a p value of ͻ0.05 was considered significant for all analyses. statistical analyses were performed using sas 9.4 software (sas institute inc, cary, nc). we thank the staff of the university of rochester infectious disease research clinic for subject enrollment and sample collection and bei resources for providing some of the reagents used in this study. this project was funded in part with federal funds from the national institute of a pneumonia outbreak 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vaccination role of memory b cells in hemagglutinin-specific antibody production following human influenza a virus infection defining antigenspecific plasmablast and memory b cell subsets in human blood after viral infection or vaccination activation dynamics and immunoglobulin evolution of pre-existing and newly generated human memory b cell responses to influenza hemagglutinin assignment of weight-based antibody units to a human antipneumococcal standard reference serum, lot 89-s allergy and infectious diseases, national institutes of health, department of health and human services, under ceirs contract no. hhsn272201400005c.we declare no conflicts of interest. key: cord-276493-hoaxv5e0 authors: jeong, gi uk; song, hanra; yoon, gun young; kim, doyoun; kwon, young-chan title: therapeutic strategies against covid-19 and structural characterization of sars-cov-2: a review date: 2020-07-14 journal: front microbiol doi: 10.3389/fmicb.2020.01723 sha: doc_id: 276493 cord_uid: hoaxv5e0 the novel coronavirus, sars-cov-2, or 2019-ncov, which originated in wuhan, hubei province, china in december 2019, is a grave threat to public health worldwide. a total of 3,672,238 confirmed cases of coronavirus disease 2019 (covid-19) and 254,045 deaths were reported globally up to may 7, 2020. however, approved antiviral agents for the treatment of patients with covid-19 remain unavailable. drug repurposing of approved antivirals against other viruses such as hiv or ebola virus is one of the most practical strategies to develop effective antiviral agents against sars-cov-2. a combination of repurposed drugs can improve the efficacy of treatment, and structure-based drug design can be employed to specifically target sars-cov-2. this review discusses therapeutic strategies using promising antiviral agents against sars-cov-2. in addition, structural characterization of potentially therapeutic viral or host cellular targets associated with covid-19 have been discussed to refine structure-based drug design strategies. in late december 2019, a newly identified coronavirus strain capable of crossing the species barrier and infecting humans was first reported in wuhan, hubei province, china, and was provisionally termed 2019 novel coronavirus zhu et al., 2020) . this novel virus was later designated as severe acute respiratory syndrome coronavirus 2 (sars-cov-2), owing to its genetic similarity with other coronavirus strains (gorbalenya et al., 2020) . it is known to cause coronavirus disease 2019 , characterized by influenza-like mild or moderate respiratory symptoms including dry cough, fever, headache, and pneumonia, as well as severe lung injury and multi-organ failure, which eventually lead to death huang c. et al., 2020) . the world health organization (who) officially declared covid-19 as a pandemic on march 11, 2020 due to the rapid global dissemination of sars-cov-2. according to the who, a total of 3,672,238 confirmed cases of covid-19 and 254,045 deaths were recorded up to may 7, 2020 in over 200 countries. moreover, effective antiviral therapeutic agents or vaccines are not yet available for covid-19. the repurposing of existing drugs designed for other viruses is the most practical strategy to treat patients with covid-19 because they have already been tested for their safety. although de novo development of antivirals is a time-, cost-, and effort-intensive endeavor, it is important to generate specific antivirals for sars-cov-2 that directly target the viral or host proviral factors (cascella et al., 2020; senanayake, 2020) . with increasing structural data of key proteins in both sars-cov-2 and the host, such as the spike glycoprotein (s), the main protease (m pro ), rna-dependent rna polymerase (rdrp), and human angiotensin-converting enzyme 2 (hace2), the structure-based design of new drugs has emerged as the most promising antiviral strategy. in this review, we have summarized the promising therapeutic potential of pre-existing drugs against covid-19. in addition, the structural characterization of potentially therapeutic viral or host cellular targets associated with covid-19 have been discussed to refine structure-based drug design strategies. sars-cov-2 is an enveloped, positive-sense, single-stranded rna virus and belongs to the genus betacoronavirus, which also includes sars-cov and mers-cov (andersen et al., 2020; lu et al., 2020; zhu et al., 2020) . the genome sequence of sars-cov-2 is more closely related to that of sars-cov (79% identity) than with that of mers-cov (∼50%) . notably, the s protein of sars-cov-2 and sars-cov are highly homologous with 76.5% amino acid sequence identity . consequently, sars-cov-2 and sars-cov are believed to bind to the same host cell entry receptor hace2 zhou et al., 2020) instead of human dipeptidyl peptidase 4 (hdpp4), which is used by mers-cov (raj et al., 2013) . sars-cov-2 has club-like spikes on its surface and a distinct replication strategy analogous to other coronaviruses. the life cycle and replication of sars-cov-2 is shown in figure 1 . viral infection is initiated by the interaction between the s protein and hace2, followed by subsequent endocytosis or membrane fusion. the s protein comprises two subunits: s1 and s2. the s1 subunit contains the receptor binding domain (rbd) and binds to n-terminal hace2, while the s2 subunit mediates virus-host membrane fusion. s proteins are cleaved by the host cell furin protease and transmembrane serine protease 2 (tmprss2) at the s1/s2 boundary and the s2 ′ position. proteolytic cleavage at the s1/s2 boundary is thought to promote tmprss2-dependent entry into the target cells (belouzard et al., 2009; hoffmann et al., 2020; walls et al., 2020) . after the release of the viral polycistronic rna into the cytoplasm, the replicase gene comprising open reading frames (orfs) 1a and 1ab is directly translated into either replicase polyprotein pp1a (∼450 kda, nsp1-11) or pp1ab (∼750 kda, nsp1-16) by a ribosomal−1 frameshift near the 3 ′ -end of orf 1a and autoproteolytically cleaved into 16 non-structural proteins (nsp1-16) by two orf1aencoded protease domains (brierley et al., 1989; herold et al., 1993; thiel et al., 2001 thiel et al., , 2003 harcourt et al., 2004; prentice et al., 2004; ziebuhr, 2004) . furthermore, the main protease m pro (also called 3cl pro ) and papain-like protease (pl pro ) participate in this extensive proteolytic cleavage. the large pp1ab polyprotein has no <11 conserved cleavage sites that are mediated by m pro , which cleaves at leu-gln↓(ser, ala, gly) (arrow indicates the cleavage site) (ziebuhr et al., 2000; hegyi and ziebuhr, 2002) . positive-strand rna viruses usually form a cytoplasmic enzyme complex called replicase-transcriptase complex (rtc) that can mediate the synthesis of the full-length genome (replication) or discontinuous mrnas (transcription) (gorbalenya et al., 2006; pasternak et al., 2006; sawicki et al., 2007) . structural and accessory proteins are subsequently translated from these transcripts, and new viruses assemble by budding into the lumen of the endoplasmic reticulum-golgi intermediate compartment (ergic) and are eventually secreted (klumperman et al., 1994; hogue and machamer, 2008) . antivirals can be broadly divided into two categories: directacting antivirals (daa) and indirect-acting antivirals (iaa). daas directly target specific viral components, such as viral polymerase, or steps in the viral life cycle without affecting other host cellular processes. the development of daas can facilitate the treatment of patients with covid-19. in contrast, iaas target host proviral factors and indirectly inhibit viral infection or replication by impeding the function or interaction of these factors. iaas have an advantage over daas because they are not susceptible to viral mutations, which are frequently found in rna viruses. however, iaas can alter the host cellular system and are not considered safe. therefore, daas targeting viral entry, proteases, and replication can serve as effective antivirals owing to their enhanced safety features. drug repurposing of preexisting antiviral agents is considered one of the most practical strategies because there is no available approved antiviral drug or vaccine for covid-19. furthermore, the de novo development of drugs typically requires over $1 billion usd and 10-17 years (cascella et al., 2020; senanayake, 2020) . drug repurposing of several approved antivirals against covid-19 has progressed into clinical trials (table 1) . however, there is a potential risk of drug-resistant mutations with the use of daa. a combination of repurposed drugs can reduce the time, cost of treatment, and risk of drug-resistance, and increase therapeutic efficacy to facilitate progression into clinical trials (cheng et al., 2019) . moreover, due to the existence of crystal structures of viral and host cellular proteins associated with sars-cov-2, such as s protein, m pro , rdrp, and hace2, structure-based drug design can be performed to develop more effective drugs with reduced off-target toxicity (schomburg and rarey, 2014 ). the cryo-electron microscopy (cryoem) structure of the extracellular domain of the s protein of sars-cov-2 revealed a homotrimeric conformation (wrapp et al., 2020) . the binding of rbd-located in the s1 subunit-to hace2 on the host cell surface initiates interaction between the virus and the host cell; therefore, the switching conformation of rbd is considered an important event for viral entry (shang et al., 2020) . cryoem figure 1 | viral life cycle of sars-cov-2. interaction between the s protein of sars-cov-2 and hace2 initiates sars-cov-2 infection. following receptor binding, the virus enters the cell by acid-dependent proteolytic cleavage of the s protein by tmprss2 or other proteases. upon fusion of the viral and host cell membranes, viral genomic rna is released in the cytoplasm. the viral rna initiates translation of co-terminal polyproteins (pp1a/ab) by−1 frameshifting. these polyproteins are subsequently cleaved into nonstructural proteins (nsps) by m pro and pl pro . several nsp proteins interact with nsp12 (rdrp) to form the replicase-transcriptase complex (rtc), which is responsible for the synthesis of full-length viral genome (replication) and sub-genomic rnas (transcription). the viral structural proteins are expressed and translocated into the endoplasmic reticulum (er). the nucleocapsid (n) protein-encapsidated genomic rna translocates with the structural proteins into the er-golgi intermediate compartment (ergic) for virion assembly. the newly synthesized virions are budded through the cell membrane and exocytosed. studies revealed that the rbd in two out of three s proteins binds to the n-terminal domain (ntd) of the neighboring protomer of the s protein. these inter-molecular interactions result in a down (closed) conformation, wherein the hace2 interaction interfaces are buried inside the structure. moreover, the rbd in the third s protein forms an up (open) conformation to facilitate binding with the n-terminal region of hace2 (figure 2a ) (wrapp et al., 2020) . the cryoem study of sars-cov-2 s showed that single rbd formed an open conformation in an asymmetric trimer. the structural comparisons between the s protein of sars-cov (pdb id 6crz) and sars-cov2 (pdb id 6vsb) showed that the major structural differences came from rbd in a closed conformation. although the rbd of s from sars-cov and sars-cov-2 were largely resembled, the sars-cov-2 rbd showed a higher binding affinity toward hace2 than sars-cov rbd shang et al., 2020) . the cyroem structure of full-length hace2 revealed a homodimeric conformation, with each monomer of hace2 binding to one rbd of the sars-cov-2 s protein ( figure 2b ) . the crystal structure of hace2 in complex with sars-cov-2 rbd (pdb id 6m0j and 6vw1) showed that sars-cov-2 rbd binds to the n-terminal region of hace2 via s19, q24, t27, f28, d30, k31, h34, e35, e37, d38, y41, q42, l45, l79, m82, y83, q325, n330, k353, d355, and r357 residues of hace2 and k417, v445, g446, y449, y453, l455, f456, y473, a475, g476, e484, f486, n487, y489, q493, g496, q489, t500, n501, g502, v503, and y505 residues of sars-cov-2 rbd ( figure 2c ) (shang et al., 2020; wrapp et al., 2020) . most of these interactions are mediated by α1 of hace2 ( figure 2c) ; moreover, an n-glycosylation chain at n90 of hace2 interacts with sars-cov-2 s protein (shang et al., 2020) . as mentioned earlier, the s1/s2 junction and s2 ′ site of the s protein are cleaved by furin and tmprss2, to enable efficient entry of sars-cov-2 into the host cell (figure 2a) . in addition to trypsin, cathepsin l, and elastase, tmprss2 is known to activate the s protein and induce virus-cell membrane fusion (matsuyama et al., 2010) . a recent study reported that tmprss2 is also essential for sars-cov-2 entry into target cells matsuyama et al., 2020) . the overall structure reveals that human ace2 forms a homodimer (orange and light-yellow) with b0at1 (dark and light gray), which is located in the transmembrane region. the two sars-cov-2 rbds are shown as dark and light green surfaces. (c) the interaction interface between rbd and ace2 is shown (pdb id 6m0j). the residues involved in the interaction between sars-cov-2 rbd and hace2 are represented with stick models in green and orange, respectively. alpha helix 1 (α1) of hace2 is also labeled. (d) the overall structure of sars-cov-2 rbd in complex with its neutralizing antibody cr3022 (pdb id 6w41). the fab regions of the heavy and light chains are shown in hot pink and pink, respectively. sars-cov-2 rbd is shown in green. (e) structural comparison of interfaces between sars-cov-2 rbd and nab or hace2. the interaction interfaces with the light chain of cr3022, heavy chain of cr3022, and hace2 are shown in pink, hot pink, and orange, respectively. (f) hinge movement of hace2 upon binding of the enzyme inhibitor. the apo form (pdb id 1r42) and inhibitor-bound form (pdb id 1r4l) are superimposed and shown in blue and red, respectively. accordingly, targeting proteins that participate in sars-cov-2 entry can be a potential therapeutic strategy. the use of neutralizing antibodies (nabs) against sars-cov-2's s protein is thought to be promising for the treatment of patients with covid-19 (pinto et al., 2020) . a nab-cr3022-known to target sars-cov rbd and prevent lung pathology, can also bind to sars-cov-2 rbd (ter meulen et al., 2006; tian et al., 2020) . the crystal structure of sars-cov-2 rbd in complex with cr3022 revealed that cr3022 forms a distinct interaction interface with sars-cov-2 rbd, and does not overlap with the interaction interface between hace2 and sars-cov-2 rbd (figures 2d,e) . although cr3022 binds to sars-cov rbd and sars-cov-2 rbd with binding affinities (kd) of 1 and 115 nm, respectively, it is unable to neutralize sars-cov-2 in vitro largely due to its inability to form the interaction interface and its low binding affinity (pinto et al., 2020; yuan et al., 2020) . however, continuous efforts are being undertaken to identify potent nabs by collecting plasma from infected individuals, and this has shown significant progress. the p2b-2f6 from sars-cov2 infected patients have overlapping residues, g446 and y449, with higher rbd binding affinity than ace2/rbd (5.14 and 4.70 nm respectively) (ju et al., 2020) . furthermore, the interaction interface of c105/rbd overlapped with the ace2 binding region, and b38 share similar binding structures with prominent neutralizing effects (barnes et al., 2020; wu et al., 2020) . also they showed recent concern of mutation in s (d614g) that might increase sars-cov-2's transmission rate and has a rare chance to affect the rbd-binding mab c105, because of the distance between the rbd region and d614 (barnes et al., 2020) . in addition to identifying nabs targeting sars-cov-2's s protein, a pilot trial to use recombinant soluble human ace2 in covid-19 patients has been initiated (clinicaltrial.gov #nct04287686). however, this trial was recently withdrawn as it was not approved by the center for drug evaluation (cde). because ace2 can counter the activation of renin-angiotensin-aldosterone system (raas) treatment with ace2 inhibitors, it can increase ace2 expression in some patients to compensate for the blocked ace2 activity (vaduganathan et al., 2020) . in some animal studies, treatment of raas inhibitor resulted in increased expression of ace2 in specific tissues (ferrario et al., 2005; soler et al., 2009) . in this regard, some researchers hypothesized that treatment of the raas inhibitor might enhance the accessibility of sars-cov-2 into cells and therefore increase the risk of severity in patients carrying covid-19 (fang et al., 2020; watkins, 2020) . however, a recent case population study showed that there was no correlation between use of raas inhibitors and increased risk of covid-19 (de abajo et al., 2020) . the ramipril, ace inhibitor showed cardiac protective effects without increased expression of ace2 (burchill et al., 2012) . these contradictory results suggested that clinical validations of raas inhibitors are needed to demonstrate its effectiveness toward covd-19. the highresolution x-ray crystal structure of apo-hace2 and hace2 in complex with its enzymatic inhibitor mln-4760 showed that inhibitor binding at the active site of hace2 can cause large hinge-bending movement (towler et al., 2004) (figure 2f) . furthermore, a structure-based drug discovery study showed that an enzymatic hace2 inhibitor can prevent sars-cov infection (huentelman et al., 2004) . therefore, hace2 inhibitors can potentially prevent sars-cov-2 infection. although the structure of human tmprss2 is not available yet, homology modeling and in silico docking studies have demonstrated the molecular mechanisms of camostat mesylate, nafamostat, and bromhexine hydrochloride in inhibiting tmprss2 (sonawane et al., 2020) . in this respect, active sitespecific inhibitors of tmprss2 can be used as potential antiviral agents against sars-cov-2. the crystal structure of sars-cov m pro -a cysteine proteaseconsists of domains 1-3. the catalytic processes of m pro are mediated by the non-canonical cys-his catalytic dyad located between domains i and ii (anand et al., 2002 (anand et al., , 2003 . the m pro protein is highly conserved among sars-cov, mers-cov, and sars-cov-2, and it shares the common substrate recognition sequence consisting of lq(s,a,g) (ziebuhr et al., 2000; hegyi and ziebuhr, 2002; dai et al., 2020) . among them, the gln in p1 of the substrate is an important common feature required for their catalytic activity. human proteases with a similar substrate specificity to that of m pro do not exist; therefore, development of m pro inhibitors is a potential therapeutic strategy for targeting sars-cov-2. sars-cov-2 m pro consists of three domains, analogous to that of m pro from other covs (figure 3a ) (dai et al., 2020; jin et al., 2020; zhang et al., 2020b) . the crystal structure of m pro revealed that it forms homodimers (dimeric protomer) through interactions between domain ii of protomer a and n-terminal residues of protomer b (figure 3a ) (zhang et al., 2020b) . homodimerization of m pro is required for its enzymatic activity. mutational studies on the dimeric interface, as well as crystal structure analysis, revealed that the interaction between two protomers is required to form the s1 pocket at the substrate binding site (figure 3b ) (anand et al., 2002; lim et al., 2014; zhang et al., 2020b) . the substrate binding site of sars-cov-2 consists of s1 ′ -s1-s2-s4 pockets lined with, h41, s46, m49, y54, f140, l141, n142, g143, c145, h163, h164, m165, e166, l167, h172, f185, d187, q189, t190, a191, and q192 residues ( figure 3b ) (dai et al., 2020; jin et al., 2020; zhang et al., 2020b) . notably, the s2 pocket of covs is typically hydrophobic and can accommodate the bulky p2 fragment (figure 3b) . several structure-based drug discovery studies have investigated the interaction of inhibitors in the substrate-binding pockets of sars-cov-2 m pro ( figure 3c ) (dai et al., 2020; jin et al., 2020; zhang et al., 2020b) . a previous study for developing broad spectrum inhibitors targeting cov m pro showed that inhibitors of sars-cov-2 contain a (s)-γ-lactam ring at p1 position to mimic glutamine and occupy the s1 pocket of sars-cov-2 m pro (zhang et al., 2020a) . a total of 103 structures of sars-cov-2 m pro in both apo and inhibitor complex forms are available in the protein data bank (pdb) database (https://www.rcsb. org/) until 27 april 2020. zhang et al. (2020b) have developed peptidomimetic α-ketoamide inhibitors targeting sars-cov-2 m pro . they also solved the crystal structure of m pro in complex with α-ketoamide 13b (pdb id 6y2g) and showed the presence of a γ-lactam ring at p1 position and cyclopropyl at p2 position ( figure 3d) . the biochemical ic 50 of sars-cov-2, sars-cov, and mers-cov m pro were found to be 0.67, 0.90, and 0.58 µm, respectively (zhang et al., 2020b) . simultaneously, dai et al. (2020) developed inhibitors with an aldehyde-substituted compound at warhead for occupying the s1 site and thus it covalently bonds with the catalytic cysteine of sars-cov-2 m pro (pdb id 6lze and 6mok) (dai et al., 2020) (figure 3e) . these compounds showed high inhibition activity with ic 50 of 53 and 40 nm in vitro and reduced sars-cov-2 infection with figure 3 | structure of sars-cov-2 viral m pro and its complex with inhibitors. (a) the crystal structure of sars-cov-2 m pro . m pro is a cysteine protease that consists of three domains and two protomers. protomer b is shown in darker colors than protomer a and each domain is shown in different colors (sky blue, split pea, and violet represent domains 1, 2, and 3, respectively). (b) substrate binding site of sars-cov-2 m pro . the substrate binding site of m pro is subdivided into s1, s1 ′ , s2, and s4 (shown in bold orange). the inhibitors bind to 17 residues shown as yellow sticks (h41, s46, m49, y56, f140, l141, n142, c145, h164, m165, e166 ec 50 of 0.53 and 0.72 µm in plaque reduction assay (dai et al., 2020) . the crystal structure of sars-cov-2 m pro in complex with the inhibitor compound n3 (pdb id 7bqy), previously designed to inhibit cov m pro , revealed that n3 occupies the substrate binding pocket and forms a covalent bond with catalytic c145 of sars-cov-2 m pro . consistently, the lactam ring at p1 position of n3 forms a hydrogen bond with h163 of sars-cov-2 m pro ( figure 3f ) (yang et al., 2005; jin et al., 2020) . x77, a potential inhibitor of sars-cov-2 m pro , also occupies the substrate binding pocket; however, it does not form covalent bonds (pdb id 6w63) ( figure 3g ). in conclusion, m pro of sars-cov-2 is a key protein that participates in the proteolytic processing of polyproteins and shows no overlapping substrate specificity with any of the known human proteases. several potent inhibitors share common structural features, including covalent bond formation with catalytic cysteine and a lactam ring at p1 position. because most inhibitors occupy the substrate binding pocket of sars-cov-2 figure 4 | cryoem structure of rdrp in complex with cofactors (nsp7 and nsp8), rna template, and remdesivir. (a) surface representation of the cryoem structure of sars-cov-2 rdrp in complex with its cofactors (two nsp8 and one nsp7) (pdb id 6m71). nsp7 and nsp8 are shown in gray and pink, respectively. the β-hairpin, niran, interface, thumb, palm, and finger of sars-cov-2 rdrp are shown in cyan, yellow, green, orange, purple, and blue, respectively. (b) a cartoon representation of the overall structure of sars-cov-2 rdrp in complex with the rna template and its inhibitor remdesivir (pdb id 7bv2). the rna template and primer strand are shown in blue and red, respectively. the red arrow indicated the direction of ntp entry. (c) magnified view of remdesivir monophosphate binding region. remdesivir covalently binds to the primer rna strand and interacts with the template rna. m pro , targeting this pocket could be an efficient and safe strategy in terms of toxicity. replication of sars-cov-2 genomic rna is mediated by a multiprotein complex consisting of several non-structural proteins, such as nsp7, nsp8, nsp12, and nsp14. the functional core of this multiprotein complex consists of rna-dependent rna polymerase (rdrp, also called nsp12) . sars-cov-2 rdrp plays an important role in the replication and transcription of viral genomic rna (figure 1 ) and its catalytic residues are highly conserved among covs (venkataraman et al., 2018; . it is because of this that the nucleotide analog remdesivir (gs-5734, gilead) was treated to target rdrp of mers-cov, sars-cov, and sars-cov-2 (warren et al., 2016; holshue et al., 2020; wang m. et al., 2020) . although the viral rdrp is a core component of viral replication, nsp7 and nsp8 are still required for full-fill transcriptional activity of rdrp (zhai et al., 2005; venkataraman et al., 2018; kirchdoerfer and ward, 2019; gao et al., 2020) . the cryoem structure of nsp12 revealed an n-terminal β-hairpin (aa 31-50), extended nidovirus rdrpassociated nucleotidyl-transferase domain (niran, aa 117-250), interface domain (aa 251-365), and rdrp domain (aa 366-920) consisting of finger, palm, and thumb subdomains (gao et al., 2020; yin et al., 2020) (figure 4a ). structural studies have demonstrated that nsp12 can recognize the rna template in a sequence-independent manner, suggesting that the enzymatic activity of rdrp is largely sequence independent. the cryoem structure of sars-cov-2 rdrp in complex with an rna template or its small molecule inhibitor, remdesivir, (figure 4b ) revealed the molecular inhibitory mechanism of remdesivir (yin et al., 2020) . remdesivir monophosphate interacts with the primer strand and uridine of the template strand by base stacking and hydrogen bonding, respectively, at the center of the catalytic active site of rdrp (yin et al., 2020) (figure 4c) . the covalent incorporation of remdesivir monophosphate into the primer strand blocks the entry of nucleotide triphosphates to the active site, and terminates the transcriptional activity of rdrp (yin et al., 2020) (figure 4b ). other nucleotide analog compounds such as favipiravir, ribavirin, eidd-1931, and eidd-2801 may exhibit a similar mechanism of action as remdesivir to inhibit rdrp with non-obligate rna chain termination (elfiky, 2020; sheahan et al., 2020; wang y. et al., 2020) . although the u.s. food and drug administration issued an emergency use authorization for remdesivir on may 1, 2020 for the treatment of suspected or laboratory-confirmed covid-19 in adults and children hospitalized with severe symptoms, the clinical efficacy of remdesivir against sars-cov-2 is not known yet. moreover, no significant clinical benefits of remdesivir against sars-cov-2 were observed in a recent randomized, double-blind, placebo-controlled, multicenter clinical trial (clinicaltrials.gov, nct04257656) . taken together, compounds that target sars-cov-2 rdrp are largely nucleotide analogs because of their ability to form covalent bonds with the viral template rna and block the catalytic active site of rdrp. zoonotic coronavirus outbreaks such as covid-19 can not only affect public health but also have a major impact on societies and the global economy. therefore, global cooperation among academic institutions, governments, and pharmaceutical companies is necessary to overcome covid-19. despite intensive worldwide efforts undertaken by researchers to contain the spread of sars-cov-2, covid-19 has attained pandemic status. considering that the development of an effective vaccine and new therapeutics are still in the early stages, repurposing fda-approved and well-characterized drugs might be a pragmatic approach. consequently, some of these drugs, such as remdesivir, have been approved for emergency use and some are being tested in clinical trials. in addition, combination treatment might be an approach which could achieve synergistic effects and reduce the risk of drug-resistant mutations. a few studies have shown that some pre-existing drugs are effective for the treatment of patients with covid-19. in this review, we described the ongoing therapeutic strategies targeting various components of the sars-cov-2 life cycle ( table 1 ). in addition, we provided structural insights into the mechanism of action of well-characterized drugs targeting the interaction between hace2 and the spike protein of sars-cov-2 for viral entry, as well as m pro and rdrp for viral replication. we believe that structural characterization can aid in developing an effective therapeutic strategy not only against covid-19 but also other viral outbreaks in the future. gj and hs conceived, designed, did the literature review, provided, and wrote the 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this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-290290-wyx9ib7s authors: sinegubova, maria v.; orlova, nadezhda a.; kovnir, sergey v.; dayanova, lutsia k.; vorobiev, ivan i title: high-level expression of the monomeric sars-cov-2 s protein rbd 320-537 in stably transfected cho cells by the eef1a1-based plasmid vector date: 2020-11-05 journal: biorxiv doi: 10.1101/2020.11.04.368092 sha: doc_id: 290290 cord_uid: wyx9ib7s the spike (s) protein is one of the three proteins forming the coronaviruses’ viral envelope. the s protein of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has a spatial structure similar to the s proteins of other mammalian coronaviruses, except for a unique receptor-binding domain (rbd), which is a significant inducer of host immune response. recombinant sars-cov-2 rbd is widely used as a highly specific minimal antigen for serological tests. correct exposure of antigenic determinants has a significant impact on the accuracy of such tests – the antigen has to be correctly folded, contain no potentially antigenic non-vertebrate glycans, and, preferably, should have a glycosylation pattern similar to the native s protein. based on the previously developed p1.1 vector, containing the regulatory sequences of the eukaryotic translation elongation factor 1 alpha gene (eef1a1) from chinese hamster, we created two expression constructs encoding sars-cov-2 rbd with c-terminal c-myc and polyhistidine tags. rbdv1 contained a native viral signal peptide, rbdv2 – human tpa signal peptide. we transfected a cho dg44 cell line, selected stably transfected cells, and performed a few rounds of methotrexate-driven amplification of the genetic cassette in the genome. for the rbdv2 variant, a high-yield clonal producer cell line was obtained. we developed a simple purification scheme that consistently yielded up to 30 mg of rbd protein per liter of the simple shake flask cell culture. purified proteins were analyzed by polyacrylamide gel electrophoresis in reducing and non-reducing conditions and gel filtration; for rbdv2 protein, the monomeric form content exceeded 90% for several series. deglycosylation with pngase f and mass spectrometry confirmed the presence of n-glycosylation. the antigen produced by the described technique is suitable for serological tests and similar applications. humanity is faced with an unprecedented challenge -the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), which causes a severe respiratory illness -coronavirus disease 2019 (covid19) pandemic. countries were sent to lockdown; people could not make informed decisions about the possibility of social contacts; the need for diagnostic tests is very high. existing tests for sars-cov2 are reviewed in [1] . at the beginning of the pandemic, pcr testing methods dominated since such test systems can be developed urgently, soon after the emergence of a new virus in the population. among the disadvantages of pcr-tests is a high sensitivity to contamination and dependence on sampling's correctness, a high proportion of falsepositive signals. unlike pcr diagnostics, serological testing gives positive results long after the event of infection, at least for several months. this testing method makes it possible to reliably determine whether a person is infected with the sars-cov-2, even in the absence of disease symptoms. we need serological tests, both in express format and screening tests based on elisa. serologic tests are also needed to detect convalescent plasma of therapeutic interest and assess emerging vaccines' effectiveness. in order for serological testing to have a more significant predictive value, mapping of the epitopes to which neutralizing antibodies appear should be carried out, as was done for sars-cov1 [2] , аnd convalescent or postvaccinal sera should be massively tested for the presence of neutralizing antibodies, for example, with a surrogate virus neutralization test based on antibody-mediated blockage of ace2-spike protein-protein interaction [3] or another that can be carried out on a relatively large scale. the use of highly specific and high-affinity viral antigens is already a big step towards improving diagnostic accuracy. the immunodominant antigen of sars-cov2 is the rbd domain of the spike protein [4] . another antigen widely used for diagnostics -the nucleocapsid (n) protein -combines high sensitivity and low specificity; therefore, it needs accurate antigen mutagenesis to remove highly conserved areas without compromising affinity. cases are described for sars-cov when the results of testing with n-protein were clarified using two subunits of spike protein [5] . the coronaviruses' spike (s) protein forms large coronal-like protrusions on the virions surface, hence the name of the family coronaviridae. the s protein plays a crucial role in receptor recognition, cell membrane fusion, internalization of viruses, and their exit from the endosomes. it is described in detail in the review [6] . it consists of s1 and s2 subunits and, in the case of the sars-cov-2 virus, has 1260 amino acids [7] . the s protein is co-translationally incorporated into the rough endoplasmic reticulum (er) and is glycosylated by n-linked glycans. glycosylation is essential for proper folding and transport of the s protein. the s protein trimer is transported from the er. interacting with the m and e proteins s protein trimer is transported to the virus's assembly site. s protein is required for cell entry but not necessary for virus assembly [8] . during their intracellular processing, s proteins of many types of coronaviruses, including sars-cov-2 and mers-cov, but not sars-cov, undergo partial proteolytic degradation at the furin signal protease recognition site with the formation of two subunits s1 and s2. apparently, most of the s protein copies on the membrane of sars-cov-2 viral particles are trimers of s2 subunits that are incapable of interacting with the receptor. the full-length s protein trimer on the viral particle's surface also undergoes complex conformational rearrangements during the formation of the rbd-receptor complex and the virus's penetration into the cell. the s protein homotrimer binds to the ace2 dimer, detailed study of this interaction is available here [9] . as part of the trimer, the spike protein's monomer "moves its head" -the s1 subunit can form the open or closed conformation; that is, it can have a raised fragment or a lowered rbd domain, this can influence the affinity of antibodies targeted to it. the s protein of sars-cov-2 amino acid sequence is variable, with more than 200 and 18 relatively frequent s protein amino acid variations. a glycan shield is formed by n-linked glycans on the s protein surface, which is likely to help viral immune escape. in a comparative study of genome-wide sequencing data of natural isolates of sars-cov-2 [10] for the detected 228 variants of the s protein, all 22 potential nglycosylation sites within the s protein's ectodomain were completely conserved, which confirms the importance of each of these sites for maintaining the integrity of the s protein oligosaccharide envelope. it should be noted that not all of the 22 potential n-glycosylation sites are occupied, for s1 and s2 subunits, obtained from transiently transfected hek293 cells [11] n-glycosylation events were experimentally confirmed only for 17 out of 22 sites, also at least one o-glycosylation site was experimentally found inside the rbd-domain area of the s1 subunit with the mucin-like structures. non-vertebrate cells may be used to produce the s protein or its fragments; in this case, n-glycans are present mostly in the form of bulky high mannose or paucimannose structures, possibly blocking the interaction of antibodies with the folded s protein [12] . computational modeling of the glycan shield, performed for the hek293-derived s protein, revealed that in the case of human cells, around 40% of the protein's surface is effectively shielded from igg antibodies [13] . the use of full-length s protein for practical serological testing is nearly impossible due to its insolubility, caused by the presence of transmembrane domain. an artificial trimer of its ectodomain has been successfully used as an antigen in serological tests; however, such complex protein cannot be obtained in large quantities in mammalian cells, apparently due to the limitation on the folding of the trimerized abundantly glycosylated protein and subsequent difficulties in its isolation and purification. it is generally believed that the sars-cov-2 s protein receptor-binding domain is a minimal proteinaceous antigen, adequately resembling the immunogenicity of the whole spike protein. this domain contains only two occupied n-glycosylation sites [11] and 1-2 occupied o-glycosylation sites. it does not contribute to the trimer formation, and its surface is mostly unshielded. isolated rbd's of the s proteins of beta-coronaviruses were produced in various expression systems. bacterial expression of the rbd from mers-cov produced no soluble target protein, refolding attempts also were unsuccessful [14] . budding yeasts pichia pastoris were the suitable host for the secretion of mers-cov rbd with at least two (from three) n-linked glycosylation sites present. similar data were obtained for the rbd from sars-cov virus -removal of all n-glycosylation sites resulted in the sharp drop of protein secretion rate in the p. pastoris yeast, in the case of full rbd domain (residues 318-536), secretion of the unglycosylated target protein was stopped completely [15] . it may be proposed that the addition of n-glycans in these sites is needed for correct folding of the rbd in the er of eukaryotic cells. the sars-cov-2 s protein rbd, expressed in e.coli, also was detected only as inclusion bodies and was found to be unreactive even on blotting [16] . hyperglycosylated yeast-derived sars-cov-2 rbd was obtained in reasonable quantities (50 mg/l in bioreactor culture) by the p. pastoris expression system and successfully used for mice immunization [17] . unfortunately, yeast-derived glycosylated proteins contain immunogenic glycans and cannot be used for immune assays with human antibodies. similarly, sars-cov-2 rbd may be produced in the nicotiana benthamiana plant, resulting in non-vertebrate n-glycans addition, potentially reactive with human antibodies [18] . most early preprints and peer-reviewed articles describing the sars-cov-2 s protein and its rbd domain production methods were focused on transient transfection of hek293 cells [11] [19] and purification of small protein lots in a very short time. for example, d. stadlbauer [20] reports more than 20 mg/l target protein titer in transiently transfected hek-293 cells. simultaneously, the scalability of transiently transfected cell lines cultivation is still questionable, and gram quantities of rbd, needed for large scale in vitro diagnostic activity, may be produced only by stably transfected cell lines. previously we have developed the plasmid vector p1.1, containing large fragments of non-coding dna from the eef1a1 gene of the chinese hamster and fragment of the epstein-barr virus long terminal repeat concatemer [21] and employed it for unusually high-level expression of various proteins in cho cells, including blood clotting factors viii [22] , ix [23] , and heterodimeric follicle-stimulating hormone [24] . cho cells were successfully used for transient sars-cov rbd expression at 10 mg/l secretion level [25] . we have proposed that sars-cov-2 rbd, suitable for in vitro diagnostics use, may be expressed in large quantities by stably transfected cho cells, bearing the eef1a1-based plasmid. p1.1-tr2-rbdv1 construction. the rbd coding sequence was synthesized according to [26] . the dna fragment encoding the rbdv1 orf with kozak consensus sequence and c-terminal c-myc and 6xhis tags were obtained by pcr using primers ad-cov-absf and ad-rbd-myc6hnher (listed in table 1 the resulting ptm vector was sequenced as described above, available from addgene, plasmid # 162783. ptm-rbdv2 construction. rbd orf was amplified using adaptor primers ad-sfr2-nhef and ad-sfr2-xmar restricted by nhei and xmai (sibenzyme, novosibirsk, russia) and cloned into ptm vector, restricted by nhei and asigi (sibenzyme, novosibirsk, russia). the resulting construct was sequenced using sq-5ch6-f and sq-mych-r primers. ptm-rbdv2 is available from addgene, plasmid # 162785 plasmids for cell transfections were purified by the plasmid midiprep kit (evrogen, moscow, russia) and concentrated by ethanol precipitation in sterile conditions. the transgene copy number in the cho genome was determined by the quantitative real-time-pcr (qpcr) as described in [22, 24] . serial dilutions of p1.1-egfp [21] or pgem-rab1 plasmids were used for calibration curves generation. the weight of one cho haploid genome was taken as 3 pg, according to [27] . genomic chinese hamster ovary dg-44 cells (thermo fischer scientific) were cultured in the procho 5 medium (lonza, switzerland), supplemented by 4 mm glutamine, 4 mm alanyl-glutamine and hypoxanthinethymidine supplement (ht) (paneco, moscow, russia). cells were grown as a suspension culture in sterile 125 ml erlenmeyer flasks with vented caps, routinely passaged 3 to 4 days with centrifugation (300 g, 5 min) and seeding density 3-4*10 5 cells/ml. the 50-80 µg of each plasmid were precipitated by the addition of 96% ethanol and 3m sodium acetate, washed with 70% ethanol, dried, and resuspended in 100 µl of sterile r-buffer, neon transfection kit (thermo seeding cell culture was grown in 125 ml erlenmeyer shake flasks with 30 ml of lonza procho 5 medium, supplemented with 4 mm glutamine, 4 mm alanyl-glutamine and 2-8 µm mtx until cell concentration exceeds 1-1.5 mln cells/ml. cell suspension was transferred to four 250 ml erlenmeyer flasks, each containing 60 ml of culture medium, and grown to the same cell density. the entire cell suspension was transferred to a single 2 l erlenmeyer flask with 1 l culture medium, final seeding density 3-4*10 5 cells/ml. cells were cultured for three days, on the fourth day of culture, daily glucose measurements were started. glucose concentration in the cell supernatant was measured by the accutrend plus system (roche, switzerland); if glucose level was below 20 mm, it was added up to 50 mm as the sterile 45% solution. the culture in 2 l flask was grown for 6 to 8 days until the cell viability, measured by trypan blue exclusion, dropped below 50%. the clonal cell line was obtained by the limiting dilution method from the cell population, cultured in 8 µm mtx. methotrexate was omitted in the culture medium for two 3 d passage before cloning. cells were additionally split by 1:1 dilution 24 hours before the cloning procedure. cells were diluted in excell-cho (merck, germany) culture medium supplemented with 4 mm glutamine, 4 mm alanyl-glutamine, ht and 10% of untransfected cho dg 44 conditioned medium resulting in seeding density 0.5 cell/well, and the suspension was seeded into 96-well plates (200 μl/well). plates were left undisturbed for 14 days at 37°c, 5% co2 atmosphere. wells with single colonies were screened by microscopy; well grown colonies were detached by pipetting and transferred to the wells of 12-well plate, containing 4 ml of the excell-cho, supplemented as described above and grown for 7 days undisturbed. product titer was measured by elisa, as described below, 6 wells with highest rbdv2 titer were used for further cultivation. best-producing clonal cell lines were transferred to 125 ml erlenmeyer flasks with the procho 5 culture medium supplemented with 4 mm glutamine, 4 mm alanyl-glutamine and 8 µm mtx and after 5 days in suspension culture, the best producing clone was determined by measuring the product titer and cell concentration. sds-page was performed with the 12.5% acrylamide in the separating gel, in reducing conditions, if not stated otherwise, with the pageruler prestained marker, 5 µl/lane (thermofisher scientific). gels were stained by the colloidal coomassie blue according to [28] , scanned by the conventional flatbed scanner in the transparent mode as 16-bit grayscale images and analyzed by the totallab tl120 gel densitometry software (nonlinear dynamics, uk). sds-page was performed as described above, protein transfer, blocking, hybridization and color development were done according to [29] using nitrocellulose transfer membrane (gvs group, bologna, italy) and towbin buffer with methanol. primary anti-c-myc antibody (sci store, moscow, russia #psm103-100) was used at the 1:2000 dilution, anti-mouse-hrp conjugate (abcam, cambridge, uk, ab6789) was used at 1:2000 dilution; membrane was developed by the dab-metal substrate and scanned by the flatbed scanner in the reflection mode. multimeric forms of the rbd were quantified by size exclusion chromatography, utilizing waters extracts were vacuum-dried and redissolved in the 0.5% trifluoroacetic acid (tfa), 3% acn solution. prepared solutions were mixed at 3:1 ratio with 20% α-cyano-4-hydroxycinnamic acid (merck) solution in 20% acn, 0.5% tfa on the target plate. solutions of intact and deglycosylated proteins were passed through the ziptip c18 microcolumns (millipore), washed and eluted according to manufacturer protocol. one and a half µl of protein solutions were mixed on the target plate with 0.5 µl of the 20% 2,5-dihydroxybenzoic acid (merck) solution in 20% acn, 0.5% tfa. mass spectra were obtained by the maldi-tof mass spectrometer ultraflextreme peptides identification was performed by the gpmaw 4.0 software (lighthouse data, denmark) and by the mascot server (matrix science, boston, usa). glycopeptides mass assignment was performed by the glycomod online software tool [30] . sandwich elisa with anti-s protein antibodies was performed using a prototype of the sars-cov-2 antigen detection kit (xema co., ltd., moscow, russia, a generous gift of dr. yuri lebedin). pre-covid-19 normal human plasma sample (renam, moscow, russia) was used for preparation of the sars-cov-2 negative serum sample. serum samples of five patients with the pcr-confirmed sars-cov-2 infection were pooled for testing and one serum sample with the borderline igg titer level was tested separately. the blood sampling protocol conformed to the local hospital human ethics committee guidelines. antibody capture elisa with human serum samples was performed according to [29] at the 100 ng per well antigens load. antigens were applied on elisa 96-well plates (corning, usa) overnight at + 4oc, in pbs, the t-test was performed using the graphpad quickcalcs web site: https://www.graphpad.com/quickcalcs/ttest1.cfm (accessed november 2020). the native n-terminal signal peptide of sars-cov-2 s protein (amino acid sequence mfvflvllplvssq) was fused to the rbd sequence (319 -541, according to yp_009724390.1) and joined with a c-terminal c-myc epitope (eqkliseedl), short linker sequence, and hexahistidine tag. n-terminal part of the rbdv1 gene was constructed according to [26] , utilizing the optimized codon usage gene structure. c-terminal tags were not optimized for codon usage frequencies. the resulting synthetic gene was cloned into the p1.1-tr2 vector plasmid, a shortened derivative of the p1.1 plasmid [21] , and used for transfection of dhfr-deficient cho dg44 cells. the resulting expression plasmid p1.1-tr2-rbdv1 [genbank: mw187858] is shown on fig 1a. the stably transfected cell population was obtained by selection in the presence of 200 nm of dhfr inhibitor methotrexate, rbd titer 0.33 mg/l was detected for 3-days culture (fig. s1 ). one-step target gene amplification was performed by increasing the mtx concentration tenfold and maintaining the cell culture for 17 days until cell viability restored to more than 85%; the resulting polyclonal cell population could secrete up to 3,0 mg/l rbd in the 3-days culture. the target protein was purified by a single imac chromatography step, utilizing the ida-based resin chelating sepharose fast flow (cytiva), ni2+ ions, and step elution by increasing imidazole concentrations (fig 1b, fig 1c) . the resulting protein production method was found to be sub-optimal due to unexpectedly low secretion rate, signs of cellular toxicity of the target gene -33 h cell duplication time, maximal cell density in shake flask of 2.3 mln сells/ml (fig s2) , and unacceptable level of contaminant proteins co-eluting with the rbdv1. at the same time, the rbdv1 protein was stable in the culture medium during the extended batch cultivation of cells for at least 7 days (fig 1d) , making the long-term feed batch cultivations a viable option for its production in large quantities. we proposed that target protein secretion rate and its purity after one-step purification could be significantly improved by a simultaneous shift of the rbd domain boundaries, exchange of the sars-cov-2 s protein native signal peptide to the signal peptide of more abundantly expressed protein, two-step genome amplification and switch from ida-based resin to the nta-based one (fig 1e) . human tissue plasminogen activator signal peptide (htpa sp, amino acid sequence mdamkrglccvlllcgavfvsas) is commonly used for heterologous protein expression in mammalian cells. it was successfully used for the expression of sars-cov s protein in the form of dna vaccine [31] and envelope viral protein gp120 [32] . in the case of mers-cov s protein rbd -fc fusion protein, various heterologous signal peptides modulate target protein secretion rate by the factor of two [14] . corrected boundaries of the sars-cov-2 rbd were determined according to the cryo-em data [pdb id: 6vxx] [33] obtained for the trimeric sars-cov-2 s protein ectodomain. initially used 319 -541 coordinates, described in the [26] include one unpaired cys residue originated from the n-terminal part of the next domain sd1 (structural domain 1), so we excluded lys319 from the n-terminus of the mature rbd protein, aiming at the maximization of signal peptide processing, and removed c-terminal aminoacids c 538 vnf 541 , which form the structure of the sd1 domain. both linker areas surrounding the folded rbd domain core remain present in the rbdv2 protein (320 -537, according to yp_009724390.1). additionally, we redesigned c-terminal tags by introducing the pro residue immediately upstream of the c-myc tag, adding the short linker sequence sagg between the c-myc tag and polyhistidine tag, and extending the polyhistidine tag up to 10 residues. we expected this structure to expose the c-myc tag properly on the protein globule's surface and move the decahistidine tag away from possible masking negatively charged protein surface areas. we constructed an expression vector ptm [genbank: mw187855], where consensus kozak sequence, htpa sp and c-myc and 10-histidine tags are coded in the polylinker. rbd coding fragment was cloned in-frame, resulting ptm-rbdv2 expression plasmid [genbank: mw187856] is shown on fig.2a . cho dg44 cells were transfected by the ptm-rbdv2 plasmid, stably transfected cell population was established at the 200 nm mtx selection pressure. target protein titer was similar to the previous plasmid design -0.9 mg/l for 3-days culture, but after one step of the mtx-driven genome amplification, it increased eleven-fold to 9.7 mg/l at 2 µm mtx ( fig 2b) and then increased by a factor of 2.5 after second amplification step at 8 µm mtx, resulting titer was 24.6 mg/l for 3-days culture (fig 3a) . a steady increase of the target protein titer was detected for the extended batch cultivation of polyclonal cell population obtained at 8 µm mtx, peaking at 50 mg/l at 8 days of cultivation in the 2 l shake flask (fig 3d, 3e) . a similar ratio of product titer increase after multi-step mtx-driven genome amplification was described for the mers-cov rbd -40-fold increase after 9 steps of consecutive increments of mtx concentration, overall amplification period length was 60 days [14] . vector plasmid ptm, used in this study, allowed a much more rapid amplification course -a 27-fold titer increase in two steps, 33 days total. this all cell populations, secreting rbd proteins, were analyzed by the quantitative pcr and it was found, that increased productivity of populations, adapted to higher concentrations of mtx corresponds to higher copy numbers of target gene (fig 3c) . higher cell productivity in the case of rbdv2 protein was not due to higher target gene copy numbers, then in the case of rbdv1. cell culture medium pro cho5 (lonza), utilized in this study, contains unknown components, blocking histagged rbd protein's interaction with the ni-nta chromatography resin. clarified conditioned medium, used for protein purification, was concentrated approximately tenfold by tangential flow ultrafiltration on the 5 kda mwco cassettes and completely desalted by diafiltration, 20 diafiltration volumes of the 10 mm imidazole-hcl, ph 8.0 solution. rbdv1 and rbdv2 proteins were purified by imac utilizing ni-nta agarose (thermo fischer scientific, usa) in the same conditions. desalted conditioned medium was applied onto the column in the presence of 10 mm imidazole; the column was washed by the solution containing elution was performed by the 300 mm imidazole solution; further column strip by the 50 mm edta-na solution revealed no detectable target protein rbdv2 in the eluate (fig 2c) . purified proteins were desalted by another round of ultrafiltration/diafiltration on the centrifugal concentrators with 5 kda mwco membranes; diafiltration solution was pbs; final concentration 3-7 mg/ml. purified proteins were flashfrozen in liquid nitrogen and stored frozen in aliquots. overall protein yield for rbdv2 was 64%, 32 mg of purified rbdv2 were obtained from 1 l shake flask culture. the apparent molecular weight of intact rbdv1 was determined as 35.3 kda, deglycosylated rbdv1 -26.1 kda, theoretical molecular weight -27647 da. rbdv2 molecular weight was determined as 35.7 kda for the intact protein, deglycosylated protein -28.5 kda, theoretical molecular mass -27459 da (fig 4a) . both protein variants possess two distinct forms of intramolecular disulfide bonds sets, visible as two closely adjacent bands in non-reducing conditions and complete absence of such band pattern in reducing conditions. previously it was reported that sars-cov-2 rbd 319-541, expressed transiently in hek-293 cells, tends to form a covalent dimer, around 30% from the total, visible as the 60 kda band on the denaturing gel in nonreducing conditions [19] . we confirmed this observation; in the case of stably transfected cho cells, covalent dimerization was also 31% according to gel densitometry data. at the same time, it should be noted that the rbdv2 protein, redesigned explicitly for mitigation of this unwanted dimerization and containing an even number of cys residues, still forms 6 % of the covalent dimer. purified rbdv2 was tested by size exclusion chromatography. the major monomer form's apparent molecular weight was determined as 32.4 kda (fig s3) , admixtures peaks apparent molecular masses corresponded well to rbd dimer, tetramer, and two high molecular mass oligomers accounting for 6% of all peak areas (fig 4b) . mass-spectrometry analysis of rbdv1 and rbdv2 revealed that both proteins' molecular masses diminished ( fig s5, s6 ). this long peptide was completely absent in both spectra of de-glycosylated proteins (table s1 -s4) . a more detailed analysis of this area of the rbd protein may be of some interest for the s protein structure-function investigation but is out of scope for the present study. purified rbd variants were used as antigens for microplates coating and subsequent direct elisa with pooled sera obtained from patients with the rt-pcr-confirmed covid-19 diagnosis, 1 weakly positive serum sample from the rt-pcr-confirmed covid-19 patient, and serum sample obtained from a healthy volunteer before december 2019 (fig 4e) . both rbd variants perform equally -all serum samples produce highly similar od readings for all dilutions tested with both antigens. here we describe a method of generating stably transfected cho cell lines, secreting large quantities of monomeric sars-cov-2 rbd, suitable for serological assays. at present, serological assays for detection of seroconversion upon sars-cov-2 infection are mostly based on two viral antigens -nucleoprotein (np) and s protein or fragments of the s protein, including the rbd. there are various reports on the specificity and sensitivity of assays based on these two antigens. in some cases, the sensitivity of clinically approved npbased assays was challenged by direct re-testing of np-negative serum samples by the rbd-based assays [34] . other studies question the specificity of np-based elisa tests, demonstrating a significant level of false-positive results for the full-length sars-cov-2 np [35] . it may be proposed that testing of serum samples with both sars-cov-2 antigens will produce the most accurate results, as was done, for example, in the south-east england population study [36] ; this conclusion was made in the microarray study of a limited number of patients serum samples [37] . it is unclear yet, which part of the s protein is the optimal antigen for serological assays; microarray analysis revealed that s2 fragment generates more false-positive results than s1 or rbd antigen variants [37] in the case of igg detection, at the same time the rbd protein generated much lower signals on covid-19 patients serum samples then s1 or s1+s2 antigens. in another microarray study it was found that igg response toward the rbd domain in the convalescent plasma samples correlates well with the response toward full-length soluble s protein [38] . in the conventional elisa test format, rbd demonstrated nearly 100% specificity and sensitivity on a limited number of sars-cov-2 patients and control serum samples [4] . as of 26.10.20, at least 104 various immunoassays for sars-cov-2 antibodies were authorized for in vitro diagnostic use in the eu [39], many of them use rbd as the antigen. a simple elisa screening test with the 96-well microplate will consume around 10 µg of the rbd antigen for 40 test samples, so even one million tests will require 250 mg of the purified rbd protein, making the antigen supply a critical step in the production of such tests. method of the generation of highly productive stably transfected cho cell line, secreting the rbd protein, may be important for ivd test manufacturers in securing the sources of rbd antigen with highly predictable properties. although the rbd fragment of the s protein from sars-cov-2 is not the most popular antigen variant in the current efforts of anti-sars-cov-2 vaccine development [40] , it may be considered as the viable candidate for a simple subunit vaccine. it demonstrated the significant protective immune response development in rodents, without signs of ade effect [41] and some rbd-based protein subunit vaccine have advanced to phase ii clinical trials. cultured cho cells are the reliable source of rbd protein for this kind of vaccines; at the productivity level achieved in our study, only 30 m 3 of cell culture supernatant will provide enough antigen material for 100 mln of typical 10 µg/vial vaccine doses. с, d -protein sequence coverage by tryptic peptides, maldi-tof analysis. glycosylated peptides found are not pictured, signal peptides are yellow, detected tryptic peptides -violet, experimentally obtained masses, [m+h]+, are stated in the boxes. e -immunoreactivity of rbdv1 and rbdv2 by elisa with pooled serum samples from pcr-positive patients -(+)pooled, single serum sample from pcr-positive patient (+) and pre-covid-19 pooled sera (-). all sera samples were analyzed in duplicates, data are mean. supporting figure s1 . cell growth and viability dynamics of initial selection and mtx-driven target gene amplification. supporting figure s2 . cell growth curve for the extended batch cultivation of rbdv1 and rbdv2producing cell populations, 2 um mtx selection pressure. supporting figure s3 . size exclusion chromatography trace of molecular mass calibrators and molecular mass calibration curve. supporting figure s4 . maldi-tof spectra traces of intact proteins in glycosylated and deglycosylated forms. supporting figure s5 . maldi-tof spectra traces of tryptic peptides mxtures from intact and deglycosylated rbdv1. supporting figure s6 . maldi-tof spectra traces of tryptic peptides mxtures from intact and deglycosylated rbdv2. supporting table s1 . peptides mass list of the rbdv1 intact protein, in-gel digestion, reduced protein. supporting table s2 . peptides mass list of the rbdv2 intact protein, in-gel digestion, reduced protein. supporting molecular and immunological diagnostic tests of covid-19: current status and challenges. iscience antigenic and immunogenic characterization of recombinant baculovirus-expressed severe acute respiratory syndrome coronavirus spike protein: implication for vaccine design a sars-cov-2 surrogate virus neutralization test based on antibody-mediated blockage of ace2-spike protein-protein interaction the receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in sars-cov-2 patients false-positive results in a 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improved elongation factor-1 alpha-based system a highly productive cho cell line secreting human blood clotting factor ix high-level expression of biologically active human follicle stimulating hormone in the chinese hamster ovary cell line by a pair of tricistronic and monocistronic vectors a 219-mer cho-expressing receptor-binding domain of sars-cov s protein induces potent immune responses and protective immunity a serological assay to detect sars-cov-2 seroconversion in humans eukaryotic genome size databases highly sensitive and fast protein detection with coomassie brilliant blue in sodium dodecyl sulfate-polyacrylamide gel electrophoresis antibodies : a laboratory manual glycomod--a software tool for determining glycosylation compositions from mass spectrometric data identification of two neutralizing regions on the severe acute respiratory syndrome coronavirus spike glycoprotein produced from the mammalian expression system extracellular matrix proteins mediate hiv-1 gp120 interactions with alpha4beta7 structure, function, and antigenicity of the sars-cov-2 spike glycoprotein testing for responses to the wrong sars-cov-2 antigen? whole nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 may cause false-positive results in serological assays estimates of the rate of infection and asymptomatic covid-19 disease in a population sample from se england analysis of sars-cov-2 antibodies in covid-19 convalescent blood using a ): p. 3581. 39. database. foundation for innovative new diagnostics. sars-cov-2 diagnostic pipeline a systematic review of sars-cov-2 vaccine candidates the sars-cov-2 receptor-binding domain elicits a potent neutralizing response without antibody-dependent enhancement we thank mr. arthur isaev (genetico, moscow, russia) and dr. alexander ivanov (institute of molecular biology russian academy of sciences, moscow, russia) for valuable comments and early access to the sars-cov-2 s protein sequence data, dr. yuri lebedin, eugenia kostrikina and xema co., ltd., for providing anti-rbd mabs and conjugates.the measurements were carried out on the equipment of the shared-access equipment centre "industrial biotechnology" the research center of biotechnology of the russian academy of sciences. dna sequencing was carried out in the inter-institutional center for collective use "genome" imb ras, organized with the support of the russian foundation of basic research.the authors would like to acknowledge all the doctors who diagnose and treat patients during the covid-19 pandemic. primers for rbdv1 cloning, restriction sites are underlined ad-cov-absf aacctcgaggccgccaccatgttcatgccttctt ad-rbd-myc6hnher gctagcctaatggtgatggtgatgatgaccggtatgcatat tcagatcctcttctgagatgagtttttgttcgaagttcacgc atttgtt primers for ptm construction, sticky ends of annealed pairs are underlinedctagtgatggtgatggtgatggtgatggtgatgaccgcctg cagacagatcctcttcgctgatcagtttttgttcaccggta primers for rbdv2 cloning, restriction sites are underlined ad-sfr2-nhef gctagcgtgcagcccaccgaatcc ad-sfr2-xmar cccgggtttgttcttcacgagattggt sequencing primers sq-5ch6-f gccgctgcttcctgtgac iresa rev aggtttccgggccctcacattg sq-mych-r gatgaccgcctgcagac key: cord-320238-qbjrlog1 authors: okba, nisreen m. a.; widjaja, ivy; van dieren, brenda; aebischer, andrea; van amerongen, geert; de waal, leon; stittelaar, koert j.; schipper, debby; martina, byron; van den brand, judith m. a.; beer, martin; bosch, berend-jan; haagmans, bart l. title: particulate multivalent presentation of the receptor binding domain induces protective immune responses against mers-cov date: 2020-05-29 journal: emerging microbes & infections doi: 10.1080/22221751.2020.1760735 sha: doc_id: 320238 cord_uid: qbjrlog1 middle east respiratory syndrome coronavirus (mers-cov) is a who priority pathogen for which vaccines are urgently needed. using an immune-focusing approach, we created self-assembling particles multivalently displaying critical regions of the mers-cov spike protein ─fusion peptide, heptad repeat 2, and receptor binding domain (rbd) ─ and tested their immunogenicity and protective capacity in rabbits. using a “plug-and-display” spytag/spycatcher system, we coupled rbd to lumazine synthase (ls) particles producing multimeric rbd-presenting particles (rbd-ls). rbd-ls vaccination induced antibody responses of high magnitude and quality (avidity, mers-cov neutralizing capacity, and mucosal immunity) with cross-clade neutralization. the antibody responses were associated with blocking viral replication and upper and lower respiratory tract protection against mers-cov infection in rabbits. this arrayed multivalent presentation of the viral rbd using the antigen-spytag/ls-spycatcher is a promising mers-cov vaccine candidate and this platform may be applied for the rapid development of vaccines against other emerging viruses such as sars-cov-2. emerging zoonotic viruses, such as severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) have been able to cross the species barrier posing a threat to the human population. mers-cov causes severe respiratory disease and fatalities in humans [1, 2] , and the virus is continuously introduced into the human population through infected dromedary camels, the viral reservoir with resulting outbreaks [3] . the wide geographical distribution of this viral reservoir, the high case-fatality rate in humans (35%), and the lack of treatment and licensed vaccines, make the virus a threat to the human population. this has put mers-cov on the recent who list of diseases having an epidemic or even pandemic potential for which countermeasures are lacking and are urgently needed [4] . vaccination is potentially one of the most effective ways to prevent the ongoing mers-cov outbreaks. several mers-cov vaccine candidates have been developed using different platforms including inactivated, live-attenuated, and subunit vaccines [5] . compared to other vaccine production platforms, recombinant subunit proteins have a higher safety profile, are relatively faster and easier to produce, and can be scaled-up in a more cost-effective manner; nonetheless, they tend to induce lower levels of protective immunity [6] . the use of self-assembling multimeric protein scaffold particles (mpsp) to present antigens in a multivalent virus-mimicking manner (size, repetitiveness, and geometry), has been shown to enhance vaccine-induced immune responses [7] [8] [9] [10] [11] , and to offer advantages over other multimeric antigen presentation platforms (reviewed in [12] ). both lumazine synthase (ls) and i3-01 (i3) can self-assemble into 60-meric particles, which can be expressed in e. coli and have been used as scaffolds for development of multimeric vaccines with improved immune responses compared to monomeric forms [13] [14] [15] . an ls-based hiv vaccine, (eod-gt8), has recently advanced to a phase i human clinical trial (nct03547245). linking of antigens to these mpsp can be achieved through several mechanisms; as e.g. genetic fusion or the syptag-spycatcher (st/sc) system [16] . while the former requires the antigen and scaffold to be produced in the same expression system, the latter allows each to be expressed in its suitable system harnessing a rapid post-translational "plug-andplay" assembly. this is advantageous, allowing scaffold-sc to be produced at scalable levels in e. coli and spytagged glycosylated antigens such as viral surface proteins to be produced in its optimal system, such as mammalian or insect cells. the antigen-st can then be multivalently displayed on the surface of the scscaffolds through the spontaneous formation of a stable isopeptide bond. this can be a platform for rapid vaccine manufacturing in case of epidemics or pandemics, to create optimized vaccines at reduced costs and also with reduced development times. the mers-cov spike (s) protein is the main target for subunit vaccine development [5] it assembles as a homotrimer and consists of an n-terminal head (s1 subunit) and a c-terminal stalk (s2 subunit). the s1 subunit mediates virus attachment and entry through its n-terminal s1 a domain and its c-terminal receptor binding domain (rbd), respectively [17, 18] . the s1 a domain binds sialic acids, a viral attachment factor, while the rbd binds to the viral receptor, dipeptidyl peptidase 4 (dpp4). following attachment and entry, the s2 subunit mediates viral fusion to the host cell through its fusion machinery; comprised of the fusion peptide (fp) and the two heptad repeats -hr1 and hr2 [19] . mers-cov neutralizing antibodies (abs) mainly recognize epitopes in the rbd of the spike head s1 subunit; and to a lower extent, epitopes in the sialic acid binding domain and the fusion-mediating more conserved s stalk (s2). nonetheless, antibodies directed against the sialic acid binding s1 a domain or the more conserved s2 subunit, although subdominant, may protect against mers-cov [20, 21] . immune focusing can enhance immune responses to subdominant regions [22] . in the current study, using ls and i3 self-assembling particles, we evaluated whether immune focusing and multivalent presentation can induce immune responses to the more sequence-conserved s2 regions: fp and hr2. furthermore, using a syptag/spycatcher system and ls particles, we tested whether immune focusing with/ without multivalent presentation of the viral rbd can lead to enhanced protection against a mers-cov challenge in rabbits. expression constructs were cloned using standard pcr methods. the gene encoding the 6,7-dimethyl-8-ribityllumazine synthase (ls; genbank accession no. wp_010880027.1) of a. aeolicus was synthesized using human-preferred codons obtained from gen-script usa, inc, as described previously [17] . the cysteine at position 37 and asparagine at position 102 of ls were mutated to alanine and glutamine, respectively. the gene encoding i3-01 (i3; pdb 5kp9, amino acid residues 19-222) derived from thermotoga maritima was synthesized using human-preferred codons obtained from genscript usa. the gene fragments encoding the δn1spycatcher (sc; uniprot accession no. afd50637.1; amino acid residues 48-139; [23] ) and spytag (st; uniprot accession no. wp_129284416.1; amino acid residues 981-994) based on the cna b-type domain-containing protein of streptococcus pyogenes were synthesized using human-preferred codons obtained from genscript usa, inc. the ls and i3 gene constructs were cloned into the pgex-2 t bacterial expression vector (sigma aldrich). to generate the hr2-ls expression vector, the hr2 region (amino acid residues 1215-1287) encoding sequence of the mers-cov s gene (accession no. nc_019843) was ligated in-frame with an n-terminal sequence encoding a cd5 signal sequence and streptag tag purification tag, and with a c-terminal sequence encoding the ls via a linker, and subsequent cloned into the pcaggs mammalian expression vector. to generate the i3-hr2 expression vector, the heptad repeat 2 encoding region (hr2, amino acid residues 1215-1287) of the mers-cov s gene was ligated in-frame with an n-terminal sequence encoding the i3-01 and a c-terminal streptag purification tag interspaced with a linker, and subsequent cloned into the pgex-2 t bacterial expression vector (sigma aldrich). to generate the fp-i3 and fp-ls expression vectors, the fusion peptide (fp; amino acid residues 884-898) encoding sequence of the mers-cov s gene was ligated in-frame with an n-terminal sequence encoding the i3-01 or ls, and a c-terminal streptag purification tag and subsequently cloned into the pgex-2 t bacterial expression vector (sigma aldrich). to generate the rbd-st expression vector, the mers-rbd (amino acid residues 358-588) encoding sequence of the mers-cov s gene was ligated inframe with an n-terminal sequence encoding a cd5 signal sequence and with a c-terminal sequence encoding the st followed by a double streptag, and subsequently cloned into the pcaggs mammalian expression vector. to generate the ls-sc expression vector, the codon optimized sc sequence equipped with an n-terminal flag-tag (dykddddk) was cloned to the n-terminus of the ls sequence in the pet15b bacterial expression vector (novagen). all protein sequences are provided in supplementary figures s1 and s2. mammalian expression of the hr2-ls and rbd-st constructs was done, as described previously [17] . in short, expression plasmids were polyethylenimine (pei)-transfected into 60% confluent hek-293 t cells for 6 h, after which transfections were removed and medium was replaced with 293 sfm ii-based expression medium (gibco life technologies) and incubated at 37°c in 5% co 2 . tissue culture supernatants were harvested 5-6 d post transfection, and expressed proteins were purified using streptactin sepharose beads (iba) according to the manufacturer's instruction. bl21 cells (novagen) were transformed with pgex-2 t expression vectors and grown in 2× yeast-tryptone medium to log phase (od 600 ∼1.0) and subsequently induced by adding iptg (isopropyl-β-d-thiogalactopyranoside) (gibco brl) to a final concentration of 1 mm. two hours later, the cells were pelleted, resuspended in 1/25 volume of 10 mm tris (ph 8.0)-10 mm edta-1 mm phenylmethylsulfonyl fluoride, and sonicated on ice (five times, 2 min each). the cell homogenates were centrifuged at 20,000 × g for 60 min at 4°c. proteins were purified from the cell lysate supernatant using streptactin sepharose beads (iba) according to the manufacturer's instruction. all purified proteins were analyzed on a 12% sds/ page gel under reducing conditions and stained with gelcodeblue stain reagent (thermo scientific). purified proteins were stored at 4°c until further use. expression of the flag-ls-sc was performed as described above with the following modifications: 1) cells were treated with 1 mg/ml lysozyme in lysis buffer (50 mm tris-hcl, 150 mm nacl, 1% triton x-100) for 1 h at room temperature prior to sonification on ice. 2) purification was performed using anti-flag® m2 affinity gel (sigma aldrich) as recommended by the manufacturer. purified proteins were dialyzed against 1x tbs buffer (50 mm tris-hcl, 150 mm nacl, ph 7.4) and stored at −80°c until further use. rabbit immunizations and challenge were carried out at viroclinics bioscience b.v. under permit no. avd277002015283-wp03, using bsl-3 containment facilities. female new zealand white rabbits (envigo, venray, the netherlands) of 11 weeks age were assigned to six groups (i-vi) of five animals each. immunizations were performed intramuscularly with either i) hr2-ls, ii) fp-ls, iii) ls, at day 0 and boosted with either i) hr2-i3, ii) fp-i3, iii) i3 on day28 or iv) pbs, v) rbd + ls, vi) rbd-ls on days 0 and 28. each animal received each time 15 µg of antigen adjuvanted with adjuplex (5%; sigma-aldrich, zwijndrecht, the netherlands) in a total volume of 500 µl. three weeks after the last vaccination (day 49 of the study), all animals were challenged intranasally under anesthesia with mers-cov (10 6 50% tissue culture infectious dose (tcid 50 ) mers-cov emc strain (accession no. nc_019843) in a volume of 1 ml divided over both nostrils). the animals were euthanized on day 4 postchallenge (day 53 of the study). serum samples were collected on days 0, 28, and 46. nasal swabs were collected on day 46 (pre-challenge) and on days 1 through 4 post-challenge. following euthanasia, lungs were examined for gross pathology and lung tissue samples were collected for virus detection, and in 10% formalin histopathology and immunohistochemistry. antigen-binding and anti-ls (scaffold) antibodies produced after vaccination were tested in the sera collected at different time points as well as in pre-challenge nasal swabs using elisa. costar high-binding 96-well elisa plates were coated overnight at 4°c with 1 µg/ ml of either recombinant ls, mers-cov s1 or s2 proteins in pbs. the plates were washed with pbs and blocked for 1 hr using 1%bsa/0.5%tween-20/pbs. following blocking, diluted samples (1:100 or serially diluted) were added and further incubated for 1 hr. the plates were then washed and and probed with an hrp-labeled goat anti-rabbit ig (1:2000, dako) secondary antibody. tmb was used for signal development and the absorbance of each sample was measured at 450 nm (od 450 ). antibody avidity was assessed using an ammonium thiocyanate (nh 4 scn)-displacement elisa. this was carried out as described above using serum dilutions containing same level of s1 absorbance units added in triplicates. following serum incubation and washing, nh 4 scn (0-5 m) was added to the wells for 15 min. the plates were then washed and further developed as described above. the concentration of nh 4 scn resulting in a 50% reduction in signal was taken as the avidity index (ic 50 ). to confirm the antigenicity of the rbd-ls particles, we tested its binding to well-characterized monoclonal antibodies binding conformational rbd epitopes [20] . human monoclonal antibodies 7.7g6, 1.6f9, 1.2g5, 1.8e5, 4.6e10 targeting the receptor binding domain of the mers-cov spike protein were produced and purified as described earlier [20] . nunc maxisorp plates (thermo scientific) were coated with the rbd-ls antigen at 100 ng /well at 4°c overnight. plates were washed three times with pbs containing 0.05% tween-20 and blocked with pbs with 5% protifar in pbs containing 0.1% tween-20 at room temperature for 2 h. four-folds serial dilutions of mabs starting at 10 µg/ml (diluted in blocking buffer) were added and plates were incubated for 1 h at room temperature. plates were washed three times and incubated with hrp-conjugated goat anti-human secondary antibody (itk southern biotech) diluted 1:2000 in blocking buffer for one hour at room temperature. hrp activity was measured at 450 nm using tetramethylbenzidine substrate (biofx) and an elisa plate reader (el-808, biotek). the presence of mers-cov neutralizing antibodies in the sera and nasal swabs of vaccinated animals was tested using a plaque reduction neutralization assay (prnt). heat -inactivated two-fold serially diluted samples (starting 1:10) were mixed 1:1 with 400 pfu of mers-cov (emc/2012) and incubated for one hour. the mix was then overlaid on huh-7 cells in 96-well plates. following one hour of incubation, the mix was removed and the cells were incubated for 8 hr. the cells were then fixed, permeabilized and stained using a mouse anti-mers-cov n protein monoclonal antibody (sino biological) followed by an hrp-labelled goat anti-mouse igg1 (southernbiotech). the signal was developed using a precipitate forming peroxidase substrate (true blue, kpl). the immunospot® image analyzer (ctl europe gmbh) was used to count the number of infected cells per well. the neutralization titre of each serum sample was determined as the reciprocal of the highest dilution resulting in a ≥50% (prnt 50 ) or ≥90% (prnt 90 ) reduction in the number of infected cells. a titre of ≥ 20 was considered to be positive. to evaluate the protective efficacy of vaccination against mers-cov challenge, nasal swabs, and homogenated lung tissues were tested for the presence of mers-cov rna using rt-qpcr for and for the presence of infectious virus by virus titration. the presence of viral rna in nasal swabs and lung tissues was tested using upe rt-qpcr as previously described [24] . rna was extracted from samples using magnapure lc total nucleic acid isolation kit (roche). rna amplification and quantification were carried out using a 7500 real-time pcr system (applied biosystems). samples with a c t value <40 were considered positive. rna dilutions extracted from a mers-cov stock of known titre was used to generate a standard curve in order to calculate the tcid 50 equivalent of rna detected in samples. concentrations of viral rna in lung tissue are expressed in as tcid 50 equivalents per gram tissue (tcid 50 eq/ g), and in the nasal swabs as tcid 50 eq/ml. the presence of mers-cov infectious viral particles in respiratory tract samples (nasal swabs and lung tissue homogenates) was detected by titration on vero cells as described previously [24] . briefly, 10-fold serially diluted samples (starting undiluted) were overlaid on vero cells and the plates were incubated for five days at 37°c and the cytopathic effect was recorded. infectious virus titres in lung tissue are expressed as tcid 50 per gram tissue (tcid 50 /g), and infectious virus titre in nose swabs are expressed as tcid 50 /ml. lung tissue samples were collected in formalin and embedded in paraffin for pathological analysis. hematoxylin-eosin staining was carried out for histopathological analysis. the presence of mers-cov nucleoprotein was detected by immunohistochemistry as previously published [24] . statistical analyses were performed using prism 7 (graphpad software inc, usa). data were compared using mann-whitney u test or student's t-test. pvalues < 0.05 were considered significant. all data are available within the article and its supplementary information or available from the authors on request. particulate multivalent antigen display can enhance immunogenicity through different mechanisms, allowing for induction of immune responses against otherwise weakly immunogenic antigens [7, 25] . we sought to design antigens capable of inducing strong immune responses against critical parts of the viral entry and fusion machinery within the mers-cov spike protein through immune focusing and multivalent presentation on self-assembling particles (figure 1 ). within the s1 subunit, the rbd is the main target for the induction of neutralizing antibodies and has been used to develop several vaccine candidates for mers-cov [5, 26] . indeed, the immunogenicity of rbd can be enhanced by its presentation on ferritin nanoparticles [27] . likewise, the fusion peptide (fp) and the hr2, which show a high degree of sequence conservation among covs relative to the rbd, play crucial roles in the cov spike-mediated fusion machinery, and can be targets for cov protective antibodies [28] [29] [30] [31] [32] . genetic fusion was chosen for fp and hr2, due to their small size, whereas the st/sc system was used for rbd display on particles to ensure correct folding of the protein. two 60-meric hyperstable self-assembling particles with icosahedral symmetry were used for multivalent display of mers-cov domains. the lumazine synthase (ls) particle, an icosahedron with a diameter of 15 nm (pmid: 23539181) and the i3-01 (i3) particle, a dodecahedron with a diameter of 25 nm (pmid: 27309817). the n-and c-termini of both scaffolds are surface exposed, providing a platform to multivalently present (antigenic) domains. two functional segments of the s2 subunit of the mers-cov spike protein were genetically fused to these nanoparticles; the fusion peptide containing region (amino acid residues 884-898) and the hr2 containing region (amino acid residues 1215-1287) ( figure 1b, supplementary figure s1 ). chimeric nanoparticles were purified after expression in eukaryotic (mammalian) or prokaryotic systems (figure 1 ). in addition, we used the spytag/spycatcher system to multivalently display the mers-cov rbd on ls nanoparticle via covalent bonding [8] . for this purpose, the spycatcher (sc) was genetically fused to ls and expressed and purified from e. coli. the spytag (st) was genetically fused to the mers-rbd (amino acid residues 358-588) and expressed and purified from hek-293 t cells ( figure 1c ). rbd-st was incubated with ls-sc in different molar ratios to assess the optimal coupling of both components. a 1:2 molar ratio of rbd-st and ls-sc allowed the optimal coupling of all of the provided rbd-st antigens to the sc-ls particles ( figure 1d ). the resulting conjugation products were used for immunization. in order to assess the effect of the particle-based multivalent antigen display on immunogenicity, a mixture of non-coupled rbd-st and ls (without sc) was taken along for immunization in the same molar ratio. all particulate preparations displaying mers-s antigenic domains (genetically fused or sc/st coupled) were analyzed by sds-page ( figure 1e, supplementary table s1 ), confirming their molecular integrity. we further confirmed the antigenicity of the rbd-ls particles by testing their capacity to bind monoclonal antibodies directed against conformational epitopes on the rbd [20] using elisa. all antibodies bound to rbd-ls in a dose dependant manner ( figure s3 ) indicating that the rbd is correctly folded confirming its antigenicity. we then evaluated the immunogenicity of the multimeric spike antigens using six groups of rabbits (n = 5 per group), which were intramuscularly immunized twice at a 4-week interval (figure 2a ). the ls/i3 and pbs immunized groups served as controls. after the first immunization, we detected antibody responses against the corresponding s subunit (s1 or s2) in the vaccinated rabbits. while the control groups remained negative ( figure 2b -e). endpoint antibody titres for the vaccinated groups are shown as geometric mean titres (gmt) in supplementary table s2 . the antibody responses were further boosted after the second immunization in all groups, while no responses were detected in the control groups, confirming the immunogenicity of the tested antigens in rabbits. anti-s2 antibody responses were detected in the hr2 and fp vaccinated groups with weak to no mers-cov neutralizing capacity ( figure 2b,c) . only hr2 vaccination induced low levels of mers-cov neutralizing antibodies (prnt 90 titres: 20 -40) in 4/ 5 rabbits; all 5 had mers-cov neutralizing antibodies at a 50% cut-off (data not shown). likewise, both the monomeric rbd (rbd + ls) and the multimeric rbd-ls were immunogenic and elicited high s1-specific antibody titres which were further boosted after the second immunization. the rbd-ls-induced s1 antibody titres were significantly higher than those induced by the monomeric rbd following the prime-as well as booster-vaccination (p = 0.0397 and p = 0.0317, respectively by mann-whitney u test) ( figure 2d ). multimeric rbd-ls vaccination elicited higher mers-cov neutralizing antibodies, a main correlate of protection, than the monomeric rbd + ls when tested for live virus neutralization using prnt 90 assay (p = 0.0109, and p = 0.0079, post-prime and boost, respectively by mann-whitney u test) ( figure 2e ). the vaccine induced antibodies were able to neutralize clade a (emc/2012 strain; figure 2e ) as well as the more recently circulating clade b (qatar15/2015 strain; supplementary figure s4 ) viruses. the spike protein of the former strain differs from the clade a emc/2012 strain in two positions; t95s and q1020r. following a single immunization, binding antibody titres were four-fold higher and neutralizing antibodies were eleven-fold higher in the coupled multimeric rbd-ls group than in the uncoupled monomeric rbd + ls (supplementary table s2 ). three weeks after the boost, binding antibody responses were seven-fold higher (p = 0.0079, mann-whitney u test) and neutralizing antibodies were ten-fold higher (p = 0.0079, mann-whitney u test) in the coupled rbd-ls group than in the uncoupled rbd + ls ( figure 2d , e supplementary table s2 ). additionally, we tested for vaccine induced mucosal immunity in the respiratory tract of vaccinated rabbits pre-challenge (day 49) using elisa. mers-cov specific antibodies were only detected in the nasal swabs of the groups vaccinated with conjugated or non-conjugated rbd (figure 2 f,g) . antibody responses detected in the rbd-ls vaccinated group were higher than those in the rbd + ls vaccinated group (p = 0.0357, student's t-test). this demonstrates that rbd-ls induces improved local mucosal immune responses compared to the monomeric rbd. thus, vaccination with the newly produced rbd-ls mers-cov mpsp vaccines induce a robust immune response. the avidity of mers-cov spike-specific antibodies in the monomeric versus the multimeric rbd vaccinated groups was analyzed at days 28 (4 weeks after prime) and 46 (3 weeks after boost) using an ammonium thiocyanate (nh 4 scn)-displacement elisa [33] . the avidity index ic 50 was determined for each vaccinated rabbit and compared between the two groups. the avidity of the s1-specific antibody responses was higher following rbd-ls vaccination compared to the monomeric rbd + ls vaccination (p < 0.0001, student's ttest) (figure 3 ), indicating that a multimeric rbd-ls vaccine can induce antibody responses of both higher quantity and quality ( figures 2d,e and 3) . in addition to evaluating anti-s (antigen) responses, we also tested for the induction on ls-specific (scaffold) antibodies. antibody responses were elicited against the ls-particle in all groups except the pbs group, indicating that the particle was accessible and not sterically hidden by antigens displayed on its surface; even when rbd was displayed on its surface using spytag:spy-catcher linkage (figure 4 ). despite that, antigenspecific responses were not adversely affected by the presence of these anti-scaffold antibodies, as demonstrated by the booster effect after the second immunization ( figure 2d,e) . nonetheless, we tested whether a heterologous scaffold boost could help in minimizing such anti-scaffold responses using an ls/i3 primeboost scheme. using this approach, we found no significant increase in anti-scaffold antibody responses compared to the homologous prime-boost scheme ( figure 4c ). this indicates that a heterologous scaffold prime-boost approach could be advantageous for limiting unnecessary anti-scaffold responses. to evaluate the protective efficacy of the immune responses induced by the different mers-cov spike mpsp vaccines, rabbits were challenged intranasally with 10 6 tcid 50 of mers-cov (strain hcov-emc/2012) and nasal swabs were collected up to 4 days post inoculation (pi) (figure 2a ). on day 4 pi, the animals were euthanized, and lung tissue samples were collected. consistent with earlier reports [34, 35] , none of the rabbits in the control group developed any clinical signs of infection upon mers-cov inoculation, and titration of infectious virus from lung tissues and nasal swabs was variable. thus, to evaluate protection, we tested for mers-cov rna by qrt-pcr, for mers-cov infectious virus by virus titration, and for mers-cov antigen (n protein) in lung tissues by immunohistochemistry (ihc). except for the rbd-ls vaccinated group, viral rna was detected in all vaccinated groups from day 1 through day 4 postchallenge at levels similar to control groups ( figures 5 and 6 ). viral rna titres were significantly reduced in the nasal swabs of the rbd-ls vaccinated groups as early as day 1 post-challenge and were undetectable by day 4, in line with the absence of detectable infectious virus particles ( figure 5 ). viral rna was also reduced in the lungs of rbd-ls-vaccinated rabbits ( figure 6 ). consistently, ihc revealed no viral antigen in the lungs of the rbd-ls vaccinated rabbits ( figure 6c ), and antigen was also not detected a, b) the percentage of serum antibodies bound following the addition of different concentration of scn was used to determine (c) the avidity index (ic 50 ). the difference in serum avidity between both groups was tested for statistical significance using a student's t-test, with asterisks indicating the level of significance. ***p ≤ 0.001, ****p ≤ 0.0001. error bars indicate mean ± s.e.m. in the rbd + ls vaccinated rabbits. overall, in contrast to the monomeric form, the antigen-focused multimeric rbd-ls vaccine was able to block mers-cov replication significantly in the nose and lungs of the infected rabbits. the efficacy of rbd-ls immunization in protecting against a mers-cov challenge, makes it a potential vaccine candidate. however, for production at industrial scale, unnecessary sequences (e.g. tags) need to be removed, preparations have to be further structurally and biochemically characterized. recombinant subunit proteins provide advantages regarding safety, costs, and speed of vaccine production, making them very attractive platforms for the development of vaccines for emerging viruses. multivalent antigen display allows for virus-mimicking presentation of antigens and has been shown to induce antibodies of high avidity and magnitude [7, 10, 11, 27, 36] ; with non-viral self-assembling mpsp providing advantages over other multimeric antigen presentation platforms [8, 12] . among the mers-cov vaccine candidates developed so far, the latter approach has been used to design two candidates, both are based on the receptor-binding domain [27, 37] , the main target for mers-cov protective antibodies [26] . one used self-assembling ferritin nanoparticles [27] and the second used canine parvovirus (cpv) vp2 structural protein forming virus like particles [37] as scaffolds. both vaccine candidates were able to induce humoral and cellular immune responses in mice, nonetheless none has been tested for its protective capacity in a viral-challenge animal model. in our study, using an immune-focusing approach to target protective epitopes and domains along with multivalent presentation on self-assembling ls particles using a spontaneous covalent linker (spy-tag/spycatcher). we report for the first time the invivo protective capacity of a multimeric mers-cov rbd particle vaccine. we used self-assembling ls and i3 particles to generate chimeric multimeric protein scaffold particle displaying critical domains in the mers-cov spike protein and evaluated their immunogenicity and protective efficacy in rabbits. multimeric fp and hr2 vaccinations induced high levels of anti-s2 antibodies, nonetheless, with low to undetectable virus neutralizing capacities and couldn't protect rabbits against virus challenge. meanwhile, multimeric rbd-ls vaccination was highly immunogenic and induced robust antibody responses of high magnitude, avidity and neutralizing capacity. following a live virus challenge, it protected upper and lower respiratory tract of rabbits as detected by decrease in viral rna titres, with an 6 ). despite producing strong antibody responses, the monomeric rbd failed to protect rabbits against mers-cov following an intranasal challenge. the presence of ls did not seem to influence the outcome, as it was included in the formulation of the monomeric form (rbd + ls), indicating that the coupling and the multimeric presentation are responsible for the enhanced response seen with the multimeric rbd-ls vaccine. the "plug-and-display" spytag/spycatcher system [8] used to generate these multimeric rbd-ls particles allows for rapid and robust production of vaccines in a cost-effective manner. this enables the development of vaccines in a timely manner, which is crucial to prevent global public health consequences of evolving, emerging and re-emerging viruses. the efficacy of rbd-ls immunization in protecting against a mers-cov challenge, makes it a potential vaccine candidate for further development. nonetheless, in case of production at an industrial scale, unnecessary sequences (e.g. tags) need to be removed, preparations have to be further structurally and biochemically characterized. when using scaffolds as antigen carriers, anti-scaffold antibody responses need to be considered to avoid their potential to compromise the targeted antigen-induced responses or to induce potential auto-antibodies against human antigens. antibody responses were induced against the ls protein scaffold used in this study. however, antigen-specific responses were boosted following the second immunization and were not adversely affected by the presence of these anti-scaffold antibodies (figure 4 ), similar to other reports [38] . since the sequence of the ls protein does not show any similarity to any human sequences, it is unlikely that they will induce unwanted auto-(antihuman) antibodies. an ls-based vaccine for hiv, in a current phase 1 clinical trial (nct03547245), can provide further evidence for the safety of this platform. nonetheless, we developed a heterologous scaffold prime-boost using ls and i3 which can help in reducing anti-scaffold responses. a challenge facing mers-cov vaccine development is the limited number of appropriate animal models for testing protection against clinical virus isolates. rabbits provide some advantages as an animal model for mers-cov. by having the mers-cov receptor dpp4 expressed in both the upper and lower respiratory tract epithelium [24] , the rabbits can be naturally infected. this allows the evaluation of both upper and lower respiratory tract mers-cov infection and in turn protection using natural field virus isolates rather than adapted strains. however, the animals are not able to develop severe infection such as that seen in severe human cases [34] . nonetheless, severe infection, thus far, has not been established consistently in any of the other animal models without genetic modification and/or virus adaptation, except for marmosets [39] . in addition to the aforementioned, rabbits are readily available and easier to handle compared to other species that can be naturally infected such as non-human primates. following the addition of mers-cov as a priority pathogen in the who r&d blueprint for action to prevent epidemics, a target product profile was developed which called for three types of mers-cov vaccines [40] . these include one for camels to prevent virus shedding and transmission, and two for humans: a two-dose vaccine for long-term protection of those at continuous high risk such as camel handlers and health-care workers, and a single-dose vaccine for rapid onset of immune responses to protect those at acute risk in outbreak settings. the rbd-ls can be used to develop the two-dose vaccine required to protect the high-risk populations, and can be further optimized using the heterologous scaffold prime/boost scheme developed in this study. nonetheless, evaluating the longevity of the induced immune responses is warranted. following the prime, rbd-ls vaccination induced antibody responses of high avidity and mers-cov neutralizing capacity. owing to the robust immune responses induced after one dose, the rbd-ls can be a candidate for developing a rapid single-dose vaccine for mers-cov, which is required for reactive use in outbreak situations [40] . additionally, this vaccine candidate was able to block mers-cov replication in the upper respiratory tract of infected rabbit, thus it could potentially be of use as a dromedary vaccine to block mers-cov transmission. however, both approaches need to be further validated. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation list of 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stick, click, and glue identification of sialic acid-binding function for the middle east respiratory syndrome coronavirus spike glycoprotein the receptor binding domain of the new middle east respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor towards a solution to mers: protective human monoclonal antibodies targeting different domains and functions of the mers-coronavirus spike glycoprotein. emerg microbes infect importance of neutralizing monoclonal antibodies targeting multiple antigenic sites on the middle east respiratory syndrome coronavirus spike glycoprotein to avoid neutralization escape application of built-in adjuvants for epitope-based vaccines structural analysis and optimization of the covalent association between spycatcher and a peptide tag lack of middle east respiratory syndrome coronavirus transmission in rabbits. viruses vaccine delivery: a matter of size, geometry, kinetics and molecular patterns advances in mers-cov vaccines and therapeutics based on the receptor-binding domain. viruses chaperna-mediated assembly of ferritin-based middle east respiratory syndrome-coronavirus nanoparticles identification of an immunodominant linear neutralization domain on the s2 portion of the murine coronavirus spike glycoprotein and evidence that it forms part of complex tridimensional structure analysis of murine coronavirus surface glycoprotein functions by using monoclonal antibodies human monoclonal antibodies against highly conserved hr1 and hr2 domains of the sars-cov spike protein are more broadly neutralizing characterization of neutralizing monoclonal antibodies recognizing a 15-residues epitope on the spike protein hr2 region of severe acute respiratory syndrome coronavirus (sars-cov) monoclonal antibodies targeting the hr2 domain and the region immediately upstream of the hr2 of the s protein neutralize in vitro infection of severe acute respiratory syndrome coronavirus antibody avidity determination by elisa using thiocyanate elution asymptomatic middle east respiratory syndrome coronavirus infection in rabbits enhanced inflammation in new zealand white rabbits when mers-cov reinfection occurs in the absence of neutralizing antibody nanoassembly routes stimulate conflicting antibody quantity and quality for transmission-blocking malaria vaccines. sci rep novel chimeric viruslike particles vaccine displaying mers-cov receptorbinding domain induce specific humoral and cellular immune response in mice self-assembling influenza nanoparticle vaccines elicit broadly neutralizing h1n1 antibodies infection with mers-cov causes lethal pneumonia in the common marmoset a roadmap for mers-cov research and product development: report from a world health organization consultation we thank the technical staff of the preclinical department of viroclinics biosciences b.v. for their excellent technical support. key: cord-291420-40xsypzt authors: nelson-sathi, shijulal; umasankar, pk; sreekumar, e; radhakrishnan nair, r; joseph, iype; nori, sai ravi chandra; philip, jamiema sara; prasad, roshny; navyasree, kv; ramesh, shikha; pillai, heera; ghosh, sanu; santosh kumar, tr; radhakrishna pillai, m. title: mutational landscape and in silico structure models of sars-cov-2 spike receptor binding domain reveal key molecular determinants for virus-host interaction date: 2020-10-01 journal: biorxiv doi: 10.1101/2020.05.02.071811 sha: doc_id: 291420 cord_uid: 40xsypzt protein-protein interactions between virus and host are crucial for infection. sars-cov-2, the causative agent of covid-19 pandemic is an rna virus prone to mutations. formation of a stable binding interface between the spike (s) protein receptor binding domain (rbd) of sars-cov-2 and angiotensin-converting enzyme 2 (ace2) of host actuates viral entry. yet, how this binding interface evolves as virus acquires mutations during pandemic remains elusive. here, using a high fidelity bioinformatics pipeline, we analysed 31,403 sars-cov-2 genomes across the globe, and identified 444 non-synonymous mutations that cause 49 distinct amino acid substitutions in the rbd. molecular phylogenetic analysis suggested independent emergence of these rbd mutants during pandemic. in silico structure modelling of interfaces induced by mutations on residues which directly engage ace2 or lie in the near vicinity revealed molecular rearrangements and binding energies unique to each rbd mutant. comparative structure analysis using binding interface from mouse that prevents sars-cov-2 entry uncovered minimal molecular determinants in rbd necessary for the formation of stable interface. we identified that interfacial interaction involving amino acid residues n487 and g496 on either ends of the binding scaffold are indispensable to anchor rbd and are well conserved in all sars-like corona viruses. all other interactions appear to be required to locally remodel binding interface with varying affinities and thus may decide extent of viral transmission and disease outcome. together, our findings propose the modalities and variations in rbd-ace2 interface formation exploited by sars-cov-2 for endurance. importance covid-19, so far the worst hit pandemic to mankind, started in january 2020 and is still prevailing globally. our study identified key molecular arrangements in rbd-ace2 interface that help virus to tolerate mutations and prevail. in addition, rbd mutations identified in this study can serve as a molecular directory for experimental biologists to perform functional validation experiments. the minimal molecular requirements for the formation of rbd-ace2 interface predicted using in silico structure models may help precisely design neutralizing antibodies, vaccines and therapeutics. our study also proposes the significance of understanding evolution of protein interfaces during pandemic. cov-2 and related sars-cov provided initial clues regarding molecular architecture of the interface. rbd comprises of 223 amino acid long peptide in the s1-region of s-protein ( table 1 ) (walls et al., 2020) . however, ace2 binding information is confined to a variable loop region within rbd called receptor binding motif (rbm). these structures elucidated key interfacial interactions responsible for enhanced binding affinity of sars-cov-2 to ace2 than sars-cov . it also suggested that few amino acid changes in rbm can remodel the interface resulting in altered binding affinities and viral transmission. however, all these studies were based on parental sars-cov-2 wuhan strain and several questions remain unanswered. what are the mutations acquired on rbd during covid-19 and what are the interfacial molecular rearrangements induced by these mutations? can we gain valuable insights regarding rbd-ace2 interface formation by analyzing these mutations? to address these questions we investigated the mutational landscape of sars-cov-2 rbd. a total of 55,485 spike proteins of sars-cov-2 were directly downloaded on 29 th june 2020 from the gisaid database. we removed the partial sequences, sequences greater than 1% unidentified 'x' amino acids and sequences from low quality genomes. further, 31,403 spike protein sequences along with wuhan reference spike protein (yp_009724390.1) were aligned using mafft (maxiterate 1,000 and global pair-ginsi) (katoh et al. 2002) . the alignments were visualized in jalview (waterhouse am et al., 2009) and the amino acid substitutions in each position were extracted using custom python script. we ignored the substitutions that were present in only one genome and unidentified amino acid x. the mutations that are present in at least two independent genomes in a particular position were further considered. these two criteria were used to avoid mutations due to sequencing errors. the mutated amino acids were further tabulated and plotted as a matrix using r script. for the maximum-likelihood phylogeny reconstruction, we have used the sars-cov-2 genomes containing rbd mutations, and 10 genomes were sampled as representatives for each known subtype with wuhan refseq strain as root. sequences were aligned using mafft (maxiterate 1,000 and global pair-ginsi), and phylogeny was reconstructed using iq-tree (nguyen et al., 2015) . the best evolutionary model (gtr+f+i+g4) was picked using the modelfinder program (kalyaanamoorthy et al., 2017) . the structural analysis of the mutated spike glycoprotein of sars-cov-2 rbd domain was done to assess the impact of interface amino acid residue mutations on binding affinity towards the human ace2 (hace2) receptor. the crystal structure of the sars-cov-2 rbd-hace2 receptor complex was downloaded from protein data bank (pdb id:6lzg) and the mutagenesis analysis to capture changes that affect viral tropism during pandemic, we searched for non-synonymous mutations in rbd sequences from sars-cov-2 genomes. using unbiased and stringent filtering criteria, we analyzed 31,403 genomes deposited in gisaid till 29th june, 2020. altogether, 444 non-synonymous mutations in rbd were identified that belong to viral genomes from 30 countries. these mutations were found to substitute 49 amino acid residues in which 23 residues lie within rbm ( table 1) . these residues include those that directly engage ace2 (g446, l455, a475, g476, e484, f490 and q493) and those that are in the near binding vicinity (figure 1a and 1b). hot spot mutations were also identified that caused recurrent substitutions of amino acid residues in the same position (n354, p384, q414, i468, s477, v483, f490, a520, p521 and a522). each rbd mutation was found to be unique to the genome; a combination of mutations was never observed in our analysis. overall, rbd mutations accounted for ~9% of the total non-synonymous mutations in s-protein. to see the evolutionary trend in rbd mutations, we compared rbds from sars-cov-2, the related sars-cov and the bat coronavirus ratg13, a suspected precursor of sars-cov-2. sars-cov-2 rbd is 73.4 % identical to sars-cov and 90.1 % identical to ratg13 ( figure 1a ). we identified several rbd mutations on residues that are unique to sars-cov-2 (n439k, v483a/f/i, e484d, f490s/l, q493l and s494p) or are conserved in all three viruses ( figure 1a ). in addition, we observed micro evolutionary reversion mutations in sars-cov-2 that interchange residues to that in sars-cov or ratg13 (r346k, n354d, n439k, l452r, e471v and s477g) ( figure 1a) . interestingly, most of the unique and reversion mutations were located in the rbm region and thus may have implications in viral tropism. we performed phylogenomic analysis to understand the evolutionary pattern of rbd variants during pandemic and observed an unbiased clustering of rbd variants among distinct sars-cov-2 subtypes likely indicating independent emergence of these mutants (figure 1c ). rbd is divided into a structured core region comprising five antiparallel β-sheets and a variable random coil region, rbm that directly binds to ace2 (figure 1b) . on the contrary, binding information on ace2 is located across long α-helices. structurally, rbm scaffold resembles a concave arch that makes three contact points with ace2 α-helix; cluster-i, ii and iii. cluster-i and cluster-iii are on two ends and cluster-ii is towards the middle of the interface (figure 2a) . we analysed the effect of observed rbm mutations on the molecular interactions at rbd-ace2 binding interface. it has been shown that differences in ace2 residues render mouse resistant to infection from sars-like coronaviruses (zhao et al., 2020) . hence, to gain insights into relevant interactions that can create stable interface in mutants, we also included rbd-mouse ace2 interface in our analysis ( figure 2b) . structure models were created for all mutants based on the information from three recently reported crystal/ cryo-em structures of sars-cov-2 rbd-ace2 bound complex (figure 2a) . comparative analysis of structures showed key differences in all three binding clusters of sars-cov-2 rbd wild type and mutant interfaces with human or mouse ace2 (figure 2c, 2d and table s1 ). in cluster-i, f486 of sars-cov-2 rbm is found buried into the hydrophobic pocket made of human ace2 residues l79, m82 and y83. a mutation of f>l in sars-cov disrupts this pocket, thus weakens the binding affinity suggesting importance of this interaction (wan et al., 2020) . in addition, n487 of sars-cov-2 rbm forms hydrogen bonds with q24 and y83 of human ace2. the hydrophobic pocket and n487-y83 interactions were completely abolished in mouse interface due to natural ace2 substitutions in l79t, m82s and y83f. but, these interactions were retained in all rbm mutants suggesting their importance in the pandemic. nevertheless, interactions of a475/ g476 of sars-cov-2 rbd with s19 of ace2 in cluster-i which were present in human and mouse were disrupted in mutants. in addition, sars-cov-2 genomes containing a475v and g476s replacements were identified in our analysis suggesting these mutations can be well tolerated. an additional hydrogen bond between y489-y83 was seen in mutants lacking a475/g476-s19 interaction. this could possibly be a compensatory mechanism to stabilize cluster-i interactions (figure 2c, 2d and table s1 ). table s1 ). a bunch of interactions in cluster-iii involving g446/y449/q498 of rbm and q42 of ace2 were present in human but abolished in mouse and rbm mutants. however, additional interactions to compensate for these were not seen. a hydrogen bond formed between g496 of rbm and k353 of human ace2 appeared significant as this was completely abolished in mouse, owing to k353h substitution, but retained in all rbm mutants. in addition, other interactions; g502-k353, y449-d38 and t500-y41 in the same cluster were maintained in human, mouse and mutants likely suggesting their supportive role ( figure 2c, 2d and table s1 ). the varying interface arrangements in mutants were consistent with the binding affinity differences (δδg). compared to wild type, δδg values of mutants ranged within ~ + 1 kcal/mol, with the lowest value close to that of sars-cov ( figure s1) . sine rbm is a variable loop, mutations on any residue could impact spatial arrangements of backbone leading to altered binding affinities. consistently, we did not find a considerable difference in binding energies between mutations on residues that are involved in ace2 interaction or are in the near vicinity. in conclusion, we could pinpoint two interfacial interactions that remain unaffected in all mutants analysed. these are interactions mediated through rbd residues n487 and g496 and are located in cluster-i and cluster-iii respectively. based on their spatial arrangement, these residues appear critical in directly anchoring the rbm loop onto ace2. this may help initiate interface formation that favours viral entry. both n487 and g496 are highly conserved in all sars-like corona viruses further reinforcing this notion. the significant remodelling in cluster-ii interactions indicates they are dispensable for anchoring but might be important for stabilizing the interface. since rbd-ace2 interface is a direct determinant of viral infectivity, along with other factors, varying interface architecture and binding affinities in sars-cov-2 rbm mutants may account for global variations in covid-19 transmission and outcome. sars-cov-2 s-protein is highly immunogenic, so recombinant vaccines and neutralizing antibodies that target the whole s-protein or rbd are currently being considered in clinics . our investigations reveal key molecular determinants and their modalities for rbd-ace2 interaction. this information might be used to design vaccines, synthetic nanobodies or small molecules that could specifically target rbm anchoring residues or their binding pockets. tepymol molecular graphics system the spike protein of sars-cov -a target for vaccine and therapeutic development sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor modelfinder: fast model selection for accurate phylogenetic estimates mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform increasing the precision of comparative models with yasara nova-a selfparameterizing force feld structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor iq-tree: a fast and effective stochastic algorithm for estimating maximum likelihood phylogenies characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov zdock server: interactive docking prediction of protein-protein complexes and symmetric multimers swiss-model: an automated protein homology-modeling server pic: protein interactions calculator ligplot: a program to generate schematic diagrams of protein-ligand interactions structure, function, and antigenicity of the sars-cov-2 spike glycoprotein receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus structural and functional basis of sars-cov-2 entry by using human ace2 enhanced receptor binding of sars-cov-2 through networks of hydrogen-bonding and hydrophobic interactions jalview version 2-a multiple sequence alignment editor and analysis workbench cryo-em structure of the 2019-ncov spike in the prefusion conformation prodigy: a web server for predicting the binding affinity of protein-protein complexes mining of epitopes on spike protein of sars-cov-2 from covid-19 patients broad and differential animal angiotensin-converting enzyme 2 receptor usage by sars-cov-2 the authors wish to acknowledge john b johnson, mahendran kr and sara jones for critical comments. the work was supported by the department of biotechnology, government of india. key: cord-315415-3aotsb2g authors: dong, jianbo; huang, betty; wang, bo; titong, allison; gallolu kankanamalage, sachith; jia, zhejun; wright, meredith; parthasarathy, pannaga; liu, yue title: development of humanized tri-specific nanobodies with potent neutralization for sars-cov-2 date: 2020-10-20 journal: sci rep doi: 10.1038/s41598-020-74761-y sha: doc_id: 315415 cord_uid: 3aotsb2g sars-cov-2 is a newly emergent coronavirus, which has adversely impacted human health and has led to the covid-19 pandemic. there is an unmet need to develop therapies against sars-cov-2 due to its severity and lack of treatment options. a promising approach to combat covid-19 is through the neutralization of sars-cov-2 by therapeutic antibodies. previously, we described a strategy to rapidly identify and generate llama nanobodies (vhh) from naïve and synthetic humanized vhh phage libraries that specifically bind the s1 sars-cov-2 spike protein, and block the interaction with the human ace2 receptor. in this study we used computer-aided design to construct multi-specific vhh antibodies fused to human igg1 fc domains based on the epitope predictions for leading vhhs. the resulting tri-specific vhh-fc antibodies show more potent s1 binding, s1/ace2 blocking, and sars-cov-2 pseudovirus neutralization than the bi-specific vhh-fcs or combination of individual monoclonal vhh-fcs. furthermore, protein stability analysis of the vhh-fcs shows favorable developability features, which enable them to be quickly and successfully developed into therapeutics against covid-19. sars-cov-2 is a coronavirus that causes the human disease covid-19, which is contagious and can rapidly spread to cause mild to severe infection, including death [cdc (https ://www.cdc.gov/coron aviru s/types .html) 1 ]. the spread of this newly emergent virus has reached a pandemic level with a significant public impact on the world, leading to more than 25 million infections and more than a 0.85 million deaths worldwide [world health organization (who) (https ://www.who.int/emerg encie s/disea ses/novel -coron aviru s-2019)]. in addition to threatening human health, covid-19 has also caused a significant socio-economic impact around the world [united nations (https ://www.undp.org/conte nt/undp/en/home/coron aviru s/socio -econo mic-impac t-of-covid -19.html)]. although there are relatively successful diagnostic methods to detect the sars-cov-2 infection in humans, there are currently no successful therapies that can interfere with virus replication. the small antiviral molecule remdesivir (gilead) which inhibits the rna-dependent rna polymerase of sars-cov-2 decreases the recovery time in patients with covid-19 2 , but it most likely cannot completely stop or prevent sars-cov-2 infections in humans. another small antiviral molecule, grl-0617, shows promise in interfering with the sars-cov-2 replication by inhibiting the papain-like protease, however, it is yet to be tested in clinical trials 3 . moreover, there are no fda-approved vaccines to prevent sars-cov-2 infections in humans, although several groups are currently in the pursuit such vaccines [who (https ://www.who.int/publi catio ns/m/item/draft -lands cape-of-covid -19-candi date-vacci nes)]. therefore, rapid development of therapeutics and preventative strategies has become an essential and urgent need to fight the covid-19 pandemic. the trimeric spike (s) proteins that protrude through the envelope of the sars-cov-2 virion mediate virus entry into the host cells by interacting with the human ace2 receptor [4] [5] [6] [7] [8] [9] . therefore, a major target for anti-sars-cov-2 neutralizing antibodies in development are to block the interaction of sars-cov-2 s1 protein with ace2. in particular, two popular strategies have been employed to discover and develop monoclonal igg antibodies that can recognize sars-cov-2 s1 protein mainly by binding to its receptor binding domain (rbd) [10] [11] [12] [13] [14] [15] . the first commonly used method is to clone the antibody v genes from the b cells of surviving covid-19 patients who have mounted a natural immune response against sars-cov-2 10, 11, 13 . this strategy has yielded a number of neutralizing monoclonal antibodies; however, it is important to note that the patients' antibody repertoire condition and the timing of blood sample collection play a critical role in its success. the other well-recognized and classic approach for antibody generation is by immunizing humanized mice 15 . additionally, new sars-cov-2 identification of vhhs binding to different epitopes of sars-cov-2 s1 protein rbd. recently, we reported the identification of llama vhhs that bind to the sars-cov-2 s1 protein rbd 19 . briefly, we used two llama vhh libraries (one naïve library and another humanized synthetic library derived from the naïve library) to screen for vhhs that bind to the sars-cov-2 s1 protein in-vitro 19 . we identified a total of 89 s1 protein binders, 64 from the naïve and 25 from the humanized libraries. out of the s1 protein binders, 19 vhhs blocked the interaction between sars-cov-2 s1 rbd and ace2 receptor, with 12 s/ace2 blockers identified from the naïve library and 7 identified from the humanized library (data not shown). furthermore, we observed that the pairwise addition of some of the vhhs caused synergistic effects on sars-cov-2 s/ace2 blocking 19 . we hypothesized that this synergistic effect is caused by binding of the vhhs to different epitopes within the s1 rbd. to test this, we performed epitope binning assays by biolayer interferometry (fig. 1a -c) or elisa (fig. 1d ) on a selected number of candidates. in the initial epitope binning assay (fig. 1a-c) , we used an s1 rbd sensor to capture 2a-fc, 1b-fc, or 3f-fc separately, followed by the incubation with our lead candidates 1b-fc, 3f-fc or 2a-fc. the vhhs were fused to human igg1 fc domains to render the fc effector functions against sars-cov-2 19 . this analysis showed that with the 2a-fc-loaded probe, the addition of 3f-fc further increased the signal compared to the 2a-fc control, while the addition of 1b-fc decreased the signal compared to the control (fig. 1a) . this indicates that 3f-fc does not compete with the 2a-fc site and it is likely that they bind to different s1 rbd epitopes. in contrast, 1b-fc competed with 2a-fc, indicating that they compete for binding to the same s1 rbd epitope (fig. 1a) . similarly, with the 1b-fc-loaded probe (fig. 1b) , 3f-fc increased the signal compared to the 1b-fc control. this shows that 3f-fc does not compete with 1b-fc. interestingly, 2a-fc also increased its signal compared to 1b-fc control. this suggests that although having a common binding region, 2a binds to a wider epitope than 1b (fig. 1b) . with 3f-fc-loaded probe, both 2a-fc and 1b-fc showed an increase of the signal compared to the 3f-fc control. this further shows that 3f-fc does not compete with either 1b-fc or 2a-fc, and likely bind to a different epitope (fig. 1c) . these results confirm our hypothesis and show that s/ace2 blocking vhhs bind to at least two separate unique epitopes within the s1 rbd. next, we performed an elisa-based epitope binning assay to assess five additional vhhs (1c, 1f, 3a, 4f, and g4) unfused to fc, but previously assessed to block the sars-cov-2 s/ace2 interaction 19 . the assessment of more vhhs would allow us to categorize several of our other vhhs into binding groups, which will aid in multi-specific antibody design and construction. in this elisa, wells were coated with sars-cov-2 s1 and incubated with bi-specific vhh-fc 1b-2a (based on previous data, 1b and 2a likely bind the same epitope) or monoclonal vhh-fc 3f-fc (based on previous data, this binds a different epitope than 1b or 2a) premixed with the vhh candidates. the resulting relative fluorescence signals obtained for each sample were calculated to reflect the percent difference from 1c, 1f, 3a, 4f, g4, and controls (3f-fc and 1b-2a-fc) signals, when the vhhs are combined with 1b-2a-fc or 3f-fc (fig. 1d) . the results show that vhh-fcs 1c, 1f, 4f, as well as the 1b-2a-fc control have almost 100% difference from 1b-2a-fc, which highly suggest that they compete for the same epitope (highlighted in red). however, g4 (highlighted in light red) may partially compete with 1b-2a-fc, whereas 3a does not likely compete for the same epitope (highlighted in green). additionally, these results show that 3a and the 3f-fc control may compete with 3f-fc (fig. 1d) , while other vhhs, including the 1b-2a-fc control resulted in a lower percent difference. we also performed additional epitope binning assays using biolayer interferometry to assess the competition of the vhh-fcs 1c, g4, and 3a to bind to s1 rbd. the vhh-fcs 1f and 4f poorly bound to the biolayer interferometry probes used for this assay and were excluded from analysis. this approach confirmed the results that we obtained by elisa and showed that 1c and g4 likely belong to group 1, and 3a belongs to group 2 in terms of the binding competition ( supplementary fig. 1 ). interestingly, g4-fc shows competition with either 1b-fc and 2a-fc when it is loaded onto the probe first (data not shown). in contrast, reversal the of loading further increased its signal compared to both 1b-fc control and fig. 1 ). taken together, we could categorize 8 vhh blockers of s/ace2 interaction into 2 major groups based on their epitope binding; group 1 consist of 6 vhhs, whereas group 2 consist of 2 vhhs (fig. 1e ). elucidation of epitopes on s1 rbd that bind to vhh-fcs. in an effort to elucidate the structural basis of the newly discovered epitope binding groups, we computationally generated structural models for 1b, 3f, and 2a vhhs and docked them with sars-cov-2 s1 rbd structure exported from pdb 6m0j using schrodinger bioluminate software [20] [21] [22] . for context, fig. 2a shows the sars-cov-2 s1 protein with the ace2 binding residues in red font. this approach generated an array of poses of each s1 rbd/vhh complex structure, which allowed us to further analyze the interfaces of those poses with a good piper cluster size and led us to identify five regions in the rbd which may interact with vhh 1b, 2a, and 3f, respectively (fig. 2a,b) [23] [24] [25] . next, we generated 5 different s1 rbd deletion mutants to validate the computationally mapped epitopes in-vitro to select the best docking model for molecular analysis. interestingly, these s1 rbd deletion regions have been shown to mediate the s1 rbd/ace2 interaction in recently published literature [10] [11] [12] [13] 26 (table 1) . we tested wild-type and all the s1 deletion mutants for their ability to bind to a tri-specific vhh-fc to check whether the proteins are folded and expressed on the cells. the results show that they are indeed expressed and folded as they all bind to the tri-specific vhh-fc, although the level of expression and/or folding might be different across the mutants based on the strength of the binding signals. the wild-type s1 rbd and the deletion 2 (del2) shows stronger binding, whereas the deletions 1 (del1), 3 (del3), 4 (del4) and 5 (del5) show weaker binding to the tri-specific vhh-fc ( supplementary fig. 2 ). then we assessed the binding profiles of the s1 rbd wild-type and the deletion mutants with selected vhh-fcs from group 1 and group 2, as well as ace2 (fig. 2c,d) . the binding of vhh-fc candidates from both group 1 and group 2, as well as ace2 to s1 rbd are affected following the removal of del1. it is possible that this result is due to a conformational change or decrease of s1 protein expression following its deletion because based on crystal structure of the rbd/ace2 complex (pdb 6m0j), the deleted domain is not part of the s1 rbd/ace2 interface. the del2 mutant, which is adjacent to a computationally-predicted epitope domain in region 1, does not prevent the binding of both group 1 and group 2 vhh-fcs to s1 rbd. in addition, it does not prevent the binding of ace2 to s1 rbd. the del3, 4, and 5 mutants all decreased binding of both group 1 and group 2 vhh-fcs to s1 rbd. however, these regions are more critical for group 1 than (d) epitope binning of vhhs were assessed using an elisa method. briefly, the sars-cov-2 s1 protein was incubated with 1b-2a-fc or 3f-fc, and binding competition was performed with the vhhs followed by the detection of the biotinylation. the experiment was performed in duplicates, and the average percent difference from the competing pairs relative to the vhh-fc alone signal are indicated in the table. the vhh associated percentages highlighted in red are likely high vhh competitors, in light red are partial competitors, and in green are likely non-competitors. (e) the two groups of vhhs categorized based on the binding to epitopes on s1 rbd. figure 2 . elucidation of epitopes on s1 rbd that bind to vhh-fcs. (a) ace2 binding residues on sars-cov-2 s1 rbd were determined by schrodinger bioluminate based on the protein-protein interactions of protein data bank (pdb) 6m0j. (schrödinger release 2020-3: bioluminate, schrödinger, llc, new york, ny, 2020. https ://www.schro dinge r.com/produ cts/biolu minat e. requires permission to be used). the residues in red are predicted ace2 interactors. the deletions generated on sars-cov-2 s1 rbd are shown with the boxed regions. (b) deletion map schematics of the s1 rbd deletion mutants. (c) the binding of vhh-fcs and ace2 to expi293 cells expressing sars-cov-2 s1 wild type (wt) or mutant proteins (del1-del5) were assessed by flow cytometry following fitc-conjugated secondary antibody treatment. an isotype control antibody and facs buffer were used as negative controls. the experiment was performed at least three times which yielded similar trends in results. a representative image of a single experiment is shown here. the graph was generated by the prism (graphpad) software (prism version 8.4.3. https ://www.graph pad.com/scien tific -softw are/prism /. requires permission to be used) (d) in the experiment shown in (c), the binding percentages relative to the s1 wt for each vhh-fc were calculated in the context of each deletion mutant. the group 1 vhh-fcs and the values with binding differences that contributed to their categorization into that group are shown in red. those values for group 2 vhh-fcs are shown in blue. (e) docking models between sars-cov-2 s1 rbd and the lead vhhs generated with schrodinger bioluminate software. (schrödinger release 2020-3: bioluminate, schrödinger, llc, new york, ny, 2020. https ://www.schro dinge r.com/produ cts/biolu minat e. requires permission to be used). scientific reports | (2020) 10:17806 | https://doi.org/10.1038/s41598-020-74761-y www.nature.com/scientificreports/ group 2 for their binding. in addition, these regions are critical for ace2 to bind to s1 rbd. taken together, the binding epitopes for group 1 is more associated with del3, 4 and 5 regions which are located at the interface of s1 rbd/ace2, while at least part of the epitopes for group 2 are shifted farther away from the s1 rbd/ace2 interface relative to the epitopes for group 1 vhhs (fig. 2c,d) . based on the binding and epitope binning data, we constructed 3d docking models that predicted the interactions between sars-cov-2 s1 rbd, ace2 and lead vhh-fcs (fig. 2e) . these models show that predicted binding epitopes for group 1 vhhs 1b and 2a are located at the s1 rbd/ace2 interface. in contrast, the epitope for group 2 vhh 3f is located away from the s1 rbd/ace2 interface (fig. 2e) . interestingly, there are binding variations seen within group 1. the binding of 2a to del1, del3, del4 and del5 have decreased more than that of 1b. this shows that epitopes for 2a and 1b are not the same even though they compete with each other and were initially characterized to be within the same binding group 1 (fig. 2c,d) . taken together, our analysis confirms that there are two major binding groups (group 1 and group 2) and we show the likely binding regions on the sars-cov-2 s1 protein for each vhh. tri-specific vhh-fcs show potent s1 rbd binding and s/ace2 blocking activity. next, we tested whether the combination of individual vhhs binding to different s1 rbd epitopes into bi-specific antibody molecules would yield synergistic effects in sars-cov-2 binding and s/ace2 blocking. as expected, the resulting bi-specific vhh-fc 1b-3f showed superior binding to s1 rbd and s/ace2 blocking compared to individual component vhh-fcs 19 . since sars-cov-2 s proteins formed trimers, we started to study whether trispecific antibodies with two binding units from group 1 and another binding unit from group 2 or vice versa would have better binding and blocking function than the bi-specific antibody [27] [28] [29] . here, we only focused on tri-specific, as any larger multi-specific molecules will likely affect developability with fc fusion proteins. we selected the vhhs from both group 1 and 2 with the most favorable binding, functional and developability features, and constructed tri-specific vhh-fcs with the computer-aided antibody design using the software bioluminate (schrodinger) that enabled their effective construction and optimization [20] [21] [22] . then, we tested the tri-specific, bi-specific and mono-specific vhh-fcs for their ability in-vitro for sars-cov-2 s1 protein binding and s/ace2 blocking (fig. 3a,d) . as expected, the multi-specific antibodies showed higher binding affinities to sars-cov-2 s1 protein rbd in-vitro, with the tri-specific vhh-fcs 3f-1b-2a (kd ~ 0.047 nm) and 1b-3f-2a (kd ~ 0.095 nm) showing more potent binding than bi-specific vhh-fc 1b-3f ( fig. 3a -c,e). the binding affinities for tri-specific vhh-fcs were higher than that of individual component vhh-fcs 1b, 3f and 2a used in combination, and the binding affinity for 1b-3f-fc was higher than that of individual component vhh-fcs 1b, and 3f used in combination (fig. 3a ). in addition, 3f-1b-2a and 1b-3f-2a showed potent blocking of the sars-cov-2 s/ace2 interaction, with ic 50 values of 0.71 nm and 0.74 nm, and full inhibition around 10 nm for both, respectively, that were far superior to using individual component vhh-fcs as combinations (ic 50 of 2.21 nm and full inhibition around 100 nm). in addition, 3f-1b-2a and 1b-3f-2a were more potent than bi-specific vhh-fc 1b-3f in blocking sars-cov-2 s/ace2 interaction (fig. 3d) . interestingly, the tri-specific vhh-fc 2a-1b-3f had lower s/ace2 blocking ability showing the physical arrangement and/or binding orientation of the vhhs in a multi-specific antibody is important for its binding and blocking (fig. 3d) . taken together, this data indicates that the tri-specific vhh-fcs have a higher synergistic potency in both binding and blocking the s1 or s1/ace2 interaction than bi-specific or mono-specific antibodies. tri-specific vhh-fcs have favorable developability features. during the computer-aided design process, we incorporated several development-enhancing features in the structures of our vhh-fcs. therefore, we analyzed the physico-chemical properties, using dls and dsf/sls methods, of our lead bi-and tri-specific antibodies to determine whether they possess favorable characteristics for large-scale manufacturing that is essential for the commercial development of the antibodies (fig. 3e) . our data revealed that the lead tri-specific vhh-fc 3f-1b-2a has lower aggregation potential based on the dls method and is thermostable based on the dsf/sls method (fig. 3e) . we tested the multispecific vhh-fcs for their ability to target sars-cov-2 in cell biological functional assays. first, we analyzed the virus neutralizing ability of our antibodies using a pseudovirus that expresses the sars-cov-2 s1 protein. the tri-specific vhh-fcs 3f-1b-2a, 1b-3f-2a, and the mono-specific combinations of vhhs (1b-fc + 3f-fc + 2a-fc) prevented the infection of human cells by the pseudoviruses (fig. 4a ). in accordance with (fig. 4a) . this pseudovirus data presented here confirm the synergistic effect of the tri-specific antibodies and most importantly, it suggests that it is likely effective in preventing the sars-cov-2 infection. as our vhh-fcs contain the fc domain of human igg1, we expected it would be able to trigger the fcdependent functions to eliminate the viruses from the body. to test this, we used a cell line that transiently expresses the sars-cov-2 s1 protein. then, we assessed the ability of our multi-specific vhh-fcs to promote antibody-dependent cellular cytotoxicity (adcc) that is an fc-dependent function of the antibodies. in addition to our lead tri-specific vhh-fc antibody 3f-1b-2a, we also tested 3a-3f-2a-fc, another tri-specific antibody we constructed with similar s1 binding and s/ace2 blocking potency (supplementary fig. 3 ). as expected, the vhh-fcs were able to induce adcc in the cells (fig. 4b) . this suggests that these vhh-fcs could bind to immune cells through their fc domain and elicit fc-dependent functions, thereby allowing multiple mechanisms of actions against sars-cov-2, including binding sars-cov-s1 and blocking s1/ace2 interactions. docking model for sars-cov-2 s1 rbd with tri-specific vhh-fc 3f-1b-2a was generated by bioluminate (schrödinger release 2020-3: bioluminate, schrödinger, llc, new york, ny, 2020. https ://www.schro dinge r.com/produ cts/biolu minat e. requires permission to be used). in the software, the sars-cov-2 rbd spike protein trimer (pdb 6x2a) was split into three monomeric forms (chain a, b and c). then, 1b/3f/rbd model structure was aligned with chain a of pdb 6x2a to create group 1 and 2a/rbd model structure was aligned with chain b of pdb 6x2a to create group 2. then, group 1, group 2 and chain c were merged to generate the final structure. the s1 rbd/vhh docking structure is represented with a surface structure (a) and ribbon structure (b). the enlarged s1 rbd/vhh docking structure is shown in right (c). in this study we developed and characterized llama-derived multi-specific nanobodies that yielded data that strongly suggests they will be effective against sars-cov-2 that causes covid-19. the covid-19 pandemic has caused widespread health and social issues around the globe, and requires therapeutics that can effectively stop and prevent the infection of sars-cov-2. several monoclonal antibodies against sars-cov-2 have been suggested and being tested as anti-viral therapies, either as individual agents or combination therapies; however, this is the first study that introduces and demonstrates the efficacy of multi-specific antibodies against sars-cov-2 to our knowledge [10] [11] [12] [13] [14] [15] 19, 30 . to successfully design and construct multi-specific vhh binders, the epitope information for each individual vhh clone is necessary. here, instead of obtaining crystal structures for each antigen/antibody complex, we utilized a different method for epitope identification. we initially performed epitope binning with biolayer interferometry and categorized s/ace2 blocking vhhs into 2 groups. the vhhs within each group competed, but there was no competition with vhhs from the other group, strongly suggesting that group 1 and group 2 vhhs are two separate binding groups. then, we computationally constructed vhh models and docked them separately to an s1 rbd structure obtained from a publicly-available crystal structure of sars-cov-2 s1 rbd/ ace2, and utilized docking structures with higher pose cluster size to predict possible epitopes for the individual vhhs. to validate the involvement of predicted epitopes in vhh/s1 rbd binding, we compared the binding ability of each vhh to wild type s1 rbd and five deletion mutants with each predicted epitope deleted. as shown in fig. 2 , both group 1 and group 2 vhhs likely bind to the regions del3, del4, and del5 which overlap with the ace2 binding interface of s1 rbd, however, at least part of the epitope for group 2 is likely located more outwards of this region and has relatively less overlap with the ace2 binding interface of s1 rbd. currently, a number of structures of s1 rbd/antibody complexes have been published. the analysis of these structures show that there are likely 2 main "hot" antibody binding regions in s1 rbd: one likely in the n-terminal region (del1) 12, 13 , and the other likely in the ace2 binding interface (del3, del4, del5) 10, 11, 26 . our selected vhh binders in tri-specific antibodies possibly cover both of these regions (fig. 5 ). based on this information, we were able to define the lead tri-specific vhh-fc format, including the linker length and the order of the vhh binders. the tri-specific antibodies are advantageous as therapeutic agents because they simultaneously bind multiple epitopes within the s1 protein rbd that increase their antigen-binding affinity and avidity (fig. 5) . the vhhs 1b and 3f that comprises the bi-specific antibody bind to two different epitopes in the s1 protein rbd 19 . in our tri-specific antibody design, we incorporated the vhh 2a that has an almost identical epitope as 1b. these vhhs could bind in different orientations to the same or similar epitopes, or to a corresponding epitope in another s1 protein in the trimer, increasing the binding and blocking potency of the tri-specific vhh-fc. in fact, this phenomenon has been previously shown for other multi-specific antibodies. for example, the cd20 targeting t cell engager antibody cd20-tcb (roche) with two cd20 binding domains (2:1 molecular format) has increased potency compared to other cd20-binding bi-specific antibodies in clinical development 31 . in agreement with this hypothesis, the resulting tri-specific vhh-fcs showed very potent characteristics in terms of the s binding and s/ace2 blocking efficacy, which are among the best in currently published anti-sars-cov-2 therapeutic antibodies ( table 1) . because of these characteristics, the tri-specific vhh-fcs could be used at low concentrations for therapeutic applications that would potentially lower their toxicity in humans. in addition, the strong binding of the antibodies to the virions would minimize the risk of antibody-dependent enhancement (ade) that is caused by sub-optimal antigen-antibody interactions and promotes enhanced viral infections 32, 33 . the multi-specific targeting approach also minimizes the loss of antibody binding to viral antigens due to the mutations of the viruses. the rna viruses are known to mutate, and in this sense coronaviruses could lose the binding to antibodies relatively easily due to structural changes in the viral components 34, 35 . however, the vhh multi-specific antibodies would still bind to the mutated virus since the other vhhs in the tri-specific antibody would bind the unmutated epitopes of the virus. another advantage of the vhh multi-specific platform is the ability to target multiple viruses. for example, it is possible to adjoin vhhs that bind to other coronaviruses such as sars-cov and mers-cov, and construct pan-coronavirus tri-specific vhh-fcs that would be effective in preventing and treating a broad spectrum of coronaviruses. our multi-specific antibody design connects human igg1 fc domain to bi-or tri-specific vhhs. having the fc domain in the antibody structure confers fc-dependent cytotoxic functions such as adcc, complementdependent cytotoxicity (cdc) and antibody-dependent cellular phagocytosis (adcp) [36] [37] [38] [39] [40] . these additional fc-dependent functions, in addition to blocking virus entry and possible virus aggregation, would equip the vhh-fcs with multiple mechanisms of action, making them more potent in neutralizing the coronaviruses. indeed, our lead tri-specific vhh-fc 3f-1b-2a show potent neutralization of sars-cov-2 pseudovirus infection in human cells. one of the questions in the field of antibody therapeutics is whether the effect of using multi-specific single molecule is better than using a combination of monoclonal antibodies that collectively target the same epitopes or not 41 . here, we show that multi-specific antibodies are more effective in blocking host-virus interactions than a combination of monoclonal antibodies. our tri-specific vhh-fc 3f-1b-2a was much more potent in blocking the sars-cov-2 s/ace2 interaction than using vhh-fcs 3f, 1b and 2a individually as a combination. it is likely that physically combining the vhhs increases overall association constants (k on values) and decreases overall dissociation constants (k off values), producing lower binding constants, thus increasing antibody affinity towards antigens. it also increases the avidity of the antibodies, making them more effective in neutralizing viruses. one of the hallmarks of a successful therapeutic antibody is its developability features 42, 43 . especially during pandemics such as covid-19 when rapid production of antibodies in high quantities is essential, the developability and manufacturability of the antibodies play even crucial roles. our design has the advantage of using scientific reports | (2020) 10:17806 | https://doi.org/10.1038/s41598-020-74761-y www.nature.com/scientificreports/ llama vhh nanobodies that have high stability 17, 18 . indeed, the biochemical and biophysical characteristics of the multi-specific vhh-fc show that they can be purified in high quantity, have better aggregation resistance, and have favorable thermostability. in addition, our antibodies have high developability because the multi-specific design combines the individual vhhs into single molecules instead of combinations, making their manufacturing easier. an alternative strategy of increasing developability of the anti-sars-cov-2 multi-specific antibodies would be to combine 4 vhhs without the addition of igg fc domain to construct tetra-specific vhhs. these molecules would have the added advantage of increased affinity and avidity towards sars-cov-2 s1 protein compared to bi-and tri-specific vhh-fcs, despite lacking the fc effector functions. these tetra-specific antibodies would be ideally suited as antibody prophylactic to prevent the sars-cov-2 infection in humans because their llama vhh-only structure would have increased thermostability, easier combination capability, and the possibility of easy large-scale manufacturing using cost-effective expression systems such as yeast 17 . one of the key features of our therapeutic antibodies is the use of computer-aided design that greatly reduces their development time and enhances their optimization efficiency. for instance, from the inception of this project, it was possible to produce, optimize and test our lead tri-specific vhh-fcs in less than 3 months. this shows that this strategy is powerful for producing novel therapeutic antibodies for time-sensitive unmet needs, and can be utilized for future outbreaks that would require rapid development of antibody therapeutics. cell lines and transfections. the cell lines used in this study were cultured in media as stated below. epitope binning (competition) assays. the initial assay was performed using gator system (probe life). after pre-wetting the sars-cov-2 s1 rbd sensors in q buffer (probe life), the sensor captured 10-30 µg/ ml of the first monoclonal vhh-fc for about 300 s, then the loaded sensor captured 10 µg/ml of the second monoclonal vhh-fc, either 1b, 3f, or 2a, which was quantified over time by gator. the follow-up assay for vhh-fcs 1b-2a and 3f was performed by elisa. a 96-well plate was coated to a final concentration of 1 µg/ml of sars-cov-2 s1 protein and placed overnight at 4ºc. to test the binding with vhhs 1c, 1f, 3a, 4f and g4, the following method was used. 1b-2a-fc or 3f-fc at 60 µg/ml were premixed with each competing c-myc tagged vhhs from periplasmic supernatant at a 1:1 ratio. after another one hour of incubation, a biotinylated anti-c-myc antibody (9e10) was added and the samples were incubated for another one hour. then, streptavidin-hrp was added followed by the treatment with amplex red (thermo fisher scientific) and 30% h 2 o 2 containing development solution. the emitted signal for each sample was detected by using a fluorescence plate reader (spectramax gemini xps). to test the binding with vhhs 1b-2a-fc and 3f-fc, the following method was used. 1b-2a-fc or 3f-fc at 50 µg/ml were premixed with competing biotinylated 1b-2a-fc or 3f-fc at a 10:1 ratio. after one hour of incubation, the biotin-streptavidin detection system as described above was used to analyze their competition. the percent difference from the competing pairs versus the vhh-fc alone signal was calculated using the following formula; % difference from vhh-fc signal = (1 − ((signal of competing pair − no antibody signal)/(signal of vhh-fc alone − no antibody signal)) × 100. cell binding assay. binding of vhh-fcs to sars-cov-2 s1 expressing cells was assessed by flow cytometry. briefly, cells were harvested and resuspended in pbs with 2% fetal bovine serum (fbs) and plated in v-bottom 96-well plates at 5 × 10 4 cells/well density. they were incubated for 1 h at room temperature with 10 µg/ml of indicated vhh-fcs, control antibodies or recombinant biotinylated human ace2 also dissolved in pbs with 2% fbs. then, the cells were washed twice with the same buffer, and incubated with fitc-conjugated goat anti-human igg (jackson immunoresearch) at 1:200 dilution or pe-conjugated streptavidin (thermo fisher, scientific reports | (2020) 10:17806 | https://doi.org/10.1038/s41598-020-74761-y www.nature.com/scientificreports/ for the detection of biotinylated ace2) at 1:500 dilution for 30 min at room temperature. cells were washed again following the secondary antibody treatment. then they were analyzed by a facscalibur cytometer (bd biosciences). cell populations were visualized as forward vs side scatter and gated to exclude dead cells. cells treated with no antibodies were used to establish background fluorescence. the resulting facs data were analyzed by the software flowjo (bd biosciences) and the graphs were generated by the software prism (graphpad). in-vitro s1 protein binding assay. the 96-well elisa plates (greiner bio-one) were directly coated with sars-cov-2 s1 protein (acro biosystems) diluted in pbs at 1 µg/ml and incubated overnight at 4 °c. then, the plates were washed with pbs containing 0.5% tween20 (pbst) and blocked with 1% bsa in pbs at room temperature for one hour. the plates were washed again with pbst and incubated with the test antibodies at room temperature for one hour. the antibodies were used at 1:5 serial dilutions. the plates were washed with pbst followed by the addition of anti-human-fc antibodies conjugated to horseradish peroxidase (hrp) (jackson immunoresearch) at 1:5000 dilution in pbst and the plates were incubated at room temperature for 1 h. following washing again by pbst, the plates were treated with elisa development buffer solution containing amplex red and 30% h 2 o 2 . the emitted binding signal for each sample was detected by using a fluorescence plate reader. the blocking was measured in relative fluorescence units (rfu) and the % inhibition was calculated as follows; % inhibition = (1 − (mean experimental value/mean of no antibody control)) × 100. s/ace2 blocking assay. the 96-well elisa plates (greiner bio-one) were coated with sars-cov-2 s1 protein (acro biosystems) and incubated overnight as stated previously. then, the plates were washed with pbst and blocked with 2% bsa in pbs at room temperature for one hour. the plates were washed again with pbst and incubated with the test antibodies at room temperature for 45 min. the antibodies were used at 1:5 serial dilutions. then, recombinant biotinylated-ace2 (acro biosystems) was directly added to the plates at 4.65 µg/ µl and incubated at room temperature for another 45 min. the plates were washed with pbst followed by the addition of streptavidin conjugated to hrp at 1:1000 dilution in pbst. the plates were incubated at room temperature for another 45 min. then, they were washed with pbst and treated with elisa development buffer. the emitted binding signal for each sample was detected by using a fluorescence plate reader. analysis of physical characteristics of vhh-fcs. the purified vhh-fcs were analyzed by the uncle system (unchained labs) for their thermostability using differential scanning fluorimetry (dsf) and static light scattering (sls), and aggregation potential using dynamic light scattering (dls) assays. the dls was measured at 25 °c and the data was analyzed using uncle analysis software. for dsf/dls assays, a temperature ramp of 1 °c/min was performed with monitoring from 25 to 95 °c. sls was measured by uncle at 266 nm and 473 nm. tm and tagg were analyzed and calculated by the uncle analysis software. with genscript biotech (piscataway, nj). briefly, pseudovirus expressing luciferase and containing sars-cov-2 s1 as the envelope glycoprotein in a lentiviral vector was produced in hek293t cells, and the virus titration was determined by elisa. hek293 cells expressing ace2 receptor and transmembrane serine protease 2 (tmprss2) were used as the target cells, and were seeded in 96-well plates. then, pseudovirus with the serial dilutions of the antibodies were mixed with the target cells. the cells were incubated for 48 h at 37 °c and an amount of 30 µl of the cell suspension was transferred to an assay plate. it was mixed with luciferase detection reagents from bio-glo™ luciferase assay system (promega) and incubated for 5-10 min at room temperature. then, the luminescence was measured by a plate reader. the background rlu was subtracted from the rlu of the experimental samples. the values for % inhibition were derived from rlu as follows; % inhibition = (1 − (mean of experimental value − mean of cells treated only with buffer)/(mean of cells treated only with sars-cov-2)) × 100. antibody-dependent cellular cytotoxicity (adcc) assay. target expi293 cells expressing s1 protein (293 sprot) were washed with rpmi media containing 10% horse serum and 40 ng/ml il-2, and plated in 96-well plates at 1 × 10 4 cells/well density. then, they were mixed with antibodies at 40 µg/ml of final concentration. then, nk-92-cd16 cells expressing gfp were added to wells at 3 × 10 4 cells/well density (effector: target-3:1) and the plates were incubated overnight 37 °c, 8% co 2 . then, the cells were washed twice and resuspended in dpbs with 2% fbs. they were assessed by flow cytometry using a facscalibur cytometer (bd biosciences). 293 sprot and gfp-nk-92-cd16 cells were each used as a reference to set up overall target cell gating and to establish the gfp positive nk-92-cd16 populations, allowing differentiation between the nk-92-cd16 effector cells and 293 sprot target cells. the gfp negative 293 sprot cell percentage was evaluated for all samples. then, cell death percentage was calculated as follows; % cell death = (1 − (antibody treated cell percentage/average of isotype control percentage)) × 100. statistical analysis. the four-parameter non-linear regression analysis from prism software version 8.4.3 was used for all binding and blocking curves, which also included the ic 50 values for the blocking assays. all error bars represented in the data are based on standard deviation, unless otherwise specified. the data generated and/or analyzed during this study are available from the corresponding author on 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neutralization of virus infectivity by antibodies: old problems in new perspectives nk-mediated antibody-dependent cell-mediated cytotoxicity in solid tumors: biological evidence and clinical perspectives complement in monoclonal antibody therapy of cancer antibody-dependent cellular phagocytosis in antiviral immune responses fc-mediated antibody effector functions during respiratory syncytial virus infection and disease engineering multi-specific antibodies against hiv-1 developability assessment during the selection of novel therapeutic antibodies structure, heterogeneity and developability assessment of therapeutic antibodies we thank all ab studio inc. and ab therapeutics inc. members for their valuable support and input in this project. all authors of this study are employees of ab studio inc. yue liu also serves as the chief executive officer of ab therapeutics inc. supplementary information is available for this paper at https ://doi.org/10.1038/s4159 8-020-74761 -y.correspondence and requests for materials should be addressed to j.d.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. key: cord-254735-8reu45yz authors: reguera, juan; santiago, césar; mudgal, gaurav; ordoño, desiderio; enjuanes, luis; casasnovas, josé m. title: structural bases of coronavirus attachment to host aminopeptidase n and its inhibition by neutralizing antibodies date: 2012-08-02 journal: plos pathog doi: 10.1371/journal.ppat.1002859 sha: doc_id: 254735 cord_uid: 8reu45yz the coronaviruses (covs) are enveloped viruses of animals and humans associated mostly with enteric and respiratory diseases, such as the severe acute respiratory syndrome and 10–20% of all common colds. a subset of covs uses the cell surface aminopeptidase n (apn), a membrane-bound metalloprotease, as a cell entry receptor. in these viruses, the envelope spike glycoprotein (s) mediates the attachment of the virus particles to apn and subsequent cell entry, which can be blocked by neutralizing antibodies. here we describe the crystal structures of the receptor-binding domains (rbds) of two closely related cov strains, transmissible gastroenteritis virus (tgev) and porcine respiratory cov (prcv), in complex with their receptor, porcine apn (papn), or with a neutralizing antibody. the data provide detailed information on the architecture of the dimeric papn ectodomain and its interaction with the cov s. we show that a protruding receptor-binding edge in the s determines virus-binding specificity for recessed glycan-containing surfaces in the membrane-distal region of the papn ectodomain. comparison of the rbds of tgev and prcv to those of other related covs, suggests that the conformation of the s receptor-binding region determines cell entry receptor specificity. moreover, the receptor-binding edge is a major antigenic determinant in the tgev envelope s that is targeted by neutralizing antibodies. our results provide a compelling view on cov cell entry and immune neutralization, and may aid the design of antivirals or cov vaccines. apn is also considered a target for cancer therapy and its structure, reported here, could facilitate the development of anti-cancer drugs. the coronaviridae is a large family of enveloped, plus-rna viruses. they are involved in respiratory, enteric, hepatic and neuronal infectious diseases in animals and humans that lead to important economic losses [1, 2] , as well as to high mortality rates in severe acute respiratory syndrome cov (sars-cov) infections [3] . the covs are a numerous group of coronaviridae. they have been clustered in the coronavirinae subfamily, which includes three approved genera, alpha-, betaand gammacoronavirus, as well as a tentative new genus, the deltacoronavirus [4] . representative cov species in each genus are alphacoronavirus 1 (comprising transmissible gastroenteritis virus (tgev), porcine respiratory cov (prcv) and related canine and feline covs), human coronavirus (hcov-229e and hcov-nl63, genus alphacoronavirus), murine coronavirus (including mouse hepatitis virus (mhv), genus betacoronavirus, cluster a), severe acute respiratory syndrome-related coronavirus (sars-related cov, genus betacoronavirus, cluster b), avian coronavirus (including infectious bronchitis virus (ibv), genus gammacoronavirus), and bulbul-cov (tentative genus deltacoronavirus) [4] . cov particles display characteristic large surface projections or peplomers (17-20 nm) comprised of homotrimers of the spike glycoprotein (s), a type i membrane protein [1, 5] . the peplomers have a globular portion connected by a protein stalk to the transmembrane domain [6] . the globular region is formed by the n-terminal s1 region, whereas the stalk corresponds to the membrane-proximal s2 region, which mediates virus fusion to host cells and adopts a helical structure characteristic of class i virus fusion proteins [7] . determinants of cov tropism locate at the s1 region [1, 8] , which mediates attachment of cov particles to cell surface molecules, initiating virus entry into cells and infection. there is considerable variability in receptor usage among the covs. most alphacoronavirus such as tgev and hcov-229e use apn [9, 10] , whereas the related hcov-nl63 uses a distinct cell entry receptor, the human angiotensin converting enzyme 2 (ace2) [11] ; sars-cov also recognizes the ace2 receptor [12] . sars and nl63 cov bind to common regions of the ace2 protein, although the structures of their receptor-binding domains (rbds) are quite distinct [11, 13] . mhv uses the cell adhesion molecule ceacam1a [14] ; a recent crystal structure showed that the mhv rbd adopts a galectin-like fold [8] . the use of alternative receptors that confer extended tropism has been described for sars-cov, mhv and tgev [1, 8] . the mammalian apns (cd13) are type ii cell surface metalloproteases whose large glycosylated ectodomain has a zinc metal ion at the active site [15] . apn is linked to many cell functions, leading it to be termed the ''moonlighting enzyme'' [15] . animal models confirmed a role for this cell surface enzyme in angiogenesis [16] . peptides and inhibitors that target apn showed a link between this protein and tumor growth and invasion [17, 18] . apn is a target for cancer chemotherapies; drugs that bind this protein have been developed to treat tumors, some of which are in clinical trials [19] . as mentioned above, apn is also a major cov cell entry receptor [1, 9, 10] . cov recognition of apn is species-specific, and specificity is associated with n-linked glycosylations in the apn protein [20] . cell tropism and immune neutralization have been extensively studied in some porcine alphacoronavirus, such as the enteropathogenic tgev and porcine respiratory cov (prcv), a nonenteropathogenic virus derived from tgev [21] . both viruses use porcine apn (papn) for cell entry. the apn-binding domain in tgev, prcv and other alphacoronavirus locates at the c-terminal portion of the s1 region [8, 22, 23] , which bears epitopes recognized by cov-neutralizing antibodies [22, 23, 24, 25] . most tgev-neutralizing antibodies cluster at antigenic site a [25, 26] , comprised within the rbd at the s1 region ( figure 1a ) [22] ; the other antigenic sites defined in the tgev s1 region (b through d) are outside the rbd ( figure 1a ) [21] . to date, there is no structural information available on antibody neutralization and apn recognition by alphacoronavirus. we determined crystal structures of the prcv rbd in complex with the papn ectodomain, and the tgev rbd in complex with the neutralizing monoclonal antibody (mab) 1af10 [25] . the rbd adopts a b-barrel fold, with a distinct protruding tip engaged in papn recognition. the structures show how these porcine alphacoronavirus recognize its cell entry papn receptor and how immune neutralization of these covs is achieved by antibody targeting of receptorbinding residues in the s protein. the mechanisms used by tgev to escape immune neutralization and the evolution of receptor recognition in the cov family are discussed. apn-binding domain and epitopes for neutralizing mabs overlap in tgev and prcv s proteins apn receptor recognition and envelope s antigenicity are well documented in tgev and related prcv. the papn-binding figure 1 . apn-binding domain and epitopes for neutralizing mabs overlap in tgev and prcv s proteins. a. scheme of tgev and prcv s proteins showing the s1, s2, transmembrane (t) and cytoplasmic (cy) regions. location of the c, b, d and a antigenic sites [21] , and the papn rbd (bar with n and c-terminal residues) [22] are shown. length is indicated for mature s1 regions. b. a short, soluble s protein variant containing the tgev rbd region binds to cell surface papn. binding of a bivalent sa-fc fusion protein (sa) to bhk-papn (open histograms), alone (left) or in the presence of the site a-specific 6ac3 mab (right), as analyzed in facs. filled histograms correspond to an unrelated fc fusion protein. c. binding of site a-specific tgevneutralizing mab to the sa protein. mab binding to plastic-bound sa protein, monitored by optical density (od). site a mabs are specific for the aa (1bb1), ab (1de7) and ac (6ac3, 1af10) subsites [24] . an anti-ha mab that binds to the ha tag in the sa protein was used as control. d. site a-specific mabs prevent sa protein binding to papn. binding of the sa protein to bhk-papn cells in panel b was monitored alone or in the presence of site a (shown in c) or site d-specific (1dg3) mab, and the binding ratio determined (see materials and methods). c, d. mean and standard deviation for three experiments. doi:10.1371/journal.ppat.1002859.g001 the cell surface aminopeptidase n (apn), a membranebound metalloprotease target for cancer therapy, is a major cell entry receptor for coronaviruses (covs), agents that cause important respiratory and enteric diseases. in some covs, the virus envelope spike glycoprotein (s) mediates attachment of the virus particles to the host apn protein and cell entry, which is blocked by antibodies that prevent cov infections. the crystal structures of the s proteins of two porcine cov in complex with the pig apn (papn) or with a neutralizing antibody shown here, reveal how some cov bind to its cell surface apn receptor and how antibodies prevent receptor binding and infection. the report uncovers a unique virus-receptor recognition mode that engages a glycan n-linked to the papn ectodomain, revealing structural determinants of the receptor-binding specificity in covs. neutralizing antibodies target viral residues used for binding to the apn receptor and entry into host cells, showing that efficient cov neutralization requires immune responses focused toward key receptor binding motifs in the virus envelope. these structural insights, together with the structure of the apn ectodomain, provide a compelling view of relevant cell membrane processes related to infectious diseases and cancer. domain was mapped within residues 506 to 655 of the mature tgev s polypeptide [22] , whereas tgev mab-resistant (mar) mutants defined four antigenic sites (c, b, d and a) [24, 26] ( figure 1a) . antigenic sites c and b are not present in the prcv s protein. antigenic site a determinants are located within the papn-binding domain at the c-terminal moiety of the tgev and prcv s1 regions ( figure 1a ) [21, 22] . we recently reported the modular dissection of the n-terminal s1 region of tgev and prcv, and the preparation of soluble s1 length variants with single antigenic sites [27] . we produced a recombinant short s protein fragment termed sa, which comprises only residues 481 to 650 of the tgev s protein that binds cell surface papn ( figure 1b ) and displays conformational epitopes for the three antigenic a subsites (aa, ab, and ac) ( figure 1c ). antibodies clustered at the aa (1bb1), ab (1de7) and ac (1af10 and 6ac3) subsites blocked binding of the soluble sa protein to papn ( figure 1d ). the sa protein therefore includes the papn-binding domain of tgev and epitopes for site aneutralizing mab. we applied x-ray crystallography to s protein variants containing the rbd of the related tgev and prcv, and have identified how these alphacoronavirus bind to the cell surface papn and its inhibition by neutralizing antibodies. we attempted crystallization of the soluble papn-binding sa protein derived from the tgev s, alone and in complex with several neutralizing mabs. crystals were prepared with the sa protein in complex with the fab fragment of the 1af10 mab [25] ; the structure of the complex was determined and refined using diffraction data extending to 3.0 å resolution (materials and methods; table 1 ). the asymmetric unit of the crystals contains two antibody-rbd complexes, one of which is shown in figure 2 . residues pro507 to val650 of the tgev s protein, previously identified as the papn-binding domain ( figure 1a ) [22] , were well defined in the crystal structure. they folded in a single domain structure, the rbd of tgev (figure 2a) . the rbd adopts a bbarrel fold formed by two b-sheets with five b-strands each (scheme in figure s1a ). n-and c-terminal ends are on the same side of the domain (terminal side), which presumably lies close to other s protein domains; at the opposite side, two b-turns (b1-b2 and b3-b4) form the tip of the barrel (figure 2a) , where the mab binds to the rbd. the immunoglobulin (ig) variable domains of the mab heavy (v h ) and light (v l ) chains contact the b1-b2, b3-b4 and b5-b6 regions of the tgev rbd ( figure 2b ), burying a virus protein surface of ,810 å 2 . the buried surface of the 1af10 mab is ,750 å 2 , with equal contribution by the v h (51%) and v l (49%) ig domains. complementarity determining regions (cdr) of the antibody heavy (h3) and light (l1 and l3) chains, the n-terminus of the light chain and the c, c9 and c0 b-strands of the v h domain contact the viral rbd tip ( figure 2b ). the cdr-h3 of the 1af10 mab is relatively long, with two-residue insertion (tyr103 h and asp104 h ) relative to other homologous h3 loops in reported mab structures (table s1 ). the rbd b1-b2 hairpin with tyr528 at its tip is at the center of the interacting surface and penetrates between the v l and v h ig domains of the 1af10 mab ( figure 2b and 2c). similar antibodyantigen recognition is described for some peptides and is common for small hapten molecules [28, 29] . the rbd b1-b2 region contributed 73% of the rbd surface buried by the 1af10 mab, and docked between the 1af10 mab variable domains ( figure 2b ). the b-turn is fully buried between the mab ig domains ( figure 2c ), forming a contact network with mab residues ( figure 2d ). the rbd residue tyr528 at the bottom of the pocket contacts mab residues trp47 h and tyr107 h , whereas its hydroxyl group is hydrogen bonded to the side chain of gln89 l and main chain carbonyl of tyr107 h ( figure 2c and 2d). these structural findings on 1af10 recognition of the rbd b1-b2 region correlate with 1af10 mab binding to peptides (mkrsgygqpia533) that include this hairpin region [24] . the rbd b3-b4 and b5-b6 regions are at the periphery of the epitope ( figure 2b ); their contribution to interaction with 1af10 is smaller than that of the b1-b2 region, representing respectively ,17% and 10% of the rbd surface buried by the mab. they contact either the v l or v h ig domains ( figure 2b ). rbd residues leu570 and trp571 at the b3-b4 loop contact the n-terminus, cdr-l1 and cdr-l3 of the v l domain, whereas the b5-b6 loop contacts the long cdr-h3 loop ( figure 2b and 2c). to characterize cov attachment to its apn receptor, we attempted crystallization of the papn ectodomain in complex with tgev and prcv s protein variants comprising their rbds (materials and methods). crystals were obtained only with a mixture of a prcv s protein (s3h) and the papn. using these crystals, we determined the structure of the prcv rbd-papn complex by molecular replacement using previously solved structures of the tgev rbd shown in figure 2 (97% sequence identity) and of the papn ectodomain (materials and methods and table 1 ). the asymmetric unit of the crystals contained two macromolecular rbd-papn complexes ( figure 3a ). the prcv rbd adopts a b-barrel fold like the tgev rbd ( figure s1 ). each papn molecule was engaged by the tip of a single prcv rbd molecule, which bears two exposed aromatic residues (tyr and trp) ( figure 3a , in red), and they bound to a membrane-distal region of the papn ectodomain ( figure 3a ). the rbd n-and cterminal ends and the remaining cov s are also distant from the papn, and are unlikely to contact the receptor molecule. based on a cryo-em structure of the sars-cov s [6] , the rbd must be also at the viral-membrane distal side of the s and therefore, the receptor binding edge must be accessible for cov binding to the apn receptor. the papn is a type ii membrane protein and the n-terminal end of the ectodomain must be near the cell membrane ( figure 3a ). the 25 n-terminal residues of the crystallized papn ectodomain are largely disordered in the structure and they might form a flexible region close to the cell membrane. the papn ectodomain is composed of four domains ( figure 3a ). domain i (orange) is made of b-strands, domain ii (yellow) adopts a thermolysin-like fold bearing a zinc ion at the catalytic site, domain iii (red) is a small b-barrel domain, and the c-terminal domain iv (green) is composed of alpha-helices (domain boundaries are shown in figure s2 ). the papn molecule structure is closely related to that of the human endoplasmic reticulum aminopeptidase-1 [30, 31] (root-mean-square deviation of 2.3 å for 791 residues sharing 33% sequence identity, based on dali server). domain ii bearing the enzyme active site is the most related domain (47% identity), whereas domain iv is the most distinct (22% identity). the zinc ion is coordinated to conserved residues at the papn active site in domain ii ( figure s2 ). the active site conformation is similar to that of other aminopeptidases ( figure s3 ). the papn crystallized in complex with the prcv rbd had an open conformation [30, 31, 32] , in which domain iv was ,20-25 å from domains i and ii; this creates a central cavity in which the zinc ion at the catalytic site is highly accessible ( figure 3a ). the mammalian apns are cell surface metalloproteases that form membrane-bound dimers [33] . the crystallized papn ectodomain also behaved as a dimer in solution ( figure s4 ). the papn dimeric assembly showed in figure 3a buried a large accessible surface (,980 å 2 ) in each monomer. the dimerization surface comprises 29 residues spread across domain iv, which are distinct from those recognized by cov ( figure s2 ). similar dimeric assemblies were observed in two crystal structures determined for the papn ectodomain alone (not shown), crystallized using distinct conditions. the papn molecular assembly shown here might thus be representative of the dimer described for mammalian apn on membrane surfaces [33] . in the crystals of the prcv rbd-papn complex, the rbd tip contacts a membrane-distal region of the papn ectodomain ( figure 3a ). the conformations of the receptor-binding loops (b1-b2 and b3-b4) at the tips of the two prcv b-barrel domains in the structure are identical ( figure s1b ), suggesting very similar rbd-papn interactions in both complexes of the asymmetric unit. the virus-receptor interaction buried ,870 å 2 of the virus protein, 60% of which corresponded to the b1-b2 region ( figure 3b ) and 30% to the b3-b4 turn ( figure 3c ). the size of the papn surface buried by the rbd was similar (,770 å 2 ), and included papn residues ranging from alpha helix 19 (a19) to 22 (a22) in domain iv, and a few domain ii residues ( figure s2 , table s2 ). the end of the papn helix a19 and helix a21 contacted the b1-b2 region of the rbd ( figure 3b ). the tyr side chain (tyr528 in tgev), which protrudes at the b-turn in prcv and tgev rbds ( figure 3b and 3d) , is almost fully buried in the complex, locating between the first n-acetyl glucosamine (nag7361) linked to papn asn736, the end of helix a19, and the first half of helix a21 ( figure 3b ). the hydroxyl group of the rbd tyr528 was hydrogen bonded to side chains of papn residues glu731 and trp737, and contributed to virus-receptor binding specificity. the preceding rbd gly527 residue was at the papn proximal side of the b-turn, hydrogen bonded to the papn asn736 main chain; at the opposite side, the rbd gln530 side chain formed a network of hydrogen bond interactions with papn nag7361 and asn736 side chain ( figure 3b ). the n-acetyl moiety of the glycan also interacted with rbd residues at the b2 and b6 strands ( figure 3b , table s2 ). the papn n-linked glycan and surrounding residues that contact the cov rbd b1-b2 region in the structure were identified as one of the apn determinants of the cov host range [20] . the second relevant virus-receptor interacting region engaged a b-turn at the beginning of the rbd b3-b4 loop ( figure 3c and 3d). the unique rbd trp571 residue, which protrudes at the turn, docked in a papn cavity formed by the coils that precede helices a22 in domain iv and a5 in domain ii ( figure 3c and s2). the bulky side chain of the rbd trp571 residue packed against papn residues his786 and pro787, and its imino group was hydrogen bonded to the main chain carbonyl of asn783 ( figure 3c ). the rbd trp571 as well as the rbd tyr528 at the b-barrel tip in tgev and prcv appear to be central residues in the virus-receptor interaction, as they contact with many papn residues and contribute also to binding specificity by mediating polar interactions with the papn (table s2) . to confirm the contribution of the prcv or tgev rbd bbarrel tip in papn receptor recognition, we analyzed binding of wild type and mutant tgev rbd proteins to cell surfaceexpressed papn ( figure 4a ). mutations in the three regions (b1-b2, b3-b4 and b5-b6) that build the receptor binding edge of the b-barrel decreased rbd binding to papn, whereas mutations outside the receptor-binding region (v617ngly) had no effect on receptor recognition. deletion of the papn asn736 glycosylation site also abolished tgev rbd binding to cell surface-expressed papn ( figure 4b ). deletion of the homologous glycan in feline apn similarly prevents cell infection by feline, canine and porcine covs, all of which share the glycan-binding tyr residue in the b1-b2 turn (see below), whereas addition of this glycan to human apn is sufficient to render it a tgev receptor [20] . we determined the crystal structures of the related tgev and prcv rbds bound to two distinct ligands. the rbds adopt bbarrel structures with small differences in the ligand binding loops (figures s1). in the rbd, each of the two highly twisted b-sheets that build the b-barrel is formed by five b-strands ( figure 5a ). the bent b-strand 5 (b5) crosses both b-sheets and has a b-bulge at asn608 ( figure 5a , magenta). at one side of the b-barrel, all bstrands are antiparallel ( figure 5a, cyan) , whereas on the opposite a dali search of structural homologs showed the greatest similarity (z score of 10) with the rbd of the ace2 receptorbinding hcov-nl63 (root-mean-square deviation of 2.4 å for 103 residues), the other alphacoronavirus rbd whose structure is known [11] . the cores of the tgev and hcov-nl63 b-barrel domains are structurally similar, but the loops at the tips ( figure 5b and 5d). the tip region of the hcov-nl63 rbd is the ace2 receptor-binding edge and has a ''bowl''-shaped conformation ( figure 5c ) that differs from the tgev rbd protruding edge. aromatic residues protrude from the b-turns at the tip of the bbarrel in tgev, whereas they are partially buried at the center of the ''bowl''-shaped edge in hcov-nl63 ( figure 5b and 5c). the distinct rbd tip conformation in ace2-binding hcov-nl63 and in apn-binding tgev might be a determinant of their distinct cell entry receptor specificities. the degree of sequence identity in the rbd region among members in the species alphacoronavirus 1 (,90% identity) suggests a structure closely related to that of tgev, including conforma-tion of the receptor-binding loops (b1-b2 and b3-b4) at the bbarrel tip ( figure 6 ). therefore, tgev, prcv, ccov and fcov must recognize the apn receptor in similar fashion. in contrast, the receptor-binding loops at the tip appear to have a different conformation from tgev in the hcov-229e rbd, which also binds to the apn. in this cov, the b1-b2 region has two cys, as in hcov-nl63, and lacks the apn-binding tyr residue in alphacoronavirus 1, although it preserves the two gly residues found in the tgev b-turn ( figure 6 ). the b3-b4 loop in hcov-229e is markedly shorter than in tgev, but it also has a trp residue. sequence identities between the rbd of tgev and ibv (gammacoronavirus) or the bulbul-cov (tentative deltacoronavirus) are relatively large (,25%), and similarities are found mostly in bstrands and at the rbd c-terminal half ( figure 6) . these data indicate a conserved rbd fold between alphacoronavirus and gammaor deltacoronavirus. there is less sequence similarity between the alphaand betacoronavirus rbd regions (,10%), which correlates with notable structural differences between their detail of the rbd b1-b2 region with the exposed tyr residue interacting with the papn. side chains of rbd and papn residues engaged in the interaction are shown as sticks with carbons in magenta or green, respectively. nag7361 glycan n-linked to papn asn736 is shown with carbons in yellow and the electron density map, determined without the glycan, shown as a blue mesh contoured at 3 sigma. c. detail of the rbd b3-b4 region with the trp residue interacting with the papn. in b and c, rbd residues are numbered following the tgev sequence shown in d, and intermolecular hydrogen bonds are shown as dashed red lines. d. structure-based sequence alignment of the tgev and prcv rbds. b-strands are marked with bars. tgev sequence is numbered. in red, 1af10 mab-(for tgev) and papn receptor-binding residues (for prcv) identified by the structures. residues absent in the rbd structures are in grey, and the thrombin recognition sequences at the end of recombinant porcine cov rbds are in lowercase letters. doi:10.1371/journal.ppat.1002859.g003 rbds [8, 11, 13] . the rbds of the sars and mhv betacoronavirus adopt folds unrelated to the b-barrel shown for alphacoronavirus. the most tgev-neutralizing mabs, including 1af10, recognize antigenic site a in the s protein, divided into the aa, ab and ac subsites [24] . to further characterize site a antigenic determinants in the tgev rbd, we mutated rbd residues targeted by the 1af10 mab ( figure 2 ) and some surrounding residues, and analyzed binding to other site a-specific mabs. the antigenicity of residues in the b1-b2 region, in the center of the epitope for 1af10 ( figure 2c ), was determined by monitoring mab binding to rbd mutants with tgev residue substitutions gly527 (g527d), tyr528 (y528a) and gly529 (g529d) ( figure 7a ). all three substitutions abolished rbd binding by the ac subsite-specific mabs 1af10 and 6ac3. the y528a rbd mutant was recognized by aa-(1bb1) and ab-specific (1de7) mabs ( figure 7a) , and mab 1de7 also bound the g529d mutant. in contrast to the antibody binding profile of the y528a rbd mutant, ala substitution of the tgev trp571 residue (w571a), a papn-binding residue in the b3-b4 loop at the periphery of the rbd epitope for 1af10 ( figure 2c ), did not affect binding by the ac-specific mabs (1af10 and 6ac3), whereas rbd recognition by 1bb1 and 1de7 mabs was greatly reduced ( figure 7a ). deletion of the b3-b4 turn (lwd572a mutant) reduced 6ac3 mab binding to the rbd markedly, with a partial reduction in 1af10 binding ( figure 7a ); this indicates that mab 6ac3 recognizes a broader epitope, which correlates with its higher tgev neutralization activity [25] . replacement with ala of rbd residues thr631 and asn632 at the b5-b6 hairpin, which contacts the 1af10 mab in the rbd-1af10 structure ( figure 2c) , reduced binding by all site a-specific mab ( figure 7a ). this might be a result of a conformational effect induced on the nearby b1-b2 region of the rbd. results for antibody binding to rbd mutants showed that site a epitopes extend across the tgev rbd tip, although there are some differences among the three a subsites ( figure 7b ). the epitopes recognized by aa-and ab-specific mabs bear the exposed tgev trp571 residue at the b3-b4 loop, whereas epitopes for the acspecific mabs center on tyr528 in the b1-b2 turn. none of the mab tested simultaneously targeted the two aromatic side chains (tyr and trp) at the tip of the tgev rbd that bind to the papn. subsite-specific residues defined by mar mutants (lys524 for aa, arg577 for ab and gly529 for ac) might be located at the periphery of their respective epitopes ( figure 7b ). ab and ac subsites appear to be relatively far apart, with the aa epitope in an intermediate position. the rbd tip, shown here as the papn-binding edge of the domain (figure 3) , is the main s protein determinant of antigenic site a, recognized by the most effective neutralizing antibodies of tgev and related cov infections [25, 26] . here we show how a group of covs attaches to the cell surface apn metalloprotease for entry into host cells, and how some covneutralizing antibodies prevent infection. the rbd-receptor complex structures determined for alphacoronavirus indicate that the conformation of the receptor binding edge in the envelope s proteins probably determines their receptor-binding specificity. the cov that bind apn analyzed here have protruding receptorbinding motifs that engage recessed surfaces on the receptor. this mode of receptor recognition is essentially opposite to that reported for cov binding to the ace2 receptor, where recessed receptor-binding motifs in the viral rbd cradle exposed surfaces of the ace2 ectodomain [11, 13] . in the case of papn, an nlinked glycan is also engaged in the virus-receptor interaction. the inherent flexibility of this glycan might facilitate the initial contact of the cov tyr residue with apn amino acids, and subsequent virus-receptor interactions could lock the bound tyr between the glycan and an a-helix ( figure 3b ). the glycan n-linked to asn736 in papn is also conserved in canine and feline apn proteins ( figure s2 ), as are the viral s protein residues that interact with this glycan in the rbd b1-b2 and the b5-b6 regions ( figure 6 ). this unique glycan-virus interaction must thus be conserved among the different covs in the species alphacoronavirus 1, in accordance with the glycan requirement reported for cell infection by ccov, fcov, and tgev/prcv [20] . the lack of this glycan in human apn ( figure s2 ) and the absence of the interacting tyr residue in the b1-b2 region of hcov-229e rbd ( figure 6 ) imply distinct virus-apn local contacts in humans. as shown for the alphacoronavirus 1 group, however, hcov-229e probably has a protruding receptor-binding edge in the envelope s, responsible for its apn-binding specificity. the structure of the rbd-1af10 complex, together with structure-guided rbd mutagenesis and mab binding data, demonstrated that the receptor-binding region is a major antigenic determinant in the envelope s protein of cov that bind apn. potent tgev-neutralizing antibodies, such as the 6ac3 mab [25] , target key apn-binding residues in the s (figure 7) , preventing infection. data from antibody neutralization-resistant tgev mar mutants nonetheless show that some substitutions can be accommodated in the receptor-binding region of alphacoronavirus, which confer the ability to escape immune neutralization, while preserving . substituted residues at the rbd tip that contact papn in the prcv rbd-papn structure are shown in figure 3 , except for the v617ngly mutant, with a glycan at rbd position 617 in the b5-b5b loop, outside the rbd tip (see figure 3d) . b. tgev rbd binding to cell surface papn glycosylation mutants. relative binding of the sa protein and the anti-ha mab to ha-tagged papn proteins with (papn) or without the glycan linked to asn736 (n736a and t738v). mean and standard deviation for three experiments. doi:10.1371/journal.ppat.1002859.g004 the receptor-binding affinity necessary for cell entry [24, 26] . our results thus demonstrate that the receptor-binding region in alphacoronavirus is under selective pressure from the immune system, as described for other viruses [34, 35, 36, 37] . it is tempting to speculate that immune pressure on exposed receptor-binding residues in the cov s could lead to conformational changes in receptor-binding edges of cov rbds. this would result either in changes in the apn-recognition mode observed with hcov-229e and tgev, or in conformational changes in the rbd tip that lead to a receptor specificity switch for cell entry, as observed for hcov-nl63 [11] . virus use of recessed binding regions, as for hcov-nl63, is a well-defined strategy for hiding conserved receptorbinding residues from antibodies [34, 36] . like hcov-nl63, sars-cov uses a recessed, although broader ace2-binding surface, which can accommodate mutations that permit crossspecies receptor recognition [13] . it remains to be understood why, despite major changes in the receptor-binding region, all these cov use metalloproteases as cell entry receptors. in the course of our studies, we also determined the crystal structure of the cell surface apn, an important target for cancer therapies. the domain architecture of apn resembles that of related aminopeptidases [30, 31, 32] . here we show a unique dimer configuration for the apn, mediated by its domain iv, the most divergent domain among m1 aminopeptidases [31] . the implication of these structural findings for apn biology will require further biochemical analysis. knowledge of the structure is leading to research on the mechanism of action of numerous anti-tumor compounds that target mammalian apn [19] ; these studies will be fundamental for improving drug specificity. the detailed view of the apn-cov interaction shown here might also lead to development of small molecules to block cov infection. we have identified the receptor-binding region as the major antigenic site in the alphacoronavirus envelope s, which could guide the design of immunogens that boost cov-neutralizing immune responses to key motifs for virus cell entry. design of soluble s proteins variants of tgev and prcv has been described [27] . the sa protein containing the rbd of tgev was derived from the sc11 strain, and contains residues b-strands are marked (bars) above or beneath their sequences. tgev sequence is numbered. ace2 receptor-binding residues reported for hcov-nl63 [11] , as well as papn receptor-binding residues for tgev (supplementary table s2 ) are colored as in c. residues absent in the rbd structures are in grey, and the thrombin recognition sequence at the end of the tgev rbd is in lowercase letters. doi:10.1371/journal.ppat.1002859.g005 481 to 650 of the tgev s, an n-terminal influenza hemagglutinin ha peptide, and either a flag mab epitope (monovalent sa-flag variant) or the human igg1 fc portion (bivalent sa-fc variant) at the c-terminal end. the engineered soluble papn contains residues 36 to 963 (ectodomain) of the cell surface protein fused to ha and flag tags at the n and c terminus, respectively [27] . the soluble s protein crystallized in complex with the papn was derived from the prcv hol87 strain (s3h in [27] ), and contains the n-terminal 426 residues of the prcv s protein and same c-terminus as the tgev-derived sa protein [27] . a recombinant membrane bound papn with an ha tag at the cterminal end was engineered for cell surface expression. thrombin recognition sequences were introduced between the tags and the viral or papn protein sequences. proteins were produced in transiently transfected 293t or stably transfected cho-lec 3.2.8.1 (cho-lec) cells as described [27] , and concentration in cell supernatants determined by elisa. proteins prepared in cho-lec cells were used in crystallization experiments. hybridoma cells secreting the tgev s mabs were grown in dmem supplemented with 10% fcs in roller bottles. proteins secreted to culture supernatants were initially purified by affinity chromatography. all protein samples were further purified by size exclusion chromatography in hepes-saline buffer (20 mm hepes, 150 mm nacl) ph 7.5. the fab fragment of the 1af10 mab was prepared by papain digestion of the purified antibody. the reaction was terminated by the addition of e64 (sigma) and the fab fragment purified by size exclusion and ion exchange chromatography using hepes-saline buffer ph 8.0. the polypeptide chains of the ig variable domains of the 1af10 mab were determined by sequencing of their cdna prepared from reverse transcribed mrna purified from hybridoma cells. binding of anti-tgev s or -ha (control) mab to wild type and mutant sa proteins was tested in 96-well plates, using purified mab or hybridoma supernatants. the sa-fc fusion proteins in serum-free (opti-mem, invitrogen) cell supernatants were bound to plastic, and mab binding monitored by optical density (od 490 nm ). at least four sa-fc protein concentrations ranging from 10 to 1 mg/ml were used in duplicate and average binding determined in each experiment. binding ratios were determined after correction for background binding. apn binding assays were also carried out with the sa-fc fusion protein comprising the tgev rbd. bhk-papn cells constitutively expressing cell surface papn were used for binding experiments comparing wild type and mutant rbds, whereas transiently transfected 293t cells were used for analysis of rbd binding to papn glycosylation mutants. binding was monitored as the percentage of stained cells with the fc fusion proteins and fitc labeled anti-fc antibodies by fluorescence-activated cell sorting (facs), as shown in figure 1b . the percentage of cells stained was determined for each protein sample and corrected for background staining. papn binding ratios for wild type and mutant rbd proteins shown in figure 4a were determined from the percentage of bhk-papn cells stained with same concentration of wild type and mutant sa-fc proteins. the binding ratios for wild type and mutant papn glycosylation mutants shown in figure 4b were determined from the percentage of sa-fc stained 293t cells expressing similar amounts of ha-tagged papn proteins. cell surface expression of the papn-ha protein was determined with the ha 12ac5 mab. the tgev rbd in complex with the 1af10 fab fragment was crystallized using the size exclusion-purified complex of a table 1 ). crystallization of the papn ectodomain in complex with porcine cov s proteins was carried out with mixtures of the receptor protein and several tgev and prcv protein variants comprising the receptor-binding region (sa, s1h and s3h in [27] ). crystals appeared only in trials performed with an equimolar mixture of papn and the s3h protein derived from the prcv s at a final protein concentration of 13 mg/ml, and with a crystallization solution of 20% peg-4k, 0.2 m lithium sulfate and 0.1 m tris buffer ph 8.5. crystals were transferred to crystallization solution containing 20% ethylene glycol and frozen for diffraction data collection at the id29 beamline (prcv rbd-papn in table 1 ). the structure of the tgev rbd-1af10 fab fragment was initially determined by the molecular replacement (mr) method using the phaser program [38] , and two search models having either the variable or constant regions of the pdb id 1aif mab structure. the 1af10 fab model structure was built manually following electron density maps determined from the mr solution, after improvement with the dm program [39] . the 1af10 fab structure was refined with the program phenix.refine [40] , which provided an excellent electron density map for building residues 507 to 650 of the tgev s, as well as four residues of a thrombin recognition site at the c-terminus. final structure refinement of the complex was carried out with data extending to 3.0 å resolution (statistics in table 1 ). three cycles of solvent correction, refinement of individual coordinates and atomic displacement parameters combined with tls were applied in each step of structure refinement with phenix.refine, which was alternated with manual adjustment of the model to the electron density maps. all residues are in allowed regions of the ramachandran plot. sa protein residues included in the structure of the tgev rbd are shown in figure 3d . the structure of the prcv rbd-papn complex was resolved by the mr method using the papn structure determined alone (manuscript in preparation) and the tgev rbd structure as search models. mr solutions were obtained for the two papn molecules (chains a and b) of the asymmetric unit and for one rbd molecule (chain e). the three molecules were adjusted manually and refined with the phenix.refine program. the second rbd molecule (chain f) bound to papn molecule b was built manually into the electron density map. the 282 residues nterminal to the prcv rbd in the s3h protein were largely disordered or degraded during crystallization, and are absent in the structure. the complex structure was refined with the program phenix.refine applying solvent correction, ncs, refinement of individual coordinates and atomic displacement parameters combined with tls ( table 1 ). the current model comprises residues 60 to 963 of the papn ectodomain with a zinc metal ion at the papn enzyme active site, and residues 283 to 426 of the prcv s, homologous to the tgev s residues 507 to 650 that defined the tgev rbd structure ( figure 3d ). all the residues are in allowed regions of the ramachandran plot. coordinates and structure factors have been deposited in the protein data bank with id codes 4f2m (tgev rbd-1af10) and 4f5c (prcv rbd-papn). buried surfaces and residues at the molecular complex interfaces were determined with the pisa server (http://www. ebi.ac.uk/msd-srv/prot_int/pistart.html). only residues with at least 10% of their surface buried at interfaces in the two independent molecules of the crystal asymmetric units are shown. figure 2d was prepared with ligplot (http://www.ebi.ac.uk/ thornton-srv/software/ligplot/), figure 5a with ribbons [41] and the other structure representations with pymol (pymol.org). structural alignments were carried out with modeller using a gap penalty of 3 [42] . accession numbers of the alphacoronavirus s proteins mentioned are q0pkz5 (tgev), q65984 (ccov), p10033 (fcov), p15423 figure 7 . determinants of tgev s antigenic site a. a. binding of tgev-neutralizing, site a-specific mabs to rbd mutants. relative binding (%) of mutants to wild type sa protein is shown for tgev sspecific mabs (top; described in figure 1c ) and a control anti-ha antibody (see materials and methods). rbd regions in which mutations locate are shown (bottom; see also figure 5d ). mean and standard deviation of data from at least three experiments. b. antigenic site a in the tgev rbd and epitopes for antibodies. surface and ribbon representation of the rbd with the 1af10 contact regions colored as in figure 2b . three antibody-binding residues (tyr528, trp571 and asn632) in the loops at the rbd tip, as well as tgev lys524, arg577 and gly529 residues associated with aa, ab and ac subsites [24] , respectively. lines indicate epitopes for mabs specific for each of the three antigenic subsites: aa in yellow, ab in green and ac in red. doi:10.1371/journal.ppat.1002859.g007 (hcov-229), q6q1s2 (hcov-nl63), b6vdw0 (bulbul-cov) and q9q9p1 (ibv). the prcv hol87 s protein sequence is reported in reference [21] . sequence identities among s proteins were determined with psiblast (http://www.ebi.ac.uk/tools/sss/ psiblast/). accession number for the papn protein is p15145. figure s1 structures of tgev and prcv rbds. a. secondary structure elements of the rbd structures. b-strands are shown with arrows and colored in blue and cyan, a b-bulge at the b-strand 5 is shown in magenta, helix with a red cylinder, coils with black lines, and disulphide bonds with green lines. b. stereo view of the superimposed asymmetric unit rbd structures of tgev (blue and cyan), complex with the 1af10 mab, and of prcv (green and red), complex with the papn protein. view as in figures 2a and 5a . locations of n and c terminal ends are indicated in lowercase letters. (tif) figure s2 mammalian apn ectodomains. sequence alignment of the porcine, canine, feline and human apn proteins with conserved residues highlighted in red. secondary structure elements of the papn structure determined in complex with the rbd of prcv are shown above the sequences. cov-binding residues and those engaged in papn dimerization are highlighted in blue and green, respectively, whereas those at the papn catalytic site are in yellow. residues coordinating the zinc ion are marked with an asterisk, and the n-linked glycosylation site recognized by cov is marked with a triangle at the papn asn736. the beginning of each of the four apn domains is indicated. (tif) figure s3 aminopeptidases active site. side chains of residues at the catalytic site of four structurally aligned zinc aminopeptidases based on domain ii are shown with stick representation, and with the coordinated zinc ion as a cyan sphere. human erap-1 (pdb code 2xdt) is shown in green, aminopeptidase n of e. coli (pdb code 2hpt) in magenta, aminopeptidase n of neisseria meningitidis (pdb code 2gtq) in blue, and papn in yellow. the glutamic acid located in the gamen motif is labeled in blue and those located at the conserved hexxhx 18 e motif are in red (sequence in figure s2 ). (tif) figure s4 dimerization of the papn ectodomain in solution. size exclusion chromatography of the soluble papn ectodomain. continuous line shows optical density (od) at 280 nm for the elution volume. papn protein was run through a superdex 200 16/60 column (ge healthcare) with hepessaline buffer ph 7.5. exclusion volume and size (kda) of molecular weight markers are indicated. determined molecular weight for the single recombinant glycosylated papn ectodomain is about 130 kda, whereas the protein elutes with a volume corresponding to ,300 kda. (tif) table s1 sequence of homologous cdr-h3 loops in known mab structures. sequence of homologous heavy chain cdr-h3 loops to that of the 1af10 mab, identified by a blast search among protein structures, whose pdb codes are shown. (tif) table s2 intermolecular contacts in the prcv rbd-papn complex structure. rbd and papn residues in close contact (#5 å ) in the two complexes of the crystal asymmetric unit, computed with the program ncont [39] . rbd residues from the b1-b2, b3-b4 and b5-b6 regions at the tip of the bbarrel domain are shown, with those engaged in hydrogen bonding in red. tgev/prcv numbering is given for the rbd residues. (tif) the molecular biology of coronaviruses encyclopedia of virology coronaviruses post-sars: update on replication and pathogenesis virus taxonomy: ninth report of the international committee on taxonomy of viruses assembly of coronavirus spike protein into trimers and its role in epitope expression architecture of the sars coronavirus prefusion spike the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor human aminopeptidase n is a receptor for human coronavirus 229e aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev crystal structure of nl63 respiratory coronavirus receptor-binding domain complexed with its human receptor angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus structure of sars coronavirus spike receptor-binding domain complexed with receptor cloning of the mouse hepatitis virus (mhv) receptor: expression in human and hamster cell lines confers susceptibility to mhv the moonlighting enzyme cd13: old and new functions to target impaired angiogenesis in aminopeptidase n-null mice the neovasculature homing motif ngr: more than meets the eye novel aminopeptidase n (apn/cd13) inhibitor 24f can suppress invasion of hepatocellular carcinoma cells as well as angiogenesis aminopeptidase n (cd13) as a target for cancer chemotherapy mutational analysis of aminopeptidase n, a receptor for several group 1 coronaviruses, identifies key determinants of viral host range genetic evolution and tropism of transmissible gastroenteritis coronaviruses major receptor-binding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein identification of a receptor-binding domain of the spike glycoprotein of human coronavirus hcov-229e residues involved in the antigenic sites of transmissible gastroenteritis coronavirus s glycoprotein mechanisms of transmissible gastroenteritis coronavirus neutralization four major antigenic sites of the coronavirus transmissible gastroenteritis virus are located on the amino-terminal half of spike glycoprotein s antigenic modules in the n-terminal s1 region of the transmissible gastroenteritis virus spike protein crystal structures of an antibody to a peptide and its complex with peptide antigen at 2.8 a crystal structures of a quorum-quenching antibody crystal structures of the endoplasmic reticulum aminopeptidase-1 (erap1) reveal the molecular basis for n-terminal peptide trimming structural basis for antigenic peptide precursor processing by the endoplasmic reticulum aminopeptidase erap1 structure of aminopeptidase n from escherichia coli suggests a compartmentalized, gated active site reconstitution of purified amphiphilic pig intestinal microvillus aminopeptidase. mode of membrane insertion and morphology the canyon hypothesis: hiding the host cell receptor attachment site on a viral surface from immune surveillance evolution subverting essentiality: dispensability of the cell attachment arg-gly-asp motif in multiply passaged foot-and-mouth disease virus structural basis of immune evasion at the site of cd4 attachment on hiv-1 gp120 structure of the measles virus hemagglutinin bound to the cd46 receptor pushing the boundaries of molecular replacement with maximum likelihood the ccp4 suite: programs for protein crystallography phenix: a comprehensive python-based system for macromolecular structure solution ribbon models of macromolecules comparative protein modelling by satisfaction of spatial restrains we thank the esrf for provision of synchrotron radiation facilities through bag-madrid projects, as well as the swiss-sls facility, n. cubells for technical help and c. mark for editorial assistance. key: cord-183197-dxmto1tu authors: zhao, tom y.; patankar, neelesh a. title: tetracycline as an inhibitor to the coronavirus sars-cov-2 date: 2020-08-13 journal: nan doi: nan sha: doc_id: 183197 cord_uid: dxmto1tu the coronavirus sars-cov-2 remains an extant threat against public health on a global scale. cell infection begins when the spike protein of sars-cov-2 binds with the cell receptor, angiotensin-converting enzyme 2 (ace2). here, we address the role of tetracycline as an inhibitor for the receptor-binding domain (rbd) of the spike protein. targeted molecular investigation show that tetracycline binds more favorably to the rbd (-9.40 kcal/mol) compared to chloroquine (-6.31 kcal/mol) or doxycycline (-8.08 kcal/mol) and inhibits attachment to ace2 to a greater degree (binding efficiency of 2.98 $frac{text{kcal}}{text{mol}cdot text{nm}^2}$ for tetracycline-rbd, 5.59 $frac{text{kcal}}{text{mol}cdot text{nm}^2}$ for chloroquine-rbd, 5.16 $frac{text{kcal}}{text{mol}cdot text{nm}^2}$ for doxycycline-rbd). stronger tetracycline inhibition is verified with nonequilibrium pmf calculations, for which the tetracycline-rbd complex exhibits the lowest free energy profile along the dissociation pathway from ace2. tetracycline appears to target viral residues that are usually involved in significant hydrogen bonding with ace2; this inhibition of cellular infection complements the anti-inflammatory and cytokine suppressing capability of tetracycline, and may further reduce the duration of icu stays and mechanical ventilation induced by the coronavirus sars-cov-2. the extreme urgency for therapeutics against the acute respiratory syndrome coronavirus 2 (sars-cov-2) drives the review of existing drugs for their ability to inhibit the function of this virus 1;2 . tetracycline has been proposed as a strong candidate against sars-cov-2 3 due to its lipophilic nature, anti-inflammatory response, as well as its ability to chelate zinc species on matrix metalloproteinases (mmps). tetracycline class antibiotics have also been shown to be effective in reducing the duration of ventilatory support and icu stay from acute respiratory distress syndrome 5 , and doxycycline has been suggested to be an important component in combination therapy for its anti-viral properties 6 . tetracycline as well as a broad band of related antibiotics have been approved by the fda 4;7 . in this work, we quantify the performance of 1 to whom correspondence may be addressed. email: tomzhao@u.northwestern.edu ; n-patankar@northwestern.edu tetracycline in inhibiting the binding of the sars-cov 2 spike protein to ace2. tetracycline is found to bind more favorably to the receptor binding domain (rbd) of the spike protein compared to doxycycline or chloroquine, which was included in this study as a baseline. the tetracycline-rbd complex also displays lower binding efficiency to the human cell receptor ace2. the sars-cov 2 rbd, ace2, tetracycline, and chloroquine molecular structures were obtained from rcsb pdb (6m0j, 2uxo, 4v2o, 2xrl) 14;15;16;17 . missing hydrogen atoms were appended, after which structural preparation and molecular docking with full ligand and protein backbone flexibility were carried out using the rosetta suite 18;19;20 . the resulting complexes were inspected manually, after which the binding affinities of the best-scoring complexes were gauged us-ing mm/pbsa calculations after 100 ns equilibrium molecular dynamics simulations 21;22;23 . the potentials of mean force (pmf) 24 along the dissociation pathway of these rbd complexes from ace2 were found in lammps 25 using steered molecular dynamics after parameterization with charmm 26 . jarzynski's equality was employed to calculate the free energy profile for each rbd complex from 10 statistically independent trajectories 27 . tetracycline exhibits higher binding affinity to the rbd in both blind and site-specific docking (-9.40 kcal/mol) compared to doxycycline (-8.08 kcal/mol) or chloroquine (-6.63 vs 6.31 kcal/mol) as delineated in table 1 . the amino acid residues of the rbd involved in hydrogen bonding with the tetracycline molecule are tyr 449, asn 501, gly 496, and tyr 505 (fig. 1) , which have been shown to be crucial for the sars-cov 2 rbd in binding to ace2 for cellular access 8 . these four residues comprise major hot spots that form persistent hydrogen bonds with ace2. meanwhile, the amino acids of rbd that interact with chloroquine in the site-specific configuration are lys 356, arg 454, arg 466 and arg 355, of which none are involved in extended hydrogen bonding with ace2. tetracyline appears to bind preferably to polar or slightly lipophilic rbd residues, which comprise the majority of amino acids that form persistent hydrogen bonds with ace2 8;9 . other tetracycline derivatives as doxycycline or minocycline are known to be more lipophilic 3;4;9 and may therefore prefer nonpolar residues 10 that are often buried beneath the solvent accessible surface area of the spike protein. indeed, the rbd residues that have highest binding affinity to doxycycline are tyr 449, gly 447, val 445, gly 496, of which only two overlap with rbd amino acids that engage in extended hydrogen bonding with ace2. on the other end of the spectrum, chloroquine targets clusters of charged residues on the rbd that do not actively participate in hydrogen bonding with the cell receptor ace2. the binding efficiency 13 (magnitude of binding energy normalized by contact interface area) of the sars-cov2 rbd-ace2 complex was found to be 7.58 kcal/(mol·nm 2 ). in the presence of the protein-ligand complex tetracycline-rbd, the binding efficiency with ace2 (2.98 kcal/(mol·nm 2 )) is significantly lower than that for chloroquine-rbd (5.59 kcal/(mol·nm 2 )) and doxycycline-rbd (5.16 kcal/(mol·nm 2 )) as displayed in table 2 . a survey of hydrogen bonding lifetimes between the important binding site residues in the rbd with ace2 8 shows that the tetracycline inhibited rbd exhibits the least hydrogen bonding activity with ace2 (fig. 2 ). this suggests that not only does tetracycline bind more favorably to the receptor binding domain of the spike protein, it also inhibits the binding of the rbd to ace2 to a greater degree. to verify this statement, steered molecular dynamics simulations were carried out to find the potential of mean force (pmf) along a singular dissociation pathway for the inhibited and uninhibited rbd-ace2 complexes. figure 3 shows that the pmf for unbinding of the tetracycline-rbd complex from ace2 was lowest of the three structures tested, which is in agreement with the binding efficiencies found from equilibrium simulations. this disruption of the rbd-ace2 interface may therefore inhibit the signaling cascade initiated during binding of the viral spike protein. binding efficiency to ace2 (kcal/(mol·nm 2 )) rbd 7.58 chloroquine-rbd 5.59 doxycycline-rbd 5.16 tetracycline-rbd 2.98 table 2 : the binding efficiency 13 (magnitude of binding energy normalized by contact interface area) of the spike protein rbd as well as the tetracycline-rbd, doxycycline-rbd and chloroquine-rbd complexes to the human cell receptor ace2. binding efficiency is lowest for the tetracycline-rbd complex, indicating that tetracycline is a more effective inhibitor. the tetracycline class of antibiotics, including tetracycline, oxytetracycline, and doxycycline may be helpful in the fight against the coronavirus sars-cov-2, due to its preferential association with the important residues in the viral receptor binding domain and the resulting strong inhibition of the rbd-ace2 complex. further experimental studies are recommended to validate how this reduction of cellular infection complements or enhances the anti-inflammatory and anti-viral properties of tetracyclines in their role as treatment for sars-cov-2. author contributions t.y.z conceived and planned the research, as well as performed calculations. n.a.p. and t.y.z. performed analysis and wrote the manuscript. silico identification of potent inhibitors of covid-19 main protease (mpro) and angiotensin converting enzyme 2 (ace2) from natural products: quercetin, hispidulin, and cirsimaritin exhibited better potential inhibition than hydroxy-chloroquine against covid-19 main protease active site and ace2 docking study of chloroquine and hydroxychloroquine interaction with rna binding domain of nucleocapsid phospho-protein -an in silico insight into the comparative efficacy of repurposing antiviral drugs therapeutic potential for tetracyclines in the treatment of covid-19 a proposed randomized, double blind, placebo controlled study evaluating doxycycline for the prevention of covid-19 infection and disease in healthcare workers with ongoing high risk exposure to covid-19 prophylaxis with tetracyclines in ards: potential therapy for covid-19-induced ards? medrxiv doxycycline as a potential partner of covid-19 therapies fda approved antibacterial drugs structural and simulation analysis of hotspot residues interactions of sars-cov 2 with human ace2 receptor heuristic molecular lipophilicity potential (hmlp): lipophilicity and hydrophilicity of amino acid side chains the anti-amyloidogenic action of doxycycline: a molecular dynamics study on the interaction with ab42 screening of chloroquine, hydroxychloroquine and its derivatives for their binding affinity to multiple sars-cov-2 protein drug targets the molecular docking study of potential drug candidates showing anti-covid-19 activity by exploring of therapeutic targets of sars-cov-2 binding efficiency of protein-protein complexes structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor crystal structures of multidrug binding protein ttgr in complex with antibiotics and plant antimicrobials cooperativity and stability of the tetracycline repressor (tetr) upon tetracycline binding rosettaligand docking with full ligand and receptor flexibility rosetta ligand docking with flexible xml protocols rosettaligand: protein-small molecule docking with full sidechain flexibility gromacs: a message-passing parallel molecular dynamics implementation 0: a package for molecular simulation and trajectory analysis. molecular modeling annual a. g_mmpbsa-a gromacs tool for high-throughput mm-pbsa calculations accurate determination of the binding free energy for kcsacharybdotoxin complex from the potential of mean force calculations with restraints. biophysical journal fast parallel algorithms for short-range molecular dynamics charmm: the biomolecular simulation program computing equilibrium free energies using non-equilibrium molecular dynamics the authors have no competing financial interests or other interests that might be perceived to influence the results and/or discussion reported in this paper. key: cord-273893-3nd6ptrg authors: lu, guangwen; hu, yawei; wang, qihui; qi, jianxun; gao, feng; li, yan; zhang, yanfang; zhang, wei; yuan, yuan; bao, jinku; zhang, buchang; shi, yi; yan, jinghua; gao, george f. title: molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 date: 2013-07-07 journal: nature doi: 10.1038/nature12328 sha: doc_id: 273893 cord_uid: 3nd6ptrg the newly emergent middle east respiratory syndrome coronavirus (mers-cov) can cause severe pulmonary disease in humans(1,2), representing the second example of a highly pathogenic coronavirus, the first being sars-cov(3). cd26 (also known as dipeptidyl peptidase 4, dpp4) was recently identified as the cellular receptor for mers-cov(4). the engagement of the mers-cov spike protein with cd26 mediates viral attachment to host cells and virus–cell fusion, thereby initiating infection. here we delineate the molecular basis of this specific interaction by presenting the first crystal structures of both the free receptor binding domain (rbd) of the mers-cov spike protein and its complex with cd26. furthermore, binding between the rbd and cd26 is measured using real-time surface plasmon resonance with a dissociation constant of 16.7 nm. the viral rbd is composed of a core subdomain homologous to that of the sars-cov spike protein, and a unique strand-dominated external receptor binding motif that recognizes blades iv and v of the cd26 β-propeller. the atomic details at the interface between the two binding entities reveal a surprising protein–protein contact mediated mainly by hydrophilic residues. sequence alignment indicates, among betacoronaviruses, a possible structural conservation for the region homologous to the mers-cov rbd core, but a high variation in the external receptor binding motif region for virus-specific pathogenesis such as receptor recognition. supplementary information: the online version of this article (doi:10.1038/nature12328) contains supplementary material, which is available to authorized users. the newly emergent middle east respiratory syndrome coronavirus (mers-cov) can cause severe pulmonary disease in humans 1,2 , representing the second example of a highly pathogenic coronavirus, the first being sars-cov 3 . cd26 (also known as dipeptidyl peptidase 4, dpp4) was recently identified as the cellular receptor for mers-cov 4 . the engagement of the mers-cov spike protein with cd26 mediates viral attachment to host cells and virus-cell fusion, thereby initiating infection. here we delineate the molecular basis of this specific interaction by presenting the first crystal structures of both the free receptor binding domain (rbd) of the mers-cov spike protein and its complex with cd26. furthermore, binding between the rbd and cd26 is measured using real-time surface plasmon resonance with a dissociation constant of 16.7 nm. the viral rbd is composed of a core subdomain homologous to that of the sars-cov spike protein, and a unique strand-dominated external receptor binding motif that recognizes blades iv and v of the cd26 b-propeller. the atomic details at the interface between the two binding entities reveal a surprising protein-protein contact mediated mainly by hydrophilic residues. sequence alignment indicates, among betacoronaviruses, a possible structural conservation for the region homologous to the mers-cov rbd core, but a high variation in the external receptor binding motif region for virus-specific pathogenesis such as receptor recognition. the recent identification of a novel coronavirus, mers-covwhich, as of may 15th 2013, had infected 40 patients with a total of 20 fatalities-has drawn worldwide attention as a potential cause of a future pandemic 5 . unlike most coronaviruses circulating in humans that only cause mild respiratory illness 6 , mers-cov possibly represents a second reported coronavirus of severely high virulence after sars-cov, which caused over 8,000 infection cases globally in 2003, with more than 800 deaths 3 . the clinical manifestations of mers-cov infection include fever, cough, acute respiratory distress syndrome and, in some cases, accompanying renal failure 1,2 , and are very similar to those caused by sars-cov. however, the novel coronavirus diverges from sars-cov in genomic sequence, and is much more closely related to the bat-derived hku4 and hku5 coronaviruses 7, 8 . consistent with phylogenetic analysis, mers-cov does not use the sars-cov receptor, angiotensin converting enzyme 2 (ace2), as its entry receptor 9 ; rather, a recent study showed that it uses human cd26 for this purpose 4 . cd26 is the third peptidase to be identified as a functional coronavirus receptor, the others being aminopeptidase n (anpep, also known as apn and cd13) 10, 11 and ace2 (ref. 12) . the recognition of cd26 by mers-cov is mediated by virus surface spike (s) protein 4 . as with other coronaviruses, the mers-cov s protein would be cleaved in host cells into s1 and s2 subunits (fig. 1a) . s1 engages the receptor 4 whereas s2, with typical sequence motifs homologous to those identified as the heptad repeats in class i enveloped viruses [13] [14] [15] , should mediate membrane fusion. the exploitation of the virus-receptor interaction and thus of the intervention strategies requires an atomic delineation of the receptor-binding properties of s1. on the basis of previous studies, the receptor attachment sites of coronavirus s1 subunits might locate to either the amino-terminal (such as in murine hepatitis virus 16 ) or the carboxy-terminal (such as in, for example, sars-cov 17 and human coronavirus nl63 (ref. 18)) domain. we therefore tested individually the binding of mers-cov s1 and its n-and c-terminal-domain proteins to cell-surface-expressed cd26 molecules. the receptor-binding capacity was attributed to the c-terminal amino acids 367-606 of mers-cov s1 (fig. 1b) . we hereby referred to this domain as rbd. the potent interaction between mers-cov rbd and cd26 was further demonstrated by surface plasmon resonance assays, in which cd26 binds to mers-cov rbd with a dissociation constant (k d ) of about 16.7 nm (k on , 1.793 10 5 m 21 s 21 ; k off , 2.99 3 10 23 s 21 ), but does not bind to the rbd of sars-cov (fig. 1c) . we crystallized mers-cov rbd and solved its structure at a resolution of 2.5 å (supplementary table 1 ). two molecules of essentially the same structure are present in the asymmetric unit. each molecule contains 208 consecutive density-traceable amino acids from v381 to l588. a dali 19 search within the protein data bank (pdb) revealed clear structural homology between mers-cov rbd and sars-cov rbd (pdb code, 2dd8; z score, 15.1). we therefore divided the mers-cov rbd structure into two subdomains: a core and an external b-sheet, using the structure of sars-cov rbd as a reference. the core subdomain reveals a five-stranded antiparallel b-sheet (b1, b3, b4, b5 and b10) in the centre. the connecting helices (four a-helices: a1-4 and two 3 10 -helices: g1 and g2) and two small b-strands (b2 and b11) further decorate the sheet on both sides, together forming a globular fold. three disulphide bonds, connecting c383 to c407, c425 to c478, and c437 to c585, respectively, stabilize the core-domain structure from the interior. at the solvent-exposed side, the rbd termini are clinched adjacent to each other (fig. 2a, b) . this subdomain fold is very similar to that of the sars-cov rbd core (a root mean squared deviation of 2.79 å for 76 ca pairs). superimposition of the two structures reveals a well-aligned centre sheet and homologous peripheral helices and strands, although several intervening loops are observed to exhibit large conformational variance (fig. 2c) . the external subdomain of mers-cov rbd is mainly a b-sheet structure with three large (b6, b8 and b9) and one small (b7) strand arranged in an antiparallel manner. it is anchored to the rbd core through the b5/6, b7/8 and b9/10 intervening loops, which touch the core subdomain like a clamp at both the top and bottom positions. two small 3 10 helices (g3 and g4) and most of the connecting loops in this subdomain locate on the interior side of the sheet, hence exposing a flat exterior sheet-face to the solvent. residues c503 and c526 form the fourth disulphide bond, linking the g3 helix to strand b6 (fig. 2a, b) . with no observable structure homology (fig. 2c) , the external subdomains of mers-cov and sars-cov rbds are topological equivalents, both being present as an 'insertion' between the equivalent core-strands (strands b5 and b10 in mers-cov, and b6 and b9 in sars-cov) (supplementary fig. 1 ). to elucidate the structural basis of the virus-receptor engagement, we further prepared the rbd-cd26 complex by in vitro mixture of the two proteins and then purification on a gel filtration column. consistent with the high binding affinity between mers-cov rbd and cd26, the complex is easily obtainable and stable ( supplementary fig. 2 ). the complex structure was solved at 2.7 å resolution (supplementary table 1 ) with one rbd binding to a single cd26 molecule in the asymmetric unit. the receptor, as shown in previous reports 20,21 , is composed of an eight-bladed b-propeller domain and an a/b hydrolase domain. mers-cov rbd binds to the side-surface of the cd26 bpropeller, recognizing blades iv and v and a small bulged helix in the blade-linker. as for the viral ligand, the entire receptor binding site locates in the external subdomain and to the solvent-exposed sheetface, qualifying the subdomain as the receptor binding motif (rbm) (fig. 3a) . overall, engagement of the receptor does not induce obvious conformational changes in rbm, although small structural variance could be observed for the tip-loops. the g2-a4 loop in the rbd core, however, unexpectedly exhibits a large conformational difference between the free and the bounded structures ( supplementary fig. 3) . we believe this is due to a crystal contact present in the free rbd structure, which is interrupted in the complex crystal by the engaging receptor. cd26 is a type ii transmembrane protein. it is present as a homodimer on the cell surface 20-22 . the dimerization of the peptidase relies on broad intermolecule contacts contributed by the hydrolase domain and the extended strands in blade iv of the b-propeller 20,21 . a lateral binding of mers-cov rbd to cd26 would therefore not disrupt cd26 dimerization. accordingly, a similar u-shaped cd26 dimer could be generated by symmetry operations of the complex structure. the viral ligand locates at the membrane-distal tip of the dimer, corresponding well to a trans interaction between the virus and the receptor (fig. 3b) . considering that the rbd n and c termini are on the same side distant from cd26, it is unlikely that the remaining s domains would contact the receptor molecule. the binding mode revealed by the complex structure is also in good accordance with a previous study showing that the virus-receptor interaction is independent of the peptidase activity of cd26 (ref. 4) . the bound rbd is far away from interfering with either the substrate/product accessing tunnels or the catalytic centre 20,21 (fig. 3b) . overall, a surface area of 1203.4 and 1113.4 å 2 in cd26 and mers-cov rbd, respectively, is buried by complex formation (fig. 4a) . scrutinization of the binding interface reveals a group of hydrophilic residues at the site, forming a polar-contact (h-bond and salt-bridge) network. these interactions are predominantly mediated by the residue side chains (including rbd y499 with cd26 r336, n501 with q286, k502 with t288, d510 with r317, e513 with q344, and d539 with k267), although cd26 l294 and rbd d510 are observed to contact rbd r542 and cd26 y322, respectively, through the mainchain oxygen atom (fig. 4b) . in addition, the bulged helix in cd26 properly positions three hydrophobic residues a291, l294 and i295 into close proximity with the rbd amino acids y540, w553 and v555, forming a hydrophobic centre at the interface (fig. 4c) . further virusreceptor contacts include v341 and i346 of cd26 packing against p515 and the apolar carbon atoms of r511 and e513 in rbd (fig. 4d) , and a cd26 n229-linked carbohydrate moiety interacting with rbd amino acids w535 and e536 (fig. 4e) . overall, the virusreceptor engagement is dominated by the polar contacts mediated by the hydrophilic residues, and mutations of those in rbd (six alanine substitutions and one y499f mutation of the cd26-interacting amino acids) completely abrogated its interaction with cd26 ( supplementary fig. 4 ). the features of these residue interactions are very similar to those mediating the interaction between adenosine deaminase (ada) and cd26 (ref. 23). by a pairwise comparison, we unexpectedly found that all those cd26 residues identified in the virus-receptor interface are also involved in ada binding, indicating a competition between ada and the virus for cd26 receptor. as the ada-cd26 interaction is shown to induce co-stimulatory signals in t cells 22 , this may indicate a possible manipulation of the host immune system by mers-cov through competition for the ada-recognition site. it is also noteworthy that those cd26 residues involved in rbd binding are highly conserved between human and bat, with only two variations (i295t and r317q), explaining the capability of mers-cov using bat cd26 for cell entry 4 ( supplementary fig. 5 ). coronaviruses can be categorized into three main genera or groups (group 1 (alpha), group 2 (beta) and group 3 (gamma) coronaviruses) 24 . both mers-cov and sars-cov belong to the betacoronavirus genus, but are classified into different lineage subgroups (subgroup 2b for dimer observed in the complex crystal. the two-fold axis is shown as an upright arrow. the transmembrane topology of cd26 is indicated with a modelled lipid-bilayer membrane. in cd26, the propeller and side openings indicated as the substrate entrance/exit tunnels are marked with arrows, and the catalytic triad residues are highlighted as spheres. colour selections are the same as in a, and the cd26 a/b hydrolase domain is shown in orange. the n and c termini are labelled. to facilitate comparison, the secondary-structure elements of sars-cov rbd (pdb code, 2dd8) are marked with spiral (helices) and arrow (strands) lines below the sequence. the cysteine residues that form disulphide bonds are labelled as in a, and residue n410 with a star. c, a structural alignment between mers-cov (magenta for core and cyan for external subdomains) and sars-cov (green) rbds. sars-cov and 2c for mers-cov) 8 . we noted that the spike sequences are of low identity among different subgroup members. for example, mers-cov and sars-cov s proteins show a sequence identity of less than 28%. nevertheless, rbds of the two coronaviruses are homologous for the core subdomain. notably, the three interior disulphide bonds in the core are well-aligned for the steric positions in the two rbd structures and well-conserved in sequence among betacoronaviruses. conversely, the external rbm region is highly variable in both length and residue composition ( supplementary fig. 6 ). consistently, no structural homology in this subdomain is observed between mers-cov and sars-cov. yet it is this subdomain that engages cellular receptors. we therefore assume that betacoronaviruses probably have a similar core-domain fold in the s protein to present the external amino acids with divergent structures for viral pathogenesis, such as receptor recognition. our work presents the fifth structure of virus s protein-receptor complexes in the coronaviridae family [16] [17] [18] 25 . taking into account both the rbd structure and the binding mode with receptors, mers-cov is related to sars-cov 17 (a single insertion functioning as rbm) but differs from porcine respiratory coronavirus 25 and nl63 (ref. 18 ) of alphacoronaviruses (multiple discontinuous rbms) ( supplementary fig. 7) . nevertheless, related structural topologies can still be observed in rbds of these coronaviruses 26 . we noted that in the rbd-receptor complex structures of both mers-cov and porcine respiratory coronavirus the binding interfaces involve a receptor n-glycan. this might represent another cross-genus similarity in the coronaviridae family, which supports a proposed common evolutionary origin of coronavirus s proteins 26 . it would therefore be interesting to investigate the contribution of the sugar moiety to the virus-receptor interaction for mers-cov in the future. vaccination remains the most useful measure to combat viral infection and transmission. a large number of antibodies show neutralization activity by targeting the rbd and thereby disrupting the virus-receptor engagement. therefore, a properly folded rbd could be an ideal immunogen for vaccination, as demonstrated for sars-cov 27 . a recent report indeed shows the presence of s-specific neutralizing antibodies in mers-cov-infected patients 28 . it may be worth attempting to test the immunization effect of mers-cov rbd in the future. protein expression, purification, crystallization and structure determination. both his-tagged cd26 and mers-cov rbd proteins were expressed in insect high five cells using the bac-to-bac baculovirus expression system (invitrogen). the recombinant proteins were then purified via nickel-chelated affinity chromatography and gel filtration. crystals were obtained by initial screening with the commercially available kits followed by optimization. the rbd structure was solved by single-wavelength anomalous diffraction and the complex structure by molecular replacement. full methods and any associated references are available in the online version of the paper. supplementary information is available in the online version of the paper. isolation of a novel coronavirus from a man with pneumonia in saudi arabia severe respiratory illness caused by a novel coronavirus world health organization. cumulative number of reported probable cases of severe acute respiratory syndrome (sars) dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc world health organization. novel coronavirus infection -update coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. microbiol genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans sars-like virus in the middle east: a truly bat-related coronavirus causing human diseases human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines human aminopeptidase n is a receptor for human coronavirus 229e aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus combating the threat of pandemic influenza: drug discovery approaches coiled coils in both intracellular vesicle and viral membrane fusion following the rule: formation of the 6-helix bundle of the fusion core from severe acute respiratory syndrome coronavirus spike protein and identification of potent peptide inhibitors crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor structure of sars coronavirus spike receptorbinding domain complexed with receptor crystal structure of nl63 respiratory coronavirus receptor-binding domain complexed with its human receptor dali server: conservation mapping in 3d crystal structure of the swine-origin a (h1n1)-2009 influenza a virus hemagglutinin (ha) reveals similar antigenicity to that of the 1918 pandemic virus processing of x-ray diffraction data collected in oscillation mode collaborative computing project number 4. the ccp4 suite: programs for protein crystallography advances in direct methods for protein crystallography pushing the boundaries of molecular replacement with maximum likelihood density modification for macromolecular phase improvement phenix: a comprehensive python-based system for macromolecular structure solution coot: model-building tools for molecular graphics procheck: a program to check the stereochemical quality of protein structures espript: analysis of multiple sequence alignments in postscript acknowledgements this work was supported by the ministry of science and technology of china (most) 973 project (grant no. 2011cb504703) and the national natural science foundation of china (nsfc, grant no. 81290342). assistance by the staff at the shanghai synchrotron radiation facility (ssrf) of china and the high energy accelerator research organization (kek) of japan is acknowledged. we thank z. fan and t. zhao for their technical assistance. g.f.g. is a leading principal investigator of the nsfc innovative research group (grant no. 81021003). we thank m. yang from tsinghua university for his help with data collection. author contributions g.f.g. designed and coordinated the study. g.l., y.h., q.w. and y.s. conducted the experiments. j.q. and f.g. collected the data sets and solved the structures. y.l., y.z., w.z., y.y. and j.y. assisted with the cell maintenance and protein preparations. g.l. and g.f.g. wrote the manuscript and j.y., j.b. and b.z. participated in the manuscript editing and discussion.author information the coordinates and related structure factors have been deposited into the protein data bank pdb under accession numbers 4kqz for the free mers-cov rbd structure and 4kr0 for the rbd-cd26 complex structure. reprints and permissions information is available at www.nature.com/reprints. the authors declare no competing financial interests. readers are welcome to comment on the online version of the paper. correspondence and requests for materials should be addressed to g.f.g. (gaof@im.ac.cn). protein expression and purification. the proteins used for crystallization and surface plasmon resonance experiments were prepared with the bac-to-bac baculovirus expression system (invitrogen). the coding sequences for mers-cov rbd (genbank accession number jx869059, spike residues 367-606), sars-cov rbd (accession number nc_004718, spike residues 306-527), human cd26 (accession number np_001926, residues 39-766) and human ace2 (accession number baj21180, residues 19-615) were individually cloned into the pfastbac1 vector. for each construct, a previously described gp67 signal peptide sequence 29 was added to the protein n terminus for protein secretion, and a hexa-his tag was added to the c terminus to facilitate further purification processes. transfection and virus amplification were conducted with sf9 cells, and the recombinant proteins were produced in high five cells. the cell culture was collected 48 h after infection and passed through a 5-ml histrap hp column (ge healthcare). after removal of most of the impurities, the recovered proteins were then pooled and further purified on a superdex 200 column (ge healthcare). finally, each collected protein was prepared in a buffer consisting of 20 mm tris-hcl (ph 8.0) and 150 mm nacl and concentrated to about 10 mg ml 21 for further use.to obtain the complex of mers-cov rbd bound to cd26, the individual proteins were in vitro mixed at a molar ratio of 1:1 and incubated at 4 uc for about 2 h. the complex was then further purified on a superdex 200 column, and concentrated to about 15 mg ml 21 for crystallization experiments.to prepare the fc chimaeric proteins, the fragment encoding mers-cov s1 (residues 1-751) or ntd (residues 1-353) or rbd (adding the s residues 1-17 of the signal peptide to its n terminus to facilitate protein secretion) was fused 59terminally to a fragment coding for the fc domain of mouse igg and ligated into the pcaggs expression vector. a mutant rbd-fc protein-expressing plasmid was also constructed by site-directed mutagenesis, for which the identified hydrophilic residues involved in cd26 binding were mutated simultaneously (y499f; n501a, k502a, d510a, e513a, d539a and r542a). the expression plasmids were then transfected into hek293t cells. the cell culture was collected 48 h after transfection and directly used in the flow cytometric assay. analytical gel filtration. mers-cov rbd, cd26 and their protein complex were individually prepared and adjusted to the same volume. the samples were then loaded onto a calibrated superdex 200 column (ge healthcare). the chromatographs were recorded and overlaid onto each other. the pooled proteins were analysed on a 12% sds-page gel and stained with coomassie blue. surface plasmon resonance assay. the biacore experiments were carried out at room temperature (25 uc) using a biacore 3000 machine with cm5 chips (ge healthcare). for all the measurements, an hbs-ep buffer consisting of 10 mm hepes, ph 7.5, 150 mm nacl, 3 mm edta and 0.005% (v/v) tween-20 was used, and all proteins were exchanged to the same buffer in advance via gel filtration. the mers-cov rbd and sars-cov rbd proteins were immobilized on the chip at about 500 response units. gradient concentrations of human cd26 (0, 5, 10, 20, 40, 80, 160, 320, 640 and 1,280 nm) or human ace2 (0, 10, 20, 40, 80, 160, 320, 640 and 1,280 nm) were then used to flow over the chip surface. after each cycle, the sensor surface was regenerated via a short treatment using 10 mm naoh. the binding kinetics were analysed with the software biaevaluation version 4.1 using the 1:1 langmuir binding model. flow cytometric assay. for the surface expression of cd26, the full-length coding sequence was cloned into the pegfp-c1 vector which yields a plasmid encoding a recombinant cd26 protein with an egfp-tag fused to its n terminus. the plasmid was transfected into the cd26-negative bhk cells using lipo2000 (invitrogen) according to the manufacturer's instructions. the cells were collected 48 h after transfection.for staining, the mock-transfected bhk cells or the cells transfected with the cd26-expressing plasmid were suspended in pbs and incubated with the individual fc-fusion protein culture or goat anti-cd26 igg (r&d systems) at room temperature for 1 h. the cells were then washed and further incubated at room temperature for about 0.5 h with anti-mouse or anti-goat secondary igg antibodies (r&d systems). after washing, the cells were analysed by flow cytometry with a bd facscalibur machine. the cells incubated only with the secondary antibodies were used as the negative controls. crystallization. all the crystals were obtained by vapour-diffusion sitting-drop method with 1 ml protein mixing with 1 ml reservoir solution and then equilibrating against 100 ml reservoir solution at 18 uc. the initial crystallization screenings were carried out using the commercially available kits. the conditions that yield crystals were then optimized. diffractable crystals of the free rbd protein were finally obtained in a condition consisting of 0.1 m ammonium tartrate dibasic, ph 7.0, and 12% peg 3,350 with a protein concentration of 10 mg ml 21 . derivative crystals were obtained by soaking rbd crystals for 24 h in mother liquor containing 2 mm kaucl 4 n2h 2 o. the complex crystals were grown in 6% (v/v) 2-propanol, 0.1 m sodium acetate ph 4.5 and 26% peg550 with a protein concentration of 15 mg ml 21 . data collection, integration and structure determination. for data collection, all crystals were flash-cooled in liquid nitrogen after a brief soaking in reservoir solution with the addition of 20% (v/v) glycerol. the native rbd data set was collected at the high energy accelerator research organization (kek) bl1a (wavelength, 1.03818 å ), whereas the diffraction data for the au derivative crystal (wavelength, 1.0382 å ) and the complex crystal (wavelength, 0.97930 å ) were collected at the shanghai synchrotron radiation facility (ssrf) bl17u. all data were processed with hkl2000 (ref. 30) . additional processing was performed with programs from the ccp4 suite 31 .the structure of rbd was determined by the single-wavelength anomalous diffraction (sad) method. the au sites were first located by shelxd 32 for the au-sad data. the identified position were then refined and the phases were calculated with sad experimental phasing module of phaser 33 . the real space constraints were further applied to the electron density map in dm 34 . the initial model was built with autobuild in phenix package 35 . additional missing residues were added manually in coot 36 . the final model was refined with phenix.refine in the phenix 35 with energy minimization, isotropic adp refinement, and bulk solvent modelling. the complex structure was solved by molecular replacement module of phaser 33 , with the solved rbd structure and previously reported cd26 structure (pdb code, 2bgr) as the search models. the atomic model was completed with coot 36 and refined with phenix.refine 35 . the stereochemical qualities of the final models were assessed with procheck 37 . the ramachandran plot distributions for the residues in the free rbd structure were 86.8, 11.8 and 1.4% for the most favoured, additionally and generously allowed regions, respectively. these values were 86.5, 13.1 and 0.5% for the rbd-cd26 complex structure. data collection and refinement statistics are summarized in supplementary table 1 . all structural figures were generated using pymol (http://www.pymol.org). secondary-structure determination. the secondary structure determination was based on the espript 38 algorithm. key: cord-262043-66qle52a authors: basit, abdul; ali, tanveer; rehman, shafiq ur title: truncated human angiotensin converting enzyme 2; a potential inhibitor of sars-cov-2 spike glycoprotein and potent covid-19 therapeutic agent date: 2020-05-20 journal: j biomol struct dyn doi: 10.1080/07391102.2020.1768150 sha: doc_id: 262043 cord_uid: 66qle52a the current pandemic of covid-19 caused by sars-cov-2 is continued to spread globally and no potential drug or vaccine against it is available. spike (s) glycoprotein is the structural protein of sars-cov-2 located on the envelope surface, involve in interaction with angiotensin converting enzyme 2 (ace2), a cell surface receptor, followed by entry into the host cell. thereby, blocking the s glycoprotein through potential inhibitor may interfere its interaction with ace2 and impede its entry into the host cell. here, we present a truncated version of human ace2 (tace2), comprising the n terminus region of the intact ace2 from amino acid position 21-119, involved in binding with receptor binding domain (rbd) of sars-cov-2. we analyzed the in-silico potential of tace2 to compete with intact ace2 for binding with rbd. the protein-protein docking and molecular dynamic simulation showed that tace2 has higher binding affinity for rbd and form more stabilized complex with rbd than the intact ace2. furthermore, prediction of tace2 soluble expression in e. coli makes it a suitable candidate to be targeted for covid-19 therapeutics. this is the first md simulation based findings to provide a high affinity protein inhibitor for sars-cov-2 s glycoprotein, an important target for drug designing against this unprecedented challenge. communicated by ramaswamy h. sarma the rapid spread of sars coronavirus 2 (sars-cov-2) demands an immediate public health emergency, and no fda approved treatment/vaccines are currently available. sars-cov-2 spike (s) protein (1267 amino acids) is essential for virus entry through binding with the host receptor angiotensin converting enzyme ii (ace2) and mediating virus-host membrane fusion (boopathi et al., 2020; sarma et al., 2020) . the s protein contains two functional domains (s1 and s2). the s1 (residues 14-685) domain performs the function of virion attachment with human ace2 receptor on epithelial membrane cell surface, followed by its internalization, hence initiating the infection (hasan et al., 2020) . this binding induces certain conformational changes in the s protein, which results the s2 (residue 686-1273) to mediate fusion with cellular membrane. the receptor-binding domain (rbd) of the sars-cov-2 s protein are highly conserved and directly involve in binding to human ace2 (yuan et al., 2020) . since, ace2 is not mutated/evolved to recognize s protein of sars-cov-2; therefore, using alternative of ace2 with more binding affinity for s protein than the wild type receptor, may inhibit entry of sars-cov-1& à2 into human cells. this strategy can play important role in devising therapeutics of sars-cov-2. several studies have proposed small compounds based inhibitors as therapeutic agents for covid-19 (aanouz et al., 2020; elmezayen et al., 2020; gupta et al., 2020; khan et al., 2020; wahedi et al., 2020) . the small compounds based drugs may not efficiently block the entire binding patch of s protein. on the other hand, the peptides based therapeutics can block the entire binding interface (rbd) of s protein (wan et al., 2020b) , as reported for hiv peptide based drug fuzeona (jenny-avital, 2003; w ojcik & berlicki, 2016) . there is growing interest in peptide based therapeutics for covid-19 treatment (pant et al., 2020) and approximately 140 peptide based drugs have been evaluated in clinical trials (fosgerau & hoffmann, 2015) . peptide based drugs have little side effects and little drug tolerance compared with chemical drugs (bruno et al., 2013) . in order to block the fusion of sars-cov-2 s protein with human cells, a recent study has reported a neck and transmembrane deficient ace2, called as soluble ace2 (sace2), that can block the entry of sars-cov-2 into the host cell (procko, 2020) , which is also found safe in healthy human subjects (haschke et al., 2013) and patients with lung disease (khan et al., 2017) . recombinant sace2 is under clinical trials for covid-19 treatment in guangdong province of china (clinicaltrials.gov #nct04287686). the study proposed that mutations in ace2 receptors interface may increase s/ace2 interaction. another study has proposed a 23 amino acid peptide, derived from ace2 (amino acid position 21-43), which can bind with sars-cov-2 s protein with a low nanomolar affinity, and can block the attachment of sars-cov-2 to human ace2 . since, the binding residues of ace2 involve in interaction with rbd are located at amino acid position 21-119 (wan et al., 2020a; yan et al., 2020) , therefore, we hypothesized that this fragment carrying all the binding residues will have better binding affinity for rbd and can hinder the interaction of sars-cov-2 with human ace2, hence blocking its entry into the epithelial cells. we designed a truncated version (tace2) of ace2 receptor covering the binding residues and performed protein-protein docking and molecular dynamic simulations to analyze its binding affinity for rbd and complex stability. the tace2 will compete with wild type human ace2 receptors for binding to sars-cov-2, as they will have more binding affinity for s protein. this will allow all sars-cov-2 viral particles to bind strongly with the tace2, blocking all its available binding sites for the host ace2 receptors, thus inhibiting its entry into the cell which will be eliminated through body defense mechanisms. we further determined the soluble expression for tace2 in e. coli, a suitable host for bulk production of tace2. the pdb structure of ace2 and rbd of sars-cov-2 s glycoprotein (pdb id: 6m17) was obtained from pdb database. in order to determine the variation in the sars-cov-2 s glycoprotein sequence reported from different regions of the globe, 61 s glycoprotein sequences of sars-cov-2 including reference sequence (nc_045512, reported from wuhan, china) were retrieved from ncbi. multiple sequence alignment of the sequences was performed through mega-x. the aligned sequences were then analyzed for amino acid variations. the pdb structures of ace2 and rbd were repaired for their missing loops and optimized for energy minimization and amino acid side chain clashes through foldx (schymkowitz et al., 2005) . side chains were optimized through foldx to remove vander waals' clashes by mutating residues with bad energy values into new rotamers with energy minimization (van durme et al., 2011) . the optimized three dimensional (3 d) structures of ace2 and rbd were used to design truncated ace2 and studying protein-protein interactions. based on protein-protein interactions between ace2 and rbd shown in ace2-rbd complex (pdb id 6m17), a truncated version of ace2 was produced by removing the c-terminus residues from amino acid position 116-768, leaving a truncated n-terminus fragment tace2, from 21-119 amino acid position. the first 20 residues of ace2 is the signal peptide (huang et al., 2003; turner & hooper, 2004) , therefore it was also removed. the structure of tace2 was produced through i-tasser, which build the model by assembling continues fragments of multiple threading templates, identified through replica exchange monte carlo (remc) simulations (yang et al., 2015) . in order to determine binding affinity of both intact and truncated ace2 with sars-cov-2 s glycoprotein, rigid body protein-protein docking tools; zdock (pierce et al., 2014) , cluspro (kozakov et al., 2017) , patchdock (schneidman-duhovny et al., 2005) and a flexible protein-protein docking tool, haddock (van zundert et al., 2016) were used. the energy function used by zdock is z score, which is a cumulative of pairwise shape complementarity function with desolvation and electrostatics. the zdock rank the top 10 predicted docking poses on the basis of z score (chen et al., 2003) . cluspro uses piper's scoring function, which contains terms of shape complementarity, electrostatics, and pairwise potentials applied on the top 1000 conformations produced and ranked on the basis of cluster size. patchdock uses patchdock score as the energy function which ranked the docked model based on desolvation energy, interface area size and geometric score (zhang et al., 1997) . haddock is a flexible docking method used for docking of protein-protein complexes. haddock drive the docking process by retrieving information from experimentally identified protein complex interfaces. the haddock scoring function consists on combination of various energies and buried surface area. the scoring of the models was performed according to the haddock score. all the generated docking poses of ace2 and spike protein were visualized through pymol (schrodinger, 2010) . based on the haddock score and the docking rmsd value, the docked complexes of ace2 and tace2 with rbd were analyzed for binding affinity dg (kcal mol à1 ) and stability using protein binding energy prediction (prodigy) server (xue et al., 2016) . the server predicts the binding affinity and stability on the basis of structural properties of the proteinprotein complexes. stability of the protein-protein complex is measured through dissociation constant k d (m). the run was performed at different temperatures ranging from 25 to 36˚c. the protein-protein docked complex with the minimum rmsd and higher binding affinity was considered for md simulation to further confirm stability of the complex. md simulation of the rbd domain in complex with intact ace2 and tace2 was performed through gromacs 5.0.4 (abraham et al., 2015) . simulation was performed by using charm 36.0 force field and tip3p cube box as water model. the protein complex in the cubic box was solvated with water molecules to provide an aqueous environment. the system was then neutralized with addition of 3 na ions followed by energy minimization for removal of conflict between the atoms. the system was then equilibrated through nvt and npt at constant temperature (300 k) and pressure (1 bar), respectively. langevin thermostat was applied to regulate temperature of the system. md simulation was then run for 20 ns. in order to determine post translation modifications (ptms) in ace2, the protein sequence was submitted to ptm-ssmp server, which combines the submitted sequence and site specific modification profile to predict ptm sites in mammalian protein (liu et al., 2018) . since, glycosylation is the most abundant and diverse posttranslational modification of proteins, therefore, we further determined the o-glycosylation sites in ace2 using netoglyc 4.0 server which specifically predict the galnac-type o-glycosylation site, unique to ser and thr (steentoft et al., 2013) . we further determined the n-glycosylation sites by using netnglyc-1.0 server using a threshold value of 0.5 (gupta et al., 2004) . in order to express the tace2 in e. coli, its soluble expressionat 37 c was determined through camsol intrinsic and camsol structurally corrected online solubility prediction tools (sormanni & vendruscolo, 2019) . camsol determines the solubility on the basis of amino acid sequence, while camsol structurally corrected tool determines the solubility profile on the basis of the structure, which accounts for amino acid distribution in the structure and their solvent exposure. both run was performed at ph 7.0. in both methods, the solubility profile scores higher than 1.0 denotes highly soluble regions, while scores lower than à1 indicates poor solubility in e. coli. in the current study, we have proposed a truncated version of ace2 that comprises the binding interface for receptor binding domain (rbd) of sars-cov-2 spike protein. recently, in-vitro binding assay have confirmed that rbd is mainly responsible for initial binding to ace2, which further mediate virus entry into the host cell (lan et al., 2020) . variation in the rbd sequence was analyzed in the sars-cov-2 genome reported from various region of the globe so far (shu et al., 2020) . the sequence alignment showed more than 99.99% homology for rbd domain, with only single variation r408i in the sars-cov-2 genome reported from india ( figure s1 ). the rest of the sars-cov-2 genome sequences submitted throughout the globe have identical rbd sequence, which indicate that the sars-cov-2 rbd is highly conserved globally. structural elucidation has also found the rbd domain as highly conserved (lan et al., 2020). in order to block the spike protein attachment to the cell, the ace2/rbd binding interface comprising residues from position 21-119 of ace2 was selected as truncated version of ace2.the structure of tace2 was built through i-taseer with c-score 1.22. the c-score value in range à5 to 2 shows correctness of the fold. the high c-score for tace2 suggest the highly likelihood of the structure. the tace2 fragment contains almost all binding residues involve in binding with rbd domain of sars-cov-2 (yan et al., 2020) , covering two complete helices (lan et al., 2020) . this suggests that rational design of a binder based on this interface with enhanced affinities to rbd may play vital role by blocking the sars-cov-2 spike protein interaction with ace2, thus inhibiting viral entry into the host cell. previously, peptides based strategies have been employed successfully to inhibit fusion of the sars-cov-1 s protein and membrane receptor (du et al., 2009) . another recent study has reported a 23 amino acid based peptide, a homologue of ace2 binding interface, which successfully bind with s protein with low nanomolar affinity . since, the binding residues for ace2 are located at distant location on rbd, thus providing a larger protein binding site, which is difficult for a small size therapeutic peptide to cover the entire binding sites on rbd. however, our proposed tace2 fragment carrying almost all the binding residues that can block the attachment of rbd with the intact ace2. rbd was docked with intact and truncated ace2 through haddock, a flexible protein-protein docking tool. the method allows the side-chains and backbone atoms of both the protein and receptor flexible during docking run . haddock scoring function (haddock score) is a linear combination of non-bonded intermolecular van der waals (vws), coulomb electrostatics energies and empirically derived desolvation energy term (vangone et al., 2017) .the haddock-score of ace2 and tace2 was à111 and à126.6, respectively, (the more negative the better). similarly the vws and electrostatic energy of tace2-rbd complex was also greater than the ace2-rbd complex, which shows higher binding affinity of tace2 for rbd than the intact ace2 (table 1 ). the rmsd value of ace2 and tace2 in complex with rbd were 0.7 and 0.8, respectively, showing the high likelihood of the docked complexes with native-one (vangone et al., 2017) . in order to further confirm these docking scores, rigid docking was also performed through patchdock, z-dock and cluspro protein-protein docking tools. the docking results obtained for ace2 was compared with tace2 in term of energy functions of each docking tool (table 2 ). all the three docking scores are higher for tace2 than that of the intact ace2, indicating high affinity of tace2 for rbd. our docking results showed that seven residues of ace2 glu23, thr27, asp30, glu35, tyr 83, asn 330 and lys 353 of ace2 interact with rbd residues lys417, lys458, asn487, tyr 489, gln493, tyr495, gly496, thr500 and gly502, respectively, which is almost similar to the binding residues profile of ace2 interface reported previously (yan et al., 2020) , with additional thr27 and glu35 reported by our docking results (figure 1(a, b) ). however, the tace2 form a different binding residues network than the intact ace2. our docking results showed that ser23, asn31, tyr30, glu36, gln40, gln76 and arg95 of tace2 are involved in binding with rbd ( figure 1(c, d) ). this seems that the truncation has produced the conformational changes in the tace2-rbd complex which results in exposure of buried binding residues , thus facilitate higher binding of tace2 to the rbd as compared to the native ace2, which are in agreement with previously reported peptides inhibiting viral attachment with the host cell (koehler et al., 2013) . since, docking methods are not reliable for predicting binding affinity between protein-protein complexes, due to their simple scoring functions (ram ırez & caballero, 2016). as binding affinity of protein-protein complex also depends on dissociation constants (k d ), ph and temperature (kastritis & bonvin, 2010) , while these parameters are not included in the benchmark of docking scoring functions. therefore, we determined the binding affinity of ace2 variants for rbd through prodigy server, which determine the binding affinity based on structural properties of the protein-protein complexes (vangone & bonvin, 2015) . the ace2 and tace2 complexes showed à10.7 and à12.7dg (kcal mol à1 ) binding affinity for rbd, respectively, at temperatures ranges from 20 to 37 c, showing higher binding affinity of tace2 for rbd than the intact ace2 (table 3) . similarly, the dissociation constant k d value of tace2-rbd complex was more than three-fold lesser than the intact ace2-rbd complex, showing that tace2 is more tightly bound to rbd. the smaller k d value indicates high stability and strong binding affinity between protein-protein complex (johnson et al., 2007) . the ace2 variants showed a significant decline in k d value when temperature was increased from 20 c to 36 c, leading to a lower k d (9.8 â 10 à10 m) for tace2 (higher affinity) than that of intact ace2 (2.6 â 10 à8 m) at 36 c.this k d value of tace2 is lesser than the previously reported k d value (47 nm) of sbp1 (an ace2 derived peptide of 23 amino acid) to rbd . the optimum stability of the complexes was found at 36˚c (table 3 ). the dramatic changes of binding kinetics might be caused by reduced stability of ace2 complex below optimum temperature 36˚c (zhao et al., 2018) . in order to determine the structural stability and dynamic behavior of intact ace2-rbd and tace2-rbd complexes, we performed md simulation for 20 ns using gromacs 5.0.4. the docking pose of each complex obtained from haddock with lowest energy was selected for md simulation run. to investigate structural stability of the complex, rmsd plot of the complex backbone was produced. a uniform rmsd plot signifying structural stability of tace2-rbd complex. the rmsd value for tace2 complex was 0.2-0.25 nm, while intact ace2 showed 0.25-3.0 nm rmsd (figure 2 ). the rmsd value of tace2-rbd complex is lesser than sbp1-rbd complex, reported previously, which is almost 0.8 nm , showing higher stability of tace2-rbd complex. root mean square fluctuation (rmsf) was determined to evaluate the residues flexibility of both ace2 and rbd in the docked complexes. the high rmsf values indicate the mobility of residue side chains in relation to their average position (kumar et al., 2014) .the rmsf plot shows the residues of rbd in tace2 complex are stable with a few peaks with rmsf more than 0.2 nm (figure 3(b) ), while rbd of ace2 complex shows many residues with rmsf above 0.35 nm (figure 3(d) ).the residues of tace2 at position 24, 30, 40, 76 and 95 showed reduction in rmsf value due to creating binding interactions with rbd (figure 3(a) ). the residues involved in binding with other protein, present lower rmsf values, reveal the most stable regions of the complex (ardalan et al., 2018) .similarly, the residues window of 470-480 of rbd showed higher fluctuation to 0.25 nm, while decrease in fluctuation at the binding residues positions (figure 3(b) ). the most violent fluctuation in the intact ace2 was observed at c-terminus, which was above 0.7 nm (figure 3(c) ). the overall rmsf values of both tace2 and rbd are below 0.2 nm, which indicate that tace2 complex with rbd is stable, which are in agreement with a previously reported rmsf value 0.4 nm, showing complex stability (maqsood et al., 2020) . the overall trajectories obtained after every 100 ps during a 20 ns md simulation run, very small backbone deviation for both the intact ace2 and tace2 complex was observed (figure 4) . however, the amino acid region 470-489 of rbd has shown backbone fluctuation highlighted as yellow (figure 4(c) ), which we suggest the region of binding site for ace2. previously, the amino acid region of the sars-cov-2 spike protein (480-488) was also reported as binding region for ace2 (ibrahim et al., 2020) . à185.2 ã the haddock score is defined as: 1.0 evdw þ 0.2 eelec þ 1.0 edesol þ 0.1 eair. ãã the z-score produced by haddock indicates standard deviations from the average cluster (the more negative the better). radius of gyration (r g) of both ace2 complexes describes overall spread of molecule during a 20 ns md run. a low rg value indicates better structural integrity and folding behavior (erva et al., 2016) . a slight increase in rg value of the intact ace2-rbd complex was observed during first 5 ns of the run, then after no further drifts till end ( figure 5 , red line), however, the tace2-rbd complex was found stable throughout the md run ( figure 5 , violet line), which indicates its structural integrity. overall, the md simulation results confirm that tace2 form a more stabilized complex with rbd and suggest its inhibitory features for sars-cov-2 spike glycoprotein. post-translational modifications (ptms) play important role in protein-protein interactions (su et al., 2017) . since, experimental methods are high-cost and time-consuming, therefore, it is necessary to theoretically predict ptms site on protein to be expressed heterologously. ptm-ssmp, which predict ptms sites on human protein based on local sequence and site specific modification profile (liu et al., 2018) . ace2 analysis through ptm-ssmp server predicted ubiquitination at position 74 and 304, phosphorylation at 606 and o-glycosylation at 720 residue position. the ptm site at 74 is important for protein degradation and have no role in ppis (lecker et al., 2006) . in transmembrane proteins, the extracellular domains may only be n-glycosylated (gupta et al., 2004) . however, there was no n-glycosylation and oglycosylation site predicted for tace2. these results conclude that there is no ptms site predicted on tace2, which is important for protein-protein interactions. interestingly, an experimental study reported that the lack of glycosylation do not affect the binding of sars-cov-1 rbd to human ace2 (chakraborti et al., 2005) , which strongly support our designed tace2 fragment, if expressed in e. coli may bind efficiently with rbd of sars-cov-2 s glycoprotein. figure 2 . rmsd plot of the ace2-rbd (red) and tace2-rbd complex (violet) backbone atoms. the tace2 complex showing less rmsd value than the intact ace2, indicating its higher complex stability than the intact ace2. since, there was no ptm site predicted in tace2, therefore, e. coli would be an ideal host for its large scale expression. e. coli is the easiest, quickest, and cheapest expression host with a fully known genome, most widely used for hetrologous expression of recombinant protein (basit et al., 2019) . since, ace2 is eukaryotic protein; therefore, its expression in its native form in e. coli will be uncertain, as most of the eukaryotic protein showed insoluble expression in e. coli, which need to be refolded invitro , which is costly and time consuming. that's why, the protein that express in soluble form in e. coli are referred as "low hanging fruit", as their bulk production is cost effective and easy to recover (maqsood et al., 2020) . both sequence and structure based solubility prediction tool using camsol software predicted expression of intact ace2 in a completely insoluble form in e. coli with intrinsic solubility score à1.027 and complete soluble expression of tace2 with a solubility score of 1.23. the software generate solubility profile with one score per residue, where regions with scores higher than 1 denote highly soluble regions, while scores lower than à1 showing poorly soluble ones (figure 6(a, b) ). these results propose e. coli as a suitable host for soluble expression of tace2 using pet28a (þ) as an expression vector, which favors single step purification. structure-based rational design of inhibitory protein with enhanced affinities to the sars-cov-2 spike glycoprotein may facilitate development of potential therapeutics. in this study, we have designed a truncated version of human angiotensin converting enzyme 2 as a potential inhibitor of spike glycoprotein. the truncated protein tace2 was extensively studied through protein-protein docking and md simulation for binding to rbd of sars-cov-2 spike glycoprotein. we found that tace2 can bind to rbd with a higher binding affinity and form more stabilized complex than the intact ace2. in addition, the tace2 sequence predicted soluble expression in e. coli, which makes it an easy target for rapid production at large scale for sars-cov-2 prevention. we believe that this study narrow down the region of interaction between sars-cov-2 s glycoprotein and human ace2 and paves the way to further enhance the binding affinity between tace2 and sars-cov-2 s glycoprotein through rational design. this will open a new path to covid-19 treatment. the authors declare no conflict of interest. moroccan medicinal plants as inhibitors of covid-19: computational investigations gromacs: high performance molecular simulations through multi-level parallelism from laptops to supercomputers novel mutant of escherichia coli asparaginase ii to reduction of the glutaminase activity in treatment of acute lymphocytic leukemia by molecular dynamics simulations and qm-mm studies truncation of the processive cel5a of thermotoga maritima results in soluble expression and several fold increase in activity health improvement of human hair and their reshaping using recombinant keratin k31 improvement in activity of cellulase cel12a of 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compounds as promising drug candidates against covid-19 receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus peptide-based inhibitors of protein-protein interactions prodigy: a web server for predicting the binding affinity of protein-protein complexes structural basis for the recognition of sars-cov-2 by full-length human ace2 the i-tasser suite: protein structure and function prediction a highly conserved cryptic epitope in the receptor-binding domains of sars-cov-2 and sars-cov determination of atomic desolvation energies from the structures of crystallized proteins the first-in-class peptide binder to the sars-cov-2 spike protein. biorxiv, 1-15 impact of temperature on heparin and protein interactions key: cord-259185-qg4jwbes authors: vadlamani, b. s.; uppal, t.; verma, s. c.; misra, m. title: functionalized tio2 nanotube-based electrochemical biosensor for rapid detection of sars-cov-2 date: 2020-09-09 journal: nan doi: 10.1101/2020.09.07.20190173 sha: doc_id: 259185 cord_uid: qg4jwbes the coronavirus disease (covid-19) is a newly emerging viral disease caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). rapid increase in the number of covid-19 cases worldwide led the who declare pandemic within a few month after the first case of infection. due to the lack of a prophylactic measure to control the virus infection and spread, early diagnosis and quarantining of infected as well as the asymptomatic individuals are necessary for the containment of this pandemic. however, the current methods for sars-cov-2 diagnosis are expensive and time consuming although some promising and inexpensive technologies are coming out for emergency use. in this work, we report the synthesis of a cheap yet highly sensitive cobalt-functionalized tio2 nanotubes (co-tnts)-based electrochemical biosensor and its efficacy for rapid detection of spike glycoprotein of sars-cov-2 by examining s-rbd protein as the reference material. a simple, low-cost, and one-step electrochemical anodization route was used to synthesize tnts, followed by an incipient wetting method for cobalt functionalization of the tnts platform, which is connected to a potentiostat for data collection. the sensor specifically detected the s-rbd protein of sars-cov-2 even at very low concentration (range of 14 nm to 1400 nm). additionally, our sensor showed linear response in the detection of viral protein with concentration. in summary, our co-tnt sensor is highly effective in detecting sars-cov-2 s-rbd protein in approximately 30 seconds, which can be explored for developing a point of care diagnostics for rapid detection of sars-cov-2 in nasal secretions or saliva samples. sars-cov-2 is currently a global pandemic on a scale that has not been experienced since the 1918 influenza pandemic. one of the reasons why this pandemic virus has spread so quickly is because many infected individuals with sars-cov-2 remain asymptomatic and involuntarily transmit the virus before they come down with symptoms. therefore, uniform surveillance and quarantining of infected as well as the asymptomatic individuals could provide an effective measure to contain the spread of sars-cov-2. however, the current methods for sars-cov-2 diagnosis are expensive and time consuming although some inexpensive technologies are getting approvals for emergency use. our manuscript reports the synthesis of a cheap yet highly sensitive cobalt-functionalized tio2 nanotubes (co-tnts)-based electrochemical biosensor for rapid detection of spike glycoprotein of sars-cov-2. our sensor is synthesized through one-step electrochemical anodization route, followed by an incipient wetting method for cobalt functionalization of tnts platform. the readout of this sensor is an electrochemical signal collected through a potentiostat, which can be adopted for use through smartphone applications and the development of a point of care diagnostics for covid-19. the current outbreak of novel coronavirus (ncov-2019 or sars-cov-2), was first detected in wuhan, china in december 2019, but quickly spread to other parts of china as well as to the entire world causing pandemic [1] . according to the who, as of 16 th august 2020, around 21,294,845 people are infected, and 761,779 people have died due to sars-cov-2 infection [2] . sars-cov-2 infection causes a variety of symptoms including fever, cough and respiratory distress, which are collectively called as coronavirus disease or covid-19 [3] . the transmission of sars-cov-2 primarily occurs from person to person through close contact or via small droplets produced during coughing, sneezing, and talking [4] [5] . the incubation period for sars-cov-2 is around 2-7 days, with no noticeable symptoms; however, the viral transmission from an infected person to a non-infected person is still possible during this asymptomatic period [6] . under the current scenario, with no vaccines in the market, global lockdown regulations are in place in order to minimize the viral spread. additionally, the pandemic has caused a severe socio-economic impact on the world economy and raised fears of a global recession [7] . currently, the real-time reverse-transcriptase polymerase chain reaction (rt-pcr) technique is the most common and reliable laboratory testing method for qualitative/quantitative sars-cov-2 detection [8] [9] followed by serum virus neutralization assay (svna) for the determination of antibody neutralization [10] and enzyme-linked immunoassays (elisa) for the detection of antibody against sars-cov-2 [11] . however, the major limitations of these laboratory based diagnostic tests is the invasive nature of the tests that often require trained personal for nasopharyngeal sample collection, along with the requirement of highly sophisticated machines, cross-reactivity with other viruses, and longer duration of testing. in order to contain the viral spread, surveillance of even . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.07.20190173 doi: medrxiv preprint asymptomatic individuals are needed, which is feasible only after the development of a simple, portable and rapid point-of-use sensor for the detection of sars-cov-2. sars-cov-2 has positive-sense, single-stranded rna (~30k bp) genome with 14 orfs that encode for structural, replication and non-structural proteins [12] . similar to its genetic cousin, human sars-cov, sars-cov-2 consists of four structural proteins viz. spike (s), envelope (e), membrane (m), and nucleocapsid (n). coronaviruses are named for the crown like spike glycoprotein, s (composed of two subunits: the s1 subunit and s2 subunit) on the surface/envelop [13] . the s1 subunit of the s protein consists of a receptor binding domain (rbd) that has a high binding affinity towards the host angiotensin-converting enzyme ii (ace2) receptor present on the human cells and the s2 subunit mediates virus-host cell fusion and entry [14] . importantly, the s protein is highly immunogenic and induces immune response to produce neutralizing antibodies as well as t-cell responses in sars-cov-2 infected individuals [15] . functionally, binding of the s-rbd to the hace2 receptor is a crucial for the entry of sars-cov-2 into the human cells. infringingly, sars-cov-2 s-rbd shares only 70% sequence identity with sars-cov s-rbd, which has been evaluated for vaccines and therapeutic drug development [16] . hence, the s-rbd of sars-cov-2 are excellent target for diagnostic and therapeutic intervenstions. electrochemical biosensors are advantageous for sensing biomolecules because of their ability to detect biomarkers with accuracy, specificity and high sensitivity [17] . electrochemical biosensors have been successfully used in medical diagnostics for the detection of viruses such as middle east respiratory syndrome coronavirus (mers-cov), [18] human enterovirus 71 (ev71) [19] , human influenza a virus h9n2 [20] , avian influenza virus (aiv) h5n1 [21] . lahyquah et al. [18] used an array of carbon electrodes modified with gold nanoparticles for the detection of mers-cov. very recently, a biosensor using gold nanoparticle decorated fto glass immobilized . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.07.20190173 doi: medrxiv preprint with ncovid-19 monoclonal antibody was reported for the detection of sars-cov-2 [22] . the functionality of the electrochemical biosensor can be further improved by nanostructuring the electrode as it increases the electrochemical reaction rate due to an increased electrode surface area to volume ratio, thereby increasing the electrode surface area to analyte fluid volume. in the work by chin et al. on the encephalitis virus, it was found that nanostructuring of carbon electrodes with carbon nanoparticles increased the current response by 63% due to an enhanced electron charge transfer kinetics [23] . similarly, we have reported that co functionalized tio2 nanotubes (ni-tnts) with higher surface-to-volume ratio can detect the biomarkers associated with tuberculosis [24] [25] . the proposed sensing mechanism involves the formation of a complex between co and the biomarker at specific bias voltage, due to the reduction of co ions and oxidation of biomarker. similarly, we hypothesized that s-rbd or sars-cov-2 can be detected through complexing of functionalized nanoparticles with the s-rbd protein and a schematic of viral detection directly from patient sample as shown in figure 1. in the current work, we have determined the potential of co-functionalized tio2 nanotubes (co-tnts) for the electrochemical detection of s-rbd protein of sars-cov-2. tnts were synthesized by simple, cost-effective, one-step electrochemical anodization route, and co functionalization was carried out using the incipient wetting method. our data showed that cobalt functionalized tnts could selectively detect the s-rbd protein of sars-cov-2 using the amperometry electrochemical technique in ~ 30 secs. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted september 9, 2020. . tnts were synthesized by electrochemical anodization of ti sheet. ti sheet of size 1.5 cm x 1.5 cm, with a tab 2 mm in width, was cut out of g1 grade ti sheet (thickness 0.01016 mm). one side of the coupon was polished with 600 grit polishing paper for 4 min to remove any surface metal oxide layer. the coupon was ultrasonicated in 1:1 solution of ethanol and acetone for 2 min. the unpolished side was masked with kapton tape to avoid any exposure to electrolyte during anodization. the electrochemical anodization was performed in a standard two-electrode configuration, using ti foil as a working electrode and platinum foil as a counter electrode with a 3 cm gap between them. the anodization was carried out using an electrolyte of composition 96.5 ml (ch2oh)2, 3 ml di h2o, and 0.505 g nh4f in a teflon beaker. the electrolyte was maintained at a subzero temperature and continuously stirred using a magnetic stirrer at a speed of 140 rpm. the anodization was carried by maintaining a constant voltage of 30 v across both the electrodes for 50 min. after anodization, the sample was rinsed in di h2o and baked in an oven at 120°c for 4 hrs. the kapton tape was removed from the sample after baking, and the sample was annealed in a tube furnace at 500°c for 3 h in a continuous flow of oxygen. the annealed tnts obtained from the furnace were functionalized with cobalt using an incipient wetting method, i.e. a wet ion exchange process. the same side of the sample that was masked earlier was again masked with kapton tape. the sample was ultrasonicated in a solution containing 2.306 g of cocl2.6h2o in 20 ml ethanol for 35 min. the sample was baked in an oven at 120°c for 4 hrs to obtain cobalt functionalized tnts. the morphology of the tnts and co-tnts were examined using dual beam scanning electron microscopy (sem, thermofisher scientific). the cobalt content in the co-tnts sample . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.07.20190173 doi: medrxiv preprint was analyzed using the eds detector attached to sem. the sem micrographs were analyzed using imagej software. the . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.07.20190173 doi: medrxiv preprint the electrochemical sensing of s-rbd protein was carried out using a custom-built co-tnt packaged printed circuit board setup. the sensor response was measured with the help of gamry reference 600+ potentiostat attached to the printed circuit board. the schematic of the whole sensing set up along with the detection methodology is shown in figure 1 . the sensor response with various s-rbd protein concentrations was determined using the amperometry technique, at a bias voltage of -0.8 v. the bias voltage was determined by conducting the cyclic voltammetry experiments in the voltage window -2 v to +2 v. all the experiments were carried out at room temperature. the scanning electron microscopy (sem) micrographs of the tnts, prepared by electrochemical anodization, are shown in figure 2a . the inset shows the side view of the tnts ( figure 2a) . the outer diameter and wall thickness of tnts were ~60 nm and ~10 nm, respectively. the average length of tnts was found to be ~1.1 µm. in our earlier work, tnts synthesized under similar conditions were found to show the crystalline anatase phase predominantly [25] . the surface morphology of the co-tnts examined under sem is shown in figure 2b . the sem micrograph reveals the presence of precipitates on top of the tnt surface. eds analysis confirmed the uniform distribution of co on top of tnts, and the co content was found to be ~ 4 wt %. we have previously shown that co exists in co +2 state in the form of co(oh)2 on co-tnts [26]. therefore, the morphology of tnts can be visualized as having a very large surface area, uniformly decorated with co +2 ions. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.07.20190173 doi: medrxiv preprint the s-rbd protein, biomarker for sars-cov-2 detection, was characterized via sds-page (under denaturizing conditions). the rbd domain of spike glycoprotein comprises of amino acids 329-521, which is a ~25 kda protein with potential n-glycosylation sites. as shown in figure 3a -b, the sds-page gel of his6-tagged s-rbd protein was either stained with simplyblue safestain (figure 3a ) or transferred to a nitrocellulose membrane and detected with 1 ug/ml of mouse anti-his monoclonal antibody (figure 3b) , followed by incubation with infrareddye-tagged secondary ir-dye680 antibody and scanning with an odyssey infrared scanner. specific bands were detected for sars-cov-2 s-rbd protein at approximately 35kda and 70kda, representative of the monomeric and dimeric forms of s-rbd protein, respectively (fig. 3b ). we detected the s-rbd at a slightly higher molecular weight (~35kda) possibly because of post-translational modifications including glycosylation. the ability of co-tnt to sense s-rbd protein of sars-cov-2 was determined by performing an amperometry experiment at a bias voltage of -0.8 v. the amperometry curves obtained at various concentrations of protein are shown in figure 4 . the sensor was exposed to protein 30 sec after beginning of experiment (marked by an arrow). the sensor response current increases sharply and rapidly as the sensor was exposed to the protein. at a protein concentration of 1400 nm (nano molar), the peak sensor current output was found to be ~0.74 µa (nano ampere). the peak current decreases to ~0.45 µa at a protein concentration of 140 nm and further decreases to ~0.23 µa at a protein concentration of 14 nm. the sensor detection time was ~ 30 sec over the concentration range of 14 nm to 1400 nm. it is hypothesized that the rapid increase in sensor response current could be attributed to the electrochemically triggered unfolding of protein that . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted september 9, 2020. . exposes its interior [27] [28] [29] and subsequent complex formation between co and the protein [30] [31] [32] . the average sensor response time, which is defined as the time taken to reach the peak current, was found to be ~ 2 sec. it is very short compared to our earlier studies on the sensor for colorectal cancer, where a sensor response time of ~200 sec was documented [33] . the shorter sensor response time indicates higher kinetics of reaction between co-tnt and the protein molecules. the sensor response (sr) was calculated at various protein concentrations based on the following equation: where !"#,%&'()*+ is the maximum current obtained when sensor is exposed to sars-cov-2 s-rbd protein and !"#,,"-) /*+) is the maximum current obtained when sensor is not exposed to the protein. the value of !"#,,"-) /*+) , which is the current obtained when sensor is not exposed to protein, was found to be ~10 pa ( figure 4 ). the sensor responses measured at different protein concentrations are shown in figure 5 . the sensor response was found to increase with an increase in the concentration of protein. moreover, the sensor response exhibited excellent linearity over the concentration range 14 to 1400 nm with a correlation coefficient of r 2 =0.99. the regressed linear calibration curve for sensor response was obtained as follows: = (0.266 ± 0.026) log( ) + (4.053 ± 0.059); r 2 =0.99 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.07.20190173 doi: medrxiv preprint where sr is the sensor response, and c is the concentration of protein in nm. using statistical analysis [34] the limit of detection for measurements made using sensor was determined to be 0.7 nm. the limit of detection can be further improved by the use of (i) co-tnt synthesized by insitu anodization technique and (ii) co-tnts of even higher length. previously, we found that co-tnt synthesized by in-situ anodization with higher sensor sensitivity compared to co-tnt synthesized by incipient wetting route towards the detection of tuberculosis biomarkers [25] . a higher sensor sensitivity corresponds to a better limit of detection and senstivity of quantitation. the increased sensitivity was attributed to the presence of co(oh)2 precipitate sites in direct contact with parent tio2 due to which direct conduction is possible. the sensor sensitivity can also be improved by using longer co-tnts as higher surface area results in a higher reaction rate; thereby, higher sensor response current can be obtained even at lower protein concentrations. in this study, we developed a co-metal functionalized tnt as a sensing material for electrochemical detection of sars-cov-2 infection through the detection of the receptor binding domain (rbd) of spike glycoprotein. we confirmed the biosensor's potential for clinical application by analyzing the rbd of the spike glycoprotein on our sensor. amperometry electrochemical studies indicated that the sensor could detect the protein in the concentration range 14 nm to 1400 nm. the relationship between sensor response and protein concentration was found to be linear with the limit of detection as low as ~0.7 nm levels. importantly, our sensor detected sars cov-2 s-rbd protein in a very short time (~30 sec) confirming its implication in developing a rapid diagnostic assay. thus, our report demonstrate the development of a simple, . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.07.20190173 doi: medrxiv preprint inexpensive, rapid and non-invasive diagnostic platform that has the potential of detecting sars-cov-2 on clinical specimens including nasal, nasopharyngeal swabs or saliva. moreover, the developed approach has the potential for diagnosis of other respiratory viral diseases by identifying appropriate metallic elements to functionalize tnts. scv: conceptualization, methodology, project administration, funding, writing -review and editing. the environmental and biological safety committee of the university of nevada, reno, approved methods and techniques used in this study. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted september 9, 2020. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.07.20190173 doi: medrxiv preprint mol. wt. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.07.20190173 doi: medrxiv preprint . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted september 9, 2020. . https://doi.org/10.1101/2020.09.07.20190173 doi: medrxiv preprint novel coronavirus. world heal organ coronavirus disease (covid-19) the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 cluster of sars among medical students exposed to single patient identification of severe acute respiratory syndrome in canada international journal of infectious diseases the sars-cov-2 outbreak : what we know the socioeconomic implications of the coronavirus pandemic (covid-19): a review molecular diagnosis of a novel coronavirus ( 2019-ncov ) causing an outbreak of pneumonia positive rt-pcr test results in patients recovered from covid-19 a sars-cov-2 surrogate virus neutralization test ( svnt ) based on antibody-mediated blockage of ace2-spike ( rbd ) protein-protein interaction diagnostic performance of seven rapid igg 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analysis of redox activity of proteins on the carbon screen printed electrodes chemical-induced unfolding of cofactor-free protein monitored by electrochemistry biochemistry dominant protein electrochemistry: application in medicine. a review detection of food decay products using functionalized one-dimensional titania nanotubular arrays detection of four distinct volatile indicators of colorectal cancer using functionalized titania determination of the lower limit of article pdf first page preview authors declare no conflict key: cord-311035-s3tkbh9r authors: procko, erik title: deep mutagenesis in the study of covid-19: a technical overview for the proteomics community date: 2020-10-21 journal: expert review of proteomics doi: 10.1080/14789450.2020.1833721 sha: doc_id: 311035 cord_uid: s3tkbh9r introduction the spike (s) of sars coronavirus 2 (sars-cov-2) engages angiotensin-converting enzyme 2 (ace2) on a host cell to trigger viral-cell membrane fusion and infection. the extracellular region of ace2 can be administered as a soluble decoy to compete for binding sites on the receptor-binding domain (rbd) of s, but it has only moderate affinity and efficacy. the rbd, which is targeted by neutralizing antibodies, may also change and adapt through mutation as sars-cov-2 becomes endemic, posing challenges for therapeutic and vaccine development. areas covered deep mutagenesis is a big data approach to characterizing sequence variants. a deep mutational scan of ace2 expressed on human cells identified mutations that increase s affinity and guided the engineering of a potent and broad soluble receptor decoy. a deep mutational scan of the rbd displayed on the surface of yeast has revealed residues tolerant of mutational changes that may act as a source for drug resistance and antigenic drift. expert opinion deep mutagenesis requires a selection of diverse sequence variants; an in vitro evolution experiment that is tracked with next-generation sequencing. the choice of expression system, diversity of the variant library and selection strategy have important consequences for data quality and interpretation. investigations of protein mutations have classically been approached by precision targeting, in which a small number of mutations are deliberately introduced and tested individually. this requires preconceived ideas or hypotheses on which residues and what changes to those residues might be relevant. when the important residues in a protein sequence are unknown, screens and selections can be used instead, in which a library of random mutations is in some way sorted to enrich for a small number of mutants with the intended phenotype. both experiments are limited in the scale of information they provide. deep mutagenesis or deep mutational scanning take advantage of next-generation sequencing to bring experimental protein mutagenesis to the realm of big data [1] . a screen or selection of a diverse library of variants is tracked by next-generation sequencing to observe how the population's genetic makeup changes. mutations with enhanced function are enriched, while deleterious mutations are depleted; the enrichment ratio comparing frequencies in the selected population with the naive library thus acts as a proxy for relative phenotype. now, the relative effects of thousands of mutations can be assessed simultaneously in a single experiment and a comprehensive mutational landscape can be calculated from experimental data. deep mutagenesis has been developed by multiple groups over the past decade [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] and has proven especially invaluable to meet three goals: assisting protein engineering, understanding mutational tolerance within a protein sequence, and predicting which mutations might be associated with adverse disease outcomes, especially in the context of cancer or drug resistance. two recent and prominent studies of sars coronavirus 2 (sars-cov-2) have used deep mutagenesis to address each of these problems [14, 15] . this special report summarizes the two studies with a focus on experimental details and caveats that will be unfamiliar to those outside the deep mutational scanning community. two deep mutagenesis studies have determined how thousands of mutations within the sars-cov-2 spike or the virus' human receptor affect their binding. the data have proven invaluable for engineering high affinity decoy receptors that are under preclinical development as a covid-19 therapy, and have revealed the scope of mutational tolerance within the spike that may have bearing on genetic drift as the virus becomes endemic and changes over time. while these two studies focused on expression and binding between the viral spike and its receptor, the underlying selection strategies used in deep mutational scans are increasingly tied to more complex phenotypes, such as selections for structural stability based on protease-sensitivity [16] , using competing ligands to engineer specificity into proteins including viral receptors [17] [18] [19] , and selections based on catalytic or biological activity [20] [21] [22] [23] . undoubtedly there are more questions related to sars-cov-2 biology and the biochemistry of its while much attention has been given to isolating monoclonal antibodies with tight affinity for the sars-cov-2 spike (s) glycoprotein [24] [25] [26] [27] [28] [29] [30] , an alternative is to use the entry receptor as a soluble decoy to neutralize infection. s is a class i viral fusion protein that is proteolytically processed into two subunits, s1 and s2, that are non-covalently associated and decorate the coronavirus envelope [31] [32] [33] . s recognizes angiotensin-converting enzyme 2 (ace2) on host cells to initiate attachment and fusion of the viral and plasma membranes [33] [34] [35] [36] [37] [38] . soluble ace2 (sace2) blocks receptor-binding sites on s [15, 37, [39] [40] [41] [42] and while escape mutations in s rapidly emerge in tissue culture in the presence of monoclonal antibodies [43] , in principle the virus has limited mechanisms to escape a soluble decoy receptor without simultaneously losing affinity for the natural receptor. the decoy receptor might also have a virucidal effect by inducing conformational changes and s1 shedding, such that virus particles are inactivated even if sace2 dissociates. however, monoclonal antibodies have superior affinity and neutralization efficacy. to improve the therapeutic potential of decoy receptors, my group used deep mutagenesis to find mutations in ace2 that enhance affinity [15] . a library of over 2,000 single amino acid substitutions in ace2 was constructed, focused on diversification of residues at the structurally defined interface with the receptor-binding domain (rbd) of s [44, 45] and also within the ace2 catalytic cleft. the library was expressed in a human cell line, with a c-myc epitope tag fused to the extracellular n-terminus of ace2 for detection of surface expressed protein. other than the presence of the epitope tag, ace2 expressed in this experimental selection system matches native ace2 in the human body. the culture expressing the ace2 library was then selected by fluorescence activated cell sorting (facs) to collect cells expressing ace2 variants with tight affinity for fluorescently labeled rbd from s of sars-cov-2 ( figure 1a ). for the artificial selection to be successful, cells must express a single protein variant from a single sequence variant, thereby providing a tight physical link between the phenotype of ace2 expressed at the plasma membrane and a single sequence within the cell. getting human cells in culture to acquire and express a single coding variant is no trivial feat, as transfection methods typically introduce many plasmid copies. different methods to solve this technical challenge have included the use of episomal plasmids that randomly partition to daughter cells during division until progeny harbor a single coding variant over time [4] , the use of engineered integration sites in the genome [9, 46, 47] , or the use of viral vectors at low multiplicities-of-infection [48, 49] . my group used carrier dna to sufficiently dilute the ace2 plasmid library such that each cell typically acquired no more than a single coding variant [11] . an episomal plasmid is used for the library so that extrachromosomal replication within the cell enhances expression of the protein under investigation. (the carrier dna, itself a modified episomal plasmid, further assists in this process [50] .) the disadvantage to this simple solution for linking a single genotype to phenotype is that the coding sequence is so diluted with carrier dna, most cells in the (a) a library of ace2 variants was expressed in human cells. full-length ace2 (tan) was tagged with a c-myc epitope at its extracellular n-terminus for detection of surface expression with a fluorescent antibody (red). sars-cov-2 rbd (pale green) genetically fused with superfolder green fluorescent protein (sfgfp; dark green) was incubated with the cell culture. facs was used to collect fluorescent cells expressing ace2 with bound rbd-sfgfp. (b) the isolated rbd of sars-cov-2 (pale green) was fused at its n-terminus to aga2p (blue) and at its c-terminus to a c-myc epitope tag. a saturation mutagenesis library of the rbd was expressed on the yeast surface. following induction of rbd expression, the yeast were incubated with dimeric, biotinylated sace2 (tan). bound ace2 was detected with fluorescent streptavidin (purple) and surface expressed rbd was detected with a fluorescent antibody (red). • in deep mutagenesis, the relative phenotypes of thousands of mutations in a protein sequence are determined in a single experiment. • the experimental mutational landscape of ace2 for binding the rbd of sars-cov-2 provides a blueprint for engineering high affinity decoy receptors. • a deep mutational scan of the sars-cov-2 rbd reveals considerable opportunity for genetic drift without loss of receptor affinity. • different expression systems for selecting ace2 or spike variants have inherent advantages and disadvantages. • there are opportunities for deep mutagenesis to provide biochemical insights on other sars-cov-2 proteins. culture do not express ace2 and facs time is wasted on sorting a large number of negative cells. this has important consequences on the data, as time spent sampling negative cells is time not spent sampling cells expressing the protein under investigation, and consequently variants in the library may be under-sampled giving poor data accuracy. undersampling becomes exceptionally concerning as the library size increases, and for this reason the library was limited to single amino acid substitutions at just 117 positions in ace2. following facs selection of the human culture to enrich a cell population with high binding activity for sars-cov-2 protein s, rna transcripts were isolated and illumina sequenced. an enrichment ratio is calculated for each mutation by dividing its frequency in the sorted cell transcripts by its frequency in the naive plasmid library [51] . illumina sequencing did not cover the full length of ace2 and instead the cdna was sequenced as a series of fragments that together provided full coverage of the diversified regions. one assumes during analysis that there are no additional mutations outside a sequenced fragment, a reasonable assumption when a mutation is found because the library was constructed to have only one amino acid substitution per plasmid. however, the assumption breaks down when no mutations are observed in the sequenced fragment, as one cannot know whether there was a mutation elsewhere outside the sequenced region. as a consequence, the wild type sequence is not directly observed and is instead only estimated. there are strategies using the introduction and analysis of silent mutations that can resolve this issue [52] . overall, there was close agreement between the mutation enrichment ratios from two independent replicates of the facs experiments, indicating that the ace2 library was well sampled and there was high confidence in the data [15] . the enrichment ratios calculated for each variant in the sorted ace2 library provide a mutational landscape that defines the relative phenotypes of thousands of ace2 mutations for binding to sars-cov-2 s [15] . the data in this experiment are qualitative and it is unclear how a log 2 enrichment ratio of, say, −2 or +3 translates to an exact change in a biophysical parameter such as k d . furthermore, mutations can impact not only binding affinity for the rbd of s but also ace2 surface expression. to filter out the contribution of mutations to expression, two populations of cells were collected by facs. in addition to collecting cells that express ace2 and tightly bind rbd, cells were simultaneously collected in the same experiment that express ace2 but have weak rbd binding. ace2 mutants that were not expressed at the cell surface would be depleted from both sorted populations, which was apparent from tracking the depletion of nonsense mutations. in this way, information was collected on how ace2 mutations impact expression and rbd binding from a single facs experiment. the deep mutational scan of ace2 revealed that mutations can indeed be found to enhance binding toward sars-cov-2 rbd (figure 2) , suitable for engineering high affinity soluble decoy receptors [15] . mutations were found at the binding interface where they enhance specific atomic contacts, and were also found distally in the second shell and beyond where they may impact ace2 conformation, folding and dynamics. a soluble ace2 variant that combines three mutations, called sace2 2 .v2.4, was found to be highly expressed, is a stable monodisperse dimer, binds sars-cov-2 s with picomolar affinity and potently neutralizes infection of a susceptible cell line by authentic virus. its properties rival affinity-matured monoclonal antibodies under commercial development for therapy and prophylaxis. despite only affinity toward sars-cov-2 being considered during the engineering process, sace2 2 . v2.4 also potently neutralizes authentic sars-cov-1, and we speculate that it will have broad activity against betacoronaviruses that use ace2 as an entry receptor. in unpublished work that has yet to be peer reviewed, we have found sace2 2 . v2.4 broadly and tightly binds bat coronaviruses that may be a source for future pandemics, supporting the concept of receptor-based decoys as antiviral biologics with exceptional breadth. as determined by yeast display, the effects of mutations in the rbd of sars-cov-2 protein s on receptor affinity are plotted in the heat map at left, with dark green indicating the mutations are deleterious and pale colors indicating the mutations are neutral. the effects of mutations in human cell-expressed ace2 on binding to soluble rbd are plotted in the heat map at right, with depleted mutations in orange, neutral mutations in white and enriched mutations in blue. positional scores are mapped to the atomic structure of rbd-bound ace2 (pdb 6m17) at center. conserved ace2 residues for rbd binding are orange, while ace2 residues that are hot spots for mutations with increased affinity are blue. rbd residues conserved for ace2 binding are green. most rbd mutations in this region of the interface are deleterious, whereas numerous mutations were found in ace2 that increased affinity. in starr et al, deep mutagenesis was applied to the sars-cov -2 spike to assess mutational tolerance for expression and ace2 interactions [14] . instead of investigating the entire trimeric s protein expressed on a cellular or viral membrane, the isolated rbd was fused to the yeast mating factor aga2p and displayed on the yeast surface [53] (figure 1b) . this is an artificial display platform that removes the rbd from its native context. n-glycosylation in yeast is also of high-mannose type and lacks the complex, terminally sialylated glycans produced by human cells [54] , which can be important when binding interactions are glycan-dependent as is seen for some antibodies targeting viral spikes [55] . however, this display platform harnesses yeast genetics to confer tremendous advantages for in vitro selection and evolution. using yeast display, large diverse libraries can be readily sorted by facs to provide highquality data. separate selections were completed at a range of different sace2 concentrations to simulate a titration experiment, from which the data could be converted to quantitative changes in apparent k d on the yeast surface ( figure 2 ). as a surrogate for how rbd mutations may impact expression of the viral spike, the effects of mutations on rbd surface display were also assessed in a standalone facs selection. quality control pathways for protein secretion in yeast can be forgiving of misfolded protein sequences [16] and there are residues of the rbd that would ordinarily be buried in the context of the full s protein; it therefore remains to be seen how closely the yeast display data will correlate with equivalent experiments in more physiologically relevant expression systems. nonetheless, the predicted effects by yeast display of some mutations were validated using full length s expressed in human cells and packaged in pseudovirus [14] . the library encoding nearly 4,000 single amino acid substitutions in the sars-cov-2 rbd was pacbio sequenced, providing long reads that match untranslated nucleotide barcodes to a specific protein variant. following facs-based selection, only the barcodes are read to determine how favorable sequence variants are enriched or deleterious sequence variants are depleted. this resolves issues with illumina sequencing failing to cover the full cdna length, and because multiple barcodes are associated with any given protein variant, there are additional internal checks for data quality and consistency. despite the limitations of a yeast display platform, the deep mutational scan of the isolated rbd provides a high quality and useful data set from which several important conclusions were drawn. first, the ace2 binding surface of sars-cov-2 rbd tolerates surprisingly high sequence diversity, even though it is a critical site for function [14] . high diversity is also seen in the ace2-binding sites of s proteins from sars-related bat coronaviruses, but this matches corresponding diversity in ace2 from ecologically diverse bat species [56] and does not necessarily mean that the rbd tolerates mutations for binding ace2 from a single species. the deep mutational scan addresses this uncertainty and is further supported by evidence showing that diverse rbd sequences from bat coronaviruses are all competent for binding human ace2 with varying affinities [38] . second, mutations were found in the rbd that enhance binding to ace2, yet there does not appear to be positive selective pressure for these variants in the human population [14] . sars-cov-2 affinity for ace2 is therefore 'good enough,' with no additional fitness benefit for higher affinity. it is worth noting that classical sars-cov-1 is also a highly infectious and virulent pathogen, despite having weaker ace2 affinity [36, 57] . the rapid spread of sars-cov -2 probably has more to do with asymptomatic and presymptomatic transmission than enhanced receptor binding. third, mutations were found within the epitopes for monoclonal antibodies but maintain high ace2 binding, and it is likely that sars-cov-2 can easily mutate to escape neutralization without losing infectivity [14] . this agrees with selection experiments of pseudovirus expressing sars-cov-2 s variants, in which escape mutants in the viral spike rapidly emerge to neutralizing antibodies in a single passage [43] . this has profound implications for antibody therapy, where the standard has become combinations of noncompeting monoclonals in a cocktail to prevent rapid resistance. it is currently unknown whether an engineered soluble decoy receptor, such as sace2 2 .v2.4, will similarly be susceptible to the emergence of viral spike variants that can discriminate between the engineered decoy and the native receptor. we hypothesize that engineered decoys will be broadly active against sars-cov-2 variants and this remains an active area of investigation. http://orcid.org/0000-0002-0028-490x papers of special note have been highlighted as either of interest (•) or of considerable interest deep mutational scanning: a new style of protein science high-resolution mapping of protein sequence-function relationships a fundamental protein property, thermodynamic stability, revealed solely from large-scale measurements of protein function deep mutational scanning of an antibody against epidermal growth factor receptor using mammalian cell display and massively parallel pyrosequencing affinity and cross-reactivity engineering of ctla4-ig to modulate t cell costimulation an engineered switch in t cell receptor specificity leads to an unusual but functional binding geometry computational design of a protein-based enzyme inhibitor experimental estimation of the effects of all amino-acid mutations to hiv's envelope protein on viral replication in cell culture a platform for functional assessment of large variant libraries in mammalian cells multiplex assessment of protein variant abundance by massively parallel sequencing mapping interaction sites on human chemokine receptors by deep mutational scanning this study established a simple and effective method for linking phenotype to a single genotype in transfected human cells. this technical accomplishment is necessary for selection and deep mutational scanning in human cells a comprehensive biophysical description of pairwise epistasis throughout an entire protein domain high-throughput profiling of influenza a virus hemagglutinin gene at single-nucleotide resolution deep mutational scanning of sars-cov-2 receptor binding domain reveals constraints on folding and ace2 binding the isolated rbd of sars-cov-2 was displayed on yeast and deep mutationally scanned to understand the mutational landscape for yeast surface expression (a surrogate for folding) and ace2 binding. the data reveal substantial sequence diversity is tolerated on the rbd surface engineering human ace2 to optimize binding to the spike protein of sars coronavirus 2 deep mutationally scanned ace2 expressed on a human cell membrane to identify substitutions that enhance binding to s of sars-cov-2. this guided the engineering of high affinity and potently neutralizing decoy receptors global analysis of protein folding using massively parallel design, synthesis, and testing a computationally designed inhibitor of an epstein-barr viral bcl-2 protein induces apoptosis in infected cells computationally designed high specificity inhibitors delineate the roles of bcl2 family proteins in cancer engineered receptors for human cytomegalovirus that are orthogonal to normal human biology single-mutation fitness landscapes for an enzyme on multiple substrates reveal specificity is globally encoded a comprehensive, highresolution map of a gene's fitness landscape comprehensive sequence-flux mapping of a levoglucosan utilization pathway in e. coli molecular determinants of chaperone interactions on mhc-i for folding and antigen repertoire selection cross-neutralization of sars-cov-2 by a human monoclonal sars-cov antibody studies in humanized mice and convalescent humans yield a sars-cov-2 antibody cocktail potent neutralizing antibodies from covid-19 patients define multiple targets of vulnerability broad neutralization of sars-related viruses by human monoclonal antibodies a human monoclonal antibody blocking sars-cov-2 infection a noncompeting pair of human neutralizing antibodies block covid-19 virus binding to its receptor ace2 isolation of potent sars-cov-2 neutralizing antibodies and protection from disease in a small animal model structure, function, and antigenicity of the sars-cov-2 spike glycoprotein structural insights into coronavirus entry sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor a pneumonia outbreak associated with a new coronavirus of probable bat origin receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars cryo-em structure of the 2019-ncov spike in the prefusion conformation angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus this study reports the original discovery of ace2 as the entry receptor for classical sars-cov-1 functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses * ace2 is shown to be a shared entry receptor for a clade of sars-associated betacoronaviruses, including diverse strains from bats and human virus sars-cov-2 susceptibility to sars coronavirus s protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor neutralization of sars-cov-2 spike pseudotyped virus by recombinant ace2-ig inhibition of sars-cov-2 infections in engineered human tissues using clinical-grade soluble human ace2 novel ace2-igg1 fusions with improved in vitro and in vivo activity against sars-cov2 antibody cocktail to sars-cov-2 spike protein prevents rapid mutational escape seen with individual antibodies sars-cov-2 spike is able to rapidly acquire mutations to escape neutralizing monoclonal antibodies in tissue culture, necessitating the combination of multiple non-competing monoclonals in a cocktail to prevent resistance structural basis for the recognition of the sars-cov-2 by full-length human ace2 structure of sars coronavirus spike receptor-binding domain complexed with receptor an improved platform for functional assessment of large protein libraries in mammalian cells mammalian cell surface display for monoclonal antibody-based facs selection of viral envelope proteins hiv vaccine design to target germline precursors of glycan-dependent broadly neutralizing antibodies structure-based design of native-like hiv-1 envelope trimers to silence non-neutralizing epitopes and eliminate cd4 binding structural architecture of a dimeric class c gpcr based on co-trafficking of sweet taste receptor subunits enrich: software for analysis of protein function by enrichment and depletion of variants hiv-1 broadly neutralizing antibody precursor b cells revealed by germline-targeting immunogen isolating and engineering human antibodies using yeast surface display the humanization of n-glycosylation pathways in yeast glycan-dependent neutralizing antibodies are frequently elicited in individuals chronically infected with hiv-1 clade b or c. aids research and human retroviruses exceptional diversity and selection pressure on sars-cov and sars-cov-2 host receptor in bats compared to other mammals structural basis of receptor recognition by sars-cov-2 key: cord-280941-ds6x0yym authors: kim, young-seok; son, ahyun; kim, jihoon; kwon, soon bin; kim, myung hee; kim, paul; kim, jieun; byun, young ho; sung, jemin; lee, jinhee; yu, ji eun; park, chan; kim, yeon-sook; cho, nam-hyuk; chang, jun; seong, baik l. title: chaperna-mediated assembly of ferritin-based middle east respiratory syndrome-coronavirus nanoparticles date: 2018-05-17 journal: front immunol doi: 10.3389/fimmu.2018.01093 sha: doc_id: 280941 cord_uid: ds6x0yym the folding of monomeric antigens and their subsequent assembly into higher ordered structures are crucial for robust and effective production of nanoparticle (np) vaccines in a timely and reproducible manner. despite significant advances in in silico design and structure-based assembly, most engineered nps are refractory to soluble expression and fail to assemble as designed, presenting major challenges in the manufacturing process. the failure is due to a lack of understanding of the kinetic pathways and enabling technical platforms to ensure successful folding of the monomer antigens into regular assemblages. capitalizing on a novel function of rna as a molecular chaperone (chaperna: chaperone + rna), we provide a robust protein-folding vehicle that may be implemented to np assembly in bacterial hosts. the receptor-binding domain (rbd) of middle east respiratory syndrome-coronavirus (mers-cov) was fused with the rna-interaction domain (rid) and bacterioferritin, and expressed in escherichia coli in a soluble form. site-specific proteolytic removal of the rid prompted the assemblage of monomers into nps, which was confirmed by electron microscopy and dynamic light scattering. the mutations that affected the rna binding to rbd significantly increased the soluble aggregation into amorphous structures, reducing the overall yield of nps of a defined size. this underscored the rna-antigen interactions during np assembly. the sera after mouse immunization effectively interfered with the binding of mers-cov rbd to the cellular receptor hdpp4. the results suggest that rna-binding controls the overall kinetic network of the antigen folding pathway in favor of enhanced assemblage of nps into highly regular and immunologically relevant conformations. the concentration of the ion fe(2+), salt, and fusion linker also contributed to the assembly in vitro, and the stability of the nps. the kinetic “pace-keeping” role of chaperna in the super molecular assembly of antigen monomers holds promise for the development and delivery of nps and virus-like particles as recombinant vaccines and for serological detection of viral infections. introduction various types of viral vaccines have been developed over the last century with a wide spectrum of efficacy and safety (1, 2) . the manufacturing of most conventional vaccines-live attenuated, inactivated, or subunit vaccines-invariably require the culturing of infectious viruses in cell substrates (3) . despite dedicated efforts, conventional cell culture often fails to produce sufficient amounts of virus for evaluating the immunogenicity, protective efficacy, and safety of viral vaccines. moreover, some emerging viruses cause high-mortality rates, without options for treatment or prophylaxis, necessitating their manipulation, and manufacture under stringent bio-safety environment (4) . not surprisingly, alternative technologies that circumvent these limitations are a high priority in the areas of vaccine development and production. nanoparticles (nps), virus-like particles (vlps), and assembly of multimeric peptides each provide attractive platforms for vaccine design (5) . virus-like particles and nps structurally resemble infectious virions, but are non-infectious due to the lack of viral genomes. recombinant surface antigens from natural virions are assembled into highly ordered conformations as empty particles devoid of genetic material. antigenic epitopes are presented on the multivalent and highly repetitive outer structure of the nps, which leads to the crosslinking of b-cell receptors and the induction of long-lasting immune responses (6) (7) (8) . by mimicking the morphology of the natural infectious virions, the regularly assembled particles are highly immunogenic, and are amenable to diagnostic and prophylactic exploitation. among the simplest targets are the vlps of non-enveloped viruses, such as hepatitis e virus or human papilloma virus, and are composed purely of viral capsid proteins (9) (10) (11) . in contrast to non-enveloped viruses, where virion assembly is exclusive to capsid proteins, enveloped viruses (e.g., coronavirus or flavivirus), require an additional membrane component for assembly into mature virions. consequently, in enveloped vlps, the assembly of matrix proteins provides a molecular scaffold, and viral antigens are embedded into lipid membranes. different types of glycoproteins may be embedded in the lipid membrane as target antigens for generating immunological responses (12) . however, this process requires multiple proteins (surface antigens and matrix proteins), and the enveloped vlps are not structurally uniform and are difficult to characterize. a promising alternative is to present the target antigens on the surfaces of self-assembled nps, which, in lieu of lipid membranes, serve as the macromolecular scaffold for the presentation of the antigens of interest. in developing np vaccines, consideration should be given regarding the selection of a robust and faithful system for np assembly that enables the cost-effective development and delivery of vaccines in a timely manner. structure-based approaches in silico and their underlying principles are relatively advanced for np assembly (13) (14) (15) . most of the approaches consider the thermodynamic stability of the final assembled nps, without due recognition for the kinetic complexities controlling regular assemblage over random interactions that lead to misfolded aggregations. therefore, it is not surprising that most engineered nps are refractory to soluble expression, which presents practical challenges in production, both at a laboratory-scale and in commercial manufacturing processes. this problem becomes augmented when expressed in bacterial hosts because of a lack of folding assistance in the bacterial cytoplasm for viral antigens. therefore, due to advantages in assisted folding, posttranslational modifications, and the possibility of generating multiple-component nps and vlps, eukaryotic hosts such as yeast, insects, and mammalian cells have been favored over bacterial hosts (16) (17) (18) . however, these systems are significantly more expensive than bacterial systems, are more time-consuming, and the down-stream processes are usually more complex. moreover, the purification of vlps from insect cell systems poses a challenge due to similar physicochemical properties between the vlps and the baculoviruses (1, 16) . bacterial systems, if available, would provide a cost-effective means to develop and deliver vaccines, as well as sero-diagnostic antigen kits used to diagnose-specific infection diseases. middle east respiratory syndrome (mers) was first reported in saudi arabia in 2012 and has caused multiple cases of infection with high mortality in europe and asia (19, 20) . mers is caused by mers-coronavirus (mers-cov), which can be transmitted from camels to humans, and from humans to other humans (21, 22) . worldwide transmission is increasing in direct household and community-wide transmission, as well as in nosocomial settings, as exemplified in a 2015 outbreak in korea (23, 24) . neither effective vaccines nor therapeutic interventions are currently available. because of this, assembly of mers-cov antigens into immunologically relevant conformation as nps would be of interest and may be helpful in developing vaccines, sero-diagnostic tools, and therapeutic monoclonal antibodies. in the current study, we present a novel bacterial np of mers-cov antigen using ferritin as a molecular scaffold for self-assembly. ferritin, which is present in most living organisms, has 24 identical subunits that spontaneously self-assemble and form np complexes with internal and external diameters of 8 and 12 nm, respectively (25, 26) . previous studies show that ferritins of helicobacter pylori from a human isolate can be used as scaffold for hiv and influenza np vaccines, using eukaryotic host cells such as human embryonic kidney cells (hek293f or hek293s) (27, 28) . likewise, bacterioferritin (fr), which self-assembles into nanocages with octahedral symmetry, has also been evaluated as a potential drug delivery system (29) . however, viral antigens of human pathogens are prone to misfolding into aggregates, which necessitates chemical refolding of the insoluble aggregates in order to regain solubility and to allow regular assembly of the antigen (30, 31) . in addition, displaying antigens on the surface of multi-molecularly assembled scaffolds in bacterial hosts remains a daunting challenge. we hypothesized that nps displaying the receptor-binding domain (rbd) of the spike protein from mers-cov could be produced in a bacterial system by harnessing the function of a molecular chaperone. conventionally, protein folding and the prevention of non-functional aggregation have been ascribed to molecular chaperones (32) (33) (34) . recently, it has been shown that rna molecules are able to provide novel functions as molecular chaperones (35) (36) (37) . based on novel findings, the concept of chaperna (chaperone + rna) function was established (38) . in this report, chaperna function was harnessed for the folding and assembly of hybrid ferritin monomers into nps using a bacterial expression system. we also demonstrated that the biophysical properties, including solubility, yield, and stability of mers-cov nps, could be improved by properly controlling the rna-binding affinity, and the concentrations of fe 2+ and salts. the chapernabased np assembly may prove to be a versatile tool for developing and delivering recombinant vaccines and for serological detection of emerging/re-emerging viruses. the expression vector pge-hrid(3) was constructed from the parental vector pge-lysrs (3) (39) . the pge-lysrs(3) vector was enzymatically cut with ndei and kpni. the pcr product of hrid, which carries the tev protease cleavage site and a 6-histidine tag at the c-terminus, was cut using the same restriction enzymes and the digested fragment inserted into the vector to generate pge-hrid(3). fr (genebank accession no. nc_000913.3) dna was synthesized by, and purchased from, cosmo genetech (korea). the dna was cleaved with sali and hindiii, and inserted into pge-hrid(3) to generate hrid(3)-fr. the receptor binding domain (rbd), n-terminal residues 367-606, of the mers-cov s protein (genbank accession no. afs88936.1), was generated by gene synthesis, cut with kpni and sali, and inserted into hrid-fr to generate pge-hrid(3)-rbd-fr. linker ssg or asg was inserted into the c-terminus of the rbd using overlapping pcr, cleaved with kpni and sai, and ligated into hrid-fr, generating pge-hrid(3)-rbd-[ssg]-fr or pge-hrid(3)-rbd-[asg]-fr, respectively. the schematic diagrams of each expression vector are illustrated in figure 1b . the genes of mutant hrid(2 m) (k19a and k23a) and hrid(9 m) (k19a, k23a, r24a, k27a, k30a, k31a, k35a, k38a, and k40a) were generated by gene synthesis, cleaved with ndei and kpni, and inserted into pge-hrid(3)-rbd-fr, generating pge-hrid(2 m)-rbd-fr and pge-hrid(9 m)-rbd-fr, respectively. the mutation sites and amino acid sequences of the mutants are shown in table s1 in supplementary material. the resulting expression vectors were transformed into the escherichia coli strain shuffle ® t7. the cells were grown in 50 ml of lb medium with ampicillin (50 µg/ml) at 30°c overnight. each type of transformant was inoculated into 500 ml of lb medium with ampicillin, grown at 30°c until an optical density (od600) of 0.6-0.8 was reached. protein expression was induced with 1 mm iptg for 12 h. each sample was harvested by centrifugation, lysed by sonication in lysis buffer (50 mm tris-hcl, ph 7.5; 10% glycerol; 2 mm 2-mercaptoethanol; and 0.1% tween-20). the soluble fraction of each lysate was purified on a ni-affinity histrap™ hp column by atka prime (ge healthcare) and concentrated with centriprep™ (merck millipore ltd.). the purified proteins were treated with tev protease to remove the fusion partner hrid. the assembled nps were purified by gel filtration on 10/300 superose™ 6 increase columns (ge healthcare). to examine the size and structure of the purified nps, microscopic evaluations using tem and cryo-em were performed. for tem analysis, a drop of the nps was placed onto a formvar/carboncoated tem grid (spl). the grid was negatively stained with 2% uranyl acetate, dried, and examined using a jem-1011 electron microscope (jeol) at an accelerating voltage of 80 kv. the particle sizes were calculated using camera-megaview iii (soft imaging system-germany) for measuring the nps in random image fields. for cryo-em, the nps were placed onto plasma-treated formvar/ carbon 200 copper grid (ems) and negatively stained with 2% uranyl acetate. the grid was accelerated at 200 kv with an fei cryotecnai f20 cryo-em microscope made available through the korean institute of science and technology. the nps were examined and photographed in high resolution. nanoparticle samples (3 ml) were placed into a dispo-h cell, and analyzed using a zeta-potential & particle size analyzer (els(37, 30, and 18°c ) and the cell lysates were separated into total (t), soluble (s), and insoluble (p) fractions by centrifugation (left panel). the solubility of each protein expressed at 18°c was measured by a gel densitometer and the data were summarized and shown in the right panel (n = 3). statistical significance (**p < 0.01, ***p < 0.001) was indicated for the samples compared with the control using a two-tailed student's t-test. (d) illustration of mers-cov rbd-fr nps using the chaperna-based hrid fusion partner. the hrid facilitated folding of the aggregation-prone rbd-fr through interaction with rna. the monomer of rbd-fr formed a properly folded trimeric structure by cleaving hrid with tev protease. eight trimers assembled and formed into mers-cov-like nps. red triangles indicate the rbd trimer on the fr nps. of the nps was measured twice at 25°c in water as a solvent with the sample accumulation time at 200 s. effect of salt and fe 2+ concentrations on np assembly and stability cultured cells (3 ml) were lysed with lysis buffer in the presence of various concentrations of nacl (0, 50, 100, 150, 200, 225, 250, 275 , and 300 mm) to evaluate the intracellular proteins. all samples were performed in triplicate. the cell lysates were separated into soluble and insoluble fractions by centrifugation, and the protein stabilities analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page). thus, the proteins from cell lysates (500 ml culture) were purified using hispur™ ni-nta resin (thermo fisher scientific) in buffer a depending on nacl concentration (0-300 mm). to evaluate the effects on fe 2+ on np formation, cells were cultured in lb media with various concentrations of fe 2+ (0, 200, 500, and 1,000 µm). np formation was examined by size exclusion chromatography (sec), sds-page, tem, and dls at the various concentrations of nacl or fe 2+ . the cells were harvested, sonicated with lysis buffer, and separated into soluble and pellet fractions by centrifugation. target proteins in the soluble fraction were purified using hispur™ ni-nta resin (thermo fisher scientific), following the manufacturer's instruction. t (total lysate), s (soluble fraction), p (pellet fraction), w (wash fraction), and e (the elution fraction were analyzed by sds-page. co-purification of the nucleic acids and proteins in the wash and elute were analyzed on a native agarose gel. the nucleic acids were visualized with ethidium bromide (etbr), and the proteins with coomassie staining. cultured cells (10 ml) were harvested using the same method described above. the cells were lysed with 500 µl of protein extraction reagent b-per™ ii (thermo scientific) and separated into soluble and pellet fractions by centrifuged 12,000 rpm for 10 m. a 200 µl aliquot of each soluble fraction was further treated with 250 µg/ml of rnase a (intron biotechnology) and incubated at 37°c for 15 min. the nuclease treated samples were clarified by centrifugation at 12,000 rpm for 15 min and the soluble supernatants and the pelleted precipitates were analyzed on an sds-page gel followed by western blot analysis. to confirm the proper folding of rbd-fr and its variant (rbd-[ssg]-fr), the binding of the purified proteins with the mers-cov receptor hddp4 was performed by elisa. fr only and phosphate-buffered saline (pbs) were used as negative controls. nunc 96-well microtiter immunoplates (thermo fisher scientific) were coated with 100 ng/well of hdpp4 proteins (abcam) and incubated at 4°c overnight. the plates were washed and blocked with 150 µl/well of blocking buffer (1% bsa) for 1 h at room temperature. rbd (ssg linker, wt, 2 m, or 9 m)-fr (100 ng/ well) were added for 2 h at 37°c. an anti-penta his antibody (100 µl/well; qiagen) was serially diluted (1/100 to 1/12,800) in tbst [50 mm tris-cl (ph 7.4), 0.05% tween-20], added to the wells, and incubated for 1 h at 37°c. a secondary goat anti-mouse igg antibody conjugated with hrp in a 100-µl volume (1:5,000, sigma-aldrich) was added and incubated for 1 h at 37°c. the plates were washed three times with tbst at the end of each step. after washing, 100 µl/well of substrate tmb solution (bd biosciences) were added to the well and the plates were incubated at 37°c for 30 min in the dark. 50 µl of stop solution (2 n h2so4) was added to the well to stop the colorimetric reaction, and the absorbance at 450 nm was measured using an elisa reader, fluostar optima (bmg labtech). . the coating antigens were removed, and the wells were blocked with pbst (5% skim milk in pbs and tween-20) for 1 h at 37°c. after 2 h, the blocking solution was removed. twofold serially diluted sera from four patients (cnnh-0709, 0809, 1009, and 1309) were added to each well and incubated at 37°c for 2 h. the antigencoated wells were incubated with peroxidase-conjugated goat anti-human igg antibody (kpl, seracare life sciences, milford, ma, usa) at 37°c for 1 h. the primary antibody was removed and 3,3′,5,5′-tetramethylbenzidine (tmb; sigma-aldrich) was added to each well as colorimetric substrate. immediately after treatment of the reactions with stopping solution (sigma-aldrich), the od was read at 450 nm. six-week-old female balb/c mice were immunized with 20 µg/ mouse of the rbd-fr, rbd-[ssg]-fr, or rbd protein generated as described above, or with commercially available mers-cov rbd protein (mers-rbd-005p; eenzyme) as antigen in bsl-2 facility in ylarc. antigens were diluted in pbs. for the first group, equal volume of mf59 adjuvant (addavax, cat. no vacadx-10) (43) was mixed by pipetting. for the other group, equal volume of antigens and alum adjuvant (thermo fisher scientific) were mixed by pipetting following the manufacturers' protocol. pbs plus adjuvant and fr were used as negative controls. the immunized mice were boosted twice with intramuscular injections on days 14 and 28. mice were anesthetized on days 27 and 41 for ocular bleeding from the orbital sinus ( figure s10 in supplementary material). immune sera were processed by centrifugation of the collected blood at 12,000 × g for 30 min. the spleen and the balf (bronchoalveolar lavage) were obtained at 7 days after the last immunization from sacrificed mice. balf was taken by washing the airways with 1 ml of pbs. t-cell population from immunized mice were analyzed by flow cytometric analysis (43, 44) . the spleens were taken at 7 days after the last immunization from the sacrificed mice. to obtain single-cell suspensions, the tissues were homogenized and passed through 70 µm cell strainers (spl). after centrifugation, erythrocytes were removed by red blood cell lysing buffer (sigma). the cells were washed and resuspended in iscove's modified dulbecco's media containing 10% fbs. for intracellular cytokine staining, the splenocytes were stimulated with 10 µg/ml rbd protein or phorbol myristate acetate/ionomycin in the presence 10 ng/ml recombinant human il-2 (biolegend) and brefeldin a (1:1,000; ebioscience) at 37°c for 5 h. after stimulation, the cells were blocked with rat anti-mouse cd16/cd32 (bd biosciences) and surface stained with anti-cd8 (fitc, clone 53-6.7; biolegend) and anti-cd4 (pe/cy7, clone gk1.5; biolegend) at 4°c for 30 min. the stained cells were fixed in facs lysing solution (bd biosciences) at room temperature for 20 min, and permeabilized with facs buffer (0.5% fbs, 0.1% nan3 in pbs) containing 0.5% saponin (sigma) at room temperature for 15 min. then, the cells were stained with anti-ifn-γ (pe, clone xmg1.2; biolegend) and anti-tnf-α (apc, clone mp6-xt22; biolegend) at room temperature for 40 min. all data were collected by bd lsr fortessa (bd biosciences) and analyzed with flowjo software (tree star inc., ashland, or, usa). competition elisa was performed to determine whether mers-cov antigen [rbd-[ssg]-fr, rbd-fr, rbd, and fr (negative control)]-immunized mouse serum inhibited binding of rbd protein to hdpp4 receptor (45, 46) . 500 ng/well hdpp4 protein (abcam) was coated on nunc 96-well microtiter immunoplates (thermo fisher scientific) and incubated overnight at 4°c. plates were washed and blocked with 150 µl/well of blocking buffer [5% skim milk in pbs and tween-20 (pbst)] for 1 h at 37°c. at the same time, mouse sera immunized with rbd, rbd-[ssg]-fr, rbd-fr, and fr were serially diluted (1/10 to 1/160) with 500 ng/well rbd protein (mers-rbd-005p; eenzyme) in tbst [50 mm tris-cl (ph 7.4), 0.05% tween-20], added to new wells, and incubated for 1 h at 37°c. 100 µl solution was added to each well at 37°c and incubated for 2 h. after that, 100 µl of anti-6xhis tag antibody conjugated with horseradish peroxidase (1:1,000, thermo fisher scientific) was added to each well and incubated for 1 h at 37°c. plates were washed three times with tbst, and 100 µl/well of substrate tmb solution (bd biosciences) was incubated at 37°c for 30 min in the dark. 50 µl of stop solution (2 n h2so4) was added to the well to stop the color reaction and measure the absorbance at 450 nm using an elisa reader fluostar optima (bmg labtech). the hrid facilitated the solubility of mers-cov rbd-fr the spike glycoprotein (s) of mers-cov was used for the generation of mers-cov-like nps. s protein forms trimers, resulting in large spikes on the virus envelope (47) . it is challenging to express the full-sized s protein (~200 kda) in e. coli. thus, the s1 domain of s protein (~80 kda), which includes the receptor-binding ability, was used. our initial attempt to express the s1 domain, either as s1 or as an s1-fr fusion protein, failed; the expression level and solubility of the protein was below the lower limit of detection by sds-page and western blotting ( figure s1 in supplementary material). we therefore used the rbd (367-606 a.a.) of the s1 protein, which has a pivotal function as illustrated in figure 1b (48, 49) . when expressed alone in e. coli, the rbd is not able to form the trimeric assembly (unpublished observation), due to the lack of the hr2 domain within the s2 domain (50) . to overcome this problem, fr was used as scaffold for the assembly. fr is a spherical np whose subunits form trimers that subsequently result in octahedral structures composed of 24 identical subunits (51) . we therefore performed computational modeling to evaluate the potential of fr as scaffold for trimer formation of the rbd. possible trimer formation was analyzed by computational modeling using modeler (13, 52) and cluspro (41, 42) . various linkers, including ssg, asg, and d6, were introduced between the rbd and fr with a goal to minimize steric hindrance between the two domains so as to enhance trimer and np formation. in silico analysis showed energy-stable trimeric models of rbd-fr, rbd-[ssg]-fr, and rbd-[asg]-fr, whereas rbd-d6-fr failed to form a trimeric structure ( figure 1a) . the rbd-[ssg]-fr was predicted to be the most stable and well-structured compared with rbd-fr and rbd-[asg]-fr. initial testing of the rbd-fr constructs without hrid fusion showed that none of the constructs were solubly expressed, even under low-temperature culture conditions ( figure 1c ) ( figure 1b) . we previously confirmed that by using chaperna, the globular domain of influenza hemagglutinin (ha) is efficiently assembled into a trimeric complex with an immunologically relevant conformation (yang et al., in press) . as shown in figure 1c , the hrid fusion significantly increased the solubility of both rbd-fr (59.1%) and rbd-[ssg]-fr (62.83%), indicating that the chaperna platform effectively increased both the solubility and the folding of its fused target proteins. because of the poor expression level and low solubility of the rbd-[asg]-fr construct (figure 1c) , further experiments were performed using only the rbd-fr and rbd-[ssg]-fr constructs. after purification of the soluble proteins ( figure s2 in supplementary material), we determined the potential effects of using hrid as a fusion partner for the self-assembly of the nps. as shown in figure s3 in supplementary material, hrid-rbd-fr failed to form nps. because of this, we performed tev protease cleavage of the hrid. removal of the hrid domain facilitated the self-assembly of the rbd-fr monomers, and also eliminated the immune response against the non-self hrid domain in balb/c mice ( figure s4 in supplementary material). after hrid cleavage, rbd-fr and rbd-[ssg]-fr were purified using sec (figure 2a) . as expected, rbd-[ssg]-fr assembled into properly formed nps (1,080 kda) more efficiently than did rbd-fr nps, which were mainly detected in the void-volume fractions, suggesting they were irregularly assembled soluble aggregates. the size of the rbd-[ssg]-fr nps was further confirmed by tem. tem images of the rbd-[ssg]-fr np structures showed hollow, spherical particles that were more compact than the rbd-fr nps. the average diameter of the rbd-[ssg]-fr nps was 28-30 nm (figure 2b) . in contrast, dls analysis of the rbd-fr np structure without the ssg linker appeared to be smaller with an average intensity diameter of 26.3 nm, and this compared with rbd-[ssg]-fr that had an average intensity distribution diameter of 30.5 nm (figure 2c) . consistent with the sec analysis, rbd-fr without a fusion partner was mostly produced in a soluble aggregated form. therefore, we identified that the protein folding did not occur properly without hrid, and the formation of nps was confirmed by both sec and sds-page analyses. as shown in figure s5 in supplementary material, the purified nps retained their stability over an extended period of time at various temperatures (25, 4 , and −20°c). thus, these results indicate that the ssg linker allowed the rbd-fr to generate properly assembled nps. it should also be noted that the efficiency of protein folding and nps formation may be further enhance through appropriate linker selection. it has been reported that ionic strength plays an important role in the stability and self-assembly of ferritins (53, 54) . we examined the effect of salt concentration on the formation and stability of the rbd-[ssg]-fr nps at various concentrations (0-300 mm). consistent with the previous studies, the stability of the protein was highly affected by the concentration of nacl in the lysis buffer by sds-page ( figure 3a ) (n = 3). the solubility of the protein significantly decreased as the concentration of nacl increased from 0 to 100 mm, with the solubility being about 8.79-fold lower at 100 mm compared with 0 mm. unlike previous studies, the solubility of the protein was gradually recovered at higher nacl concentrations (>100 mm); the solubility at 300 mm was 1.45-fold higher than at 0 mm. furthermore, the yield of soluble of protein per liter of culture increased in a salt concentrationdependent manner (figure 3b) . to further investigate the effect of salt concentration, the physicochemical and morphological properties of the rbd-[ssg]-fr protein were examined by sec, tem, and dls. in 50 mm nacl, most of the protein was aggregated during the purification process, and the purified protein failed to form spherical structures, but instead, existed predominantly as 45 kda monomers (figures 3c,d; figure s6 in supplementary material). in contrast, the protein that was lysed in 0 mm nacl and purified in 200 mm nacl, developed well-structured nps according to tem and dls analyses (figures 3c,d; figure s6 in supplementary material). however, based on sec analysis, at high-salt concentrations (>250 mm), the protein failed to form stable structures with the proteins being eluted predominantly in the void volume, suggesting they were soluble aggregates under the high-salt concentrations ( figure 3c) . transmission electron microscopy images under the various salt concentrations clearly supported the conclusion, showing that the tendency for aggregation was dependent on the salt concentration ( figure 3d) . taken together, the results underscored the importance of salt concentration on the solubility of monomers and the quality of multimeric assembly of hybrid nps. ferritin has an intrinsic ability to interact with fe 2+ to form ferritin-iron cores (55) . thus, it was worth investigating the effect of fe 2+ on the assembly and stability of rbd-[ssg]-fr nps. cells were grown in lb medium with various concentrations of fe 2+ . as shown in figure 4a , the yield of purified protein was significantly increased from cultures with 500 µm fe 2+ , reflecting a 2.7-fold increase compared with similar cultures 0 µm fe 2+ . the cell growth and purification yield at 1,000 µm fe 2+ were slightly decreased, presumably due to the toxicity of ferric acid. np formation under the various concentrations of fe 2+ was analyzed by sec ( figure 4b) . consistent with the previous results, the proteins were eluted mainly in the fractions expected for the size of assembled nps (1,080 kda). of note, the ratio between nps and soluble aggregates in the sec analysis showed that np formation was facilitated at high concentrations of fe 2+ (figure 4b) . the formation of rbd-[ssg]-fr nps at an fe 2+ concentration of 1,000 µm was confirmed by tem ( figure 4c ) and dls ( figure 4d) . the tem analysis clearly showed that the morphology of the proteins was more compact, and probably highly stable, when assembled at high fe 2+ concentrations (500 µm) than at lower concentrations (0 µm) ( figure 4c ). as shown in figure 4d , the average diameter of nps examined by dls was 25.1 nm at high fe 2+ concentration (500-1,000 µm) and 27.7-32.2 nm at lower concentration (0-200 µm). these results suggest that both fe 2+ and salts concentrations influenced the efficiency and quality of the regular assembly of hybrid ferritin monomers into nps. our previous studies show that an rna-protein interaction is crucial for transducing the chaperone function of rna into the folding of client proteins (38) . consistent with that, our present study showed that rna facilitated the folding of its interacting proteins. the solubility of hrid(wt)-rbd-fr was 5.69-fold higher than rbd-fr without hrid fusion (figure 1) , strongly supporting the previous studies. in addition, the solubility of rbd alone was completely insoluble (figure 5b ; figure s7 in supplementary material). it has been shown that the positively charged residues of lysine moieties in hrid contribute to trna binding (56) . in the current study, the trna binding induced the intrinsically disordered protein (idp) status of hrid to form alpha-helical structures ( figure 5a) . thus, two rna-binding (table s1 in supplementary material). the total e. coli lysate (t) was fractionated into the soluble fraction (s) and the pellet fraction (p) by centrifugation. as expected, both rbd and rbd-fr without fusion to hrid domain, were refractory to being produced as soluble proteins ( figure 5b) . interestingly, the solubility of the rna-binding mutants did not decrease, but actually increased to 75.3% for the 2 m mutant and 93.4% for the 9 m mutant compared with wild-type protein at 60.1% (figure 5b) . considering that hrid is relatively unstructured in the absence of trna binding, the results are consistent with previous reports that the fusion with idps promotes the solubility of target proteins (57) (58) (59) . following purification of wild-type hrid-rbd-fr (hrid(wt)-rbd-fr), electrophoretic mobility shift assays showed that greater amounts of nucleic acids were co-purified with hrid(wt)-rbd-fr protein than with the mutant hrid-rbd-frs (2 and 9 m) under non-denaturing conditions ( figure 5c ). the relative ratio of nucleic acid based on etbr staining and proteins based on coomassie staining in the eluted fraction confirmed the reduced affinity of mutants to nucleic acids. to test if rna had a role in maintaining the stability of the target proteins, the lysates were treated with rnase a to eliminate rna, and the solubility of each protein was analyzed by sds-page and western blotting. the soluble fractions of the lysates (s) were incubated at 37°c in the presence and absence of rnase a and the samples were further separated into soluble fraction (ss′) and insoluble fraction (sp′) by centrifugation. as shown in the left panel of figure 6 , rnase a treatment completely abolished the effect of rna on protein solubility as compared with the control (rnase a−) or with samples prior to rnase treatment. parallel experiments with the 2 and 9 m mutants showed much less rna co-purified with the proteins, confirming the reduced affinity to nucleic acids and the complete depletion of rna by rnase a treatment (figure 6, left panel) . remarkably, the solubility of hrid(wt)-rbd-fr was greatly reduced by depletion of rna as reflected in the ratio of [ss′]/ [sp′] [0.1 and 0.4 for rnase (+) and rnase (−), respectively] by both coomassie staining and western blot analyses (figure 6 , right panel). however, the solubility of the mutants (2 and 9 m), was not significantly affected by rnase a treatment, probably due to their lower affinity to rna (figure 6, right panel) . taken together, the results demonstrate that hrid(wt)-rbd-fr maintained a strong affinity for rna, and that affinity was pivotal for maintaining the solubility of the protein. to further define the rna dependence of solubility of the ferritin hybrids (figure 6) , we investigated if the rna binding had a role in the formation of nps. rbd-fr and the various hrid-rbd-fr (wt, 2, and 9 m) proteins were purified by nickel-affinity chromatography ( figure s2 in supplementary material) and their physicochemical properties analyzed by sec (figure 7a) , tem (figure 7b) , and dls ( figure 7c) . the soluble yields of rbd-fr (hrid fusion) was approximately 1.6 mg/l of culture, representing greater than 1,000-fold higher levels than its hrid (−) counterpart (~15 μg/l culture), again confirming the role of hrid as a robust enhancer for solubility and assembly. it was striking to note that the two mutant proteins, despite high solubility (figure 5b) , were detected at disproportionately higher amounts in the void fractions of sec, indicating that they failed to form nps of a defined size, and existed predominantly as soluble aggregates ( figure 7a) . however, hrid(wt)-rbd-fr predominantly formed nps of a defined size (~1,080 kda). it is also interesting to note that there was a slight shift of the rna-binding mutants (2 and 9 m) in the elution pattern, suggesting a larger size of nps compared with wild-type nps. overall, the ratio between soluble aggregates in the void volume and the nps of defined size clearly showed that rna binding was crucial for assembly of the monomers into nps. as a control, rbd-fr (without hrid fusion) existed predominantly as soluble aggregates ( figure s8 in supplementary material). consistent with these results, em analysis confirmed well-structured nps by hrid(wt)-rbd-fr, compared to largely aggregated structures by the mutant proteins ( figure 7b) . even if multi-molecular structure was formed, the structure becomes unstable, mostly as soluble aggregates. consistently, the intensity distribution diameter of the wild-type protein, as estimated by dls analysis, was 25 nm compared with larger sizes of hrid(2 m) at 34.2, 519.2 nm and hrid(9 m) at 52, 717.7 nm (figure 7c ; figure s9 in supplementary material). it is conceivable that soluble aggregates may shield the exposed 6-histidine tag, resulting in a decreased binding affinity to nickel resins and elution in earlier fractions compared with wt protein ( figure s2 in supplementary material). taken together, the data demonstrate that rna binding prevented aggregation into irregular conformations and guided the self-assembly of the hybrid ferritin monomers into nps of a stable structure. the immunological properties of ferritin nps were analyzed by elisa. the hddp4 (human dpp4) receptor has been previously identified as the receptor for mers-cov human infection (46) . therefore, using hdpp4 as a coating antigen, elisa-binding assays between rbd nps and the receptor were performed (figure 8) . fr without rbd fusion failed to bind, and was similar to the pbs negative control. strikingly, the binding ability increased in the same order as the rna-binding ability (hrid(wt) > 2 m > 9 m), with highest absorbance observed in the wt with the ssg linker (hrid(wt)-rbd-[ssg]-fr). the results show that the conformation of rbd in the wt nps better resembled the protective antigen of mers-cov rbd from 293 cells, compared with the rna-binding mutants 2 and 9 m. again, judicious choice of linker between the ferritin carrier and the antigen was important for receptor binding and was reflected in its importance for np assembly into a stable conformation (figure 2) . finally, the elisa results for np against human patients was investigated using the sera from four mers-cov-infected patients (figure 9) . six different proteins, including five recombinant nps (hrid(wt)-rbd-fr, hrid(2 m)-rbd-fr, hrid(9 m)-rbd-fr, hrid(wt)-rbd-[ssg]-fr, and fr), and mers-cov rbd protein were compared by elisa using them as capture antigens. strong elisa signals were detected for the four recombinant nps and mers-cov rbd from 293 cells (positive control). the wt form consistently showed a higher response than the rnabinding mutants (hrid(wt) > 2 m > 9 m), with hrid(wt)-rbd-[ssg]-fr being the best binder among constructs tested. these results address to the utility of the e. coli assembled mers-cov rbd-fr nps as useful tools for sero-diagnosis of mers-cov infection. taken together, the results confirmed the immunologically relevant conformation of the mers-cov rbd displayed on the hybrid ferritin particles, and the crucial role of rna in controlling the kinetic pathway for the assembly of viral antigen monomers into stable nps. to evaluate the immunogenicity of ferritin-based nps, balb/c mice (n = 5) were immunized with rbd, rbd-fr, and rbd-[ssg]-fr nps antigens. the trnas were found to be removed from the hrid protein during the purification process. before immunization, potential rna contamination in the purified proteins was determined by gel electrophoresis. as shown in figure s11 in supplementary material, rna was below detection level, if any, after several purification steps, compared with the proteins purified in the first step. previously, mf59-adjuvated and alum-adjuvated mers-cov antigen have been reported to increase the antibody and t-cell responses in mice (44, 60) . thus, the first group and second group were immunized twice with 20.0 µg of antigen containing the equal volume of alum figure s2 in supplementary material and size exclusion chromatography was used to explore hdpp4 receptor-binding affinity to the protein. all data are shown as mean ± sd from triplicate samples. fr alone and phosphate-buffered saline were used as negative controls. figure s2 in supplementary material were used as coating antigens. fr alone and infected cell lysates were used as negative and positive controls, respectively. virus-infected sera from four patients were serially diluted from 1:100 (twofold dilution). all data are presented as mean ± sd of duplicate samples. higher than rbd, respectively. the antibody responses by rbd-fr and rbd-[ssg]-fr nps were much stronger than the rbd in all antibody subtypes tested (igg1, igg2a, and igg2b) (figures 10b-d) . as a test of mucosal immune responses, the rbd-specific iga antibody levels from balf were also analyzed by elisa (figure 10e) . mf59 adjuvanted rbd-[ssg]-fr nps presented significantly higher od values than rbd and fr (negative control). these results suggested that rbd-[ssg]-fr nps induces local mucosal immune response stronger than rbd. in addition, it was confirmed that antibody responses of igg, igg1 (th1), igg2a, and igg2b (th2) against mf59-adjuvated antigens were higher than those from alum-adjuvated antigens. in contrast, pbs and fr control groups failed to, or only weakly induce an antibody response against rbd protein. these results suggest that fr-based nps significantly enhance various antibody responses than monomeric antigens. the cellular immune responses were investigated in mice immunized with protein (rbd, rbd-fr, rbd-[ssg]-fr) and fr (negative control). splenocytes of mice (n = 4) were harvested 1 week after the last immunization, stimulated with | nanoparticles-immunized mouse serum inhibited interaction between middle east respiratory syndrome-coronavirus receptor-binding domain (rbd) and hdpp4 receptor. competition enzyme-linked immunosorbent assay showed that anti-rbd mouse sera (1:10, from mice immunized with rbd-[ssg]-fr, rbd-fr, and rbd) blocked binding between rbd (5 µg/ml) and hdpp4 receptor (5 µg/ml). fr-immunized mouse serum (1:10) was used as a negative control. all sera were serially diluted from 1:10 (twofold dilution). all data are presented as mean ± sd (n = 5) and p-values were obtained using student's two-tailed tests (***p < 0.001). figure 10 | immune responses in receptor-binding domain (rbd) nanoparticles (nps) immunized mice (n = 5). endpoint titer of igg (a), igg1 (b), igg2a (c), and igg2b (d) antibody binding to middle east respiratory syndrome-coronavirus rbd were detected using mice serum after two immunizations. rbd-specific antibodies were detected after immunizations of rbd nps, rbd, fr with adjuvant (alum and mf59) using enzyme-linked immunosorbent assay. (e). rbd-specific iga antibodies were detected using balf (diluted 1:8) after immunization of protein with mf59. od, optical density. each endpoint titer was shown by individual. all error bars were shown as mean ± sd (n = 5) and all p-values were obtained using student's two-tailed tests (**p < 0.01, ***p < 0.001). rbd protein, and analyzed for cytokines by flow cytometry. in the rbd-immunized group, ifn-γ and tnf-α-producing cd4 + t-cell responses were detected at low levels. however, ifn-γ and tnf-α-producing cd4 + t cells were significantly increased in rbd-fr and rbd-[ssg]-fr-immunized groups compared with rbd and fr-immunized group ( figure s12 in supplementary material) . these results demonstrated that the rbd nps vaccination induced antigen-specific cd4 + t cells that produced ifn-γ and tnf-α upon antigen stimulation. anti-nps serum effectively blocked rbd protein binding to the hdpp4 receptor middle east respiratory syndrome-coronavirus infection is mediated by the interaction of rbd and the host receptor hdpp4 (45, 46) . as a correlate of protection, a competition elisa was performed to investigate whether antibodies generated from nps immunization were able to interfere with the binding to hdpp4. thus, after incubation of rbd protein with mouse serum (1:10), the binding of serum-mixed samples to hdpp4 protein was measured. as shown in figure 11 , rbd-[ssg]-fr, rbd-fr, and rbd-immunized sera strongly abolished the binding of rbd to hdpp4 receptor (93.3, 82.2, and 75.67%, respectively). interestingly, the relative efficiency of interference correlates with that of np assemblage (figure 11 ). in contrast, the fr-immunized mouse serum (negative control) failed to inhibit the interaction. taking together, these results demonstrate that immunization of nps greatly stimulates mers-covspecific antibody response that effectively interferes with the cellular receptor binding, suggesting its possibility as a vaccine. however, protection efficacy should ultimately be tested in a live virus challenge model. having key immunologic features, like a highly repetitive nanostructure, provides a designing principle for nps in inducing potent and long-lasting antibody responses. for vlps of non-enveloped viruses, assembly is made purely by capsid proteins. for enveloped viruses, however, additional membrane components and matrix proteins are required to display the target antigens on the surface of assembled vlps. a promising alternative is to present target antigen on the surfaces of selfassembled nps, which, in lieu of lipid membranes and matrix proteins, serve as a macromolecular scaffold for the presentation of antigens of interest (61) . ferritins, as a substitute for matrix proteins and membranes, have been used as scaffold for the regular assembly of target antigens. however, ferritin-based nps have been produced only in host cells of mammalian or insect origin (28, 62) . previously, we showed that influenza ha could be assembled in a soluble, trimeric, and immunologically relevant conformation by exploiting chaperna activity (63) . the present study is the first report of using rnas as molecular chaperone for supra-molecular structures. here, we present a novel bacterial system for np assembly of hybrid ferritin displayed surface antigens from mers-cov. the nps reacted strongly with sera derived from mers-cov-infected patients (figure 9 ) confirming their utility in sero-diagnosis of infection. moreover, the antisera, generated from immunization of mice, were able to interfere with the binding to the cellular receptor hdpp4 (figure 8 ), in part of essential protective immune responses. the efficiency of receptor-binding inhibition (figure 11) , as well as the ability for inducing the mucosal responses (figure 10e) , correlated with the regular assembly of nps as examined by dls or em (figure 2) , confirming that presentation of antigenic epitopes on a multivalent and highly repetitive structure is indeed important for the quality of immune responses. overall, the quality of nps and consequent immune responses were governed by the rna-mediated assembly of antigens. we hypothesized that chaperna function could be harnessed for presenting target antigens as highly repetitive nanostructures ( figure 1d) . the hrid is the n-terminal domain of hlysrs and was previously identified as a nucleic acid-binding domain ( figure 5 ) (63) . in this report, the hrid was exploited as a transducer for chaperna function (tcf) by serving as a docking-tag for cellular rna for the folding/assembly of the hybrid fr containing client antigen proteins [rbd of mers-cov (figure 1d) ]. the advantage of using hrid as a tcf could be many fold. first, hrid is small (8.3 kda), monomeric, and was flexible enough to allow the access of site-specific protease for the removal of hrid ( figure s3 in supplementary material). of note, hrid belongs to idps, which switches into stabile α-helixes upon binding with trnas. second, the bound rna, due to its highly negative charge, may resist uncontrolled intermolecular interactions among monomers into amorphous aggregation. finally, even the naked hrid (in the absence of rna binding), due to its intrinsically flexible nature, may not pose physical hindrance to multiple interactions among monomers, enabling assembly into stable super-structures, upon removal of the hrid. thus, the potential "pace-making" function harnessed with the rna molecule, allows a regular assembly of monomers as highly repetitive nanostructures. consequently, in the current study, hybrid fr was produced in soluble forms, could be purified by one-step affinity chromatography, and most remarkably, assembled into nps of defined sizes upon removal of the hrid ( figure s2 in supplementary material). consistent with the principles of design, the loss of rna binding by hrid significantly hampered the regular assembly of the ferritin monomers and increased the amount of non-functional misfolded proteins as soluble aggregates (figure 7) . thus, the overall yield, as well as the quality of nps, were dependent on the chaperna function transduced by the hrid, which in turn was mediated by interaction with cellular rnas (likely to be trnas). the driving and controlling factors for de novo assembly of biomolecules are poorly understood. historically, host factors like groel/s were initially discovered as molecular chaperones for supporting viral growth in e. coli and supporting the assembly of viral capsid proteins (64, 65) . moreover, groel/s also cooperates with rbcx in plant cells for the assembly of multicomponent rubisco, which is the most abundant protein in the biosphere responsible for photosynthesis (66) . therefore, it is intriguing that rna could provide such a robust folding/ assembly of a supra-molecular structure. we recently confirmed that the present strategy could be successfully applied to the assembly of bacterially synthesized monomers of norovirus into vlps composed of 180 monomers (unpublished observation, seong, b.l.). whether rna can substitute for, or collaborate with pre-existing protein-based molecular chaperones remains an exciting avenue for future investigations. it should be noted that the defined versatile functions are being expanded for rna molecules. as an engineered system for harnessing chaperna function, the present report may prove to be the tip of an iceberg for pivotal function of rna molecules as chaperones for the folding and supra-molecular assembly of proteins in living organisms (36, 38) . various factors were identified as important for efficient assembly of mers-cov nps. as an extrinsic factor, the binding affinity of hrid to cellular rnas was crucial for the assembly and the quality of the assembled nps (figure 7) . as intrinsic factors, the concentration of salts and fe 2+ also influenced the assembly and stability of nps (figures 3 and 4) . the ionic strength played an important role in the stability and self-assembly of ferritins, and aggregation increased with increasing concentrations of nacl (54) . the assembly of the hybrid mers-cov nps revealed an interesting change in salt dependence, with 200-225 mm nacl buffer as optimal condition as confirmed by em and dls analyses (figure 3) . the change in salt dependence was probably due to the presence of electrostatic interactions among rbd domains (54, 67) . the dependence on fe 2+ was not surprising considering that ferritin has an intrinsic ability to interact with fe 2+ to form ferritin-iron cores (55) . based on our experience, to enhance the quality of nps, it is advisable to control fe 2+ concentrations, both during the culturing of the bacterial cells and during the purification of the soluble monomer proteins (figure 4) . first, the yield of the purified protein was increased in the presence of 500 µm fe 2+ (figure 4b) , up to 2.7-fold greater compared with the control conditions lacking fe 2+ . second, the ratio between nps and soluble aggregates in sec showed that nps formation was facilitated at high concentrations of fe 2+ , and resulted in a more compact morphology under em (figures 4b,c) . thus, both the overall yield and the quality of nps were governed by their intrinsic ability to interact with fe 2+ . finally, our data show that the presence and the nature of the linker between the ferritin and the rbd antigen was also important to the assembly of nps. it is possible that a linker with flexibility and sufficient length would accommodate the steric requirements for assembly of multimeric nps. however, it is difficult to precisely predict the effect of the linker, and therefore it is advisable to screen multiple constructs during the early stages of testing the assembly of nps displaying antigens of interest. in conclusion, the chaperna-based antigen assembly platform holds promise for the development and delivery of np-based vaccines to enhance rbd-specific antibody responses, and the serological detection of emerging viruses. various types of designing principles have advanced the structure-based approaches to np assembly (61, 68) . however, most of the in silico methods consider the thermodynamic stability of the final assembled nps, but not necessarily the kinetic pathways leading to their successful folding into regular assemblages. consequently, most nps are refractory to soluble expression and fail to assemble as designed, resulting in significant, and practical challenges in the manufacturing process. the chaperna-mediated folding and the "pace-keeping" assembly of monomers into higher ordered structures will enable faithful production of np and vlp-based vaccines against emerging and re-emerging viral infections. this study was carried out in accordance with the recommenda figure 7 | elucidation of rna-mediated nanoparticle (np) formation of receptor-binding domain (rbd)-fr. (a) size exclusion chromatography analysis of rbd-fr nps purified from the tev protease-cleaved hrid(wt, 2, or 9 m)-rbd-fr. the fractions (11-12 ml) estimated as nps were further analyzed by transmission electron microscopy (b) and dynamic light scattering (c) exploiting virus-like particles as innovative vaccines against emerging viral infections traditional and new influenza vaccines vaccine manufacturing: challenges and solutions management of accidental exposure to ebola virus in the biosafety level 4 laboratory vaccine delivery using nanoparticles vaccine delivery: a matter of size, geometry, kinetics and molecular patterns virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development the influence of antigen organization on b cell responsiveness structure of the hepatitis e virus-like particle suggests mechanisms for virus assembly and receptor binding self-assembly of human papillomavirus type 1 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mers-cov and its receptor cd26 structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor stability of a 24-meric homopolymer: comparative studies of assembly-defective mutants of rhodobacter capsulatus bacterioferritin and the native protein evaluation of comparative protein structure modeling by modeller-3 salt-dependent aggregation and assembly of e coli-expressed ferritin ferritin protein nanocages use ion channels, catalytic sites, and nucleation channels to manage iron/oxygen chemistry a peptide from the extension of lys-trna synthetase binds to transfer rna and dna sweeping away protein aggregation with entropic bristles: intrinsically disordered protein fusions enhance soluble expression intrinsically disordered proteins and intrinsically disordered protein regions intrinsically unstructured proteins and their functions identification of an ideal adjuvant for receptor-binding domain-based subunit vaccines against middle east respiratory syndrome coronavirus protein/peptide-templated biomimetic synthesis of inorganic nanoparticles for biomedical applications self-assembling influenza nanoparticle vaccines elicit broadly neutralizing h1n1 antibodies harnessing an rna-mediated chaperone for the assembly of influenza hemagglutinin in an immunologically relevant conformation host participation in bacteriophage lambda head assembly properties of a mutant of escherichia coli defective in bacteriophage lambda head formation (groe). i. initial characterization coupled chaperone action in folding and assembly of hexadecameric rubisco modeling the influence of salt on the hydrophobic effect and protein fold stability rational design of an epstein-barr virus vaccine targeting the receptor-binding site key: cord-314574-3e6u4aza authors: tian, xiaolong; li, cheng; huang, ailing; xia, shuai; lu, sicong; shi, zhengli; lu, lu; jiang, shibo; yang, zhenlin; wu, yanling; ying, tianlei title: potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody date: 2020-02-17 journal: emerg microbes infect doi: 10.1080/22221751.2020.1729069 sha: doc_id: 314574 cord_uid: 3e6u4aza the newly identified 2019 novel coronavirus (2019-ncov) has caused more than 11,900 laboratory-confirmed human infections, including 259 deaths, posing a serious threat to human health. currently, however, there is no specific antiviral treatment or vaccine. considering the relatively high identity of receptor-binding domain (rbd) in 2019-ncov and sars-cov, it is urgent to assess the cross-reactivity of anti-sars cov antibodies with 2019-ncov spike protein, which could have important implications for rapid development of vaccines and therapeutic antibodies against 2019-ncov. here, we report for the first time that a sars-cov-specific human monoclonal antibody, cr3022, could bind potently with 2019-ncov rbd (kd of 6.3 nm). the epitope of cr3022 does not overlap with the ace2 binding site within 2019-ncov rbd. these results suggest that cr3022 may have the potential to be developed as candidate therapeutics, alone or in combination with other neutralizing antibodies, for the prevention and treatment of 2019-ncov infections. interestingly, some of the most potent sars-cov-specific neutralizing antibodies (e.g. m396, cr3014) that target the ace2 binding site of sars-cov failed to bind 2019-ncov spike protein, implying that the difference in the rbd of sars-cov and 2019-ncov has a critical impact for the cross-reactivity of neutralizing antibodies, and that it is still necessary to develop novel monoclonal antibodies that could bind specifically to 2019-ncov rbd. very recently, a novel coronavirus which was temporarily named "2019 novel coronavirus (2019-ncov)" emerged in wuhan, china [1] . as of 1 february 2020, 2019-ncov has resulted in a total of 11,821 laboratory-confirmed human infections in china, including 259 deaths, and 132 exported cases in 23 countries outside of china (https://www.who.int/emergencies/ diseases/novel-coronavirus-2019/situation-reports). currently, there is no vaccine or effective antiviral treatment against 2019-ncov infection. based on the phylogenetic analysis (gisaid accession no. epi_isl_402124) [2] , 2019-ncov belongs to lineage b betacoronavirus and shares high sequence identity with that of bat or human severe acute respiratory syndrome coronavirus-related coronavirus (sarsr-cov) and bat sars-like coronavirus (sl-cov) (figure 1(a) ). in previous studies, a number of potent monoclonal antibodies against sars coronavirus (sars-cov) have been identified [3] [4] [5] [6] [7] . these antibodies target the spike protein (s) of sars-cov and sl-covs, which is a type i transmembrane glycoprotein and mediates the entrance to human respiratory epithelial cells by interacting with cell surface receptor angiotensin-converting enzyme 2 (ace2) [8] . more specifically, the 193 amino acid length (n318-v510) receptor binding domain (rbd) within the s protein is the critical target for neutralizing antibodies [9] . some of the antibodies recognize different epitopes on rbd; e.g. the sars-cov neutralizing antibodies cr3014 and cr3022 bound noncompetitively to the sars-cov rbd and neutralized the virus in a synergistic fashion [5] . we predicted the conformation of 2019-ncov rbd as well as its complex structures with several neutralizing antibodies, and found that the modelling results support the interactions between 2019-ncov rbd and certain sars-cov antibodies (figure 1(b) ). this could be due to the relatively high identity (73%) of rbd in 2019-ncov and sars-cov (figure 1(c) ). for instance, residues in rbd of sars-cov that make polar interactions with a neutralizing antibody m396 as indicated by the complex crystal structure [10] are invariably conserved in 2019-ncov rbd (figure 1(d) ). in the structure of sars-cov-rbd-m396, r395 in rbd formed a salt bridge with d95 of m396-vl. concordantly, the electrostatic interaction was also observed in the model of 2019-ncov-rbd-m396, forming by r408 (rbd) and d95 (m396-vl). this analysis suggests that some sars-cov-specific monoclonal antibodies may be effective in neutralizing 2019-ncov. in contrast, the interactions between antibody f26g19 [11] or 80r [12] and the rbd in 2019-ncov decreased significantly due to the lack of salt bridges formed by r426-d56 in sars-cov-rbd-f26g19 or d480-r162 in sars-cov-rbd-80r, respectively. furthermore, while most of the 80r-binding residues on the rbd of sars-cov are not conserved on rbd of 2019-ncov ( figure 1(c) ), it is unlikely that the antibody 80r could effectively recognize 2019-ncov. therefore, it is urgent to experimentally determine the cross-reactivity of anti-sars-cov antibodies with 2019-ncov spike protein, which could have important implications for rapid development of vaccines and therapeutic antibodies against 2019-ncov. in this study, we first expressed and purified 2019-ncov rbd protein. we also predicted the conformations of 2019-ncov rbd and its complex with the putative receptor, human ace2. comparison of the interaction between the complex of ace2 [13] and sars-cov rbd and homology model of ace2 and 2019-ncov rbd revealed similar binding modes (data not shown). in both complexes, β5-β6 loop and β6-β7 loop form extensive contact, including at least seven pairs of hydrogen bonds, with the receptor. notably, r426 on the forth α helix in sars-cov rbd builds a salt bridge with e329 and a hydrogen bond with q325 on ace2. however, the arginine (r426 in sars-cov rbd) to asparagine (n439) mutation in 2019-ncov rbd abolished the strong polar interactions, which may induce a decrease in the binding affinity between rbd and the receptor. interestingly, a lysine (k417 in 2019-ncov rbd) replacement of valine (v404 in sars-cov rbd) on β6 formed an extra salt bridge with d30 on ace2, which may recover the binding ability. these data indicate that the rbd in s protein of 2019-ncov may bind to ace2 with a similar affinity as sars-cov rbd does. indeed, we measured the binding of 2019-ncov rbd to human ace2 by the biolayer interferometry binding (bli) assay, and found that 2019-ncov rbd bound potently to ace2. the calculated affinity (k d ) of 2019-ncov rbd with human ace2 was 15.2 nm (figure 1(f) ), which is comparable to that of sars-cov spike protein with human ace2 (15 nm) [14] . these results indicate that ace2 could be the potential receptor for the new coronavirus, and that the expressed 2019-ncov rbd protein is functional [2] . next, we expressed and purified several representative sars-cov-specific antibodies which have been reported to target rbd and possess potent neutralizing activities, including m396 [3] , cr3014 [4] , cr3022 [5] , as well as a mers-cov-specific human monoclonal antibody m336 developed by our laboratory [15] , and measured their binding ability to 2019-ncov rbd by elisa (figure 1(e)) . surprisingly, we found that most of these antibodies did not show evident binding to 2019-ncov rbd. to confirm this result, we further measured the binding kinetics using bli. an irrelevant anti-cd40 antibody was used as a negative control. similarly, the antibody m396, which was predicted to bind 2019-ncov rbd (figure 1(d) ), only showed slight binding at the highest measured concentration (2.0 µm). further studies are needed to solve the high-resolution structure of 2019-ncov rbd and understand why it could not be recognized by these antibodies. notably, one sars-cov-specific antibody, cr3022, was found to bind potently with 2019-ncov rbd as determined by elisa and bli (figure 1(e,f) ). it followed a fast-on (k on of 1.84 × 10 5 ms −1 ) and slow-off (k off of 1.16 × 10 −3 s −1 ) binding kinetics, resulting in a k d of 6.3 nm (figure 1(f) ). this antibody was isolated from blood of a convalescent sars patient and did not compete with the antibody cr3014 for binding to recombinant s protein [5] . to further elucidate the binding epitopes of cr3022, we measured the competition of cr3022 and human ace2 for the binding to 2019-ncov rbd. the streptavidin biosensors labelled with biotinylated 2019-ncov rbd were saturated with human ace2 in solution, followed by the addition of the test antibodies in the presence of ace2. as shown in figure 1 (g), the antibody cr3022 did not show any competition with ace2 for the binding to 2019-ncov rbd. these results suggest that cr3022, distinct from the other two sars-cov antibodies, recognizes an epitope that does not overlap with the ace2 binding site of 2019-ncov rbd. the rbd of 2019-ncov differs largely from the sars-cov at the c-terminus residues (figure 1(c) ). our results implied that such a difference did not result in drastic changes in the capability to engage the ace2 receptor, but had a critical impact on the cross-reactivity of neutralizing antibodies. some of the most potent sars-cov-specific neutralizing antibodies (e.g. m396, cr3014) that target the receptor binding site of sars-cov failed to bind 2019-ncov spike protein, indicating that it is necessary to develop novel monoclonal antibodies that could bind specifically to 2019-ncov rbd. interestingly, it was reported that the antibody cr3022 completely neutralized both the wild-type sars-cov and the cr3014 escape viruses at a concentration of 23.5 μg/ml, and that no escape variants could be generated with cr3022 [5] . furthermore, the mixture of cr3022 and cr3014 neutralized sars-cov in a synergistic fashion by recognizing different epitopes on rbd [5] . these results suggest that cr3022 has the potential to be developed as candidate therapeutics, alone or in combination with other neutralizing antibodies, for the prevention and treatment of 2019-ncov infections. we expect more cross-reactive antibodies against 2019-ncov and sars-cov or other coronaviruses to be identified soon, facilitating the development of effective antiviral therapeutics and vaccines. no potential conflict of interest was reported by the author (s). genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan a pneumonia outbreak associated with a new coronavirus of probable bat origin potent cross-reactive neutralization of sars coronavirus isolates by human monoclonal antibodies human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets. the lancet human monoclonal antibody combination against sars coronavirus: synergy and coverage of escape mutants potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus coronavirus spike proteins in viral entry and pathogenesis a 193-amino acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme 2 structure of severe acute respiratory syndrome coronavirus receptorbinding domain complexed with neutralizing antibody structural insights into immune recognition of the severe acute respiratory syndrome coronavirus s protein receptor binding domain structural basis of neutralization by a human anti-severe acute respiratory syndrome spike protein antibody, 80r structure of sars coronavirus spike receptor-binding domain complexed with receptor unexpected receptor functional mimicry elucidates activation of coronavirus fusion junctional and allelespecific residues are critical for mers-cov neutralization by an exceptionally potent germline-like antibody key: cord-297072-f5lmstyn authors: struck, anna-winona; axmann, marco; pfefferle, susanne; drosten, christian; meyer, bernd title: a hexapeptide of the receptor-binding domain of sars corona virus spike protein blocks viral entry into host cells via the human receptor ace2 date: 2012-01-16 journal: antiviral res doi: 10.1016/j.antiviral.2011.12.012 sha: doc_id: 297072 cord_uid: f5lmstyn in vitro infection of vero e6 cells by sars coronavirus (sars-cov) is blocked by hexapeptide tyr-lys-tyr-arg-tyr-leu. the peptide also inhibits proliferation of coronavirus nl63. on human cells both viruses utilize angiotensin-converting enzyme 2 (ace2) as entry receptor. blocking the viral entry is specific as alpha virus sindbis shows no reduction in infectivity. peptide (438)ykyryl(443) is part of the receptor-binding domain (rbd) of the spike protein of sars-cov. peptide libraries were screened by surface plasmon resonance (spr) to identify rbd binding epitopes. (438)ykyryl(443) carries the dominant binding epitope and binds to ace2 with k(d) = 46 μm. the binding mode was further characterized by saturation transfer difference (std) nmr spectroscopy and molecular dynamic simulations. based on this information the peptide can be used as lead structure to design potential entry inhibitors against sars-cov and related viruses. the sars-associated corona virus (sars-cov) has been identified as the causative agent of severe acute respiratory syndrome (sars) which emerged as an alerting epidemic in winter of 2002-2003 resulting in over 8000 infected cases with approximately 10% deaths ksiazek et al., 2003; marra et al., 2003; peiris et al., 2003; rota et al., 2003; who, 2004) . sars-cov infects human host cells by an initial interaction of its spike glycoprotein (s) and the receptor on human cells, angiotensinconverting enzyme 2 (ace2) (dimitrov, 2003; holmes, 2003; li et al., 2003) . functional characterization of the s protein suggests that the receptor-binding domain (rbd) is located between amino acid residues 303 and 537 (xiao et al., 2003) . flow cytometry indicated that amino acids 270-510 are the minimal receptor binding region of the s glycoprotein (babcock et al., 2004) . further studies located the rbd from residues 318 to 510. the rbd fused to the fc region of human igg 1 (rbd-fc) binds ace2 with higher affinity (k d $ 10 nm) than does the full length s1-ig chimera (li et al., 2005b; wong et al., 2004) . the crystal structure of residues 306-527 of the s1 in complex with the receptor ace2 reveals that a loop within the rbd (residues 424-494) makes all the contacts to ace2 and is referred to as the receptor-binding motif (rbm). six tyrosine residues are involved in direct binding to the receptor (li et al., 2005a) . studying the virus adaptation to humans the spike protein also seems to play a major role in the species specificity of coronavirus infection. especially, the introduction of a threonine residue at position 487 and an asparagine instead of a charged lysine residue at position 479 of the spike protein seem to be responsible for its high affinity to human ace2 (holmes, 2005; li et al., 2005b; qu et al., 2005) . yi et al. demonstrated that a single amino acid substitution (r441a) in a full-length spike protein dna vaccine failed to induce neutralizing antibodies (nabs) and that the same mutation yielded pseudoviruses that were unable to enter the human cells (yi et al., 2005) . furthermore, the rbd-fc bearing the same r441a mutation shows no affinity to ace2 and is not capable of blocking s protein-mediated pseudovirus entry (he et al., 2006) . the interaction of sars-cov with its receptor ace2 is an attractive drug target as epitopes of the rbd on the spike protein may serve as leads for the design of effective entry inhibitors (du et al., 2009) . another drug target is the fusion process of the spike protein with the host cell membrane that is characterized by the presence of two heptad repeat (hr) regions, hr1 and hr2, which are postulated to form a fusion-active conformation similar to those of other typical viral fusion proteins (sainz et al., 2006; van der hoek et al., 2004; yuan et al., 2004) . peptides were synthesized on solid phase using a fmoc-protecting group strategy on a fmoc-pal-peg-ps resin (applied biosystems) with o-(benzotriazol-1-yl)-n,n,n 0 ,n 0 -tetramethyluronium tetrafluoroborate (tbtu, iris biotech) as activator. a mos x496 synthesizer (advanced chemtech) and a liberty microwave synthesizer (cem) were used for peptide syntheses starting with 20 lmol amino groups each. after each coupling step the growing peptide was capped with an acetyl residue by 10% acetic anhydride in dmf. cysteine residues were substituted by serines to avoid dimerization. using trifluoroacetic acid (tfa), triisopropylsilan and h 2 o (95:5:2, v/v), peptides were cleaved off the resin leaving an amide at the c-terminus. the cocktail was applied twice for 90 and 60 min, respectively. preparative rp-hplc was carried out on a biocad 700e instrument (perseptive biosystems) using a h 2 o/acetonitrile gradient (0.1% tfa) on a vp250/21 nucleodur c18 pyramid 5 l column (macherey & nagel). peptides were characterized by maldi-tof mass spectrometry on a biflex iii instrument (bruker daltonics) in reflector mode using 2,5dihydroxybenzoic acid (dhb) or a-cyano-4-hydroxycinnamic acid (cca) as a matrix. peptide rbd-11b (ykyryl, y438-l443) and related peptides were further characterized by 1d-and 2d-nmr spectroscopy (not shown). spr studies were carried out using a biacore 3000 or biacore t100 instrument. for all experiments a temperature of 25°c, flow rate of 5 ll/min (biacore 3000) or 30 ll/min (t100), and a tbs running buffer (25 mm tris, 0.2 m nacl, 5 lm zncl 2 at ph 8) were used. the carboxymethylated sensorchip surface of a cm5 chip (biacore) was activated by nhs/edc followed by immobilization of rhace2 (r&d systems) in acetate-buffer (ph 3.5, biacore). rhace2 was obtained in tbs that had to be changed for the immobilization of the enzyme to pbs containing additional 5 lm zncl 2 (ph 7.4) using a slide-a-lyzer mini unit (pierce biotechnology) with a molecular weight cut-off of 3500 at 4°c for at least 12 h. sensorgrams of rbd-11 and rbd-14 were recorded with a chip that had 54 fmol of ace2 immobilized, that of rbd-15 on a chip with 74 fmol, those of rbd-11b and of the rbd-11b related peptide library on a chip with 100 fmol, respectively. carboxyl groups of the activated chip surface that had not reacted with the protein were capped with ethanolamine (biacore). 2.3. saturation transfer difference (std) nmr spectroscopy 2.3.1. sample preparation nmr samples were prepared in deuterated tris-buffered saline (d-tbs) containing 25 mm perdeuterotris(hydroxymethyl)aminomethane (tris-d 11 ), 0.2 m nacl and 5 lm zncl 2 (ph 7.8) in deuterium oxide (d 2 o, 99.9%). tbs of the commercial rhace2 (r&d systems) was changed to d-tbs in slide-a-lyzer mini units (pierce biotechnology) with a molecular weight cut-off of 3500 twice for at least 12 h at 4°c. ykyryl was added from 3 mm stock solution in d-tbs with sample volume adding up to 100 ll in a 3 mm shigemi nmr micro tube with c(ace2) = 0.83 lm and peptide concentration between 14.9 and 222 lm (18-288 fold excess over ace2). all std spectra were recorded at a temperature of 295 k with a spectral width of 15 ppm on a bruker avance drx 700 mhz spectrometer equipped with a 5 mm inverse triple resonance cryoprobe. selective saturation of the protein was achieved by a train of gauss-shaped pulses of 50 ms length each, truncated at 1%, and separated by a 1 ms delay leading to a total length of saturation time of 4 s. the on-resonance irradiation of the protein was performed at a chemical shift of à0.5 ppm. off-resonance irradiation was set at 40 ppm. total scan number in the std experiments was 4096. nmr spectra were multiplied by an exponential linebroadening function of 1.0 hz prior to fourier transformation. water suppression was achieved by an excitation sculpting pulse sequence. spectra processing was performed using topspin 2.1 software (bruker). the sars-cov inhibition assay was performed as described previously (vassilatis et al., 2003) . in brief, vero cells in 24-well plates were infected in the biosafety level 4 laboratory (bni hamburg) with sars-cov (frankfurt isolate) at a multiplicity of infection (moi) of 0.01. the inoculum was removed after 1 h and replaced with fresh medium complemented with different concentrations of compound. the virus rna concentration in the supernatant was measured by real-time pcr after 2 days. rna was prepared from 140 ll supernatant using diatomaceous silica (pfaff et al., 1994) . quantitative real-time reverse transcription-pcr (rt-pcr) was performed with the purified rna according to a published protocol . in vitro transcripts of the target region were used in the pcr to generate standard curves for quantification of the virus rna. the md simulations were carried out with the software maestro/desmond on an hp z600 workstation (one quadcore cpu), using the opls_aa/2005 force field. the starting structure was placed in a water box with orthorhombic boundary conditions and salt concentration of 200 mmol/l (spc solvent model, 18,145 water molecules, 78 â 88 â 102 å). md simulations over 11,000 ps were performed to equilibrate the system at 300 k. the simulation in equilibrium was performed over 3000 ps at 300 k, with the nose-hoover thermostat method and a relaxation time of 2.0 ps. the recording interval was 0.12 ps. before starting md simulations the system was minimized three times over 2000 steps, respectively. the period of the md after equilibration with constant potential energy was used for the analysis. table 1 synthetic peptide library of sixteen 12mer peptides comprising rbd-residues n318-t509 of sars-cov spike protein. the peptides rbd-11, rbd-14 and rbd-15 show binding to ace2. residues of the spike protein (s) amino acid sequence a library of linear peptides was synthesized via solid phase synthesis using fmoc strategy containing sixteen 12mer rbd peptides (rbd-1 to rbd-16) which together comprised residues n318-t509 (cf. table 1 ). cysteine residues were substituted by serine amino acids to avoid dimerization of the peptides. the compounds were used to identify binding motifs in interaction with the human receptor ace2 by surface plasmon resonance (spr) binding studies. this method allows the determination of the binding specificity, as table 2 synthetic peptide library of fourteen 6mer peptides comprising rbd-residues n435-e452 and a471-s500 of sars-cov spike protein. the k d of rbd-11b (ykyryl) is 46 lm. the on-and off-rates are 3.7 â 10 3 m à1 s à1 and 0.17 s à1 , respectively. peptide rbd-11c (rylrhg) shows k d = 770 lm, k on = 3.1 â 10 2 m à1 s à1 and k off = 0.24 s à1 . residues of the spike protein (s) amino acid sequence well as the association and dissociation rates of ligands interacting with protein receptors. the receptor protein ace2 was immobilized on the sensor chip and the peptides were passed over the sensor surface. the affinity of the interaction is determined from the level of binding at equilibrium as a function of sample concentration and can also be determined from analysis of the binding kinetics. spr screening of the sixteen 12mer rbd peptides resulted in positive responses of three peptides, i.e. rbd-11 (y438-r449 -ykyrylrhgklr), rbd-14 (s474-t485 -sywplndygfyt) and rbd-15 (t486-v497 -ttgigyqpyrvv), respectively. all other rbd peptides showed no or insignificantly small response signals and were thus not interacting with ace2. to analyze whether the interaction of the peptides rbd-11, rbd-14 and rbd-15 is based on a specific binding event, the spr sensorgrams of each compound were recorded at several different concentrations. the signal at equilibrium measured in response units [ru] obtained from the sensorgrams was plotted against the concentration of ligand passed over the sensor surface (cf. fig 1) . assuming a one site binding model the specific saturable binding affinity of the ligand peptides is calculated to k d = 85 ± 14 lm for rbd-11, k d = 450 ± 36 lm for rbd-14 and k d = 672 ± 168 lm for rbd-15. the spr sensorgrams were also used to determine kinetic parameters of binding, the association rate constant k on [s à1 m à1 ] and the dissociation rate constant k off [s à1 ]. rbd-11 shows a fivefold higher association rate constant k on = 1.8 â 10 3 s à1 m à1 compared to rbd-14 and rbd-15 with k on = 3.7 â 10 2 s à1 m à1 and 3.2 â 10 2 s à1 m à1 , respectively. the dissociation rate of all three peptides is comparable with k off = 0.12 s à1 (rbd-11), 0.16 s à1 (rbd-14) and 0.15 s à1 (rbd-15), respectively. the dissociation constant k d = k off /k on resulting from analysis of the kinetic data of the peptides are k d = 67 lm (rbd-11), 430 lm (rbd-14) and 450 lm (rbd-15) in excellent agreement with the dissociation constants obtained from thermodynamic data analysis. the on-rates suggest that the bioactive conformation of peptide rbd-11 is more similar to its solution conformation than for rbd-14 and rbd-15. peptides rbd-11, rbd-14 and rbd-15 contain 60% of the residues, namely y440, y442, y475, n479, y484, t486, t487, g488 and y491 that were identified by x-ray crystal structure analysis to be in contact with ace2 (li et al., 2005a) . several mutations in the viral protein were necessary to change the host organisms from wild animals, like civet cats, to humans, i.e. s487t and k479n (holmes, 2005; li et al., 2005b; qu et al., 2005) . these amino acids are included in the peptides rbd-14 and rbd-15. in a screening of a library hu in all spectra water was suppressed by an excitation sculpting pulse sequence and spectra were acquired in d-tbs at 295 k in a 100 ll (hwang and shaka, 1995) . (b) determination of binding affinity from std nmr titration data. the titration curve of the aromatic protons hd,d of tyr442 is shown. the k d value was determined from the std nmr titration by using the one site binding model. the resulting k d value is 60 ± 25 lm. (c) std nmr epitope mapping of rbd-11b. the spots indicate the range of relative std% for the protons that were saturated according to their proximity to the human receptor protein ace2. a strong binding is detected for the aromatic protons of the three tyrosines. the highest degree of saturation of 4.1 std% (absolute) obtained for he,e of tyr440 was set to 100% relative std. could inhibit the binding of rbd to ace2 and the plaque formation of sars-cov in vero cells with an ec 50 value of 41.6 lm . ho et al. showed biological activities of small peptides derived from s protein to inhibit the spike protein and ace2 interaction. among others peptide sp-8 (residues 483-494, fytttgi-gyqpy), which overlaps in sequence with the identified peptides rbd-14 and rbd-15, shows inhibitory activity at a 1 million fold excess relative to ace2 (ho et al., 2006) . however, the 20mer peptide p4 comprising residues 470-489 (palncywplndygfyttsgi) of zheng et al. overlapping with peptide rbd-14 shows no inhibition in a cytopathic effect (cpe)-based assay . in contrast, as mentioned above, the partially overlapping peptides a471-l503 and sp-8 (ho et al., 2006) showed virus inhibition in their respective assays. to further characterize the binding epitope we synthesized a second peptide library of fourteen hexamer peptides comprising residues n435-e452 (rbd-11a to rbd-11e) and a471-s500 (rbd-14a to rbd-14d and rbd-15a to rbd-15e) with an overlap of three amino acids in each sequence (cf. table 2 ). these peptides were used to further locate strongly interacting amino acids by spr and saturation transfer difference (std) nmr spectroscopy. spr screening was performed with ligand concentrations of up to 500 lm in tbs and 48 fmol of immobilized ace2. rbd-11b (y438-l443; ykyryl) showed the highest spr response signal of 51 ru at a concentration of 250 lm. also the flanking peptides rbd-11a (n435-y440; nynyky) and rbd-11c (r441-g446; rylrhg) showed significant binding of 5 ru (c = 250 lm) and 34 ru (c = 500 lm), respectively. the spr response of peptides rbd-14a (a471-w476; alnsyw) and rbd-14b (s474-n479; sywpln) was comparable to that of rbd-11a (5 and 7 ru, respectively) (cf. fig. 2) . the other hexapeptides gave no significant spr signal. therefore, only peptide rbd-11b (y438-l443; ykyryl) was further investigated by spr and std nmr experiments. a concentration dependent spr affinity plot was performed with 110 fmol of receptor protein immobilized. fig. 1 shows the concentration dependent binding of rbd-11b (ykyryl) by spr resulting in a dissociation constant k d = 46 ± 14 lm which is two-fold higher affinity than that of peptide rbd-11. the binding kinetics are k on = 3.7 â 10 3 s à1 m à1 and k off = 0.17 s à1 and also result in a dissociation constant of k d = 46 lm. the two-fold increase of the binding affinity compared to rbd-11 results from a two-fold higher on-rate which is probably due to a better defined conformation in solution such that binding can occur more rapidly. the off-rate is the same as found for the dodecapeptide rbd-11. std nmr spectroscopy is a well-established method to characterize ligand-protein interactions meyer, 1999, 2001) . here it was used to determine dissociation constant and binding epitope of the interaction of rbd-11b with a soluble construct of the receptor protein ace2 in deuterated tris-buffered d 2 o. fig. 3 shows the 1 h std nmr spectrum of rbd-11b (ykyryl) and receptor ace2 at a 72 fold excess of the ligand over the protein. dependence of the std amplification factor on the concentration of rbd-11b yields the k d value. a one site binding model fits the experimental data well and gives k d values in the low micromolar range: hd,d 0 of the tyr442 results in k d = 60 ± 25 lm. the binding epitope was determined from std spectra at a 144 fold excess of the ligand. it shows that all tyrosine residues have a close contact to ace2. furthermore, the ha atom of arg441 also has a close proximity to the receptor surface. the ha atoms of tyr440 and leu443 interact also strongly with the cellular receptor. the positively charged groups of lysine and arginine side chains also show moderately strong interactions with the surface. the c-terminal leucine seems to be involved in a hydrophobic contact via its side chain. the highest level of saturation of 4.1% std (absolute) is found for the he,e of tyr440. the x-ray crystal structure analysis of the complex of rbd (residues 318-510) with ace2 shows only contacts of tyr440 and tyr442 of the rbd with ace2 (li et al., 2005a) . however, in the isolated hexapeptide ykyryl, tyr438 is also binding to ace2. according to mutation studies arg441 of sars-cov spike protein is important for binding affinity (he et al., 2006) . this fact is confirmed by std nmr analysis. the side chain protons hd,d of arg441 exhibit 0.7% absolute std, which suggests an ionic interaction of the guanidinium group with the receptor molecule. the x-ray structure of the complex revealed no direct contact between arg441 of rbd and ace2. thus in the free form the hexapeptide adopts a different binding mode and conformation compared to the case when integrated into the rbd. lys439 also makes a contact via its positively charged group evidenced by the std effect on the he,e protons that receive a saturation of 0.6%. protons of leu443 methyl groups show a std effect of 0.6% and 0.9% std, respectively, indicating a contact of the methyl groups to the receptor. the biological activity of the lead structure rbd-11b (y438-l443; ykyryl) was assayed with respect to its ability to inhibit virus replication in cell culture (drosten et al., 2004) . veroe6 cells were infected with the sars-cov isolate frankfurt. as described previously, growth of the virus in vero cells is not associated with a cytopathic effect (vassilatis et al., 2003) . cells were infected with a virus titer of 0.01 moi. the inoculum was removed after 1 h and replaced with fresh medium complemented with different concentrations of peptide rbd-11b in a one-time dose. two days post infection virus rna concentration in the supernatant was measured by real-time pcr (cf. fig. 4 ). there was no evidence for toxicity of the compound in the concentration range tested with mtt cell proliferation assay on subconfluent cells. after two days a onetime dose of the peptide rbd-11b (ykyryl, 10.5 mm) reduced the virus rna level compared to the untreated control by a factor of 10. further, the inhibition of virus proliferation by the peptide is concentration dependent. after two days, relative to an untreated control virus rna is reduced by a factor of 600 at a 14 mm concentration of the peptide. the final amount of virus rna is in fact lower than the initially added amount. these data suggest that the hexapeptide blocks indeed the binding site necessary for the initial viral attachment to the human receptor ace2 and effectively inhibits viral entry into vero cells. thus, the virus is not capable to replicate and viral particles are exposed to the degradation process. these results are in good agreement with the occupation of the receptor's binding site given the peptide's dissociation constant of k d = 46 lm. at peptide concentrations of 7 mm the occupation of binding sites is 99.3%, at 10.5 mm it is 99.6% and at 14 mm 99.7%, respectively. the peptide inhibits the infection of the corona virus specifically. this is proven by the fact that rbd-11b does not inhibit infection of vero cells with the alpha virus sindbis, which cause high fever in humans. (tesh, 1982) furthermore, the peptide was also tested in an inhibition assay with another corona virus nl63 in llc-mk2 and caco2 cells. nl63 corona virus causes severe colds in human and uses ace2 also as a functional receptor. (van der hoek et al., 2004; wu et al., 2009 ) inhibitory effects were observed for both cell lines at a concentration of 14 mm. for caco2 cells inhibition is also observed at a peptide concentration of 7 mm. we have synthesized various hexapeptides closely related to peptide rbd-11b (ykyryl) including an alanine scan library and analyzed by spr the importance of individual amino acids for binding to ace2. the alanine scan reveals that the tripeptide motif 439 kyr 441 is essential for the binding (cf. fig. 5) . the other binding curves show that the positively charged side chains of the amino acids lys439 and arg441 may be important for the receptor binding. no binding was observed for the peptides ydyryl and ykydyl with the negatively charged aspartate replacing the positively charged lys and arg. changing positively charged residues to uncharged residues (k439s, r441s) reduces the binding affinity but does not abolish it. these results indicate a clear role of lys439 and arg441 in the binding process in agreement with the std nmr data shown above. the binding mode of peptide rbd-11b to the human receptor ace2 was also analyzed by docking of the peptide to the binding site followed by a molecular dynamics (md) simulation that was recorded for 3000 ps in equilibrium using the opls_aa/2005 force field as realized in the program desmond (schrödinger) (jorgensen amino acid sequence , 1996) . the conformation of peptide rbd-11b in the crystal structure of the rbd of the viral spike protein in complex with the receptor ace2 was used as starting structure (li et al., 2005a) . the protein peptide complex was placed in a water box with 18,145 water molecules and a sodium chloride concentration of 0.2 mol/l. the peptide shows high dynamics during the md but stays always in contact with the receptor surface. fig. 6 shows the starting and the final conformation of the complex. the side chains of amino acids tyr440 and tyr442 interact with ace2 in the crystal structure. at the end of the md simulation lys439, tyr440 and arg441 show interactions with the receptor. the binding mode of hexapeptide rbd-11b to ace2 differs from the binding mode of the hexapeptide as part of the full length s protein. the positively charged residue of lys439 forms ionic interactions with the carboxyl group of glu23 of ace2. the average distance from the lysine e-nitrogen atom and an oxygen atom of the carboxyl group over the course of the md simulation is 3.08 ± 0.74 å. the guanidinium group (ng) of arg441 interacts with the carboxyl group of asp30 of the receptor with an average distance of 3.42 ± 0.91 å. the crystal structure shows the first three carbohydrate units of a high mannose n-glycan on the receptor surface resolved. in the md simulation tyr440 shows contact to the glycan molecule by interaction of its aromatic residue with the second n-acetylglucosamine and the mannose residue. the average distance of the interaction of the aromatic ring with the n-acetylglucosamine is 5.11 ± 0.88 å and with the mannose 5.97 ± 0.77 å, respectively. during the md simulations the peptide shifted 5.4 å at the c-terminus and 17.2 å at the n-terminus from its initial position to its final pose (fig. 7) . the results of the md studies support the affinity data obtained from the alanine scan, which indicates that the tripeptide motif 439 kyr 441 is important for the binding to ace2. sars belongs to the major new emerging virus diseases. in 2003 it caused a major outbreak with about 1000 deaths. it is a respiratory disease with about 10% mortality that is caused by a variant of the common coronaviruses (sars-cov). rigid political measures helped to contain the epidemic within a short period of time. so far no specific treatment against sars is available. in search for molecules that can specifically block the attachment of the virus to the human cell, we analyzed binding modes of viral peptides to the human receptor. we identified and characterized the focal point of the viral protein that is used by the virus for its attachment to the human cell. we found a hexapeptide in the receptor-binding domain (rbd) of the s protein of sars-cov that carries a significant portion of the binding affinity of the virus to the human cell. the s protein mediates the attachment of the virus to its functional receptor ace2. the attachment of the virus to ace2 does not interfere with the natural function of the receptor. therefore, it is easy to block the attachment site of the virus in the upper respiratory tract as a preventive measure against sars. we could clearly demonstrate that hexapeptide tyr-lys-tyr-arg-tyr-leu reduces viral infection of epithelial cells, as found in the upper respiratory tract, by a factor of 600. this peptide was shown to be specific against coronaviruses that attach to the ace2 receptor. its mode of action is specific as it does not interfere with other infections by viruses that utilize different receptors, like alpha virus sindbis. combination of several biophysical methods, e.g. spr, std nmr and molecular dynamics simulations, were used to characterize the specific binding mode of the inhibitory peptide. although there is currently no sars outbreak the need of an antiviral drug, e.g. based on the hexapeptide, is still present. a viral reservoir is present in wild animals like bats and civet cats and a new epidemic is likely someday. amino acids 270-510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor the secret life of ace2 as a receptor for the sars virus evaluation of advanced reverse transcription-pcr assays and an alternative pcr target region for detection of severe acute respiratory syndrome-associated coronavirus identification of a novel coronavirus in patients with severe acute respiratory syndrome the spike protein of sars-cov -a target for vaccine and therapeutic development a single amino acid substitution (r441a) in the receptorbinding domain of sars coronavirus spike protein disrupts the antigenic structure and binding activity design and biological activities of novel inhibitory peptides for sars-cov spike protein and angiotensin-converting enzyme 2 interaction sars-associated coronavirus structural biology: adaptation of sars coronavirus to humans screening and identification of linear b-cell epitopes and entry-blocking peptide of severe acute respiratory syndrome (sars)-associated coronavirus using synthetic overlapping peptide library water suppression that works -excitation sculpting using arbitrary wave-forms and pulsed-field gradients development and testing of the opls all-atom force field on conformational energetics and properties of organic liquids a novel coronavirus associated with severe acute respiratory syndrome structure of sars coronavirus spike receptor-binding domain complexed with receptor angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus receptor and viral determinants of sars-coronavirus adaptation to human ace2 characterization of ligand binding by saturation transfer difference nmr spectroscopy group epitope mapping by saturation transfer difference nmr to identify segments of a ligand in direct contact with a protein receptor coronavirus as a possible cause of severe acute respiratory syndrome selective recognition of cyclic rgd peptides of nmr defined conformation by alpha iib beta 3, alpha v beta 3, and alpha 5 beta 1 integrins identification of two critical amino acid residues of the severe acute respiratory syndrome coronavirus spike protein for its variation in zoonotic tropism transition via a double substitution strategy inhibition of severe acute respiratory syndrome-associated coronavirus (sars-cov) infectivity by peptides analogous to the viral spike protein arthritides caused by mosquito-borne viruses identification of a new human coronavirus the g protein-coupled receptor repertoires of human and mouse ligplot: a program to generate schematic diagrams of protein-ligand interactions sars-lessons from a new disease a 193-amino acid fragment of the sars coronavirus s protein efficiently binds angiotensinconverting enzyme 2 crystal structure of nl63 respiratory coronavirus receptor-binding domain complexed with its human receptor the sars-cov s glycoprotein: expression and functional characterization single amino acid substitutions in the severe acute respiratory syndrome coronavirus spike glycoprotein determine viral entry and immunogenicity of a major neutralizing domain suppression of sars-cov entry by peptides corresponding to heptad regions on spike glycoprotein synthetic peptides outside the spike protein heptad repeat regions as potent inhibitors of sars-associated coronavirus key: cord-263167-es806qhz authors: rogers, thomas f.; zhao, fangzhu; huang, deli; beutler, nathan; burns, alison; he, wan-ting; limbo, oliver; smith, chloe; song, ge; woehl, jordan; yang, linlin; abbott, robert k.; callaghan, sean; garcia, elijah; hurtado, jonathan; parren, mara; peng, linghang; ramirez, sydney; ricketts, james; ricciardi, michael j.; rawlings, stephen a.; wu, nicholas c.; yuan, meng; smith, davey m.; nemazee, david; teijaro, john r.; voss, james e.; wilson, ian a.; andrabi, raiees; briney, bryan; landais, elise; sok, devin; jardine, joseph g.; burton, dennis r. title: isolation of potent sars-cov-2 neutralizing antibodies and protection from disease in a small animal model date: 2020-06-15 journal: science doi: 10.1126/science.abc7520 sha: doc_id: 263167 cord_uid: es806qhz countermeasures to prevent and treat covid-19 are a global health priority. we enrolled a cohort of sars-cov-2-recovered participants, developed neutralization assays to interrogate antibody responses, adapted our high-throughput antibody generation pipeline to rapidly screen over 1800 antibodies, and established an animal model to test protection. we isolated potent neutralizing antibodies (nabs) to two epitopes on the receptor binding domain (rbd) and to distinct non-rbd epitopes on the spike (s) protein. we showed that passive transfer of a nab provides protection against disease in high-dose sars-cov-2 challenge in syrian hamsters, as revealed by maintained weight and low lung viral titers in treated animals. the study suggests a role for nabs in prophylaxis, and potentially therapy, of covid-19. the nabs define protective epitopes to guide vaccine design. the novel coronavirus disease (covid-19) has had devastating global health consequences and there is currently no cure and no licensed vaccine. neutralizing antibodies (nabs) to the causative agent of the disease, severe acute respiratory syndrome coronavirus-2 (sars-cov-2), represent potential prophylactic and therapeutic options and could help guide vaccine design. indeed, a nab to another respiratory virus, respiratory syncytial virus (rsv), is in widespread clinical use prophylactically to protect vulnerable infants (1). furthermore, nabs prevent death from the emerging ebola virus in macaques, even when given relatively late in infection, and thus have been proposed for use in humans in outbreaks (2, 3). generally, nabs with outstanding potency ("super-antibodies") (4) can be isolated by deeply mining antibody responses of a sampling of infected donors. outstanding potency together with engineering to extend antibody halflife from weeks to many months brings down the effective costs of abs and suggests more opportunities for prophylactic intervention. at the same time, outstanding potency can permit anti-viral therapeutic efficacy that is not observed for less potent antibodies (4). here, we present the isolation of highly potent nabs to sars-cov-2 and demonstrate their in vivo protective efficacy in a small animal model, suggesting their potential utility as a medical countermeasure. to interrogate the antibody response against sars-cov-2 and discover nabs, we adapted our pipeline to rapidly isolate and characterize monoclonal antibodies (mabs) from convalescent donors (fig. 1) . briefly, a cohort of previously swabpositive sars-cov-2 donors was recruited for peripheral blood mononuclear cell (pbmc) and plasma collection. in parallel, we developed both live replicating and pseudovirus neutralization assays using a hela-ace2 (angiotensin-converting enzyme-2) cell line that gave robust and reproducible virus titers. convalescent serum responses were evaluated for neutralization activity against sars-cov-1 and sars-cov-2, and eight donors were selected for mab discovery. single antigen-specific memory b cells were sorted, and their corresponding variable genes were recovered and cloned using a high-throughput production system that enabled antibody expression and characterization in under two weeks. promising mabs were advanced for further biophysical characterization and in vivo testing. two platforms were established to evaluate plasma neutralization activity against sars-cov-2, one using replicationcompetent virus and another using pseudovirus (psv). vero-e6 cells were first used as target cells for neutralization assays, but this system was relatively insensitive at detecting replicating virus compared to a hela cell line that stably expressed the cell surface ace2 receptor ( fig. s1a ). the hela-ace2 target cells gave reproducible titers and were used for the remainder of the study. in certain critical instances, hela-ace2 and vero cells were compared. the live replicating virus assay used the washington strain of sars-cov-2, usa-wa1/2020 (bei resources nr-52281) and was optimized to a 384-well format to measure plaque formation. in parallel, a psv assay was established for both sars-cov-1 and sars-cov-2 using murine leukemia virus (mlv)-based psv (5). the assay used single cycle infectious viral particles bearing a firefly luciferase reporter for high-throughput screening. unlike mlv-psv, which buds at the plasma membrane, coronaviruses assemble in the er-golgi intermediate compartment, so the c terminus of the sars-cov-1 spike protein (s protein) contains an er retrieval signal (6). the alignment of sars-cov-1 and sars-cov-2 s proteins showed that this er retrieval signal is conserved in sars-cov-2 (fig. s1b). to prepare high titers of infectious mlv-cov-1 and sars-cov-2 psv particles, various truncations of sars-cov-1 and sars-cov-2 s protein were expressed in which the er retrieval signal was removed to improve exocytosis of the virus. pseudovirion versions carrying sars-cov1-sδ28 and sars-cov2-sδ18s protein efficiently transduced ace2-expressing target cells, but not control hela or a549 cells (fig. s1c). control vsv-g pseudotyped virions showed a similar transduction efficiency in all target cells. luciferase expression in transduced cells proved to be proportional to viral titer over a wide range ( fig. s1d ). in parallel to the development of neutralization assays, a cohort was established in san diego, california, of 17 donors who had previously been infected with sars-cov-2 ( fig. 2a, fig. s2a , and table s1). the cohort was 47% female and the average age was 50 years. infection was determined by a positive sars-cov-2 pcr test from a nasopharyngeal swab. all donors also had symptoms consistent with covid-19, and disease severity ranged from mild to severe, including intubation in one case, although all donors recovered. donor plasma were tested for binding to recombinant sars-cov-2 and sars-cov-1 s and receptor binding domain (rbd) proteins, for binding to cell surface expressed spikes and for neutralization in both live replicating virus and pseudovirus assays (fig. 2, b to d, and fig. s2b ; 3 donors cc6, cc12 and cc25 that are further pursued below are highlighted). binding titers to sars-cov-2 s protein varied considerably, reaching ec50s at serum dilutions of around 10 4 , with titers against the rbd about an order of magnitude less. titers against sars-cov-1 s protein were notably less than for sars-cov-2 s protein and titers against sars-cov-1 rbd were only detected in a small number of donors. neutralizing titers in the psv assay varied over a wide range for sars-cov-2 ( fig. 2d and fig. s2a ) and were low or undetectable against sars-cov-1. importantly, rbd binding and psv neutralization were well correlated (fig. 2e) . there was also a positive correlation between cell surface spike binding and live replicating virus neutralization ( fig. s2c ). the titers in the psv assay and the replicating virus assay were largely similar (figs. s2 and s3). in most later measurements, the psv assay was preferred owing to its higher throughput. cryopreserved pbmcs from eight donors were stained for memory b cells markers (cd19+/igg+) and both avi-tag biotinylated rbd and sars-cov-2 s antigen baits before singlecell sorting. s+ and s+/rbd+ memory b cells were present at an average frequency of 2.0% and 0.36%, respectively, across the eight donors ( fig. s4a ). in total, 3160 antigen-positive (ag+) memory b cells were sorted to rescue native heavy and light chain pairs for mab production and validation (fig. s4b). a total of 2045 antibodies were cloned and expressed, which represents, on average, a 65% pcr recovery of paired variable genes and >86% estimated recovery of fully functional cloned genes ( fig. s4c ). the bulk-transformed ligation products for both the heavy chain and light chain were transfected and tested for binding to rbd and s protein, and for neutralization in the sars-cov-2 pseudovirus assay using hela-ace2 target cells ( fig. s5 ). the majority of transfected pairs resulted in igg expression (92%). of these, 43% showed binding only to s protein while 5.9% bound to both s and rbd proteins and 0.1% bound only to rbd. the supernatants were also screened for binding to an unrelated hiv antigen (bg505 sosip) to eliminate non-specific or polyreactive supernatants. the supernatants were next evaluated for neutralization activity using sars-cov-2 and sars-cov-1 pseudoviruses. strikingly, a small proportion of the binding antibodies showed neutralization activity and that activity was equally distributed between rbd+/s+ and s+ only binders despite a much larger number of s+ only binding supernatants as exemplified by the three donors cc6, cc12 and cc25, (fig. 3a) . these data indicate that viral infection generates a strong response against the non-rbd regions of s protein, but only a small proportion of that response is neutralizing. in contrast, there are fewer rbd binding antibodies but a larger proportion of these neutralize sars-cov-2 pseudovirus. antibodies that tested positive for neutralization in the high-throughput screening were sequence confirmed and advanced for expression at large scale for additional characterization. a total of 33 antibodies were prioritized for in depth characterization from the 3 donors, cc6, cc12 and cc25. within that subset, we identified 25 distinct lineages, with 23 containing a single member (table s2) . vh1 and vh3-gene families were notably prominent in these abs and there was a diversity of cdr3 lengths (fig. 3 , b and c). there was one prominent example of a clonally expanded lineage, with 8 recovered clonal members that averaged 4.3% and 2.8% mutations from germline at the nucleotide level in the heavy chain and light chain, respectively (fig. 3d ). the remaining clones were relatively unmutated, averaging just above 1% mutation at the nucleotide level suggesting that these antibodies were primed by the ongoing covid infection and likely not recalled from a previous endemic human coronavirus (hcov) exposure. all antibodies that were expressed at scale were evaluated in standard elisa-based polyreactivity assays with solubilized cho membrane preparations, ssdna and insulin (7, 8) , and none were polyreactive ( fig. s6 ). the antibody hits that were identified in the high-throughput screening were next evaluated for epitope specificity by biolayer interferometry (bli) using s and rbd proteins as capture antigens. the antigens were captured on anti-his biosensors before addition of saturating concentrations (100 μg/ml) of antibodies that were then followed by competing antibodies at a lower concentration (25 μg/ml). accordingly, only antibodies that bind to a non-competing site would be detected in the assay. among the antibodies evaluated, the results reveal three epitope bins for rbd (designated as rbd-a, rbd-b, and rbd-c) and three epitope bins for the s protein (designated as s-a, s-b, and s-c) ( fig. 4a and fig. s7 ). interestingly, the mab cc12.19 appears to compete with antibodies targeting two different epitopes, rbd-b and s-a ( fig. s7 ), which might indicate that this mab targets an epitope spanning rbd-b and s-a. to evaluate epitope specificities further, we next assessed binding of the antibodies to extended rbd-constructs with subdomains (sd) 1 and 2, including the independently folding rbd-sd1 and rbd-sd1-2, and the n-terminal domain (ntd) ( fig. 4b and fig. s8 , a and b). none of the antibodies showed binding to the ntd. cc12.19 binds to all the other constructs, which supports the epitope binning data described in fig. 4a . the other antibodies grouped in the s-a epitope bin that compete with cc12. 19 show either no binding to rbd or rbd-sd constructs (cc12.20 and cc12.21) or do show binding to rbd-sd1 and rbd-sd1-2 but not rbd (cc12.23). these data suggest two competing epitopes within the s-a epitope bin; one that is confined to the non-rbd region of s protein and the other that includes some element of rbd-sd1-2. this interpretation will require further investigation by structural studies. we next evaluated the mabs for neutralization activity against sars-cov-2 and sars-cov-1 pseudoviruses. the neutralization ic50 potencies of these antibodies are shown in fig. 4c and their associated maximum plateaus of neutralization (mpns) are shown in fig. 4d . a comparison of neutralization potencies between pseudovirus ( fig. s8c ) and live replicating virus ( fig. s8d ) is also included. notably, the most potent neutralizing antibodies were those directed to rbd-a epitope including two antibodies, cc6.29 and cc6.30, that neutralize sars-cov-2 pseudovirus with an ic50 of 2 ng/ml and 1 ng/ml, respectively (fig. 4c ). in comparison, antibodies directed to rbd-b tended to have higher ic 50 s and many plateau below 100% neutralization. despite this trend, cc6.33 is directed against rbd-b and showed complete neutralization of sars-cov-2 with an ic 50 of 39 ng/ml and also neutralized sars-cov-1 with an ic50 of 162 ng/ml. this was the only antibody that showed potent neutralization of both pseudoviruses. the antibodies that do not bind to rbd and are directed to non-rbd epitopes on s protein all show poor neutralization potencies and mpns well below 100%. to evaluate whether the rbd-a epitope might span the ace2 binding site, we next performed cell surface competition experiments. briefly, antibodies were premixed with biotinylated s (fig. 4e ) or rbd (fig. 4f) proteins at a molar ratio of 4:1 of antibodies to target antigen. the mixture was then incubated with the hela-ace2 cell line and the percent competition against ace2 receptor was recorded by comparing percent binding of the target antigen with and without antibody present ( fig. s8e ). the antibodies targeting the rbd-a epitope compete best against the ace2 receptor and the neutralization ic50 correlates well with the percent competition for ace2 receptor binding for both s protein (fig. 4e ) and for rbd (fig. 4f ). we also assessed the affinity of all rbd-specific antibodies to soluble rbd by surface plasmon resonance (spr) and found a poor correlation between affinity and neutralization potency ( fig. 4g and fig. s9 ). however, the correlation is higher when limited to antibodies targeting the rbd-a epitope. the lack of a correlation between rbd binding and neutralization for mabs contrasts with the strong correlation described earlier for serum rbd binding and neutralization. overall, the data highlight epitope rbd-a as the preferred target for eliciting neutralizing antibodies and that corresponding increases in affinity of mabs to rbd-a will likely result in corresponding increases in neutralization potency. sars-cov-2 has shown some propensity for mutation as it has circulated worldwide as evidenced for example in the emergence of the d614g variant (9). we investigated the activity of 5 nabs against 6 viral variants that have been reported. the 3 sera studied above neutralized all the variants s10a ). all 5 nabs neutralized the d614g variant. however, one variant with a mutation in the ace2 binding site (g476s) did show effectively complete resistance to one of the nabs and another variant (v367f) showed a 10-fold higher ic 50 than the wa-1 strain ( fig. s10b ). to investigate the relationship between in vitro neutralization and protection in vivo against sars-cov-2, we selected two mabs for passive transfer/challenge experiments in a syrian hamster animal model based on a summary of the nab data (table s3 and fig. s11 ). the experimental design for the passive transfer study is shown in fig. 5a . in the first experiment, we tested nab cc12.1, which targets the rbd-a epitope and has an in vitro ic50 neutralization of 0.019 μg/ml against pseudovirus and in the second we tested nab c12.23, which targets the s-b epitope with an ic 50 neutralization of 22 μg/ml. in both experiments an unrelated antibody to dengue virus, den3, was used as a control. the anti-sars-cov-2 nabs were delivered at 5 different concentrations to evaluate dose-dependent protection starting at 2 mg/animal (average of 16.5 mg/kg) at the highest dose and 8 μg/animal at the lowest dose. the den3 control antibody was delivered at a single dose of 2 mg/animal. sera were collected from each animal 12 hours post ip infusion of the antibody and all animals were subsequently challenged with a dose of 1x10 6 pfu of sars-cov-2 (usa-wa1/2020) by intranasal administration 12 hours post antibody infusion (fig. 5a) . syrian hamsters typically clear virus within one week after sars-cov-1 infection (10) . accordingly, the hamsters were weighed as a measure of disease due to infection. lung tissues were collected to measure viral load on day 5. a data summary is presented in fig. 5b and fig. s12a for animals that received cc12.1, which targets the rbd-a epitope. the control animals that received den3 lost on average 13.6% of body weight at 5 days post virus challenge. in comparison, the animals that received the neutralizing rbd-a antibody at a dose of 2 mg (average of 16.5 mg/kg) or 500 μg (average of 4.2 mg/kg) exhibited no weight loss. however, animals that received a dose of 125 μg (average of 0.9 mg/kg) had an average 8% loss of body weight, while animals that received a dose of 31 μg/ml (0.2 mg/kg) and 8 μg/ml (0.06 mg/kg) lost 15.8% and 16.7% of body weight, respectively. we note these animals showed a trend for greater weight loss than control animals but this did not achieve statistical significance (table s4) . given concerns about antibody-mediated enhanced disease in sars-cov-2 infection, this observation merits further attention using larger animal group sizes. the weight loss data are further corroborated by quantification of lung viral load measured by real-time pcr (fig. 5c ) and showed a moderate correlation to weight loss. the data indicate comparable viral loads between the three higher doses (2 mg, 500 μg, and 125 μg) of nabs. in contrast, equivalent viral loads were observed between the control group receiving den3 and the low dose groups receiving 31 μg and 8 μg of nab. in contrast to the nab to rbd-a, the less potent and incompletely neutralizing antibody to the s-b epitope showed no evidence of protection at any concentration compared to the control animals ( fig. s12b) . to determine the antibody serum concentrations that may be required for protection against disease from sars-cov-2 infection, we also measured the antibody serum concentrations just prior to intranasal virus challenge (fig. 5d) . the data highlight that an antibody serum concentration of approximately 22 μg/ml of nab (1160 x psv neutralization ic50) enables full protection and a serum concentration of 12 μg/ml (630 x psv neutralization ic 50 ) is adequate for 50% reduced disease as measured by weight loss. the effective antibody concentration required at the site of infection to protect from disease remains to be determined. sterilizing immunity at serum concentrations that represent a large multiplier of the in vitro neutralizing ic 50 is observed for many viruses (11) . using a high-throughput rapid system for antibody discovery, we isolated more than 1000 mabs from 3 convalescent donors by memory b cell selection using sars-cov-2 s or rbd recombinant proteins. about half of the mabs isolated could be expressed and also bind effectively to either s and/or rbd proteins. only a small fraction of these abs was neutralizing, highlighting the value of deep mining of responses to access the most potent abs (4). a range of nabs were isolated to different sites on the s protein. the most potent abs, reaching single digit ng/ml ic50s in psv assays, are targeted to a site that, judged by competition studies, overlaps the ace2 binding site. only one of the abs, directed to rbd-b, neutralized sars-cov-1 psv, as may be anticipated given the differences in ace2 contact residues between the two viruses (fig. s13) and given that the selections were performed with sars-cov-2 target proteins. abs directed to the rbd but not competitive with soluble ace2, (although they may be competitive in terms of an array of membrane-bound ace2 molecules interacting with an array of spike proteins on a virion), are generally less potent neutralizers and tend to show incomplete neutralization, plateauing at around or less than 50% neutralization. the one exception is the cross-reactive rbd-b antibody above. similar lower potency and incomplete neutralization are observed for abs to the s protein that are not reactive with recombinant rbd. the cause(s) of these incomplete neutralization phenomena is unclear but presumably originates in some spike protein heterogeneity, either glycan, cleavage or conformationally based. in any case, the rbd-a nabs that directly compete with ace2 are clearly the most preferred for prophylactic and therapeutic applications, and as reagents to define nab epitopes for vaccine design. we note that, even for a small sampling of naturally occurring viral variants, two were identified that showed notable resistance to individual potent nabs to the wa-1 strain and neutralization resistance will need to be considered in planning for clinical applications of nabs. cocktails of nabs may be required. in terms of nabs as passive reagents, the efficacy of a potent anti-rbd nab in vivo in syrian hamsters is promising in view of the positive attributes of this animal model (12) and suggests that human studies are merited. nevertheless, as for any animal model, there are many limitations, including, in the context of antibody protection, differences in effector cells and fc receptors between humans and hamsters. the failure of the non-rbd s-protein nab to protect in the animal model is consistent with its lower potency and, likely most importantly, its inability to fully neutralize challenge virus. in the context of human studies, improved potency of protective nabs by enhancing binding affinity to the rbd epitope identified, improved half-life and reduced fc receptor binding to minimize potential antibody dependent enhancement (ade) effects, should they be identified as concerning, are all antibody engineering goals to be considered. as observed for heterologous b cell responses against different serotypes of flavivirus infection, there is a possibility, but no current experimental evidence, that subtherapeutic vaccine serum responses or subtherapeutic nab titers could potentially exacerbate future coronavirus infection disease burden by expanding the viral replication and/or cell tropism of the virus. if ade is found for sars-cov-2 and operates at sub-neutralizing concentrations of neutralizing antibodies as it can for dengue virus (13) then it would be important, from a vaccine standpoint, to carefully define the full range of nab epitopes on the s protein as we have begun here. from a passive antibody standpoint, it would be important to maintain high nab concentrations or appropriately engineer nabs. the nabs described have remarkably little shm, typically one or two mutations in the vh gene and one or two in the vl gene. such low shm may be associated with the isolation of the nabs relatively soon after infection, and perhaps before affinity-maturation has progressed. low shm has also been described for potent nabs to ebola virus, respiratory syncytial virus (rsv), middle east respiratory syndrome coronavirus (mers-cov) and yellow fever virus (14) (15) (16) (17) and may indicate that the human naïve repertoire is often sufficiently diverse to respond effectively to many pathogens with little mutation. of course, nab efficacy and titer may increase over time as described for other viruses and it will be interesting to see if even more potent nabs to sars-cov-2 evolve in our donors in the future. what do our results suggest for sars-cov-2 vaccine design? in the first instance, the results suggest a focus on the rbd and indeed strong nab responses have been described by immunizing mice with a multivalent presentation of rbd (18) . the strong preponderance of non-neutralizing antibodies and very few nabs to s protein that we isolated could arise for a number of reasons including: (i) the recombinant s protein that we used to select b cells is a poor representation of the native spike on virions. in other words, there may be many nabs to s but we failed to isolate them because of the selecting antigen, (ii) the recombinant s protein that we used is close to native but non-neutralizing antibodies bind to sites on s that do not interfere with viral entry, (iii) the s protein in natural infection disassembles readily generating a strong ab response to "viral debris" that is non-neutralizing because the antibodies recognize protein surfaces that are not exposed on the native spike. importantly, the availability of both neutralizing and non-neutralizing antibodies generated in this study will facilitate evaluation of s protein immunogens for presentation of neutralizing and non-neutralizing epitopes and promote effective vaccine design. the design of an immunogen that improves on the quality of nabs elicited by natural infection may well emerge as an important goal of vaccine efforts (19) . in summary, we describe the very rapid generation of neutralizing antibodies to a newly emerged pathogen. the antibodies can find clinical application and will aid in vaccine design. (e) correlation between psv sars-cov-2 neutralization and rbd subunit elisa binding area-under-the-curve (auc). auc was computed using simpson's rule. the 95% confidence interval of the regression line is shown in light grey and was estimated by performing 1,000 bootstrap re-samplings. r 2 and p values of the regression are also indicated. cc participants from whom mabs were isolated are specifically highlighted in dark blue (cc6), pine green (cc12) and hot pink (cc25). biophysical properties of the clinical-stage antibody landscape minimally mutated hiv-1 broadly neutralizing antibodies to guide reductionist vaccine design severe acute respiratory syndrome coronavirus infection of golden syrian hamsters the antiviral activity of antibodies in vitro and in vivo simulation of the clinical and pathological manifestations of coronavirus disease 2019 (covid-19) in golden syrian hamster model: implications for disease pathogenesis and transmissibility dengue antibody-dependent enhancement: knowns and unknowns longitudinal analysis of the human b cell response to ebola virus infection junctional and allele-specific residues are critical for mers-cov neutralization by an exceptionally potent germline-like antibody infants infected with respiratory syncytial virus generate potent neutralizing antibodies that lack somatic hypermutation longitudinal dynamics of the human b cell response to the yellow fever 17d vaccine the sars-cov-2 receptor-binding domain elicits a potent neutralizing response without antibody-dependent enhancement rational vaccine design in the time of covid-19 pre-fusion structure of a human coronavirus spike protein cryo-em structure of the 2019-ncov spike in the prefusion conformation rational design of envelope identifies broadly neutralizing human monoclonal antibodies to hiv-1 recombinant hiv envelope trimer selects for quaternary-dependent antibodies targeting the trimer apex efficient generation of monoclonal antibodies from single human b cells by single cell rt-pcr and expression vector cloning r01 (ai132317, ai073148 to d.n.) and k99 (ai145762 to r.k.a.) awards, the iavi neutralizing antibody center, the bill and melinda gates foundation (opp 1170236 to i.a.w. and d.r.b.), (opp 1183956 to j.e.v.) and (opp1196345/ inv-008813 to ds and drb) key: cord-273891-7w334xgt authors: kirchdoerfer, robert n.; wang, nianshuang; pallesen, jesper; wrapp, daniel; turner, hannah l.; cottrell, christopher a.; corbett, kizzmekia s.; graham, barney s.; mclellan, jason s.; ward, andrew b. title: receptor binding and proteolysis do not induce large conformational changes in the sars-cov spike date: 2018-03-31 journal: biorxiv doi: 10.1101/292672 sha: doc_id: 273891 cord_uid: 7w334xgt severe acute respiratory syndrome coronavirus (sars-cov) emerged in 2002 as a highly transmissible pathogenic human betacoronavirus. the viral spike glycoprotein (s) utilizes angiotensin-converting enzyme 2 (ace2) as a host protein receptor and mediates fusion of the viral and host membranes, making s essential to viral entry into host cells and host species tropism. as sars-cov enters host cells, the viral s undergoes two proteolytic cleavages at s1/s2 and s2’ sites necessary for efficient membrane fusion. here, we present a cryo-em analysis of the trimeric sars-cov s interactions with ace2 and of the trypsin-cleaved s. surprisingly, neither binding to ace2 nor cleavage by trypsin at the s1/s2 cleavage site impart large conformational changes within s or expose the secondary cleavage site, s2’. these observations suggest that s2’ cleavage does not occur in the s prefusion conformation and that additional triggers may be required. viral and host membranes, making s essential to viral entry into host cells and host species 23 tropism. as sars-cov enters host cells, the viral s undergoes two proteolytic cleavages at 24 s1/s2 and s2ʹ′ sites necessary for efficient membrane fusion. here, we present a cryo-em 25 analysis of the trimeric sars-cov s interactions with ace2 and of the trypsin-cleaved s. 26 surprisingly, neither binding to ace2 nor cleavage by trypsin at the s1/s2 cleavage site impart 27 large conformational changes within s or expose the secondary cleavage site, s2´. these 28 observations suggest that s2´ cleavage does not occur in the s prefusion conformation and that 29 additional triggers may be required. 30 3 severe acute respiratory syndrome coronavirus (sars-cov) emerged in humans in 2002 31 and rapidly spread globally causing 8,096 cases and 774 associated deaths in 26 countries 32 through july 2003 1 . sars-cov reappeared in a second smaller outbreak in 2004, but has since 33 disappeared from human circulation. however, closely related coronaviruses, such as wiv1, 34 currently circulate in bat reservoirs and are capable of utilizing human receptors to enter cells 2 . 35 the more recent emergence of middle east respiratory syndrome coronavirus (mers-cov) 1 and 36 the likelihood of future zoonotic transmission of novel coronaviruses to humans from animal 37 reservoirs make understanding the coronavirus infection cycle of great importance to human 38 health. 39 coronaviruses are enveloped viruses possessing large, trimeric spike glycoproteins (s) 40 required for the recognition of host receptors for many coronaviruses as well as the fusion of 41 viral and host cell membranes for viral entry into cells 3 . during viral egress from infected host 42 cells, some coronavirus s proteins are cleaved into s1 and s2 subunits. the s1 subunit is 43 responsible for host-receptor binding while the s2 subunit contains the membrane-fusion 44 machinery. during viral entry, the s1 subunit binds host receptors in an interaction thought to 45 expose a secondary cleavage site within s2 (s2´) adjacent to the fusion peptide for cleavage by 46 host proteases 4-7 . this s2´ proteolysis has been hypothesized to facilitate insertion of the fusion 47 peptide into host membranes after the first heptad repeat region (hr1) of the s2 subunit 48 rearranges into an extended α-helix 8-10 . subsequent conformational changes in the second heptad 49 repeat region (hr2) of s2 form a six-helix bundle with hr1, fusing the viral and host 50 membranes and allowing for release of the viral genome into host cells. coronavirus s is also the 51 target of neutralizing antibodies 11 , making an understanding of s structure and conformational 52 transitions pertinent for investigating s antigenic surfaces and designing vaccines. the sars-cov s1 subunit is composed of two distinct domains: an n-terminal domain 54 (s1 ntd) and a receptor-binding domain (s1 rbd) also referred to as the s1 ctd or domain b. 55 each of these domains have been implicated in binding to host receptors, depending on the 56 coronavirus in question. however, most coronaviruses are not known to utilize both the s1 ntd 57 and s1 rbd for viral entry 12 . sars-cov makes use of its s1 rbd to bind to the human 58 angiotensin-converting enzyme 2 (ace2) as its host receptor 13,14 . 59 recent examination using cryo-electron microscopy (cryo-em) has illuminated the 60 prefusion structures of coronavirus spikes [15] [16] [17] [18] [19] [20] [21] [22] . initial examination of hcov-hku1 s showed 61 that the receptor-binding site on the s1 rbd was occluded when the rbd was in a 'down' 62 conformation and it was hypothesized that conformational changes were required to access this 63 site 16 . subsequent studies of the highly pathogenic human coronavirus s proteins of sars-64 cov 15,22 and mers-cov 17,22 showed that these viral s1 rbd do indeed sample an 'up' 65 conformation where the receptor-binding site is accessible. these structural studies also located 66 the positions of the s1/s2 and s2´ cleavage sites on the prefusion spike. the s1/s2 site lies 67 within a surface exposed loop in the second subdomain of s1 16 . however, the s2´ site lies closer 68 to base of the spike and though this region is located on the surface of the spike, cleavage at this 69 site is prevented by surrounding protein elements 17 . 70 to examine the hypothesized conformational transitions induced by proteolysis and 71 receptor binding, we used single-particle cryo-em to determine structures of s in uncleaved, 72 s1/s2 cleaved and ace2-bound states. three-dimensional classification of the s1 rbd 73 positions and corresponding atomic protein models revealed that neither ace2-binding nor 74 trypsin cleavage at the s1/s2 boundary induced substantial conformational changes in the cov may use a distinct mechanism of fp2 membrane insertion. 110 as observed in the previous sars-cov and mers-cov s structures 15,22 , the trimeric s 111 adopts two distinct conformations related to each of the s1 rbd. the 'down' conformation caps 112 the s2 helices and makes extensive contacts with the s1 ntd. the 'up' conformation of the s1 113 rbd exposes the s1 rbd receptor-binding site. it has been previously reported that for wild-114 type sars-cov s, 56% of the particles contained three 'down' rbd conformations while 44% 115 contained a single 'up' s1 rbd conformation 22 . to examine the conformation of the s1 rbd 116 among our sars-cov s 2p ectodomains, we used a local masking and 3-d sorting strategy 17 to 117 more accurately classify the conformations as being either 'down' or 'up' at each of the three s1 118 rbd positions within the trimer. this analysis revealed that the majority of the s 2p proteins 119 were in the single-'up' conformation (58%) with lesser amounts of double-and triple-'up' 120 conformations (39% and 3% respectively) and with no all-'down' conformation observed. the 7 increased propensity to adopt the 'up' s1 rbd conformation may indicate a difference in the 122 coronavirus s containing the 2p mutations, however other differences in sample preparation 123 cannot be ruled out. 124 125 ace2 and s1 c-terminal domains 126 to examine the structure of sars-cov s bound to its receptor, ace2, we combined 127 sars-cov s 2p ectodomain with an excess of soluble human ace2 with subsequent 128 purification by size-exclusion chromatography and immediate cryo-em specimen preparation. 129 initial sorting of particle heterogeneity indicated spikes could be split into ace2-bound (45%) 130 and unbound (55%) classes. using a similar masking and 3-d sorting strategy as above we sorted 131 the unbound s class further into classes with s1 conformations of one or two 'up' s1 rbds (fig 132 3 and supplementary tables 2-4 and supplementary fig. 2-4) . we did not observe an all-'down' 133 class nor a three 'up' s1 rbd class indicating a low prevalence of these conformations among 134 the unbound spikes. expanding our 3d sorting strategy, we classified our ace2-bound particles 135 at each s1 rbd position and identified single, double and triple ace2-bound s. we were 136 further able to identify s1 rbd conformations at the non-ace2 occupied rbd positions to 137 represent each population of s1 rbd conformations among ace2-bound s. 138 as hypothesized by previous structural work 15-17,22 , the s1 rbd recognizes ace2 with 139 an 'up' s1 rbd conformation. the proportion of total 'up' s1 rbd conformations within the 140 ace2-bound and -unbound classes is nearly identical within this dataset (58% 'up' s1 rbd), 141 similar to the proportion of total 'up' s1 rbd in the sars s 2p ectodomain dataset (48%). this 142 strongly suggests that binding of a single ace2 receptor does not induce adjacent s1 rbds to 143 transition from a 'down' to 'up' conformation. hence, ace2 is more likely to bind to an already 144 8 'up' s1 rbd rather than inducing the conformational changes that are required for the s1 rbd 145 to become accessible to ace2. 146 it is noteworthy that despite prolonged co-incubation and an excess of ace2, we had 147 difficulties in saturating the s1 rbd with ace2 in the context of trimeric s ectodomain. this 148 poor saturation is illustrated by the small proportion of triple-bound ace2 and the majority of 149 spikes that are unbound by receptor. in contrast, isolated recombinant s1 rbd easily binds 150 ace2 and is capable saturating ace2 on target cells to block s-mediated entry 14 . our observed 151 sub-stoichiometric ace2 binding to trimeric spikes is consistent with the difficulty in using 152 soluble ace2 receptor to neutralize sars-cov s pseudotyped onto vsv 25 . the reduced 153 binding of ace2 to trimeric spikes is likely due to the incomplete exposure and conformational 154 flexibility of the s1 rbd. incomplete neutralization with soluble receptor was not encountered 155 for mhv which binds ceacam1a via its s1 ntd, which does not undergo conformational 156 changes 19,26 . 157 similar to recently published mers-cov s structures 17 , the ace2-bound rbd adopts a 158 much more extended and rotated conformation compared to s1 rbd modeled in previous 159 sars-cov s structures 22 . this difference is likely due to poor density in the hinge regions 160 between the s1 rbd and subdomain 1 (sd-1) in these previous reconstructions 15,22 rather than 161 the presentation of a unique receptor-bound conformation. indeed, the bound ace2 receptor and 162 s1 rbd for all reconstructions here show poorer density quality than the less mobile regions of 163 the sars-cov s (fig 4) . to improve the density for ace2-bound s1 rbd, we used focused 164 refinement on this region to overcome the flexibility of these domains relative to the rest of s. 165 this yielded a 7.9 å resolution reconstruction with improved local density quality (fig 4b and c) . 166 we successfully placed the crystal structure of the sars-cov s1 rbd bound to ace2 167 9 (2ajf.pdb 23 ) into this density as a rigid body indicating that the previously determined crystal 168 structure accurately recapitulates the conformation between the ace2-bound s1 rbd in the 169 trimeric spike. 170 the ace2-bound, s1 rbd extends upwards and rotates away from contacts with nearby 171 amino acids. hence, any conformational changes induced by receptor binding to the s1 rbd are 172 more likely to be caused by the absence of the s1 rbd contacts in the 'up' conformation, rather 173 than the formation of additional contacts (supplemental figure 5) . this model provides a flexible 174 mechanism for how different coronavirus spikes can bind to different protein receptors with their 175 s1 rbd and facilitate fusion with host cells. moreover, movements of the s1 rbd to the 'up' fig. 6 ). nearing the end of the time course additional lower molecular weight 214 bands are observed which we interpret to be degradation of the s1 subunit. regardless of which 215 construct was used or whether ace2 was bound to the s ectodomains, there is no prominent 216 band that corresponds to a s2ʹ′ cleavage product (approximately 52 kda). 217 to analyze the cleavage products in detail, we performed cryo-em analysis on the 218 trypsin-cleaved sars-cov s 2p ectodomain. using all-particles and c3 symmetry yielded a 219 reconstruction at 3.3 å resolution (fig. 5, supplementary tables 1 and 4 and supplementary fig. 220 7). the short loop containing the s1/s2 cleavage site is disordered in the uncleaved spike 221 reconstruction and remains disordered in the trypsin cleaved reconstruction. moreover, 222 examination of the structure models indicates no significant differences between the trypsin-223 cleaved and uncleaved sars-cov s (fig. 5b) . fine sorting of s1 rbd positions of the trypsin-224 cleaved s reveals a very similar distribution of 'up' s1 rbd conformations available for receptor 225 binding as in the uncleaved samples, although we additionally observe a small proportion of s1 226 rbd in the all-'down' conformation (fig. 5c) . these results indicate that trypsin-cleavage at 227 s1/s2 does not impart large conformational changes on the sars-cov s and justifies the 228 removal of s1/s2 cleavage sites for the production of more homogeneous material as vaccine 229 immunogens. this suggests that although cleavage at s1/s2 may remove an obstacle for 230 conformational changes leading to fusion, s1/s2 cleavage alone does not produce significant 231 conformational changes. terminal helix of s2 hr1 (fig 6) . exposure of this site for cleavage may require remodeling of 237 this penultimate loop or hr1 beyond the conformation observed in the prefusion state. we 238 hypothesize that additional triggers beyond cleavage at the s1/s2 site or protein-receptor binding 239 are needed to transition the spike from its prefusion state to a yet to be observed intermediate. changes and that the s2′ proteolysis does not occur in the s prefusion state (fig. 7) . this grids were loaded onto a titan krios and data was collected using leginon 32 at a total dose of 65 302 e -/å 2 . frames were aligned with motioncor2 (ucsf) 33 implemented in the appion workflow 34 . 303 particles were selected using dog picker 35 . images were assessed and particle picks were 304 masked using em hole punch 36 . the ctf for each image was estimated using gctf 37 . 305 electron microscopy data processing 306 initial particle stacks were cleaned using multiple rounds of 2d classification in 307 relion 38 . good particles were selected as resembling prefusion coronavirus spikes. for the 308 sars s 2p and trypsin-treated sars s 2p, all particles from the clean stacks were used for 309 reconstruction with c3 symmetry. all datasets were extensively sorted using 3d classification to 310 examine heterogeneity in the s1 rbds as described previously 17 . briefly, 3d masks were 311 defined to encompass the possible heterogeneity at each s1 rbd position. the density within 312 these masks was then removed from unfiltered, unsharpened reconstructions. we then used 313 relion_project with image subtraction to create a particle stack containing only the signal arising 314 from the masked density. finally, we used focused 3d classification to identify compositional 315 and conformational states at each s1 rbd position. all 3d reconstructions were produced with 316 relion 38 and final refinements were performed with a six-pixel soft-edge solvent mask. post-317 processing was applied to each reconstruction to apply b-factor sharpening and amplitude 318 corrections as well as to calculate local resolution maps. 319 coordinate models were built for several of the high-resolution reconstructions using 320 5i08.pdb 16 , 2ajf.pdb 23 and 5x4s.pdb 22 as template models with reference to a recently sars and mers: recent 341 insights into emerging coronaviruses proceedings of 344 the national academy of sciences of the united states of america mechanisms of coronavirus cell 347 entry mediated by the viral spike protein two-step conformational changes in a coronavirus envelope 350 glycoprotein mediated by receptor binding and proteolysis receptor-bound porcine epidemic diarrhea virus spike 353 protein cleaved by trypsin induces membrane fusion proteolytic processing of middle east respiratory syndrome coronavirus 356 spikes expands virus tropism inhibitors of cathepsin l prevent severe acute respiratory syndrome 359 coronavirus entry the coronavirus spike protein is 362 a class i virus fusion protein: structural and functional characterization of the fusion core 363 complex structure of influenza 365 haemagglutinin at the ph of membrane fusion tectonic conformational changes of a coronavirus spike glycoprotein 368 promote membrane fusion sars immunity and vaccination recombination, reservoirs, and the modular spike: mechanisms 373 of coronavirus cross-species transmission angiotensin-converting enzyme 2 is a functional receptor for the sars 376 coronavirus a 193-amino acid fragment of the 378 sars coronavirus s protein efficiently binds angiotensin-converting enzyme 2. the 379 cryo-electron microscopy structures of the sars-cov spike glycoprotein 381 reveal a prerequisite conformational state for receptor binding pre-fusion structure of a human coronavirus spike protein immunogenicity and structures of a rationally designed prefusion mers-386 proceedings of the national academy of sciences of the united states cryo-em structure of porcine delta coronavirus spike protein in the pre-389 fusion state cryo-electron microscopy structure of a coronavirus spike glycoprotein 391 trimer glycan shield and epitope masking of a coronavirus spike protein 393 observed by cryo-electron microscopy glycan shield and fusion activation of a deltacoronavirus spike glycoprotein 396 fine-tuned for enteric infections cryo-em structures of mers-cov and sars-cov spike glycoproteins 398 reveal the dynamic receptor binding domains structure of sars coronavirus spike receptor-401 binding domain complexed with receptor peptide forms an extended bipartite fusion platform that perturbs membrane order in a 405 calcium-dependent manner vesicular stomatitis virus pseudotyped with severe acute respiratory 408 syndrome coronavirus spike protein n-terminal domain of the murine coronavirus 411 receptor ceacam1 is responsible for fusogenic activation and conformational changes 412 of the spike protein activation of the sars coronavirus spike 414 protein via sequential proteolytic cleavage at two distinct sites protease-mediated 418 enhancement of severe acute respiratory syndrome coronavirus infection physiological and molecular triggers for sars-cov 422 membrane fusion and entry into host cells host cell proteases: critical determinants of coronavirus 425 tropism and pathogenesis discovery of a rich gene pool of bat sars-related coronaviruses provides new 428 insights into the origin of sars coronavirus automated molecular microscopy: the new leginon system motioncor2: anisotropic correction of beam-induced motion for improved 433 cryo-electron microscopy appion: an integrated, database-driven pipeline to facilitate em image 435 processing dog picker 437 and tiltpicker: software tools to facilitate particle selection in single particle electron 438 microscopy emhp: an accurate automated hole 440 masking algorithm for single-particle cryo-em image processing real-time ctf determination and correction accelerated cryo-em structure 445 determination with parallelisation using gpus in relion-2 coot: model-building tools for molecular graphics atomic-accuracy models from 4.5-a cryo-electron microscopy data with 451 density-guided iterative local refinement phenix: a comprehensive python-based system for macromolecular 454 structure solution computational resources for electron microscopy 461 at the scripps research institute are supported by nih grant od021634 466 processed electron microscopy data. r.n.k and c.a.c. built and refined atomic models 457 we gratefully acknowledge travis nieusma, charles bowman, jean-christophe ducom and bill 458 anderson for microscopy and computational support. we also thank lauren holden for a critical 459 reading of this manuscript. this work was supported by grants from nih/niaid to a.b.w and 460 key: cord-258902-h0wrs01h authors: liu, xianglei; drelich, aleksandra; li, wei; chen, chuan; sun, zehua; shi, megan; adams, cynthia; mellors, john w.; tseng, chien-te; dimitrov, dimiter s. title: enhanced elicitation of potent neutralizing antibodies by the sars-cov-2 spike receptor binding domain fc fusion protein in mice date: 2020-09-22 journal: vaccine doi: 10.1016/j.vaccine.2020.09.058 sha: doc_id: 258902 cord_uid: h0wrs01h the development of an effective vaccine against sars-cov-2 is urgently needed. we generated sars-cov-2 rbd-fc fusion protein and evaluated its potency to elicit neutralizing antibody response in mice. rbd-fc elicited a higher neutralizing antibodies titer than rbd as evaluated by a pseudovirus neutralization assay and a live virus based microneutralization assay. furthermore, rbd-fc immunized sera better inhibited cell-cell fusion, as evaluated by a quantitative cell-cell fusion assay. the cell-cell fusion assay results correlated well with the virus neutralization potency and could be used for high-throughput screening of large panels of anti-sars-cov-2 antibodies and vaccines without the requirement of live virus infection in bsl3 containment. moreover, the anti-rbd sera did not enhance the pseudotyped sars-cov-2 infection of k562 cells. these results demonstrate that fc fusion can significantly improve the humoral immune response to recombinant rbd immunogen, and suggest that rbd-fc could serve as a useful component of effective vaccines against sars-cov-2. the coronavirus disease 2019 (covid-19) outbreak has become a global pandemic responsible for over 20 million confirmed cases and over 0.7 million deaths worldwide as of aug 11, 2020. severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the novel betacoronavirus, is the etiological agent of covid-19. vaccines are a highly effective strategy in preventing the spread of infectious diseases with advantages of low production and distribution cost; stark reduction in morbidity; and minimal negative long-term effects on overall health. currently, no approved vaccines are available for covid19 patients although tremendous scientific and industrial efforts have been dedicated. several approaches to the development of covid-19 vaccines have emerged including dna vaccines, rna vaccines, viral vector vaccines, recombinant subunit vaccines, inactivated and attenuated virus vaccines [1] . as of aug 10, 2020, 28 candidate vaccines are undergoing clinical evaluations with more than 139 vaccines in preclinical development stages (draft landscape of covid-19 candidate vaccines, who). among those, cansino's recombinant adenovirus type-5 vectored vaccine has achieved the most fast clinical progress as the first-in-human trial vaccine, reporting tolerability and high immunogenicity after 28 days postvaccination [2] . been demonstrated to be an appropriate immunogen capable of eliciting neutralizing antibodies [3] . both viruses are phylogenetically close to sars-cov-2 [4] , thus supporting the rationale of using the sars-cov-2 s protein as a subunit vaccine. subunit vaccines contain recombinant antigen proteins with strong immunogenicity capable of efficiently stimulating the host immune system. advantages of subunit vaccines include easy manufacture, low cost, and overall safety, since they do not contain genetic material and have a low probability to induce severe adverse reactions. for example, subunit vaccines are reported to be safer than other vaccines such as virus like particles, inactivated whole viruses and an rdna expressed s protein, which have been shown to induce the cytokine th2-type immunopathology in sars-cov [5] . several subunit vaccines based on the full-length sars-cov-2 s proteins are under-development, with five candidates in clinical trials. however, the full-length s protein immunogens contain many non-neutralizing epitopes that could result in enhanced infection through the antibody-dependent enhancement (ade) effect, which may occur through the fcγr-mediated internalization of antibody-bound virions in fcγr-expressing cells [6, 7] . the risk of ade, observed for sars-cov, mers-cov, hiv-1, zika and dengue virus vaccination, has become a major concern in vaccine development [8] [9] [10] . the sars-cov-2 receptor-binding domain (rbd) is the protruding site of the s protein that mediates viral cell fusion during the initial infection event through binding of the human receptor angiotensin-converting enzyme 2 (hace2) [11, 12] . based on its highly homology to sars-cov, sars-cov-2 rbd is corroborated to contain immune dominant epitopes capable of eliciting antibodies that can neutralize viral infection and block viral entry by competing hace2 binding. the antigenic regions on sars-cov-2 were also confirmed by computational studies exploring t cell and b cell epitopes [13] . compared to the full-length s protein based subunit vaccine, reducing the immunogen to only the neutralizing epitope containing rbd can not only specifically mount neutralizing antibody titers, but could also mitigate the risk of ade, which in most cases are mediated by the non-neutralizing antibodies [14] . therefore, sars-cov-2 rbd has been selected as a primary target for vaccine design [15, 16] . ravichandran et al recently found that compared to full-length s, sars-cov-2 rbd immunogen elicited a higher titer of neutralizing antibodies with 5-fold higher affinity [17] . yang et al also showed that rbd can induce a potent antibody response in the immunized mice, rabbits and non-human primates [18] . zang et al (preprint) have showed that the anti-rbd sera from mice did not promote (both pseudotyped and authentic) sars-cov-2 infection of fcγ receptor-bearing cells [19] . given those advantages, we chose the sars-cov-2 rbd as a subunit vaccine. to boost the vaccination-induced antibody response, we fused rbd to the igg1 fc. the fc fragment can serve as a vaccine adjuvant by promoting cellular and humoral immune responses, probably by facilitating antigen delivery and presentation through interacting with fcγ receptors on antigenpresenting cells. in addition, the fc-fusion can also improve recombinant immuogens solubility and stability and extend their in vivo half-life after injection by interacting with human neonatal fcγ-receptor (hfcrn) [20] . to stimulate anti-rbd antibody titers, we also utilized a well-accepted, highly effective adjuvant, mf59 tm . mf59 has been proven to increase neutralizing antibody production and boost the th2 and th1 immune responses. several vaccines containing mf59 as adjuvants are undergoing clinical trials (phases i-iii). notably, mf59-adjuvanted seasonal influenza vaccine (fluad™) has already been licensed, showing acceptable safety and tolerability, and improved immunogenicity [21] . the gene of sars-cov-2 rbd domain (residues 330-532) was synthesized by idt (coralville, iowa), then cloned in frame to human igg1 fc or 6×his tag in the mammalian cell expression plasmid. these proteins were expressed in the expi293 tm expression system (thermo fisher scientific) and purified by protein a resin (genscript) or ni-nta resin (thermo fisher scientific). the purified sars-cov-2 rbd and rbd-fc were analyzed by sds-page and western blot (wb). protein concentration was measured spectrophotometrically (nanovue, ge healthcare), 2 μg of proteins were separated by nupage ® 4-12% bis-tris gels (life technologies), protein purity was estimated as >95%. for western blot, the separated proteins were transferred to nitrocellulose membranes. after blocking at room temperature using 5% non-fat milk in pbst for 1h, the blots were incubated for 2h at room temperature with 100 nm hace2-mfc (mouse fc, sino biological, beijing, china). after three washes, the blots were incubated with anti-mouse igg-horseradish peroxidase (hrp) conjugated secondary antibody (1:1000, sigma-aldrich) for 1h at room temperature. the membranes were reacted with enhanced ecl chemiluminescence reagent (millipore, billerica, usa) and exposed by bio-rad detection system. protein deglycosylation mix ii (neb) was used for deglycosylation reaction following manufacturer's instructions. briefly, 2 μg proteins were mixed with deglycosylation mix buffer 1 (non-denaturing reaction) or buffer 2 (denaturing reaction, with an additional incubation at 75°c for 10min), then incubated with protein deglycosylation mix ii for reaction (25°c for 30 min, and then 37°c for 16 h). the deglycosylated proteins were analyzed by sds-page. the mw of enzymes in protein deglycosylation mix ii is approximately 36, 100, 147 and 231 kda. genes of hace2 (origene, rockville, md) and the full length s protein of sars-cov-2 (codon optimized and synthesized by idt) were subcloned into our in-house mammalian cell expression plasmid and used to construct stable cell line 293t-ace2 and 293t-s, which were cultured in dulbecco's modified eagle's medium (dmem, gibco) with 10% fetal bovine serum (fbs), 1% penicillin/streptomycin (p/s) and 200 μg/ml zeocin (thermo fisher). for the determination of 293t-s cell line, 500 nm hace2-fc (sino biological, beijing, china) were incubated with cells for 30min at 4 o c. cells were washed and then incubated with pe conjugated anti-human fc antibody (sigma-aldrich) for 30min at 4 o c. bound antibodies were detected by flow cytometry using bd lsr ii (san jose, ca). for the 293t-ace2 cell line, 500 nm rbd-his followed by pe conjugated anti-his antibody (sigma-aldrich) were used for analysis. four groups of 8-10 week old female balb/c mice (n=5) were immunized twice (day 0 and day 14) subcutaneously with rbd proteins (10 μg/mouse) with or without adjuvant mf59. group 1 was immunized with rbd-fc fusion, group 2 was immunized with rbd-fc fusion in emulsion with mf59, group 3 was immunized with rbd in emulsion with mf59, group 4 served as a control and was immunized subcutaneously with dulbecco's phosphate-buffered saline dpbs (gibco tm ). sera were collected before (pre-vaccination), after 13 days, and after 27 days vaccination. for evaluation of affinity of rbd and rbd-fc to hace2, both proteins were coated on a 96-well plate (costar) at 200 ng/well in pbs overnight at 4 o c. the plate was blocked using 3% skim milk for 1h at room temperature (rt). we then added serially diluted hace2-mfc (mouse fc, sino biological, beijing, china) and incubated for 2h at rt. the plates were washed 4 times with 0.05% tween in phosphate buffered saline (pbst). anti-mouse igg-horseradish peroxidase (hrp) conjugated secondary antibody (sigma-aldrich) was added to the plate followed by incubation for 1h at rt. after another 4 washes with pbst, the plate was incubated with a 3,3',5,5'tetramethylbenzidine substrate solution (tmb, sigma-aldrich) for 3min. the reaction was stopped using 1 m h 2 so 4 followed by reading absorbance of each well at 450 nm. for detection of anti-rbd or anti-(s1+s2) antibodies in mice serum, the sars-cov-2 rbd proteins or s1+s2 (sino biological, beijing, china) were coated at 200 ng/well in pbs overnight at 4 o c. after blocking, serially diluted mouse serum were added and incubated for 2h at rt. for antibody isotyping, the bound rbd-specific antibodies were detected by anti-mouse igg, igm, iga hrp conjugated secondary antibody (sigma-aldrich), respectively. for competitive elisa, ~20 nm (4 μg/ml) biotinylated hace2 (sino biological, beijing, china) was incubated with serially diluted mouse serum, and the mixtures were added to rbd coated wells. after washing, bound hace2 was detected by streptavidin-hrp secondary antibody (sigma-aldrich). the pseudovirus neutralization assay was performed based on previous protocols. briefly, hiv-1 backbone based pseudovirus was packaged in 293t cells by co-transfecting with plasmid encoding sars-cov-2 s protein and plasmid encoding luciferase expressing hiv-1 genome (pnl4-3.luc.re) using polyethylenimine (pei). pseudovirus-containing supernatants were collected 48 h later and concentrated using lenti-x™ concentrator kit (takara, ca). pseudovirus neutralization assay was then performed by incubation of sars-cov-2 pseudovirus with serially diluted mice serum for 1h at 37 °c, followed by addition of the mixture into pre-seeded 293t-ace2 cells. the mixture was then centrifuged at 1000 × g for 1h at rt. the medium was replaced 4 hrs later. after 24h, luciferase expression was determined by bright-glo kits (promega, madison, wi) and read using biotek synergy multi-mode reader (winooski, vt). the 50% pseudovirus neutralizing antibody titer (nt 50 ) was calculated using the graphpad prism 7. the standard live virus-based microneutralization (mn) assay was used. briefly, serially five-fold (start from 1:5) and duplicate dilutions of mice serum were incubated with 100 pfu of sars-cov-2 at room temperature for 2h before transferring into designated wells of confluent vero e6 cells (atcc, crl-1586) grown in 96-well microtiter plates. vero e6 cells cultured with medium with or without virus were included as positive and negative controls, respectively. after incubation at 37 o c for 4 days, individual wells were observed under the microscopy for the status of virusinduced formation of cytopathic effect (cpe). the titer of mice serum (nt 100 ) was expressed as the lowest dilution folds capable of completely preventing virus-induced cpe in 100% of the wells. to test mice serum mediated inhibition of cell fusions, the β-gal reporter gene based quantitative cell fusion assay was used. briefly, 293t-s cells were infected with t7 polymerase-expressing vaccinia virus (vtf7-3), while 293t-ace2 cells were infected with vaccinia virus (vcb21r lac-z) encoding t7 promotor controlled β-gal. two hours after infection, cells were incubated with fresh medium and transferred to 37°c for overnight incubation. the next day, 293t-s cells were pre-mixed with serially diluted mice serum at 37°c for 1h followed by incubation with 293t-ace2 cells at a 1:1 ratio for 3h at 37°c. then cells were then lysed, and the β-gal activity was measured using β-galactosidase assay kit (substrate cprg, g-biosciences, st. louis, mo) following the manufacturer's protocols. fusion inhibition percentage (sample reading, f) was normalized by maximal fusion (reading, f max ) of 293t-s and 293t-ace2 cells in the absence of inhibitors using this formula: fusion inhibition % = [(f max -f)/(f max -f blank )] × 100%, in which f blank refers to the od reading of 293t-s and 293t incubation wells. fusion inhibition percentage was plotted against serum dilution folds from which ic 50 was calculated in graphpad prism 7. fcγrii expressing cell lines k562 (atcc, ccl-243) were used to perform ade assays. briefly, the mouse serum were serially diluted, mixed with sars-cov-2 pseudovirus, and incubated at 37℃ for 1h. then, the mixtures were added to the pre-seeded plates with k562 cells. the following infection and culturing steps were carried out as described above in the pseudovirus neutralization assay. pseudovirus infected k562 or 293t-ace2 cells were set as the negative and positive controls, respectively. all experiments were conducted in duplicate, and data were averaged and presented as the mean ± standard deviation (sd). significant differences were determined by one-way analysis of variance followed by tukey's test, using the graphpad prism (version 7) package. statistical significance was defined as p < 0.05. recombinant sars-cov-2 rbd (without fc) and rbd-fc proteins were produced in expi293 tm mammalian cells, and then verified by sds-page and western blot. rbd-fc showed a homogenous band (fig. 1a) while the rbd exhibited relatively heterogeneous bands due to varying extents of glycosylation, which was also found by yang et al [18] . the rbd protein showed a single band upon deglycosylation, confirming the heterogeneous bands resulting from glycosylation (fig. 1b) . besides, western blot results showed that rbd with different glycoforms can react with hace2 (fig. s1) . elisa binding to hace2 further validated the qualities of both rbd proteins (fig. 1c) . rbd antigens produced in this study were also used for panning against our in-house phage antibody libraries to retrieve high affinity binders [22] . four groups of 8-10 week old female balb/c mice (n=5) were immunized subcutaneously at day 0 and boosted at day 14 with rbd-fc, rbd-fc in emulsion with mf59 (rbd-fc+mf59), and rbd in emulsion with mf59 (rbd+mf59) for each group at a dose of 10 μg of proteins per mouse. the fourth group injection was of dpbs, which served as the negative control (fig. 1d) . on day 0 (pre-immunization), day 13 and day 27, mouse sera were collected and analyzed for rbd binding, pseudovirus and live virus neutralization, and cell-cell fusion inhibition. anti-rbd sera from each trial group were firstly evaluated for rbd binding as measured by elisa (fig. 2a) . the anti-rbd antibody in post immune mouse sera were also isotyped by anti-mouse igg, igm and iga antibodies, respectively (fig. s2) . the rbd antibody titers were calculated as the dilution folds that remain 50% of maximal binding signal (ec 50 ). the recombinant rbd (his tag) was used as the detection antigen to avoid the interference of antihuman fc antibody titers in mice. the impact of his tag on the detection of rbd binding titer is marginal ( figure. s2c) . results showed that for the sera collected at 13 and 27 days post immunization, the anti-rbd antibodies were mostly composed of the igg isotype with only marginally detectable igm (fig. s2a) and no detectable iga isotype (fig. s2b) . the low igm titer detected at day 13 and 27 may correlate to the fact that igm is typically rapidly mounted post infection (within one week) followed by isotype switching into igg isotype [23] . the lack of iga titer may result from iga usually deriving from mucosa immunity, leading to low titers in sera [24] . for the igg isotype antibodies, the pre-immunization sera showed no binding to rbd, while the day 13 sera from all three rbd immunized groups exhibited varying extents of binding to the rbd. interestingly, the rbd binding titer elicited by the rbd+m59 group on day 13 was much less than titers of the rbd-fc (titer 1:36) and rbd-fc+m59 (titer 1:66) groups, indicating the immune stimulation roles of fc fusion. however, for post-boosting sera on day 27, the rbd binding titers of rbd+mf59 group was significantly mounted to 1:368. in contrast, on day 27 the titers of rbd-fc (titer 1:603) and rbd-fc+mf59 (titer 1:1130) groups were only improved by 17-fold compared to those of day 13, indicating distinct humoral response kinetics against rbd and rbd-fc immunogens. interestingly, although to a lesser extent than before receiving the booster, the rbd-fc and rbd-fc+mf59 groups exhibited 1.6 and 3 folds higher titers, over the rbd+mf59 group (p<0.05) respectively, assuring the enhancing role of fc in elicitation antibody response by the rbd immunogen. it is also intriguing that the rbd-fc+mf59 group exhibited slightly higher titers than the rbd-fc group, probably due to the adjuvant role of mf59. interestingly, we also correlated the rbd binding titer to the full-length s ectodomain binding titer for the day 27 sera. the elisa showed that the rbd binding sera also bound to s1+s2 with similar titers (fig. s2d) , which suggest that the rbd recognition antibody in the sera can also bind to full length s. hace2 blocking is a surrogate indicator for anti-sars-cov-2 antibody neutralizing activity. to preliminarily infer the neutralizing titers of post-immunization mouse serum, we performed the hace2 competitive elisa, in which serially diluted mouse sera in the presence of the biotinylated hace2 were added into rbd coated plates. bound hace2 was detected by the streptavidin-hrp secondary antibody. results showed the three rbd immunogen groups developed discernable hace2 competitive titers on day 13 compared to the pbs control group; further significantly boosted to 1:34, 1:65, 1:31 for the rbd-fc, rbd-fc+mf59, rbd+mf59 groups respectively on day 27 (fig. 2b) . consistent with the above rbd binding titer, the rbd-fc and rbd-fc+mf59 groups sera showed 1.1 folds and 2.1 folds higher competitive titers respectively than the rbd+mf59 group (p<0.05), supporting the role of fc in mounting neutralization titers. the competitive elisa results gave the specific hace2 blocking titers elicited by rbd immunogens, which presumably predict their neutralization activity [25] . next we exploited the sars-cov-2 s pseudotyped hiv-1 to evaluate the neutralization activity of those anti-rbd sera. sars-cov-2 pseudovirus was packaged by co-transfecting hek 293t cells with pcdna3.1-s plasmid encoding codon-optimized full-length sars-cov-2 s protein and pnl4-3.luc.re plasmid containing luciferase expressing hiv-1 genome. serially diluted mouse sera were pre-incubated with pseudovirus followed by infection of 293t cells stably expressing hace2 (293t-ace2). as shown in fig. 3a , on day 13 all the rbd immunized groups sera showed substantial 50% neutralizing antibody titers (nt 50 ) compared to the pre-immune sera on day 0 (titer 1:63, 1:76, 1:10), which were largely boosted to 1:486, 1:2243, 1:165 for the rbd-fc, rbd-fc+mf59 and rbd+mf59 groups respectively. intriguingly, unlike the marginal differences for the rbd binding and hace2 competitive titers across the sera of the three rbd immunized groups on day 27, the pseudovirus neutralization titers were significantly distinct with the rbd-fc+mf59 group showing highest titers, 4.6-fold higher than the rbd-fc group and 13.6fold than the rbd+mf59 group (p<0.05). in addition to the pseudovirus neutralization, we also evaluated the live virus neutralization potency of those mouse anti-rbd sera (day 27) by using a microneutralization (mn) assay. in this assay, the cytopathic effect (cpe) of vero e6 was observed after 4 days incubation with live virus, which was pre-mixed with the anti-rbd sera. the neutralization titer of mice serum (nt 100 ) was expressed as the lowest dilution folds capable of completely preventing virus-induced cpe in 100% of the wells. consistently, the nt 100 of serum in rbd-fc+mf59 group (1:25) was higher than those of the rbd-fc and rbd+mf59 groups (1:5) (fig. 3b) . these results clearly demonstrated that fc fusion could significantly augment the elicitation of neutralizing antibody titers by rbd immunogens, and the adjuvant mf59 can further stimulate the antigenicity of the rbd-fc fusion proteins. interestingly, we found that the pseudovirus neutralization titer positively correlated with the hace2 competition titer (fig. 3c) , demonstrating the utility of our hace2 competition elisa in predicting neutralization titers in convalescent plasma therapy and in detecting of the presence of neutralizing antibodies in serological tests during the covid19 pandemic. to further evaluate whether the anti-rbd sera could prevent sars-cov-2 s-mediated cell-cell fusion, we established a quantitative cell fusion assay using β-galactosidase (β-gal) as a reporter gene. we constructed 293t cell lines stably overexpressing sars-cov-2 s (293t-s) and hace2 (293t-ace2) respectively (fig. s3) . in this assay, the 293t-s cells were infected with t7 polymerase-expressing vtf7-3 vaccinia virus and the 293t-ace2 cells were infected with t7 promotor controlled β-gal expressing vcb21r vaccinia virus. therefore, β-gal expression is only allowed after cell-cell fusion, which can be quantified by monitoring β-gal activity. serially diluted mouse sera were pre-mixed with infected 293t-s cells followed by incubation with infected 293t-ace2 cells. as shown in fig. 4a , the pre-vaccination sera at day 0 and the pbs control mouse sera did not inhibit the cell-cell fusion. however, the day 27 rbd-fc+mf59 sera showed obvious cell-cell fusion inhibition with a 50% fusion inhibition antibody titers (ic 50 ) of 1:97, which was 3.9-fold higher than the inhibition titers of the rbd-fc (1:25) and 4.9-fold higher than the rbd+mf59 (1:20) group (p<0.05). interestingly, in this assay, the rbd-fc and the rbd+mf59 groups did not show significant differences (p>0.05). we also observed a nearly perfect correlation of the cell-cell fusion inhibition titer with the pseudovirus neutralization titer (r 2 =0.9872, p<0.0001, fig. 4b ), which may be attributed to theirmechanism of action. anti-rbd antibodies typically neutralize virus by blocking viral entry. for sars-cov-2, virus entry and cell-cell fusion share similar mechanisms. both viral entry and cell-cell fusion are initiated by the s protein binding to the receptor ace2, followed by s1 subunit triggered susceptibility to protease cleavage, which causes s1 dissociation and conformational change of the s2 subunit. then, the fusion peptide (fp) in s2 is exposed for anchoring into host cell membrane and heptad repeats (hr1 and hr2) establishes the six helical-bundle resulting in the membrane fusion between viral and host cells [26] . molecules, including antibodies, interfering with any of the above processes can block viral entry as well as cell-cell fusion [11] . based on their similar mechanisms, the inhibitory activity of antibodies for cell-cell fusion can be a highly relevant predictor of the antibody neutralizing activity. this is further supported by our and others' sars-cov-2 neutralizing antibodies, which shows both potent inhibition of s mediated cell-cell fusion and neutralization of sars-cov-2 [27] . although highly correlated, there are differences between these two assays in terms of different environments on the cell and viral surface such as s protein conformation/ density, accessibility of proteases. due to the high correlation to the virus neutralization, this method allows for high-throughput screening and is therefore well suited for the characterization of cell-cell fusion mediated by sars-cov-2 or other viruses. in addition, this assay is highly effective in screening potential neutralizing antibodies with fast speed since this assay can be finished within one day without the requirement of in biosafety level 3 facilities while the virus neutralization assays typically require several days which is of particular relevance in the context of the global pandemic, which urgently needs vaccines and candidate antibody drugs. from the mechanism, one can also envision that rbd antibodies showing ace2 competition is sufficient, but not necessary for virus neutralization and cell-cell fusion, since antibodies disturbing other entry steps, rather than ace2/rbd binding, can also neutralize virus and inhibit cell-cell fusion, as exampled by the antibody 47d11 [27] . in this regard, one pertinent result from this study is that we found the extent of the correlation of ace2 competition elisa titer with the neutralizing titer (r 2 =0.8204, p=0.0129, fig. 3c ) and competition elisa titer with cell-cell fusion inhibition titer (r 2 =0.8854, p=0.0051, fig. 4c ) were lower than that of neutralizing titer to cell-cell fusion inhibition titer (r 2 =0.9872, p<0.0001, fig. 4b ). finally, we evaluated whether the anti-rbd mouse sera can enhance sars-cov-2 infection of fcγrii expressing k562 cells [28] . the results showed that sars-cov-2 pseudovirus alone cannot infect the k562 cells (fig. s4) . in addition, treatment with serially diluted (ranging from 1:250 to 1:10 7 ) anti-rbd sera did not enhance sars-cov-2 pseudovirus infection, indicating that the anti-rbd sera may not promote ade. after washing, the binding was detected by hrp conjugated anti-mouse igg antibody. (b) 200 ng of rbd were coated and 5-fold serially diluted mice serum were added in the presence of ~20 nm biotinylated hace2 followed by pbst washing. for detection, streptavidin-hrp secondary antibody was used. experiments were performed in duplicate and the error bars denote ± sd, n =2. statistical significance was defined as *: p<0.05. safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored covid-19 vaccine: a dose-escalation, open-label, nonrandomised, first-in-human trial the spike protein of sars-cov--a target for vaccine and therapeutic development a highly conserved cryptic epitope in the receptor binding domains of sars-cov-2 and sars-cov immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection fc receptors in antibody-dependent enhancement of viral infections the potential for antibody-dependent enhancement of sars-cov-2 infection: translational implications for vaccine development molecular mechanism for antibody-dependent enhancement of coronavirus entry tortoises, hares, and vaccines: a cautionary note for sars-cov-2 vaccine development sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor the secret life of ace2 as a receptor for the sars virus immunoinformatics-aided identification of t cell and b cell epitopes in the surface glycoprotein of 2019-ncov implications of antibodydependent enhancement of infection for sars-cov-2 countermeasures receptor-binding domain as a target for developing sars vaccines characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine antibody signature induced by sars-cov-2 spike protein immunogens in rabbits a vaccine targeting the rbd of the s protein of sars-cov-2 induces protective immunity immunization with the receptor-binding domain of sars-cov-2 elicits antibodies cross-neutralizing sars-cov-2 and sars-cov without antibody-dependent enhancement fc-based recombinant henipavirus vaccines elicit broad neutralizing antibody responses in mice vaccines with mf59 adjuvant expand the antibody repertoire to target protective sites of pandemic avian h5n1 influenza virus potent neutralization of sars-cov-2 by human antibody heavy-chain variable domains isolated from a large library with a new stable scaffold antibody response of mice to lactate dehydrogenase-elevating virus during infection and immunization with inactivated virus the effects of secretory iga in the mucosal immune system a sars-cov-2 surrogate virus neutralization test based on antibody-mediated blockage of ace2-spike protein-protein interaction cell entry mechanisms of sars-cov-2 a human monoclonal antibody blocking sars-cov-2 infection dengue virus neutralization in cells expressing fc gamma receptors inhibition of cell-cell fusion (a) by mice serum, correlation analysis for fusion inhibition assay with pseudo-nertralization (b) and competitive elisa (c). (a) a β-galactosidase (β-gal) reporter gene-based quantitative cell-cell fusion assay was used, in which t7 polymerase expressing 293t-s pre-incubated with mice serum followed by mixing with t7 promotor controlled β-gal expressing 293t-ace2 cells correlation analysis between cell-cell fusion inhibition (ic 50 ) and pseudo-neutralization antibody titers (nt 50 ) for sera of day 13 and day 27. (c) correlation analysis between cell-cell fusion inhibition (ic 50 ) and competitive elisa (ec 50 ) for sera of day 13 and day 27. correlation and linear regression analyses were performed in graphpad prism using pearson's correlation coefficients. statistical significance was calculated using the two-tailed test conflict of interest statement. the authors declare no conflict of interest we would like to thank the members of the center for antibody therapeutics doncho zhelev, du-san baek, liyong zhang, and xiaojie chu for their helpful discussions. this work was supported by the university of pittsburgh medical center. key: cord-278869-7zr1118b authors: ravichandran, supriya; coyle, elizabeth m.; klenow, laura; tang, juanjie; grubbs, gabrielle; liu, shufeng; wang, tony; golding, hana; khurana, surender title: antibody repertoire induced by sars-cov-2 spike protein immunogens date: 2020-05-13 journal: biorxiv doi: 10.1101/2020.05.12.091918 sha: doc_id: 278869 cord_uid: 7zr1118b multiple vaccine candidates against sars-cov-2 based on viral spike protein are under development. however, there is limited information on the quality of antibody response generated following vaccination by these vaccine modalities. to better understand antibody response induced by spike protein-based vaccines, we immunized rabbits with various sars-cov-2 spike protein antigens: s-ectodomain (s1+s2) (aa 16-1213), which lacks the cytoplasmic and transmembrane domains (ct-tm), the s1 domain (aa 16-685), the receptor-binding domain (rbd) (aa 319-541), and the s2 domain (aa 686-1213 as control). antibody response was analyzed by elisa, surface plasmon resonance (spr) against different spike proteins in native conformation, and a pseudovirion neutralization assay to measure the quality and function of the antibodies elicited by the different spike antigens. all three antigens (s1+s2 ectodomain, s1 domain, and rbd) generated strong neutralizing antibodies against sars-cov-2. vaccination induced antibody repertoire was analyzed by sars-cov-2 spike genome fragment phage display libraries (sars-cov-2 gfpdl), which identified immunodominant epitopes in the s1, s1-rbd and s2 domains. furthermore, these analyses demonstrated that surprisingly the rbd immunogen elicited a higher antibody titer with 5-fold higher affinity antibodies to native spike antigens compared with other spike antigens. these findings may help guide rational vaccine design and facilitate development and evaluation of effective therapeutics and vaccines against covid-19 disease. one sentence summary sars-cov-2 spike induced immune response the ongoing pandemic of sars-cov-2 has resulted in more than 2 million human cases 42 and 125,000 deaths as of 15 th april 2020. therefore, development of effective vaccines for commercially available sars-cov-2 spike protein and subdomains: the spike s1+s2 ectodomain 71 (aa 16-1213), the s1 domain (aa 16-685), rbd domain (aa 319-541), and the s2 domain (aa 686-72 1213) as a control, which is devoid of rbd (fig. 1a, suppl. fig. 1 ). theese spike proteins were 73 either produced in hek 293 mammalian cells (s1 and rbd) or insect cells (s1+s2 ectodomain 74 and s2 domain). the purified s1+s2 ectodomain, the s1 domain, and the rbd proteins retained 75 the functional activity as demonstrated in spr assay using human ace2 protein, the sars-cov-76 2 receptor (fig. 1b) . the s1+s2 ectodomain, s1 domain and rbd (black, blue and red binding 77 curves, respectively) demonstrated high-affinity interaction with human ace2. the control s2 78 domain protein (green curve), lacking the rbd, did not bind to human ace2, demonstrating 79 specificity of this receptor-binding assay (fig.1b) . female new zealand white rabbits were immunized twice intra-muscularly at a 14-day 81 interval with 50 g of the purified proteins mixed with emulsigen adjuvant. sera were collected 82 before (pre-vaccination) and after the first and second vaccination and analyzed for binding 83 antibodies in elisa and spr, in a pseudovirion neutralization assay, and by gfpdl analysis. igg to various spike proteins and domains in elisa (s1+s2; black, s1; blue, rbd; red, and s2; 88 green) (fig. 1c) . representative titration curves to spike ectodomain (s1+s2) and to the rbd in 89 igg-elisa are shown in suppl. fig. 2 . end-point titers of the serum igg were determined as the 90 reciprocal of the highest dilution providing an optical density (od) twice that of the negative 91 control (fig. 1c ). all four immunogens elicited strong igg binding to the spike ectodomain 92 (s1+s2). binding to the individual domains (s1, s2, and rbd) was specific, in that sera generated 93 by s2 vaccination bound to s2, but not to s1 or rbd, and vice-versa (fig. 1c ). spr allows following antibody binding to captured antigens in real-time kinetics, including 95 total antibody binding in resonance units (max ru) and affinity kinetics (suppl. fig. 3 ). in elisa, 96 the antigens directly coated in the wells can be partially denatured increasing the likelihood of 97 presenting epitopes that are not seen on the native form of the protein by the polyclonal serum igg. on the other hand, in our spr, the purified recombinant spike proteins were captured to a ni-nta 99 sensor chip to maintain the native conformation (as determined by ace2 binding) to allow 100 comparisons of binding to and dissociation from the four proteins. importantly, the protein density 101 captured on the chip surface is low (200 ru) and was optimized to measure primarily monovalent 102 interactions, so as to measure the average affinity of antibody binding in the polyclonal serum (8, 103 13). additionally, while elisa measured only igg binding, in spr, all antibody isotypes 104 contributed to antibody binding to the captured spike antigen. in the current study, all rabbit sera 105 contained anti spike antibodies that were at least 86% igg (data not shown). serial dilutions of 106 post-vaccination serum were analyzed for binding kinetics with different spike proteins (suppl. 107 fig. 3 ). the spike ectodomain (s1+s2) generated antibodies that predominantly bound to s1+s2 108 6 (black bar), followed by the s1 protein (blue bar), and 3-fold lower antibody binding to the rbd 109 and the s2 domain (red and green bars, respectively) (fig. 1d ). the s1 domain antigen induced 110 antibodies that bound with similar titers (max ru values) to the s1+s2, s1 and rbd proteins 111 (black, blue and red bars, respectively), and did not show reactivity to the s2 domain (green bar). however, the antibody reactivity of rabbit anti-s1 serum to s1+s2 domain was 3-fold lower than 113 the antibodies in the rabbit anti-s1+s2 serum. rbd immunization generated similar high-titer 114 antibody binding to s1+s2, s1 and rbd (black, blue and red bars, respectively), (fig. 1d) . in 115 contrast, the s2 domain induced antibodies that primarily bound to homologous s2 antigen (green 116 bars) and only weakly binding to the s1+s2 ectodomain (black bars), and no binding to either s1 117 or rbd (fig. 1d ). antibody off-rate constants, which describe the fraction of antigen-antibody complexes 119 that decay per second, were determined directly from the serum sample interaction with sars-120 cov-2 spike ectodomain (s1+s2), s1, s2, and rbd using spr in the dissociation phase only for 121 sensorgrams with max ru in the range of 20-100 ru (suppl. fig. 3 ) and calculated using the 122 biorad proteon manager software for the heterogeneous sample model as described before(11). these off rates provide additional important information on the affinity of the antibodies following 124 vaccination with the different spike proteins that are likely to have an impact on the antibody 125 function in vivo, as was observed previously in studies with influenza virus, rsv and ebola virus 126 (13-15). surprisingly, we observed significant differences in the affinities of antibodies elicited by 127 the four spike antigens (fig. 1e) . specifically, the rbd induced 5-fold higher affinity antibodies 128 (slower dissociation rates) against s1+s2 (black), s1 (blue) and rbd (red) proteins, compared 129 with the post-vaccination antibodies generated by other three immunogens (fig. 1e ). this region may not be highly exposed on the virions or infected cells but is clearly immunogenic 150 in the soluble recombinant spike ectodomain. in addition, the rabbit anti-s1+s2 antibodies bound 151 diverse epitopes spanning the rbd and to a lesser degree to the n-terminal domain (ntd) and the 152 c-terminal region of s1, and the n-terminus of s2, including the fusion peptide ( fig. 2b and suppl. 153 table 1 ). the s1 domain elicited very strong response against the c-terminal region of s1 protein 154 and a diverse antibody repertoire recognizing the ntd and rbd/rbm regions ( fig. 2c and suppl. 155 table 1 ). the recombinant rbd induced high-titer antibodies that were highly focused to the 156 rbd/rbm (fig. 2e , and suppl. table 1 ). in contrast, the recombinant s2 immunogen after two 157 immunizations in rabbits elicited antibodies primarily targeting the c-terminus of the s2 protein 158 (cd-hr2). . table 1 ). structural depiction of these antigenic sites on the sars-cov-2 spike (suppl. table 1 ). the other epitopes identified in our study cover less conserved sequences 182 between the two sars-cov viruses that are unique to the sars-cov-2 spike and were not 183 identified in the in-silico approach by grifoni et al. 184 surprisingly, the s2 domain doesn't appear to elicit as many neutralizing antibodies as 185 rbd or s1. although s2 contains the fusion peptide, it does not appear to be as immunogenic, 186 compared with s1 or rbd, in generating binding antibodies to the intact spike (s1+s2) 187 ectodomain, as observed in both igg elisa and spr. even though we characterized the purified 188 proteins in various assays, there is a possibility that the structure of the antigens used in the study 189 is different from the corresponding authentic spike protein on the surface of sars-cov-2 virion 190 particle. one unexpected finding in this study was the higher affinity of antibodies elicited by the 192 rbd compared with the other spike antigens (s1+s2 ectodomain, s1 and s2 domains). in earlier anti-spike reactivity of post-immunization rabbit sera. serial dilutions of post-second vaccination 250 rabbit sera were evaluated for binding to various spike proteins and domains (s1+s2; black, s1; 251 blue, rbd; red, and s2; green) in elisa. representative titration curves are shown in fig. s2 . to spike protein and domains from sars-cov-2 (s1+s2; black, s1; blue, rbd; red, and s2; (e) antibody off-rate constants, which describe the fraction of antigen-antibody complexes that 261 decay per second, were determined directly from the serum/ sample interaction with sars-cov-262 2 spike ectodomain (s1+s2), s1, s2, and rbd using spr in the dissociation phase only for the recombinant sars-cov-2 proteins were purchased from sino biologicals (s1+s2 302 ectodomain; 40589-v08b1, s1; 40591-v08h, rbd; 40592-v08h or s2; 40590-v08b). recombinant purified proteins used in the study were either produced in hek 293 mammalian 304 cells (s1 and rbd) or insect cells (s1+s2 ectodomain and s2 domain). female new zealand white rabbits (charles river labs) were immunized twice intra-308 muscularly at 14-days interval with 50 g of purified proteins mixed with emulsigen adjuvant. sera were collected before (pre-vaccination) and after 1 st and 2 nd vaccination and analyzed for 310 binding antibodies in elisa, spr, neutralization assay and gfpdl analysis. 1hr, plates were washed as before and opd was added for 10min. absorbance was measured at 320 492 nm. end titer was determined as 2-fold above the average of the absorbance values of the 321 naïve serum samples. the end titer is reported as the last serum dilution that was above this cutoff. proteon manager software (version 3.1). all spr experiments were performed twice and the 339 researchers performing the assay were blinded to sample identity. in these optimized spr 340 conditions, the variation for each sample in duplicate spr runs was <5%. the maximum resonance 341 units (max ru) data shown in the figures was the ru signal for the 10-fold diluted serum sample. antibody off-rate constants, which describe the fraction of antigen-antibody complexes that decay 343 per second, are determined directly from the serum/ sample interaction with sars cov-2 spike 344 ectodomain (s1+s2), s1, s2, and rbd using spr in the dissociation phase only for the 345 sensorgrams with max ru in the range of 20-100 ru and calculated using the biorad proteon 346 manager software for the heterogeneous sample model as described before(11). off-rate constants 347 were determined from two independent spr runs. the datasets generated during and/or analyzed during the current study are available from the 388 corresponding author on reasonable request. a crucial role of angiotensin converting enzyme 2 (ace2) in sars 394 coronavirus-induced lung injury cryo-em structure of the 2019-ncov spike in the prefusion conformation structural basis for the recognition of sars-cov-2 399 by full-length human ace2 structure, function, and 401 antigenicity of the sars-cov-2 spike glycoprotein sars-cov-2 vaccines: status report. immunity antigenic fingerprinting of h5n1 avian influenza 405 using convalescent sera and monoclonal antibodies reveals potential vaccine and diagnostic 406 targets vaccines with mf59 adjuvant expand the 409 antibody repertoire to target protective sites of pandemic avian h5n1 influenza virus human antibody 412 repertoire after vsv-ebola vaccination identifies novel targets and virus-neutralizing igm 413 antibodies antigenic fingerprinting following primary rsv infection in young children identifies novel antigenic sites and reveals unlinked 416 evolution of human antibody repertoires to fusion and attachment glycoproteins as03-adjuvanted h5n1 vaccine promotes antibody diversity 420 and affinity maturation, nai titers, cross-clade h5n1 neutralization, but not h1n1 cross-subtype 421 neutralization mf59 adjuvant enhances diversity and affinity of antibody-mediated 424 immune response to pandemic influenza vaccines high-affinity 427 h7 head and stalk domain-specific antibody responses to an inactivated influenza h7n7 vaccine after priming with live attenuated influenza vaccine longitudinal human antibody repertoire against complete viral proteome from ebola virus survivor reveals protective sites for vaccine design intravenous immunoglobulin for adults with influenza a or b infection (flu-ivig): a double-blind, 437 randomised, placebo-controlled trial antigenic fingerprinting of respiratory syncytial virus (rsv)-a-infected 440 hematopoietic cell transplant recipients reveals importance of mucosal anti-rsv g antibodies 441 in control of rsv infection in humans the 443 covid-19 vaccine development landscape characterization of the receptor-445 binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as 446 a viral attachment inhibitor and vaccine a sequence homology and bioinformatic approach can predict candidate targets for immune responses to sars-cov-2 h5 n-terminal beta sheet promotes oligomerization of h7-ha1 that induces better 452 antibody affinity maturation and enhanced protection against h7n7 and h7n9 viruses compared 453 to inactivated influenza vaccine 456 differential human antibody repertoires following zika infection and the implications for 457 serodiagnostics and disease outcome 2020) with a single receptor-binding domain (rbd) in the up conformation, wherever available using ucsf chimera software. the rbd region is shaded in red (residues 319-541) on every structure key: cord-275185-9br8lwma authors: zeng, hao; wang, dongfang; nie, jingmin; liang, haoyu; gu, jiang; zhao, anne; xu, lixin; lang, chunhui; cui, xiaoping; guo, xiaolan; zhou, changlong; li, haibo; guo, bin; zhang, jinyong; wang, qiang; fang, li; liu, wen; huang, yishan; mao, wei; chen, yaokai; zou, quanming title: the efficacy assessment of convalescent plasma therapy for covid-19 patients: a multi-center case series date: 2020-10-06 journal: signal transduct target ther doi: 10.1038/s41392-020-00329-x sha: doc_id: 275185 cord_uid: 9br8lwma convalescent plasma (cp) transfusion has been indicated as a promising therapy in the treatment for other emerging viral infections. however, the quality control of cp and individual variation in patients in different studies make it rather difficult to evaluate the efficacy and risk of cp therapy for coronavirus disease 2019 (covid-19). we aimed to explore the potential efficacy of cp therapy, and to assess the possible factors associated with its efficacy. we enrolled eight critical or severe covid-19 patients from four centers. each patient was transfused with 200–400 ml of cp from seven recovered donors. the primary indicators for clinical efficacy assessment were the changes of clinical symptoms, laboratory parameters, and radiological image after cp transfusion. cp donors had a wide range of antibody levels measured by serology tests which were to some degree correlated with the neutralizing antibody (nab) level. no adverse events were observed during and after cp transfusion. following cp transfusion, six out of eight patients showed improved oxygen support status; chest ct indicated varying degrees of absorption of pulmonary lesions in six patients within 8 days; the viral load was decreased to a negative level in five patients who had the previous viremia; other laboratory parameters also tended to improve, including increased lymphocyte counts, decreased c-reactive protein, procalcitonin, and indicators for liver function. the clinical efficacy might be associated with cp transfusion time, transfused dose, and the nab levels of cp. this study indicated that cp might be a potential therapy for severe patients with covid-19. in december 2019, the outbreak of coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) emerged in wuhan, china, and has rapidly spread around the world. 1 covid-19 can manifest on a spectrum of illness from mild disease to severe respiratory failure requiring intensive care unit admission. the world health organization (who) has declared covid-19 a pandemic on march 11, 2020. as of may 17, 2020, it had caused a total of 4,525,497 cases of infection and resulted in 307,395 deaths globally. 2 epidemiology and etiology for covid-19 are rapidly evolving, giving us a greater understanding of those at risk and elucidating more potential therapy targets. 3 in addition to supportive care, such as oxygen support and extracorporeal membrane oxygenation, several drugs for this disease are still being researched, such as remdesivir, lopinavir/ritonavir, arbidol, and darunavir. 4, 5 however, up to now, no approved vaccine or specific antiviral agents has been proved to be effective to prevent or treat sars-cov-2 infection due to the absence of evidence. passive immunity delivered as neutralizing antibodies (nabs) from convalescent plasma (cp) may offer an alternative therapeutic approach for covid-19. 6 cp therapy has been empirically used in other epidemics, including sars, middle east respiratory syndrome (mers), and 2009 influenza a (h1n1). 7-10 a metaanalysis of 32 reports on sars coronavirus infection and severe influenza revealed a statistically significant reduction of mortality after administration of cp, especially when cp was given early after symptom onset. 8 however, in a case series on influenza a (h5n1) virus infection, nonsignificant benefits following the intervention of cp were reported, 11 and no association of cp therapy with an increased survival was observed in 84 patients with ebola virus disease. 12 it is possibly due to the unknown levels of nabs in the infused plasma, which may obscure the effects of cp. 10 in this current pandemic, preliminary studies suggested the effectiveness of cp with no severe adverse events to treat patients infected with sars-cov-2. [13] [14] [15] [16] [17] the results from a pilot study applying cp transfusion for 10 severe patients showed that administration of cp with nab titers above 1:640 led to improvement in clinical symptoms and pulmonary lesions. 14 these findings indicate that cp transfusion may be a promising therapy in the treatment for covid19. nonetheless, due to the limitations of the study design and small sample size, current evidence on the efficacy and safety of cp therapy for covid-19 is still limited. moreover, the quality control of cp and individual variation in patients in different studies make it rather difficult to evaluate the efficacy and risk of cp therapy. thus, more supporting evidence (such as multi-level assessment of specific antibodies in cp, indications for cp treatment, and selection of transfusion timing) is called for with wider adoption of cp for covid-19 in multi-centers and regions. herein, we performed a retrospective observational study involving eight critical or severe patients with covid-19 from four designated hospitals in the southwest region of china, aiming to explore the potential efficacy and safety of cp therapy, and to provide more evidence for the quality control of donated plasma and reasonable clinical application of cp transfusion. clinical characteristics of the patients a total of 8 patients (4 males and 4 females) with critical or severe covid-19 were enrolled. the median age was 65.0 years (iqr, 63.0-67.0 years). the median time from symptom onset to hospital admission was 4.0 days (interquartile interval (iqr), 3.0-8.5 days). the most common symptoms during hospitalization were cough (7/8), shortness of breath (5/8) , and fever (4/8), while patients had fewer manifestations of dyspnoea (two cases), diarrhea (two cases), headache (one case), and fatigue (one case). five patients had coexisting chronic diseases at admission, including type ii diabetes, hypertension, chronic obstructive pulmonary disease (copd), and coronary heart disease (chd). table 1 listed the drug treatments prior to and after cp transfusion. all 8 patients received combination therapy of various antiviral treatment and other supportive care. the most commonly used antivirals were interferon alfa-1b (8/8), lopinavir/ritonavir (7/ 8), and arbidol (7/8). darunavir and hydroxychloroquine sulfate were also administered for three and two patients, respectively. antibiotic or antifungal agents were used when patients had coinfection. five patients were given corticosteroids at the appropriate situation. chest computed tomography (ct) scans demonstrated that all patients presented bilateral multiple ground-glass opacity or partial consolidation at the time of admission, with primary involvement of subpleural lesions. characteristics of convalescent plasma donors in total, seven donors (5 males and 2 females) from the participating hospitals who had recovered from sars-cov-2 infection donated 300-400 ml of cp ( table 2 ). the median age was 37.0 years (iqr, 34.0-42.5 years). these donors donated the cp at the median day of 11.0 (iqr, 9.5-17.5 days) from discharge. all of 7 donors were mild or moderate patients during a hospital stay with no comorbidities. we measured sars-cov-2 specific antibodies using four platforms of immunological tests. the sars-cov-2 specific antibody titers were detected by magnetic chemiluminescence enzyme immunoassays (mclia) which targeted at the combination of nucleoprotein (np) and receptor binding domains of spike protein (s-rbd) specific antigens, as well as by enzyme-linked immunosorbent assays (elisa) which determined anti-np and anti-s-rbd specific igg antibodies separately. the igg titers detected by mclia ranged from 1:160 to 1:1280, and the igm mclia titers were less than or equal to 1:50 in six donors, except donor 4 (1:320). the elisa results showed that the anti-s-rbd and anti-np specific igg titers were in a range of 1:640-1:2560 and 1:320-1:5120, respectively. we measured the inhibitory activity of receptor binding (rbia) of the cp samples by a receptor-binding assay, finding the 50% inhibitory titer (it50) values ranging from 1:3 to 1:74. importantly, the neutralizing activity of these plasma samples, which offer the most informative assessment of antiviral activity of patient sera against viral infection, was measured by a pseudovirus based neutralization assay. the nabs of the donated plasma also showed variable levels (nab titer (nat50) range, 1:255-1:1576), and only three cp donors (donor 4, 5, and 7) had nat50 values greater than 1:640. the results of correlation analyses as shown in fig. 1a indicated that there was positive correlation between igg mclia titer and s-rbd specific igg elisa titer (r = 0.94, p = 0.029). nat50 was positively correlated with s-rbd and np specific igg elisa titers, respectively (r = 0.87, p = 0.019; r = 0.93, p = 0.007, respectively). however, the positive association between igg mclia titer and nat50 did not show statistical significance (r = 0.77, p = 0.071). notably, it50 was neither related to nat50, nor correlated to igg titers. comparing the antibody levels of cp collected at different time, we found that the cp donated greater that 21 days had higher levels of s-rbd igg elisa titer and igg mclia titer than cp which collected less than or equal to 21 days (fig. 1b) . the detailed information about cp treatment for the 8 patients were shown in table 3 . these patients were administered one or two transfusions of cp. two transfusions were administered with an interval less than 24 h. abo-compatible and cross-matched cp were administered at the discretion of the attending clinicians and according to plasma availability. patients received cp transfusion between 9 and 34 days following the onset of symptoms, with three of them given within 21 days from symptom onset. five of eight patients received two doses of 100-200 ml of cp within 24 h (totally 300 or 400 ml), while the other three cases only received one dose of 200 ml. clinical response of cp transfusion adverse effects of cp transfusions. no adverse events were observed in the eight patients after cp transfusion. clinical characteristics. as the patients have been treated by antiviral drugs and oxygen support before cp therapy, the body temperature, heart rate, and systolic pressure were normal even prior to cp transfusion, and kept unchanged within 5 days after cp transfusion as indicated in table 4 . individual patient's change in the category of oxygen support during hospitalization are shown in fig. 2 . six of eight patients showed an improvement in the category of oxygen support within 5 days from cp treatment. obvious improvement was observed in patients who were receiving high-flow nasal cannula oxygenation (n = 3), or noninvasive positive pressure ventilation (nippv, n = 3) prior to cp treatment. it is notable that patient 1, 2, and 5 rapidly shifted highflow supplemental oxygen or nippv to low-flow supplemental oxygen within 24 h after cp transfusion. pulmonary lesions on chest ct examinations. chest ct scans showed that pulmonary lesions improved at varying degrees in six out of eight patients. a partial resolution of pulmonary lesions was observed in patient 2, 3, and 4 on 1st day, in patient 6 and 7 on 3rd day, in patient 4 on 5th day, and in patient 1 on 8th day after plasma transfusion, respectively. representative chest ct images of patient 1-3 were shown on fig. 3 . laboratory results. we monitored the development of the virusspecific igg and igm antibodies by mclia prior to and after cp transfusion in all patients except patient 5. in 5 of 7 patients, the igg titer increased within 2 days posttransfusion, with patient 4, 7, and 8 presenting the most obvious increment (fig. 4a) . the igm level was observed lower than igg for all patients, and waved in a small range after cp transfusion (fig. 4b) . sars cov-2 viral load, estimated by the cycle threshold (ct) value from reverse transcriptase-polymerase chain reaction (rt-pcr), was positive in five patients before cp transfusion (for other three cases, the data of ct values was not available). the ct value was decreased to a negative level in patient 2 and 3 on posttransfusion day 1, patient 4 on day 2, patient 8 on day 5, and patient 1 on day 7 (fig. 4c, d) , which was basically consistent with the improvement of pulmonary lesions indicated by ct scans mentioned above. the result of arterial blood gas analysis showed that the ratio of the partial pressure of arterial oxygen (pao 2 ) to fraction of inspired oxygen (fio 2 ) (pao 2 /fio 2 ) (median, 259.0; iqr, 163.0-283.0) prior to transfusion immediately increased one day after transfusion (median, 312.0; iqr, 246.0-409.0), and five patients were indicated a tendency of improvement of pao 2 /fio 2 in the following 5 days after cp therapy (table 4 and fig. 4e ). lymphocytopenia, which is a prominent feature of critically ill patients with covid-19, was also observed in this study, with the median lymphocyte counts of 0.4 (iqr, 0.2-1.5) ( table 4 ). within 5 days following plasma transfusion, the lymphocyte counts showed an increase in 6 out of 8 patients (fig. 4g ). the changes of white blood cell count (fig. 4f) and neutrophil count were similar with an overall downward trend, except that patient 4, 7, and 8, who had the complications of bacterial or fungal pneumonia, presented an increase after cp therapy. as for the inflammatory biomarkers, the increased creactive protein (crp) and procalcitonin before plasma transfusion were observed a declining trend following cp treatment for 4 out of 5 patients, and for 5 of 6 patients, respectively (fig. 4h , i). proinflammatory cytokines, including interleukin-6 (il-6) and tumor necrosis factor-α (tnf-α) demonstrated an increase for 5 of 8 patients (fig. 4j) , and for 5 of 6 patients ( supplementary fig. s1a) , respectively, as compared to the status before cp therapy. other inflammatory cytokines, such as interferon-γ (ifn-γ), il-2, il-10, and il-17a, showed various alterations in each patient after cp treatment (supplementary fig. s1b -e). we also observed tendencies of increment of the ratios of proinflammatory cytokines and antiinflammatory cytokine (il-6/il-10, and il-6/il-4) in four patients (table 4 ). concerning the parameters indicative of liver function, the alanine aminotransferase (alt), aspartate aminotransferase (ast), and total bilirubin (tbil) tended to decrease after cp therapy, except for an increase of all these indicators in patient 4, and elevated alt and ast in patient 8. the coagulation profile of patients was also monitored following cp treatment, indicating that 5 out of 6 patients kept the normal level of prothrombin time, while abnormally elevated d-dimer prior to plasma transfusion (median, 1.2; iqr, 0.5-4.0) still increased within 5 days after plasma treatment in 4 of 6 patients ( supplementary fig s1f) . outcome of patients treated with cp. all patients were discharged from the hospital with a median length of stay of 28.0 days (iqr, 24-32.5 days), except for patient 4, who remained hospitalized for further treatment of underlying diseases as of april 8, 2020. assessment of possible factors associated with clinical effects. to assess the factors which might affect the clinical effects, we compared the clinical features between patients who received cp transfusion on different time, with different doses, and with different nab titers (table 5) . although the results were not statistically analyzed due to limited samples in each group, patients who received cp transfusion before 21 days from symptom onset tended to show a more rapid negative conversion of viral nucleic acid, and shorter hospital stays compared to patients who were transfused after 21 days. concerning the doses of plasma, we found that the viral nucleic acid in patients transfused with 400 ml of cp had a tendency to turn faster to a negative level than that in patients who received 200 ml of cp. when comparing to patients treated by cp with nat50 ≤ 1:640, the viral rna tended to be decreased to an undetectable level in less time, and the increment of igg mclia level indicated by the igg titer ratio (the titer at day 1 after cp transfusion divided by the value before cp transfusion) tended to be higher in patients receiving cp with nat50 > 1:640. but the hospitalization was longer for patients receiving high nat50, mainly because of patient 4 who remained hospitalized for treating for severe complications including acute respiratory distress syndrome (ards), this retrospective observational study explored the potential efficacy and safety of cp treatment in 8 patients who were critically or severely ill with covid-19. one or two doses of cp with a total of 300-400 ml was well tolerated by all patients without any adverse effects. improved clinical conditions as indicated by improvement of oxygen support and chest ct imaging were observed in most patients after cp treatment. the viral load as estimated by the ct value also declined to undetectable level within 7 days post transfusion. it has been suggested that cp served as a method of passive immunity therapy 18, 19 could significantly reduce the mortality of patients with sars infection. 8, 9 one possible mechanism for the efficacy of cp therapy is the nabs from cp which may lead to the clearance of viraemia. 20, 21 our results showed that only plasma from donor 4, 5, and 7 had relatively high neutralizing activity (nat50 > 1:640). this is consistent with a recent finding that the majority of cp donors had relatively modest neutralizing activity and a small proportion of donors had high neutralization activity. 22 it is not surprising since all donors were previously moderate or mild patients, and there is evidence that mild patients frequently had a lower level of sars-cov-2 specific antibodies than severe patients. 23 assessing the effects of neutralizing activity of cp on the patients' clinical efficacy, we found that patients treated by cp with high nat50 (>1:640) had more obvious improvement than patients receiving low nat50 value (≤1:640) of cp, including shorter negative conservation time of viral rna, and higher increment of igg level after cp transfusion. in line with other publications, 14 our results indicated that cp with high concentration of nabs may contribute to the clearance of the virus. based on the fact that cp donors who usually recovered from mild infection may not generate adequate protective antibodies, and the levels of plasma neutralizing activity required to prevent sars-cov-2 re-infection are currently unknown, more studies are necessary to assess the minimum threshold of nab titers necessary to prevent sars-cov-2 reinfection. in addition to pseudovirus based neutralization test, this study also employed multiple sars-cov-2 serology tests and receptorbinding assay. the results demonstrated that cp donors had a wide range of antibody levels measured across multiple platforms. pseudotyped virus assay, an alternative of neutralization test which is considered as the optimal assay to determine the antiviral activity of antibodies, could measure how effectively donor plasma or serum can inhibit virus infection of target cells. 24 but it is not feasible to implement neutralization test or pseudotyped virus assay as a measurement of antiviral antibodies for general population investigation. by contrast, serology tests are more convenient and practical. here we examined the correlations between serology test results and neutralization assay in the cp samples, which is seldomly explored in other studies. our results indicated that s-rbd and np specific igg elisa titers had a significant strong correlation with nab level, and igg mclia titer showed a modest correlation with neutralization activity. however, the inhibitory activity of receptor binding of the cp samples had a low degree of association with neutralization activity. these findings may provide some clues about that elisa or mclia assays may serve as a surrogate for pseudovirus neutralization assay to predict the degree of neutralization activity present in recovered patients or vaccine recipients. studies with larger sample size are necessary to further explore these alternative serology tests which could help to refine the cp selection, as well as inform immunogenicity of vaccines against sars-cov-2. the treatment timing is considered as another important factor associated with the effectiveness of cp therapy. 8 viraemia reaches to the peak in the first week of infection for most viral illnesses. patients usually develops a primary immune response by days 10-14, which is followed by virus clearance. 6, 7 the largest study involved the cp treatment of 80 patients in hong kong with sars found that the better clinical outcome was observed among patients who were given cp before day 14 of illness and among cases who were pcr positive and seronegative for coronavirus at the time of plasma infusion. 7 a recent study on covid-19 demonstrated that cp therapy could not reduce the mortality rate in critically ill patients with end-stage disease. 25 thus, to obtain the greatest benefit from cp, treatment should be fig. 1 sars-cov-2 specific antibody levels of cp samples measured by serology tests, receptor-binding assay, and pseudovirus based neutralization assay. a the correlations among anti-sars-cov-2 specific igg and igm titers detected by commercial mclia kits, anti-s-rbd and anti-np specific igg titers determined by in-house elisa assays, inhibition activity measured by a receptor-binding assay, and neutralizing antibody titer measured by a pseudovirus based neutralization assay. b comparisons of antibody levels between cp samples collected before and after 21 days from symptom onset. mclia magnetic chemiluminescence enzyme immunoassay, elisa enzyme-linked immunosorbent assay, rbd receptor binding domains, np nucleoprotein, it50 inhibitory titer which was calculated with the dilution of plasma that inhibits 50% rbd-fc binding to receptor ace2, nat50 neutralizing antibody titer which was calculated with the highest dilution of plasma that resulted in a 50% reduction of virus infection, gmt geometric mean titer, ci confidence interval table 3 . detailed information about patients receiving convalescent plasma treatment the efficacy assessment of convalescent plasma therapy for covid-19. . . zeng et al. administered early in the course of the disease (e.g., before sars-cov-2 seroconversion). in our study, only three patients were given cp before 21 days from illness onset, and all patients had developed sars-cov-2-specific igg before cp transfusion. these three patients tended to show a more rapid negative conversion of viral rna, and shorter hospital stays compared to other patients who were transfused after 21 days. the late administration of cp may result in the fact that the patients with critical illness and complications did not show obvious clinical improvement. specifically, patient 1 who was given cp transfusion on the 30th day of infection, and had suffered from bacterial pneumonia prior to cp therapy, showed latest conversion of virus nucleic acid on posttransfusion day 7. on the other hand, patient 2 was observed rapid decrease of ct value on posttransfusion day 1 and obviously promoted clinical manifestation after receiving cp early after disease onset (day 12). our results support that cp treatment in potentially critically ill patients with covd-19 early in the course of disease may be more effective. most patients with severe covid-19 were featured by substantially elevated levels of proinflammatory cytokines, which was characterized as cytokine release syndrome. [26] [27] [28] our study also observed abnormally high levels of proinflammatory cytokines (especially il-6) in some patients prior to cp therapy. notably, inflammatory cytokines, including il-6 and tnf-α, proinflammatory/anti-inflammatory ratios (il-6/il-10, and il-6/il-4) unexpectedly kept increased within 5 days of cp treatment in almost half of patients, which was not compatible with another study on cp treatment for covid-19. 13 it is probably because that increased systemic cytokine production may lead to the pathophysiology of severe covid-19, including ards and multiple organ failure, 28 and it might be unable to attenuate the inflammatory damage soon after cp transfusion. elevated il-6 level was found to be a stable indicator of poor outcome in patients with severe covid-19 with pneumonia and ards. 23 moreover, lymphopenia has been proven to be an effective and reliable indicator of the clinical severity in covid-19 patients 29 early administration of cp containing nabs may not only inhibit viral entry and replication, but also consequently blunt an early proinflammatory pathogenic endogenous response and restore the immune system. 4, 30 thus, we suggest that cp be given at an early stage in patients at high risk of subsequent deterioration (for instance, persistently abnormal inflammatory cytokines, and lymphopenia) for maximizing efficacy to prevent cytokine storms. besides, to prevent from worsening disease outcome, it is beneficial to monitor the abovementioned prognostic biomarkers for patients at high risk of developing ards or multiple organ failure, especially for those with chronic diseases, such as hypertension, diabetes, and copd. based on our findings, the dose of infused cp might play a role on its therapeutic effect, as demonstrated by the result that the viral nucleic acid in patients transfused with 400 ml of cp tended to turn faster to undetectable than that in patients who received 200 ml of cp. while a study about the cp therapy in sars patients found that there was no correlation between clinical outcome and the volume of infused plasma. 7 future large-scale studies are needed to investigate the association between the dose of cp transfusion and its clinical efficacy. there are some limitations that should be noted in this study. first, this study was a case series with small sample size, and the outcome of the cp treated patients was not compared with a control group of patients who did not receive the intervention. second, the patients received other therapies (including antiviral agents, antibiotics or antifungal drugs, and corticosteroids), making it impossible to discriminate the specific contribution of cp to the clinical course or outcomes. moreover, cp was administered 9-34 days after admission in this study. the association between the transfusion timing and clinical outcomes should be further clarified. in addition, patients in the current study were given different doses of cp. it is unclear whether the doses and the titers of antibodies were associated with the treatment efficacy. despite these limitations, this study provided more evidence to support that cp therapy might be a promising option to treat covid-19 patients, which is also supported by the recent issue by fda of emergency use authorization for cp as potential promising covid-19 treatment. 31 overall, this study not only provided more evidence on the potential efficacy and safety of cp therapy, but also contributed to the quality control of donated plasma and reasonable clinical application of cp transfusion. in conclusion, our preliminary study indicated that cp might be a potential therapy for severe patients with covid-19. we observed improvement of clinical features without the occurrence of serious adverse reactions following cp transfusion. further welldesigned randomized clinical trials are needed to evaluate the efficacy and safety of cp transfusion, and to explore best donation candidates with high virus-specific antibodies, and indications for cp therapy (e.g., optimal transfusion time point, early warning indicators, and transfused dose). this study was performed from february 17, 2020, to april 10, 2020, at four centers, chongqing public health medical center, chongqing three gorges central hospital, yongchuan hospital of chongqing medical university, and affiliated hospital of north this study was approved by the ethical committee of chongqing public health medical center (approval number, 2020-030-01-ky). all patients signed a written informed consent before any procedure was carried out. if patients cannot make rational decisions, the consents were signed by their family members on behalf of the patients. this study was conducted in accordance with the helsinki declaration as revised 1981. donors for convalescent plasma transfusion cp was obtained from donors who had recovered from covid-19 infection. the recovery status was defined as follows: (1) aged between 18 and 55 years; (2) at least 3 weeks following symptom onset; (3) afebrile status for at least 3 days; (3) significant improvement in respiratory symptoms; (4) two consecutively negative results of sputum sars-cov-2 of real-time rt-pcr assay (one-day sampling interval). persons who met all criteria were eligible for plasma donation. written informed consent was obtained from each donor. plasma preparation apheresis was performed using haemonetics mcs + ln90 00-220e blood cell separator (haemonetics, boston, ma, usa). convalescence plasma for treatment was collected from 7 donors. a 200 or 400 ml of abo-compatible plasma sample was collected from each donor, and each sample was divided and stored as 100 or 200 ml aliquots at 4°c without any detergent or heat treatment. the cp was then treated with methylene blue and light treatment for 30 min in the medical plasma virus inactivation cabinet (shanghai blood technology co., ltd, shanghai, china). the plasma samples were tested negative for hepatitis b virus, hepatitis c virus, hiv, syphilis, and blood type irregular antibody. as a routine check with plasma donation, the cp was also confirmed negative for residual sars-cov-2 by rt-pcr. rt-pcr detection of sars-cov-2 rna throat swab samples were collected from patients for extracting sars-cov-2 rna using the rna viral kit (daan, guangdong, china). the real-time rt-pcr assay was performed using commercials kit specific for sars-cov-2 nucleic acid detection (liferiver, shanghai, china; shengxiang, sansure biotech, hunan, china) approved by the china national medical products administration (approve numbers, 20203400057 for liferiver, and 20203400064 for shengxiang). two target genes, including open reading frame1ab (orf1ab) and nucleocapsid protein (n), were simultaneously amplified using the real-time rt-pcr assay. each transcript provided a ct value, which is the number of cycles required for the fluorescent signal. a higher ct value is correlated with a lower viral load. a ct value less than 40 was defined as a positive result, and a ct value of 40 or more was defined as a negative test. all procedures involving clinical specimens and sars-cov-2 were performed in a biosafety level 3 laboratory. the collected cp and serum samples from the donors and patients were inactivated at 56°c for 30 min and stored at −20°c before testing, and serially diluted before determination. igg and igm against sars-cov-2 were tested using mclia kits supplied by bioscience co. (tianjing, china) (approved by the china national medical products administration; approval numbers, 20203400183 (igg) and 20203400182 (igm)), according to the manufacturer's instructions. mclia for igg or igm detection was developed based on a double-antibody sandwich immunoassay. the recombinant antigens containing the nucleoprotein and a peptide from the spike protein of sars-cov-2 were conjugated with fitc and immobilized on anti-fitc antibody-conjugated magnetic particles. the tests were conducted on an automated magnetic chemiluminescence analyzer (axceed 260, bioscience, tianjing, china) according to the manufacturer's instructions. the mclia titers of specific igg and igm antibodies were defined as the highest dilution giving a chemiluminescence value of more than or equal to 1. all tests were performed under strict biosafety conditions. detection of specific igg levels against sars-cov-2 s-rbd and np sars-cov-2 np and s-rbd specific igg antibodies in plasma were measured by in-house elisa separately. purified np and s-rbd antigens were coated onto maxisorp elisa plates (corning costar, acton, ma, usa) in 0.1 m carbonate buffer (ph 9·6) at concentration of 0.2 μg/ml overnight at 4°c, respectively. plates were washed 4 fig. 4 changes of laboratory results before and at day 1-5 after convalescent plasma transfusion. a, b sars-cov-2 specific igg and igm levels, respectively, determined by mclia. c, d cycle threshold (ct) values of orf1ab-gene and n-gene, respectively. a ct value of 40 was defined as undetectable. e pao 2 /fio 2 (normal range: 400-500 mmhg). f white blood cell count (normal range: 3.5-9.5). g lymphocyte count (normal range: 1.1-3.2). h c-reactive protein (normal range: <8). i procalcitonin (normal range: <0.1). j il-6 (normal range: 0-5.30) times with phosphate-buffered saline (pbs) containing 0.1% vol/vol tween-20 (pbst) and blocked with 1% bovine serum albumin for 2 h at 37°c. the plates were then washed with pbst. the serum samples were diluted to 80-fold into pbs as initial concentration, and then serial 2-fold diluted until 81920-fold. the serial dilutions of serum samples were added to the plate wells and incubated, followed by wash and incubation with anti-human igg hrpconjugated coat secondary antibody (abcam, cambridge, uk). after 4 washes, plates were developed by tetramethylbenzidine substrate (tiangen biotech co., beijing, china) at room temperature in the dark. the absorbance was measured at 450 nm using a microplate reader (molecular devices co., sunnyvale, ca, usa) after adding the stop solution (2 m sulphuric acid). all samples were run in duplicate. the titers of np and s-rbd specific igg antibodies were defined as the highest dilution giving an absorbance value of more than 2.1 times that of the negative control. receptor-binding assay inhibitory effects of the cp samples on rbd-fc binding to receptor angiotensin-converting enzyme 2 (ace2) were tested using an elisa-based assay. recombinant soluble human ace2 (sino biological) was coated at 2 µg/ml to 96-well elisa plates (corning costar) in 0.1 m carbonate buffer (ph 9.6) at 4°c overnight. plates were washed 4 times with 0.1% vol/vol pbst and blocked with 0.1% bovine serum albumin for 2 h at 37°c. 80 ng/ml recombinant sars-cov-2 spike rbd-mfc (sino biological) was mixed with the presence or absence of serially diluted cp or serum samples 1:1 and incubated at 37°c for 1 h, then add the 100 μl mixed solution to the wells. incubated at 37°c for 10 min, 100 μl of the hrp conjugated goat anti-mouse igg (zsgb-bio) was added to the wells. after incubation at 37°c for 1 h, 100 μl of the substrate tmb was added to the wells. developed at room temperature in the dark for 5 min, it was terminated with the stop solution (2 m sulphuric acid). the absorbance was measured at 450 nm. all samples were run in duplicate. the 50% inhibitory titer (it50) was defined as the dilution of serum or plasma that inhibits 50% rbd-fc binding to receptor ace2 using a linear interpolation algorithm. pseudovirus based neutralization assay the neutralization of plasma samples was measured by a pseudovirus-based neutralizing assay as described previously. 34 in brief, pseudovirus was incubated with serial dilutions of the plasma samples (six dilutions in a 3-fold step-wise manner) in duplicate for 1 h at 37°c, together with the virus control and cell control wells in hexaplicate. then, freshly huh-7 cells (japanese collection of research bioresources [jcrb], 0403) were added to each well. following 24 h of incubation in a 5% co 2 environment at 37°c, the luminescence was measured using a microplate luminometer (perkinelmer, ensight). the nab titers (nat50) were defined as the 50% inhibitory dilution (id50) which was calculated with the highest dilution of plasma that resulted in a 50% reduction of relative light units compared with virus control. clinical data collection and efficacy assessment clinical information of the patients before and after cp transfusion was retrieved from the hospital electronic medical records system, including: (1) basic clinical data: age, sex, days of admission from symptom onset, presenting symptoms, comorbidities, and other treatments; (2) cp transfusion information: time and dose of cp infusion, complications prior to cp therapy, and adverse effects; (3) clinical features, laboratory data, and chest ct imaging. adverse events and serious adverse events associated with cp transfusion were assessed by the clinician. the primary indicators for efficacy assessment were the changes of clinical symptoms, laboratory parameters, and radiological image after cp intervention. clinical outcomes include discharge and hospitalization. statistical analysis continuous variables were summarized as median and iqr or range. spearman correlation analyses were used to calculate the correlations among log 2 -transformed anti-sars-cov-2 specific igg and igm mclia titers, anti-s-rbd and anti-np specific igg elisa titers, it50, and nat50 of cp. graphs were plotted using graphpad prism 7.0 (graphpad software, san diego, ca, usa). correlation analysis was performed using spss 22.0 (spss inc., chicago, il, usa). two-tailed p value of less than 0.05 was considered statistically significant. the efficacy assessment of convalescent plasma therapy for covid-19. . . zeng et al. clinical features of patients infected with 2019 novel coronavirus in wuhan novel coronavirus (covid-19) situation discovering drugs to treat coronavirus disease 2019 (covid-19) review of emerging pharmacotherapy for the treatment of coronavirus disease 2019 compassionate use of remdesivir for patients with severe covid-19 convalescent plasma as a potential therapy for covid-19 use of convalescent plasma therapy in sars patients in hong kong the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory 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plasma as potential promising covid-19 treatment, another achievement in administration's fight against pandemic the cytokine release syndrome (crs) of severe covid-19 and interleukin-6 receptor (il-6r) antagonist tocilizumab may be the key to reduce the mortality lymphopenia predicts disease severity of covid-19: a descriptive and predictive study collecting and evaluating convalescent plasma for covid-19 treatment: why and how? vox sang the authors declare that the data supporting the findings of this study are available within the paper and its supplementary materials. the online version of this article (https://doi.org/10.1038/s41392-020-00329-x) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. key: cord-281793-tj4m01s4 authors: ho, mitchell title: perspectives on the development of neutralizing antibodies against sars-cov-2 date: 2020-05-20 journal: antib ther doi: 10.1093/abt/tbaa009 sha: doc_id: 281793 cord_uid: tj4m01s4 sars-cov-2 gains entry to human cells through its spike (s) protein binding to angiotensin-converting enzyme 2 (ace2). therefore, the receptor binding domain (rbd) of the s protein is the primary target for neutralizing antibodies. selection of broad-neutralizing antibodies against sars-cov-2 and sars-cov is attractive and might be useful for treating not only covid-19 but also future sars-related cov infections. broad-neutralizing antibodies, such as 47d11, s309, and vhh-72, have been reported to target a conserved region in the rbd of the s1 subunit. the s2 subunit required for viral membrane fusion might be another target. due to their small size and high stability, single-domain antibodies might have the ability to be administered by an inhaler making them potentially attractive therapeutics for respiratory infections. a cocktail strategy combining two (or more) antibodies that recognize different parts of the viral surface that interact with human cells might be the most effective. covid-19 is caused by the new coronavirus sars-cov-2 (initially called 2019-ncov) [1, 2] . as of may 16, 2020, there are 4,632,903 confirmed cases and 311,739 death worldwide with 188 countries affected (https://coronavirus.jhu.edu/ma p.html). it is widely believed that neutralizing antibodies can be used to treat covid-19 by reducing sars-cov-2 infectivity [3] . in the current issue of antibody therapeutics, two articles related to covid-19 have been published through a fast track peer review, revision, and production [4, 5] . while most publications in our journal have been focused on cancer therapies, it was not the first time that antibody therapeutics published papers that were relevant to antibody development for viral diseases. in one previous study, a human papillomavirus vaccine using a novel viruslike particle was shown to induce antibody response in figure 1 . development of neutralizing antibodies for treating covid-19. in the receptor binding stage, the s1 subunit of sars-cov-2 binds human ace2 on the host cell surface. antibodies that bind the rbd domain on the s1 subunit might block the interaction of the rbd and the ace2. crossreactive antibodies (e.g., 47d11, s309, and vhh-72) that bind highly conserved epitopes on the rbds of sars-cov and sars-cov-2 could have broad neutralization activities against viral infection. in the viral fusion stage, after the cleavage of s1 subunit, the viral fusion peptide (fp) on the s2 subunit inserts into the host cell membrane, inducing the conformational change of the s2 subunit, which forms a six-helix bundle (6-hb) with the hr1 and hr2 trimers. antibodies (e.g., 1a9 against sars-cov) that target the hr domains might block viral fusion. ab, antibody. the bat coronavirus ratg13, indicating that the bat coronavirus might be the origin of the sars-cov-2 [10] . furthermore, sars-cov-2 might be the result of a recombination between bat (ratg13) and pangolin coronaviruses, as particularly indicated in the s protein sequence [10] . the receptor binding domain (rbd) of the sars-cov-2 s protein contains several novel residues that might be introduced through recombination with the pangolin coronavirus, indicating a possible critical step in the evolution of the ability of sars-cov-2 to infect humans [10] . the structures of sars-cov-2 s protein trimer [11] and human ace2 [12] have been rapidly solved using modern cryoelectron microscopy (cryo-em). the affinity of the sars-cov-2 rbd for human ace2 appears stronger than the sars-cov rbd. the structural analysis of the rbd-ace2 complex reveals some of the key mutations on the rbd, such as f486 and n501, that form stronger contacts with human ace2 [12] . interestingly, these residues can be found in the pangolin coronavirus [10] . in the review paper from dr zhiqiang an's group in the university of texas health science center at houston, ku et al. summarized current findings on the structures and functions of sars-cov-2 viral proteins. they describe potential strategies for repurposing drugs for the treatment of covid-19 and the current development of vaccines, plasma therapies, and neutralizing antibodies [4] . their review highlights the potential viral targets, screening methods, in vitro and in vivo models, as well as discussing potential antibody-dependent enhancement (ade) and fc engineering for developing neutralizing antibodies for treating covid-19 patients. in particular, their review describes major screening strategies for the discovery and development of sars-cov-2 neutralizing antibodies and provides several representative examples using these methods. antibody sources may include memory or plasma b cells from recovered patients, phage, yeast and ribosome libraries, or from mouse, rabbit, monkey, and llama immunizations. most antibodies are tested for their ability to block s protein (or rbd) binding to ace2 and preventing spike-mediated membrane fusion. antibody activity is tested either by using a pseudovirus-based neutralization assay or by a live virus-based neutralization assay. animal models such as human ace2 transgenic mice are also summarized in their review article. in the research paper from chengdu medical college and ablink biotech co., ltd in china, zeng et al. isolated a human monoclonal antibody (named "rrbd-15") that inhibits the interaction of the rbd of sars-cov-2 and the ace2 and neutralizes the pseudovirus infection [5] . the group used a competitive screening strategy to isolate human antibodies from a phage display library. in the first round of phage panning, they followed the standard procedure by screening phage on immobilized rbd. after the first-round enrichment of rbd binders, in the 2nd and 3rd rounds, they immobilized ace2 and added the mixture of free rbd and a phage pool enriched from the 1st round. phage that bind rbd at epitopes different from the ace2-binding site were captured by the immobilized ace2 to form a "sandwich" complex. phage that competed with ace2 for rbd were in the supernatant along with presumably unknown amounts of nonbinders or nonspecific binders. the phage that bind the ace2 site on the rbd were then isolated by magnetic beads using the histidine tag on the rbd. the key for success using this strategy is the ratio of immobilized ace2, free rbd, and phage concentrations in solution. therefore, they used a low concentration of rbd (1 μg/ml) close to the ec 50 value of rbd binding to ace2 and a low concentration 1 × 10 10 pfu of the phage they enriched from 1st round of panning on rbd. standard phage panning protocols used about 100 times more phage or about 1 × 10 12 pfu. in this way, they expected to reduce nonspecific binding of phage. antibody therapeutics, 2020 111 several important questions are raised in the development of neutralizing antibodies for treating covid-19. could such antibodies be cross-reactive with other sars-related covs (sarsr-covs)? could such cross-reactive antibodies have neutralizing activities for all sarsr-covs? what would be ideal targets or epitopes for cross-neutralizing antibodies? selection of cross-neutralizing antibodies would be useful for treating not only current covid-19 patients but also future sarsr-cov infections. the rbd of the s protein is the primary target for neutralizing antibodies. many known neutralizing antibodies, including s230, m396, and 80r, are specific for sars-cov rbd but fail to bind sars-cov-2 even at the concentration up to 1 μm [11] . polyclonal antibodies from mice immunized with a stabilized sars-cov s protein can inhibit sars-cov-2 entry into target cells. this suggests that immunity against one sarsr-cov can potentially provide protection against related sars-cov [13] . in contrast, another study showed that polyclonal rabbit anti-sars-cov s1 antibodies (t62) inhibited entry of sars-cov, but not sars-cov-2 pseudovirus [14] . in addition, sera from recovered sars and covid-19 patients show only modest cross-neutralization, suggesting that recovery from one sarsr-cov infection might not protect against the other. a recent report showed that none of the monoclonal antibodies isolated from sars-cov-2 infected individuals by single b-cell sorting were cross-reactive with the rbd of sars-cov [15] . in a research article published in our journal, zeng et al. did not test the cross-reactivity of the rrbd-15 human antibody for sars-cov rbd [5] . in the review article, ku et al. discusses two unique cross-reactive antibodies, cr3022 and 47d11, which bind highly conserved epitopes in the rbd [4] . cr3022 is an antibody that binds both sars-cov and sars-cov-2 rbds, but it cannot neutralize sars-cov-2 as it does sars-cov [16] . more recently, wang et al. identified the 47d11 monoclonal antibody that can neutralize both sars-cov and sars-cov-2 infection [17] . the 47d11 antibody was isolated from transgenic mice immunized sequentially with purified s proteins of different coronaviruses (hcov-oc43, sars-cov, and mers-cov). the transgenic mice produce chimeric immunoglobulins with human variable regions and rodent constant regions. four of 51 antibodies specific for the s protein of sars-cov show cross-reactivity with the s1 subunit of sars-cov-2. among them, the 47d11 antibody exhibits the cross-neutralizing activity of sars-cov-2 and sars-cov in cell culture. interestingly, 47d11 binds the rbd but does not block the interaction of rbd and ace2, indicating that 47d11 might bind a highly conserved epitope of the rbd distinct from the ace2 binding site. previous studies indicate that sars-cov rbd antibodies that block the interaction of the rbd and ace2 are not cross-reactive with sars-cov-2 rbd [11] . cr3022 also binds a highly conserved epitope on the rbd and binds both sars-cov and sars-cov-2 rbds [16] , but unlike 47d11, it does not have the cross-neutralizing activity against sars-cov-2. the structure complex of 47d11 and the rbd (or the s1/s protein) would reveal a novel conserved site on the rbd for broad-neutralizing antibodies against sarsr-covs. in addition to 47d11, another human antibody (s309) isolated from memory b cells of a sars survivor infected in 2003 neutralizes sars-cov-2 [18] . interestingly, s309 recognizes a glycan-containing epitope on the rbd in both the open and closed s states. the cryo-em structure of the complex of s309 and sars-cov-2 s protein indicates that the antibody engages an epitope distinct from the ace2 binding motif and would not clash with ace2 for its binding to s protein. the glycan recognition of s309 implies the importance of the n-glycans in sars-cov-2 s protein. furthermore, antibody cocktails containing s309 further improved sars-cov-2 neutralization and might be useful for preventing or mitigating virus escape mutants. this supports the idea that antibody cocktails could be more effective than single antibody therapy. besides human neutralizing antibodies, a single domain camelid antibody (vhh-72), commonly called "nanobody", was isolated from a llama immunized with sars-cov s and mers-cov s. this nanobody showed reactivity with sars-cov-2 s protein by binding to a highly conserved epitope on the rbd partially overlapping the cr3022 binding site [19] . however, unlike cr3022, the bivalent vhh-72-fc fusion protein not only prevents the binding of ace2 but also has neutralizing activity against sars-cov-2 pseudovirus. the neutralizing effects of 47d11 and vhh-72 suggest that co-immunizing animals with s proteins from sars-cov-2 and other coronavirus may produce potent broad-neutralizing antibodies against sars-cov-2 by targeting the rbd. single domain antibodies have unique binding features [20, 21] , so they can bind novel viral conformational epitopes including highly conformational and/or buried sites unreachable by conventional antibodies. besides their unique binding features, other advantages include the construction of multivalent/multispecific molecules and thermostability/chemostability [22] . for respiratory infection, single domain antibodies might be particularly attractive because they might be administered as an inhaler directly to the site of infection [19] . both 47d11 and s309 are fully human igg molecules, whereas vhh-72 is a single domain antibody derived from a llama heavy chain antibody that has not been humanized yet. for therapeutic applications, the camelid-specific sequences in the framework may need to be mutated to their human heavy chain variable domain equivalent [23] . its suitability for prophylactic and therapeutic treatments remains to be determined. it might be useful to analyze the mutations of sars-cov-2 as it spreads worldwide, so neutralizing antibodies can be effective for multiple strains of the virus [24] [25] [26] . up to now, most of the mutations found in the rbd of sars-cov-2 are of low frequency [26] . the g476s mutation was located in the binding interface of the rbd with the ace2, although it occurred in early samples and diminished in late samples, indicating that the virus with mutations in the critical functional site might not have advantage for its survival or spread. while the rbd is the focus for the development of neutralizing antibodies against sars-cov-2, the function of non-rbd regions is poorly understood. recently, an antibody (4a8) isolated from convalescent covid-19 patients shows the binding on the n-terminal domain (ntd) of the sars-cov-2 s protein and exhibits high neutralization potency sars-cov-2. the structural analysis has confirmed its binding to the ntd, not the rbd which directly interacts with ace2, demonstrating a new vulnerable epitope in the s1 subunit as a target of neutralizing antibodies for treating covid-19 [27] . antibodies that target the s protein beyond the s1 subunit have rarely been reported. the s2 subunit, in particular heptad repeat (hr) loops including hr1 and hr2 domains, required for membrane fusion might be another target. the 1a9 antibody is the only known monoclonal antibody that binds the hr2 domain on s2 subunit of sars-cov [28] (fig. 1) . a more recent study showed that sars-cov-2 had a superior plasma membrane fusion capacity compared with that of sars-cov [29] . the x-ray crystal structure of six-helical bundle (6-hb) core of the hr1 and hr2 domains in the sars-cov-2 s protein s2 subunit has also been solved [29] . a lipopeptide (ek1c4) that disturbs viral 6-hb formation by binding the hr1 domain can inhibit the fusion of sars-cov-2 as well as other human coronavirus including sars-cov and mers-cov, suggesting that a broad inhibitor targeting the hr region should be tested for the treatment of infection by current and future sarsr-covs. besides widely studied protein targets, glycan targets might be worth exploring as well. the s protein of sars-cov-2 is highly glycosylated [11] . isolation of the s309 neutralizing antibody that recognizes a glycan-containing epitope on the rbd indicates that glycosylation on the s protein would affect the development of neutralizing antibodies targeting sars-cov-2 [18] . on the host cell, the primary target is the viral entry protein, ace2. it has been proposed that recombinant ace2 might be used as a potential inhibitor to block the virus entry [30] . the ace2-human igg1 fc fusion has been engineered. the fusion protein can neutralize pseudoviruses that express the s proteins of sars-cov-2 or sars-cov in cell culture [31] . heparan sulfate proteoglycans (hspgs) provide the initial sites for the virus to make primary contact with the cell surface [32] . my laboratory research has been focused on the biology of hspgs, in particular glypicans, and their roles as targets in cancer therapy [33] . using one of our human antibodies (hs20) specific for heparan sulfate oligosaccharides with high affinity [34] [35] [36] , we and collaborators previously showed that the hs20 antibody can block the attachment of pathogenic polyomaviruses on cells [37] . interestingly, treatment of the cells with heparinase or exogenous heparin prevents the binding of the s protein to host cells and inhibits sars pseudovirus infection [32] , suggesting that in addition to ace2, hspgs might be essential for sars-cov entry into host cells. blocking the hspgs on human cells by therapeutic antibodies is worth investigating as another potential strategy for treating covid-19. many antibodies capable of neutralizing specifically either sars-cov or sars-cov-2, but not both, have been identified and reported through many methodologies. a very few and very special sars-cov and sars-cov-2 crossneutralizing antibodies have also been documented, including 47d11, s309, and vhh-72, among which the former two, which are human monoclonal antibodies, are ace2 nonblockers, whereas the third one, which is a llama single domain antibody, is an ace2 blocker. competitive phage display panning might be a new way to identify both ace2 blockers and nonblockers. even though zeng et al. only reported the identification of one antibody, rrbd-15, that blocked ace2 and neutralized sars-cov-2 [5] , the same phage display competitive panning strategy could also be used to identify antibodies that do not block the interaction between s protein and ace2 yet still neutralize viral infections. more importantly, with s proteins of both sars-cov and sars-cov-2 as competitors, this competitive panning strategy could be utilized to identify both ace2 blocking and nonblocking antibodies against both viruses with functional profiles similar to 47d11, s309, and vhh-72, respectively. in the last 20 years, human coronaviruses have infected humans and causes three major outbreaks due to sars-cov, mers-cov, and sars-cov-2. an urgent and important challenge in modern medicine is whether we could identify a so-called "universal" target or strategy for inhibiting sarsr-cov or even all coronaviruses. the molecular mechanisms of sars-cov-2 infection are not yet fully understood. more research on the sars-cov-2 biology is urgently needed. current challenges in developing neutralizing antibodies against sars-cov-2 include mutations in less conserved region of s1 subunit, possibly antigen drift, immunodominant epitope, ade potentially induced by nonneutralizing antibodies, or increased affinity of viral s protein for ace2 [26] . it is important to identify and develop neutralizing antibodies, such as 47d11, s309, and vhh-72, against highly conserved region of rbd or s1, to combat not only various strains of sars-cov-2 but also broadly against other sarsr-covs. furthermore, a single monoclonal antibody therapy might not be enough. there might be different strains of the virus that cannot be recognized by the antibody. mutations in the virus can lead to escape variants [38] . multiple strategies and combination of multiple mechanisms are highly expected as described in mers [39] and sars [38] antibody development. a combination of two (or more) antibodies that recognize different parts including both neutralizing and nonneutralizing epitopes (e.g., rbd, ntd, hr, and glycan) of the viral surface that interact with human cells might be the most effective. future therapeutic applications could include cocktail therapy by combining early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study the race is on for antibodies that stop the new coronavirus antibody therapies for the treatment of covid-19 isolation of a human monoclonal antibody specific for the receptor binding domain of sars-cov-2 using a competitive phage biopanning strategy induction of neutralizing antibodies by human papillomavirus vaccine generated in mammalian cells construction and next-generation sequencing analysis of a large phage-displayed vnar single-domain antibody library from six naive nurse sharks renin-angiotensin-aldosterone system inhibitors in patients with covid-19 receptor recognition by the novel coronavirus from wuhan: an analysis based on decadelong structural studies of sars coronavirus the proximal origin of sars-cov-2 cryo-em structure of the 2019-ncov spike in the prefusion conformation structural basis for the recognition of sars-cov-2 by full-length human ace2 structure, function, and antigenicity of the sars-cov-2 spike glycoprotein characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov human neutralizing antibodies elicited by sars-cov-2 infection a highly conserved cryptic epitope in the receptor-binding domains of sars-cov-2 and sars-cov a human monoclonal antibody blocking sars-cov-2 infection structural and functional analysis of a potent sarbecovirus neutralizing antibody structural basis for potent neutralization of betacoronaviruses by single-domain camelid antibodies crystal structure of a shark single-domain antibody v region in complex with lysozyme a cold-blooded view of adaptive immunity ancient species offers contemporary therapeutics: an update on shark vnar single domain antibody sequences, phage libraries and potential clinical applications general strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold phylogenetic network analysis of sars-cov-2 genomes patient-derived mutations impact pathogenicity of sars-cov-2 spike mutation pipeline reveals the emergence of a more transmissible form of sars-cov-2 a potent neutralizing human antibody reveals the n-terminal domain of the spike protein of sars-cov-2 as a site of vulnerability monoclonal antibodies targeting the hr2 domain and the region immediately upstream of the hr2 of the s protein neutralize in vitro infection of severe acute respiratory syndrome coronavirus inhibition of sars-cov-2 (previously 2019-ncov) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion therapeutic strategies in an outbreak scenario to treat the novel coronavirus originating in wuhan neutralization of sars-cov-2 spike pseudotyped virus by recombinant ace2-ig inhibition of sars pseudovirus cell entry by lactoferrin binding to heparan sulfate proteoglycans glypicans as cancer therapeutic targets isolation of antibodies to heparan sulfate on glypicans by phage display human monoclonal antibody targeting the heparan sulfate chains of glypican-3 inhibits hgf-mediated migration and motility of hepatocellular carcinoma cells epitope mapping by a wnt-blocking antibody: evidence of the wnt binding domain in heparan sulfate infectious entry and neutralization of pathogenic jc polyomaviruses human monoclonal antibody combination against sars coronavirus: synergy and coverage of escape mutants towards a solution to mers: protective human monoclonal antibodies targeting different domains and functions of the mers-coronavirus spike glycoprotein antibodies (including multiple single domain antibodies) that target different epitopes via different mechanisms. i would like to thank chin-hsien tai, yaping sun, bryan fleming, and jessica hong (nci) for reading the manuscript, and alan hoofring and ethan tyler (nih medical arts design section) for making the figure. the content of this publication does not necessarily reflect the views or policies of the department of health and human services nor does mention of trade names, commercial products, or organizations imply endorsement by the u.s. government. the hs20 human monoclonal antibody is available for licensing, in a wide range of fields of use, from the national cancer institute. if you are interested in obtaining a license, please contact dr. mitchell ho. the author is supported by the intramural research program of nih, nci (z01 bc010891, zia bc010891, and nci ccr antibody engineering program). none declared. key: cord-268894-amfv3z2y authors: nguyen-contant, phuong; embong, a. karim; kanagaiah, preshetha; chaves, francisco a.; yang, hongmei; branche, angela r.; topham, david j.; sangster, mark y. title: s protein-reactive igg and memory b cell production after human sars-cov-2 infection includes broad reactivity to the s2 subunit date: 2020-07-21 journal: biorxiv doi: 10.1101/2020.07.20.213298 sha: doc_id: 268894 cord_uid: amfv3z2y the high susceptibility of humans to sars-cov-2 infection, the cause of covid-19, reflects the novelty of the virus and limited preexisting b cell immunity. igg against the sars-cov-2 spike (s) protein, which carries the novel receptor binding domain (rbd), is absent or at low levels in unexposed individuals. to better understand the b cell response to sars-cov-2 infection, we asked whether virus-reactive memory b cells (mbcs) were present in unexposed subjects and whether mbc generation accompanied virus-specific igg production in infected subjects. we analyzed sera and pbmcs from non-sars-cov-2-exposed healthy donors and covid-19 convalescent subjects. serum igg levels specific for sars-cov-2 proteins (s, including the rbd and s2 subunit, and nucleocapsid [n]) and non-sars-cov-2 proteins were related to measurements of circulating igg mbcs. anti-rbd igg was absent in unexposed subjects. most unexposed subjects had anti-s2 igg and a minority had anti-n igg, but igg mbcs with these specificities were not detected, perhaps reflecting low frequencies. convalescent subjects had high levels of igg against the rbd, s2, and n, together with large populations of rbdand s2-reactive igg mbcs. notably, igg titers against the s protein of the human coronavirus oc43 in convalescent subjects were higher than in unexposed subjects and correlated strongly with anti-s2 titers. our findings indicate cross-reactive b cell responses against the s2 subunit that might enhance broad coronavirus protection. importantly, our demonstration of mbc induction by sars-cov-2 infection suggests that a durable form of b cell immunity is maintained even if circulating antibody levels wane. importance recent rapid worldwide spread of sars-cov-2 has established a pandemic of potentially serious disease in the highly susceptible human population. key questions are whether humans have preexisting immune memory that provides some protection against sars-cov-2 and whether sars-cov-2 infection generates lasting immune protection against reinfection. our analysis focused on preand post-infection igg and igg memory b cells (mbcs) reactive to sars-cov-2 proteins. most importantly, we demonstrate that infection generates both igg and igg mbcs against the novel receptor binding domain and the conserved s2 subunit of the sars-cov-2 spike protein. thus, even if antibody levels wane, long-lived mbcs remain to mediate rapid antibody production. our study also suggests that sars-cov-2 infection strengthens preexisting broad coronavirus protection through s2-reactive antibody and mbc formation. the high susceptibility of humans to sars-cov-2 infection, the cause of covid-19, reflects the 24 novelty of the virus and limited preexisting b cell immunity. igg against the sars-cov-2 spike 25 (s) protein, which carries the novel receptor binding domain (rbd), is absent or at low levels in 26 unexposed individuals. to better understand the b cell response to sars-cov-2 infection, we 27 asked whether virus-reactive memory b cells (mbcs) were present in unexposed subjects and 28 whether mbc generation accompanied virus-specific igg production in infected subjects. we 29 analyzed sera and pbmcs from non-sars-cov-2-exposed healthy donors and covid-19 30 convalescent subjects. serum igg levels specific for sars-cov-2 proteins (s, including the rbd 31 and s2 subunit, and nucleocapsid [n] ) and non-sars-cov-2 proteins were related to 32 measurements of circulating igg mbcs. anti-rbd igg was absent in unexposed subjects. most 33 unexposed subjects had anti-s2 igg and a minority had anti-n igg, but igg mbcs with these 34 specificities were not detected, perhaps reflecting low frequencies. convalescent subjects had high 35 levels of igg against the rbd, s2, and n, together with large populations of rbd-and s2-reactive 36 igg mbcs. notably, igg titers against the s protein of the human coronavirus oc43 in 37 convalescent subjects were higher than in unexposed subjects and correlated strongly with anti-s2 38 titers. our findings indicate cross-reactive b cell responses against the s2 subunit that might 39 enhance broad coronavirus protection. importantly, our demonstration of mbc induction by 40 sars-cov-2 infection suggests that a durable form of b cell immunity is maintained even if 41 circulating antibody levels wane. 42 43 importance 44 the betacoronavirus sars-cov-2, the causative agent of a respiratory disease termed 56 covid-19, emerged in china in late 2019 and rapidly spread worldwide (1). a pandemic was 57 declared in march 2020 and global deaths from covid-19 now exceed 500,000. the rapid 58 increase in cases in many countries has challenged healthcare systems and shutdowns and 59 quarantine measures introduced to slow virus spread have caused major disruptions to society and 60 economies (2). sars-cov-2 infection produces a wide spectrum of outcomes. a proportion of 61 infections, likely more than 20%, remain asymptomatic. most clinical cases develop mild to 62 moderate respiratory symptoms, but up to 20% progress to a more severe disease with extensive 63 pneumonia (3, 4). 64 when sars-cov-2 emerged and began to spread, the severity of the threat was primarily 65 attributed to the novelty of the virus to the human immune system and, consequently, a lack of 66 preexisting immune memory to quickly clear virus and limit disease progression. four types of 67 common cold coronavirus are endemic in humans, the alphacoronaviruses 229e and nl63 and the 68 betacoronaviruses oc43 and hku1. however, limited relatedness between key structural proteins 69 of these human coronaviruses (hcovs) and those of sars-cov-2 suggested that significant 70 cross-reactive immunity was unlikely (5, 6). initial studies of non-sars-cov-2-exposed 71 individuals found negligible levels of igg against the sars-cov-2 spike (s) protein, the viral 72 attachment protein that binds the receptor angiotensin converting enzyme 2 (ace2) on host cells 73 to initiate infection (7). more recently, however, studies have provided evidence of sars-cov-74 2-reactive b and t cell memory in unexposed subjects that could confer some protection against 75 sars-cov-2 or modulate disease pathogenesis. 76 sera from non-sars-cov-2-exposed individuals have been screened for igg binding to 77 the s1 and s2 subunits of the sars-cov-2 s protein. the membrane-distal s1 subunit contains 78 the receptor binding domain (rbd) for receptor recognition, and the membrane-proximal s2, 79 which has higher homology among coronaviruses than does s1 (6, 8), mediates membrane fusion 80 to release viral rna into the host cell. in two large cohorts of unexposed subjects, approximately 81 10% had igg that bound s2, but not s1 or the rbd. approximately 4% of subjects had igg against 82 the sars-cov-2 nucleocapsid (n) protein, which is highly conserved among coronaviruses (9, 83 10) . although n is an internal viral protein and not a target of neutralizing antibodies (abs) , 84 coronavirus infections typically elicit strong anti-n ab production (11). the idea that circulating 85 hcovs elicit igg that cross-reacts with sars-cov-2 is supported by the finding that sars-cov-86 2 infection increases igg titers against the s proteins of multiple hcovs (12). in t cell studies, 87 cd4 + t cells in up to 50% of non-sars-cov-2-exposed donors responded to epitopes in s and 88 non-s proteins of sars13) . notably, s-reactive cd4 + t cells in unexposed subjects 89 were mostly reactive to the conserved s2 subunit, consistent with cross-reactivity to circulating 90 hcovs (8). sars-cov-2-reactive cd8 + t cells were also detected in unexposed donors, but the 91 response was less marked than for cd4 + t cells (13). 92 are also likely to be present in non-sars-cov-2-exposed individuals. indeed, mbcs might be 94 more important than preexisting cross-reactive abs as a source of protection against sars-cov-95 2. igg mbcs are more broadly reactive than abs generated against the same antigen, they persist 96 after circulating ab levels wane, and they are readily activated to generate strong ab responses or 97 seed germinal centers for additional rounds of affinity maturation (14). concurrent early 98 production of virus-specific igm and igg in the response to sars-cov-2 infection suggests a 99 response mediated by igg mbcs as well as naïve b cells (9, (15) (16) (17) . this picture is supported by to extend our understanding of the b cell response to sars-cov-2 infection, the current 116 study compared ab and mbc immunity to sars-cov-2 in unexposed individuals and individuals 117 in the convalescent phase of infection. in particular, we were interested in the presence of sars-118 cov-2-reactive mbcs in unexposed subjects that could confer some protection against sars-119 cov-2, and formation of mbcs by sars-cov-2 infection to provide durable protection against 120 igg mbcs reactive to the novel rbd and the conserved s2 subunit of the s protein. mbcs are thus likely to be available to mediate rapid protective ab responses if circulating ab 123 levels wane and reinfection occurs. our study also draws attention to preexisting sars-cov-2-124 cross-reactive b cell memory to the s2 subunit in sars-cov-2-naïve subjects. we speculate that 125 the strong response to s2 after sars-cov-2 infection reflects preexisting s2-reactive mbc 126 activation and strengthens broad coronavirus protection. convalescent subjects sampled 4-9 weeks after symptom onset. reactivity was measured against 135 the s (including the rbd and s2 subunit) and n proteins of sars-cov-2 and the s proteins of 136 the human alphacoronavirus 229e and betacoronavirus oc43. the h1 influenza virus 137 hemagglutinin and tetanus toxoid (ttd) were included as control antigens that humans are 138 commonly exposed to through infection and vaccination. 139 serum igg levels were measured by elisa. approximately one-third of non-sars-cov-140 2-exposed subjects in the healthy donor cohort had low levels of serum igg against the s and n 141 proteins of sars-cov-2, likely reflecting cross-reactivity with seasonal hcovs ( figure 1a ). 142 notably, 86% of unexposed subjects had igg against the highly conserved s2 subunit of the s 143 protein. it is possible that inherent features of the bulky s reagent used in our analysis reduced 144 binding by anti-s2 abs. igg that bound the highly novel rbd was not detected in unexposed 145 subjects. all non-sars-cov-2-exposed subjects had igg against s proteins of the hcovs 229e 146 and oc43, indicating previous infection, and against the control proteins h1 and ttd ( figures 1c-147 1f). 148 response to the s2 subunit. levels of igg against s, rbd, s2 and n were markedly higher in 150 convalescent subjects than unexposed subjects, indicating strong induction of these abs by sars-151 cov-2 infection ( figure 1a) . in a small number of convalescent subjects, high anti-s igg titers 152 were associated with low levels of anti-n igg. indeed, more than 40% of convalescent subjects 153 had anti-n igg levels within the range in unexposed subjects, questioning the reliability of using 154 anti-n igg measurement to identify previous sars-cov-2 infection. 155 notably, serum igg titers against s2 were consistently higher than against the rbd in 156 convalescent subjects, perhaps reflecting the novelty of the rbd and a response dependent on 157 naive b cell activation ( figure 1b) . interestingly, titers of igg were higher against the s protein 158 of the hcov oc43 in convalescent subjects than in unexposed subjects, but this was not the case 159 for the s protein of hcov 229e (or for the control proteins h1 and ttd) ( figures 1c-1f ). the cov-2 infection ( figure 1g ). the particularly strong correlation between igg titers against oc43 163 s and the sars-cov-2 s2 suggests a cross-reactive response to the s2 subunit. 164 since the healthy donor samples in our analysis were collected 6-10 years before the 165 emergence of sars-cov-2, we considered the possibility that a recently circulating hcov could 166 have been responsible for the higher anti-oc43 s igg titers in the convalescent subjects. to 167 exclude this possibility, we measured anti-oc43 s igg titers in sera collected from 20 healthcare 168 workers in 2020. the healthcare workers cared for hospitalized sars-cov-2 patients, but all were 169 negative for igg against sars-cov-2 s and rbd, consistent with the effectiveness of personal 170 protective equipment and appropriate work practices. oc43 s-reactive igg levels in healthcare 171 worker sera were similar to those in non-sars-cov-2-exposed healthy donor sera and 172 significantly lower than those in sera from convalescent subjects ( figure 1c ). taken together, our 173 results indicate that sars-cov-2 infection generates a strong igg response that cross-reacts with 174 the s2 of human betacoronaviruses. 175 reactivity to the rbd and s2 subunit. pbmcs from non-sars-cov-2-exposed subjects and 177 convalescent subjects were analyzed for mbcs reactive to sars-cov-2 proteins. circulating proportion of unexposed subjects suggested that igg mbcs with the same specificity had also 190 been formed. however, these mbcs were not detected, possibly because of very low frequencies 191 in the circulation. in contrast, igg mbcs reactive to the s proteins of the hcovs oc43 and 229e 192 and the control proteins h1 and ttd were detected in nearly 50% or more of non-sars-cov-2-193 exposed subjects, consistent with the higher levels of serum igg against these antigens ( figure 2e -194 2h) . as expected, sars-cov-2 rbd-reactive mbcs were not detected in unexposed subjects. 195 in marked contrast to non-sars-cov-2-exposed subjects, the vast majority of 196 convalescent subjects had circulating igg mbcs reactive to the sars-cov-2 s, rbd, and s2, 197 indicating strong induction by sars-cov-2 infection of mbcs reactive to novel and conserved 198 regions of the s protein ( figure 2a) . notably, numbers of igg mbcs reactive to the s protein of 199 the hcov oc43 were higher in convalescent subjects than in unexposed subjects ( figure 1e generates igg mbcs reactive to the sars-cov-2 s2 that cross-react with the s2 of human 207 betacoronaviruses. interestingly, only a small proportion of the convalescent subjects generated 208 detectable n-reactive igg mbcs, even though most subjects produced high levels of anti-n igg 209 in serum (figures 2c, 2d) . it is unclear whether this reflects a real difference between s-and n-210 reactive mbc formation or an effect of the sampling time. overall, we demonstrate that sars-211 cov-2 infection induces strong s-reactive mbc formation that would be expected to provide 212 lasting protection against reinfection and potentially broad protection against betacoronaviruses. 213 our goals in this study were to investigate sars-cov-2-reactive b cell memory in 216 unexposed subjects that could provide some protection against sars-cov-2 infection, and the 217 generation of b cell memory by sars-cov-2 infection that could provide lasting protection 218 against re-infection. in particular, we were interested in igg mbcs, which respond to cognate 219 antigens with rapid, vigorous, and high-affinity ab production. importantly, mbcs are long-lived 220 cells that continue to provide strong protection when circulating ab levels wane. our approach 221 was to analyze circulating igg as well as igg mbcs from the sars-cov-2-naïve and sars-222 cov-2-convalescent subject groups. our key findings are as follows: (i) the presence of igg 223 reactive to the s2 subunit of sars-cov-2 in most unexposed subjects, likely reflecting cross-224 reactivity to hcovs, (ii) markedly increased levels of igg against the sars-cov-2 s and n 225 proteins, including reactivity to the rbd and s2 subunit of s, in convalescent subjects, (iii) 226 increased igg binding to the s protein of the oc43 hcov, but not 229e hcov, in convalescent 227 subjects, reflecting greater cross-reactivity between s2 subunits of betacoronaviruses, (iv) strong 228 formation of igg mbcs reactive with the rbd and s2 subunit of the sars-cov-2 s protein in 229 convalescent subjects, and (v) formation of igg mbcs reactive with the s protein of oc43, but 230 not 229e, in convalescent subjects, consistent with s2 subunit cross-reactivity between 231 approximately one-third of our cohort of non-sars-cov-2-exposed subjects had low 233 levels of igg against the sars-cov-2 s and n proteins. the anti-n igg likely reflects infection 234 with hcovs, which have low level (20-30%) homology with the sars-cov-2 n protein (10). 235 however, a protective function for anti-n abs has not been established (21). notably, 86% of 236 unexposed subjects had igg against the s2 subunit, reflecting homology with hcovs, but none 237 had igg against the highly novel sars-cov-2 rbd (6, 8, 22) . abs that target the s2 subunit have 238 been shown to have virus neutralizing activity, raising the possibility that preexisting anti-s2 igg 239 confers some protection against sars-cov-2 (23). the processes that generate anti-s2 igg are 240 also likely to generate s2-reactive igg mbcs and these might provide more significant protection 241 than low levels of anti-s2 abs. however, s2-reactive mbcs (or s-reactive and n-reactive mbcs) 242 were not detected in non-sars-cov-2-exposed subjects. taken together with the identification 243 of s-reactive mbcs in unexposed healthy donors (19), it is likely that s2-reactive mbcs were 244 below the limit of detection in our assays. most mbcs are resident in lymphoid tissues, except for 245 mbcs against frequently seen immunogenic antigens (for example, the influenza h1 or ttd in 246 this study), and are at very low frequencies in circulation in steady state (24, 25) . 247 anti-rbd, -s, and -n igg levels were markedly higher in the convalescent subjects than 248 in non-sars-cov-2-exposed subjects, indicating strong induction by sars-cov-2 infection. 249 perhaps notably, the majority of convalescent subjects had higher igg titers against the s2 than 250 against the rbd. this is particularly surprising because of the accessibility of the rbd to b cells 251 and the expected immunodominance over the s2 subunit (26, 27). our demonstration of strong 252 anti-s2 igg production is consistent with the activation of a preexisting population of igg mbcs 253 against the conserved s2 subunit in the absence of mbcs reactive to the novel rbd. however, we 254 cannot exclude inherent differences in the stability or antigenicity of rbd and s2 reagents as an 255 explanation. in convalescent subjects, igg levels against the s protein of hcov oc43 (but not 256 229e) were significantly higher than in non-sars-cov-2-exposed subjects and correlated 257 strongly with anti-s2 igg levels. these findings support stronger b cell cross-reactivity between 258 the s2 subunits of sars-cov-2 and human betacoronaviruses than alphacoronaviruses (8). 259 importantly, we demonstrate that sars-cov-2 infection generates rbd-reactive and s2-260 reactive igg mbcs. recently, long et al. (4) found that levels of sars-cov-2-reactive abs, 261 including neutralizing abs, start to decrease within 8-12 weeks of infection, especially when the 262 infection is asymptomatic. since mbc populations are maintained for many years, perhaps 263 decades, our findings indicate that mbcs generated by sars-cov-2 infection will be available 264 to rapidly generate protective abs if waning ab levels allow re-infection to occur (28). notably, 265 three convalescent subjects in our analysis had undetectable rbd-reactive igg, but nevertheless 266 had rbd-reactive igg mbcs. this might reflect mbc production by germinal centers that 267 remained active after recovery from infection (29). the proportion of subjects with mbcs reactive 268 to the hcovs oc43 and 229e was greater for the convalescent group than the unexposed group, 269 likely reflecting the increase in s2-reactive mbcs in the convalescent group and cross-reactivity 270 with hcovs. s2-reactive mbc expansion by sars-cov-2 infection could enhance protection 271 against a broad range of coronaviruses (23). n-reactive mbc formation in convalescent subjects 272 was less than expected given the large number of subjects with high titers of n-reactive igg, but 273 additional sampling times are required to confirm this observation. 274 in conclusion, our analysis investigated ab and mbc immunity to sars-cov-2 in 275 unexposed subjects and individuals soon after recovery from sars-cov-2 infection. findings 276 emphasized the novelty of the sars-cov-2 s protein rbd in unexposed subjects. however, igg 277 reactive to the s2 was widespread in unexposed subjects and likely resulted from exposure to 278 hcovs. although our approach was unable to directly identify s2-reactive mbcs in the 279 unexposed subjects, we suggest that these cells are present and strongly contribute s2-reactive igg 280 early in the response to sars-cov-2 infection. the igg response in sars-cov-2 convalescent 281 subjects was also strong against the rbd and, less consistently, against the n protein. importantly, 282 sars-cov-2 convalescent subjects had generated rbd-reactive and s2-reactive igg mbcs. the may, 2020 and consisted of 22 pcr-confirmed patients and 5 non-pcr-confirmed subjects who 300 were contacts of confirmed cases or displayed covid-19-like symptoms. the convalescent 301 subjects were sampled 4-9 weeks after symptom onset. symptoms reported (percent of subjects) 302 were fever (67%) cough (74%), sore throat (48%), stuffy/runny nose (56%), difficulty breathing 303 (52%), fatigue (85%), headache (67%), body aches (67%), nausea/vomiting (19%), and 304 diarrhea/loose stool (41%). 305 (isolate wuhan-hu-1) were expressed in-house in hek293 cells using pcaggs plasmid 307 constructs kindly provided by florian krammer (icahn school of medicine at mount sinai) (7). 308 baculovirus-expressed s2 subdomain and hek293 cell-expressed n protein were obtained from 309 sino biological (chesterbrook, pa) and raybiotech (peachtree corners, ga), respectively. 310 baculovirus-expressed s proteins from seasonal hcovs oc43 and 229e were obtained from sino 311 biological. in-house hek293 cell-expressed hemagglutinin from egg-derived h1n1 312 mabtech stockholm, sweden) and p-nitrophenyl phosphate substrate (thermo fisher) were 337 subsequently added to detect bound antigen-specific abs. absorbance was read at 405 nm after 338 color development. a weight-based concentration method was used to quantify antigen-specific 339 ab levels in test samples as described previously (30, 31) . sera from healthy donors and 340 convalescent subjects with high titers for test antigens were used to establish human serum 341 standards. the cutoff for assay positivity was set at approximately 2x the mean od value for 342 negative wells. 343 statistical analyses. the medians with (q1, q3) were summarized by subject group and 344 compared by the wilcoxon rank-sum test. spearman correlation analysis together with 345 corresponding robust regression models was used to assess monotonic associations among ab 346 responses. multiple test adjustment was not applied for this explorative study and thus a p value 347 < 0.05 was considered significant for all analyses. statistical analyses were performed using 348 software sas 9.4 (sas institute inc, cary, nc). cov-2-exposed and covid-19 convalescent subjects. sera were collected from ( 2 proteins in non-sars-cov-2-exposed and covid-19 convalescent subjects. pbmcs for mbc 522 analysis were collected from (i) 21 healthy donors sampled from 2011-14 (hd) and (ii) 26 523 covid-19 convalescent subjects sampled 4-9 weeks after symptom onset (conv). pbmcs were 524 stimulated in vitro to induce mbc differentiation into ab-secreting cells. antigen-specific 525 a pneumonia outbreak associated with a new coronavirus of probable bat origin sars-cov-2 vaccines: status report clinical features of patients infected with 369 2019 novel coronavirus in wuhan clinical and 372 immunological assessment of asymptomatic sars-cov-2 infections genome composition and 376 divergence of the novel coronavirus (2019-ncov) originating in china phylogenetic analysis 379 and structural modeling of sars-cov-2 spike protein reveals an evolutionary distinct and 380 proteolytically sensitive activation loop a serological assay to detect 386 sars-cov-2 seroconversion in humans reimer 390 presence of sars-cov-2 reactive t cells in covid-19 patients and healthy donors infectious diseases (except hiv/aids) pre-existing and de novo humoral immunity to sars-cov-398 2 in humans characterization 404 of a novel coronavirus associated with severe acute respiratory syndrome antibody response of patients with severe acute respiratory syndrome (sars) targets the 408 viral nucleocapsid virological assessment of hospitalized 412 patients with covid-2019 targets of t 416 cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and 417 unexposed individuals b cell responses: cell interaction dynamics and decisions antibody responses to sars-cov-2 in patients with covid-19 kinetics of sars-cov-2 specific igm and igg responses in covid-19 430 patients covid-19 serology at population scale: sars-cov-2-specific 435 antibody responses in saliva. infectious diseases (except hiv/aids) deep 439 sequencing of b cell receptor repertoires from covid-19 patients reveals strong 440 convergent immune signatures broad neutralization of sars-related viruses by human monoclonal antibodies convergent antibody responses to sars-cov-2 in convalescent individuals contributions of the structural proteins of severe acute respiratory syndrome 458 coronavirus to protective immunity human monoclonal 466 antibodies against highly conserved hr1 and hr2 domains of the sars-cov spike 467 protein are more broadly neutralizing the transcription factor t-bet resolves memory b cell 472 subsets with distinct tissue distributions and antibody specificities in mice and humans broad dispersion and lung localization of virus-475 specific memory b cells induced by influenza pneumonia a sequence 478 approach can predict candidate targets for immune 479 responses to sars-cov-2 the receptor binding domain of the viral spike protein is 484 an immunodominant and highly specific target of antibodies in sars-cov-2 patients cutting edge: 487 long-term b cell memory in humans after smallpox vaccination role of memory b cells in hemagglutinin-489 specific antibody production following human influenza a virus infection broad hemagglutinin-specific memory b cell 495 expansion by seasonal influenza virus infection reflects early-life imprinting and 496 adaptation to the infecting virus assignment of weight-based antibody units to a human antipneumococcal 499 standard reference serum, lot 89-s individual hd and conv subjects in order of ascending titers against s. the assigned cutoff for 509 positivity is shown by the shaded bar. (b) proportions of serum igg against the sars-cov c) serum igg concentrations against the s protein 511 of the hcov oc43 in conv, hd, and hcw subjects. (d-f) serum igg concentrations against 512 the s protein of the hcov 229e (d), the influenza virus h1 hemagglutinin (e), and ttd (f) in 513 conv and hd subjects. (g) correlation between serum igg concentrations against the s2 subunit 514 of sars-cov-2 and the s protein of the hcov oc43 001; ns [not significant]) for comparisons of serum igg concentrations between subject 516 groups was determined by the wilcoxon rank-sum test. correlations were tested by spearman 517 correlation analysis with corresponding robust regression models quantitation of mbc-derived ab (igg)-secreting cells (mascs) or mbc-derived polyclonal mpabs) provided a measure of the abundance of specific igg mbcs. (a) igg mbcs reactive 527 to the sars-cov-2 spike (s), receptor binding domain (rbd), and nucleocapsid (n) in conv 528 subjects. mbc numbers were determined by enumeration of igg mascs by elispot essay after 529 in vitro mbc stimulation. the assigned cutoff for positivity is shown by the shaded bar mbcs reactive to the influenza virus h1 hemagglutinin and ttd in conv subjects. mbc 531 numbers were determined by enumeration of igg mascs. (c) proportions of igg mbcs reactive 532 to the sars-cov-2 rbd, s2, and n for individual conv subjects. (d) comparison of serum 533 igg concentrations (upper panels) and igg mbc numbers cov-2 s (left-hand side) and n (right-hand side) proteins. serum igg was measured by elisa dilution curves are shown for 536 individual conv subjects; curves for 4 subjects are shown in different colors to identify particular 537 response patterns key: cord-260334-xo8ruswo authors: new, r.r.c.; moore, b.d.; butcher, w.; mahood, r.; lever, m.s.; smither, s.; o'brien, l.; weller, s.a.; bayliss, m.; gibson, l.c.d.; macleod, c.; bogus, m.; harvey, r.; almond, n.; williamson, e.d. title: antibody-mediated protection against mers-cov in the murine model() date: 2019-07-09 journal: vaccine doi: 10.1016/j.vaccine.2019.05.074 sha: doc_id: 260334 cord_uid: xo8ruswo murine antisera with neutralising activity for the coronavirus causative of middle east respiratory syndrome (mers) were induced by immunisation of balb/c mice with the receptor binding domain (rbd) of the viral spike protein. the murine antisera induced were fully-neutralising in vitro for two separate clinical strains of the mers coronavirus (mers-cov). to test the neutralising capacity of these antisera in vivo, susceptibility to mers-cov was induced in naive recipient balb/c mice by the administration of an adenovirus vector expressing the human dpp4 receptor (ad5-hdpp4) for mers-cov, prior to the passive transfer of the rbd-specific murine antisera to the transduced mice. subsequent challenge of the recipient transduced mice by the intra-nasal route with a clinical isolate of the mers-cov resulted in a significantly reduced viral load in their lungs, compared with transduced mice receiving a negative control antibody. the murine antisera used were derived from mice which had been primed sub-cutaneously with a recombinant fusion of rbd with a human igg fc tag (rbd-fc), adsorbed to calcium phosphate microcrystals and then boosted by the oral route with the same fusion protein in reverse micelles. the data gained indicate that this dual-route vaccination with novel formulations of the rbd-fc, induced systemic and mucosal anti-viral immunity with demonstrated in vitro and in vivo neutralisation capacity for clinical strains of mers-cov. the middle east respiratory disease syndrome (mers) first emerged in 2012 in saudi arabia [1, 2] . since then, there have been an estimated 2311 laboratory-confirmed cases with 811 deaths, reported from a total of 27 countries in the eastern mediterranean region and from 12 countries elsewhere [1] . saudi arabia, however, remains the main focus of infection and a disease outbreak in south korea involving 186 cases was traced back to an index case who had travelled from saudi arabia. whilst the incidence of mers cases in saudi arabia peaked in 2014, there are still a significant number of cases reported from the country and in the period september 2012-may 2018, there were 1844 cases including 716 deaths with a case fatality rate of 38.8% [1] . mers coronavirus (mers-cov) is a member of the betacoronavirus genus [3] and as for other betacoronaviruses, bats may provide a natural reservoir for the virus [3, 4] , but high levels of antibodies to mers-cov in dromedary camels [5] suggest that the dromedary camel is the principal source for animal-to-human transmission of mers-cov [6] . however, evidence of human-to-human transmission comes from the reporting of outbreaks in countries remote from saudi arabia such as the uk, europe, usa, and china where small outbreaks have also occurred [1] . mers-cov is an enveloped, positive-sense, single-stranded rna virus [3] . the virus possesses an envelope-anchored trimeric spike protein which binds to the human receptor dipeptidyl peptidase 4 (dpp4 or cd26) and gains host cell entry by the fusion of viral and host membranes [7] . the spike protein comprises an s1 sub-unit and a membrane fusion s2 sub-unit. in the coronaviruses, the s1 sub-units are further divided into n-terminal and c-terminal subdomains and for mers-cov, it is the c-terminal sub-domain that comprises the receptor-binding domain (rbd) [8] . the rbd also incorporates a receptor-binding motif at its c-terminal and the crystal structures of mers-cov rbd [8] and of the rbd bound to the extracellular domain of human dpp4 have been reported [9] . the rbds of the coronaviruses represent vaccine and therapeutic targets and the rbd of mers-cov as a vaccine antigen has been demonstrated to induce neutralising antibody [10] and to protect mice transduced with a viral vector expressing hdpp4, or nonhuman primates from viral challenge [11] [12] [13] [14] [15] . there are significant ongoing efforts to develop vaccines for mers-cov infection, predominantly involving live attenuated viral vectors such as adenovirus, modified vaccinia ankara or measles [16] to induce anti-viral immunity and some of these vaccines are already in clinical trials. here, we were interested to determine the relative importance of inducing systemic and/or mucosal immunity in vaccination to protect against mers-cov, an infection predominantly of the respiratory tract and lungs. to this end, we have used a dual route immunisation regimen in balb/c mice to induce both systemic and mucosal immunity, to generate rbd-specific murine antisera. initially, we immunised balb/c mice sub-cutaneously (s.c.) with rbd-fc in the mf59 adjuvant to induce rbd-specific igg. subsequently, we have immunised further groups of balb/c mice by s.c. priming and per oral (p.o.) boosting with the rbd-fc, to induce both systemic igg and mucosal iga responses. to do this, we have used novel formulations of rbd-fc coated onto microcrystals formed from histidine or glutamine and also incorporating calcium phosphate for sub-cutaneous priming [17] , whilst the formulation for oral boosting comprised rbd-fc in reverse micelles dispersed in a self-emulsifying oil phase, which has been optimised from previous formulations [18, 19] . the advantages of these formulations are that they are very stable under extremes of temperature [20] . furthermore, on translation to the clinic, only one injected priming dose would be required, followed by a p.o. booster dose; the latter could be self-administered in capsule form. we have compared the relative abilities of the two sources of antiserum to neutralise clinical isolates of mers-cov in vitro. to do this, we have used two clinical strains of mers-cov (erasmus medical center 2012 or emc2012 and london1-2012:), each of which were derived from severely-ill individuals who had contracted the virus in the middle east in 2012. subsequent sequencing of the polymerase gene from these isolates indicated them to be newly-emerged members of the betacoronavirus genus with a close sequence homology and phylogenetic relationship to the bat coronaviruses hku4 and hku-5 [21, 22] . mice are not naturally susceptible to mers-cov infection, but susceptibility can be induced by the administration of an adenovirus vector which induces expression of the human receptor (hdpp4/cd26) for the virus in vivo for a limited time, providing a non-lethal murine model of the disease [23] . we have used this transduced mouse model to test the capacity of the antiserum derived from the dual route immunisation to neutralise mers-cov in vivo, by passive transfer prior to challenge with the emc2012 strain and we have demonstrated a significant reduction in viral load in lung tissue in transduced mice. the rbd was synthesised and expressed according to methods adapted from du et al. [10] . in brief, a single dna fragment containing an in-frame fusion of the coding sequences for the human il2 signal peptide, the rbd and human igg1-fc was synthesised. this was transferred into the plasmid pef-dest51 (invitrogen) so that the target sequence was expressed as a secreted protein with a c-terminal human igg1 fc tag. this construct was transfected by cationic transfection into human embryo kidney (hek) cells in suspension (fshek) or adherent hek cells stably expressing the sv40 large t antigen (293ft), using serum-free media and incubated for 4-7 days. small scale purifications of rbd-fc were performed using protein a chromatography. for this, medium from the transfected cells was treated with ammonium sulphate to precipitate the protein, prior to dialysis and resuspension in buffers for binding to protein a beads. the latter were washed and eluted with buffer containing 1 m urea. protein concentration was determined by uv absorbance spectroscopy and purity was estimated by sds-page with coomassie staining and subsequent optical densitometry using a syngene g:box imaging system. the rbd-fc was incorporated on glutamine calcium phosphate (cap) microcrystals for s.c. immunisation, using methodology adapted from [17] . briefly, aqueous mixtures of rbd-fc with sodium orthophosphate and glutamine were precipitated as cap protein-coated microcrystals (cap-pcmc), by addition to of a 19fold excess of isopropanol containing dissolved calcium chloride. the resultant suspension contained self-assembled microcrystals comprising a glutamine core with the rbd-fc protein embedded in a thin surface layer of cap (now termed rbd-fc-pcmc). the pcmc were isolated by vacuum filtration and dried to a powder. protein content and integrity was determined by elisa and sds-page. for oral dosing, the rbd-fc was incorporated in mineral oil with added excipients using methodology adapted from [18, 19] . the oral formulation comprised rbd-fc with the mucosal adjuvant cholera toxin b sub-unit (ctb), retinoic acid (ra), vitamin d, e and trehalose debehenate (tdb), a synthetic analog of the mycobacterial trehalose dimycolate [25] and imiquimod, a tlr 7/8 agonist [26] . specific pathogen-free female balb/c mice (6-8 weeks of age) were obtained from a commercial breeder and used throughout this study. on receipt, mice were randomised for allocation to cages and given free access to food, water and environmental enrichment. mice were fully acclimatised to the animal housing facility for at least five days prior to any procedure. all animal procedures were performed in accordance with uk legislation as stated in the uk animal (scientific procedures) act 1986. the institutional animal care and use committee approved the relevant project licence. naïve mice were randomised for allocation to a treatment group (typically 5 per group) and immunised in one of two regimens: either with a s.c. priming dose followed by two s.c. doses, given at 10 and 31 days after the prime; alternatively, mice received a s.c. priming dose followed by an oral or s.c. booster dose 21 days after the prime (table 1) . for s.c. immunisation, mice received 2.5 lg of rbd-fc-pcmc in 0.1 ml pbs injection volume, whereas for all per oral (p.o.) dosing, mice received 25 lg of rbd-fc in a total volume of 0.1 ml mineral oil (mo), by oral gavage. where rbd-fc was administered s.c. in the conventional adjuvants mf59 or alhydrogel, mf59 (novartis, us) was used in a 1:1 ratio by volume with rbd-fc in pbs, whilst alhydrogel (brenntag biosector, denmark) was used in a 1:5 ratio with rbd-fc by volume in pbs. at selected intervals after dosing, mice were blood-sampled from the tail vein for assay of specific antibody titre. at the end of the immunisation schedule, individual mice were terminally anaesthetised for collection of blood by cardiac puncture, then culled prior to removal of small and large intestines for collection of faecal pellets for extraction of iga. titres of rbd-fc-specific antibody in serum samples were determined by elisa. in brief, test sera were bound to microtitre plates pre-coated with rbd-fc and antibody binding was detected with an hrpo-labelled secondary antibody to mouse igg, igg1, igg2a or iga (bio-rad). a standard curve for calibration comprising the relevant murine ig isotype (sigma) captured with an anti-fab reagent, was included on each plate. plates were developed by the addition of 2,2 0 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (abts) substrate (sigma) and optical density (od) was read at 414 nm (multiskan plate reader). for assay of antibody in faecal samples, faecal pellets were extracted in supplemented pbs as described previously [20] . in brief, 10 ml of cold pbs was prepared, supplemented with 1 tablet of complete mini protease inhibitor cocktail (sigma) and 5 ll tween 20 were added. to 0.1 g faecal pellets, 1 ml of supplemented pbs was added and left at room temperature for 5 min. samples were vortexed for approximately 30 s, incubated on ice for a further 20 min. and then centrifuged (15,000g, 5 min.). supernatants were retained and stored at à80°c pending assay. the faecal extracts were assayed for specific igg and iga content, by elisa, as for serum samples. antibody concentrations in all samples were determined from the relevant standard curves using ascent software with fourparameter logistic curve-fitting and reported in ng/ml or lg/ml serum or faecal extract, as appropriate. to determine if the antibody induced by immunisation with to rbd-fc was neutralising for mers-cov in vitro, plaque assays were performed. for this, two strains of mers-cov were used: london1-2012 (genbank accession number kc164505.2) [22] and erasmus medical center (emc2012 genbank accession number jx869059) (21) . the london1-2012 strain was obtained from the national collection of pathogen viruses, phe porton, salisbury, uk and the emc2012 strain was kindly provided by the erasmus university medical center rotterdam, the netherlands. both strains were prepared in serum-free media (gibco) at a multiplicity of infection (moi) of 0.01, equivalent to 10 3 plaque-forming units (pfu). the murine antiserum for testing was prepared at a dilution range from undiluted to 1:10 in pbs. virus was incubated overnight (4°c) with murine antiserum, negative control antibody (nibsc, uk) or media, prior to infection of a confluent monolayer of vero e6 cells (ecacc, salisbury uk) with 200 ll of the mixture. the neutralising ability of the murine antiserum was tested in duplicate or triplicate at each dilution. after incubation (1 h, 37°c), an overlay comprising a 1:1 dilution of carboxymethyl cellulose with serum-free media was added to the cells and incubation continued for a further 4 days (37°c) prior to fixing (7.4% formaldehyde) and staining (0.2% crystal violet) with enumeration of the number of plaques per ml. mice are not naturally susceptible to infection with mers-cov, since they lack the human dpp4 receptor. to induce transient susceptibility in balb/c mice, we used an ad5 construct (oxford genetics) to express the human dpp4 receptor (ad5hdpp4), as previously described (23) . mice were administered the ad5hdpp4 construct (2.5 â 10 8 pfu in 50 ll) by the intra-nasal (i.n.) route under light sedation with inhalational isofluorane and then monitored by serial blood sampling for serum levels of hdpp4/cd26 by elisa (thermoscientific). at peak levels of expression of hdpp4 (days 5-7), mice were lightly sedated as before and challenged by the i.n. route with mers-cov (emc2012 strain) at 10 4 pfu in 50 ll per mouse. mice were weighed prior to challenge on each subsequent day to monitor changes in body weight during infection. to test the in vivo neutralising capacity of murine antiserum raised to the rbd-fc construct, naïve mice (n = 10 per treatment group) were passively immunised by the i.p. route at 24 h. prior to i.n. challenge with the mers co-v (emc2012 strain), as described above. the murine antiserum, pooled from 4 mice who had been primed with rbd-fc pcmc and boosted orally (regimen 2, treatment group 2), was delivered at a dilution of 1:10 in pbs and delivered in a total volume of 100 ll per mouse. a further group of 10 mice received a purified polyclonal human igg at a single dose level (150 lg/mouse in 100 ll i.p.), which had been raised to inactivated mers-cov. control mice received a non-specific human igg at a single dose-level (200 lg/mouse in 100 ll, i.p.). both sets of human igg (specific and non-specific) were raised in a bovine transchromosomal model and purified prior to use. a further group of 10 negative control mice were included, which received pbs in place of either the ad5dpp4 construct or the mers-cov-specific antibody, and were also challenged i.n. with mers-cov (emc2012 strain) at 10 4 pfu/mouse. to determine the protection afforded by the passive immunisation, pairs of mice from each treatment group were culled on days 1-8 after challenge and their lungs were removed and weighed and then rapidly frozen (à80°c) prior to the determination of viral load. pairs of lungs from each of 2 mice per treatment group were individually thawed and homogenised in serum-free media (2 ml). rna was extracted from 140 ll of each homogenate using the qiaamp viral rna kit (qiagen), following the manufacturers' instructions. real-time pcr was conducted on duplicate 5 ll the amount of virus in tested samples was determined in duplicate using the standard curve and reported as pfu/g lung tissue. all data were analysed using graph pad prism software v.6 and expressed as mean ± s.e.m. statistical comparisons were made using one-way anova or unpaired t-test. the rbd-fc protein was expressed in both adherent 293ft and suspension human embryo kidney (hek) cells, but with greater expression in adherent cells (fig. 1) . purification of protein from adherent cells with protein a was very effective, yielding protein which was >99% pure, with molecular weight of approximately 100 kda (fig. 1a) . the use of 1 m urea for elution was optimum, as it was sufficient to solubilise the protein without denaturing it, yielding rbd-fc in optimum yield (0.2 mg/ml) and predominantly in a dimeric form (fig. 1b) . this method of protein purification was therefore selected for forward use. rbd-fc, formulated for either sub-cutaneous (s.c.) or per oral (p.o.) immunisation, was tested for immunogenicity and the formulations optimised in an iterative approach. initially, a s.c. dosing regimen was used in which rbd-fc was formulated in either alhydrogel or mf59 to deliver 2.5 lg of protein on each of three occasions at 0, 10 and 31 days. mice were monitored for 26 days after the final boost and igg titre determined ( fig. 2a) . at day 70, the total igg titres achieved with rbd-fc in alhydrogel or mf59 did not differ significantly. to determine if the presentation of rbd-fc in either alhydrogel or mf59 influenced the ability to develop virus-neutralising antibody, antisera were selected from 2 mice in each immunisation group and tested in a plaque assay for neutralisation of both the emc2012 and london1-2012 strains of mers-cov ( fig. 2b and c) . all four sera gave some neutralisation of viral activity, although at a 1:20 dilution, sera 136 and 169 were most potent, against both viral strains. sera 136 and 169 were derived from the treatment group immunised with mf59adjuvanted rbd-fc, whereas sera 132 and 150 were derived from alhydrogel-adjuvanted rbd-fc ( fig. 2a) . based on this pilot data, we subsequently used mf59 as the conventional adjuvant for rbd-fc, to compare with some novel formulations. having demonstrated to proof-of-principle that the rbd-fc, when delivered in mf59 can induce a high titre of antibody with neutralising activity, we next investigated how to tailor an rbd-fc vaccine optimally to induce both systemic and mucosal immunity, with the aim also of reducing to a 2-dose immunisation regimen and increasing functional antibody. for this, we selected novel formulations in which rbd-fc protein was presented as rbd-fc-pcmc for s.c. priming and incorporated into mineral oil (mo) for p.o. boosting. we compared the serum igg response achieved from this 2-dose dual route immunisation with that induced to rbd-fc delivered in mf59 in a 2-dose s.c. regimen (fig. 3) . at 1 month after the booster dose, at day 49, there was no significant difference in the serum igg titres achieved, so that the 2-dose dual-route immunisation with rbd-fc-pcmc for s.c. priming and incorporated in mo with excipients for p.o. boosting, was just as immunogenic as the 2-dose s.c. immunisation with rbd-fc in mf59 (fig. 3a) . at day 49, the serum response to rbd-fc in the dual-route regimen was predominantly igg1 biased, whereas s.c. dosing with rbd-fc in the presence of mf59 induced both igg1 and igg2a (fig. 3b) . since dual route immunisation effectively induced serum igg to rbd-fc, it was of interest to determine whether it could also effectively induce mucosal immunity. in this study, the rbd-fc-specific iga response was determined in serum and in faecal pellet extracts from individual animals on day 49. in this case, the rbd-specific iga responses of mice immunised in the 2-dose dual route regimen were compared with that of mice immunised by the oral route twice, and with mice immunised by the s.c. route in mf59 twice, on exactly the same days (0,21) (fig. 4) . this comparison showed that s.c. immunisation in mf59 did not induce serum iga. however s.c. priming with rbd-fc-pcmc with p.o. boosting effectively induced rbd-fc-specific iga and was not inferior to oral priming and boosting in this effect in either serum (fig. 4a ) or faecal extracts (fig. 4b ). however mice primed and boosted orally did not develop rbd-specific systemic igg (data not shown). additionally, day 49 sera from mice in all treatment groups were tested for their ability to neutralise either strain of mers-cov in vitro (table 2) . from this it can be seen that sera from 4 out of 5 mice in the dual route regimen were fully neutralising neutralisation of mers-cov in vitro. in in vitro for both strains (emc2012 and london1-2012), when tested at 1:60 dilution ( fig. 5a and b) . in order to test whether the in vitro neutralising activity translated into viral neutralisation in vivo, sera from these 4 mice (highlighted in table 2 ) were pooled in equal aliquots at 1:10 dilution to enable a subsequent passive transfer study. in order to design the passive transfer study, it was necessary to define the duration of expression of cd26 in murine lungs in vivo, following induction with the ad5hdpp4 construct. mice dosed with ad5hdpp4 i.n. at t 0 were culled in pairs and lung homogenates prepared and assayed for cd26 expression. cd26 in lung tissue was expressed in a time-dependent manner, with levels peaking at day 3 and declining to day 17 (fig. 6a) , setting a sufficient window to use the model for the determination of the protection against viral challenge afforded by the passive transfer of mers-specific antibody. to determine the protection afforded by the passive transfer of murine antiserum raised in the dual route immunisation regimen, against infection, susceptibility to mers-cov was induced at t 0 with i.n. administration of ad5hdpp4 to groups of 10 mice. passive transfer by the i.p. route of the pooled serum sample derived from the 4 mice highlighted in table 2 , which had previously been shown to be neutralising in vitro (table 2 ) was conducted 5 days later and mice were challenged after a further 24 h with mers-cov emc2012. additional groups of mice, which had been transduced with ad5hdpp4, were passively immunised with a mers-cov specific human igg and a non-specific human igg. at 1-8 days after challenge, pairs of mice were culled for the determination of viral load in lungs, which was determined to peak at 3 days p.i. (data not shown). at 3 days p.i., the pooled murine antiserum significantly reduced viral titres in lungs, to the same extent as the specific human igg, and contrasting with the negative control human igg, demonstrating significant in vivo neutralising activity (fig. 6b ). fig. 6 . a. expression of cd26 was induced in lung tissue by the administration of ad5hdpp4 (2.5 â 10 8 pfu) to mice by the i.n. route at t 0 . subsequently, mice were culled in pairs on the days shown and their lungs assayed for the expression of cd26. the plot shows the time-course of cd26 expression from 3 to 17 days post-induction. all data points were normalised for background values from control mice. 6b: content of mers-cov (emc2012 strain) in murine lungs (pfu/g tissue) determined by rt-pcr at day 3 post-infection, (equivalent to day 4 after passive transfer with murine antisera to rbd-fc which had previously been shown to neutralise the emc2012 strain in vitro). mice received either a mers-cov-specific human igg (150 lg) or non-specific human igg (200 lg) in 100 ll /mouse i.p.; or murine antisera to rbd-fc, which had been pooled from 4 murine donors and which was delivered at 1:10 dilution (100 ll/mouse i.p.). negative control mice received pbs in place of ad5hdpp4 or antiserum all mice were challenged with mers-cov emc2012 i.n. at 10 4 pfu/mouse. statistical significance was determined at the p < 0.05 level by one way anova and unpaired t-test. no significant differences in body weight were detected between treatment groups challenged with mers-cov, which was attributed to the short time period of the study. mers is a serious endemic respiratory infectious disease for which there is no licensed vaccine, although there are several vaccines in clinical trial currently including adenovirus-vectored delivery of the spike protein and sub-units [27] , dna vaccines and nanoparticle-delivered sub-unit approaches [28] . we were interested in determining the advantage of r a vaccine which could induce mucosal as well as specific systemic immunity to the key target, the rbd protein, in order to achieve optimum protective efficacy. vaccination to induce effective immunity at mucosal surfaces, should prime the immune system to respond rapidly to invading pathogens such as mers-cov. previous studies have used adenovirus delivery of the mers spike protein with intra-muscular (i.m.) or intra-gastric (i.g.) delivery to induce neutralising systemic igg, but not iga; further, whilst i.m. delivery also induced specific t-cell immunity, i.g. delivery did not [29] . others have shown that intra-nasal delivery of a live attenuated adenovirus-vectored subunit vaccines does induce specific mucosal as well as systemic immunity, although translation of this approach to the clinic may raise safety issues [30] . here, we have relied on novel formulations of a sub-unit protein to enable dual route vaccination (parenteral and oral) to induce mucosal as well as systemic immunity. in this study, we have achieved the expression and purification of a recombinant rbd-fc protein in milligram quantities. we have also demonstrated that when formulated as a sub-unit vaccine, the construct induced murine antibody which effectively neutralised two different clinical strains of mers-cov in vitro. additionally, we have shown that these murine antisera, when passively transferred into naïve mice transduced to express the hdpp4 /cd26 receptor, conferred protection against viral challenge in the recipients, with significantly reduced viral loads in the lung tissue of the recipient mice. whilst the use of conventional adjuvants such as mf59 or alhydrogel to formulate the rbd-fc protein resulted in high titres of specific igg in serum, the mf59 formulation did not induce specific iga in serum. in order to promote both systemic and mucosal immunity to rbd-fc, we have formulated this protein for injected priming and p.o. boosting, entailing the optimisation of the cap pcmc and of the reverse micelles in oil emulsion, respectively. this has enabled the achievement of a vaccination regimen comprising only two doses and rapidly inducing rbd-fc-specific systemic and mucosal immunity. whilst the pcmc formulation of rbd-fc was as effective as rbd in mf59 in inducing a primary igg response, we have shown that an oral formulation of rbd-fc in mineral oil with selected immunostimulants was as effective as mf59 when used as a booster immunisation. additionally, we have shown that non-invasive oral priming and boosting is as effective at inducing a specific mucosal response, measured as specific iga in serum and faeces, as is injected priming with rbd-fc in the pcmc formulation together with oral boosting, leading to the exciting concept of a potential orally-dosed sub-unit vaccine for mers. whilst both alhydrogel and the combination of pcmc and oral formulations are th2-polarising, as evidenced by the predominantly igg1 titres raised to rbd-fc, the influence of mf59 on the response to rbd-fc was a mixed th-2/th1 effect, with a significant induction of specific igg1 and igg2a. to counter a viral infection, it would be expected that a th1 response would be most appropriate. however, the fact that neutralising antibody to rbd-fc was raised under either th1 or th2-polarising influences, suggests that either isotype can be protective and primes the immune system sufficiently and would allow for cross-presentation to occur on subsequent exposure to the virus [31] . in this study, we have not examined the induction of a cell-mediated memory response to the rbd-fc protein, although this will play a significant role in protection against the virus. currently, we are presenting the rbd protein in our formulations with an fc tag, derived from human igg1 and useful in purifying the protein. the fc tag may contribute additional adjuvantising activity by engaging antigen-presenting cells in the vaccinee [32] and it may aid mucosal immunity since the fc receptor, an mhc1 transmembrane protein, is also expressed at mucosal surfaces e.g. in the respiratory tract [33] . vaccination of the zoonotic host, the dromedary camel, may also effectively curb outbreaks of mers in endemic regions and limit the risk of viral recombination [34] and significant progress with an orthopox-vectored vaccine for mers has recently been reported [35] . the potential use of a sub-unit vaccine for mers in camel vaccination could be aided by varying the sequence of the rbd protein [36] and substituting the human fc tag with an alternative tag recognised by the camel, to design approaches tailored for animal vaccination, bearing in mind that a single dose vaccine would be ideal in this context. however, future work in our laboratory will also address the value of retaining or removing the fc tag from the rbd protein for clinical or veterinary iterations of the vaccine. in this study we have determined vaccine efficacy by demonstrating in vitro and in vivo neutralising ability of murine antisera raised in the dual route two-dose regimen against two virulent clinical strains of mers-cov which have greater than 99% genome sequence homologyy. in future work, it will be worthwhile to test the efficacy of this approach against other clinical isolates of mers-cov. this is the first report of a dual route dosing regimen applied to a sub-unit vaccine for mers-cov. future development of this approach would require the direct testing of efficacy in the immunised transduced mouse model. as well as giving a direct readout of vaccine efficacy, this will enable the identification of the immune correlates of protection, ready for transitioning this candidate vaccine into more extensive pre-clinical testing and clinical development. the authors declare that all the data supporting the findings of this study are available within the paper. ksa mers-cov investigation team. hospital outbreak of middle east respiratory syndrome coronavirus genomic characterization of a newly 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a mouse model for middle east respiratory syndrome real-time reverse transcription-pcr assay panel for middle east respiratory syndrome coronavirus cutting edge: mincle is essential for recognition and adjuvanticity of the mycobacterial cord factor and its synthetic analog trehalose-dibehenate the tlr7 agonists imiquimod and gardiquimod improve dc-based immunotherapy for melanoma in mice chadox1 and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice novel chimeric virus-like particles vaccine displaying mers-cov receptor-binding domain induce specific humoral and cellular immune response in mice systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type 5 or 41 vector-based vaccines carrying the spike protein of middle east respiratory syndrome coronavirus a tetravalent dengue vaccine based on a complex adenovirus vector provides significant protection in rhesus monkeys against all four serotypes of dengue virus intracellular recycling and cross-presentation by mhc class i molecules fc-fusion proteins: new developments and future perspectives fc-fusion proteins and fcrn: structural insights for longer-lasting and more effective therapeutics co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels recombinant receptor-binding domains of multiple middle east respiratory syndrome coronaviruses (mers-covs) induce cross-neutralizing antibodies against divergent human and camel mers-covs and antibody escape mutants the authors acknowledge with thanks the expert technical assistance of lin eastaugh, louise thompsett and vicky roberts. this work was supported by sbri awards 972228 and 971527 from innovate uk to rrcn and na, respectively and on independent research commissioned from na and funded by the nihr policy research programme, [971527]. the views expressed in the publication are those of the author(s) and not necessarily those of the nhs, the nihr, the department of health, 'arms' length bodies or other government departments. the authors declare no conflict of interests. supplementary data to this article can be found online at https://doi.org/10.1016/j.vaccine.2019.05.074. key: cord-015235-lv8mll28 authors: kim, hyun; hong, yeongjin; shibayama, keigo; suzuki, yasuhiko; wakamiya, nobutaka; kim, youn uck title: functional analysis of the receptor binding domain of sars coronavirus s1 region and its monoclonal antibody date: 2014-04-16 journal: genes genomics doi: 10.1007/s13258-014-0186-9 sha: doc_id: 15235 cord_uid: lv8mll28 severe acute respiratory syndrome (sars) is caused by the sars coronavirus (cov). the spike protein of sars-cov consists of s1 and s2 domains, which are responsible for virus binding and fusion, respectively. the receptor-binding domain (rbd) positioned in s1 can specifically bind to angiotensin-converting enzyme 2 (ace2) on target cells, and ace2 regulates the balance between vasoconstrictors and vasodilators within the heart and kidneys. here, a recombinant fusion protein containing 193-amino acid rbd (residues 318–510) and glutathione s-transferase were prepared for binding to target cells. additionally, monoclonal rbd antibodies were prepared to confirm rbd binding to target cells through ace2. we first confirmed that ace2 was expressed in various mouse cells such as heart, lungs, spleen, liver, intestine, and kidneys using a commercial ace2 polyclonal antibody. we also confirmed that the mouse fibroblast (nih3t3) and human embryonic kidney cell lines (hek293) expressed ace2. we finally demonstrated that recombinant rbd bound to ace2 on these cells using a cellular enzyme-linked immunosorbent assay and immunoassay. these results can be applied for future research to treat ace2-related diseases and sars. severe acute respiratory syndrome (sars) is a fatal emerging infectious disease caused by the sars coronavirus (cov) (baker 2004; rota et al. 2003) . sars-covlike virus has been isolated from horseshoe bats in china, and this has been postulated to be the natural reservoir for the virus (lau et al. 2005; li et al. 2005b ). although there have been no recent sars outbreaks, serious concerns remain about its re-emergence from host animals and its potential application as a bioterrorism agent (chan et al. 2006; peiris and yuen 2004) . sars-cov mediates infection of target cells via the spike (s) protein, which is a type one transmembrane glycoprotein divided into two functional domains of s1 (15-680 a.a.) and s2 (681-1255 a.a.) (he et al. 2004a; li et al. 2005a ). infection of sars-cov is initiated by binding of the s protein to the angiotensin-converting enzyme 2 (ace2) functional receptor expressed on target cells (li et al. 2003) . a 193-amino acid fragment (residues 318-510) within the s1 subunit of the s protein has been characterized as the minimal receptor-binding domain (rbd) . in early studies, mice immunized with inactivated sars-cov were used to generate monoclonal antibodies capable of blocking infectivity. of these, several antibodies were directed against the s protein (berry et al. 2004) . the s protein serves as the main antigen that elicits protective immune responses, including neutralizing antibodies in infected humans and animals (bisht et al. 2004; buchholz et al. 2004; greenough et al. 2005; hofmann et al. 2004) . several studies have demonstrated that rbd of the s region is a major target for neutralizing sars-cov antibodies (he et al. 2004b (he et al. , 2005 zhou et al. 2004) . ace2, also called aceh (ace homologue), is an integral membrane protein and a zinc metalloprotease of the ace family that also includes somatic and germinal ace (komatsu et al. 2002; tipnis et al. 2000) . ace2 has been implicated in the pathology of hartnup's disease, a disorder of amino acid homeostasis, and it has been recently revealed that ace2 controls intestinal inflammation and diarrhea via its function in amino acid transport; thus, regulating the gut microbiome (kuba et al. 2013) . mouse ace2 has about a 40 % amino acid identity to the n-and c-terminal domains of mouse somatic ace. the predicted mouse ace2 protein sequence consists of 798 amino acids, including an n-terminal signal peptide, a single catalytic domain, a c-terminal membrane anchor, and a short cytoplasmic tail. ace2 is a newly described rennin-angiotensin (ras) system component that is sensitive to chloride ion concentration (donoghue et al. 2000) . it is a membrane-bound enzyme that acts as a monocarboxypeptidase and is an essential regulator of heart function. within the ras, ace2 competes with ace because it is capable of hydrolyzing the inactive decapeptide angiotensin i (ang i) into the nonapeptide ang (1-9); thus, decreasing the amount of ang i available for pressor ang ii generation by ace. similarly, ace2 degrades the vasoconstrictor ang ii into vasodilator ang (1-7), which may also be produced from ang (1-9) hydrolysis by ace (donoghue et al. 2000; vickers et al. 2002; zhong et al. 2010) . the antagonistic relationship between ace and ace2 modulates the balance between ang ii (vasopressor) and ang 1-7 (vasodilator), which plays a significant role regulating renal and cardiovascular functions. sars-cov infections and the s protein decrease ace2 expression, and s protein injections into mice worsen acute lung failure in vivo . in contrast, ace2 and the type 2 ang ii receptor protect mice from severe acute lung injury, but other components of the ras (including ace, ang ii, and ang ii type 1a receptor) promote disease occurrence ). these findings suggest a possible therapeutic role for ace2 in acute lung injury, which affects many people worldwide every year. expression of ace2 is more restricted than ace, which is widely distributed on the endothelial cells of the arteries, arterioles, and venules in the heart and kidneys (tipnis et al. 2000; oudit et al. 2003) . ace2 is also expressed in the vascular smooth muscle cells of the intrarenal arteries, the renal tubular epithelium, coronary blood vessels (donoghue et al. 2000) and adult leydig cells of the testis (douglas et al. 2004) . other investigators have shown that ace2 expression occurs on the surface of lung alveolar epithelial cells and enterocytes of the small intestine (hamming et al. 2004) . ace2 mediates binding between a vero e6-ace2 expressing cell line and the recombinant s protein expressed on the surface of cho cells, even under high stringency washing conditions (chou et al. 2005) . ace2 was recently reported at various levels in various tissues that also express ace mrna. because ace2 is a functional receptor for the sars-cov, its tissue distribution seems to be of great importance and appears to be species specific. ace2 may also contribute to programmed hypertension. here, we report that a recombinant rbd fusion protein induced a high titer of rbd-specific monoclonal antibodies, and effectively operated the antigen protein. our cellular enzyme-linked immunosorbent assay (elisa) and competitive binding assay using a polyclonal ace2 antibody indicated that our prepared recombinant rbd fusion protein binds to various tissues as well as nih3t3 and hek293 cells through ace2. we think that our prepared recombinant rbd and its monoclonal antibody can be developed to prevent sars and disease pathogenesis. the pgex-4t-1 plasmid was purchased from ge (ge healthcare life sciences, uppsala, sweden) to express the rbd-gst fusion protein. goat anti-mouse igg alkaline phosphatase and 4-nitrophenyl phosphate (npp) were provided by sigma (st. louis mo, usa) for the elisa. dulbecco's modified eagle's medium (dmem) was purchased from gibco (grand island, ny, usa) for hybridoma cell culture, and hat and ht were obtained from sigma for selecting the hybridoma cells. sp2/0 myeloma, hek293, and nih3t3 cells were provided by the american type culture collection (rockville, md, usa) for preparing the hybridoma cell and cellular elisa, respectively. various tissues were lysed from balb/c mice purchased from slc (tokyo, japan) for detecting ace2 expression. alexa fluor 488 reactive dye was purchased from invitrogen (carlsbad, ca, usa) to obtain confocal images. all other chemicals used were of the best grade available from commercial sources. the pcr fragments encoding 193-a.a rbd sequences were amplified was done with two primers, srar6 (5 0 -gtccg cgaattcaacatcaccaacctgtg) and sars7 (5 0 -cttcggctcgagcacggtggcgggcgcgt) against codon-optimized sars spike protein gene in pcdna3.1 (a gift of dr. m. farzan, harvard medical school, ma) as template in polymerase chain reaction (pcr). the pcr fragments were digested with ecori and xhoi and ligated into the same sites of the pgex 4t-1 vector (ge healthcare bio-sciences corp, nj, usa). as result, the expression plasmid carries an n-terminal glutathione-stransferase (gst)-tag/thrombin/lac promoter. the nucleotide sequence analysis was performed using the dye terminator cycle sequencing ready reaction kit with an abi 373 dna sequencer. the recombinant plasmid was transformed into competent e. coli bl 21 codon plus, and grown with constant shaking in 29 yt broth (20 g tryptone, 10 g yeast extract, 10 g nacl/l) in the presence of ampicillin (50 lg/ml). five ml of cell suspension was inoculated into 50 ml 29 yt fresh media/250 ml flask for induction of the recombinant protein and was incubated at 37°c until optical density reached 0.6. the culture suspensions were further incubated for 4 h at 37°c in the presence of 0.5 mm isopropyl-b-d-thio-galactoside with vigorous shaking (180 rpm). four ml of bacterial culture was harvested by centrifugation at 4°c, and the pellet was resuspended with 4 ml of reduced sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) sample buffer. the reactions were heated at 95°c for 5 min, and only the supernatant was applied to 13 % sds-page gels using a mini-protein electrophoresis apparatus (bio-rad hercules, ca, usa). the gel was soaked in 0.2 m cold kcl solution for 10 min until the protein bands appeared as a gray color, then the bands were cut with a razor for homogenizing. the chopped gel and 0.5 ml pbs were added to a microtube for homogenizing, and about 30 strokes were done to crush the gel. the tube was centrifuged for 30 min at 15,0009g to remove the gel piece and then filtered with a 0.2 lm filter. purity was confirmed by 13 % sds-page and used to immunize mice to prepare a monoclonal antibody. the purified rbd fusion protein was mixed with an equal volume of complete freund's adjuvant (sigma) and injected intraperitoneally. the antigen-adjuvant mixture was injected into female balb/c mice (8 weeks old). the first injection was followed by three booster injections at 3-or 4-week intervals. the final injection was administered without adjuvant 3-4 days before cell fusion. after confirming the antibody titer in tail blood from immunized mice, b cells were separated from the spleen for fusion with myeloma cells. feeder cells were prepared 1 day before fusion from a 15 week-old mouse. the abdominal skin was carefully removed and feeder cells were collected by centrifugation. the fusion experiments were performed as follows. spleen cells were released by tearing the removed spleen with forceps and the rough side of a slide glass, and the cells were collected in a 15 ml centrifuge tube. the spleen cells and sp2/0-ag-14 mouse myeloma cells were mixed in a 10:1 ratio, and 1 ml of 50 % polyethylene glycol 4000 in serumfree dmem was added slowly. the fusion process was allowed to continue for 1 min at 37°c and centrifuged for 2 min at 1009g. then, 4.5 ml dmem was added slowly for the first 3 min, and 5 ml was added over the next 2 min. the fused cells were brought up to 50 ml with dmem, and collected by centrifugation at 1009g for 5 min. the cells were carefully resuspended in 35 ml of selective hat medium [dme supplemented with 20 % fetal bovine serum (fbs), antibiotics, and hat] by swirling, and then incubated under 8 % co 2 for 30 min. each 100 ll of cell suspension was transferred to 96-well plates, and incubated under 8 % co 2 in an incubator. about 2 weeks after the fusion, culture supernatants were collected and screened by elisa. positive clones were transferred to 6-well plates, and frozen in liquid nitrogen. all positive clones were frozen first and cloned by limiting dilution after thawing. hybridoma cells (1 9 10 7 ) were intraperitoneally injected into a balb/c mouse to collect ascites and purify the monoclonal antibody. after 2 weeks, the drained ascites were centrifuged for 30 min at 15,0009g to remove residual cells and insoluble aggregates and then applied to a protein g-agarose column (hitrap 5 ml, ge healthcare life sciences). the column was washed with phosphatebuffered saline (pbs) until the absorbance of unbound proteins decreased to background, and then the antibody was eluted with 0.1 m glycine-hcl, ph 2.5. the eluted antibody was neutralized by adding 1 m tris and dialyzed against pbs overnight. a 96-well micro titer plate (costar, boston, ma, usa) was coated with 50 ll (5 lg/ml) of purified rbd fusion and gst proteins at 4°c overnight and then was washed three times with deionized distilled water. the wells were blocked with 250 ll blocking buffer (borate buffered saline genes genom (2014) 36:387-397 389 containing 5 % skim milk, 1 mm edta, 0.05 % nan 3 , and 0.05 % tween 20) for 30 min at room temperature. after three washes with blocking buffer, 50 ll of antiserum or cell supernatant was added and incubated for 2 h at room temperature. the wells were washed three times with water, and then blocked with blocking buffer for 30 min at room temperature. a goat anti-mouse igg antibody coupled with alkaline phosphatase (sigma, 100 lg/ml in blocking buffer) was incubated at room temperature for 2 h to bind the first antibody. the 96-well plates were washed with water and detected with 100 ll of 1 mg/ml (3 mm) p-npp in 0.05 m na 2 co 3 and 0.05 mm mgcl 2 . the reaction was stopped with 25 ll of 0.5 m naoh and measured at 405 nm using a microtiter plate reader (bio-rad). for the cellular elisa, various tissues were separated from the balb/c mouse and treated with ammonium sulfate to remove the erythrocytes. the cells were washed and cultured with 10 % fbs/dmem media, then transferred to a 96-well plate at 5 9 10 5 cells/well for immobilizing cells to the plate. the wells were washed with pbs, and 150 ll of 1 % paraformaldehyde was added (ethanol:methanol = 1:1). the plate was incubated for 30 min at -20°c and washed with water to react the rbd or ace2 antibody (rabbit igg polyclonal antibody, cat # 18-661-15167, genway biotech, ca, usa). paraformaldehyde fixation has been used to attach various primary tissue cells and established cell lines to plastic wear or slide glass. we used paraformaldehyde fixation because we worried about detachment of primary tissue cells and suspension cells from vessel during various experimental procedures. for example, in our cellular elisa, intact cell was attached to plastic wear in the first step, and then several reactions such as rbd or ab were successively followed. the rbd fusion protein or tissue cell lysates were suspended in reduced sds-page sample buffer and heated, then resolved on 12 % sds-page. the gels were electrophoretically transferred to pvdf membranes using a mini protein ii transfer chamber (bio-rad). the membranes were blocked overnight at 4°c in pbs containing 5 % skim milk, and then washed three times with pbs containing 0.1 % tween 20 (pbs-t). sequentially, the membrane was washed two times with pbs-t and incubated with 5 lg/ml rbd or ace2 antibody for 1 h. after washing twice with pbs-t, the membrane was reacted with goat anti-mouse igg antibody coupled with alkaline phosphatase for 2 h at room temperature. the membranes were washed five times with pbs-t and five times with distilled water, then developed using 1 mg/ml (3 mm) (npp in 0.05 m na 2 co 3 , 0.05 mm mgcl 2 . immunofluorescence assay nih3t3 and hek293 cells were cultured and transferred to a four-chambered flask for fluorescence labeling and imaging. after washing with pbs, paraformaldehyde solution (4 %) was added to the cells and incubated for 30 min at 4°c to fix the cells to the wells. after three washes with pbs, 0.2 % triton x-100 was added to the plate for 30 min at 4°c. the plate was washed three times with pbs, and then 1 % bsa (in pbs) was added and incubated for 1 h at room temperature. after washing with pbs, the rbd fusion protein was incubated for 1 h at room temperature to bind with the ace2 molecules on the cell membranes. after washing three times with pbs, the monoclonal rbd antibody was reacted with the rbd-ace2 binding reactant. finally, secondary antibody (goat anti-mouse igg coupled with alexa 488-green) was treated for 1 h at room temperature and sequentially with dapi for 30 s. cell imaging was performed using an olympus fv1000 confocal microscope equipped with a four-laser system (multi ar laser, hene g laser, hene r laser, and ld405/440 laser diode) with transmitted light, differential interference contrast, and complete integrated image analysis software system (olympus america inc., melville, ny, usa). the excitation and emission wavelengths for alexa fluor 488 (green) and dapi (red) were 488/520 and 543/570 nm, respectively. composite digital images were then converted to tiff format, imported into adobe photoshop (adobe photoshop cs2, version 9.0; adobe systems inc., san jose, ca, usa), and color balance was adjusted for presentation. expression and purification of the rbd-gst fusion protein and preparation of the monoclonal rbd antibody pcr products of 579 bp (193 a.a.) long rbd sequences were ligated to the pgex 4t-1 expression vector and expressed in e. coli bl 21 (fig. 1a) . main bands of 48-50 kda (fig. 1a , lane 1) and 25 kda (fig. 1a, lane 2) were detected in e. coli extracts, which transformed rbd-gst and gst dna, respectively. as shown fig. 1b , the two main bands were purified for immunization into five mice each. after the final injection of purified antigen, the antibody titer was checked in tail blood. after 3-4 days, mouse spleen cells were lysed and fused with myeloma cells to prepare hybridoma cells. after limiting dilutions for 4 weeks, we obtained seven rbd fusion and five gst positive colonies, respectively. among the seven positive colonies against the rbd-gst fusion protein, four colonies reacted only with the rbd but not the gst protein. the two colonies in the best condition were expanded for intraperitoneal injection to obtain a large amount of antibody. the antibodies were purified from ascites, and assayed by elisa and western blotting as shown fig. 1c , d. the results indicated that the selected monoclonal antibody recognized the rbd but not the gst region of the rbd-gst fusion protein (fig. 1d) . cells were separated from the heart, spleen, and liver to confirm binding to the cell surface. these cells were fixed to 96-well plates with paraformaldehyde, and treated using the same procedure as for the general elisa. as shown fig. 2a , three kinds of cells (heart, spleen, and liver) reacted to the rbd surface molecule. this binding phenomenon was confirmed by western blotting analysis with the same cell lysates (fig. 2b) . the rbd fusion protein band appeared at *50 kda but not the gst protein in all three panels for the positive and negative controls. these three blotted membranes were successively treated with purified rbd fusion protein and rbd monoclonal antibody. tissue cell lysates of heart or spleen revealed proteins at *60 kda. but, liver cells showed a smeared, faint band (fig. 2b) . this experiment indicated that our prepared rbd fusion protein bound to molecule(s) on the tissue cell surface. various cells were prepared from a balb/c mouse, and the total cell lysates were loaded onto a 13 % gel for western blotting analysis (fig. 3) . similar to fig. 2b , about a 60 kda band was shown in various cell extracts (fig. 3a) . among the various cells, heart and lung extracts showed strong bands that were two or three fold more intense than some studies have reported that the sars virus is related to ace. thus, we tested ace and ace2 molecules as rbd receptors, and their antibodies were used as a blocking agent for rbd binding. as shown fig. 3b , the ace2 antibody was pretreated for 30 min before the rbd reaction to block the rbd receptor, and the rbd antibody was added to detect residual rbd binding (fig. 3b) . the 60 kda band almost disappeared in the tissue cell lanes, but not from the rbd protein lane. we knew that this 60 kda band corresponded to the ace2 molecule that was detected at about the 60 kda position with an ace2 antibody (fig. 3c) . these results indicate that rbd bound to the ace2 molecule in various cells. to confirm whether the rbd would bind to established cell lines, hek293 and nih3t3 cells were fixed in 96-well plates as shown in fig. 2a . the cells were successively treated with rbd and rbd antibody. as shown fig. 4a , the rbd protein bound to both cell types to the same extent. the same amounts of rbd protein and ace2 antibody were simultaneously incubated with the cells for 30 min and detected with the rbd antibody ( fig. 4b; column 3). in this competitive assay, the ace2 antibody suppressed 60-70 % of rbd binding in both cell lines within 30 min. we showed that these cell lines express ace2 molecule l and that the molecules were the rbd receptor molecules as shown in the various mouse tissues. but, in this reaction, inhibition decreased 20-30 % after 3-4 h incubation (data not shown). it seemed that the ace2 antibody may easily separate from the ace2 molecule or was degraded. next, we examined whether rbd binding was blocked by the ace2 antibody in a western blot using mouse tissue cell lysates with a pair of membranes. the ace2 molecule appeared as three or more bands in hek293 cells and as two bands in nih3t3 cell lysates (fig. 4c, left panel) . the other transferred membrane was pretreated with ace2 antibody before rbd and rbd antibody to block the rbd fig. 2 cellular enzyme-linked immunosorbent assay and western blot analysis of mouse tissues. a various mouse tissues were cultured and then fixed with paraformaldehyde. after reacting with receptorbinding domain (rbd)-specific monoclonal antibodies, alkaline phosphatase-conjugated goat anti-mouse igg and npp substrate were successively added to each well and measured in a dynex spectrophotometer at 405 nm. b to confirm the rbd receptor, whole cell lysates from mouse organ tissue cells were loaded on to 13 % sodium dodecyl sulfate polyacrylamide gel electrophoresis and immuno-blotted using the rbd monoclonal antibody. each lane indicated as follows: s spleen; l liver; h heart; g glutathione-stransferase (gst); r, rbd-gst binding sites (fig. 4c, right panel) . expectedly, ace2 molecules in hek293 cells were blocked with the ace2 antibody but not in nih3t3 cells. we confirmed this by confocal immunofluorescence image using the two cell lines that were fixed and treated with rbd and the rbd antibody (fig. 5) . as a result, the rbd receptor (ace2) was observed in both hek293 and nih3t3 cells using the rbd antibody. a cocaine monoclonal antibody was used as the negative control in this experiment. in this study, the reason why we used elisa or immunoflourescence instead of immunoprecipitation or frozen section staining is that we want to investigate nih3t3 or hek293 cell lines and primary tissues cell at same condition and environment. the s protein of sars-cov is able to induce protective antibodies from infected animals (bisht et al. 2004; buchholz et al. 2004 ). the rbd (residues 318-510) in the s1 region of the s protein induces highly potent neutralizing antibodies against the sars-cov (he et al. 2004a, b) . here, we expressed the rbd of the s protein and immunized a mouse to prepare monoclonal antibodies. the rbd protein was prepared with the gst fused form and purified fusion protein without separating the two kinds of proteins, because this fusion protein was efficiently expressed in this system and easily separated from a gel. another benefit is that the fused protein efficiently provided monoclonal antibodies due to its suitable molecular size (see fig. 1 ). we also prepared a plural gst antibody for confirming the rbd specific binding experiments (data not shown). we have demonstrated with a cellular elisa assay that rbd bound to cell membranes and that the anti-rbd antibody worked well with its antigen. the first essential step of cov infection is interaction of the s protein via the rbd with a specific cellular receptor. there are many reports that rbd plays a role infecting host cells. the rbds on the s proteins of other covs such as mouse hepatitis virus, transmissible gastroenteritis virus, and human coronavirus also contain major antigenic determinants capable of binding to host cells (bonavia et al. 2003; godet et al. 1994) . some studies have reported that rbd-specific antibodies block receptor binding and virus entry (he et al. 2004a) . therefore, the rbd of the s protein may serve as an important target site for developing sars vaccines and immunotherapeutics. in this study, we conducted two kinds of experiments to demonstrate the results of previous reports. the first was cellular binding of rbd using a cellular elisa as an intact cell instead of a traditional protein or antigen, and the second was western blot analysis using the same cell lysates as in the cellular elisa. in the cellular elisa data, we demonstrated that the binding was due to rbd not gst, because the rbd fusion protein included the gst protein. this was shown by using the anti-gst antibody (data not shown), and the rbd binding receptor clearly appeared as a 60 kda protein in the spleen and heart but was smeared in the liver lysate (fig. 2b) . the smeared band in the liver may indicate degradation by proteolysis or something that occurred during extract preparation, because this band was shown in other preparations (fig. 3a) . there are many reports of sars s or rbd protein binding to ace2 on host cell membrane proteins using an antibody against sars-cov and by other methods. various neutralizing monoclonal antibodies against sars-cov recognize overlapping sets of residues relative to the ace2 molecule (zhu et al. 2007) . ace2 is predominantly expressed in the heart, kidneys, and testes and at lower levels in a wide variety of tissues, particularly the colon and lungs (lew et al. 2008) . it seems that this rbd region plays a critical role attaching the host ace2 molecule. this phenomenon was also supported by chimeric monoclonal antibodies that bind to the ace2 rbd of the sars s protein (berry et al. 2010) . experimental support for ace2-rbd binding was provided from a study of sars antibody fragments competing with ace2 for binding to the rbd (prabakaran et al. 2006; sui et al. 2004; hwang et al. 2006) . the crystal structure of the rbd-ace2 complex has been identified, providing detailed information concerning rbd structure and function (li et al. 2005a ). the structure revealed that the rbd can be further divided into two separate subdomains. one is the rbd core and the other is the rbd loop (rpm) (a.a. 424-494). the rbd loop is the region that directly contacts the ace2 molecule. in contrast, the rbd core contacts accessory proteins (li et al. 2005a) . we showed competitive binding of the rbd with the anti-ace2 antibody (see figs. 3, 4, 5) . the same weights of tissue were homogenized and lysed by reducing sds-page sample buffer for western blotting analysis. ace2 was detected by the anti-ace2 antibody in all of our organ tissue samples, which cross-reacts with the mouse/human ace2 molecule. ace2 contains seven potential n linked glycosylation sites and is therefore likely to be glycosylated. overexpressed ace2 migrates at 120 kda compared with the deglycosylated polypeptide that migrates at 85 kda (lew et al. 2008) . very low levels of ace2 were detected in plasma by western blot analysis and in multiples smaller than the full-length enzyme. this likely resulted from proteolytic cleavage (lew et al. 2008 ). an approximately 90-kda immunoreactive band was present in the whole-cell lysate, and a slightly smaller band was detected in the conditioned medium of ace2-transfected cells, indicating that full-length ace2 is processed in cho cells to generate a secreted form (donoghue et al. 2000) . we report, for the first time, that our prepared recombinant rbd bound to various mouse tissues and established after reacting with the receptor binding domain (rbd) and rbd monoclonal antibody, alkaline phosphataseconjugated goat anti-mouse igg was added to each well. the plates were developed with npp substrate solution, and then read at 405 nm in a dynex spectrophotometer. b, c competition assay by cellular elisa (b) and western blot (c). prior to treatment with the rbd protein, the angiotensin-converting enzyme 2 (ace2) polyclonal antibody was treated for 30 min to block the rbd binding site. then, the rbd-specific monoclonal antibody and each secondary antibody were sequentially added to the cells cell lines. the ace2 molecule was detected at approximately 60 kda and expressed strongly in the heart and lungs of mice. this is different from humans in which there is stronger expression in the kidneys than that in the lungs. rbd binding to ace2 molecule was interfered with by pre-treatment with the ace2 antibody in mouse tissues (see fig. 3 ). we confirmed these results in nih3t3 and hek293 cells by cellular elisa and a competitive assay (see fig. 4a , b). the anti-ace2 antibody suppressed rbd-ace2 binding about 70-80 % within 30 min, but the inhibition decreased to 30-50 % after a 2-3 h incubation (data not shown). it seemed that the ace2 antibody separated from the ace2 molecules after a long incubation or degraded during incubation. we confirmed the molecular weight of ace2 in hek293 and nih3t3 cells for the competitive assay in gels. expressed ace2 was 120 kda, but it differs in tissue and cell lysates (donoghue et al. 2000; lew et al. 2008 ). in our lysates, ace2 molecules occurred in three or more bands in hek 293 cells and in two or more bands in nih3t3 cells. this seemed to be a proteolytic cleavage pattern resulting from the different tissue lysate preparations (see fig. 4c , left panel). the ace2 molecules were completely blocked by the ace2 antibody before treatment with the rbd protein in hek293 cells, but not in nih3t3 cells (see fig. 4c , right panel). we suggest that the rbd binding site on ace2 molecules in nih3t3 cells may have been destroyed or changed due to cleavage of the ace2 molecules during sample preparation. this suggestion was supported by our confocal microscope images results in which nih3t3 cells stained well with the rbd fusion protein as in the hek293 cells. here, we first confirmed that ace2 was expressed in various mouse cells such as heart, lungs, spleen, liver, intestine, and kidneys using a commercial ace2 polyclonal antibody. we also confirmed that the mouse fibroblast (nih3t3) and human embryonic kidney cell lines (hek 293) expressed ace2. we finally demonstrated that recombinant rbd bound to ace2 on these cells. we also demonstrated that our preparing rbd elicited high antibody titers in its immunized mice. therefore, it appears to be an ideal immunizing antigen for generating monoclonal antibodies as well as a possible vaccine candidate. we have also found that the rbd binds to various tissues through ace2. these results can be applied for future research to help treat ace2 related diseases and sars. fig. 5 confocal microscope of established cell lines. nih3t3 and hek293 cells were reacted with the receptor binding domain (rbd) protein, and then the rbd monoclonal antibody and secondary alexa 488 antibody were sequentially added to the each cell line, respectively. an anti-cocaine monoclonal antibody was used for the negative control. green color indicates rbd binding to the cell surface. nucleus was specifically stained by dapi (blue color) characterisation of neutralising monoclonal antibody to the sars-coronavirus neutralizing epitopes of the sars-cov s-protein cluster independent of repertoire, antigen structure or mab technology severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice identification of a receptor-binding domain of the spike glycoprotein of human coronavirus hcov-229e contributions of the structural proteins of severe acute respiratory syndrome to protective immunity sars: clinical presentation, transmission, pathogenesis and treatment options a novel cell-based binding assay system reconstituting interaction between sars-cov s protein and its cellular receptor a novel angiotensin-converting enzyme-related carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 the novel angiotensin-converting enzyme (ace) homolog, ace2, is selectively expressed by adult leydig cells of the testis major receptorbinding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein development and characterization of a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibody that provides effective immunoprophylaxis in mice tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis receptor binding domain of sars-cov spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (sars) coronavirus: implication for developing sars diagnostics and vaccines identification of a critical neutralization determinant of severe acute respiratory syndrome (sars)-associated coronavirus: importance for designing sars vaccines s protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients structural basis of neutralization by a human anti-severe acute respiratory syndrome spike protein antibody, 80r angiotensin-converting enzyme 2 protects from severe acute lung failure molecular cloning, mrna expression and chromosomal localization of mouse angiotensinconverting enzyme-related carboxypeptidase (mace2) a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury multiple functions of angiotensin-converting enzyme 2 and its relevance in cardiovascular diseases severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats angiotensin-converting enzyme 2 catalytic activity in human plasma is masked by an endogenous inhibitor angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus structure of sars coronavirus spike receptor-binding domain complexed with receptor bats are the natural reservoirs of sars-like coronaviruses the role of ace2 in cardiovascular physiology severe acute respiratory syndrome structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody characterization of a novel coronavirus associated with severe acute respiratory syndrome potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association a human homolog of angiotensin-converting enzyme: cloning and functional expression as a captopril-insensitive carboxypeptidase hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase a 193-amino acid fragment of the sars coronavirus s protein efficiently binds angiotensin converting enzyme 2 angiotensin converting enzyme 2 suppresses pathological hypertrophy, myocardial fibrosis and cardiac dysfunction an exposed domain in the severe acute respiratory syndrome coronavirus spike protein induces neutralizing antibodies potent cross-reactive neutralization of sars coronavirus isolates by human monoclonal antibodies key: cord-253438-k8iqv1jb authors: li, yujun; wang, haimin; tang, xiaojuan; fang, shisong; ma, danting; du, chengzhi; wang, yifei; pan, hong; yao, weitong; zhang, renli; zou, xuan; zheng, jie; xu, liangde; farzan, michael; zhong, guocai title: sars-cov-2 and three related coronaviruses utilize multiple ace2 orthologs and are potently blocked by an improved ace2-ig date: 2020-10-27 journal: j virol doi: 10.1128/jvi.01283-20 sha: doc_id: 253438 cord_uid: k8iqv1jb the ongoing coronavirus disease 2019 (covid-19) pandemic has caused >20 million infections and >750,000 deaths. severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the etiological agent of covid-19, has been found closely related to the bat coronavirus strain ratg13 (bat-cov ratg13) and a recently identified pangolin coronavirus (pangolin-cov-2020). here, we first investigated the ability of sars-cov-2 and three related coronaviruses to utilize animal orthologs of angiotensin-converting enzyme 2 (ace2) for cell entry. we found that ace2 orthologs of a wide range of domestic and wild mammals, including camels, cattle, horses, goats, sheep, cats, rabbits, and pangolins, were able to support cell entry of sars-cov-2, suggesting that these species might be able to harbor and spread this virus. in addition, the pangolin and bat coronaviruses, pangolin-cov-2020 and bat-cov ratg13, were also found able to utilize human ace2 and a number of animal-ace2 orthologs for cell entry, indicating risks of spillover of these viruses into humans in the future. we then developed potently anticoronavirus ace2-ig proteins that are broadly effective against the four distinct coronaviruses. in particular, through truncating ace2 at its residue 740 but not 615, introducing a d30e mutation, and adopting an antibody-like tetrameric-ace2 configuration, we generated an ace2-ig variant that neutralizes sars-cov-2 at picomolar range. these data demonstrate that the improved ace2-ig variants developed in this study could potentially be developed to protect from sars-cov-2 and some other sars-like viruses that might spillover into humans in the future. importance the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is the etiological agent of the currently uncontrolled coronavirus disease 2019 (covid-19) pandemic. it is important to study the host range of sars-cov-2, because some domestic species might harbor the virus and transmit it back to humans. in addition, insight into the ability of sars-cov-2 and sars-like viruses to utilize animal orthologs of the sars-cov-2 receptor ace2 might provide structural insight into improving ace2-based viral entry inhibitors. in this study, we found that ace2 orthologs of a wide range of domestic and wild animals can support cell entry of sars-cov-2 and three related coronaviruses, providing insights into identifying animal hosts of these viruses. we also developed recombinant ace2-ig proteins that are able to potently block these viral infections, providing a promising approach to developing antiviral proteins broadly effective against these distinct coronaviruses. cov-2 whu01, pangolin-cov-2020, bat-cov ratg13, and sars-cov bj01. purified rbd proteins were then used to perform surface staining of 293t cells transfected with each of the 16 ace2 orthologs or a vector plasmid control (fig. 2) . all of the rbd proteins showed binding to a number of ace2 orthologs. unexpectedly, although the sars-fig 1 sars-cov-2 and ace2 contact residues are conserved among four sars-like viruses and 16 ace2 orthologs, respectively. (a) interactions between the sars-cov-2 receptor binding domain (rbd, red) and ace2 (blue) involve a large number of contact residues (pdb accession no. 6m0j). rbd residues ͻ5 å from ace2 atoms and ace2 residues ͻ5 å from rbd atoms are shown. (b) the sequences of the sars-cov-2 whu01, a pangolin coronavirus identified in manis javanica (pangolin-cov-2020), a bat coronavirus identified in r. affinis (bat-cov ratg13), and the sars-cov bj01 are aligned, with residues different from the corresponding ones in sars-cov-2 highlighted in blue. the stars indicate rbd residues ͻ5 å from ace2 atoms. the yellow lines indicate the rbm region. n-linked glycosylation motifs are indicated in green. (c) sequences of ace2 orthologs from the 16 indicated species are aligned, with only residues ͻ5 å from rbd atoms shown here. the numbering is based on human ace2 protein, and the residues different from the corresponding ones in human ace2 are highlighted in blue. cov-2 and sars-cov rbds differ significantly in the rbm region and ace2-contact residues, both rbds bound to 11 ace2 orthologs with 10 identical ones. moreover, the pangolin-cov-2020 rbd, which differs from sars-cov-2 rbd with only one amino acid within the rbm region, kept nine sars-cov-2 rbd-interacting ace2 orthologs and gained three additional interacting ones, including that of rat, mouse, and chicken. the rbd of bat-cov ratg13 then showed a binding profile significantly different and narrower than the other three rbds. note that human ace2 and ace2 orthologs of some domestic animals, including camels, cattle, horses, goats, sheep, cats, and rabbits, support efficient binding to all the four tested rbds, suggesting that these ace2 orthologs might be generally functional for supporting cell entry of the four tested viruses. a wide range of ace2 orthologs can support entry of the four coronaviruses. to evaluate spike protein-mediated entry of these coronaviruses, we generated retrovirus-based luciferase reporter pseudoviral particles (pp) enveloped with one of six different spike proteins, including a wild-type sars-cov-2 spike (sars-cov-2 whu01 fig 2 a wide range of ace2 orthologs support binding to rbd proteins of sars-cov-2 and three related coronaviruses. (a) 293t cells were transfected with adjusted amounts of the indicated ace2-ortholog plasmids to have similar expression levels of the ace2 ortholog proteins. cells were then stained with an rbd-mouse igg2 fc fusion protein of sars-cov-2 whu01, pangolin-cov-2020, bat-cov ratg13, or sars-cov bj01, followed by staining with an alexa 488-goat anti-mouse igg secondary antibody. rbd-ace2 binding was detected using flow cytometry. (b) percentages of cells positive for rbd binding in panel a are presented as a heatmap according to the indicated color code. (c) expression levels of the indicated ace2 orthologs were detected using western blotting. the data shown are representative of two independent experiments performed by two different people with similar results. pp), a furin site deletion mutant of sars-cov-2 spike (sars-cov-2 δfurin pp), a wild-type sars-cov spike (sars-cov bj01 pp), a sars-cov spike carrying the pangolin-cov-2020 rbd (pangolin-rbd/bj01 pp), a wild-type bat-cov spike (bat-cov ratg13 pp), and a bat-cov spike carrying the pangolin-cov-2020 rbd (pangolin-rbd/ratg13 pp). these reporter pseudoviruses were used to infect 293t cells expressing each of the 16 ace2 orthologs. a vesicular stomatitis virus protein g (vsv-g)-pseudotyped reporter retrovirus whose entry is independent of ace2 was used as a control virus. as expected, all the orthologs that supported rbd binding were also functional on supporting pseudovirus infection. again, ace2 orthologs of humans and most domestic mammals, including camels, cattle, horses, goats, sheep, cats, and rabbits, supported entry of all the tested pseudoviruses ( fig. 3a to g). it is of note that, although furin-cleaved and uncleaved sars-cov-2 spike trimers have significant structural difference (23) , infection with sars-cov-2 δfurin pseudovirus produced stronger reporter signals but an almost identical pattern of ace2-ortholog usage as the wild-type pseudovirus ( fig. 3a and b) . these data are consistent with the findings that both furin-cleaved and uncleaved sars-cov-2 spike trimers can adopt an ace2-binding-competent conformation albeit at different frequency (17 to 50%), and furin cleavage reduces overall stability of the spike protein (23) (24) (25) . the ability of ace2 orthologs to support sars-cov-2 infection was further confirmed by infection assays using sars-cov-2 live virus (fig. 3h) . these data indicate that humans and these domestic animals might be generally susceptible to infections of the four distinct coronaviruses. ace2-ig variants that have soluble ace2 domain truncated at residue 740 but not 615 potently block sars-cov-2 entry. recombinant rbd and soluble ace2 proteins have been shown to potently block sars-cov entry (26, 27) . to investigate whether similar approaches could also be applied to sars-cov-2, we first produced mouse igg2a fc fusion proteins of rbd (rbd-ig) and soluble ace2 (ace2-ig) variants (fig. 4a) . specifically, the rbd variants include wild-type rbds of sars-cov, pangolin-cov-2020, and sars-cov-2, and four mutants of sars-cov-2 rbd that were expected to bind ace2 better via additional possible aromatic-stacking (f486w, y505w) or salt-bridge (k417r, g496d) interactions. the ectodomain of cell-surface ace2 spanning its residues 18 to 740 contains an enzymatic domain (18-615) and a collectrin-like domain (cld). previous crystal-structure studies showed that soluble ace2 protein truncated at its residue 615, preceding the cld domain, express well and forms stable complex with the rbd of sars-cov and sars-cov-2, respectively (21, 22) . therefore, the ace2-ig variants include human ace2 truncated at its residue 615 (615-wt) and 740 (740-wt), respectively, and mutants of the 615-and 740-version ace2-ig proteins that were expected to inactivate ace2s' enzymatic activity (nn) or bind sars-cov-2 rbd better via additional possible hydrophobic (y83w, h34y, m82k) or salt-bridge (d30e) interactions. we then evaluated these proteins for their potency of blocking sars-cov-2 δfurin pseudovirus infection ( fig. 4b and c) . among all the rbd-ig variants, the y505w mutant of sars-cov-2 rbd showed modestly improved potency over the wild type, and wild-type rbd of pangolin-cov-2020 showed the best neutralization activity among all the tested rbds (fig. 4b ). among the tested ace2-ig variants, interestingly, all the 740-version variants showed significantly better potency than the 615-version variants (two-tailed two-sample t test, p ͻ 0.001; fig. 4c ). in addition, d30e mutants of both 615-and 740-version proteins showed improved potency over the corresponding wild types (two-sample t test, p ͻ 0.01), and the 740-d30e variant outperformed all the rbd-ig and ace2-ig variants. the 740-d30e variant of ace2-ig is a broadly neutralizing immunoadhesin. we further tested the 740-wt and 740-d30e variants of ace2-ig for their neutralization activities against sars-cov-2, sars-cov, pangolin-cov-2020, and bat-cov ratg13 pseudotypes (fig. 5) . interestingly, the d30e mutation improved the protein's neutralization activity against sars-cov-2 and bat-cov ratg13 pseudoviruses (two-sample t test, p ͻ 0.01; fig. 5a and c) but not pangolin-rbd/bj01 or sars-cov pseudovirus (two-sample t test, p ͼ 0.05; fig. 5b and d). the d30 residue of human ace2 was consistently found to form a salt-bridge interaction with the k417 residue of sars-cov-2 rbd in multiple released structures of sars-cov-2 rbd in complex with ace2 (20, 21) (fig. 5e ). the residue 417 of bat-cov ratg13 is also a lysine, while the same residue is an arginine for pangolin-cov-2020 rbd and a valine for sars-cov rbd. thus, the mechanism of the d30e-mediated improvement is likely that the mutation enhances the salt-bridge interaction between the residue 30 of the ace2 and residue 417 of sars-cov-2 and bat-cov ratg13 rbds. these data suggest that the 740-d30e variant of ace2-ig is a broadly neutralizing immunoadhesin against sars-cov-2, sars-cov, pangolin-cov-2020, and bat-cov ratg13. an antibody-like ace2-ig variant further improves neutralization potency by ϳ10-fold. to obtain a more potent ace2-ig that may serve as an anti-sars-cov-2 drug candidate, we generated more variants and used human igg1 domains to replace the mouse counterparts in the ace2-ig fusion proteins. two new variants, which have an antibody like configuration and contain four or six soluble ace2 domains in a single molecule, are named as ace2-ig-v3 and ace2-ig-v4, respectively (fig. 6a ). we first tested these variants for their neutralization potency against sars-cov-2 pseudovirus. both of the new variants showed pronounced improvements over ace2-ig-v1.1, the original 740-d30e dimer variant ( fig. 6a to c). specifically, ace2-ig-v3 and ace2-ig-v4 have estimated 50% inhibitory concentration (ic 50 ) values of 40 to 70 pm and ic 90 values of 250 to 730 pm, representing ͼ10-fold improvement on ic 50 and ͼ25-fold improvement on ic 90 compared to those of ace2-ig-v1.1 ( fig. 6b and c) . because the ace2-ig-v4 configuration significantly impairs protein yield during production (data not shown), we chose to proceed with ace2-ig-v3 to test its neutralization potency against sars-cov-2 live virus (fig. 6d) . consistent with the pseudovirus neutralization data, ace2-ig-v3 at 0.16 g/ml showed a more potent inhibition of sars-cov-2 live virus infection than ace2-ig-v1 at 0.8 g/ml. moreover, ace2-ig-v3 at 0.8 g/ml (1.85 nm) already completely abolished viral nucleocapsid protein (np) immunofluorescent signal. these data demonstrate that ace2-ig-v3 is a markedly improved ace2-ig variant as a potent entry inhibitor against sars-cov-2 virus. in this study, we investigated the ability of sars-cov-2 to utilize animal orthologs of ace2 for cell entry. we observed that ace2 orthologs of a wide range of domestic animals, including camels, cattle, horses, goats, sheep, cats, and rabbits, efficiently supported binding and entry of this virus, suggesting that these domestic mammals might be susceptible to this viral infection ( fig. 2b and fig. 3g ). consistent with this, during preparation of the manuscript, two studies independently reported laboratory ). sars-cov-2 and ratg13 have a k417 residue at their spike proteins, while pangolin-cov has an r417 residue and sars-cov has a v417 residue at their spike proteins, respectively. thus, a stabilized salt bridge interaction between e30 of the ace2-ig protein and k417 of the virus spike protein is likely responsible for the d30e mutation-mediated neutralization enhancement. the data shown are representative of two or three experiments independently performed by two different people with similar results, and data points in panels a to d represent the means ϯ the sd of three or four biological replicates. and natural infection of cats by sars-cov-2 (28, 29) . therefore, it is necessary to further investigate the susceptibility of these domestic animals to sars-cov-2. in addition to the species investigated in this study, farmed mink has been found susceptible to sars-cov-2 and able to transmit the virus back to humans (30, 31) . the currently available information therefore suggests that surveillance of livestock and farmed mammals in marketplaces for sars-cov-2 infection might be necessary. pangolins have been proposed as potential intermediate or natural hosts of sars-cov-2 (11) (12) (13) . it has been proposed that sars-cov-2 might originate from recombination of a pangolin-cov-like coronavirus and a ratg13-like coronavirus (13, 32) . thus, the intermediate host of sars-cov-2 should also be susceptible to both of the "parental viruses." here, we found that pangolin (manis javanica) ace2 does not support binding or entry of bat-cov ratg13, a coronavirus known to have the highest genome sequence identity (96.2%) to sars-cov-2 so far, suggesting a lower likelihood of malayan pangolins (manis javanica) being intermediate hosts for sars-cov-2. on the other hand, we also found that sars-cov-2, sars-cov, and pangolin-cov-2020 (11) can all efficiently utilize pangolin (manis javanica) ace2 for cellular binding and entry, supporting the hypothesis that pangolins might be natural hosts of sars-cov-like coronaviruses (11, 12) . it is also noteworthy that pangolin-cov-2020 can also efficiently utilize human ace2, as well as a wide range of domestic-and wild-animal ace2 orthologs for cell entry (fig. 2b and 3g) , indicating that this virus has very broad host range and high risk of spillover into human population in the future. animal models are essential for preclinical evaluation of efficacy and potential toxicity of candidate prophylactic vaccines or therapeutics for covid-19, as well as for studying the transmission, pathogenesis, and immunology of this disease. in this study, rabbit ace2 was found to efficiently support binding and entry of all the four coronaviruses ( fig. 2b and 3g ). it is therefore worth exploring whether rabbit, a commonly used laboratory species, could serve as a common model animal for studying covid-19, sars, and diseases caused by other sars-related coronaviruses. adaptation of viruses to infect mice is another way of developing small animal models of covid-19. we found in this study that pangolin-cov-2020, whose rbm only differs from that of sars-cov-2 with one amino acid, could efficiently utilize mouse ace2 for binding and cell entry (fig. 1b, 2b, and 3g) . these data suggest that mice might be susceptible to this viral infection, and thus this pangolin coronavirus could be used as a surrogate to sars-cov-2 for in vivo studies in wild-type mice. effective vaccines or targeted therapeutics against sars-cov-2 infections are not yet available (15) . ace2-ig that has soluble ace2 truncated at its residue 615 had been proposed as a candidate therapeutic for sars-cov-2 infection in the beginning of the covid-19 pandemic (33) . two recently posted experimental studies by lui et al. (34) and case et al. (35) have also investigated the use of this 615-version ace2-ig to block sars-cov-2 infection and showed ic 50 values of ϳ1 and 29 g/ml, respectively. consistent with these studies, we got in our study an estimated ic 50 value of ϳ4 g/ml for this variant (615-wt in fig. 4c ). it is noteworthy that our studies here have identified three key improvements over the 615-version ace2-ig. first, the 740-wt ace2-ig variant showed a ͼ20-fold potency improvement over the 615-wt variant, and all the 740version variants showed markedly enhanced neutralization potency over the 615version variants (fig. 4c) . the reason for this improvement is not very clear yet, but it is possible that the cld domain, included in the 740 version, stabilizes an orientation of the ace2 enzymatic domain favorable to s-protein binding, or that the cld has an independent anti-sars-cov-2 activity. second, likely because sars-cov-2 has adapted in animals (e.g., pangolins) whose ace2 orthologs have a glutamic acid at position 30, the d30e mutation further improves the 740-version ace2-ig's neutralization potency against sars-cov-2. moreover, the d30e mutation also enables the protein to neutralize all the four distinct sars-like coronaviruses (fig. 5) . third, by utilizing an antibodylike structure to build an ace2 tetramer, we generated a variant ace2-ig-v3 that has at least 10-fold additional improvement on neutralization potency against sars-cov-2 pseudotype as well as live virus (fig. 6 ). through these changes, we have therefore successfully improved the originally very modest immunoadhesin inhibitor (ic 50 ϸ 18 nm) to be a very potent entry inhibitor (ic 50 ϸ 60 pm) against sars-cov-2. recently, a clinical-grade recombinant soluble ace2 protein has been shown to block sars-cov-2 infection in engineered human organoid (36) . a phase 2 clinical trial to investigate this protein, injected twice daily, as a treatment for covid-19 patients has already started recruiting patients in multiple countries (https://clinicaltrials.gov/ct2/ show/nct04335136). considering that fusion with igg fc improves soluble ace2 protein's half-life in mice from ͻ2 h to over a week (37, 38) and that we have markedly improved the neutralization potency of ace2-ig, we therefore optimistically expect that the improved ace2-ig variants described in this study could potentially be developed to provide effective protections from sars-cov-2 and other sars-like viruses that might spillover into humans in the future, as well as sars-cov-2 variants that emerge over the course of the current pandemic. cells. 293t cells and vero cells were kindly provided by stem cell bank, chinese academy of sciences, confirmed mycoplasma-free by the provider, and maintained in dulbecco modified eagle medium (dmem; life technologies) at 37°c in a 5% co 2 -humidified incubator. growth medium was supplemented with 2 mm glutamax-i (gibco, catalog no. 35050061), 100 m nonessential amino acids (gibco, catalog no. 11140050), 100 u/ml penicillin and 100 g/ml streptomycin (gibco, catalog no. 15140122), and 10% fbs (gibco, catalog no. 10099141c). 293t-based stable cells expressing human ace2 were maintained under the same culture condition as 293t, except that 3 g/ml of puromycin was added to the growth medium. 293f cells for recombinant protein production were generously provided by yu j. cao (school of chemical biology and biotechnology, peking university shenzhen graduate school) and maintained in smm 293-tii serum-free medium (sino biological, catalog no. m293tii) at 37°c, 8% co 2 , in a shaker incubator at 125 rpm. plasmids. dna fragments encoding spike proteins of sars-cov-2 whu01 (genbank accession no. mn988668.1), sars-cov bj01 (genbank ay278488.2), pangolin-cov (national genomics data center gwhabkw00000000; https://bigd.big.ac.cn/search/?dbidϭgwh&qϭgwhabkw00000000&pageϭ1) (11) , and bat-cov ratg13 (genbank mn996532) (8), were synthesized by the beijing genomic institute (bgi, china) and sangon biotech (shanghai, china) and then cloned into pcdna3.1(ϩ) plasmid or pcaggs plasmid between ecori and xhoi restriction sites. plasmids encoding recombinant rbd and soluble ace2 variants were generated by cloning each of the gene fragments into a pcaggs-based mouse-igg2a or human igg1 fc fusion protein expression plasmid between noti and bspei sites. the retroviral reporter plasmids encoding a gaussia luciferase or a green fluorescent protein (gfp) reporter gene were constructed by cloning the reporter genes into pqcxip plasmid (clontech), respectively. dna fragments encoding c-terminally s-tagged ace2 orthologs were synthesized in puc57 backbone plasmid by sangon biotech (shanghai, china). these fragments were then cloned into pqcxip plasmid (clontech) between sbfi and noti restriction sites. igg fc fusion protein production and purification. 293f cells at the density of 6 ϫ 10 5 cells/ml were seeded into 100 ml of smm 293-tii serum-free medium (sino biological, catalog no. m293tii) 1 day before transfection. the cells were then transfected with 100 g of plasmid in complex with 250 g of pei max 4000 (polysciences, inc., catalog no. 24765-1). cell culture supernatants were collected at 48 to 72 h posttransfection. recombinant fc fusion proteins are purified using protein a-sepharose cl-4b (ge healthcare, catalog no. 17-0780-01), eluted with 0.1 m citric acid at ph 4.5, and neutralized with 1 m tris-hcl at ph 9.0. buffers were then exchanged to phosphate-buffered saline (pbs), and proteins were concentrated by 30-kda cutoff amicon ultra-15 centrifugal filter units (millipore, catalog no. ufc903096). flow cytometry for detecting interactions of rbd-ig proteins with cell surface ace2 orthologs. 293t cells were seeded at 20% density in 48-well plates at 12 to 15 h before transfection. cells in each well were then transfected with 0.5 l of lipofectamine 2000 (life technologies, catalog no. 11668019) in complex with 200 ng of plasmid encoding one of the 16 ace2 orthologs or a d30e mutant of the human ace2. culture medium was changed at 6 h after transfection. cells were then detached with 5 mm edta (life technologies, catalog no. 15575020) at 36 h posttransfection. the cells were then stained with 5 g/ml rbd-ig proteins at 37°c for 10 min, washed three times, and then stained with 2 g/ml alexa488-conjugated goat anti-mouse igg secondary antibody (invitrogen, catalog no. a-11001) at room temperature for 20 min. after another three washes, cells were analyzed by attune nxt flow cytometer (thermo fisher), and signals of 10,000 fsc/ssc-gated cells were collected for each sample. western blot to detect s-tagged ace2 (ace2-s-tag) or c9-tagged spike (spike-c9-tag) expression in 293t cells. 293t cells were seeded at 20% density in 6-well plates at 12 to 15 h before transfection. cells in each well were then transfected with 2 g of plasmid in complex with 5 l of lipofectamine 2000 (life technologies, catalog no. 11668019). at 36 h after transfection, the cells were lysed, and 10 g of total protein was used for western blotting. ace2-s-tag expression was detected by using 6.2, a mouse anti-s-tag monoclonal antibody (invitrogen, catalog no. ma1-981), and a horseradish peroxidase (hrp)-conjugated goat anti-mouse igg fc secondary antibody (invitrogen, catalog no. 31437). beta-actin was used as an internal control. spike-c9-tag expression was then detected by using 1d4, a mouse anti-c9-tag monoclonal antibody (invitrogen, catalog no. ma1-722), and the hrp-conjugated goat anti-mouse igg fc secondary antibody (invitrogen, catalog no. 31437). mlv retroviral vector-based coronavirus-spike and vsv-g pseudotypes were produced using a previously described protocol (23) with some modifications. 293t cells were seeded at 30% density in 150 mm dish at 12 to 15 h before transfection. cells were then transfected with 67.5 g of pei max 4000 (polysciences, inc., catalog no. 24765-1) in complex with 11.25 g of plasmid encoding a coronavirus spike protein or vsv-g, 11.25 g of plasmid encoding murine leukemia virus) gag and pol proteins, and 11.25 g of a pqcxip-based gfp or luciferase reporter plasmid. eight hours after transfection, cell culture medium was refreshed and changed to growth medium containing 2% fetal bovine serum (fbs; gibco, catalog no. 10099141c) and 25 mm hepes (gibco, catalog no. 15630080). cell culture supernatants were collected at 36 to 48 h posttransfection, spun down at 3,000 ϫ g for 10 min, and filtered through 0.45-m filter units to remove cell debris. coronavirus spike-pseudotyped viruses were then concentrated 10 times at 2,000 ϫ g using 100-kda cutoff amicon ultra-15 centrifugal filter units (millipore, catalog no. ufc910024). pseudovirus infection of 293t cells expressing ace2 orthologs. 293t cells were seeded at 20% density in polylysine precoated 48-well plates 12 to 15 h before transfection. cells in each well were then transfected with 0. coronavirus pseudovirus neutralization assay. coronavirus spike protein-pseudotyped luciferase reporter viruses were prediluted in dmem (2% fbs, heat inactivated) containing titrated amounts of rbd-ig or ace2-ig variant proteins. an fc fusion protein of an anti-influenza hemagglutinin (ha) antibody, f10-scfv (25) , was used as a control protein here. virus-inhibitor mixtures were then added to ace2-expressing 293t or hela cells in polylysine (sigma, catalog no. p4832-50ml) precoated 96-well plates and incubated overnight at 37°c. the cells were then washed with serum-free medium and incubated in 150 l of dmem (2% fbs) at 37°c. cell culture supernatants were collected for gaussia luciferase assay at 48 h postinfection. sars-cov-2 live virus infection of ace2 ortholog-expressing 293t cells. 293t cells expressing one of the 16 ace2 orthologs were inoculated with sars-cov-2 live virus at 800 50% tissue culture infective dose(s) (tcid 50 ) and incubated for 1 h at 37°c. the cells were then washed with serum-free medium and incubated in 150 l of dmem (2% fbs) at 37°c for an additional 24 h. the cells were then fixed with 4% paraformaldehyde in pbs, permeabilized with 0.5% triton x-100, and sequentially stained with 1:200-diluted rabbit anti-sars-cov-2 nucleocapsid polyclonal antibody (sino biological, catalog no. 40588-t62) at 37°c for 30 min, 4 g/ml of alexa fluor 568 goat anti-rabbit igg (invitrogen, catalog no. a-11011) at 37°c for 20 min, and 0.5 g/ml of dapi (4=,6=-diamidino-2-phenylindole; sigma-aldrich, catalog no. d9542-5mg) at room temperature for 10 min. stained cells were then examined under fluorescence microscope (ix73 microscope; olympus). sars-cov-2 live virus neutralization by ace2-ig variants. sars-cov-2 live virus at 800 tcid 50 were prediluted in dmem (2% fbs, heat inactivated) containing titrated amounts of ace2-ig variant proteins and incubated at 37°c for 1 h. virus-inhibitor mixtures were then added to ace2-expressing 293t or hela cells in polylysine-precoated 48-well plates and incubated for 1 h at 37°c. the cells were then washed with serum-free medium and incubated in 400 l of dmem (2% fbs) at 37°c for 24 to 40 h. cells were then fixed for immunofluorescence staining of viral nucleocapsid proteins as described above. data collection and analysis. all the experiments were repeated two to four times. all of the infection assays for fig. 3 were independently performed by two different people, and all the data are reproducible in different hands. the key neutralization assays of fig. 4 to 6 were independently performed by two or three different people, and all of the data are reproducible in different hands. attune nxt software (thermo fisher) was used to collect and analyze flow cytometry data. image lab software (bio-rad) was used to collect sds-page and western blot image data. cell sens software (olympus) was used to collect fluorescence microscopy data. ice software (berthold technologies) was used to collect luciferase assay data. graphpad prism 6.0 software was used for figure preparation and statistical analyses. statistical analysis. data expressed as mean values ϯ the standard deviations (sd). statistical analyses were performed using two-sided two-sample student t test using graphpad prism 6.0 software when applicable. differences were considered significant at p ͻ 0.01. data availability. the study did not generate unique data sets or code. our research resources, including methods, plasmids, and protocols, are available upon reasonable request to qualified academic the complete genome sequence of severe acute respiratory syndrome coronavirus strain hku-39849 (hk-39) genomic characterization of the severe acute respiratory syndrome coronavirus of amoy gardens outbreak in hong kong epidemiology and cause of severe acute respiratory syndrome people's republic of china fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin isolation and characterization of viruses related to the sars coronavirus from animals in southern 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long-lasting hypertension control and organ protection in mouse models of systemic renin angiotensin system activation key: cord-102920-z5q3wo7v authors: sang, eric r.; tian, yun; gong, yuanying; miller, laura c.; sang, yongming title: integrate structural analysis, isoform diversity, and interferon-inductive propensity of ace2 to refine sars-cov2 susceptibility prediction in vertebrates date: 2020-06-28 journal: biorxiv doi: 10.1101/2020.06.27.174961 sha: doc_id: 102920 cord_uid: z5q3wo7v the current new coronavirus disease (covid-19) has caused globally near 0.4/6 million confirmed deaths/infected cases across more than 200 countries. as the etiological coronavirus (a.k.a. sars-cov2) may putatively have a bat origin, our understanding about its intermediate reservoir between bats and humans, especially its tropism in wild and domestic animals, are mostly unknown. this constitutes major concerns in public health for the current pandemics and potential zoonosis. previous reports using structural analysis of the viral spike protein (s) binding its cell receptor of angiotensin-converting enzyme 2 (ace2), indicate a broad sars-cov2 susceptibility in wild and particularly domestic animals. through integration of key immunogenetic factors, including the existence of s-binding-void ace2 isoforms and the disparity of ace2 expression upon early innate immune response, we further refine the sars-cov2 susceptibility prediction to fit recent experimental validation. in addition to showing a broad susceptibility potential across mammalian species based on structural analysis, our results also reveal that domestic animals including dogs, pigs, cattle and goats may evolve ace2-related immunogenetic diversity to restrict sars-cov2 infections. thus, we propose that domestic animals may be unlikely to play a role as amplifying hosts unless the virus has further species-specific adaptation. these findings may relieve relevant public concerns regarding covid-19-like risk in domestic animals, highlight virus-host coevolution, and evoke disease intervention through targeting ace2 molecular diversity and interferon optimization. erupting in china last december, the novel coronavirus disease (covid-19) has become a worldwide pandemic and caused near 0.4 million confirmed deaths and 6 million infected cases across 200 countries by the end of may 2020 [1, 2] . the etiological virus, designated as severe acute respiratory syndrome coronavirus 2 (sars-cov2) has been identified [3] and related to the viruses previously causing sars or middle east respiratory syndrome (mers) in humans in 2003 and 2012, respectively [4] . these three human-pathogenic coronaviruses putatively evolve from bat coronaviruses, but have different animal tropisms and intermediate reservoirs before transmission to humans [4, 5] . as civet cats and camels were retrospectively determined as reservoirs for sars and mers respectively, there is no conclusion about what animal species passing sars-cov2 to humans [4, 5] . investigations indicated that canivora animals including raccoon dogs, red foxes, badgers and minks as well swine, at a less extent, are susceptible to sars virus infections [6, 7] . although the viral nucleic acids and antibodies to mers were detectable in multiple ruminant species including sheep, goat, and donkeys, the virus inoculation studies did not result in a productive infection for mers disease in these domestic ruminants, nor in horses [8, 9] . as a group of obligate pathogens, viruses need to engage cell receptors for entering cells and race with the host immunity for effective replication and spreading to initiate a productive infection [10] . in this context, the spike proteins protruding on the coronavirus surface are responsible for cell receptor binding and mediating viral entry [5] [6] [7] . for example, mers-cov adopts the dipeptidyl peptidase 4 (dpp4, a.k.a. cd26) and sars-cov uses angiotensin-converting enzyme 2 (ace2) as primary receptors for cell attachment and entry [4] [5] [6] [7] [8] [9] . several groups have reported that sars-cov2 uses the same ace2 receptor as sars-cov, but exerts higher receptor affinity to human ace2, which may ascribe to the efficacy of sars-cov2 infection in humans [11, 12] . after cell attachment via the receptor binding domain (rbd) in the n-terminal s1 region of the s protein, the c-terminal s2 region thus engages in membrane fusion. further cleavage of s2 from s1 by a furinlike protease will release and prime the virus entering the recipient cells. several furin-like proteases, especially a broadly expressed trans-membrane serine protease 2 (tmprss2), are adopted for priming sars-cov entry [11, 12] . compared with sars-cov, studies showed that sars-cov2 spike protein also evolutionarily obtains an additional furin-like proteinase cleavage site within the s1/s2 junction region for efficient release from the cell surface and entry into the cells [3, [11] [12] [13] . because tmprss2 is widely expressed, the tissue-specific expression of ace2 has been shown to determine sars-cov2 cell tropism in humans [11, 12] . namely, human nasal secretory cells, type ii pneumocytes, and absorptive enterocytes are ace2-tmprss2 double positive and highly permissive to sars-cov2 infection [14, 15] . for cross-species animal tropism, the potential infectivity of sars-cov2 in both wild and domestic animals raises a big public health concern after the prevalence of sars-cov2 infections in humans [16, 17] . this concern involves two aspects: (1) screening to identify the animal species that serve as a virus reservoir originally passing sars-cov2 to humans; and (2) the existing risk of infected people passing the virus to animals, particularly domestic species, thus potentially amplifying the zoonotic cycle to worsen sars-cov2 evolution and prevalence [16, 17] . by diagnosis of animals in close contact with covid-19 patients or screening of animal samples in some covid-19 epidemic zones, studies detected that domestic cats and dogs could be virally or serologically positive for sars-cov2 [18] [19] [20] [21] [22] [23] [24] , as was a reported infection in a zoo tiger [25] . using controlled experimental infection of human sars-cov2 isolates, several studies demonstrated that ferrets, hamsters, domestic cats and some non-human primate species are susceptible to human sars-cov2 strains [18] [19] [20] [21] [22] [23] [24] [25] . obviously, it is impractical to test sars-cov2 susceptibility experimentally in all animal species. by adoption of a structural simulation based on published structures of the viral s-rbd/ace2 complex, studies have predicted a broad spectrum of vertebrate species with high potential for sars-cov2 susceptibility, which, if true, entails unexpected risks in both public and animal health, and warrants further critical evaluation [26] [27] [28] . ace2 is a key enzyme catalyzing angiotensin (agt) further conversion into numeral active forms of agt1-9, which are hormonal mediators in the body's renin-angiotensin system (ras) [29, 30] . thus, ace2 plays a regulatory role in the blood volume/pressure, body fluid balance, sodium and water retention, as well as immune effects on apoptosis, inflammation, and generation of reactive oxygen species (ros) [29, 30] . in this line, the expression of ace2 is also inter-regulated by immune mediators pertinent to its systemic function. multiple physio-pathological factors, including pathogenic inflammation, influence on ras through action on ace2 expression [29] [30] [31] . interferon (ifn) response, especially that mediated by type i and type iii ifns, comprises a frontline of antiviral immunity to restrict viral spreading from the initial infection sites, and therefore primarily determines if a viral exposure becomes controlled or a productive infection [32] . several recent studies revealed that human ace2 gene behaves like an interferon-stimulated gene (isg) and is stimulated by a viral infection and ifn treatment; however, mouse ace2 gene is not [15, 33, 34] . therefore, to determine the cell tropism and animal susceptibility to sars-cov2, the cross-species ace2 genetic and especially epigenetic diversity in regulation of ace2 expression and functionality should be evaluated [26] [27] [28] [29] [30] [31] [32] [33] [34] . in this study, through integration of structural analysis and key immunogenetic factors that show species-dependent differences, we critically refine the sars-cov2 susceptibility prediction to fit recent experimental validation [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] . along with showing a broad susceptibility potential across mammalian species based on structural analysis [26] [27] [28] , our results further reveal that domestic animals including dogs, pigs, cattle and goats may evolve previously unexamined immunogenetic diversity to restrict sars-cov2 infections. protein and promoter sequence extraction and alignment: the amino acid sequences of ace2 proteins and dna sequences of the proximal promoters of each ace2 genes were extracted from ncbi gene and relevant databases (https://www.ncbi.nlm.nih.gov/gene). ace2 genes and corresponding transcripts have been well annotated in most representative vertebrate species. in most cases, the annotations were double verified through the same gene entries at ensembl (https://www.ensembl.org). the protein sequences were collected from all non-redundant transcript variants and further verified for expression using relevant rna-seq data (ncbi geo profiles). the proximal promoter region spans ~2.5 kb before the predicted transcription (or translation) start site (tss) of ace2 or other genes. the protein and dna sequences were aligned using the multiple sequence alignment tools of clustalw or muscle through an embl-ebi port (https://www.ebi.ac.uk/). other sequence management was conducted using programs at the sequence manipulation suite (http://www.bioinformatics.org). sequence alignments were visualized using jalview (http://www.jalview.org) and megax (https://www.megasoftware.net). sequence similarity calculations and plotting were done using sdt1.2 (http://web.cbio.uct.ac.za/~brejnev). other than indicated, all programs were run with default parameters. phylogenic analysis: the phylogenic analysis and tree visualization were performed using megax and an online program, evoview. the evolutionary history was inferred using the neighbor-joining method. percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) was also performed. the evolutionary distances were computed using the p-distance method and in units of the number of amino acid differences per site. other than indicated, all programs were run with default parameters as the programs suggested. structural simulation and analysis: the structure files of human ace2 protein and its interaction with sars-cov2 s-rbd were extracted from the protein data bank under the files of 6m17 and 6m0j. the residual mutation and structure simulation were performed using ucsf chimera and pymol available at https://www.cgl.ucsf.edu/chimera/ and https://pymol.org/, respectively. structural visualization were using pymol. the binding affinity energy (δg), dissociation constant (kd) and interfacial contacts between s-rbd and each ace2 were calculated using an prodigy algorithm at https://bianca.science.uu.nl/prodigy/. profiling transcription factor binding sites in ace2 promoters and pwm scoring: the regulatory elements (and pertinent binding factors) in the ~2.5 kb proximal promoter regions was examined against both human/animal tfd database using a program nsite (version 5.2013, at http://www.softberry.com). the mean position weight matrix (pwm) of key cis-elements in the proximal promoters were calculated using pwm tools through https://ccg.epfl.ch/cgibin/pwmtools, and the binding motif matrices of examined tfs were extracted from jaspar core 2018 vertebrates (http://jaspar.genereg.net/). for expression confirmation, several sets of rna-seq data from ncbi gene databases, and one of ours generated from porcine alveolar macrophages (bioproject with an accession number of srp033717), were analyzed for verification of the differential expression of ace2 genes in most annotated animal species. especially, the expression of porcine ace2 isoforms and relevant other genes in the porcine lung macrophage datasets. significantly differentially expressed genes (degs) between two treatments were called using an edger package and visualized using heatmaps or bar charts as previously described [58]. 3.1. vertebrate ace2 orthologs share an functional constraint but experience intra-species diversification in livestock with unknown selective pressure sequence comparison among ace2 orthologs across 30 representative vertebrate species shows a pairwise identity range at 57-85% ( fig. 1a and supplemental fig. s1 and excel sheet), which is 15-27% higher than the average value generated through a similarity analysis at 30-70% on gene orthologs at a genome-wide scale [35] . this indicates that ace2 exerts a similar and basic function cross-species, consistent with its systemic and regulatory role as a key enzyme in ras, an essential regulatory axis underlying the body circulatory and execratory systems in vertebrates [29] [30] [31] . a comparison of evolutionary rates of major genes within ras including angiotensinogen (agt), ace, and several receptors of the processed angiotensin hormones showed that ace2 actually evolves slightly faster than ace [36, and unpublished data] . this implies that ace2 may bear pressure for ras adapting evolution per a species-dependent physiological and pathological requirement [29] [30] [31] . this evolutionary adaptability of ace2 genes is demonstrated by the existence of numerical genetic polymorphisms [37] and several transcript isoforms particularly in humans and major livestock species ( fig. 1b and supplemental fig. s1 and excel sheet). we identified (and verified by rna-seq annotation) four transcripts of ace2 isoforms in humans (fig. 1b ) that primarily differ in the c-terminal 50 residues within the collectrin domain. particularly, 1-2 short ace2 isoforms were identified in dogs, pigs, cattle, and goats in addition to the longer ace2 consensus to the human's (designated as -s or -l, respectively after the animal common names in fig. 1b and thereafter). these livestock ace2-s isoforms have a 70-130 residual truncation at their n-terminal peptidase domains, which also span the region interacting with sars-cov spike protein. the selective mechanisms driving the evolution of these short ace2 isoforms in livestock are unknown, but may relate to previous pathogenic exposure or unprecedented physiopathological pressure. to support this reasoning, short ace2 isoforms are detected in both domestic bos taurus and hybrid cattle, but not in the wild buffalo and bison; and ace2 isoforms from each species are generally paralogous and sister each other within a clade in the phylogenic tree (fig. 1b ). phylogenic analysis of vertebrate ace2 orthologs/paralogs reveals a general relationship aligning to the animal cladistics (fig. 1b) . in this context, homologs from the fish, frog and chicken conform to a primitive clade. all ungulate homologs form into parallel clades next to each other. the homologs from the glires, primates and carnivores cluster into a big clade (marked with yellow triangle node), which contains all the sars-cov2 susceptible species that have been verified via natural exposure or experimental infections (fig. 1b , marked with red/orange circles). we examined and merged several previous studies about the prediction of sars-cov2 susceptibility in vertebrates based on the simulated structural analysis of s-rbd-ace2 complex [26] [27] [28] . as numerous vertebrate species were predicted to be high or low potential (fig. 1b , labeled as red h or green l) for sars-cov2 susceptibility, incongruence between the predicted sars-cov2 susceptibility and infected validation is apparent in pangolin, ferret, tiger, cat and horseshoe bat, indicating that some other factors besides ace2-rbd affinity should be considered [15, [32] [33] [34] . we, therefore, refined the prediction matrix to include the rbd-binding evasion of some ace2 orthologs identified in major livestock species and the interferon-stimulated ace2 expression underlying sars-cov2 infections [15, [32] [33] [34] . several recent studies have elegantly demonstrated the structural interaction of the viral s protein or its rbd in complex with human ace2 receptor [38, 39] . showing that the contacting residues at the rbd/ace2 interface ( fig. 2a) involve at least 19 residues in ace2 (fig. 2b , listed in the table cells and referred to the aligned residual positions in human ace2) and 10 residues in the sars-cov2 rbd (fig. 2b , blue circles with residue labels above the table) [28, 38, 39] . the cross-species residual identity (%) of these interacting residues in ace2 are dispersed in a broader range (32-100%) than the whole ace2 sequence identity rate at 57-85% [35] , indicating a faster evolution rate of this virus-interacting region. notably, the s-binding region spans a large part of the n-terminal peptidase domain and s-binding may competitively block a majority of active sites of the enzyme (fig. 2c ). using a similar structural analysis procedure [27, 28] , we modeled the ace2 structures of animal species of interest and simulated their interaction with sars-cov2 s-rbd based on a published rbd-human ace2 structure (protein data bank file 6m0j) [38] . fig. 3 demonstrates the s-rbd interaction with the simulated structures of ace2 long isoforms from the dog, pig and cattle, respectively. the major changes of the rbd-ace2 interacting interfaces are from the residual exchanges in ace2 from other species compared with human ace2 (fig. 2b -2d, highlighted in red). in addition, the exchange of n90t (in pigs) and n322y (in cattle and sheep) would destroy the n-glycosylation site in human ace2. ace2 from goat (supplement fig. s1 ) exhibits identical amino acid exchanges as in cattle in the rbd-ace2 interfacial contacts. in contrast,when compared with human ace2, ace2 from cats (supplement fig. s1 ) conserves all relevant glycosylation sites in human ace2 [28, 38] . we also calculated the interfacial contacts using parameters of protein-protein interaction including the predictable binding affinity energy (δg), dissociation constant (kd) and number of different interfacial contacts within the s-rbd and ace2 contact. although the exact numbers may differ from previous reports [38] , they provide a very comparable matrix generated using the same algorithm ( fig. 3e ) [40] . data show that the ace2 of most domestic animals, including that from mouse and rat (species known to be unsusceptible to human sars-cov2), have a binding affinity (δg) at -11.2 to -12.8 kcal/mol. this is within the binding affinity range (11.2-12.9 kcal/mol) between the rbd and the ace2 from known susceptible species (fig. 3e , underlined in the left part of the table). this indicates that other factors, conceivably from genetic divergence and/or natural immunity, also contribute to sars-cov2 susceptibility in animal species. therefore, an effective prediction matrix should include the critical immunogenetic factors to further determine virus susceptibility in addition to the sequence/structural similarity of ace2 receptors ( fig. 1 and fig. s1 ) [15, 34, 37] . we detected several short ace2 isoforms in the domestic animals including dog, pig, goat and cattle that have an n-terminal truncation spanning 10-13 key residues in the contacting network to s-rbd but retain the enzyme active sites (fig. 4a ). most of the splicing isoforms of ace2 genes, such as in zebrafish, cats and humans, share a common proximal promoter and encode ace2 proteins containing all 19 key rbd-interacting residues [38, 39] . however, these short ace2-s isoforms in domestic animals truncate for 71 (cattle/goat ace2-s) or 132 (dog/pig ace2-s) residues at their n-termini compared with the long ace2 isoforms in the same species ( fig. 1 and fig. s1 ). therefore, these short ace2 isoforms destroy 10-13 key residues in the contacting network to s-rbd but likely retain ace2 enzymatic function in ras. paired structural comparison between the human ace2 structure (extracted from 6m17) with each simulated ace2-s structure from the pig, dog, and cattle/goat, reveals that all these ace2-s orthologs from domestic animals, particularly the porcine one, show high structural similarity to the human ace2 except for the nterminal truncations ( fig. 4b-4d ). this indicates that these short ace2 isoforms in domestic animals have little chance to be engaged by the viral s-binding, and predict an unexpected evolutionary advantage to allay potential covid-19 risk resulting from viral engagement and functional distortion on the classical long ace2 isoforms in these animal species [37, 41] . sars-cov2 infection induces a weak ifn response but a production of a high amount of inflammatory cytokines including interleukin (il)-6 and chemokine cxcl10 in most severe covid-19 patients [42] [43] [44] [45] . studies of sars and mers showed that these pathogenic coronaviruses share similar viral antagonisms, including the endoribonuclease (endou) encoded by nonstructural protein 15 (nsp15), which directly blunts cell receptors responding to viral dsrna and in turn weaken the acute antiviral response [46] . several recent studies revealed that sars-cov2 seems more cunning in not only evading or antagonizing but also in exploiting the ifn response for efficient cell attachment [15, 42, 43, 46] . as a key enzyme in ras, the expression of ace2 gene has been primarily investigated for physiological response to circulatory regulations, and a response to pathological inflammation is also expected [29] [30] [31] . however, the expression of the ace2 gene was highly responsive to both viral infection and host ifn response, i.e. human ace2 gene seems an unstudied ifn-stimulated gene (isg) [15, 33] . surprisingly, the isg propensity of ace2 genes is species-dependent, for example: the mouse ace2 gene is less ifn responsive which may partly explain the mouse insusceptibility to sars-cov2 infection [15] . to categorize the different ifn-inductive propensity of ace2 genes in vertebrates, particularly in major livestock species, we profiled the regulatory cis-elements and relevant transcription factors in the proximal promoter regions of each ace2 genes (2.5 kb before tss or atg). figure 5 illustrates major regulatory cis-elements located in ace2 genes from major livestock animals and several reference animal species. data show that animal ace2 gene promoters are evolutionally different in containing ifn-or virus-stimulated response elements (isre, prdi, ifrs, and/or stat1/3 factors) and cis-elements responsive to pro-inflammatory mediators. all these cis-elements recruit corresponding transcription factors (tf) to mediate differential ace2 responses to antiviral ifns and inflammation that is associated with covid-19 disease [2, 3, 47] . we discover that ace2 genes obtain species-different isg propensity responsive to ifn and inflammatory stimuli. in most (if not all) of the sars-cov2 susceptible species the ace2 genes obtained the ifn response between the typical robust and tunable ifn-stimulated genes (isg) [48] . in general, the robust isgs (isg15 is an example here) are stimulated in the acute phase of viral infection and play a more antiviral role; in contrast, the later responsive tunable isgs (irf1 is an example) contribute more to anti-proliferation of ifn activity [48] . in addition, unlike the promoter of the short ace2 isoforms in cattle and goats, which share most common promoter regions with their paralogous long isoforms, the short ace2 isoforms of dogs (dog-s) and pigs (pig-s) have distinct proximal promoter regions (and different ifn responsivity) to the paralogous long ace2 isoforms ( fig. 5 and fig 6) . results indicate that the short ace2 isoforms in pigs and dogs diversify from their long paralogs at both the levels of genetic coding and epigenetic regulation to adapt to some evolutionary pressure, such as that from pathogenic interaction (fig. 7) [37,49]. the position weight matrix (pwm) stands as a position-specific scoring model for the binding specificity of a transcription factor (tf) on the dna sequences [50]. using pwm toolsets online (https://ccg.epfl.ch/cgi-bin/pwmtools), we evaluate mean pwm of key cis-elements in the proximal promoters of ace2 genes that containing binding sites for canonical ifn-dependent transcription factors, which include isre/stat, irf1. irf3/7 and irf8, as well as c/ebp representing a core transcription factor for pro-inflammation. these ifn-dependent transcription factors, particularly irf3/7 and isre/stat for ifn stimulation, are differentially enriched in the promoter regions of ace2 genes in a species-dependent way. higher enrichment of isre/stat1/3 and/or irf3/7 binding sites are detected in most sars-cov2/covid19 susceptible species (indicated with solid orange or red circles, respectively). in contrast, the pwm for irf1 and c/ebp, which regulate inflammation, are less differentiated in ace2 promoters from animal species, indicating that ace2 genes are more universally regulated by inflammation than that by the viral infection or ifninduction in a species-dependent way (fig. 6 ). as compared with the promoters of a typical human robust isg15 and tunable irf1 genes, this data indicate that ace2 genes (particularly the primate ones) are not typical robust or tunable isgs as represented by isg15 or irf1, but respond differently to viral infection (through irf3/7) or ifn auto-induction (via isre/stat) in a speciesdependent manner ( fig. 6) [48]. higher enrichment of isre/stat1/3 and/or irf3/7 corresponds to sars-cov2 susceptibility in experimentally validated mammalian species especially primates, but not to the phylogenically distant species such as zebrafish, which has very low potential for sars-cov2 susceptibility due to the high disparity of ace2 structures ( fig. 1 and fig. s1 ). in addition, the proximal promoters of the pig and dog ace2-s genes differ much in their ifn-responsive elements to most ace2 promoters in mammalians ( fig. 5 and fig. 6 ). however, they are phylogenically sister to the ace2 promoters from the primitive vertebrates (frog, chicken and zebrafish) (fig. 7, phylogenic tree) . this indicates that the expression of these short ace2 isoforms is more conservative than the long ace2 isoforms, which represent a more recent evolution obtaining ace2 epigenetic regulation by ifn-signaling (fig. 7) [49]. studies show that affinity adaption of the viral s-rbd and ace2 receptor determines the cellular permissiveness to the virus [28, 38, 39] . sars-cov2 not only adapts a high binding affinity to human ace2 for cell attachment, but also antagonizes host antiviral interferon (ifn) response and utilizes ifn-stimulated property of human ace2 gene to boost spreading [15, 38, 39, 49] . in addition to structural analysis of simulated s-rbd-ace2 interaction, we propose that several immunogenetic factors, including the evolution of s-binding-void ace2 isoforms in some domestic animals, the species-specific ifn system, and epigenetic regulation of ifn-stimulated property of host ace2 genes, contribute to the viral susceptibility and the development of covid-19-like symptoms in certain animal species [15, 38, 39, 49] . a computational program in development that incorporates this multifactorial prediction matrix and in vitro validation of sars-cov2 susceptibility in major vertebrate species will be necessary to address public concerns relevant to sars-cov2 infections in animals (fig. 7) . it will also lead to the development of better animal models for anti-covid19 investigations [21] . in addition, several ifn-based therapies for treatment of covid19 have been proposed and are in the process of clinic trails [51-54]. considering the viral stealth of ifnstimulated property of human ace2, a timely and subtype-optimized ifn treatment should be delivered rather than a general injection of typical human ifn-α/β subtypes [51-54]. in this line, domestic livestock like pigs and cattle have a most evolved ifn system containing numerous unconventional ifn subtypes. some of these unconventional ifn subtypes, such as some porcine ifn-ω exert much higher antiviral activity than ifn-α even in human cells and most ifn-λ retain antiviral activity with less pro-inflammatory activity, could be utilized for developing effective antiviral therapies [55, 56] . in summary, a predication matrix, which integrates the structural analysis of s-rbd-ace2 interfacial interface and the species-specific immunogenetic diversity of ace2 genes, was used to predict the sars-cov2 susceptibility and fit current knowledge about the infectious potential already validated in different animal species (fig. 7) . more extensive validation experiments are needed to further improve this prediction matrix. our current results demonstrate several previously unstudied immunogenetic properties of animal ace2 genes and imply some domestic animals, including dogs, pigs and cattle/goats, may obtain some immunogenetic diversity to confront sars-cov2 infection and face less covid-19 risk than may have been previously thought. however, immediate biosecurity practices should be applied in animal management to reduce animal exposure to the virus and prevent potential species-specific adaptation (fig. 7) . for livestock breeding programs that targeting disease resistance to respiratory viruses, the genetic and epigenetic diversity of ace2 genes as well antiviral isgs are highly recommended [48,49,55,56]. in conclusion, sars-cov2 evolves to fit well with human (and non-human primates) ace2 receptor through the structural interfacial affinity, immunogenetic diversity and epigenetic expression regulation, which results in a highly infectious efficacy [1] [2] [3] 15, 28, 38, 39] . most mammals, especially those that belong to glires, primates and carnivores, have a higher potential for sars-cov2 susceptibility but in a species-different manner based on the s-binding-void ace2 isoforms and the difference of the ifn-inductive propensity of the major ace2 genes. most ungulate animals appear have a low susceptibility potential with horses and sheep having a high potential (fig. 7) . default parameter setting. the prediction of sars-cov2 susceptibility is based on the sequence similarity of each ace2 to human ace2 in the s-rbd binding region and simulated using a published human ace2-rbd structure (6m0j) and refers to two recent publications using similar procedures but different structural models [27, 28] . compared with the currently available experimental data, incongruence of the predicted sars-cov2 susceptibility is clearly demonstrated in pangolin, ferret, tiger, cat and horseshoe bat, indicating that some other factors besides ace2-rbd affinity should be considered. interfacial contacts of the sars-cov2 s-rbd with ace2 orthologs of major livestock species. most domestic animals ace2 including that from mouse and rat (species known not to be susceptible to sars-cov2) have a binding affinity (δg) at -11.2 to -12.8 kcal/mol that is within the range (11.2-12.9 kcal/mol) between the rbd and the ace2 from the known susceptible species (underlined in the left part of the table), indicating that some other factors, especially those from genetic divergence and natural immunity, contribute to the sars-cov2 susceptibility of different animal species. the phylogenic tree of identified ace2 orthologs/variants from different species was built with a neighbor-joining approach and visualized using an evoview program under default parameter setting. the prediction of sars-cov2 susceptibility is based on the sequence similarity of each ace2 to human ace2 in the s-rbd binding region and simulated using a published human ace2-rbd structure (6m0j) and refers to two recent publications using similar procedures but different structural models [27, 28] . compared with the currently available experimental data, incongruence of the predicted sars-cov2 susceptibility is clearly demonstrated in pangolin, ferret, tiger, cat and horseshoe bat, indicating that some other factors besides ace2-rbd affinity should be considered. we emphasize to integrate other factors, including the rbd-binding evasion of some short ace2 orthologs identified in some major livestock species and the recently identified ace2-interferon association [15] , to refine the sars-cov2 susceptibility prediction. y489 y505 predicted permissiveness by sequence similarity and ace2-rbd binding energy figure 2 : prediction of sars-cov2 susceptibility in major livestock species based on the conservation of key interacting residues and binding capacity between the viral spike (s) protein on the host ace2 receptor. (a) sars-cov-2 uses the cell receptor, angiotensin-converting enzyme 2 (ace2) for entry and the serine protease tmprss2 and furin for s protein priming. (b) as tmprss2 is broadly expressed and active with a furin-like cleavage activity, the affinity adaption of the s receptor binding domain (rbd) and ace2 receptor determines the viral permissiveness. the contacting residues of human ace2 (a distance cutoff 4.5 å) at the sars-cov-2 rbd/ace2 interfaces are shown, and the contacting network involves at least 19 residues in ace2 (listed in the table cells and referring to the aligned residual positions in human ace2) and 10 residues in the sars-cov-2 rbd (blue circles with residue labels), which are listed and connected with black lines (indicating hydrogen bonds) and red line (represents salt-bridge interaction). the cross-species residual identity (%) of these interacting residues in ace2 are listed in a broad range (32-100%) [26] [27] [28] . (c) we also detected several short ace2 isoforms (underlined) in the domestic animals including dog, pig, goat and cattle, which have a n-terminal truncation spanning 10-13 key residues in the contacting network to s-rbd but keeping the enzyme active sites (indicated by yellow triangles), thus resulting in little engagement by the viral s protein and predicting an unexpected evolutionary advantage for relieving potential covid-19 risk caused by the viral engagement and functional distortion on the classical long ace2 isoforms in these animal species. the ncbi accession numbers of the ace2 orthologs are listed as in fig. 1 s-rbd with ace2 orthologs of major livestock species simulated using the human ace2/cov2-rbd structure (6m0j). most residues involved in binding are highlighted as magenta (ace2) or orange (s) sticks and labeled as one-letter amino-acid codes plus residual numbers in bold or regular font respectively for s or ace2 residues. the dotted/blue lines indicate intermolecular salt bridge or hydrogen bonds between interacting residues (generated and visualized with ucsf chimera and pymol from protein data bank file 6m0j). (b) to (d) rbd interaction with the simulated structures of ace2 long isoforms from the dog, pig and cattle, respectively. amino acid exchanges in ace2 from another species compared with human ace2 are highlighted in red. e) prediction of binding affinity energy (δg), dissociation constant (kd) and interfacial contacts of the sars-cov2 s-rbd with ace2 orthologs of major livestock species. most domestic animals ace2 including that from mouse and rat (species known not to be susceptible to sars-cov2) have a binding affinity (δg) at -11.2 to -12.8 kcal/mol that is within the range (11.2-12.9 kcal/mol) between the rbd and the ace2 from the known susceptible species (underlined in the left part of the table), indicating that some other factors, especially those from genetic divergence and natural immunity, contribute to the sars-cov2 susceptibility of different animal species. in contrast to most splicing isoforms such as in cats and humans, which share a common proximal promoter and encode ace2 proteins with similar sequences containing all 19 key rbd-interacting residues, these short ace2-s isoforms in domestic animals truncate for 71 (cattle/goat ace2-s) or 132 (dog/pig ace2-s) residues at their n-termini compared with human ace2 or the long ace2 isoforms in these species, thus destroying 10-13 key residues in the contacting network to s-rbd but retaining all enzyme active sites (yellow triangles in the blue ace2 domain bar). this results in little chance to be engaged by the viral s protein binding and predicts an unexpected evolutionary advantage to relieve potential covid-19 risk caused by the viral engagement and functional distortion on the classical long ace2 isoforms in these animal species. (b), (c) and (d) paired structural comparison between the human ace2 structure (6m17) with each simulated ace2-s structure from pig (b), dog (c) and cattle/goat (d). human ace2 structure are in green, and each compared animal ace2-s structure in magenta. the n-terminal residues of both compared structures are in cyan (arrows indicating n-termini of the ace2-s isoforms) and shared c-termini are in red. putative proximal promoter region 10 9 8 7 6 5 4 3 2 1 0 (ace2-p) figure 5 . categorizing ace2 genes based on regulatory cis-elements predicted in their proximal promoter regions (<2 kb before tss or atg). the regulatory elements (and pertinent binding factors) in the ~2 kb proximal promoter regions were examined against both human/animal tfd database using a program nsite (version 5.2013, at http://www.softberry.com), including ace2 genes identified in major livestock animals and several reference animal species. data show that animal ace2 gene promoters are evolutionally different in containing ifn-or virus-stimulated response elements (isre, prdi, ifrs, and/or stat1/3 factors) and cis-elements responsive to proinflammatory mediators, which mediate different ace2 responses to antiviral interferons (ifns) and inflammation associated with covid-19 disease. legend: ○, gata-1 regulating constitutive expression; acute (◊) or secondary (◊) ifn-stimulated response element (isre) and prdi that interact with irf, isgf3 and stat factors, respectively; □, cis-elements interacting with factors to mediate immune/ inflammatory responses including c/ebp, nf-kb, nf-il6, and p53; •, cis-elements reacting with other factors significant in other developmental/physiological responses. the promoter features of two typical human interferon-stimulated genes (isg), the robust isg15 and tunable irf1 are shown as references to indicate that ace2 genes obtain species-different isg propensity responsive to ifn and inflammatory stimuli. covid-19 dashboard by the center for systems science and 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genes evolutionary constraints on structural similarity in orthologs and paralogs a genomic survey of angiotensin-converting enzymes provides novel insights into their molecular evolution in vertebrates ace2 receptor polymorphism: susceptibility to sars-cov-2, hypertension, multi-organ failure, and covid-19 disease outcome structural basis for the recognition of sars-cov-2 by full-length human ace2 structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor prodigy: a web server for predicting the binding affinity of protein-protein complexes imbalanced host response to sars-cov-2 drives development of covid-19 weak induction of interferon expression by sars-cov-2 supports clinical trials of interferon lambda to treat early covid-19 key: cord-261877-4y37676n authors: xu, cong; wang, yanxing; liu, caixuan; zhang, chao; han, wenyu; hong, xiaoyu; wang, yifan; hong, qin; wang, shutian; zhao, qiaoyu; wang, yalei; yang, yong; chen, kaijian; zheng, wei; kong, liangliang; wang, fangfang; zuo, qinyu; huang, zhong; cong, yao title: conformational dynamics of sars-cov-2 trimeric spike glycoprotein in complex with receptor ace2 revealed by cryo-em date: 2020-06-30 journal: biorxiv doi: 10.1101/2020.06.30.177097 sha: doc_id: 261877 cord_uid: 4y37676n the recent outbreaks of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and its rapid international spread pose a global health emergency. the trimeric spike (s) glycoprotein interacts with its receptor human ace2 to mediate viral entry into host-cells. here we present cryo-em structures of an uncharacterized tightly closed sars-cov-2 s-trimer and the ace2-bound-s-trimer at 2.7-å and 3.8-å-resolution, respectively. the tightly closed s-trimer with inactivated fusion peptide may represent the ground prefusion state. ace2 binding to the up receptor-binding domain (rbd) within s-trimer triggers continuous swing-motions of ace2-rbd, resulting in conformational dynamics of s1 subunits. noteworthy, sars-cov-2 s-trimer appears much more sensitive to ace2-receptor than sars-cov s-trimer in terms of receptor-triggered transformation from the closed prefusion state to the fusion-prone open state, potentially contributing to the superior infectivity of sars-cov-2. we defined the rbd t470-t478 loop and residue y505 as viral determinants for specific recognition of sars-cov-2 rbd by ace2, and provided structural basis of the spike d614g-mutation induced enhanced infectivity. our findings offer a thorough picture on the mechanism of ace2-induced conformational transitions of s-trimer from ground prefusion state towards postfusion state, thereby providing important information for development of vaccines and therapeutics aimed to block receptor binding. coronaviruses are a family of large, enveloped, positive-stranded rna viruses that cause upper respiratory, gastrointestinal and central nervous system diseases in humans and other animals (song et al., 2018; walls et al., 2019) . in the past few decades, new evolved coronaviruses have posed a global threat to public health, including the outbreaks of the severe acute respiratory syndrome coronavirus (sars-cov) in 2002 -2003 and the middle east respiratory syndrome coronavirus (mers-cov) in 2012 which had caused thousands of infection, and the mortality rate of them was about 10% and 34.4%, respectively (rabaan et al., 2020) . the recent coronavirus disease 2019 pandemic is caused by a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (sars-cov-2). on june 29, 2020, there had been 9,962,193 laboratory-confirmed sars-cov-2 infections globally, leading to 498,723 deaths. to date, there is no approved therapeutics or vaccines against sars-cov-2 and other human-infecting coronaviruses. as in other coronaviruses, the spike (s) glycoprotein of sars-cov-2 is a membranefusion machine that mediates receptor recognition and viral entry into cells and is the primary target of the humoral immune response during infection (rabaan et al., 2020; tang et al., 2020) . the s protein is a homotrimeric class i fusion protein that forms large protrusions from the virus surface and undergoes a substantial structural rearrangement to fuse the viral membrane with the host-cell membrane once binds to a host-cell receptor (bosch et al., 2003; li, 2016) . the s protein ectodomain consists of a receptor-binding subunit s1 and a membrane-fusion subunit s2 (tang et al., 2020; walls et al., 2020; wrapp et al., 2020) . two major domains in coronavirus s1 have been identified, including an n-terminal domain (ntd), and a c-terminal domain (ctd) also called receptor binding domain (rbd). following the rbd, s1 also contains two sub-domains (sd1 and sd2) . the s2 contains a variety of motifs, starting with the fusion peptide (fp). the fp describes a short segment, conserved across the viral family and composed of mostly hydrophobic residues, which inserts in the hostcell membrane to trigger the fusion event (epand, 2003; tang et al., 2020) . recent cryoelectron microscopy (cryo-em) studies on the stabilized ectodomain of sars-cov-2 s protein revealed a closed state of s trimer with three rbd domains in "down" conformation (walls et al., 2020) , as well as an open state with one rbd in the "up" conformation, corresponding to the receptor-accessible state (walls et al., 2020; wrapp et al., 2020) . unlike in mers-cov s protein (pallesen et al., 2017) , the two or three rbd "up" conformation has not been detected for sars-cov-2 s trimer. sars-cov-2 s and sars-cov s share 76% amino acid sequence identity, yet, they bind the same host-cell receptor-human angiotensin-converting enzyme 2 (ace2) (hoffmann et al., 2020; wang et al., 2020; zhou et al., 2020) . it is usually considered that the transition process towards the postfusion conformation is triggered when the s1 subunit binds to a hostcell receptor; receptor binding destabilizes the prefusion trimer, resulting in shedding of the s1 subunit and transition of the s2 subunit to a stable postfusion conformation (walls et al., 2017b) . the available crystal structures of the rbd domain of sars-cov-2 interacting with the extracellular peptidase domain (pd) of ace2, together with the cryo-em structure of rbd domain associated with the full length ace2 provided important information on the rbd-ace2 interaction interface, revealing that the residues s438 to q506, known as the receptorbinding motif (rbm), within rbd directly interact with ace2 (lan et al., 2020; wang et al., 2020; yan et al., 2020) . however, a complete picture of ace2 associating with the sars-cov-2 trimeric s protein is still missing, and it remains elusive on how ace2 binding induces sars-cov-2 s trimer conformational destabilization to facilitate transitions towards the postfusion state. here, we present cryo-em structures of sars-cov-2 s trimer in a tightly closed state, and the s trimer in complex with the receptor ace2 (termed sars-cov-2 s-ace2) at 2.7 å and 3.8 å resolution, respectively, in addition to a s trimer structure in the unliganded open state. the tightly closed ground prefusion state with originally dominant population may indicate a conformational masking mechanism of immune evasion for sars-cov-2 spike. our data suggested there is one rbd in the "up" conformation and is trapped with ace2 in the s-ace2 complex; ace2 can greatly shift the conformational landscape of s trimer, and trigger continuous swing motions of ace2-rbd in the context of the s trimer resulting in conformational dynamics in s1 subunits. we demonstrated the rbm t470-t478 loop and residue y505 as viral determinants for specific recognition of sars-cov-2 rbd by ace2. our findings provide a blueprint for the understanding of the mechanisms of ace2-induced conformational dynamics and resulted conformational transitions of the s trimer towards postfusion state, which may benefit anti-sars-cov-2 drug and vaccine development. prefusion stabilized ectodomain trimer of sars-cov-2 s glycoprotein was produced from hek293f cells using the strategy also adopted in other studies (fig. s1a ) (kirchdoerfer et al., 2018; miroshnikov et al., 1998; pallesen et al., 2017; tortorici et al., 2019; walls et al., 2017a; walls et al., 2020; walls et al., 2016; walls et al., 2019; wrapp et al., 2020) , and was subjected to cryo-em single-particle analysis ( fig. s2a-b ). our initial reconstruction suggested a preferred orientation problem associated with the s trimer (highly preferred "side" orientation but lacking tilted top views, fig. s2c ), which is also the case for the influenza hemagglutinin (ha) trimer (but highly preferred "top" orientation) (tan et al., 2017) . to overcome this problem, we adopted the recently developed tilt stage strategy in data collection with additional data collected at 30º and 40º tilt angles (tan et al., 2017) . this allowed us to obtain a cryo-em structure of sars-cov-2 s trimer in a closed state at 2.7 å resolution (with imposed c3 symmetry, termed s-closed) (figs. 1a, and s2-s3, movie 1). excitingly, after overcoming the preferred orientation problem, our s-closed map very well resolved the peripheral edge of the ntd domain ( fig. 1a-c) , which was less well resolved in the recent reports (walls et al., 2020; wrapp et al., 2020) . this enabled us to build a more complete model of the sars-cov-2 s trimer containing the previously missing loop regions (including q14-p26, k77-f79, y144-n164, q173-n185, r246-s247, and s255-a262, fig.1b, s2g) ; additionally, the s469-c488 loop in the rbm subdomain was also captured in our structure ( fig. 1d) . interestingly, compared with the recent closed state sars-cov-2 s trimer structure (walls et al., 2020) , our map represents an uncharacterized tightly closed conformation. for instance, the upper portion of s1 subunit especially ntd and rbd depicts an anti-clockwise rotation of 9.4º and 3.7º, respectively (fig. 1e ). accompanying this rotation, there is a slight inward tilt leading the peripheral edge of ntd exhibiting a 12.4 å inward movement for ca of t124 (fig. s2g ). these motions can be propagated to the central helix (ch) of s2 subunit, generating a clockwise rotation of 1.1º (fig. 1e ). this central portion clockwise rotation associating with the outer potion opposite anti-clockwise rotation in reality twists the complex in a more compact conformation. indeed, the average interaction interface between protomers increased from ~4,671.7 å 2 in their structure to 6,837.2 å 2 in our structure (fig. 1f ). taken together, our map represents a tightly closed state of the sars-cov-2 s trimer, not captured before. furthermore, when comparing our sars-cov-2 s-closed structure with the closed state sars-cov s trimer cryo-em structure (gui et al., 2017) , there is an anti-clockwise rotation of 10.5º and 8.7º in ntd and rbd, respectively, and a clockwise rotation of 4.3º in ch region from their structure to our s-closed structure, associating with a rbd inward shift towards the central axis (rmsd of 6.7 å, fig. s2h ). collectively, our s-closed structure appears more compact than that of sars-cov s trimer (6,837.2 å 2 vs. 4,874.0 å 2 in interaction interface, fig. 1f ). altogether, our study revealed a tightly closed conformation of sars-cov-2 s trimer, not observed in the homologous sars-cov s neither, extending the detected conformational space of sars-cov-2 spike protein. the tightly closed state with stably packed fusion peptide may represent the ground prefusion state of sars-cov-2 s trimer the hydrophobic fusion peptide, immediately after the s2' cleavage site and essential for host-cell membrane fusion, is highly conserved among sars-cov-2, sars-cov, and mers-cov s proteins (tang et al., 2020) . still, the majority of fp is missing in the available sars-cov-2 s trimer structures. thus, how it folds and where it locates within s trimer of the virus and how it can be activated remain unclear. here, our s-closed map enabled us to capture the entire fp of sars-cov-2 including the previously undetected l828-q853 fragment, which locates on the flank surface of s trimer, surrounded by hr1 of s2 subunit from the same protomer, and sd1/sd2 of s1 subunit from the clockwise neighboring protomer (fig. 1g-h) . the fp fragment is well ordered, forming two small helixes (y837-g842, l849-f855) and connecting loops (fig. 1g-h) . this observation further substantiates the notion that our sclosed structure with inactivated fp most likely represents the ground prefusion state. further interaction analysis revealed that sd2 and hr1 can form hydrogen bonds/salt bridges with the fp fragment, and sd2 plays a key role in this interaction involving in 6 predicted hydrogen bonds/salt bridges (table s2) . noteworthy, among the 6 sd2-fp interactions, d614 from sd2 contributes to the formation of 4 hydrogen bonds/salt bridges, majorly through its sidechain atoms, with k835, y837 and k854 of fp, suggesting d614 may be essential in the interaction with and stabilization of fp (fig. 1i and table s2 ). this could be related to the recent reports suggesting that the d614g mutation of sars-cov-2 s enhanced viral infectivity (more in discussion) (korber et al., 2020) . interestingly, it appears that before being activated, fp could serve as a linkage that wraps around the neighboring protomers in their s1/s2 interface and simultaneously connects s1 with s2, this way to coordinately lock the s trimer in the tightly closed ground prefusion state ( fig. 1g-h) . moreover, in this dataset the dominant population of the particles (~94%) is in the tightly closed state; although performed multiple rounds of 3d classification, eventually we found only a minor population (6%) of the particles is in the open state (fig. s3 ). our observations indicate that the open state sars-cov-2 s might be intrinsically dynamic and only exist transiently to expose the rbd domain. interestingly, the dominant population of the sars-cov-2 s trimer is in the ground prefusion state with inactivated fp and all the rbd domains buried, which may result in "conformational masking" preventing antibody binding and neutralization, similar to that described for hiv-1 envelope (env) (kwong et al., 2002; munro et al., 2014) . the population distribution of closed and open state of sars-cov-2 s varies among different studies (walls et al., 2020; wrapp et al., 2020) , which is reminiscent of observations made with sars-cov s and mers-cov s trimers. this observed variation could be potentially due to subtle difference in chemical condition used by different research groups (gui et al., 2017; kirchdoerfer et al., 2018; pallesen et al., 2017; song et al., 2018; walls et al., 2019; yuan et al., 2017) . to gain a thorough picture on how the receptor ace2 binding induces conformational dynamics of the sars-cov-2 s trimer and triggers transition towards the postfusion state, we determine the cryo-em structure of sars-cov-2 s trimer in complex with human ace2 pd domain to 3.8 å resolution (termed sars-cov-2 s-ace2, figs. 2a, s4a-e, and s5). further focused-refinements improved the resolution of the s trimer portion of the map to 3.3 å, and the connectivity in the ace2-rbd portion of the map, respectively (fig. s4e, s5) . we then built a pseudo atomic model of the complex with combined map information (fig. 2b ). to the best of our knowledge, the structure of sars-cov-2 s-ace2 complex has not been reported before. in this dataset we additionally captured an unliganded s trimer in the open state with one rbd up (resolved to 6.0 å resolution, termed s-open), but did not detect the closed state . we should mention that our bio-layer interferometry (bli) assay revealed a relatively rapid disassociation kinetics between ace2 and the s trimer (koff = 4.56x10 -3 s -1 , fig. s1e ). we thus determined the complex structure in the presence of trace amount of cross linker glutaraldehyde (methods). additionally, we also determined the s-ace2 complex structure without cross linker at 5.3 å resolution, and the two maps are in comparable conformation, suggesting that addition of cross linker did not change the conformation of the complex (fig. s4g ). we then used the s-ace2 map at 3.8 å resolution for detailed structural analysis. to inspect the conformational changes from the closed state to the unliganded open state, we first overlaid our s-open with our s-closed structures together. in the s-open structure, the only up rbd domain from protomer 1 (termed rbd-1) shows a 71.0º upwards/outwards rotation, resulting in an exposed rbm region accessible for ace2 binding (fig. 2c ). this rbd-1 rotation can be propagated to the underneath sd1, inducing a downwards movement of sd1 (fig. 2c) . we also noticed a considerable clockwise rotation of 9.4º, 11.2º, and 12.9º in ntd for protomer 1, 2, and 3, respectively, and anti-clockwise rotations in the ch of corresponding s2 subunit, greatly untwisting the s trimer from the tightly closed state (fig. 2d ). associated with this s1 untwisting, there is a downwards/outwards movement of ntds in the scale of ~10 å (fig. 1d, right panel) . these combined untwisting motion could release the original protomer interaction strength, beneficial for the transient raising up of the rbd. moreover, our local resolution analysis on the s-open map also suggested that other than rbd-1, the consecutive rbd-2 also exhibits considerable dynamics (fig. s4d ). our sars-cov-2 s-ace2 structure revealed that the s trimer binds with one ace2 through the only up rbd domain, while the other two rbds remain in the down conformation ( fig. 2a-b) , suggesting ace2 binding to sars-cov-2 strictly requires the up conformation of rbd. unlike the observations made with sars-cov and mers-cov s trimers, we did not detect s trimer with two rbd domains up with bound ace2 (kirchdoerfer et al., 2018; pallesen et al., 2017) . though our s-ace2 and s-open structures generally resemble each other especially in the s2 region, there are noticeable differences in the s1 region. specifically, after ace2 binding, the up rbd-1 from the s-open state can be pushed tilting downwards slightly, with the angle to the horizontal plane of s trimer reduced from 73.2º to 68.6º in ace2 bound state (fig. 2e ). this ace2 binding induced motion of rbd-1 could be propagated to the neighboring rbd-2 and the consecutive rbd-3 (rmsd: 1.95 å, fig. 2f ), collectively disturbing the allosteric network of the fusion machinery. indeed, the neighboring protomer interaction interface was reduced from the original ~6837.2 å 2 in the s-closed state to 4898.4~5791.2 å 2 in the ace2 bound state (fig. 2g ). altogether, these s1 subunits untwisting and rbd-1 tilting motions could destabilize the prefusion state of s trimer, prepared for the subsequent conformational transitions towards the postfusion state. interestingly, our s-ace2 structure showed that the core region of the up rbd-1 and the rbm t470-f490 loop of the neighboring rbd-2 could form aromatic interactions with the involvement of y369/f374 from rbd-1 and f486/y489 from rbd-2 ( fig. 2h ), potentially enhancing interactions between neighboring s1 subunits, thus beneficial for subsequent simultaneous release of s1 subunits. this interaction was not detected in the counterpart of the homologous sars-cov s-ace2 structure, likely due to longer distance between the adjacent "up" and "down" rbds in that structure (kirchdoerfer et al., 2018; song et al., 2018) . noteworthy, the originally stably packed fp from protomer 3 surrounded by sd1/sd2 of the neighboring protomer 1 in the s-closed structure is now mostly missing in the s-ace2 structure, which is also the case in the s-open structure. this is mostly caused by the s trimer untwisting-motion induced downwards shift of sd1 (fig. 2c , i). indeed, the b34/b37 strands within sd1 shift downwards for up to 5.4 å; consequently, the c590 and t588 from b37 and the connecting loop could clash with the y837 and l841 of the originally packed a8 helix of fp ( fig. 2i ), potentially resulting in destabilization and activation of the fp motif from protomer 3. since the untwisting/downwards-shift motions of s1 subunits are allosterically coordinated within the s trimer in its opening process, the density corresponding to fps in protomer 1 and 2 are also missing, indicating a coordinated activation mechanism of fp, which may be one of the key elements prepared for the subsequent fusion of s trimer. according to our sars-cov-2 s-ace2 cryo-em structure, the overall ace2-rbd interaction interface is comparable to that of the crystal structures of the rbd domain of sars-cov-2 s interacting with the ace2 pd domain (fig. 3a ) (lan et al., 2020; wang et al., 2020) , i.e. our structure revealed 19 residues of rbd are in contact with 17 residues of ace2 with a distance cut-off of 4 å (table s3 ). sequence alignment demonstrated that the rbm t470-f490 loop is the most diversified region between sars-cov-2 and sars-cov s proteins (fig. s6 ). in line with this, structural comparison revealed that the conformation of the rbm t470-f490 loop in our sars-cov-2 s-ace2 structure is very distinct from that in the sars-cov rbd-ace2 crystal structure ( fig. 3b ) (li et al., 2005) . noteworthy, the rbm t470-f490 loop can originally be resolved in our s-closed structure, but is mostly missing in our s-open structure, indicating the t470-f490 loop may be activated in the open state. in our s-ace2 structure, a portion of this loop forms contact with the n-terminal helix of ace2 (fig. 3a ), for instance, a475 within this loop could interact with s19/t27 of ace2 (table s3 ), suggesting that the rbm t470-f490 loop may play an important role in receptor recognition. moreover, the s-ace2 structure indicated that the q498-y505 region located in the other edge of rbm could also form close contact with ace2, i.e. y505 could form hydrogen bonds/contacts with to further define the subdomains/residues critical for rbd binding to ace2, we designed and produced three sars-cov-2 rbd mutant proteins, each of which had a single subdomain substituted with the counterpart of sars-cov. these rbd mutants were termed rbd-(core), rbd-(rbm-r2) and rbd-(rbm-r3), which harbored r319 to n437 of the core region, l452 to k462, and t470 to t478 of the rbm from sars-cov, respectively (figs. 3c and s6). results from ace2-binding enzyme linked immunosorbent assay (elisa) showed that the binding activity of the three rbd mutants towards anti-rbd polyclonal antisera and the crossreactive monoclonal antibody 1a10 was comparable to that of the wildtype sars-cov-2 rbd protein ( fig. 3c) , indicating that the mutations did not significantly affect the overall conformation of the rbds. the mutants rbd-(core) and rbd-(rbm-r2) bound ace2 as efficiently as the wildtype rbd; in contrast, rbd-(rbm-r3) completely lost ace2-binding ( fig. 3c ). these results pinpoint the rbm-r3 region (residues 470-teiyqagst-478) as the critical viral determinant for specific recognition of sars-cov-2 rbd by the ace2 receptor. additionally, we constructed three single-point mutants of sars-cov-2 rbd protein, rbd (q498a), rbd (v503a), and rbd (y505a). our elisa ace2-binding assay showed that the mutation y505a was sufficient to completely abolish the binding of ace2, while the other two mutations did not show such effect (fig. 3d) , demonstrating that the residue y505 of sars-cov-2 rbd is a key amino acid required for ace2 receptor binding. our sars-cov-2 s-ace2 map showed well defined density for the s trimer region, but relatively lower local resolution in the associated ace2-rbd region (fig. s4c) , suggesting considerable conformational heterogeneity of ace2-rbd as well as relative dynamics between ace2-rbd and the remaining part of s trimer with respect to each other. this is in line with the report showing that in sars-cov s trimer, the associated ace2-rbd is relatively dynamic, showing three major conformational states with the angle of ace2-rbd to the surface of s trimer at ~51º, 73º, and 111º, respectively (song et al., 2018) . to better delineate the conformational space of the ace2 engaged sars-cov-2 s trimer, we performed multi-body refinement in relion 3.1 (fernandez-leiro and scheres, 2017) . principal component analysis of the movement revealed that approximately 68% of the movement of the complex is described by the first three eigenvectors representing swing motions in distinct directions relative to the s trimer (fig. 4a) . eigenvector 1 describes a swing motion of ace2-rbd towards rbd-2 direction with the angular range of 12.2º, eigenvector 2 corresponds to the swing motion of ace2-rbd towards the original location of rbd-1 with the angular range of 11.9º, and eigenvector 3 describes the swing motion of ace2-rbd along the ntd-1 to ntd-3 direction with the angular range of 9.8º (fig. 4b) . histograms of the amplitudes along the three eigenvectors are unimodal, indicative of continuous motions (fig. 4c ). as the dynamic motions in the complex are formed by linear combination of all eigenvectors, these data suggested that ace2-rbd processes on top of the s trimer in a noncorrelated manner. moreover, multi-body analysis on the non-cross-linked sars-cov-2 s-ace2 data showed similar swing motions (fig. s4h) , indicating the presence of cross linker did not disturb the mode of ace2-rbd motions within the s trimer. additionally, compared with the homologous sars-cov s-ace2 complex, which shows discrete movements of ace2-rbd in one direction (similar to our eigenvector 2 direction) (song et al., 2018) , ace2 binding to sars-cov-2 s induces more complex combined continuous swing motions of ace2-rbd within the complex. putting together, our observations suggest that ace2 receptor binding to sars-cov-2 s triggers considerable conformational dynamics in s1 subunits that could destabilize the prefusion s trimer. indeed, the b-factor distribution of our s-ace2 complex demonstrated that ace2 binding induces strikingly enhanced dynamics in the s1 region including rbd and ntd domains (fig. 4d) , facilitating the release of the associated ace2-s1 component and transitions of the s2 subunit towards a stable postfusion conformation. indeed, we found a notable drop in the interaction surface between s1 and s2 subunits from the s-closed state (8982.3 å 2 ) to the s-ace2 state (6521.7 å 2 ). it has been suggested that the large number of n-linked glycans covering the surface of the spike protein of sars-cov and mers-cov could pose challenge to antigen recognition, thus may help the virus evade immune surveillance yuan et al., 2017) . similar to sars-cov s, sars-cov-2 s also comprises 22 n-linked glycosylation, with 13 glycans in the s1 subunit and the other 9 in the s2 subunit ( fig. 5a ) (walls et al., 2020; watanabe et al., 2020) . in our s-closed structure, we resolved the density for 18 n-linked glycans per protomer (fig. 5a-b and s2i ), including two undetected glycans at site n17 and n149 located in the ntd (fig. 5b) , while the three glycans located in the flexible c-terminal region are missing as in other studies (walls et al., 2020; wrapp et al., 2020) . similar to mers-cov and sars-cov s trimers (walls et al., 2017b; walls et al., 2019; yang et al., 2015) , sars-cov-2 s trimer also forms a glycan hole at proximity of the s1/s2 cleavage site and the fusion peptide (near the s2' cleavage site, fig. 5b) . although there is an extra glycan at n657 site near the s1/s2 cleavage site in sars-cov-2 s, the hole region is still more sparsely glycosylated than the rest of the protomer. this glycan hole might be important for permitting the access of activating host proteases and for allowing membrane fusion to take place without obstruction (walls et al., 2017b; walls et al., 2019; yang et al., 2015) . moreover, after ace2 binding, our s-ace2 structure revealed that the density corresponding to glycan at n165 site is weaker in protomer 1, while the other resolved glycans in the s-closed state can also be visualized in the s-ace2 structure (fig. 5c ). the outbreak of covid-19 caused by sars-cov-2 virus has become pandemic. several structures of sars-cov-2 spike rbd domain bound to ace2 have been reported (lan et al., 2020; wang et al., 2020; yan et al., 2020) . however, the complete architecture of sars-cov-2 trimeric s in complex with ace2 remains unavailable, leading to an incomplete understanding of the nature of this interaction and of the resulted conformational transitions of the s trimer towards postfusion and virus entry. in the present study, we determined an uncharacterized tightly closed state of sars-cov-2 s trimer revealing the stably packed fusion peptide, most likely representing a previously undetected ground prefusion state of s trimer. the tightly closed s trimer with originally dominant population may indicate a conformational masking mechanism of immune evasion for sars-cov-2 spike. importantly, we captured the complete architecture of sars-cov-2 s trimer in complex with ace2. we found the presence of ace2 could dramatically shift the conformational landscape of the s trimer, and after engagement the continuous swing motions of ace2-rbd in the context of the s trimer could generate considerable conformational dynamics in s1 subunits resulting in a significant decrease in s1/s2 interface area. furthermore, our structural data combined with biochemical analysis revealed that the rbm t470-t478 loop and residue y505 play vital roles in the binding of sars-cov-2 rbd to ace2 receptor. our findings depict a new role of fp in stabilizing s trimer and the mechanism of fp activation, expand the detected conformational space of the s trimer, and provide structural basis on the sars-cov-2 spike d614g mutation induced enhanced infectivity. based on the data, we put forward a mechanism of ace2 binding-induced conformational transitions of sars-cov-2 s trimer from the tightly closed ground prefusion state transforming towards the postfusion state (fig. 6) . in the receptor-free sars-cov-2 s, the majority of the s trimers is in the tightly closed ground prefusion state with inactivated fp, and only a minor population of the particles is in the transient open state with one rbd up representing the fusion-prone state, forming a dynamic balance between the two states (step 1). however, the presence of ace2 and subsequent trapping of the rbd (discussed later) could overcome the energy barrier, break the balance and shift the conformational landscape towards the open state with an untwisting/downwards-shift motion of the s1 subunits, leading to unpacked/activated fps, weakened interactions among the protomers, and an up rbd. in step 2, once the receptor ace2 grasp the up rbd, the rbd will be trapped in the up conformation, and the associated ace2-rbd together shows combined continuous swing motions on the topmost surface of the s trimer. these motions and dynamics could disturb the allosteric network and release the constrains imposed on the fusion machinery, beneficial for the releasing of the ace2-s1 component, thereby allowing the s trimers to refold and fuse the viral and host membranes (step 3). the dominantly populated conformation (94%) for the unliganded sars-cov-2 s trimer is in the tightly closed state (more compact than that of sars-cov s trimer) with all the rbd domains buried, resulting in conformational masking preventing antibody binding and neutralization at sites of receptor binding. this sars-cov-2 conformational masking mechanism of neutralization escape suggested here could affect all antibodies that bind to the receptor binding site, similar to that described for hiv-1 env (kwong et al., 2002; munro et al., 2014) . while for mers-cov or sars-cov s trimer, the closed state is less populated (5.4% and 27.6%, respectively, indicating the conformational masking mechanism may be less effective for the two viruses (gui et al., 2017; pallesen et al., 2017) . interestingly, our findings also suggest that unliganded s trimer proteins of sars-cov-2 are inherently competent to transiently display conformation with one rbd up ready for receptor ace2 binding; ace2 facilitates the capture of pre-existing s trimer open conformation that are spontaneously sampled in the unliganded spike, rather than triggering a trimer opening event. therefore, the spontaneously sampled s trimer conformations may serve a functional role in infectivity. intriguingly, our data also suggest that the sars-cov-2 s trimer is very sensitive to (gui et al., 2017; song et al., 2018) . this demonstrates that the sars-cov-2 s trimer is much more sensitive to the ace2 receptor than sars-cov s in terms of receptor-triggered transformation from the closed prefusion state to the fusion-prone open state, which might have contributed to the observed superior infectivity of sars-cov-2 as compared to that of sars-cov. noteworthy, the mutation sars-cov-2 spike d614g has gained urgent concern; the mutated genotype g614 began spreading in early february, and it was detected to reach at a frequency of ~72% in early june according to gisaid public repository (daniloski et al., 2020; korber et al., 2020) . moreover, it has been reported that the d614g mutation promotes the infectivity of sars-cov-2 and enhances viral transmissibility in multiple human cell types (daniloski et al., 2020; hu et al., 2020; zhang et al., 2020) . however, the structural basis of d614g enhanced infectivity has not been fully understood yet. here our s-closed structure in the ground prefusion state showed that d614 heavily involves in the interaction with fp through its side chain atoms (fig. 1i , table s2 ). this interaction could contribute greatly to the linkage between neighboring protomers as well as between s1 and s2 subunits. however, the mutation of d614 to g without side chain could eliminate most of the hydrogen bonds/salt bridges the d614 originally forms with fp, hence greatly reduce its interaction with fp potentially leading to a coordinated unpacking/activation of fps. therefore, d614g mutation could (1) reduce the constrains between neighboring protomers as well as s1/s2 interactions within s trimer, and (2) lower the energy barrier for the conformational transformation from closed prefusion state to fusion-prone open state, leading to even more sensitive sars-cov-2 s trimer to ace2 binding. collectively, these factors may contribute to the enhanced infectivity and viral transmissibility of the g614 strain. in summary, our data revealed the unliganded sars-cov-2 s trimer to be intrinsically transforming between two distinct pre-fusion conformations, whose relative occupancies could be dramatically remodeled by receptor ace2. these findings support a dynamics-based mechanism of immune evasion and ligand recognition (munro et al., 2014) . thus, our study delineates the properties of the sars-cov-2 spike glycoproteins that simultaneously allow the retention of function and the evasion of the humoral immune response. we also delineated that the substantial conformational dynamics of s1 subunits induced by ace2 binding could trigger the transition of the spike protein towards postfusion state prepared for viral entry and infection. collectively, our findings suggest that stabilization of the tightly closed ground prefusion state of s trimer with inactivated fps might be a general and effective means of inhibiting sars-cov-2 entry, and an understanding of the properties of the sars-cov-2 s trimer that permit neutralization resistance will guide attempts to create vaccines as well as therapeutics that target receptor binding. we are grateful to the staffs of the ncpss electron microscopy facility, database and cryo-em maps have been deposited in the electron microscopy data bank, https://www.ebi.ac.uk/pdbe/emdb/ (accession nos. ***), and the associated models have been deposited in the protein data bank, www.rcsb.org (accession nos. **, **, and **). the authors declare that they have no conflict of interest. to express sars-cov-2 s glycoprotein ectodomain, the mammalian codon-optimized gene coding sars-cov-2 (wuhan-hu-1 strain, genbank id: mn908947.3) s glycoprotein ectodomain (residues m1-q1208) with proline substitutions at k986 and v987, a "gsas" substitution at the furin cleavage site (r682-r685) was cloned into vector pcdna 3.1+. a cterminal t4 fibritin trimerization motif, a tev protease cleavage site, a flag tag and a his tag were cloned downstream of the sars-cov-2 s glycoprotein ectodomain (fig. s1a) before bli experiments, sars-cov-2 s trimer protein was biotinylated using the ez-link™ sulfo-nhs-lc-lc-biotin kit (thermo fisher) and then purified using zeba™ spin desalting column (thermo fisher), according to manufacturer's protocols. to determine binding affinity of ace2, bli assay was carried out using an octet red 96 instrument (pall fortébio, usa). briefly, biotinylated sars-cov-2 s trimer protein was loaded onto streptavidin (sa) biosensors (pall fortébio). s-trimer-bound biosensors were dipped into wells containing varying concentrations of ace2 protein and the interactions were monitored over a 500-sec association period. finally, the sensors were switched to dissociation buffer (0.01 m pbs supplemented with 0.02% tween 20 and 0.1% bovine serum albumin) for a 500-sec dissociation phase. data was analyzed using octet data analysis software version 11.0 (pall fortébio). the purified sars-cov-2 s glycoprotein ectodomain and human ace2 pd domain were mixed at a molar ratio of 1:3 and were incubated on ice for 2 hours. the mixture was purified by filtration chromatography using a superose 6 increase 10/300 gl column (ge healthcare) pre-equilibrated with 20 mm tris-hcl ph 7.5, 200 mm nacl, 4% glycerol. for cross linking complex, the buffer of purified sars-cov-2 s glycoprotein ectodomain and human ace2 pd domain were exchanged to 20 mm hepes ph 7.5, 200 mm nacl; then sars-cov-2 s and human ace2 were mixed at a molar ratio of 1:3. after incubation on ice for 2 hours, the complex was cross linked by 0.1% glutaraldehyde, which is commonly used in cryo-em studies of fragile macromolecular complexes (kastner et al., 2008; patel et al., 2018) . the glutaraldehyde was neutralized by adding 20 mm tris-hcl ph 7.5 after incubated on ice for 1 hour. the mixture was run over a superose 6 increase 10/300 gl column (ge healthcare) in 20 mm tris-hcl ph 7.5, 200 mm nacl, 4% glycerol. the complex peak fractions were concentrated and assessed by sds-page and negative-staining electron microscopy. for the ns sample, a volume of 5 µl of sars-cov-2 s-ace2 sample was placed on a plasma cleaned copper grid for one minute. excess sample on the grid was blotted off using filter paper, and a volume of 5 µl of 0.75% uf (sigma-aldrich) was added to wash the grid. after blotting, another volume of 5 µl of 0.75% uf was placed on the grid again for one minute to stain. grids were visualized under a tecnai g2 spirit 120 kv transmission electron microscope (thermo fisher scientific), and micrographs were taken using an eagle camera with a nominal magnification of 67,000×, yielding a pixel size of 1.74 å. 41,827 particles were autopicked in eman2 (bell et al., 2016) . after 2d classification, we selected good averages with 13,047 particles for initial model building, which were performed in relion 3.0 (zivanov et al., 2018) . to prepare the cryo-em sample of sars-cov-2 s trimer, a 2.2-µl aliquot of this sample was applied to a plasma cleaned holey carbon grid (r2/1, 200 mesh; quantifoil) or graphene oxide-lacey carbon grid (300 mesh, emr). the grid was blotted with vitrobot mark iv (thermo fisher scientific) and then plunged into liquid ethane cooled by liquid nitrogen. to prepare the cryo-em sample of s-ace2 complex with or without cross linking, we used graphene oxide-lacey carbon grid (300 mesh, emr), and adopted the same vitrification procedure as for the s trimer. cryo-em movies of the samples were collected on a titan krios electron microscope (thermo fisher scientific) operated at an accelerating voltage of 300 kv with a nominal magnification of 22,500x (table s1 ). the movies were recorded on a k2 summit direct electron detector (gatan) operated in the super-resolution mode (yielding a pixel size of 1.02 å after 2 times binning), under low-dose condition in an automatic manner using serialem (mastronarde, 2005) . each frame was exposed for 0.15 s and the total accumulation time was 6.45s, leading to a total accumulated dose of 50 e -/å 2 on the specimen. to solve the problem of preferred orientation associated with sars-cov-2 s trimer, we additionally collected tilt datasets with the stage tilt at 30° or 40°, while the other conditions remained the same. single particle analysis was mainly executed in relion 3.1 (fernandez-leiro and scheres, 2017) . all images were aligned and summed using motioncor2 software (zheng et al., 2017) . after ctf parameter determination using ctffind4 (rohou and grigorieff, 2015) , particle auto-picking, manual particle checking, and reference-free 2d classification, particles with s trimer features were maintained for further processing. for receptor-free s trimer sample, 226,082 particles were picked from non-tilt micrographs, and 118,420 remained after 2d classification (fig. s3) . these particles went through 3d auto-refine using available sars-cov-2 s trimer cryo-em map (emdb: 21452) lowpass filtered to 40 å resolution as initial model (walls et al., 2020) . these particles were refined into a closed state map of s trimer with imposed c3 symmetry. we then re-extracted the particles using the refinement coordinates to re-center it. after ctf refinement and polishing, these particles were refined with c3 symmetry again. noteworthy, the euler angle distribution of the map suggested the dataset is lacking tilted top views (fig. s2c left panel) . indeed, when refine the dataset without imposing 3-fold symmetry, the top view of the map appeared distorted indicating a preferred orientation problem associated with the sample. to overcome the preferred orientation problem, we additionally collected tilt data, and boxed out 198,737 particles from 40º tilt micrographs and 16,010 particles from 30º tilt micrographs. after 2d classification, 184,661 particles remained. we then used goctf software to determine the defocus for each of the tilt particle, and these particles were re-extracted with corrected defocus (su, 2019) . after combining the tilt with non-tilt particles, we refined the dataset without imposing symmetry, then performed two rounds of 3d and 2d classifications to further cleanup the dataset, and obtained a dataset of 151,505 particles, of which 62,368 particles were from the tilt data. we then carried out heterogeneous refinement in cryosparc (punjani et al., 2017) , and obtained a closed state map from 142,345 particles and an open state reconstruction with 9,160 particles ( fig s3) . after ctf refinement and bayesian polishing, the closed state map was refined to 2.7 å resolution with c3 symmetry, while the open state map was at 12.8 å resolution and hardly to improve the resolution, indicating an intrinsic dynamic nature of the open state. the overall resolution was determined based on the gold-standard criterion using an fsc of 0.143 (scheres and chen, 2012) . for the sars-cov-2 s-ace2 cross-linked dataset, 298,127 particles were picked from original micrographs, and 138,632 particles remained after 2d classification (fig. s5) . these particles were refined with an initial model built from our negative staining data. we then reextracted the particles to re-center them. these particles went through a 3d-2d classification step resulting in a further cleaned up dataset of 77,440 particles. we refined these particles into a map of ace2 bound s trimer complex. we then used this map as initial model to refine the originally picked 298,127 particles for one round to re-extract and re-center the particles. after 2d classification, 207,742 particles remained. after 2 rounds of 3d-2d cleaning step, 136,412 particles were left for further structure determination. after heterogeneous refinement in cryosparc, class 1 resembled an ace2-free open state of s trimer, and classes 2-5 adopted s-ace2 engaged conformation. for class 1, after further 2d classification, we refined the 24,502 cleaned up particles into a s-open map at 6.0 å resolution using non-uniform refinement in cryosparc. among the other four classes with bound ace2, we sorted out good particles for classes 2-4 by 2d classification and combined them with class 5 exhibiting good structural details, resulting in a dataset of 68,987 particles. after refinement, bayesian polishing, and ctf refinement, we reconstructed a 3.8 å resolution sars-cov-2 s-ace2 map. the s trimer portion without the up rbd was rather stable, could be locally refined to 3.3 å using local refinement in cryosparc with non-uniform refinement option chosen. the ace2 associated with the up rbd was subtracted and refined in relion to obtain a more 8.4 å map with better connectivity. multi-body refinement in relion 3.1 was applied to analyze the motion of the complex. for sars-cov-2 s-ace2 w/o crosslinking dataset, we followed similar classification and cleaning up strategy and obtained 81,820 particles. through heterogeneous refinement and 2d classification in cryosparc, we reconstructed a 5.3 å resolution sars-cov-2 s-ace2 map from 32,866 particles using non-uniform refinement, and an unliganded open state map of 11.2 å resolution from 15,149 particles, with the population of 68.4% and 31.6%, respectively. multi-body refinement was also applied to analysis the mobility of the complex. to build the pseudo atomic model for our sars-cov-2 s-closed structure, we used the available atomic model of sars-cov-2 s (pdb: 6vxx) as initial model (walls et al., 2020) . we first refined the model against our map using phenix.real_space_refine module in phenix (adams et al., 2010) . for the missing loop regions in s1 subunit, we either built the homology model based on sars-cov s structure (pdb: 6crw) (kirchdoerfer et al., 2018) through swiss-model webserver (waterhouse et al., 2018) , or built the loop manually according to the density in coot (emsley and cowtan, 2004) . for the fp region, we first built the homology model by modeller tool within chimera by using mers-cov s structure (pdb: 6nb3) as template (pettersen et al., 2004; sali, 1995; walls et al., 2019) , then used rosetta to refine this region against the density map (dimaio et al., 2015) . eventually, we used phenix.real_space_refine again for the protomer and s-trimer model refinement against the map. for the sars-cov-2 s-ace2 structure, we used the sars-cov-2 rbd-ace2 crystal structure (pdb: 6m0j) as initial model for the ace2 and the associated up rbd portion, and our s-closed model as initial model for the remaining portion. these models were firstly refined against the corresponding focused map using rosetta and phenix (dimaio et al., 2015) , then combined together in coot. we then refined the combined model against our 3.8 å resolution sars-cov-2 s-ace2 map using rosetta and phenix. for the s-open structure, we used the model of sars-cov-2 s-ace2 as initial model with ace2 removed, and refined against the map using rosetta. we used phenix.molprobility to evaluate the models, and calculated b-factors by atom displacement refinement function in phenix.real_space_refine. we used ucsf chimera and chimerax for figure generation (goddard et al., 2018; pettersen et al., 2004) , and also for rotation, translation, rmsd, and vdw contact measurement. interaction surface analysis was conducted by pisa server (krissinel and henrick, 2007) . to uncover the amino acids important for ace2 receptor recognition, ace2 ecotodomain (residues q18 to s740) gene, with an n-terminal il10 signal peptide, tagged with human igg1fc and his tag at the c-terminus, were cloned into the pcdna 3.4 vector. codonoptimized rbd (residues v320 to g550) gene fragment, with an n-terminal il10 signal peptide, tagged with his tag at the c-terminus, were cloned into the pcdna 3.4 vector. three sars-cov-2 rbd mutants were constructed. for mutant rbd (core), amino acids r319 to n437 of core region in the sars-cov-2 rbd were substituted by the corresponding region of sars-cov strain tor2 (genbank id: aap41037.1). for mutants rbd (rbm-r2) and rbd (rbm-r3), residues l452 to k462, and residues t470 to t478 of rbm region in the sars-cov-2 rbd were mutated into the corresponding regions of sars-cov strain tor2, respectively. for single point mutations of rbd (q498a), rbd (v503a), and rbd (y505a), rbd residues q498, v503, and y505 were substituted by ala, respectively. all mutant plasmids were constructed using the mutexpress tm ii fast mutagenesis kit v2 (vazyme, china) according to the manufacturer's instruction. the proteins were generated using hek 293f expression system and purified as described above. anti-rbd polyclonal antibody and monoclonal antibody (mab) 1a10 were prepared by immunizing balb/c mice with recombinant sars-cov-2 rbd fused with a c-terminal mouse iggfc tag (sino biological inc, beijing, china) using previously described protocols (qu et al., 2020) . the purified rbd mutants were tested by elisa for reactivity with the receptor ace2. briefly, elisa plates were coated with 100 ng/well of the purified rbd mutants in pbs at 37°c for 2 hours and then blocked with 5% milk in pbs-tween20 (pbst). next, the plates were incubated with 50 ng/well of ace2-hfc fusion protein, 50 µl/well of culture supernatant of hybridoma 1a10, or 50 µl/well of mouse anti-rbd sera (diluted at 1/1000) at 37°c for 2 h. after washing, the corresponding secondary antibodies, horseradish peroxidase (hrp)conjugated anti-human igg1 (abcam, usa) or hrp-conjugated anti-mouse igg (sigma, usa), were added and incubated at 37°c for 1 h. after washing color development, absorbance at 450 nm was determined. cryo-em data processing procedure for sars-cov-2 s trimer in the presence of ace2. amino acid sequence alignment of sars-cov-2 s to sars-cov s. the secondary structure elements were defined based on an espript (robert and gouet, 2014) algorithm and are labeled based on our sars-cov-2 s-closed structure. the rbd domain is labeled in green frames, and the subdomains of rbm are also labeled. contacting residues at the sars-cov-2 rbd-ace2 interface (distance cutoff of 4 å) y453 h34 l455 k31 f456 t27, d30, k31 y473 t27 a475 s19, t27 g476 s19, q24 f486 l79, m82, y83 n487 q24, y83 y489 t27, f28, k31 f490 k31 q493 k31, h34 s494 h34 y495 h34 g496 k353 q498 y41 t500 y41, d355, r357 n501 k353 g502 k353, g354 y505 k353, g354, a386, r393 phenix: a comprehensive python-based system for macromolecular structure solution high resolution single particle refinement in eman2 the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex the d614g mutation in sars-cov-2 spike increases transduction of multiple human cell types atomic-accuracy models from 4.5-a cryo-electron microscopy data with density-guided iterative local refinement coot: model-building tools for molecular graphics fusion peptides and the mechanism of viral fusion a pipeline approach to single-particle processing in relion ucsf chimerax: meeting modern challenges in visualization and analysis cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor the d614g mutation of sars-cov-2 spike protein enhances viral infectivity and decreases neutralization sensitivity to individual convalescent sera grafix: sample preparation for single-particle electron cryomicroscopy stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis spike mutation pipeline reveals the emergence of a more transmissible form of sars-cov-2 inference of macromolecular assemblies from crystalline state hiv-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor structure, function, and evolution of coronavirus spike proteins structure of sars coronavirus spike receptor-binding domain complexed with receptor automated electron microscope tomography using robust prediction of specimen movements engineering trimeric fibrous proteins based on bacteriophage t4 adhesins conformational dynamics of single hiv-1 envelope trimers on the surface of native virions immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen structure of human tfiid and mechanism of tbp loading onto promoter dna ucsf chimera--a visualization system for exploratory research and analysis cryosparc: algorithms for rapid unsupervised cryo-em structure determination a new class of broadly neutralizing antibodies that target the glycan loop of zika virus envelope protein sars-cov-2, sars-cov, and mers-cov: a comparative overview deciphering key features in protein structures with the new endscript server ctffind4: fast and accurate defocus estimation from electron micrographs comparative protein modeling by satisfaction of spatial restraints prevention of overfitting in cryo-em structure determination cryo-em structure of the sars coronavirus spike glycoprotein in complex with its host cell receptor ace2 goctf: geometrically optimized ctf determination for single-particle cryo-em addressing preferred specimen orientation in single-particle cryo-em through tilting coronavirus membrane fusion mechanism offers a potential target for antiviral development structural basis for human coronavirus attachment to sialic acid receptors crucial steps in the structure determination of a coronavirus spike glycoprotein using cryo-electron microscopy function, and antigenicity of the sars-cov-2 spike glycoprotein cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion unexpected receptor functional mimicry elucidates activation of coronavirus fusion structural and functional basis of sars-cov-2 entry by using human ace2 site-specific analysis of the sars-cov-2 glycan shield swiss-model: homology modelling of protein structures and complexes cryo-em structure of the 2019-ncov spike in the prefusion conformation structural basis for the recognition of sars-cov-2 by full-length human ace2 two mutations were critical for bat-to-human transmission of middle east respiratory syndrome coronavirus cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains the d614g mutation in the sars-cov-2 spike protein reduces s1 shedding and increases infectivity motioncor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy a pneumonia outbreak associated with a new coronavirus of probable bat origin new tools for automated high-resolution cryo-em structure determination in relion-3 key: cord-308428-zw26usmh authors: walter, justin d.; hutter, cedric a.j.; garaeva, alisa a.; scherer, melanie; zimmermann, iwan; wyss, marianne; rheinberger, jan; ruedin, yelena; earp, jennifer c.; egloff, pascal; sorgenfrei, michèle; hürlimann, lea m.; gonda, imre; meier, gianmarco; remm, sille; thavarasah, sujani; zimmer, gert; slotboom, dirk j.; paulino, cristina; plattet, philippe; seeger, markus a. title: highly potent bispecific sybodies neutralize sars-cov-2 date: 2020-11-10 journal: biorxiv doi: 10.1101/2020.11.10.376822 sha: doc_id: 308428 cord_uid: zw26usmh the covid-19 pandemic has resulted in a global crisis. here, we report the generation of synthetic nanobodies, known as sybodies, against the receptor-binding domain (rbd) of sars-cov-2 spike protein. we identified a sybody pair (sb#15 and sb#68) that can bind simultaneously to the rbd, and block ace2 binding, thereby neutralizing pseudotyped and live sars-cov-2 viruses. cryo-em analyses of the spike protein in complex with both sybodies revealed symmetrical and asymmetrical conformational states. in the symmetric complex each of the three rbds were bound by both sybodies, and adopted the up conformation. the asymmetric conformation, with three sb#15 and two sb#68 bound, contained one down rbd, one up-out rbd and one up rbd. bispecific fusions of the sybodies increased the neutralization potency 100-fold, as compared to the single binders. our work demonstrates that linking two binders that recognize spatially-discrete binding sites result in highly potent sars-cov-2 inhibitors for potential therapeutic applications. the ongoing pandemic arising from the emergence of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in 2019, demands urgent development of effective antiviral therapeutics. several factors contribute to the adverse nature of sars-cov-2 from a global health perspective, including the absence of herd immunity [1] , high transmissibility [2, 3] , the prospect of asymptomatic carriers [4] , and a high rate of clinically severe outcomes [5] . despite intense development efforts, a vaccine against sars-cov-2 remains unavailable [6, 7] , making alternative intervention strategies paramount. in addition to offering relief for patients suffering from the resulting covid-19 disease, therapeutics may also reduce the viral transmission rate by being administered to asymptomatic individuals subsequent to probable exposure [8] . finally, given that sars-cov-2 represents the third global coronavirus outbreak in the past 20 years [9, 10] , development of rapid therapeutic strategies during the current crisis could offer greater preparedness for future pandemics. akin to all coronaviruses, the viral envelope of sars-cov-2 harbors protruding, club-like, multidomain, homotrimeric spike proteins that provide the machinery enabling entry into human cells [11] [12] [13] . the spike ectodomain is segregated into two regions, termed s1 and s2. the outer s1 subunit of sars-cov-2 is responsible for host recognition via interaction between its c-terminal receptor-binding domain (rbd) and human angiotensin converting enzyme 2 (ace2), present on the exterior surface of airway cells [13, 14] . while there is no known host-recognition role for the s1 n-terminal domain (ntd) of sars-cov-2, it is notable that s1 ntds of other coronaviruses have been shown to bind host surface glycans [11, 15] . in contrast to the spike subunit s1, the s2 subunit contains the membrane fusion apparatus, and also mediates trimerization of the ectodomain [11] [12] [13] . prior to host recognition, spike proteins exist in a metastable pre-fusion state, wherein the s1 subunits lay atop the s2 region and their rbds oscillate between up and down conformations that are, respectively, capable and incapable of receptor binding [11, 16, 17] . upon processing at the s1/s2 and s2' cleavage sites by host proteases as well as engagement to the receptor, the s2 subunit undergoes dramatic conformational changes from the pre-fusion to the post-fusion state. such structural rearrangements are associated with fusion of the viral envelope with host membranes, thereby allowing release of the rna genome into the cytoplasm of the host cell [18, 19] . coronavirus spike proteins are highly immunogenic [20] , and several experimental approaches have sought to target this molecule for the purpose of virus neutralization [21] . the high specificity, potency, and modular nature of antibody-based antiviral therapeutics have shown exceptional promise [22] [23] [24] , and the isolated, purified rbd has been a popular target for the development of antibodies directed against the spike proteins of pathogenic coronaviruses [25] [26] [27] [28] . however, binders of the isolated rbd may not effectively engage the aforementioned pre-fusion conformation of the spike protein, which could account for the poor neutralization ability of recently described single-domain antibodies that were raised against the rbd of sars-cov-2 spike protein [29] . therefore, to more easily identify molecules with qualities befitting a drug-like candidate, it would be advantageous to validate rbdspecific binders in the context of the full, stabilized, pre-fusion spike assembly [12, 30] . single domain antibodies based on the variable vhh domain of heavy-chain-only antibodies of camelids -generally known as nanobodies -have demonstrated great potential in several studies [31] . nanobodies are small (12) (13) (14) (15) , stable, and inexpensive to produce in large amounts in bacteria and yeast [32] , yet they bind targets in a similar affinity range as conventional antibodies. due to their minimal size, they are particularly suited to reach hidden epitopes such as crevices of target proteins [33] . we recently designed three libraries of synthetic nanobodies, termed sybodies, based on elucidated structures of nanobody-target complexes (fig. 1a) [34, 35] . sybodies can be selected against any target protein within twelve working days, which is considerably faster than the generation of natural nanobodies, which requires the repetitive immunization during a period of two months prior to binder selection by phage display [35] . a considerable advantage of our platform is that the selection of sybodies is carried out under defined conditions -in the case of coronavirus spike proteins, this offers the opportunity to generate binders recognizing the metastable pre-fusion conformation [12, 13] . finally, due to the feasibility of inhaled therapeutic nanobody formulations [36] , virus-neutralizing sybodies could offer a convenient, fast and direct means of prophylaxis. here, we identified a series of sybodies, which bind to two non-overlapping epitopes at the rbd of sars-cov-2. when fused to generate a bispecific binder format, the sybodies potently neutralize viral entry of both pseudotyped and live viruses. cryo-em analyses confirmed simultaneous binding of two sybodies and revealed a novel asymmetric spike conformation with one up rbd, one up-out rbd and one down rbd. sybodies were selected using two rbd constructs fused to additional domains (fc of mouse igg1 and vyfp, respectively). our "target swap" selection approach (fig. s1 ) resulted in two enriched pools for each of the three sybody libraries (concave, loop and convex, fig. 1a ). an off-rate selection step was performed using the pre-enriched purified sybody pool after phage display round 1 as competitor (see materials and methods). after two rounds of phage display, strong enrichment by factors ranging from 10 to 263 were determined by qpcr (table s1 ). elisa screening was performed using rbd-vyfp (rbd), commercially acquired spike ectodomain containing wild-type s1 and s2 (ecd), and maltose binding protein (mbp) as negative control. elisa analysis revealed very high hit rates for the rbd and the ecd, ranging from 81 % to 100 % and 66 % to 96 %, respectively (fig. s2 , table s1 ). at a later stage, we also performed elisas using engineered pre-fusion-stabilized spike ectodomain, containing two stabilizing proline mutations (s-2p) [12] (fig. s2 ). while most elisa signals for the ecd and s-2p were highly similar, we found around 40 sybodies with stronger binding to ecd than to s-2p, which can be explained by the fact that the s-2p forms a stable trimer, whereas the ecd lacked stabilizing proline mutations as well as the c-terminal foldon trimerization motif and therefore may be predominantly dissociated into monomers with increased internal epitope accessibility. in addition, the ecd might partially or completely adopt a post-fusion state, whereas s-2p is expected to be stabilized in the trimeric pre-fusion state [12, 13] . 72 elisa-positive sybodies were sequenced (12 for each of the 6 selection reactions numbered from sb#1-72, see also fig. s1 ). sequencing results of 70 out of 72 sybody clones were unambiguous. out of these 70 clones, 63 were found to be unique and belonged to the concave (23), loop (22) and convex (18) sybody libraries (fig. s2, fig. s3 , table s2 ). there were no duplicate binders identified in both selection variants, indicating that the two separate selection 4 streams gave rise to completely different sybody populations. two other research groups also used our sybody libraries to generate binders against the sars-cov-2 rbd [37, 38] . interestingly, there is no sequence overlap amongst binder hits in these three independent sybody generation campaigns. this demonstrates that the sybody libraries are highly diverse and suggests that identical binders must be the result of over-enrichment, likely occurring towards the end of the binder selection process (i.e., during phage display). although the high sybody sequence diversity was not unexpected due to the very large size of the sybody libraries, this unique and autonomous multi-institute sybody selection campaign clearly demonstrates that it is possible to get access to an enormous variety of binders via independent selection experiments. the 63 selected unique sybodies were individually expressed in e. coli and purified via ni-nta affinity chromatography and size exclusion chromatography. ultimately, 57 sybodies revealed appropriate biochemical features with respect to solubility, yield, and monodispersity, in order to proceed with further characterization. for an in vitro kinetic analysis of sybody interactions with the viral spike, we employed grating-coupled interferometry (gci) [39] to probe sybody binding to immobilized rbd-vyfp. first, the 57 purified sybodies were subjected to an off-rate screen, which revealed six sybodies (sb#14, sb#15, sb#16, sb#42, sb#45, and sb#68) with strong binding signals and comparatively slow off-rates. binding constants were then determined by measuring on-and off-rates over a range of sybody concentrations, revealing affinities for rbd within a range of 20-180 nm using a langmuir 1:1 model for data fitting (fig. s4a ). next, we evaluated the ability of the 57 purified sybodies to compete with ace2 binding by elisa. to this end, binding of purified rbd to immobilized hace2 was measured in the presence or absence of an excess of each purified sybody ( fig. 2a) . nearly all sybodies were found to inhibit rbd-hace2 interaction. the signal decrease relative to unchallenged rbd was modest for most sybodies, with an average signal reduction of about 50 %. however, five sybodies (sb#14, sb#15, sb#16, sb#42, and sb#45) reduced rbd-attributable elisa signal to near-background levels, implying that these binders were able to almost entirely abolish the interaction between rbd and hace2. notably, these five hace2-inhibiting sybodies were among the six aforementioned highest affinity rbd binders. we sought to determine if our set of sybodies recognized separate epitopes on the rbd surface. elisa experiments demonstrated that incubation of sb#68 with s-2p only slightly diminished the ability of the spike from binding to immobilized sb#15, whereas pre-incubation with sb#14, sb#15, sb#16, sb#42, or sb#45 almost completely prevented the interaction of the spike protein with immobilized sb#15 (fig. s5 ). this suggested that sb#15 and sb#68 can bind simultaneously to the spike. therefore, we characterized sb#15 and sb#68 in more detail and performed gci measurements with the rbd (as a repetition of the initial experiments), as well as s-2p and an even further stabilized version of the spike protein containing six prolines (hexapro [40] ), termed here s-6p (fig. 1b, fig. s4b ). in contrast to the data generated using rbd, for which the langmuir 1:1 model was used to fit the data, the experimental data for s-2p and s-6p could only be fitted adequately using a heterogenous ligand model, which accounts for a high and a low affinity binding site. as our cryo-em analysis revealed binding of three sb#15 molecules and two sb#68 molecules to a highly asymmetric spike trimer (see below), the heterogenous ligand model could be justified. in the case of sb#15, the higher binding affinities (kd1) for s-2p and s-6p (12 nm and 15 nm, respectively) were found to be similar to the one determined for the rbd (14 nm). in contrast, kd1 of sb#68 was more than 10-fold stronger for s-2p and s-6p (9 nm and 6 nm, respectively) than for rbd (120 nm) (fig. 1b, fig. s4b ). to investigate if both sybodies can also bind simultaneously in the context of the trimeric full-length spike protein, we used gci to monitor binding events of the sybodies injected either alone or in combination (fig. 1c) . when we analyzed the sybodies against coated rbd, the maximal binding signals for sb#15 (12 pg/mm 2 ) and sb#68 (10 pg/mm 2 ) were approximately additive when both sybodies were co-injected (21 pg/mm 2 ), clearly showing that both sybodies can bind simultaneously. interestingly, when the same analysis was performed using s-2p and s-6p, the binding signals of the co-injections (64 pg/mm 2 for s-2p and 50 pg/mm 2 for s-6p) were clearly greater than the sum of the binding signals of sb#15 and sb#68 when injected individually (26 pg/mm 2 and 27pg/mm 2 for s-2p and 18 pg/mm 2 and 24 pg/mm 2 for s-6p). this suggests cooperative binding of the two sybodies to the full-length spike protein, but not of the isolated rbd. to investigate interference of sb#15 and sb#68 with ace2 binding in detail, we performed an ace2 competition experiment using gci. to this end, s-2p was coated on a gci chip and sb#15 (200 nm), sb#68 (200 nm) and the non-randomized convex sybody control (sb#0, 200 nm) were injected alone or together with ace2 (100 nm) to monitor binding (fig. 2b) . indeed, sb#0 did not bind when injected alone and consequently did not disturb ace2 binding when co-injected. conversely, both sb#15 and sb#68 were found to dominate over ace2 in the association phase during co-injection, and the resulting curves are highly similar to what was observed when these two sybodies were injected alone. this experiment unequivocally demonstrates a strong competition of ace2 binding by the two sybodies using s-2p as target. ace2 competition by sb#68 to this extent was surprising in view of the initial ace2 elisa competition experiment ( fig. 2a) . however, the seeming discrepancy can be explained by our observation that the affinity of sb#68 for s-2p (used in the gci experiment) is more than 10 times stronger than for the isolated rbd (used in the elisa experiment). to determine the inhibitory activity of the identified sybodies, we conducted in vitro neutralization experiments. towards this aim, we employed engineered vesicular stomatitis viruses (vsv) that were pseudotyped with sars-cov-2 spikes [41] . interestingly, only the high affinity sybodies (sb#14 and sb#15), which also efficiently blocked receptor binding, exhibited potent neutralizing activity with ic50 values of 2.8 µg/ml (178 nm) and 2.3 µg/ml (147 nm), respectively (fig. 3a , table 1 ). in contrast, sb#16 and sb#45 inhibited pseudotyped vsvs only to a limited extent. in agreement with the high affinity of sb#68 for soluble spike and its ability to compete with ace2 in the context of s-2p as determined by gci, the ic50 values were similar to those observed for sb#15 (2.3 µg/ml, 138 nm). since sb#15 and sb#68 can bind simultaneously to the rbd and the full-length spike protein, we mixed sb#15 and sb#68 together to investigate potential additive or synergistic neutralizing activity of these two independent sybodies. indeed, consistent with the binding assays, the simultaneous presence of both sybodies resulted in improved neutralization profiles with ic50 values reaching 1.7 µg/ml (53 nm) (fig. 3a , table 1 ). note that no neutralization of the pseudotype virus was observed in a control experiment using a nanobody directed to mcherry at the highest concentration (100 µg/ml), thus validating the specificity of the identified sybodies. in addition to the individual sybodies, we also explored potential avidity effects of sybodies genetically fused to human igg1 fc domains. the respective sybody-fc constructs (sb#14-fc, sb#15-fc, sb#16-fc, sb#45-fc and sb#68-fc) exhibited vsv pseudotype ic50 values in the range of 0.6 to 3.9 µg/ml (8 nm to 50 nm) and were therefore clearly improved over the respective values of the sybodies alone, which ranged from 2.3 to 20 µg/ml (138 nm to 1250 nm) ( table 1 ). this suggests that the bivalent arrangement of the fc fusion constructs resulted in a discernible avidity effect. it is interesting to note that for some sybodies the gain of neutralization potency was much higher (e.g. for sb#16, the ic50 values for single sybody versus fc-fused sybodies were 1250 nm versus 8 nm), whereas for others it was only modest (e.g. for sb#68, the respective values were 138 nm versus 50 nm). this indicates that the avidity effect strongly depends on the binding epitope. next, the neutralizing activity of the various sybodies was assessed with live sars-cov-2 (strain münchen-1.1/2020/929) [42] employing a 50% neutralization dose (nd50) assay (table 1) . sybodies which exhibited the least potent neutralization activities in the pseudotyped vsv assays (sb#14, sb#16 and sb#45), did not block sars-cov-2 infection. in sharp contrast, sb#15 and sb#68 successfully inhibited sars-cov-2 cell entry, with nd50 values of 37.4 and 34.6 µg/ml, respectively. with the exception of sb#14, the overall neutralization data obtained with live sars-cov-2 virus corroborated the findings obtained with the pseudotyped vsv system, although the sybodies were less potent against live sars-cov-2. the binding and neutralization data, as well as the structural data presented below, highlighted that sb#15 and sb#68 are (i) the most potent neutralizing sybodies; (ii) bind to non-overlapping epitopes on the rbd surface; and (iii) exhibit synergistic virus neutralizing effects. these findings provided the basis to investigate whether fusing both sybodies would further improve the neutralization potency. towards this aim, we engineered three constructs consisting of sb#15 and sb#68 fused via a flexible linker (ggggs) of various length (repetitions of 2x, 4x or 6x) (fig. 4a ). the resulting bi-specific sybodies were accordingly designated gs2, gs4 and gs6, respectively. the binding kinetics of these three bispecific sybodies were then analyzed by gci using coated s-6p (fig. 4b) , and binding affinities were found to range between 218 pm to 330 pm (using a langmuir 1:1 fitting model). this pronounced improvement of the affinity of the bispecific sybodies over the individual binders indicated that the two sybodies of the fused construct bind simultaneously to the spike protein, thereby resulting in a strong avidity effect. in agreement with the improved affinity, all three engineered bispecific constructs displayed highly potent neutralizing activities against both pseudotyped virus and live sars-cov-2 (ic50 values of gs2: 0.03 µg/ml (1 nm), gs4: 0.02 µg/ml (0.7 nm) and gs6: 0.04 µg/ml (1.3 nm) (fig. 4c , table 1 ). for live sars-cov-2 virus, nd50 values of gs2: 1.6 µg/ml (54 nm), gs4: 0.79 µg/ml (26 nm) and gs6: 1.0 µg/ml (32 nm) were determined (table 1) . collectively, these data show that fusing sb#15 and sb#68 via flexible linkers results in bispecific sybodies with dramatically improved neutralization activity (by a factor of about 100 times compared to the single binders). to gain structural insights into how sb#15 and sb#68 recognize the rbd, we performed single particle cryo-em analysis of the spike protein in complex with the sybodies. to generate complexes, sybodies (alone or in combination) were mixed with spike protein at a molar ratio of 1.3:1 (sybody:spike monomer), prior to a final purification step using size-exclusion chromatography. in total, three cryo-em datasets were collected, allowing a glimpse of the spike protein either simultaneously bound to both sybodies, or associated to sb#15 or sb#68 alone ( fig. s6-8 , table s3 ). the highest resolution was obtained for the spike protein in complex with both sybodies (fig. s6 ). in contrast, the structures with the individual sybodies were determined based on fewer particles and mainly served to unambiguously assign the binding epitopes of sb#15 (fig. s7 ) and sb#68 (fig. s8 ). although the global resolution of the spike protein in complex with both sybodies is around 3 å, the local resolution of the rbds with bound sybodies was only in the range of 6-7 å, presumably due to conformational flexibility (fig. s6) . therefore, we did not build full models of the sybodies and provide details only on their interaction surface with the rbds. however, the cryo-em density is good enough to describe the general epitope location and the distinct conformations adopted by the rbds. for better assessment and visualization, we fitted homology models of the respective sybodies into the densities ( fig. s9 -s11). the sybody homology models were based on pdb:3k1k [43] in case of the concave sb#15 and pdb:5m13 [34] for the convex sb#68. analysis of the spike/sb#15/sb#68 particles after 3d classification revealed that the spike protein adopts two distinct conformations (fig. s6 ). the first conformation (30% of particles) has a three-fold symmetry, with three rbds in the up conformation (3up) and two sybodies bound to each of the rbds, confirming that sb#15 and sb#68 bind simultaneously (fig. 5a, fig. s6c , f and s9a). according to the spike structure obtained with sb#15 alone (detailed analysis below, fig. s7 and s10), sb#15 binds to the top of the rbd. its binding epitope consists of two regions (residues 444-448 and 491-507) and thereby strongly overlaps with the ace2 binding site (fig. 5b ). in contrast, sb#68 binds to the side of the rbd ( fig. s8 and s11d-e) and recognizes a conserved epitope [44] clearly distinct from the ace2 interaction site, which includes residues 369-381 and 408-411 and is buried if the rbd is in its down conformation. although the binding epitope of sb#68 is clearly distinct from the one of ace2, there would be a steric clash between the sb#68 backside loops and ace2, if ace2 docks to the rbd (fig. 5b ). this accounts for sb#68's ability to compete with ace2 as evident from gci analyses (fig. 2b ). the second resolved conformation (20 % of particles) of the spike/sb#15/sb#68 complex is asymmetric with the rbds in three distinct states, and was obtained at a global resolution of 3.3 å (fig. 5c, fig. s6c , g and s9b). in this case, three sb#15 and two sb#68 were bound. the first rbd was in the up conformation, having sb#15 and sb#68 bound in an analogous fashion as in the symmetric 3up structure. the second rbd adopted a down state with only sb#15 bound. this conformation of sb#15bound rbd appears to act as a wedge, pushing the third rbd outward and away from the three-fold symmetry axis (fig. 5d ). the third rbd was in an up-out conformation with sb#15 and sb#68 bound. however, the density for sb#68 was very weak, indicating either a very high flexibility or a substoichiometric occupancy. we refer to this novel asymmetric spike conformation as a 1up/1upout/1down state (fig. 5c ). virtually the same asymmetric 1up/1up-out/1down spike conformation was observed for the spike/sb#15 complex, reinforcing our interpretation that wedging by sb#15 is responsible for the outward movement of the second up-rbd (fig. s10 ). however, according to our analysis, comprising 8 only a limited number of images (fig. s7d ), sb#15 alone was unable to induce the 3up conformation, suggesting that adoption of the 3up state requires the synergistic action of both sybodies to populate this symmetric conformation. finally, analysis of the spike/sb#68 complex dataset revealed two distinct populations ( figure s8 and s11). the most abundant class showed an 1up2down conformation without sybody bound, which is identical to the one obtained for the spike protein alone [12, 13] . the second structure featured two rbds in an up conformation with bound sb#68. density for the third rbd was very weak, presumably due to high intrinsic flexibility, hindering the interpretation of its exact position and conformation. we therefore refer to this conformation as an 2up/1flexible state. structural comparisons revealed that sb#68 cannot access its epitope in the context of the 1up2down conformation, due to steric clashes with the neighboring rbd (fig. s11b ). in order to bind, at least two rbds need to be in the up conformation. in summary, both sybodies stabilized the up conformation of the rbds. notably, without sybodies, s-2p predominantly assumes an equilibrium between the 3down and the 1up2down conformation [12, 13] . upon addition of sb#15, the conformational equilibrium was shifted towards an asymmetric 1up/1up-out/1down state, whereas addition of sb#68 favored an asymmetric state with rbds adopting a 2up/1flexible conformation. when added together, the sybodies appear to synergistically act to stabilize two states: a predominant 3up state, as well as the asymmetric 1up/1up-out/1down state. in this work, we have demonstrated the ability of our rapid in vitro selection platform to generate sybodies directed to the sars-cov-2 rbd. the biochemical characterization of these sybodies led to the identification of a high-affinity subset of binders, which were further analyzed in depth using structural, biochemical and functional methods. thereby, we found a pair of sybodies, sb#15 and sb#68, which bind simultaneously to the rbd. both sybodies were found to compete with ace2 binding, albeit likely through different mechanisms. while the binding epitope of sb#15 directly overlaps with the one of ace2, this is not the case for sb#68, which interferes with ace2 through a steric clash at the sybody backside (fig. 5b ). in agreement with their similar affinities for the s-2p spike protein, sb#15 and sb#68 exhibited similar neutralization efficiencies in the range of 2.3 -2.8 µg/ml (140 nm). we noted a moderate synergistic effect in the virus neutralization test when both individual sybodies were mixed together, resulting in an improved ic50 of 1.7 µg/ml (53nm). this synergy can be explained by the concerted action of the sybodies to compete with ace2 docking via epitope blockage and steric clashing. cryo-em analyses revealed distinct binding epitopes for the two sybodies sb#15 and sb#68. the s-2p spike protein we used for cryo-em was shown to predominantly adopt the 3down and 1up/2down conformations [12, 13] , whereas the s-2p/sb#15/sb#68 complex adopts either a novel 1up/1upout/1down or a 3up conformation. the structures further revealed that sb#68 can only bind to the up-rbd. the inability of sb#68 (and to some degree also sb#15) to bind to the down-rbd resulted in conformational selection of spike protein with at least two up rbds, thereby shifting the conformational equilibrium of the spike. it is interesting to note that the binding epitope of sb#68 is highly conserved between sars-cov-1 and sars-cov-2, because it constitutes an interaction interface that, upon binder engagement, stabilizes the rbd in the down conformation. the same conserved epitope is also recognized by the human antibodies cr3022 (isolated from a sars-cov-1 infected patient and showing cross-specificity against sars-cov-2) and ey6a (vice versa) [44, 45] (fig. 6) . hence, the binding epitope of sb#68 is less likely to be remodelled due to drug-induced selection pressures, thereby limiting the evolution of sars-cov-2 escape mutants if sb#68 were to be used as a therapeutic antiviral drug. despite sharing a similar epitope on the rbd, cr3022 and ey6a do not display an obvious direct steric clash with ace2 and in contrast to sb#68 do not compete directly with ace2 binding (fig. 6 ). since cr3022 and ey6a have strong neutralizing capacity, inhibition mechanisms in addition to ace2 blockage could exist, which may also apply for sb#15 and sb#68. however, for the ey6a antibody it has been proposed that surface glycans on ace2 may interact with ey6a and at least partially account for its neutralizing effect [44] . akin to the cr3022 and ey6a antibodies, our sybodies share the ability to stabilize spike conformations with 2-or 3-up rbds. thereby, the spike protein may be destabilized, resulting in the premature and unproductive transitions to the irreversible post-fusion state. this mechanism was dubbed "receptor mimicry" in a study on a neutralizing antibody s230, which only bound to up-rbds and thereby triggered fusogenic conformational changes of sars-cov-1 spike [19] . however, since we obtained well-resolved cryo-em structures with sb#15 and sb#68 bound to the spike after incubating the complex for more than 3 hours, we consider the mechanism of receptor mimicry less plausible in our case. yet, it is important to note that recent investigations of nonengineered sars-cov-2 spike protein extracted from membranes by detergents revealed unique structural features not found in the stabilized pre-fusion spike, including a stronger compaction of the spike trimer and the pre-dominance of the 3-down rbd conformation [46] . further, the study highlighted a high propensity of the native sars-cov-2 spike to spontaneously transit to the postfusion state without interacting with ace2. therefore, it is still possible that the sybodies (and in particular sb#68) accelerate these spontaneous spike inactivation process in the context of live sars-cov-2 virus, without affecting the pre-fusion stabilized soluble spike protein used for cryo-em analyses. the recent months have brought about a large number of publications on neutralizing antibodies [47] [48] [49] [50] , nanobodies [37, 38, 51, 52] and other binder scaffolds [53] . for the smaller scaffolds, in particular in case of nanobodies, fusion of binders via flexible linkers emerged as a promising strategy to improve neutralization efficiencies by exploiting avidity effects in the context of the trimeric spike protein. however, strategies to exploit genetically fused nanobodies so far included only identical binders recognizing the same epitope on the rbds [54] . a crucial issue regarding development of reliable therapeutics against enveloped rna viruses such as sars-cov-2 is their ability to rapidly develop resistance mutations. recently, the emergence of resistance against monoclonal antibodies targeting the sars-cov-2 spike-rbd was investigated in vitro [50] . while drug-resistant viruses indeed emerged rapidly when such antibodies with overlapping epitopes were administered either individually or in combination, escape mutants were not generated when treated with cocktails of non-competing antibodies. because the neutralizing sybody pair (sb#15/sb#68) identified in this study was found to simultaneously bind to two spatially-distinct epitopes on the spike-rbd (of which one is highly conserved among sarbecoviruses [44] ), we anticipate that our rationally engineered single-format bispecific constructs, which displayed highly potent neutralization profiles, may also exhibit high resistance barriers. although monoclonal antibodies (mabs) hold great promise in modern medicine, their manufacture remains tedious, time-consuming and expensive. in addition, the administration of mabs must be performed by medical professionals at hospitals, which further hampers their fast and global availability. conversely, single domain antibodies and their derivative multi-component formats can be produced easily, quickly, and inexpensively in bacteria, yeast, or mammalian cell culture. furthermore, the biophysical properties of single domain antibodies make them feasible for development in an inhalable formulation, thereby not only enabling direct delivery to nasal and lung tissues (two key sites of sars-cov-2 replication), but also offering the potential of self-administration. overall, we present a robust platform to generate highly potent multi-specific biomolecules against coronaviruses. in particular, the rapid selection of sybodies and their swift biophysical, structural and functional characterization, provide a foundation for the accelerated reaction to potential future pandemics. finally, our recently described flycode technology can be utilized for deeper interrogation of sybody selection pools, in order to facilitate discovery of exceptional sybodies possessing very slow off-rates or recognizing rare epitopes [55] . a gene encoding sars-cov-2 residues pro330-gly526 (rbd, genbank accession qhd43416.1), downstream from a modified n-terminal human serum albumin secretion signal [56] , was chemically synthesized (geneuniversal). this gene was subcloned using fx technology [57] into a custom mammalian expression vector [58] , appending a c-terminal 3c protease cleavage site, myc tag, venus yfp [59] , and streptavidin-binding peptide [60] onto the open reading frame (rbd-vyfp). 100-250 ml of suspension-adapted expi293 cells (thermo) were transiently transfected using expifectamine according to the manufacturer protocol (thermo), and expression was continued for 4-5 days in a humidified environment at 37°c, 8% co2. cells were pelleted (500g, 10 min), and culture supernatant was filtered (0.2 µm mesh size) before being passed three times over a gravity column containing nhsagarose beads covalently coupled to the anti-gfp nanobody 3k1k [43] , at a resin:culture ratio of 1ml resin per 100ml expression culture. resin was washed with 20 column-volumes of rbd buffer (phosphate-buffered saline, ph 7.4, supplemented with additional 0.2m nacl), and rbd-vyfp was eluted with 0.1 m glycine, ph 2.5, via sequential 0.5 ml fractions, without prolonged incubation of resin with the acidic elution buffer. fractionation tubes were pre-filled with 1/10 vol 1m tris, ph 9.0 (50 µl), such that elution fractions were immediately ph-neutralized. fractions containing rbd-vyfp were pooled, concentrated, and stored at 4°c. purity was estimated to be >95%, based on sds-page (not shown). yield of rbd-vyfp was approximately 200-300 μg per 100 ml expression culture. a second purified rbd construct, consisting of sars-cov-2 residues arg319-phe541 fused to a murine igg1 fc domain (rbd-fc) expressed in hek293 cells, was purchased from sino biological (catalogue number: 40592-v05h, 300 µg were ordered). purified full-length spike ectodomain (ecd) comprising s1 and s2 (residues val16-pro1213) with a c-terminal his-tag and expressed in baculovirus-insect cells was purchased from sino biological (catalogue number: 40589-v08b1, 700 µg were ordered). the prefusion ectodomain of the sars-cov2 spike protein containing two stabilizing proline mutations (s-2p) (residues 1-1208) [12] , was transiently transfected into 50x10 8 suspension-adapted expicho cells (thermo fisher) using 3 mg plasmid dna and 15 mg of pei max (polysciences) per 1l procho5 medium (lonza) in a 3l erlenmeyer flask (corning) in an incubator shaker (kühner). one hour post-transfection, dimethyl sulfoxide (dmso; applichem) was added to 2% (v/v). incubation with agitation was continued at 31°c for 5 days. 1l of filtered (0.22 um) cell culture supernatant was clarified. then, a 1ml gravity flow strep-tactin®xt superflow® column (iba lifescience) was rinsed with 2 ml buffer w (100 mm tris, ph 8.0, 100 mm nacl, 1 mm edta) using gravity flow. the supernatant was added to the column, which was then rinsed with 5 ml of buffer w (all with gravity flow). finally, six elution steps were performed by adding each time 0.5 ml of buffer bxt (50mm biotin in buffer w) to the resin. all purification steps were performed at 4°c. to remove amines, all proteins were first extensively dialyzed against rbd buffer. proteins were concentrated to 25 µm using amicon ultra concentrator units with a molecular weight cutoff of 30 -50 kda. subsequently, the proteins were chemically biotinylated for 30 min at 25°c using nhs-biotin (thermo fisher, #20217) added at a 10-fold molar excess over target protein. immediately after, the three samples were dialyzed against tbs ph 7.5. during these processes (first dialysis/concentration/biotinylation/second dialysis), 20%, 30%, 65% and 44% of the rbd-vyfp, rbd-fc, ecd and s-2p respectively were lost due to adsorption to the concentrator filter or due to aggregation. biotinylated rbd-vyfp, rbd-fc and ecd were diluted to 5 µm in tbs ph 7.5, 10 % glycerol and stored in small aliquots at -80°c. biotinylated s-2p was stored at 4°c in tbs ph 7.5. sybody selections with the three sybody libraries concave, loop and convex were carried out as previously detailed [35] . in short, one round of ribosome display was followed by two rounds of phage display. binders were selected against two different constructs of the sars-cov-2 rbd; an rbd-vyfp fusion and an rbd-fc fusion. mbp was used as background control to determine the enrichment score by qpcr [35] . in order to avoid enrichment of binders against the fusion proteins (yfp and fc), we switched the two targets after ribosome display (fig. s1 ). for the off-rate selections we did not use non-biotinylated target proteins as described [35] because we did not have the required amounts of purified target protein. instead, we employed a pool competition approach. after the first round of phage display, all three libraries of selected sybodies, for both target-swap selection schemes, were subcloned into the psb_init vector (giving approximately 10 8 clones) and expressed in e. coli mc1061 cells. the resulting three expressed pools were subsequently combined, giving one sybody pool for each selection scheme. these two final pools were purified by ni-nta affinity chromatography, followed by buffer exchange of the main peak fractions using a desalting pd10 column in tbs ph 7.5 to remove imidazole. the pools were eluted with 3.2 ml of tbs ph 7.5. these two purified pools were used for the off-rate selection in the second round of phage display at concentrations of approximately 390 µm for selection variant 1 (competing for binding to rbp-fc) and 450 µm for selection variant 2 (competing for binding to rbp-yfp). the volume used for off-rate selection was 500 µl, with 0.5% bsa and 0.05% tween-20 added to pools immediately prior to the competition experiment. off-rate selections were performed for 3 minutes. elisas were performed as described in detail [35] . 47 single clones were analyzed for each library of each selection scheme. since the rbd-fc construct was incompatible with our elisa format due to the inclusion of protein a to capture an α-myc antibody, elisa was performed only for the rbd-vyfp (50 nm) and the ecd (25 nm) and later on with the s-2p (25 nm). of note, the three targets were analyzed in three separate elisas. as negative control to assess background binding of sybodies, we used biotinylated mbp (50 nm). 72 positive elisa hits were sequenced (microsynth, switzerland). the 63 unique sybodies were expressed and purified as described [35] . in short, all 63 sybodies were expressed overnight in e.coli mc1061 cells in 50 ml cultures. the next day the sybodies were extracted from the periplasm and purified by ni-nta affinity chromatography (batch binding) followed by sizeexclusion chromatography using a sepax srt-10c sec100 size-exclusion chromatography (sec) column equilibrated in tbs, ph 7.5, containing 0.05% (v/v) tween-20 (detergent was added for subsequent kinetic measurements). six out of the 63 binders (sb#4, sb#7, sb#18, sb#34, sb#47, sb#61) were excluded from further analysis due to suboptimal behavior during sec analysis (i.e. aggregation or excessive column matrix interaction). to generate the bispecific sybodies (sb#15-sb#68 fusion with variable glycine/serine linkers), sb#15 was amplified from psb-init_sb#15 (addgene #153523) using the forward primer atatatgctcttcaagtcaggttc and the reverse primer tatatagctcttcaagaaccgccaccgccgctaccgccaccacctgcgctcacagtcac, encoding 2x a ggggs motif, followed by a sapi cloning site. sb#68 was amplified from psb-init_sb#68 (addgene #153527) using forward primers 1 (atatatgctcttcttctcaagtccagctggtgg), 2 (atatatgctcttcttctggtggtggcggtagcggcggtggcggtagtcaagtccagctggtgg) or 3 (atatatgctcttcttctggtggtggcggtagcggcggtggcggttctggtggtggcggtagcggcggtggc ggtagtcaagtccagctggtgg) each combined with the reverse primer tatatagctcttcctgcagaaac. the forward primers start with a sapi site (compatible overhang to sb#15 reverse primer), followed by non, 2x or 4x the ggggs motif. the pcr product of sb#15 was cloned in frame with each of the three pcr products of sb#68 into psb-init using fx-cloning [57] , thereby resulting in three fusion constructs with linkers containing 2x, 4x or 6x ggggs motives as flexible linkers between the sybodies (called gs2, gs4 and gs6, respectively). the three bispecific fusion constructs gs2, gs4 and gs6 were expressed and purified the same way as single sybodies [35] . the high affinity sybodies were cloned and produced as human igg1 fc-fusions by absolute antibody, where they are commercially available. purified recombinant hace2 protein (mybiosource, cat# mbs8248492) was diluted to 10 nm in phosphate-buffered saline (pbs), ph 7.4, and 100 μl aliquots were incubated overnight on nunc maxisorp 96-well elisa plates (thermofisher #44-2404-21) at 4°c. elisa plates were washed three times with 250 μl tbs containing 0.05% (v/v) tween-20 (tbst). plates were blocked with 250 μl of 0.5% (w/v) bsa in tbs for 2 h at room temperature. 100 μl samples of biotinylated rbd-vyfp (25 nm) mixed with individual purified sybodies (500 nm) were prepared in tbs containing 0.5% (w/v) bsa and 0.05% (v/v) tween-20 (tbs-bsa-t) and incubated for 1.5 h at room temperature. these 100 μl rbd-sybody mixtures were transferred to the plate and incubated for 30 minutes at room temperature. 100 μl of streptavidin-peroxidase (merck, cat#s2438) diluted 1:5000 in tbs-bsa-t was incubated on the plate for 1 h. finally, to detect bound biotinylated rbd-vyfp, 100 μl of development reagent containing 3,3′,5,5′-tetramethylbenzidine (tmb), prepared as previously described [35] , was added, color development was quenched after 3-5 min via addition of 100 μl 0.2 m sulfuric acid, and absorbance at 405 nm was measured. background-subtracted absorbance values were normalized to the signal corresponding to rbd-vyfp in the absence of added sybodies. purified sybodies carrying a c-terminal myc-his tag (sb_init expression vector) were diluted to 25 nm in 100 µl pbs ph 7.4 and directly coated on nunc maxisorp 96-well plates (thermofisher #44-2404-21) at 4°c overnight. the plates were washed once with 250 µl tbs ph 7.5 per well followed by blocking with 250 µl tbs ph 7.5 containing 0.5% (w/v) bsa per well. in parallel, chemically biotinylated prefusion spike protein (s-2p) at a concentration of 10 nm was incubated with 500 nm sybodies for 1 h at room temperature in tbs-bsa-t. the plates were washed three times with 250 µl tbs-t per well. then, 100 µl of the s-2p-sybody mixtures were added to the corresponding wells and incubated for 3 min, followed by washing three times with 250 µl tbs-t per well. 100 µl streptavidin-peroxidase polymer (merck, cat#s2438) diluted 1:5000 in tbs-bsa-t was added to each well and incubated for 10 min, followed by washing three times with 250 µl tbs-t per well. finally, to detect s-2p bound to the immobilized sybodies, 100 µl elisa developing buffer (prepared as described previously [35] ) was added to each well, incubated for 1 h (due to low signal) and absorbance was measured at 650 nm. as a negative control, tbs-bsa-t devoid of protein was added to the corresponding wells instead of a s-2p-sybody mixture. kinetic characterization of sybodies binding onto sars-cov-2 spike proteins was performed using gci on the wavesystem (creoptix ag, switzerland), a label-free biosensor. for the off-rate screening, biotinylated rbd-vyfp and ecd were captured onto a streptavidin pcp-sta wavechip (polycarboxylate quasi-planar surface; creoptix ag) to a density of 1300-1800 pg/mm 2 . sybodies were first analyzed by an off-rate screen performed at a concentration of 200 nm (data not shown) to identify binders with sufficiently high affinities. the six sybodies sb#14, sb#15, sb#16, sb#42, sb#45, and sb#68 were then injected at increasing concentrations ranging from 1.37 nm to 1 μm (three-fold serial dilution, 7 concentrations) in 20 mm tris ph7.5, 150 mm nacl supplemented with 0.05 % tween-20 (tbs-t buffer). sybodies were injected for 120 s at a flow rate of 30 μl/min per channel and dissociation was set to 600 s to allow the return to baseline. in order to determine the binding kinetics of sb#15 and sb#68 against intact spike proteins, the ligands rbd-vyfp, s-2p and s-6p were captured onto a pcp-sta wavechip (creoptix ag) to a density of 750 pg/mm 2 , 1100 pg/mm 2 and 850 pg/mm 2 respectively. sb#15 and sb#68 were injected at concentrations ranging from 1.95 nm to 250 nm or 3.9 nm to 500 nm, respectively (2-fold serial dilution, 8 concentrations) in tbs-t buffer. sybodies were injected for 200 s at a flow rate of 80 μl/min and dissociation was set to 600 s. in order to investigate if sb#15 and sb#68 bind simultaneously to the rbd, s-2p and s-6p, both binders were either injected alone at a concentration of 200 nm or mixed together at the same individual concentrations at a flow rate of 80 μl/min for 200 s in tbs-t buffer. to measure binding kinetics of the three bispecific fusion constructs, gs2, gs4 and gs6, s-6p was captured as described above to a density of 1860 pg/mm 2 and increasing concentrations of the bispecific fusion constructs ranging from 1 nm to 27 nm (3-fold serial dilution, 4 concentrations) in tbs-t buffer at a flow rate of 80 μl/min. because of the slow off-rates, we performed a regeneration protocol by injecting 10 mm glycine ph 2 for 30 s after every binder injection. for ace2 competition experiments, s-2p was captured as described above. then sb#15, sb#68 or sb#0 (non-randomized convex sybody control) were either injected individually or premixed with ace2 in tbs-t buffer. sybody concentrations were at 200 nm and ace2 concentration was at 100 nm. all sensorgrams were recorded at 25 °c and the data analyzed on the wavecontrol (creoptix ag). data were double-referenced by subtracting the signals from blank injections and from the reference channel. a langmuir 1:1 model was used for data fitting with the exception of the sb#15 and sb#68 binding kinetics for the s-2p and the s-6p spike, which were fitted with a heterogeneous ligand model as mentioned in the main text. pseudovirus neutralization assays have been previously described [30, 41, 61] . briefly, propagationdefective, spike protein-pseudotyped vesicular stomatitis virus (vsv) was produced by transfecting hek-239t cells with sars-cov-2 sdel 18 (sars-2 s carrying an 18 aa cytoplasmic tail truncation) as described previously [62] . the cells were further inoculated with glycoprotein g trans-complemented vsv vector (vsv*g(luc)) encoding enhanced green fluorescence protein (egfp) and firefly luciferase reporter genes but lacking the glycoprotein g gene [63]. after 1 h incubation at 37 °c, the inoculum was removed and the cells were washed once with medium and subsequently incubated for 24 h in medium containing 1:3000 of an anti-vsv-g mab i1 (atcc, crl-2700 tm ). pseudotyped particles were then harvested and cleared by centrifugation. for the sars-cov-2 pseudotype neutralization experiments, pseudovirus was incubated for 30 min at 37 °c with different dilutions of purified sybodies, sybdody fusions or sybody-fc fusions. subsequently, s protein-pseudotyped vsv*g(luc) was added to vero e6 cells grown in 96-well plates (25'000 cells/well). at 24 h post infection, luminescence (firefly luciferase activity) was measured using the one-glo luciferase assay system (promega) and cytation 5 cell imaging multi-mode reader (biotek). the serial dilutions of control sera and samples were prepared in quadruplicates in 96-well cell culture plates using dmem cell culture medium (50 µl/well). to each well, 50 µl of dmem containing 100 tissue culture infectious dose 50% (tcid50) of sars-cov-2 (sars-cov-2/münchen-1.1/2020/929) were added and incubated for 60 min at 37°c. subsequently, 100 µl of vero e6 cell suspension (100,000 cells/ml in dmem with 10% fbs) were added to each well and incubated for 72 h at 37 °c. the cells were fixed for 1 h at room temperature with 4% buffered formalin solution containing 1% crystal violet (merck, darmstadt, germany). finally, the microtiter plates were rinsed with deionized water and immune serum-mediated protection from cytopathic effect was visually assessed. neutralization doses 50% (nd50) values were calculated according to the spearman and kärber method. freshly purified s-2p was incubated with a 1.3-fold molar excess of sb#15 alone or with sb#15 and sb#68 and subjected to size exclusion chromatography to remove excess sybody. in analogous way, the sample of s-6p with sb#68 was prepared. the protein complexes were concentrated to 0.7-1 mg ml -1 using an amicon ultra-0.5 ml concentrating device (merck) with a 100 kda filter cut-off. 2.8 μl of the sample was applied onto the holey-carbon cryo-em grids (au r1.2/1.3, 300 mesh, quantifoil), which were prior glow discharged at 5 -15 ma for 30 s, blotted for 1-2 s and plunge frozen into a liquid ethane/propane mixture with a vitrobot mark iv (thermo fisher) at 15 °c and 100% humidity. samples were stored in liquid nitrogen until further use. screening of the grid for areas with best ice properties was done with the help of a home-written script to calculate the ice thickness (manuscript in preparation). cryo-em data in selected grid regions were collected in-house on a 200-kev talos arctica microscope (thermo fisher scientifics) with a post-column energy filter (gatan) in zero-loss mode, with a 20-ev slit and a 100 μm objective aperture. images were acquired in an automatic manner with serialem on a k2 summit detector (gatan) in counting mode at ×49,407 magnification (1.012 å pixel size) and a defocus range from −0.9 to −1.9 μm. during an exposure time of 9 s, 60 frames were recorded with a total exposure of about 53 electrons/å 2 . on-the-fly data quality was monitored using focus [64] . for the s-2p/sb#15/ sb#68 complex dataset, in total 14,883 micrographs were recorded. beaminduced motion was corrected with motioncor2_1.2.1 [65] and the ctf parameters estimated with ctffind4.1.13 [66] . recorded micrographs were manually checked in focus (1.1.0), and micrographs, which were out of defocus range (<0.4 and >2 μm), contaminated with ice or aggregates, and with a low-resolution estimation of the ctf fit (>5 å), were discarded. 637,105 particles were picked from the remaining 12,454 micrographs by cryolo 1.7.5 [67] , and imported in cryosparc v2.15.0 [68] for 2d classification with a box size of 300 pixels. after 2d classification, 264,082 particles were imported into relion-3.0.8 [69] and subjected to a 3d classification without imposed symmetry, where an ab-initio generated map from cryosparc low-pass filtered to 50 å was used as reference. two classes resembling spike protein, revealed two distinct conformations. one class shows a symmetrical state with all rbds in an up conformation (3up) and both sybodies bound to each rbd (78,933 particles, 30%). in the asymmetrical class (52,839 particles, 20%) the rbds adopt one up, one up-out and one down conformation (1up/1up-out/1down), where both sybodies are bound to rbds up and up-out state, while only sb#15 is bound to the down rbd. the 3up class was further refined with c3 symmetry imposed. the final refinement, where a mask was included in the last iteration, provided a map at 7.6 å resolution. six rounds of per-particle ctf refinement with beamtilt estimation and re-extraction of particles with a box size of 400 pixels improved resolution further to 3.2 å. the particles were then imported into cryosparc, where non-uniform refinement improved the resolution to 3 å. the asymmetrical 1up/1up-out/1down was refined in an analogous manner with no symmetry imposed, resulting in a map at 7.8 å resolution. six rounds of per-particle ctf refinement with beamtilt estimation improved resolution to 3.7 å. a final round of non-uniform refinement in cryosparc yielded a map at 3.3 å resolution. local resolution estimations were determined in cryosparc. all resolutions were estimated using the 0.143 cut-off criterion [70] with gold-standard fourier shell correlation (fsc) between two independently refined half-maps [71] . the directional resolution anisotropy of density maps was quantitatively evaluated using the 3dfsc web interface (https://3dfsc.salk.edu) [72] . a similar approach was performed for the image processing of the s-2p/sb#15 complex. in short, 2,235 micrographs were recorded, and 1,582 used for image processing after selection. 66,632 particles were autopicked via cryolo and subjected to 2d classification in cryosparc. 57,798 selected particles were used for subsequent 3d classification in relion-3.0.8, where the symmetrical 3up map, described above, was used as initial reference. the best class comprising 22,055 particles (38%) represented an asymmetrical 1up/1up-out/1down conformation with sb#15 bound to each rbd. several rounds of refinement and ctf refinement yielded a map of 4.0 å resolution. for the dataset of the s-6p/sb#68 complex, in total 5,109 images were recorded, with 4,759 used for further image processing. 344,976 particles were autopicked via cryolo and subjected to 2d classification in cryosparc. 192,942 selected particles were imported into relion-3.0.8 and used for subsequent 3d classification, where the symmetrical 3up map, described above, was used as initial reference. two distinct classes of spike protein were found. one class (24,325 particles, 13%) revealed a state in which two rbds adopt an up conformation with sb#68 bound, whereby the density for the third rbd was poorly resolved representing an undefined state. several rounds of refinement and ctf refinement yielded a map of 4.8 å resolution. two other classes, comprising 44,165 particles (23%) and 84,917 particles (44%), were identical. they show a 1up/2down configuration without sb#68 bound to any of the rbds. both classes were processed separately, whereby the class with over 80k particles yielded the best resolution of 3.3 å and was used for further interpretation. a final non-uniform refinement in cryosparc further improved resolution down to 3.1 å. defocus range (μm) -0.9 to -1.9 -0.9 to -1.9 -0.9 to -1.9 -0.9 to -1.9 -0.9 to -1.9 pixel size (å) the plasmids encoding for the six highest affinity binders are available through addgene (addgene #153522 -#153527). purified sb-fc constructs can be commercially obtained from absolute antibody. the three-dimensional cryo-em density maps are available for the reviewers upon request. all cryo-em data will be deposited in the electron microscopy data bank and include the cryo-em maps, both half-maps, the unmasked and unsharpened refined maps and the mask used for final fsc calculation. raw cryo-em data will be deposited in the electron microscopy public image archive (empair). inhibi on of the rdb-ace2 interac on by sybodies. (a) elisa inhibi on screen. individual purified sybodies (500 nm, sybody number shown on x-axis) were incubated with bio nylated rbd-vyfp (25 nm) and the mixtures were exposed to immobilized ace2. bound rbd-vyfp was detected with streptavidin-peroxidase/tmb. each column indicates backgroundsubtracted absorbance at 405 nm, normalized to the signal corresponding to rbd-vyfp in the absence of sybody (dashed red line). (b) compe on of sybodies and ace2 for spike binding inves gated by gci. s-2p was immobilized on the gci chip and sb#15 (200nm), sb#68 (200nm) and non-randomized control sybody sb#0 (200 nm) were injected alone or premixed with ace2 (100 nm). neutraliza on of viral entry using pseudotyped vsvs. (a) rela ve infec vity in response to increasing sybody concentra ons was determined. the black curve shows data when a mixture of sb#15 and sb#68 was added. (b) same assay as in (a) with sybodies fused to human fc to generate bivalency. error bars represent standard devia ons of three biological replicates. sybody selec on strategy against sars-cov-2 rbds. a total of six independent selec on reac ons were carried out, including a target swap between ribosome display and phage display rounds. enriched sybodies of phage display round 1 of all three libraries were expressed and purified as a pool and used to perform an off-rate selec on in phage display round 2. for each of the six independent selec on reac ons, 47 clones were picked at random and analyzed by elisa. micro ter plate wells were coated with inidividual sybodies, incubated with bio nylated constructs (receptor-binding domain, rbd; spike ectodomain, ecd; pre-fusion spike, s-2p; maltose-binding protein, mbp), and then detected with streptavidin-peroxidase/tmb. a nonrandomized sybody was used as nega ve control (wells h6 and h12, respec vely). sybodies that were sequenced are marked with the respec ve sybody name (sb_#1-72). please note that iden cal sybodies that were found 2-3 mes are marked with the same sybody name (e.g. sb_#41). loop 0.180 phylogene c tree of rbd sybodies. a radial tree was generated in clc 8.1.3. figure s4 kine c characteriza on of sybodies by gci. (a) rbd-vyfp and ecd were immobilized as indicated and the six top sybodies were injected at increasing concentra ons ranging from 1.37 nm to 1 μm. data were fi ed using a langmuir 1:1 model. (b) in depth affinity characteriza on of sb#15 and sb#68. rbd-vyfp and s-6p were immobilized as indicated and sb#15 and sb#68 were injected at concentra ons ranging from 1.95 nm to 250 nm for sb#15 and 3.9 nm to 500 nm for sb#68. for rbd, data were fi ed using a langmuir 1:1 model. for s-6p, the data were fi ed with the heterogeneous ligand model, because the 1:1 model was clearly not appropriate to describe the experimental data. corresponding data for s-2p is shown in main fig. 1c . simultaneous binding of sb#15 and sb#68. compe on elisa experiment in which sb#15 was coated on the elisa plate and rbd binding was assesses in the absence of presence of tag-less sybodies as indicated in the x-axis. to determine the background signal, buffer devoid of protein was added. herd immunity -estimating the level required to halt the covid-19 epidemics in affected countries the reproductive number of covid-19 is higher compared to sars coronavirus estimation of the reproductive number of novel 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conformational states of sars-cov-2 spike protein. science structures of human antibodies bound to sars-cov-2 spike reveal common epitopes and recurrent features of antibodies ultrapotent human antibodies protect against sars-cov-2 challenge via multiple mechanisms structural basis of a shared antibody response to sars-cov-2. science antibody cocktail to sars-cov-2 spike protein prevents rapid mutational escape seen with individual antibodies neutralizing nanobodies bind sars-cov-2 spike rbd and block interaction with ace2 an alpaca nanobody neutralizes sars-cov-2 by blocking receptor interaction de novo design of picomolar sars-cov-2 miniprotein inhibitors an ultra-high affinity synthetic nanobody blocks sars-cov-2 infection by locking spike into an inactive conformation engineered peptide barcodes for in-depth analyses of binding protein libraries a highly efficient modified human serum albumin signal peptide to secrete proteins in cells derived from different mammalian species a versatile and efficient high-throughput cloning tool for structural biology x-ray structure of a calcium-activated tmem16 lipid scramblase a variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications one-step purification of recombinant proteins using a nanomolar-affinity streptavidin-binding peptide, the sbp-tag. protein expression and purification structural basis for potent neutralization of betacoronaviruses by single-domain camelid antibodies a human monoclonal antibody blocking sars-cov-2 infection a vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-species type i interferon focus: the interface between data collection and data processing in cryo-em motioncor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy ctffind4: fast and accurate defocus estimation from electron micrographs sphire-cryolo is a fast and accurate fully automated particle picker for cryo-em cryosparc: algorithms for rapid unsupervised cryo-em structure determination new tools for automated high-resolution cryo-em structure determination in relion-3. elife optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy prevention of overfitting in cryo-em structure determination addressing preferred specimen orientation in single-particle cryo-em through tilting we thank rony nehmé and andré heuer (creoptix ag, wädeswil, switzerland) for the acquisition, fitting and interpretation of a first set of gci measurements using the wavesystem. we thank florence projer, david hacker and kelvin lau (protein production and structure core facility, epfl, switzerland) for the production of the pre-fusion spike protein. we are grateful to jason mclellan (the university of texas at austin, u.s.) for having provided the pre-fusion-stabilized soluble spike expression vectors for s-2p and s-6p. we thank michael fiebig (absolute antibody) for providing us with purified sb-fc. we thank raimund dutzler and marta sawicka (university of zurich) for freezing cryo-em grids. michiel punter (university of groningen) is acknowledged for it help. key: cord-281005-6gi18vka authors: singh, praveen kumar; kulsum, umay; rufai, syed beenish; mudliar, s. rashmi; singh, sarman title: mutations in sars-cov-2 leading to antigenic variations in spike protein: a challenge in vaccine development date: 2020-09-01 journal: j lab physicians doi: 10.1055/s-0040-1715790 sha: doc_id: 281005 cord_uid: 6gi18vka objectives the spread of severe acute respiratory syndrome coronavirus-2 (sars-cov-2) virus has been unprecedentedly fast, spreading to more than 180 countries within 3 months with variable severity. one of the major reasons attributed to this variation is genetic mutation. therefore, we aimed to predict the mutations in the spike protein (s) of the sars-cov-2 genomes available worldwide and analyze its impact on the antigenicity. materials and methods several research groups have generated whole genome sequencing data which are available in the public repositories. a total of 1,604 spike proteins were extracted from 1,325 complete genome and 279 partial spike coding sequences of sars-cov-2 available in ncbi till may 1, 2020 and subjected to multiple sequence alignment to find the mutations corresponding to the reported single nucleotide polymorphisms (snps) in the genomic study. further, the antigenicity of the predicted mutations inferred, and the epitopes were superimposed on the structure of the spike protein. results the sequence analysis resulted in high snps frequency. the significant variations in the predicted epitopes showing high antigenicity were a348v, v367f and a419s in receptor binding domain (rbd). other mutations observed within rbd exhibiting low antigenicity were t323i, a344s, r408i, g476s, v483a, h519q, a520s, a522s and k529e. the rbd t323i, a344s, v367f, a419s, a522s and k529e are novel mutations reported first time in this study. moreover, a930v and d936y mutations were observed in the heptad repeat domain and one mutation d1168h was noted in heptad repeat domain 2. conclusion s protein is the major target for vaccine development, but several mutations were predicted in the antigenic epitopes of s protein across all genomes available globally. the emergence of various mutations within a short period might result in the conformational changes of the protein structure, which suggests that developing a universal vaccine may be a challenging task. since the rapid outbreak of 2019 novel coronavirus (2019-ncov, later named sars-cov-2 or severe acute respiratory syndrome coronavirus 2) in wuhan, china, the world health organization on january 30, 2020, declared the sars-cov-2 epidemic as a public health emergency of international concern. the enduring pandemic has caused nearly 5 million detected cases of coronavirus disease 2019 illness and claimed over 3,25,563 lives worldwide as of may 20, 2020 according to covid-19 resource center johns hopkins. 1 however, so far, no proven therapeutic or effective vaccine candidate has been found. 2 for developing a drug or vaccine, the protein profiling and/or genomic information of the pathogen is extremely crucial. to understand genetic landscape of sars-cov-2 virus, scientists have worked tirelessly and the complete genome sequences of virus isolates are published. now, many isolates have been sequenced completely or partially and are available in the database for scientific community. 3 it is found that the genome size of sars-cov-2 varies from 29.8 kb to 29.9 kb. specific genetic characteristic in its genome have also been found. 4 the genome consists of four structural proteins including spike protein (s), envelope (e), membrane (m), and nucleocapsid (n) proteins. 3 of the four glycoproteins, the m protein is reported to have role in determining the shape of the virus envelope and stabilizing the nucleocapsids, the n protein is involved in processes related to the viral genome, the viral replication cycle, and the cellular response of host cells to viral infections. the e protein which is the smallest protein in the sars-cov-2 structure plays the role in the production and maturation of this virus. 4 however, the glycoprotein s is the core transmembrane monomer of approximately 180 kda size with two subunits s1 and s2. this glycoprotein mediates membrane fusion and finally facilitates virus entry (receptor-binding and entry of virion into the target cells). 5 the receptor binding domain (rbd) (residues 319-541) of the subunit s1 is known to interact with angiotensin-converting enzyme 2 (ace-2), which provides tight binding to the peptidase domain of ace-2. 6 this gives an impression of rbd being an important element of virus-receptor interaction and has an essential role in virus-host range, tropism, and infectivity. the rbd sequences of different sars-cov-2 strains that are circulating globally were initially thought to be conserved; however, with the availability of sequencing data several mutations have been reported. 7, 8 these findings emphasize the argument, that there may be correlation of the mutated strains circulating in a particular geographic setting with higher mortality rates and transmission patterns beside other combating factors. 9 the mutation rate in ribonucleic acid (rna) viruses is intensely high which can be million times higher than that of their hosts and this results in virulence modulation and evolutionary capability for better viral adaptation. 7 genetic depiction of virus mutations can thus offer valuable insights for assessing the fitness of drug resistance, immune escapism, and pathogenesis. due to its receptor binding property, the s protein is also supposed to be immunogenic and a putative target for developing the neutralizing antibodies and vaccines. it is reported that single-point mutations in the conserved amino acid residues in the rbd region completely abolishes the capacity of fulllength s protein to induce neutralizing antibodies. 10 thus, virus mutation studies can be crucial for designing new vaccines and antiviral drugs. in this study, we aimed to predict the mutations in the spike protein (s) of sars-cov-2 genomes available in the database (whole genome sequences as well as partial coding sequences of spike protein) and analyze the effect of each mutation on the antigenicity of the predicted epitopes. this information may be helpful in predicting the transmission and infectivity of various sars-cov-2 strains circulating worldwide. entrez direct (edirect) utilities were used to access the ncbi's nucleotide database by using in-house developed bash scripts to batch download the data. we used query keyword or phrase as "severe acute respiratory syndrome coronavirus 2" and "spike coding sequences (cds)" by applying esearch and efetch utilities implemented in bash scripts to download the dataset for genome and spike proteins that were available till may 1, 2020. a total of 1,325 complete draft genomic sequences of sars-cov-2 and additional 279 cds having partial genomes coding spike protein (total 1,604 cds of spike protein) were downloaded in fasta format available globally from ncbi database (►fig. 1). multiple sequence alignment (msa) of all the 1,325 complete genome sequences as well as 1,604 cds was performed using clustalw-mpi with default parameters. 11 generated snps were identified by in-house developed bash scripts to batch process the data using blade server (dell poweredge fc640 server) with 256 gb ram and 40 core processor with 2.30 ghz. after msa, each genome and spike protein were marked based on the location and clustering was done based on 100% similarity for ease of visualization and analysis. visualization was performed by using jalview 2.11.1.0. 12 the output snp alignment generated from msa was used to assemble a maximum likelihood phylogenetic tree using raxml (randomized axelerated maximum likelihood raxml 8.2.12). 13 phylogenetic trees were visualized using the interactive tree of life (itol) v5 with their respective metadata. 14 emboss antigenic software was used to predict the antigenic regions and the epitopes in the 88 unique spike proteins based on antigenic scores using the formula: where f(ag) = antigenic frequency; f(s) = surface frequency and antigenic score ≥ 1.0 is considered potentially antigenic. [15] [16] [17] the data for different epitopes were analyzed and the epitopes with high antigenicity were superimposed on the structure of spike protein. overall, 1,197 snps were found in 1,325 complete genome datasets. on the basis of similarity these were classified in 782 clusters. however, among cds of spike proteins (1,325 complete genomes and 279 partial cds) a total 140 snps in 88 clusters were found. further snp analysis resulted in identification of snps in a gene stretch of 21,563-25,384 bp in the s gene, encoding the spike (s) protein. the most predominant snp predicted in the gene encoding s protein was 23402a>g in 48.2% of overall genomes under study. in addition to several single mutations in the s gene of all available genomes, we also predicted double mutations such as 22436g>t, 22439c>g, 22444c>a, 22445c>t (corresponding to four amino acid antigenic drift aldp -> sves at position 292-295) and 21723t>g (l54w); 21726t>a (f55i) in two different genomes from the united states. list of all the snps in the rbd, antigenic sites, and double mutations among strains is shown in ►table 1. one deletion (21994-21996deltta) was also found in an indian strain (mt012098.1). no copy number variants were observed in this virus. the alignment of 1,604 spike proteins extracted from 1,325 complete genomes and 279 partial cds was performed and after clustering based on 100% identity, we identified 88 unique sequences with 88 hypervariable sites within these protein sequences. based on the variable sites the phylogeny was inferred showing two major clades a and b with many subclades in the s protein of sars-cov-2 circulating worldwide (►fig. 2). furthermore, the evaluation of the antigenicity of spike protein predicted 14 highest scoring antigenic epitopes (antigenic scores ≥ 1.0) due to variations in each (at positions l54f, l54w, f55i, s71f, d111n, f157l, l293m, l293v, d294e, d294i, a419s, v367f, a348v, and a653v). out of these, amino acid changes were noted at positions a348v, v367f and a419s in the rbd with v367f and a419s being novel. other speculated mutations in the putative epitopes lying within rbd showing less antigenicity were t323i, a344s, r408i, g476s, v483a, h519q, a520s, a522s, and k529e out of which t323i, a344s, a522s, and k529e are also novel. in addition, regions outside rbd (4-19, 1,215-1,256, 1,039-1,071, 123-146, 607-629, 1,123-1,136 , 857-867, 535-541) also infer high antigenicity (based on predicted antigenic score in the range 1.167-1.261) with variations in s protein of different genomes from similar locations as shown in ►fig. 3. the antigenic epitopes are depicted in the protein structure of spike protein (►fig. 4). two mutations a930v and d936y were observed in the heptad repeat domain 1 (hr1) and one mutation d1168h in heptad repeat domain 2 (hr2). several studies have shown that mutations within the spike protein influence virus-host interaction. 18 among the four proteins, viz., m, s, n and e, the m protein is known to play a significant role in virus assembly, role of e protein involves the production and maturation of this virus, the n protein is involved in processes related to viral replication cycle, and cellular response of host cells to viral infections, and s protein is the major target for neutralizing antibodies as it mediates the fusion and facilitates viral entry into host. 4, 5 in the present study, we found that although multiple genetic variants were identified in the same country, yet there were some unique mutations found in a particular country, which suggests that diversity of s protein mutations might have significant role in the pathogenicity of this virus in countries with high or low mortality rates, as proposed by others also. 19 we predicted 140 snps in the s protein with a>g and vice versa in sars-cov-2 genomes submitted from india, t>a and c>t from china and the interchange of all four nucleotides (c>t, t>a, a>g, g>t, c>g, c>a, g>c, t>c, a>t, g>a) in genomes submitted from the united states. the data from the united states is significant. however, it might be because maximum genomes were submitted from the united states only. the snp profile revealed that the s protein mutations were predominant at specific positions only. these mutations are expected to make the virus more capable to escape from the host immune and might help in natural selection and evolution of the sars-cov-2, as reported by andersen et al. 20 it is important to mention that double mutations in the s protein were found only in the strains from the united states but not in genomes from other regions. these double mutations probably could have helped in the increased virulence of the virus. 21 it has also been noticed that the death toll is comparatively higher in the united states than in other regions included in the study. 1 this might probably indicate that the prevalence of several mutated strains within the provinces would have either reduced or increased its severity. it may also help in understanding the antigenic and immunogenic changes but the correlation of mutation with regional virulence could not be established due to statistical imbalance of the available genomes in the database at the time the study was done. moreover, extensive research is required to correlate the mutations with the severity of the disease and mortality. out of 88 snp clusters, d614g was found in 34 (38.6%) sars-cov-2 genomes. the amino acid change in 23403a>g variant (p.d614g) involves a change of large acidic residue d (aspartic acid) into small hydrophobic residue g (glycine). this observation is important, as this large difference in both size and charge may help compromise the binding affinity of antibodies against s protein, due to electrostatic interactions in the tertiary structure of protein group. this may hinder the developments of vaccines and might potentiate the virus for antigenic drifts. 22 the effect of deletion variant (figured in one sars-cov-2 genome from india) on the viral phenotypes needs further investigation. the high frequency of genetic mutations in rna viruses is well known but in the genomes of sars-cov-2, we found a series of single amino acid variations. this can affect the virus evolution and emergence of the new strains. 21 the mutations in the rbd found in our study predicted conformational changes in the s1 domain of spike protein. the mutations in rbd play an important role while designing new drugs, as suggested in a recent study. 23 these mutations might affect the interaction of viral rbd with the host receptor. our study revealed 12 mutations of which six were novel mutations (►table 1). out of the six novel mutations two were exhibiting high antigenicity while others were in the less antigenic region. the amino acid change observed in the antigenic epitopes were from positively charged to uncharged amino acids (r->i, h->q), and negatively charged to uncharged (d->n, d->g, d->y) amino acids. we also found mutations in negative to positively charged (d->h) amino acids. these replacements might influence the tertiary structure of the proteins and facilitate the increased virulence by escaping host immune response. 24 the sequences in the hr1 (residues 902-952) and hr2 (residues 1,145-1,184) regions tend to form dimeric or trimeric helix bundles. 25 as the s protein of coronavirus are homodimers or homotrimers, these hr regions may undergo oligomerization and result in the conformational change of s protein during virus-host cell fusion. 26 these regions show different conformations in different fusion states and are known to be the most conserved among other regions in s protein of sars-cov. 25, 27 however, a previous study shows variations in hr1 domain which forms helical bundles with hr2 to facilitate fusion and entry of virus into the host and hypothesizes that the mutation a1168v in hr2 domain along with a930v mutation in hr1 domain confers peptide entry inhibitor resistance in mouse hepatitis coronaviruses. 28 hence the mutation a930v in hr1 domain and d1168h in hr2 domain found in our study might be relevant in explaining the pathogenesis of sars-cov-2. with the rapid spread of this virus and limitation of specific therapy, studies are being focused on exploring the potential of neutralizing antibodies (as in plasma therapy) against vulnerable epitopes of s protein. 29 our study predicts an interactive web-based dashboard to track covid-19 in real time a review of sars-cov-2 and the ongoing clinical trials genomic characterization of a novel sars-cov-2 coronavirus envelope protein: current knowledge cryo-em structure of the 2019-ncov spike in the prefusion conformation structure of mouse coronavirus spike protein complexed with receptor reveals mechanism for viral entry emerging sars-cov-2 mutation hot spots include a novel rna-dependent-rna polymerase variant preliminary identification of potential vaccine targets for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies real estimates of mortality following covid-19 infection single amino acid substitutions in the severe acute respiratory syndrome coronavirus spike glycoprotein determine viral entry and immunogenicity of a major neutralizing domain clustalw-mpi: clustalw analysis using distributed and parallel computing jalview version 2-a multiple sequence alignment editor and analysis workbench raxml-vi-hpc: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models interactive tree of life (itol) v4: recent updates and new developments emboss: the european molecular biology open software suite a semi-empirical method for prediction of antigenic determinants on protein antigens new hydrophilicity scale derived from high-performance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and x-ray-derived accessible sites potential therapeutic targeting of coronavirus spike glycoprotein priming sars-cov-2 viral spike g614 mutation exhibits higher case fatality rate the proximal origin of sars-cov-2 why are rna virus mutation rates so damn high? electrostatic interactions in protein structure, folding, binding, and condensation exploring the genomic and proteomic variations of sars-cov-2 spike glycoprotein: a computational biology approach lineage-specific differences in the amino acid substitution process interaction between heptad repeat 1 and 2 regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion mechanisms of viral membrane fusion and its inhibition coronavirus escape from heptad repeat 2 (hr2)-derived peptide entry inhibition as a result of mutations in the hr1 domain of the spike fusion protein a highly conserved cryptic epitope in the receptor binding domains of sars-cov-2 and sars-cov most of the vaccines strategies against covid-2 are focusing on the predicted epitopes of sars-cov-2 spike protein. this protein is also proposed to be the most potent and specific drug target and for designing neutralizing antibodies. our findings indicate that vaccine designing against sars-cov-2 could be a challenging task. even though both rna based, and peptide-based vaccines are being developed in more than seven laboratories, our observations may be useful in the efficacy analysis of these vaccine candidates. none.conflict of interest p.k.s. and u.k. are research officers in a department of biotechnology funded unrelated project (bt/pr23016/ ner/95/581/2017). key: cord-274280-x5s4l0pp authors: yang, jinsung; petitjean, simon j. l.; koehler, melanie; zhang, qingrong; dumitru, andra c.; chen, wenzhang; derclaye, sylvie; vincent, stéphane p.; soumillion, patrice; alsteens, david title: molecular interaction and inhibition of sars-cov-2 binding to the ace2 receptor date: 2020-09-11 journal: nat commun doi: 10.1038/s41467-020-18319-6 sha: doc_id: 274280 cord_uid: x5s4l0pp study of the interactions established between the viral glycoproteins and their host receptors is of critical importance for a better understanding of virus entry into cells. the novel coronavirus sars-cov-2 entry into host cells is mediated by its spike glycoprotein (s-glycoprotein), and the angiotensin-converting enzyme 2 (ace2) has been identified as a cellular receptor. here, we use atomic force microscopy to investigate the mechanisms by which the s-glycoprotein binds to the ace2 receptor. we demonstrate, both on model surfaces and on living cells, that the receptor binding domain (rbd) serves as the binding interface within the s-glycoprotein with the ace2 receptor and extract the kinetic and thermodynamic properties of this binding pocket. altogether, these results provide a picture of the established interaction on living cells. finally, we test several binding inhibitor peptides targeting the virus early attachment stages, offering new perspectives in the treatment of the sars-cov-2 infection. i n december 2019, a novel coronavirus (cov) was determined to be responsible for an outbreak of potentially fatal atypical pneumonia, ultimately defined as coronavirus disease-19 , in wuhan, china. this novel cov, termed severe acute respiratory syndrome (sars)-cov-2, was found to share similarities with the sars-cov that was responsible for the sars pandemic that occurred in 2002. the resulting outbreak of covid-19 has emerged as a severe pandemic. the genome of sars-cov-2 shares about 80% identity with that of sars-cov and is about 96% identical to the bat coronavirus batcov ratg13 (ref. 1 ). cov entry into host cells is mediated by its transmembrane spike (s) glycoprotein that forms homotrimers protruding from the viral surface 2 (fig. 1a) . the s glycoprotein comprises two functional subunits responsible either for binding to the host cell receptor (s1 subunit including the receptor-binding domain (rbd)) or for fusion of the viral and cellular membranes (s2 subunit). recent studies claimed that the angiotensinconverting enzyme 2 (ace2), previously identified as the cellular receptor for sars-cov, also acts as a receptor of the new coronavirus (sars-cov-2) 3 (fig. 1b) . in the case of sars-cov, the s glycoprotein on the virion surface mediates receptor recognition (fig. 1c) and membrane fusion 4, 5 . recently, the highresolution cryo-electron microscopy structure obtained on the full-length human ace2 in the presence of the rbd of the s glycoprotein of sars-cov-2 suggests simultaneous binding of two s-glycoprotein trimers to an ace2 dimer 3 . the s2 subunit is further cleaved by host proteases located immediately upstream of the fusion peptide 6 , leading to the activation of the glycoprotein that undergoes extensive irreversible conformational changes facilitating the membrane fusion process. altogether, the information obtained so far highlights the fact that cov entry into susceptible cells is a complex process that requires the concerted action of receptor binding and proteolytic activation of the s glycoprotein at the host cell surface to finally promote virus-cell membrane fusion. however, so far, direct evidence about the dynamics of the binding of the s1-to the ace2 receptor at the single-molecule level is missing. here, we analyze the biophysical properties of the sars-cov-2 s-glycoprotein binding, on model surfaces and on living cells, to ace2 receptors using force-distance (fd) curve-based atomic force microscopy (fd-curve-based afm) (fig. 1c) . we extract the kinetics and thermodynamics of the interactions established in vitro, and compare the binding properties of both the s1 subunit and rbd. next, we test short ace2-derived peptides targeting the viral s glycoprotein as potent binding inhibitor peptides and observe a significant reduction in the binding properties. results s1 subunit specifically binds to purified ace2 receptors. as sars-cov-2 binding to ace2 receptors is thought to play a key role in the first binding step at the cellular membrane 3 fig. 1 probing sars-cov-2 binding to the ace2 host receptor. a schematic of a sars-cov-2 particle, an enveloped ssrna virus expressing at its surface the spike glycoprotein (s) that mediates the binding to host cells. b structural studies have previously obtained a complex between the receptor-binding domain (rbd, a subunit of the s glycoprotein) and the angiotensin-converting enzyme 2 (ace2) receptor. c schematic of probing sars-cov-2 binding using atomic force microscopy (afm). the initial attachment of sars-cov-2 to cells involves specific binding between the viral s glycoprotein and the cellular receptor, ace2. the interactions are monitored by afm on model surfaces, where the ace2 receptor is attached to a surface and the s1 subunit or the rbd onto the afm tip, and on a549 living cells expressing or not fluorescently labeled ace2. used fd-curve-based afm to evaluate at the single-molecule level the binding strength of the interaction established between the glycosylated s1 subunit and ace2 receptors on model surfaces (fig. 2a) . to mimic cell-surface receptors in vitro, ace2 receptors were covalently immobilized onto gold surfaces coated with ohand cooh-terminated alkanethiols using carbodiimide conjugation (see methods). these model surfaces were imaged by afm, and the thickness of the grafted layer was validated by a scratching experiment, revealing a deposited layer of 6.1 ± 0.4 nm (mean ± s.d., n = 3) (see methods and supplementary fig. 1 ). to study the interaction between the s1 subunit and the immobilized ace2 receptors, we covalently grafted either the purified full s1 subunit or rbd only to the free end of a long polyethylene glycol (peg) 24 spacer attached to the afm tip [7] [8] [9] . to investigate the properties of the binding complex, force-distance (fd) curves were recorded by repeatedly approaching and withdrawing the s1 subunit or rbd-functionalized tip from the ace2 model surface (fig. 2a, b) . specific adhesion events were observed on 4-5% of the retraction fd curves at rupture distances >15 nm, which corresponds to the extension of the peg linker ( fig. 2c and supplementary fig. 2 ), and is in line with studies carried out for other virus-cell-surface receptor systems 8,10-12 . to confirm the specificity of these interactions, we conducted additional independent control experiments using (i) an afm tip only functionalized with the peg linker or (ii) toward oh-/coohterminated alkanethiol surfaces missing the receptor. the binding frequency observed during those control experiments is significantly lower, thereby confirming the specificity of the s1 subunit/rbd-ace2 complexes under our experimental conditions (fig. 2c) . exploring the dynamics of s1 subunit-ace2 interaction. single-molecule force-probing techniques, such as fd-based afm, measure the strength of a bond under an externally applied force, enabling to get insights into the binding free-energy landscape. according to the bell-evans model 13, 14 , an external force stressing a bond reduces the activation-energy barrier toward dissociation and, hence, reduces the lifetime of the ligandreceptor pair 15 (fig. 2d) . the model also predicts that far-fromequilibrium, the binding strength of the ligand-receptor bond is proportional to the logarithm of the loading rate (lr), which describes the force applied on the bond over time. to investigate the kinetics of the probed complex, fd curves were recorded at various retraction rates and contact times ( fig. 2e-h) . dynamic force spectroscopy (dfs) plots were obtained for both s1 subunit (fig. 2e) and rbd (fig. 2f) x u = 0.79 ± 0.04 nm rbd fig. 2 probing s-glycoprotein binding to the ace2 host receptor on model surface. a binding of s-glycoprotein subunit (s1 or rbd) is probed on an ace2-coated surface. b retraction part of four force-distance curves showing either nonadhesive or specific adhesive curves. c box plot of specific binding probabilities (bp) measured by afm between the functionalized tip (s1, rbd, or peg) and the grafted surface (ace2 or oh-/ cooh-terminated alkanethiol (bare surface)). one data point belongs to the bp from one map acquired at 1 µm/s retraction speed. the square in the box indicates mean, the colored box indicates the 25th and 75th percentiles, and the whiskers indicate the highest and the lowest values of the results. the line in the box indicates median. n = 12 (s1, rbd), 18 (peg), and 9 (s1, rbd vs. bare surface) maps examined over 4 (s1, rbd), 6 (peg), and 3 (s1, rbd vs. bare surface) independent experiments. d bell-evans model describing a virus-receptor bond as a two-state model. the bound state is separated from the unbound state by a single energy barrier located at distance x u . k off and k on represent the dissociation and association rate, respectively. e, f dynamic force spectroscopy (dfs) plot showing the distribution of the rupture forces as a function of their loading rate (lr) measured either between the s1 subunit and the ace2 receptor (n = 1052 data points) (e) or between the rbd and the ace2 receptor (n = 1490 data points) (f). the error bar indicates s.d. of the mean value for a single interaction (0-200 pn). the solid line represents the fit of the data with the bell-evans fit. experiments were reproduced at least four times with independent tips and samples. g, h the bp is plotted as a function of the contact time for s1 subunit and rbd on ace2 model surfaces, and data points were fitted using a least-squares fit of a monoexponential growth. one data point belongs to the bp from one map acquired at 1 µm/s retraction speed for the different contact times. experiments were reproduced three times with independent tips and samples. p values were determined by two-sample t test in origin. the error bar indicates s.d. of the mean value. source data are provided as a source data file. virus-receptor bonds 8, 10, 11, 16, 17 . to determine whether single-or multiple-bond rupture between s1/rbd and ace2 is taking place, bond strengths (every single gray data point in fig. 2e , f) were analyzed through distinct discrete ranges of lrs, plotted as force histograms and further fitted with multipeak gaussian distribution, as established previously 11, 16 (supplementary figs. 3 and 4) . using this distribution, we are able to determine the most probable unbinding force of each force peak (maximum of rupture force distribution; black dots plotted over mean lr of this range in fig. 2e , f), and can determine if single or multiple interactions were taking place. the presence of multiple parallel unbinding events is first observed in the distribution of rupture forces with the presence of multiple gaussian fits. the histograms show that most probably only single interactions were taking place; thus, the bell-evans model 15 was used to fit the data enabling to interpret the binding complex as a simple two-state model, in which the bound state is separated from the unbound state by a single energy barrier (fig. 2d) . from the slope of the fit, we estimated the length scale of the energy barrier (x u ). we obtained very close values, x u = 0.81 ± 0.05 nm and 0.79 ± 0.04 nm for both the s1 subunit and rbd, showing that we are probing similar bonds (fig. 2e , f). the kinetic off-rate (k off ) or dissociation rate is obtained from the intercept of the fit (at lr = 0) yielding k off values of 0.008 ± 0.005 s −1 and 0.009 ± 0.006 s −1 for s1 subunit and rbd, respectively. these values are in good agreement with reported values obtained by surface plasmon resonance for the s glycoprotein (k off = 0.003 s −1 ) 18 and the rbd subunit (k off = 0.008 s −1 ) binding to ace2 receptors 19 . assuming that the receptor-bond complex can be approximated by a pseudo-first-order kinetics, we also estimated the kinetic on-rate (k on ) from our single-molecule force spectroscopy experiments 11 (fig. 2g, h) . this association rate is extracted from the binding probability (bp) measured at various contact times, and depends on the effective concentration described as the number of binding partners (ligand + receptor) within an effective volume v eff accessible under free-equilibrium interaction. v eff can be approximated by a half-sphere with a radius including the linker, the viral glycoprotein (s1 subunit or rbd) and the ace2 receptor. for both the s1 subunit and rbd, we observed that the binding frequency increased exponentially with contact time, and we extracted an interaction time of~0.250 ms, leading to a k on of 6.4 × 10 4 m −1 s −1 and 8.0 × 10 4 m −1 s −1 , respectively. finally, the dissociation constant k d is calculated as the ratio between the k off and the k on , yielding values around 120 nm for both complexes. this value corresponds to a highaffinity interaction, confirming the specificity of the complexes established by sars-cov-2 with the ace2 cell-surface receptor, which in turn results in a long lifetime of the virus attachment to the cell surface. other interaction studies between sars-cov (80% sequence homology to sars-cov-2) and ace2 reported specific, high-affinity association values also in the nm range 20 . for comparison, a variety of examples for low-as well as highaffinity interactions between other virus-receptor pairs are summarized in dimitrov et al. 21 and include influenza a-sa (mm) or hiv-1-cd4 (nm) interactions. for single-molecule interactions, the bond lifetime τ can be directly related to the inverse kinetic off-rate (τ = k off −1 ), resulting here in a τ of 125 ms for the s1 subunit and 111 ms for the rbd, respectively. of course, at the virion level, the overall bond lifetime will increase with the multivalence of the interaction. by definition, highaffinity interaction has a long lifetime as the dissociation constant k d is defined as the ratio between k off and k on . for high-affinity interactions, the k d is in the nm range, leading to k off « k on and therefore maintaining the interaction in its bond state for very long times, making the development of anti-binding molecules targeting this interaction more difficult. finally, we also used optical biolayer interferometry (bli) to confirm the kinetic parameters characterizing this interaction, and obtained very close affinities in the same nm range as afm experiments ( supplementary fig. 5 ). taken together, our in vitro experiments confirm that sars-cov-2 binding to the ace2 receptors is mediated by the rbd-ace2 interface as our experimental conditions did not highlight any significant difference between s1 subunit and rbd binding. validation of the interaction on living cells. next, we wanted to investigate whether the interaction probed on isolated receptors is also established in physiologically relevant condition. to this end, we performed binding assays on living a549 cells (human adenocarcinoma alveolar basal epithelial cells). while this cell line is widely used as a type ii pulmonary epithelial cell model, it has been shown recently that those cells are incompatible with sars-cov-2 infection 22 . interestingly, ace2 expression positively correlated with the differentiation state of epithelia. although undifferentiated cells (cultured at low confluency) only express little ace2, overexpression of ace2 in undifferentiated a549 cells facilitated virus entry 23 . we transiently transfected ace2-egfp in a549 cells (a549-ace2) and probed s1-subunit binding to those cells as well as to a549 cells (serving as internal control) ( fig. 3a and supplementary fig. 6 ). confocal images showed ace2-egfp receptors homogeneously distributed in small domains at the surface of a549 cells (fig. 3b ). guided by fluorescence (fig. 3c) , we chose areas in which both cell types, i.e., transfected (a549-ace2, green fluorescence) and nontransfected (a549, no fluorescence) cells, were in proximity to one another. having both a549 cell types in one image area served as a direct control to evaluate whether interactions measured by the functionalized tip were indeed due to specific binding to fluorescent ace2-egfp receptors, and to evaluate the extent of other types of interactions (fig. 3c-e) . in such area, we simultaneously recorded a height image (fig. 3d ) and the corresponding adhesion map (fig. 3e) , which were reconstructed from fd curves recorded for each topographic pixel. the retraction part of fd curves showed specific adhesion events mainly on a549-ace2 cells, with a significantly higher bp (fig. 3f ), as exemplified with the presented adhesion map that shows 20.1% of adhesive pixels on the a549-ace2 cell versus 13.5% on the control cells ( fig. 3e and supplementary fig. 7 ). specific binding forces (and corresponding lr) were extracted from force vs. time curves recorded on a549-ace2 cells (fig. 3g) and overlaid on the dfs plot obtained on purified ace2 receptors (fig. 3h ). to explore a wide range of lr, we probed the interaction at various frequencies and amplitudes (see methods). we observed a very good alignment between the data obtained on purified receptors and on living cells confirming the physiological relevance of our results obtained on model surfaces. s1 subunit binding to the cell involves other receptors. our fdbased afm experiments performed on living cells put in evidence that the s1 subunit interacts even on control cells with a frequency ≈10% although the expression level of ace2 should be very low as the cells are not differentiated. nevertheless, some evidence pointed out that human cov s glycoproteins possess sialic acid (sa)-binding sites and in particular to 9-o-acetylsialogycans 24 , and that integrins could also be a receptor for the sars-cov-2 (ref. 25 ), which possesses a rgd motif close to the ace2-binding site. to evaluate whether these other receptors could be involved during the early binding steps to the cell surface, we performed additional experiments by injecting 9-oacetyl-sialogycans to block interaction with cell-surface sa, or added cyclo-rgd (crgd) to compete with the interactions with integrins. after sa injection, the binding frequency was reduced on a549 cells down to~7% and to~10% on ace2-transfected cells ( fig. 3f and supplementary fig. 8 ). for integrins, injection of crgd only reduces the binding frequency of~1-2% on both cell types, which is in good agreement with the fact that integrins are mostly expressed on the bottom of the cell 26 . altogether, these data obtained on cells by afm represent to date the best evidence that s1-ace2 complex is established in physiologically relevant conditions and underlines the complex situation with multiple cell-surface receptors accounting for the whole interaction. inhibition of s1-subunit binding using ace2-derived peptides. human recombinant soluble ace2 (hrsace2) is currently being considered for treatment of covid19 (refs. 27,28 ). however, ace2 is involved in many key cellular processes, such as bloodpressure regulation and other cardiovascular functions. therefore, hrsace2 treatment could lead to dysregulation of those vital processes and subsequently cause deleterious side effects for treated patients. to avoid any interference of the ace2 homeostasis, we wanted to test whether small ace2-derived peptides can also interfere with sars-cov-2 binding, by blocking binding article sites on the s glycoprotein. to this end, we synthetized four different peptides (sequences provided in supplementary fig. 9 ), which have been selected to mimic the regions of ace2 that interact with the s1 subunit as determined by the crystal structure 29 , and we tested their binding inhibition properties using our single-molecule force spectroscopy approach (fig. 4a, b) . we first measured the bp between the s1 subunit and the ace2 in the absence of peptide (0 µm), with a contact time of 250 ms, as reference, and then injected our ace-derived peptides at three different concentrations (1, 10, and 100 µm). for the four peptides, we observed a progressive reduction of the bp as a function of the concentration confirming a specific inhibition. in addition, for each peptide, we noticed a reduction of >50% of the probed interactions already for the 1-10 µm concentration, suggesting a 50% inhibitory concentration (ic 50 ) in the µm range. the peptide shows the highest inhibition of the s1-ace2 complex formation with a measured reduction in the bp of~76%. the peptide shows a similar inhibition potential (~74%), suggesting that the additional amino acids do not influence the overall affinity of the peptide for the s1 subunit, as also confirmed by molecular dynamics (md) simulations showing that although the peptide 22-57 is longer, less h bonds are established between the peptide and the rbd domain ( supplementary fig. 10 ). overall, these results are in good agreement with the structural insights because these peptides are derived from the n-terminal helix of the ace2 and therefore form with the rbd interface an important network of hydrophilic interactions (including nine hydrogen bonds and a salt bridge). within the ace2-rbd complex, the [351−357] fragment is also part of a "hot binding spot" that results in our test by a good score with a reduction of 60% of the initial specific bp. finally, the [22-44-g-351-357] peptide was also synthetized and tested based on the fact that in the crystal structure, the distance between s44 and l351 is close enough to be filled by a single amino acid. a glycine residue was added between the two fragments because the two ace2 fragments have opposite directionality, and glycine has a high propensity to form reverse turns. nevertheless, under our experimental conditions, we did not notice any strong improvement in the binding inhibition. altogether, our in vitro assays at the single-molecule level provide direct evidence that ace2derived peptides are strong candidates to potentially inhibit sars-cov-2 binding to ace2 receptors (fig. 4c) . finally, we tested whether the [22−57]-binding inhibition peptide could also prevent s1-subunit binding in the cellular context (fig. 5) . the interaction between the s1 subunit and the confluent layer of a coculture of a549 and a549-ace2 cells was probed before and after addition of the peptide at 100 µm. before injection, cells overexpressing the ace2 receptors (a549-ace2) show higher bp (9.4 ± 1.6% vs. 19.4 ± 7.3%, for a549 and a549-ace2, respectively) (mean ± s.d., n = 4) (fig. 5a-d) , in good agreement with our previous observation (fig. 3f) . after injection of the [22−57] ace2-derived peptide, we observed a significant decrease of the bp on both cell types (fig. 5e, f) . in particular, the bp on a549-ace2 cells significantly drops (~70%), reaching a level close to the one of the control cells. taking into account that undifferentiated a549 cells express little ace2 and are poorly infected by cov 23 , this result supports the biological relevance of our ace2-derived peptide acting as potential inhibitor capable of efficiently blocking sars-cov-2 binding. in conclusion, we investigated the interaction established between the sars-cov-2 s glycoprotein and the ace2 receptor using single-molecule force spectroscopy. we demonstrated a specific binding mechanism between the s1 subunit and the ace2 receptor. by comparing the binding of the s1 subunit and the rbd toward the ace2 receptor, our experiment evidenced that both domains interact with the same kinetic and thermodynamic properties toward the ace2 receptor, highlighting that sars-cov-2 binding to ace2 is dominated by the rbd/ace2 interface. our measurements show that under our physiologically relevant conditions, the rbd binds the ace2 receptor with an intrinsic high affinity (~120 nm), which could even be further stabilized at the whole-virus level, thanks to possible multivalent bonds between the s-glycoprotein trimer and ace2 dimer. based on the available crystal structures of the molecular complex, we examined how several ace2-derived peptide fragments could interfere with the s1-ace2 complex formation. while all tested peptides show binding inhibition properties, peptides mimicking the n-terminal helix of the ace2 receptor show the best results. both and peptides exhibit an anti-binding activity with ic 50 in the µm range, resulting in a >70% decrease in the bp observed by afm on purified receptor and >70% on living cells. on the cellular model, we observed that the bp drops to the level of the control cells (undifferentiated a549 cells) that are poorly infected by cov 23 . therefore, those peptides appear as strong therapeutic candidates against the sars-cov-2 infection. cell culture and transfection. a549 cells (atcc® ccl-185) were grown in ham's f-12 nutrient mix with 10% fetal bovine serum, penicillin (100 u ml −1 ), and streptomycin (100 µg ml −1 ) (gibco) at 37°c in a humidified atmosphere with 5% co 2 . pcdna3.1(+) ace2-egfp was transfected using lipofectamine ltx (invitrogen) according to the manufacturer's protocol. in brief, 2 μg of fig. 3 probing s-glycoprotein binding to the ace2 host receptor on living cells. a binding of s-glycoprotein subunit 1 (s1) is probed on a549 and a549-ace2 cells. b confocal microscopy (z stack) of a549-ace2-egfp (green) cell transduced with plasma membrane bfp (blue). c overlay of egfp and dic images of a mixed culture of a549 and a549-ace2-egfp cells. d, e force-distance (fd)-based afm topography image (d) and the corresponding adhesion map (e) in the specified area in (c). the frequency of adhesion events is indicated. f box plot of the binding probability between s1 and a549 cells (gray) or a549-ace2 cells (green) without and after injection of cyclic rgd (crgd, checked boxes) or sialic acid (sa, dashed boxes), respectively. the square in the box indicates mean, the colored box indicates the 25th and 75th percentiles, and the whiskers indicate the highest and the lowest values of the results. the line in the box indicates median. g force versus time curves showing either a nonadhesive curve (bottom) or specific adhesive curves acquired at different lrs (lr1-lr3). h dfs plot showing the distribution or the rupture forces measured either between the s1 subunit and the ace2 on model surfaces (black dots, extracted from fig. 2e) , and between the s1 subunit and ace2-overexpressing a549 cells acquired at three different lrs (blue and red dots) (n = 403). blue dots belong to a data set acquired in fast-force volume mode, with a retraction velocity of 20 µm s −1 (lr1). red dots belong to data sets acquired in peak force tapping mode with 0.125 khz peak force frequency and 375-nm amplitude (lr2) or at 0.25 khz and 750 nm (lr3), respectively. the error bar indicates s.d. of the mean value. histograms of force distribution on a549-ace2 cells for lr1-lr3 are shown on the side. for experiments without injection of crgd or sa, data are representative of at least n = 11 cells from n = 6 independent experiments. the data for blocking experiments with crgd or sa were acquired for at least n = 4 cells from n = 2 independent experiments. p values were determined by twosample t test in origin. source data are provided as a source data file. pcdna3.1(+) ace2-egfp was transfected to a549 cells (60-mm plate) using 6 μl of lipofectamine ltx and 2 μl of plus reagent (invitrogen). functionalization of afm tips. pfqnm-lc and msct-d cantilevers (bruker) were used to probe the interaction between s1 subunit (genscript, #u5377fc120) or rbd protein (genscript, #u5377fc120) and ace2 protein (sino biological, 90211-c02h). nhs-peg 24 -ph-aldehyde linkers were used to functionalize afm tips as previously described 30 . briefly, the cantilevers were immersed in chloroform for 10 min and further cleaned in a uv radiation and ozone (uv-o) cleaner (jetlight), and immersed overnight in an ethanolamine solution (3.3 g of ethanolamine in 6.6 ml of dmso). they were washed with dmso and ethanol three times, respectively. ethanolamine-coated cantilevers were immersed in nhs-peg 24 -ph-aldehyde solution (3.3 mg of it was diluted in 0.5 ml of chloroform and 30 μl of triethylamine) and finally washed 3 times with chloroform and dried with nitrogen. for afm tips functionalized with s1-subunit protein, 50 µl of s1-subunit protein solution (0.1 mg/ml) was put onto the cantilevers placed on parafilm (bemis na) and 2 µl of fresh nacnbh 3 solution (6 wt% vol-1 in 0.1 m naoh(aq)) was mixed in the protein solution. the cantilevers were incubated in the solution for 1 h on ice. then, 5 µl of 1 m ethanolamine solution was carefully added to the protein solution and incubated 10 min to quench the reaction and finally washed three times with pbs. for afm tips derivatized with the rbd protein, 100 µl of a 100 µm trisnitrilotriacetic amine 540 trifluoroacetate (toronto research chemicals, canada) (tris-nta) solution was put onto them placed on parafilm, and 2 µl of fresh nacnbh 3 solution was mixed in the protein solution. they were incubated in the solution for 1 h on ice. then, 5 µl of 1 m ethanolamine solution in the protein solution was added and incubated for 10 min. the mixture of 50 µl of rbd solution (0.1 mg ml −1 ) and 2.5 µl of 5 mm nicl 2 were put onto them and they were incubated for 2 h. after incubation, they were washed in pbs solution three times. preparation of ace2-coated model surfaces. ace2 protein (sino biological, 90211-c02h) was immobilized using nhs-edc chemistry. gold-coated surfaces were first rinsed with ethanol, dried with a gentle stream of nitrogen gas, cleaned for 15 min by uv and ozone treatment (jetlight), and incubated overnight in an alkanethiol solution (99% 11-mercapto-1-undecanol 1 mm (sigma aldrich) and 1% 16-mercaptohexadecanoic acid 1 mm (sigma aldrich) in ethanol). the chemically activated samples were rinsed with ethanol, dried with nitrogen gas, and immersed for 30 min in the solution of 100 mg of chemically activated dimethylaminopropyl carbodiimide (sigma aldrich) and 40 mg of n-hydroxysuccinimide in 4 ml of milliq water. finally, the surfaces were rinsed with milliq water, incubated with ace2 protein (0.1 µg µl −1 in pbs) on parafilm (bemis na), and washed in pbs. fd-based afm on model surfaces. fd-based afm on model surfaces was performed in pbs at room temperature using functionalized msct-d probes (bruker, nominal spring constant of 0.030 n/m and actual spring constants calculated using thermal tune) 31 . a bioscope resolve afm (bruker) operated in the force-volume (contact) mode (nanoscope software v9.1) was used. areas of 5 × 5 µm were scanned, ramp size set to 500 nm, and set point force of 500 pn, with a resolution of 32 × 32 pixels and a line frequency of 1 hz. dfs analysis (using a constant approach speed of 1 µm/s and variable retraction speeds of 0.1, 0.2, 1, 5, 10, and 20 µm/s) and kinetic on-rate estimation (measuring the bp for different hold times of 0, 50, 100, 150, 250, 500, and 1000 ms) were performed. regarding dfs experiments, data including lrs and disruption forces were extracted using nanoscope analysis (v2.0, bruker). origin software (originlab) was used to display the results in dfs plots to fit histograms of rupture force distributions for distinct lr ranges, and to apply various force spectroscopy models, as described 8, 16 . for kinetic on-rate analysis, the bp (fraction of curves showing binding events) was determined at a certain hold time (t) (the time the tip is in contact with the surface). those data were fitted and k d calculated as described previously 32 . in brief, the relationship between interaction time (τ) and anti-binding effects of ace2-derived peptides on s1-subunit binding. a efficiency of blocking peptides is evaluated by measuring the binding probability of the interaction between the s1 subunit and ace2 receptor on model surface before and after incubation of the functionalized afm tip with the four different peptides at increasing concentration (1-100 µm). b histograms, with the corresponding data points overlaid in dark gray, showing the binding probability without peptide (0 µm) and upon incubation with 1, 10, or 100 µm of ace2-derived peptides ( , , [22-44bp is described by the following equation: where a is the maximum bp and t 0 the lag time. origin software is used to fit the data and extract τ. in the next step, k on was calculated by the following equation, with r eff the radius of the sphere, n b the number of binding partners, and n a the avogadro constant the effective volume v eff (4πr eff 3 ) represents the volume in which the interaction can take place. this results in a half-sphere, since only half of the s1 molecules can interact with its corresponding receptor on the substrate. peptides and competition-binding assays. to assess the influence of peptides on the s1-subunit-ace2 interaction, binding probabilities were measured before and after tip incubation with 1, 10, and 100 µm of peptide. briefly, a first map was recorded as described above (i.e., force-volume mode, 1 µm/s approach and retraction speed, ramp size of 500 nm, an applied force of 500 pn, resolution of 32 × 32 pixels, line frequency of 1 hz, and hold time of 250 ms), then the peptide at the appropriate concentration was injected, and a new map was recorded. all the peptides ( , [351] [352] [353] [354] [355] [356] [357] , , and [22-44-g-351-357] ) were synthesized by genscript (hong kong). those peptides are designed according to the sequence of the ace2 receptor in complex with the rbd domain of the s1 glycoprotein. fd-based afm and fluorescence microscopy on living cells. an afm (bioscope resolve, bruker) coupled to a confocal microscope (zeiss lsm-980) was used to acquire correlative images. the afm was equipped with a 150-µm piezoelectric scanner. the afm and the microscope were equipped with a cell-culture chamber allowing maintaining the temperature (37 ± 1°c). to keep cells alive, the humidified (95 ± 2% relative humidity) synthetic air (80% n 2 and 20% o 2 ) was supplemented with 5% co 2 and filled continuously around the cell plate allowing to diffuse into cell-culture media 16 . fluorescence images were recorded using a waterimmersion lens (×63, na 1.20, zeiss c-apochromat). pfqnm-lc cantilevers (bruker) were used to record afm images (~25 μm 2 ) at imaging forces of~500 pn. the cantilevers were oscillated either at 0.25-khz peak force frequency with a 750nm amplitude, 0.125 khz with a 375-nm amplitude in the peakforce tapping mode, or at 20 μm s −1 retraction speed in fast-force-volume mode. the sample was scanned using 256 pixels per line (256 lines) and a frequency of 0.125 hz. to study the involvement of other receptors, cells were treated with either 1 mm of 9-oacetyl-sialogycans or 500 μm of crgd. afm images and fd curves were analyzed using nanoscope analysis software, origin, gwyddion, and imagej. optical images were analyzed using zen software (zeiss). the fluorescence intensity was measured with zen software (zeiss). the same size of the area was taken on a549-ace2 and a549 cells. the average intensity of the area was calculated with zen software. the statistical analysis was performed with prism (graphpad). plasma membrane staining. plasma membrane-cfp bacmam 2.0 (invitrogen) was used to check the co-localization of ace2 protein and plasma membrane according to the manufacturer's protocol. in brief, 2 μl of plasma membrane-cfp bacmam 2.0 per 10,000 cells was added on the cell-culture dish 16 h (37°c) before imaging. z-stack image was recorded by confocal lsm-980 (zeiss) using a waterimmersion lens (×63, na 1.20, zeiss c-apochromat) and 445-and 488-nm laser line. affinity measurements using bli. affinity between the s1 subunit or rbd and ace2 was also investigated by bli, using a blitz® device equipped with aminereactive second-generation (ar2g) biosensors (pall fortebio). after hydrating the biosensor for 10 min and performing an initial baseline (1 min), the biosensor surface was chemically activated (5 min) by a freshly prepared 20 mm edc and 10 mm nhs (in milliq water) solution. then, ace2 (0.025 µg µl −1 in acetate buffer, ph 4) was loaded onto the biosensor during 3 min and the reaction quenched with ethanolamine 1 m (ph 8). after another baseline step (1 min in pbs), binding of s1 subunit or rbd (0.1 mg ml −1 ) was measured for 5 min. finally, the dissociation step (5 min) was performed in pbs. data processing and analysis were run using a routine provided by graphpad prism. md simulation between ace2 peptides and s glycoprotein. the pdb (code: 6m0j) 29 was used to perform a md simulation between ace2-derived peptides and the sars-cov-2 spike protein complex. md simulations were performed utilizing the gromacs package 33, 34 and carried out using the amber99sb-ildn 35 force fields in tip3p water 36 . the simulation system consisted of a peptide, a protein, and water (about 20,000 molecules) in a cubic box that extended 10 nm from the protein. appropriate amounts of sodium/chlorine ions were added in the system. for starting the simulation, the environment had to be developed as follows. the steepest descent algorithm was performed either up to 50,000 steps or by 100 kj mol −1 nm −1 . then, the environment of the system changed at 300 k (nvt ensemble) and subsequently at 300 k and 1 bar (npt ensemble). after developing the environment, the particle mesh ewald 37 method was used to calculate the longrange electrostatic interactions. short-range dispersion interactions were described by a lennard-jones potential with the cutoff of 1 nm. after reaching the equilibrium of temperature and pressure, mds were conducted for 60 ns at 300 k and 1 bar. the lincs algorithm 38 was applied to constrain the covalent bonds with hydrogen atoms. the time step of the simulations was set to 2 fs. the interactions above 10 å were regarded as nonbond. to determine whether a hydrogen bond exists between a peptide and a protein in the md models, a geometrical criterion was adopted, in which the formation of a hydrogen bond was defined by both atom distance and bond orientation. for example, assuming donor d, hydrogen h, and acceptor a consists of d-h ··· a configuration. then when the distance between donor d and acceptor a was shorter than 3.5 å as well as the bond angle h-d ··· a smaller than 60.0°, it has been regarded as a hydrogen bond. the hydrogen bonds are counted for 55-60 ns while running the simulations. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. a pneumonia outbreak associated with a new coronavirus of probable bat origin structural insights into coronavirus entry structural basis for the recognition of sars-cov-2 by full-length human ace2 coronavirus spike proteins in viral entry and pathogenesis proteolytic activation of the sars-coronavirus spike protein: cutting enzymes at the cutting edge of antiviral research host cell proteases: critical determinants of coronavirus tropism and pathogenesis imaging g protein-coupled receptors while quantifying their ligand-binding free-energy landscape glycan-mediated enhancement of reovirus receptor binding combining confocal and atomic force microscopy to quantify single-virus binding to mammalian cell surfaces multivalent binding of herpesvirus to living cells is tightly regulated during infection multiple receptors involved in human rhinovirus attachment to live cells monitoring early fusion dynamics of human immunodeficiency virus type 1 at singlemolecule resolution models for the specific adhesion of cells to cells sensitive force technique to probe molecular adhesion and structural linkages at biological interfaces dynamic strength of molecular adhesion bonds nanomechanical mapping of first binding steps of a virus to animal cells influenza virus binds its host cell using multiple dynamic interactions cryo-em structure of the 2019-ncov spike in the prefusion conformation structural basis of receptor recognition by sars-cov-2 angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus virus entry: molecular mechanisms and biomedical applications isolation and characterization of sars-cov-2 from the first us covid-19 patient ace2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia structural basis for human coronavirus attachment to sialic acid receptors a potential role for integrins in host cell entry by sars-cov-2 mechanical forces guiding staphylococcus aureus cellular invasion angiotensinconverting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target inhibition of sars-cov-2 infections in engineered human tissues using clinical-grade soluble human ace2 structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor linking of sensor molecules with amino groups to aminofunctionalized afm tips calculation of thermal noise in atomic force microscopy determination of the kinetic on-and off-rate of single viruscell interactions gromacs: a message-passing parallel molecular dynamics implementation 0: a package for molecular simulation and trajectory analysis improved side-chain torsion potentials for the amber ff99sb protein force field comparison of simple potential functions for simulating liquid water a smooth particle mesh ewald method lincs: a parallel linear constraint solver for molecular simulation synthesis of a new series of sialylated homo-and heterovalent glycoclusters by using orthogonal ligations synthesis of 9-o-acyl-and 4-o-acetyl-sialic acids modeller: generation and refinement of homology-based protein structure models synthesis of 9-o-acetyl-2-α-o-propargyl-sc. 2-α-o-propargyl sc was synthesized by the protocol described by dashkan et al. 39 . this molecule was selectively acetylated at the 9-position following the procedure of ogura et al. 40 .reporting summary. further information on research design is available in the nature research reporting summary linked to this article. the source data underlying figs. 2c, e-h, 3f, h, 4b, c, 5a and supplementary figs. 2, 6, 7 are provided as a source data file. all other relevant data are available from the corresponding authors upon reasonable request. source data are provided with this paper.received: 9 july 2020; accepted: 18 august 2020; this work was supported by the universitécatholique de louvain, the foundation louvain, and the fonds national de la recherche scientifique (frs-fnrs). this project received funding from the european research council under the european union's horizon 2020 research and innovation program (grant agreement no. 758224) and from the fnrs-welbio (grant # cr-2019s-01). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the paper. s.p., a.c.d., and d.a. are research fellow, postdoctoral researcher, and research associate at the fnrs, respectively. q.z., w.c., and s.p.v. are grateful to china scholarship council. j.y., s.j.l.p., m.k., a.c.d., and d.a. conceived the project, planned the experiments, and analyzed the data. j.y., s.j.l.p., and s.d. conducted the afm experiments. q.z. performed md simulation and structure predictions. s.p., m.k., and p.s. conducted and analyzed the blitz experiments. w.c. and s.p.v. conceived and synthesized the sa derivative. all authors wrote the paper. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/10.1038/s41467-020-18319-6.correspondence and requests for materials should be addressed to d.a.peer review information nature communications thanks the anonymous reviewers for their contribution to the peer review of this work. peer reviewer reports are available.reprints and permission information is available at http://www.nature.com/reprintspublisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-253447-4w6caxwu authors: zeng, xin; li, lingfang; lin, jing; li, xinlei; liu, bin; kong, yang; zeng, shunze; du, jianhua; xiao, huahong; zhang, tao; zhang, shelin; liu, jianghai title: blocking antibodies against sars-cov-2 rbd isolated from a phage display antibody library using a competitive biopanning strategy date: 2020-04-20 journal: biorxiv doi: 10.1101/2020.04.19.049643 sha: doc_id: 253447 cord_uid: 4w6caxwu the infection of the novel coronavirus sars-cov-2 have caused more than 150,000 deaths, but no vaccine or specific therapeutic antibody is currently available. sars-cov-2 relies on its spike protein, in particular the receptor binding domain (rbd), to bind human cell receptor angiotensin-converting enzyme 2 (ace2) for viral entry, and thus targeting rbd holds the promise for preventing sars-cov-2 infection. in this work, a competitive biopanning strategy of a phage display antibody library was applied to screen blocking antibodies against rbd. high-affinity antibodies were enriched after the first round using a standard panning process in which rbd-his recombinant protein was immobilized as a bait. at the next two rounds, immobilized ace2-fc and free rbd-his proteins were mixed with the enriched phage antibodies. antibodies binding to rbd at epitopes different from ace2-binding site were captured by the immobilized ace2-fc, forming a “sandwich” complex. only antibodies competed with ace2 for recognizing rbd at the same or similar epitopes can bind to the free rbd-his in the supernatant and be subsequently separated by the ni-nta magnetic beads. top 1 lead from the competitive biopanning of a synthetic antibody library, lib ab1, was produced as the full-length igg1 format. it was proved to competitively block the binding of rbd to ace2 protein, and potently inhibit sars-cov-2 pseudovirus infection of ace2-overexpressing hela cells with ic50 values of 12nm. nevertheless, top 1 lead from the standard biopanning of lib ab1, can only bind to rbd in vitro but not have the blocking or neutralization activity. our strategy can efficiently isolate the blocking antibodies of rbd, and it would speed up the discovery of neutralizing antibodies against sars-cov-2. the recent outbreak of a novel coronavirus disease (covid-19) has emerged from a public health emergency of international concern to global pandemic. its pathogen, sars-cov-2, is a newly identified β-coronavirus. coronavirus got the family name from the spike(s) protein on the viral particle. the highly glycosylated s protein stays compact in trimeric state, recognizes receptor on the host cell membrane, and then undergoes a series of conformation changes, proteolysis events and membrane fusion to complete viral entry. for vaccines, clinical diagnosis, early prevention and medication, the s protein is the most significant target. the primary sequences of s protein between severe acute respiratory syndrome coronavirus (sars-cov) and sars-cov-2 share about 76% identities and 86% similarities, which indicates high possibility of structural homology and the same infection pathway. sars-cov and sars-cov-2 recognized the same host cell receptor ace2 for mediating viral entry into host cells. it was reported that sars-cov s protein trimer bound to ace2 at 1:1 in ratio [1, 2] . before infection, rbd of each sars-cov s monomer was partially buried in the inactive "down" conformation and not able to bind ace2 due to steric clash. once infection started, one monomer turned "up" its rbd to expose enough space to ace2, inducing further conformational open and loose for proteolysis [1, 3] . atomic-level structural analysis suggested that the spatial interaction and interface between sars-cov-2 rbd and ace2 was mostly in accordance with the sars-cov case [4] . besides, a cryo-em structure of sars-cov-2 s protein trimer published recently showed that one of the three rbds was in "up" conformation and naturally exposed the whole interaction interface [5] , while the classic closed symmetric trimer still existed [6] . that might explain why sars-cov-2 is much more contagious than sars-cov and causing tricky problems worldwide. no effective cure or vaccine is currently available for covid-19. based on structure information above, blocking sars-cov-2 rbd is a rational therapeutic approach. here we developed a competitive biopanning strategy to efficiently isolate blocking antibodies from phage display antibody libraries. several high-affinity antibodies targeting sars-cov-2 rbd and blocking its binding to ace2 were isolated, and the top 1 lead exhibited a neutralization activity of sars-cov-2 pseudotyped vsv infection. recombinant proteins ace2-his was purchased from novoprotein (shanghai, china). ace2-hfc and sars-cov-2 rbd-his were purchased from sino biological (beijing, china). sars-cov-2 rbd-mfc was expressed using ablink biotech's hek 293f expression system. a synthetic human fab antibody library ab1 (libab1) was constructed according to a procedure previously described [7] . human germline immunoglobulin variable segments vh3-30 and vl1-16 were employed as templates, the complementarity-determining regions l3 (cdr-l3) and h3 (cdr-h3) was diversified by the designed mutagenic oligonucleotides. the oligonucleotides were synthesized using the trimer phosphoramidites mix z (glen research) containing codons for 12 amino acids in the following molar ratios: 20% each y, s &g, 6% each t & a, and 4% each p, h, r, f, w, v & l. the number of positions denoted by z in cdr-l3 (qq (z)n plt) and -h3 (ar (z) n (a/g/d/y) fdy) was varied from 3 to 12 and 8 to 12, respectively. the library size is estimated to be 1×10 12 . antibodies against rbd were screened at the first round using a standard biopanning protocol [8] . briefly, rbd-his was coated on 96-well maxisorp plates at 4°c overnight. after the coating buffer was decanted, the plate was blocked with 1% polyvinyl alcohol (pva) at room temperature for 1 hour. 100μl of phage libraries (10 13 pfu/ml) was added per well for 2-hour binding. after washing eight times with pt buffer (0.05% tween-20 in pbs), bound phages were eluted with 100mm hcl (100μl per well), followed by 5-min incubation. the eluent was transferred into a 1.5ml microfuge tube and neutralized with 1m tris-hcl (ph 8.0). half the neutralized phage solution was mixed with 1ml of actively growing e. coli neb 5-alpha f' (od600 = 0.8) in 2×yt media containing 10μg/ml tetracycline and incubated at 37°c for 1 hour. 10 10 pfu of m13k07 helper phages were added next and incubated for another 1 hour. the infected bacteria were amplified in 50ml 2×yt medium containing 50μg/ml carbenicillin and 25μg/ml kanamycin, shaking at 200rpm and growing overnight at 37°c. the next day, phages were harvested in precipitant with peg/nacl solution and resuspended in pbs buffer for the following rounds of panning. after the first round of the standard biopanning, a competitive biopanning protocol that included steps of competitive binding, magnetic separation, elution and amplification ( fig.1) , was applied to isolate the epitope-specific antibodies. briefly, 100μl of ace2-hfc protein (5μg/ml) was coated on the 96-well maxisorp plates. the wells were washed and blocked with 1% pva, and then the mixture of antibody library (10 10 pfu per well) and free rbd-his protein (100ng per well) was added. after a 2-hour competitive binding, the supernatant was transferred into a 1.5ml microfuge tube containing the pre-washed ni-nta magnetic beads (genscript) and incubated on a shaker at room temperature for 1 hour. beads were collected using the magnetic separation rack and washed by the pt buffer for 8 times. bound phages were eluted with 100mm hcl (100μl per tube) after 5-min incubation. beads were collected using the magnetic separation rack, and the supernatant was transfer into a tube for neutralization. half the neutralized phage solution was mixed with 1ml of actively growing neb alpha f' cells and amplified as the standard biopanning protocol. 10μl of the bacterial culture before infection with helper phages was taken, diluted, and grown on the lb plates containing 50μg/ml carbenicillin at 37°c overnight. the single clones were picked up next day for the phage elisa assay. fig.1 schematic presentation of a competitive biopanning strategy. a specific binder of target protein was added during the binding step for the selection of blocking antibodies. in this work, the immobilized ace2-hfc captured rbd-his and the antibodies binding rbd at different epitopes, forming a complex like a "sandwich". however, when an antibody recognized the same or similar epitopes within rbd as the ace2 did, it could block rbd-ace2 interaction. the antibodies would bind to the free rbd-his in the supernatant and be subsequently separated by the ni-nta magnetic beads. single clones were inoculated into 400μl 2×yt medium containing 50μg/ml carbenicillin, 25μg/ml kanamycin and 10 10 pfu/ml helper phages in 96-deep-well plates and incubated overnight at 37°c and 250rpm. the plates were centrifuged at 4,000rpm and the supernatant was applied for phage elisa. the 96-well maxisorp plates were coated overnight at 4°c with rbd-mfc (1μg/ml, 100μl per well). after blocking with 1% pva, plates were incubated with 50μl bacterial supernatant containing phages for 2 hours at room temperature. after six times of wash with pt, bound phages were detected using an hrp-conjugated anti-m13 antibody (sino biological) and tetramethyl benzidine (tmb) as substrate. absorption at 450nm was measured. vh and vl of the positive phage were subcloned respectively into the pfusess-chig-hg1 and pfusess-clig-hk (invitrogen). antibodies were transiently expressed in freestyle™ hek 293-f cells (life technologies) using 293fectin transfection reagent according to manufacturer's instructions. after transfection, cells were grown in the serum-free medium for an additional 5 days. the supernatant was collected and purified on a mabselect protein a column (ge healthcare). eluted igg was dialyzed against pbs and stored at -80°c. recombinant human ace2-his (5μg/ml, 100μl per well) was coated on 96-well maxisorp plates, followed by a pre-incubated mixture of the anti-rbd antibody titrated into a constant amount of rbd-mfc (1µg/ml). rbd binding to ace2 was detected using hrp conjugated anti-mouse fc antibody. the neutralization effects of antibodies on sars-cov-2 pseudovirus were performed by the genscript inc. (nanjing, china) under a research service contract. briefly, 20,000 of the human ace2-overexpressing hela monoclonal cells were seeded into each well of a 96-well plate. sars-cov-2 pseudovirus and antibodies were incubated at ambient temperature for 1 hour. the mixture was transferred into wells and incubated with cells at 37°c, 5% co2 for 24 hours. the culture medium was freshly replaced, and cells were incubated for another 24 hours. the culture medium was removed, and cells were rinsed with pbs. 50µl lysis buffer was added and further incubated at ambient temperature for 40 minutes. 40µl supernatant was transferred to a sterile un-clear 96-well plate with the bio-glo luciferase substrate added, and the luminescence signal was measured with envision. the dose response curves were plotted with the relative luminescence unit against the antibody concentration. the assay results were processed by microsoft office excel 2013 and graphpad prism 6. high-affinity antibodies were identified by the phage elisa after 2 rounds of the competitive biopanning, 48 clones were randomly selected. their properties of binding to rbd were measured using phage elisa. positive binding was defined as an od450 reading two or more times higher than the negative control (pva alone). 18 clones showed positive signals (fig.2) . after the dna sequencing, these clones were summarized into 5 groups of unique antibodies. identification of positive clones to immobilized antigen in competitive manner. taken od450 readings as measurement, data of each group fluctuated within 20%. the highest-ranking one was named rrbd-15 in this work. rrbd-15, the top1 lead with the highest od450 reading isolated from the competitive biopanning, and rrbd-16, the top 1 lead isolated from the standard biopanning at round 3, were expressed as full-length igg1 antibodies using the 293f expression system. their binding and blocking abilities against rbd were compared. both rrbd-15 and rrbd-16 had high affinities for rbd, with ec50 at 3.8nm and 5.3nm, respectively. only rrbd-15 blocked the binding of rbd to ace2 with an ic50 at 3.0nm, while rrbd-16 did not. as a positive control, the recombinant ace2-hfc (100µg/ml) totally inhibited the infection of ace2-overexpressing hela monoclonal cells with sars-cov-2 pseudovirus. the antibody rrbd-15 showed a significant neutralization activity against the sars-cov-2 pseudovirus with ic50 values of 12.2nm. however, the antibody rrbd-16 had no neutralization effect of the pseudovirus and there were no significant differences between the highest concentration antibody group and the blank group without antibody addition. two sars-cov-2 rbd-specific antibodies selected from different strategies showed different neutralization activities. the antibody rrbd-15 competed with ace2 could neutralize sars-cov-2 pseudovirus, but rrbd-16 couldn't. rbd-ace2 interaction initiated viral infection of both sars-cov and sars-cov-2. their rbds share high sequence identities (73%) and structure homology, so the well-established sars-cov antibodies were firstly assumed short-cut therapeutic candidates for sars-cov-2. however, the real scenario is much more problematic. several independent peer-reviewed studies as well as preprinted ones have proved that all structurally known sars-cov specific antibodies, including s230, 80r, m396 and f26g19, have no cross-reactivity of sars-cov-2 [4, 5, 9] . these antibodies all compete with ace2 to bind sars-cov rbd, but their epitopes only have limited overlaps of the full ace2-rbd interface, which could be the reason of lacking cross-reactivity. cr3022 is a special case with 86% conserved key residues in the epitope between sars-cov-2 and sars-cov. its cross-reactivity was remarkable, but one site loss of n-glycan results in 1~2 magnitude reduction of binding affinity to sars-cov-2 rbd [9] . as in human life, rbd-specific monoclonal antibodies derived from covid-19 recovered individuals indicated similar patterns of no cross-reactivities with either sar-cov or mers-cov [10] . in general, structural and functional analysis suggests that targeting sars-cov-2 rbd could be a direct and promising therapeutic strategy, while focusing on previous sars-cov antibodies is not very ideal or efficient. no sars-cov-2 rbd-specific monoclonal antibody has been reported from human antibody libraries (up to april 17 th , 2020). in the meantime, sars-cov-2 spreads unexpectedly fast around the world, and a new study just shifted its basic reproductive number (r0) from 2.2 to 5.7 [11] . a rapid and effective method of obtaining the sars-cov-2 neutralizing antibodies is much required. naï ve antibody libraries derived from natural immune systems have their capacity limits, while synthetic libraries with higher diversity have more opportunities to isolate binders especially for novel infectious antigens. compared to a naï ve antibody library of 10 8~1 0 9 diversity, a synthetic library with additional artificial randomization on cdrs can reach diversity as high as 10 12~1 0 13 . when the recombinant rbd and ace2 proteins were ready, it took 3 weeks to isolate, produce and verify the antibodies in this study. using the standard biopanning method, we enriched rbdspecific phages from our synthetic lib ab1 but not from our naï ve antibody libraries (data not shown). unfortunately, the top1 lead rrbd-16 from the standard biopanning of lib ab1 couldn't block the rbd-ace2 interaction (fig. 4) , although it did bind to rbd with an ec50 of 5.3nm (fig. 3) . the clinical potential and applications of an antibody often depends on its binding epitopes of the target protein. a high-affinity antibody against the target protein can be screened from a phage display antibody library using the standard biopanning process, but its binding epitopes are identified by some extra steps, such as epitope mapping and competitive elisa. we therefore developed a new competitive biopanning strategy to efficiently isolate isotype-specific antibodies from libraries. as expected, the top1 lead rrbd-15 successfully bind to rbd in compete with ace2 both in solution and in pseudovirus, and its binding affinity is quite high in 1~10nm differing from measuring methods. in conclusion, our strategic discovery of human monoclonal antibodies against sars-cov-2 rbd may fill the blanks of antibody-related pharmaceutical development and shed light on new treatments in need of global health concerns. cryo-em structure of the sars coronavirus spike glycoprotein in complex with its host cell receptor ace2 unexpected receptor functional mimicry elucidates activation of coronavirus fusion cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding structural and functional basis of sars-cov-2 entry by using human ace2 cryo-em structure of the sars-cov-2 spike in the prefusion conformation structure, function, and antigenicity of the sars-cov-2 spike glycoprotein a single-framework synthetic antibody library containing a combination of canonical and variable complementarity-determining regions identifying specificity profiles for peptide recognition modules from phage-displayed peptide libraries potent human neutralizing antibodies elicited by sars-cov-2 infection high contagiousness and rapid spread of severe acute respiratory syndrome coronavirus 2 we thank chengdu zicheng yibo biotechnology co., ltd for providing the laboratory consumables and bovine serum. this work was supported by sichuan science and technology program (2018rz0019), the program of sars-cov-2 protection (cyhx202032, kezhi people's air-defense equipment co., ltd) and the program of sars-cov-2 antibody discovery (jl2020c-01, ablink biotech co., ltd). key: cord-024319-isbqs7hg authors: zeng, xin; li, lingfang; lin, jing; li, xinlei; liu, bin; kong, yang; zeng, shunze; du, jianhua; xiao, huahong; zhang, tao; zhang, shelin; liu, jianghai title: isolation of a human monoclonal antibody specific for the receptor binding domain of sars-cov-2 using a competitive phage biopanning strategy date: 2020-04-30 journal: antib ther doi: 10.1093/abt/tbaa008 sha: doc_id: 24319 cord_uid: isbqs7hg the infection of the novel coronavirus sars-cov-2 has caused more than 200,000 deaths, but no vaccine or therapeutic monoclonal antibody is currently available. sars-cov-2 relies on its spike protein, in particular the receptor binding domain (rbd), to bind human cell receptor angiotensin-converting enzyme 2 (ace2) for viral entry, and thus targeting rbd holds the promise for preventing sars-cov-2 infection. in this work, a competitive biopanning strategy of a phage display antibody library was applied to screen blocking antibodies against rbd. high-affinity antibodies were enriched after the first round using a standard panning process in which rbd-his was immobilized as a bait. at the next two rounds, immobilized ace2-fc and free rbd-his were mixed with the enriched phage antibodies. antibodies binding to rbd at epitopes different from ace2-binding site were captured by the immobilized ace2-fc, forming a “sandwich” complex. only antibodies competed with ace2 can bind to the free rbd-his in the supernatant and be subsequently separated by the ni-nta magnetic beads. top 1 lead from the competitive biopanning of our synthetic antibody library, lib ab1, was produced as the full-length igg1 format. it was proved to competitively block the binding of rbd to ace2 and potently inhibit sars-cov-2 pseudovirus infection with ic(50) values of 12 nm. nevertheless, top 1 lead from the standard biopanning can only bind to rbd in vitro, but not have the blocking or neutralization activity. our strategy can efficiently isolate the blocking antibodies of rbd, and it would speed up the discovery of neutralizing antibodies against sars-cov-2. the recent outbreak of a novel coronavirus disease (covid-19) has emerged from a public health emergency of international concern to global pandemic. its pathogen, sars-cov-2, is a newly identified β-coronavirus. coronavirus got the family name from the spike (s) protein on the viral particle. the highly glycosylated s protein stays compact in a trimeric state, recognizes receptor on the host cell membrane, and undergoes a series of conformation changes, proteolysis events and membrane fusion to complete viral entry. for vaccines, clinical diagnosis, early prevention and medication, the s protein is the most significant target. the primary sequences of s protein between severe acute respiratory syndrome coronavirus (sars-cov) and sars-cov-2 share about 76% identities and 86% similarities, which indicates high possibility of structural homology and similar infection pathway. sars-cov and sars-cov-2 recognize the same host cell receptor ace2 for mediating viral entry into host cells. it was reported that sars-cov s protein trimer bound to ace2 at 1:1 in ratio [1, 2] . before infection, rbd of each sars-cov s monomer was partially buried in the inactive "down" conformation and not able to bind ace2 due to steric clash. once infection started, one rbd monomer turned "up" to expose enough space to ace2, inducing further conformational open and loose for proteolysis [1, 3] . atomic-level structural analysis suggested that the spatial interaction and interface between sars-cov-2 rbd and ace2 was mostly in accordance with the sars-cov case [4] . besides, a cryo-em structure of sars-cov-2 s protein trimer published recently showed that one of the three rbds was in "up" conformation and naturally exposed the whole interaction interface [5] , while the classic closed symmetric trimer still existed [6] . that might explain why sars-cov-2 is much more contagious and problematic than sars-cov worldwide. no effective cure or vaccine is currently available for covid-19. based on structure information above, blocking sars-cov-2 rbd is a rational therapeutic approach. here we developed a competitive biopanning strategy to efficiently isolate blocking antibodies from phage display antibody libraries. several high-affinity antibodies targeting sars-cov-2 rbd and blocking its binding to ace2 were isolated, and the top 1 lead exhibited a neutralization activity of sars-cov-2 pseudotyped vsv infection. recombinant proteins ace2-his was purchased from novoprotein (shanghai, china). ace2-hfc and sars-cov-2 rbd-his were purchased from sino biological (beijing, china). sars-cov-2 rbd-mfc was expressed using ablink biotech's hek 293f expression system. a synthetic human fab antibody library ab1 (libab1) was constructed according to a procedure previously described [7] . human germline immunoglobulin variable segments vh3-30 and vl1-16 were employed as templates, the complementarity-determining regions l3 (cdr-l3) and h3 (cdr-h3) was diversified by the designed mutagenic oligonucleotides. the oligonucleotides were synthesized using the trimer phosphoramidites mix z (glen research) containing codons for 12 amino acids in the following molar ratios: 20% each y, s &g, 6% each t & a, and 4% each p, h, r, f, w, v & l. the number of positions denoted by z in cdr-l3 (qq (z)n plt) and -h3 (ar (z) n (a/g/d/y) fdy) was varied from 3 to 12 and 8 to 12, respectively. the library size is estimated to be 1×10 12 . antibodies against rbd were screened at the first round using a standard biopanning protocol [8] . briefly, rbd-his was coated on 96-well maxisorp plates at 4°c overnight. after the coating buffer was decanted, the plate was blocked with 1% polyvinyl alcohol (pva) at room temperature for 1 hour. 100μl of phage libraries (10 13 pfu/ml) was added per well for 2-hour binding. after washing eight times with pt buffer (0.05% tween-20 in pbs), bound phages were eluted with 100mm hcl (100μl per well), followed by 5-min incubation. the eluent was transferred into a 1.5ml microfuge tube and neutralized with 1m tris-hcl (ph 8.0). half the neutralized phage solution was mixed with 1ml of actively growing e. coli neb 5-alpha f' (od600 = 0.8) in 2× yt media containing 10μg/ml tetracycline and incubated at 37°c for 1 hour. 1×10 10 pfu of m13k07 helper phages were added next and incubated for another 1 hour. the infected bacteria were amplified in 50ml 2× yt medium containing 50μg/ml carbenicillin and 25μg/ml kanamycin, shaking at 200rpm and growing overnight at 37°c. the next day, phages were harvested in precipitant with peg/nacl solution and resuspended in pbs buffer for the following rounds of panning. after the first round of the standard biopanning, a competitive biopanning protocol that included steps of competitive binding, magnetic separation, elution and amplification ( fig.1) , was applied to isolate the epitope-specific antibodies. briefly, 100μl of ace2-hfc protein (5μg/ml) was coated on the 96-well maxisorp plates. the wells were washed and blocked with 1% pva, and then the mixture of antibody library (1×10 10 pfu per well) and free rbd-his protein (100ng per well) was added. after a 2-hour competitive binding, the supernatant was transferred into a 1.5ml microfuge tube containing the pre-washed ni-nta magnetic beads (genscript) and incubated on a shaker at room temperature for 1 hour. beads were collected using the magnetic separation rack and washed by the pt buffer for 8 times. bound phages were eluted with 100mm hcl (100μl per tube) after 5-min incubation. beads were collected using the magnetic separation rack, and the supernatant was transfer into a tube for neutralization. half the neutralized phage solution was mixed with 1ml of actively growing neb alpha f' cells and amplified as the standard biopanning protocol. 10μl of the bacterial culture before infection with helper phages was taken, diluted, and grown on the lb plates containing 50μg/ml carbenicillin at 37°c overnight. the single clones were picked up next day for the phage elisa assay. single clones were inoculated into 400μl 2× yt medium containing 50μg/ml carbenicillin, 25μg/ml kanamycin and 10 10 pfu/ml helper phages in 96-deep-well plates and incubated overnight at 37°c and 250rpm. the plates were centrifuged at 4,000rpm and the supernatant was applied for phage elisa. the 96-well maxisorp plates were coated overnight at 4°c with rbd-mfc (1μg/ml, 100μl per well). after blocking with 1% pva, plates were incubated with 50μl bacterial supernatant containing phages for 2 hours at room temperature. after six times of wash with pt, bound phages were detected using an hrp-conjugated anti-m13 antibody (sino biological) and tetramethyl benzidine (tmb) as substrate. absorption at 450nm was measured. vh and vl of the positive phage were subcloned respectively into the pfusess-chig-hg1 and pfusess-clig-hk (invivogen). antibodies were transiently expressed in freestyle™ hek 293-f cells (life technologies) using 293fectin transfection reagent according to manufacturer's instructions. after transfection, cells were grown in the serum-free medium for an additional 5 days. the supernatant was collected and purified on a mabselect protein a column (ge healthcare). eluted igg was dialyzed against pbs and stored at -80°c. recombinant human ace2-his (5μg/ml, 100μl per well) was coated on 96-well maxisorp plates, followed by a pre-incubated mixture of the anti-rbd antibody titrated into a constant amount of rbd-mfc (1µg/ml). rbd binding to ace2 was detected using hrp conjugated anti-mouse fc antibody. the neutralization effects of antibodies on sars-cov-2 pseudovirus were performed by the genscript inc. (nanjing, china) under a research service contract. briefly, 20,000 of the human ace2-overexpressing hela monoclonal cells were seeded into each well of a 96-well plate. sars-cov-2 pseudovirus and antibodies were incubated at ambient temperature for 1 hour. the mixture was transferred into wells and incubated with cells at 37°c, 5% co 2 for 24 hours. the culture medium was freshly replaced, and cells were incubated for another 24 hours. the culture medium was removed, and cells were rinsed with pbs. 50µl lysis buffer was added and further incubated at ambient temperature for 40 minutes. 40µl supernatant was transferred to a sterile un-clear 96-well plate with the bio-glo luciferase substrate added, and the luminescence signal was measured with envision. the dose response curves were plotted with the relative luminescence unit against the antibody concentration. the assay results were processed by microsoft office excel 2013 and graphpad prism 6. high-affinity antibodies were identified by the phage elisa rbd had a high affinity to ace2 with an ec 50 of around 1µg/ml (fig. s1 ). thus 1µg/ml of rbd was applied in our competitive biopanning strategy to ensure the immobilized ace2 can completely capture the "sandwich binding" complex. during the standard biopanning, phages were always applied at a concentration of 1×10 12 pfu. however, we changed it to 1×10 10 pfu per well during the competitive biopanning to reduce the non-specific binding of phages to magnetic beads. after 2 rounds of the competitive biopanning, 48 clones were randomly selected. their properties of binding to rbd were measured using phage elisa. positive binding was defined as an od450 reading two or more times higher than the negative control (pva alone). 18 clones showed positive signals (fig.2) . after the dna sequencing, these clones were summarized into 5 groups of unique antibodies. rrbd-15, the top1 lead with the highest od450 reading isolated from the competitive biopanning, and rrbd-16, the top 1 lead isolated from the standard biopanning at round 3, were expressed as full-length igg1 antibodies using the 293f expression system. their binding and blocking abilities against rbd were compared. both rrbd-15 and rrbd-16 had high affinities for rbd, with ec 50 at 3.8nm and 5.3nm, respectively. only rrbd-15 blocked the binding of rbd to ace2 with an ic 50 at 3.0nm, while rrbd-16 did not. as a positive control, the recombinant ace2-hfc (100µg/ml) totally inhibited the infection of ace2-overexpressing hela monoclonal cells with sars-cov-2 pseudovirus. the antibody rrbd-15 showed a significant neutralization activity against the sars-cov-2 pseudovirus with ic 50 values of 12.2nm. however, the antibody rrbd-16 had no neutralization effect of the pseudovirus and there were no significant differences between the highest concentration antibody group and the blank group without antibody addition. rbds share high sequence identities (73%) and structure homology, so the well-established sars-cov antibodies were firstly assumed short-cut therapeutic candidates for sars-cov-2. however, the real scenario is much more problematic. several independent peer-reviewed studies as well as preprinted ones have proved that all structurally known sars-cov specific antibodies, including s230, 80r, m396 and f26g19, have no cross-reactivity of sars-cov-2 [4, 5, 9] . the lack of cross-reactivity of current mabs may result from complicated factors. these antibodies all compete with ace2 to bind sars-cov rbd, but their epitopes only have limited overlaps of the several key residue mutations from sars-cov s to sars-cov-2 s don't alter the binding of ace2, but slight changes could be enough to break antibody recognition. cr3022 is a special case with 86% conserved key residues in the epitope between sars-cov-2 and sars-cov. its cross-reactivity was remarkable, but just one site loss of n-glycan results in 1~2 magnitude reduction of binding affinity to sars-cov-2 rbd [9] . in humans, rbd-specific monoclonal antibodies derived from covid-19 recovered individuals indicated similar patterns of no cross-reactivities with either sars-cov or mers-cov [10] . the findings using polyclonal antibodies are ambiguous. sera from sars-cov s-immunized mice, not rabbits or sars recovered patients, showed modest neutralization activity against sars-cov-2 [6, 11] , while sera from covid-19 recovered patients had no effect on sars pseudovirus [11] . in general, structural and functional analysis suggests that targeting sars-cov-2 rbd could be a direct and promising therapeutic strategy, while focusing on previous sars-cov antibodies is not very ideal or efficient. no sars-cov-2 rbd-specific monoclonal antibody has been reported from human antibody libraries (up to april 17 th , 2020). in the meantime, sars-cov-2 spreads unexpectedly fast around the world, and a new study just shifted its basic reproductive number (r 0 ) from 2.2 to 5.7 [12] . a rapid and effective method of obtaining the sars-cov-2 neutralizing antibodies is much required. naïve antibody libraries derived from natural immune systems have their capacity limits, while synthetic libraries with higher diversity have more opportunities to isolate binders especially for novel infectious antigens. compared to a naïve antibody library of 10 8~1 0 9 diversity, a synthetic library with additional artificial randomization on cdrs can reach diversity as high as 10 12~1 0 13 . when the recombinant rbd and ace2 proteins were ready, it took 3 weeks to isolate, produce and verify the antibodies in this study. using the standard biopanning method, we enriched rbd-specific phages from our synthetic lib ab1, but not from our naïve antibody libraries (data not shown). unfortunately, the top1 lead rrbd-16 from the standard biopanning of lib ab1 could not block the rbd-ace2 interaction (fig. 4) , although it bound to rbd with an ec 50 of 5.3nm (fig. 3) . the clinical potential and applications of an antibody often depends on its binding epitopes of the target protein. a high-affinity antibody against the target protein can be screened from a phage display antibody library using the standard biopanning process, but its binding epitopes are identified by some extra steps, such as epitope mapping and competitive elisa. we therefore developed a new competitive biopanning strategy to efficiently isolate isotype-specific antibodies from libraries. as expected, the top1 lead rrbd-15 successfully bind to rbd in compete with ace2 both in solution and in pseudovirus, and its binding affinity is quite high in 1~10nm differing from measuring methods. further experimental plan is scheduled to verify the neutralization effect in the live viruses and animal models, together with cross-reactivity assays of other disease-related coronaviruses. in conclusion, our strategic discovery of human monoclonal antibodies against sars-cov-2 rbd may fill the blanks of antibody-related pharmaceutical development and shed light on new treatments in need of global health concerns. fig.1 schematic presentation of a competitive biopanning strategy. a specific binder of target protein was added during the binding step for the selection of blocking antibodies. in this work, the immobilized ace2-hfc captured rbd-his and the antibodies binding rbd at different epitopes, forming a complex like a "sandwich". however, when an antibody recognized the same or similar epitopes within rbd as the ace2 did, it could block rbd-ace2 interaction. the antibodies would bind to the free rbd-his in the supernatant and be subsequently separated by the ni-nta magnetic beads. two sars-cov-2 rbd-specific antibodies selected from different strategies showed different neutralization activities. luminescence signal on y-axis indicated relative proportions of pseudovirus entry into target cells. the antibody rrbd-15 competed with ace2 could neutralize sars-cov-2 pseudovirus, but rrbd-16 could not. cryo-em structure of the sars coronavirus spike glycoprotein in complex with its host cell receptor ace2 unexpected receptor functional mimicry elucidates activation of coronavirus fusion cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding structural and functional basis of sars-cov-2 entry by using human ace2 cryo-em structure of the sars-cov-2 spike in the prefusion conformation structure, function, and antigenicity of the sars-cov-2 spike glycoprotein a single-framework synthetic antibody library containing a combination of canonical and variable complementarity-determining regions identifying specificity profiles for peptide recognition modules from phage-displayed peptide libraries a highly conserved cryptic epitope in the receptor-binding domains of sars-cov-2 and sars-cov potent human neutralizing antibodies elicited by sars-cov-2 infection characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov high contagiousness and rapid spread of severe acute respiratory syndrome coronavirus 2 we thank chengdu zicheng yibo biotechnology co., ltd for providing the laboratory consumables and bovine serum. this work was supported by sichuan science and technology program (2018rz0019), the program of sars-cov-2 protection (cyhx202032, kezhi people's air-defense equipment co., ltd) and the program of sars-cov-2 antibody discovery (jl2020c-01, ablink biotech co., ltd). key: cord-256156-mywhe6w9 authors: clausen, thomas mandel; sandoval, daniel r.; spliid, charlotte b.; pihl, jessica; perrett, hailee r.; painter, chelsea d.; narayanan, anoop; majowicz, sydney a.; kwong, elizabeth m.; mcvicar, rachael n.; thacker, bryan e.; glass, charles a.; yang, zhang; torres, jonathan l.; golden, gregory j.; bartels, phillip l.; porell, ryan; garretson, aaron f.; laubach, logan; feldman, jared; yin, xin; pu, yuan; hauser, blake; caradonna, timothy m.; kellman, benjamin p.; martino, cameron; gordts, philip l.s.m.; chanda, sumit k.; schmidt, aaron g.; godula, kamil; leibel, sandra l.; jose, joyce; corbett, kevin d.; ward, andrew b.; carlin, aaron f.; esko, jeffrey d. title: sars-cov-2 infection depends on cellular heparan sulfate and ace2 date: 2020-09-14 journal: cell doi: 10.1016/j.cell.2020.09.033 sha: doc_id: 256156 cord_uid: mywhe6w9 we show that sars-cov-2 spike protein interacts with both cellular heparan sulfate and angiotensin converting enzyme 2 (ace2) through its receptor binding domain (rbd). docking studies suggest a heparin/heparan sulfate-binding site adjacent to the ace2 binding site. both ace2 and heparin can bind independently to spike protein in vitro and a ternary complex can be generated using heparin as a scaffold. electron micrographs of spike protein suggests that heparin enhances the open conformation of the rbd that binds ace2. on cells, spike protein binding depends on both heparan sulfate and ace2. unfractionated heparin, non-anticoagulant heparin, heparin lyases, and lung heparan sulfate potently block spike protein binding and/or infection by pseudotyped virus and authentic sars-cov-2 virus. we suggest a model in which viral attachment and infection involves heparan sulfate-dependent enhancement of binding to ace2. manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin presents new therapeutic opportunities. the covid-19 pandemic, caused by the novel respiratory coronavirus 2 (sars-cov-2), has swept across the world, resulting in serious clinical morbidities and mortality, as well as widespread disruption to all aspects of society. as of september 1, 2020, the virus has spread to 215 countries, causing more than 25.4 million confirmed infections and at least 851,000 deaths (world health organization). current isolation/social distancing strategies seek to flatten the infection curve to avoid overwhelming hospitals and to give the medical establishment and pharmaceutical companies time to develop and test antiviral drugs and vaccines. currently, only one antiviral agent, remdesivir, has been approved for adult covid-19 patients (beigel et al., 2020) and vaccines may be 12-18 months away. understanding the mechanism for sars-cov-2 infection and its mechanism of infection could reveal other targets to interfere with viral infection and spread. the glycocalyx is a complex mixture of glycans and glycoconjugates surrounding all cells. given its location, viruses and other infectious organisms, must pass through the glycocalyx to engage receptors thought to mediate viral entry into host cells. many viral pathogens have evolved to utilize glycans as attachment factors, which facilitates the initial interaction with host cells, including influenza virus, herpes simplex virus, human immunodeficiency virus, and different coronaviruses (sars-cov-1 and mers-cov) (cagno et al., 2019; koehler et al., 2020; stencel-baerenwald et al., 2014) . several viruses interact with sialic acids, which are located on the ends of glycans found in glycolipids and glycoproteins. other viruses interact with heparan sulfate (hs) (milewska et al., 2014) , a highly negatively charged linear polysaccharide that is attached to a small set of membrane or extracellular matrix proteoglycans (lindahl et al., 2015) . in general, glycan-binding domains on membrane proteins of the virion envelope mediate initial attachment of virions to glycan receptors. attachment in this way can lead to the engagement of protein receptors on the host plasma membrane that facilitate membrane fusion or engulfment and internalization of the virion. j o u r n a l p r e -p r o o f 5 like other macromolecules, hs can be divided into subunits, which are operationally defined as disaccharides based on the ability of bacterial enzymes or nitrous acid to cleave the chain into disaccharide units (esko and selleck, 2002) . the basic disaccharide subunit consists of α1-4 linked d-glucuronic acid (glca) and α1-4 linked n-acetyl-d-glucosamine (glcnac), which undergo various modifications by sulfation and epimerization as the copolymer assembles on a limited number of membrane and extracellular matrix proteins (only 17 heparan sulfate proteoglycans are known) (lindahl et al., 2015) . the variable length of the modified domains and their pattern of sulfation create unique motifs to which hs-binding proteins interact (xu and esko, 2014) . different tissues and cell types vary in the structure of hs, and hs structure can vary between individuals and with age (de agostini et al., 2008; feyzi et al., 1998; han et al., 2020; ledin et al., 2004; vongchan et al., 2005; warda et al., 2006; wei et al., 2011) . these differences in hs composition may contribute to the tissue tropism and/or host susceptibility to infection by viruses and other pathogens. in this report, we show that the ectodomain of the sars-cov-2 spike (s) protein interacts with cell surface hs through the receptor binding domain (rbd) in the s1 subunit. binding of heparin to sars-cov-2 s protein shifts the structure to favor the rbd open conformation that binds ace2. spike binding to cells requires engagement of both cellular hs and ace2, suggesting that hs acts as a coreceptor priming the spike for ace2 interaction. therapeutic unfractionated heparin (ufh), non-anticoagulant heparin and hs derived from human lung and other tissues blocks binding. ufh and heparin lyases also block infection of cells by s protein pseudotyped virus and authentic sars-cov-2. these findings identify cellular hs as a necessary co-factor for sars-cov-2 infection and emphasizes the potential for targeting s protein-hs interactions to attenuate virus infection. the trimeric s proteins from sars-cov-1 and sars-cov-2 viruses are thought to engage human ace2 with one or more rbd in an "open" active conformation (fig. 1a ) (kirchdoerfer et al., 2018; walls et al., 2020; wrapp et al., 2020) . adjacent to the ace2 binding site and exposed in the rbd lies a group of positively-charged amino acid residues that represents a potential site that could interact with heparin or heparan sulfate ( fig. 1a and suppl. fig. s1 ). we calculated an electrostatic potential map of the rbd (from pdb id 6m17 (yan et al., 2020) ), which revealed an extended electropositive surface with dimensions and turns/loops consistent with a heparin-binding site (fig. 1b) (xu and esko, 2014) . docking studies using a tetrasaccharide (dp4) fragment derived from heparin demonstrated preferred interactions with this electropositive surface, which based on its dimensions could accommodate a chain of up to 20 monosaccharides ( fig. 1b and 1c ). evaluation of heparin-protein contacts and energy contributions using the molecular operating environment (moe) software suggested strong interactions with the positively charged amino acids r346, r355, k444, r466 and possibly r509 (figs. 1a, 1d, and 1e) . other amino acids, notably f347, s349, n354, g447, y449, and y451, could coordinate the oligosaccharide through hydrogen bonds and hydrophobic interactions. notably, the putative binding surface for oligosaccharides is adjacent to, but separate from the ace2 binding site, suggesting that a single rbd could simultaneously bind both cell surface hs and the ace2 protein receptor. the putative hs binding site is partially obstructed in the "closed" inactive rbd conformation, while fully exposed in the open state (suppl. fig. s1 ). the amino acid sequence of s protein rbd of sars-cov-2 s is 73% identical to the rbd of sars-cov-1 s (fig. 1f) , and these domains are highly similar in structure with an overall cα r.m.s.d. of 0.929 å (fig. 1g) . however, an electrostatic potential map of the sars-cov-1 s j o u r n a l p r e -p r o o f 7 rbd does not show an electropositive surface like that observed in sars-cov-2 (fig. 1h ). most of the positively charged residues comprising this surface are conserved between the two proteins, with the exception of sars-cov-2 k444 which is a threonine in sars-cov-1 (fig. 1f ). additionally, the other amino acid residues predicted to coordinate with the oligosaccharide are conserved with the exception of asn354 in sars-cov-2, which is a negatively charged glutamate residue in sars-cov-1. sars-cov-1 has been shown to interact with cellular hs in addition to its entry receptors ace2 and transmembrane protease, serine 2 (tmprss2) (lang et al., 2011) . our analysis suggests that the putative heparin-binding site in sars-cov-2 s may mediate an enhanced interaction with heparin or hs compared to sars-cov-1, and that this change evolved through as few as two amino acid substitutions, thr lys444 and glu asn354. to test experimentally if the sars-cov-2 s protein interacts with heparin/hs, recombinant ectodomain and rbd proteins were prepared and characterized. initial studies encountered difficulty in stabilizing the s ectodomain protein, a problem that was resolved by raising the concentration of nacl to 0.3 m in hepes buffer. under these conditions, the protein could be stored at room temperature, 4 o c or at -80 o c for at least two weeks. sds-page showed that each protein was ~98% pure ( j o u r n a l p r e -p r o o f 8 recombinant s ectodomain and rbd proteins were applied to a column of heparin-sepharose. elution with a gradient of sodium chloride showed that the rbd eluted at ~0.3 m nacl, with a shoulder that eluted with higher salt (fig. 2a) . recombinant s ectodomain also bound to heparin-sepharose, but it eluted across a broader concentration of nacl. the elution profiles suggest that the preparations contained a population of molecules that bind to heparin, but that some heterogeneity in affinity for heparin occurs, which may reflect differences in glycosylation, oligomerization or the number of binding sites in the open conformation. the rbd protein from sars-cov-2 also bound in a saturable manner to heparin-bsa immobilized on a plate (fig. 2b ). the rbd domain from sars-cov-1 showed significantly reduced binding to heparin-bsa and a higher k d value (640 nm [95% c.i.; 282 -1852 nm] for sars-cov-1 rbd vs. 150 nm [95% c.i. 123 -173 nm]) for sars-cov-2 rbd), in accordance with the difference in electropositive potential in the proposed hs binding regions (fig. 1h) . a monomeric form of sars-cov-2 s ectodomain protein also bound in a saturable manner to heparin immobilized on a plate (suppl. fig. s3a ). the trimeric protein bound to heparin-bsa with an apparent k d value of 3.8 nm [95% c.i. 3.1 -4.6 nm] (fig. 2c ). binding of recombinant s ectodomain, mutated to lock the rbds into a closed (mut2) or that favors an open (mut7) conformation, showed that the heparin binding site in the rbd domain is accessible in both conformations (fig. 2d ). however, the k d value for mut7 is lower (4.6 nm [95% c.i. 3.8 -5.5 nm] vs. 9.9 nm [95% c.i. 8.7 -11.3 nm] for mut2), which is in line with the partial obstruction of the site in the closed conformation (suppl. fig. s1 ). as expected, only trimer with an open rbd conformation bound to ace2 (fig. 2e ). in contrast to spike protein, ace2 did not bind to heparin-bsa (fig. 2c) . ace2 also had no effect on binding of s protein to heparin-bsa at all concentrations that were tested (fig. 2c , inset). biotinylated ace2 bound to immobilized s protein (suppl. fig. s3b ) and a ternary complex of heparin, ace2 and s protein could be demonstrated by titration of s protein bound to immobilized heparin-bsa with ace2 (fig. 2f ). binding of ace2 under these conditions j o u r n a l p r e -p r o o f 9 increased in proportion to the amount of s protein bound to the heparin-bsa. collectively, these findings show that (i) spike protein can engage both heparin and ace2 simultaneously and (ii) that the heparin binding site is somewhat occluded in the closed conformation, but it can still bind heparin albeit with reduced affinity. the simultaneous binding of ace2 to spike protein and heparin suggested the possibility that heparin binding might affect the conformation of the rbd, possibly increasing the open conformation that can bind ace2. to explore this possibility, spike protein was mixed with ace2 (6-fold molar ratio) with or without dp20 oligosaccharides derived from heparin (9-fold molar ratio). the samples were then stained and analyzed by transmission electron microscopy, and the images were deconvoluted and sorted into 3d reconstructions to determine the number of trimers with 0, 1, 2, or 3 bound ace2 (fig. 2g -h and suppl. fig. s3c-d) . the different populations were counted and the percentage of particles belonging to each 3d class was calculated. two time points were evaluated after mixing ace2 and trimeric s: at 15 min 29,600 and 31,300 particles were analyzed in the absence or presence of dp20 oligosaccharides, respectively; at 60 min, 17,000 and 21,000 particles were analyzed in absence or presence of dp20 oligosaccharides, respectively. at both time points, the presence of dp20 increased the total amount of ace2 protein bound to spike . after 15 minutes in the absence of dp20 very few of the trimers had conformations with 1 or 2 bound ace2 (5% each), whereas the inclusion of dp20 oligosaccharides greatly increased the proportion of trimers bearing one (37%) or two (21%) ace2, with a proportional drop in the unbound conformers from 90% in the absence of heparin to 42% in its presence (fig. 2g ). extending the incubation to 60 minutes resulted in a mixture of trimers containing 1 (45%), 2 (11%) and 3 ace2 (13%) in the absence of heparin. inclusion of dp20 further increased the proportion of bound spike trimers bearing 2 (19%), and 3 (27%) ace2 (fig. 2h) . the imaging studies suggest that, under these j o u r n a l p r e -p r o o f 10 experimental conditions, heparin may stabilize the ace2 interaction, increasing the proportion of spike bound to ace2 as well as the occupancy of individual spikes. the sars-cov-2 spike protein depends on cellular heparan sulfate for cell binding. to extend these studies to hs on the surface of cells, s ectodomain protein was added to human h1299 cells, an adenocarcinoma cell line derived from type 2 alveolar cells (fig. 3a ). spike ectodomains bound to h1299 cells, with half-maximal binding achieved at ~75 nm. treatment of the cells with a mixture of heparin lyases (hsase), which degrades cell surface hs, dramatically reduced binding ( fig 3a) . the s ectodomain also bound to human a549 cells, another type 2 alveolar adenocarcinoma line, as well as human hepatoma hep3b cells (fig. 3b ). removal of hs by enzymatic treatment dramatically reduced binding in both of these cell lines as well (fig. 3b ). recombinant rbd protein also bound to all three cell lines dependent on hs (fig. 3c) . a melanoma cell line, a375, was tested independently and also showed hs dependent binding ( fig 3d) . the extent of binding across the four cell lines varied ~4-fold. this variation was not due to differences in hs expression as illustrated by staining of cell surface hs with mab 10e4, which recognizes a common epitope in hs ( we also measured binding of the s ectodomain and rbd proteins to a library of mutant hep3b cells, carrying crispr/cas9 induced mutations in biosynthetic enzymes essential for synthesizing hs (anower et al., 2019) . inactivation of ext1, a subunit of the copolymerase required for synthesis of the backbone of hs, abolished binding to a greater extent than enzymatic removal of the chains with hsases ( fig. 3f and suppl. fig. s4 ), suggesting that the hsase treatment may underestimate the dependence on hs. targeting ndst1, a glcnac n-j o u r n a l p r e -p r o o f 11 deacetylase-n-sulfotransferase that n-deacetylates and n-sulfates n-acetylglucosamine residues, and hs6st1 and hs6st2, which introduces sulfate groups in the c6 position of glucosamine residues, significantly reduced binding (figs. 3f and suppl. fig. s4 ). although experiments with other sulfotransferases have not yet been done, the data suggests that the pattern of sulfation of hs affects binding to s and rbd. to further examine how variation in hs structure affects binding, we isolated hs from human kidney, liver, lung and tonsil. the samples were depolymerized into disaccharides by treatment with hsases, and the disaccharides were then analyzed by lc-ms (experimental methods). the disaccharide analysis showed that lung hs has a larger proportion of ndeacetylated and n-sulfated glucosamine residues (grey bars) and more 2-o-sulfated uronic acids (green bars) than hs preparations from the other tissues (fig. 4a ). the different hs preparations also varied in their ability to block binding of rbd to h1299 cells (fig. 4b ). interestingly, hs isolated from lung was more potent compared to kidney and liver hs, consistent with the greater degree of sulfation of hs from this organ (suppl. table 1 ). hs from tonsil was as potent as hs from lung, but the overall extent of sulfation was not as great, supporting the notion that the patterning of the sulfated domains in the chains may affect binding. unfractionated heparin is derived from porcine mucosa and possesses potent anticoagulant activity due to the presence of a pentasaccharide sequence containing a crucial 3-o-sulfated nsulfoglucosamine unit, which confers high affinity binding to antithrombin. heparin is also very highly sulfated compared to hs with an average negative charge of -3.4 per disaccharide (the overall negative charge density of typical hs is -1.8 to -2.2 per disaccharide). mst cells, which were derived from a murine mastocytoma, make heparin-like hs that lacks the key 3-o-sulfate group and anticoagulant activity (gasimli et al., 2014; montgomery et al., 1992) . the 12 anticoagulant properties of heparin can also be removed by periodate oxidation, which oxidizes the vicinal hydroxyl groups in the uronic acids, resulting in what is called "split-glycol" heparin (casu et al., 2004) . all of these agents significantly inhibited binding of the s protein to h1299 and a549 cells ( fig. 4c and 4d ) yielding ic 50 values in the range of 0.01-0.12 µg/ml (suppl . table 1 ). interestingly, the lack of 3-o-sulfation, crucial for the anticoagulant activity of heparin, had little effect on its inhibition of s binding. in contrast, cho cell hs (containing 0.8 sulfates per disaccharide) only weakly inhibited binding (ic 50 values of 18 and 139 µg/ml for a549 and h1299, respectively) (suppl. table 1 ). these data suggest that inhibition by heparinoids is most likely charge dependent and independent of anticoagulant activity per se. the experiments shown in fig. 2g -h indicate that binding of heparin to spike protein can increase binding to ace2. to explore if hs, ace2 and spike interact at the cell surface, we investigated the impact of ace2 expression on s protein cell binding. initial attempts were made to measure ace2 levels by western blotting or flow cytometry with different mabs and polyclonal antibodies, but a reliable signal was not obtained in any of the cell lines tested (a375, a549, h1299, and hep3b). nevertheless, expression of ace2 mrna was observed by rt-qpcr (suppl. fig. 5a ). transfection of a375 cells with ace2 cdna resulted in robust expression of ace2 (fig. 5a) , resulting in an increase in s ectodomain protein binding by ~4fold (fig. 5b) . interestingly, the enhanced binding was hs-dependent, as illustrated by the loss of binding of s protein after hsase-treatment (fig. 5b ). crispr/cas9 mediated deletion of the b4galt7 gene, which is required for glycosaminoglycan assembly (suppl. fig. s5b ), also reduced binding of spike protein (fig. 5b ) despite the overexpression of ace2 (fig. 5a ). to explore the impact of diminished ace2 expression, we examined spike protein binding to a549 cells and in two crispr/cas9 gene targeted clones c3 and c6 bearing biallelic mutations in ace2 (suppl. fig. s5c ). binding of s ectodomain protein was greatly reduced in the ace2 -/-j o u r n a l p r e -p r o o f 13 clones and the residual binding was sensitive to hsases (fig. 5c ). these findings show that binding of spike protein on cells requires both hs and ace2, consistent with the formation of a ternary complex (figs. 2f-h). assays using purified components provide biochemical insights into binding, but they do not recapitulate the multivalent presentation of the s protein as it occurs on the virion membrane. thus, to extend these studies, pseudotyped vesicular stomatitis virus (vsv) was engineered to express the full-length sars-cov-2 s protein and gfp or luciferase to monitor infection. vero e6 cells are commonly used in the study of sars-cov-2 infection, due to their high susceptibility to infection. spike protein binding to vero cells also depends on cellular hs as binding was sensitive to hsases, heparin and split-glycol heparin (fig. 6a ). interestingly, hsase treatment reduced binding to a lesser extent than the level of reduction observed in a549, heparin very potently reduced infection more than ~4-fold at 0.5 µg/ml and higher concentrations (fig. 6g) . in contrast, studies of sars-cov-1 s protein pseudotype virus showed that hsase-treatment actually increased sars-cov-1 infection by more than 2-fold, suggesting that hs might interfere with binding of sars-cov-1 in this cell line (fig. 6h ). infection of h1299 and a549 cells by sars-cov-2 s pseudotype virus was too low to obtain j o u r n a l p r e -p r o o f 14 accurate measurements, but infection of hep3b cells could be readily measured (fig. 6i ). hsase and mutations in ext1 and ndst1 dramatically reduced infection 6-to 7-fold. inactivation of the 6-o-sulfotransferases had only a mild effect unlike its strong effect on s protein binding (fig. 3f) , possibly due to the high valency conferred by multiple copies of s protein on the pseudovirus envelope. hep3b cells were not susceptible to infection by sars-cov-1 s protein pseudotyped virus, but was infected by mers-cov s protein pseudotyped virus and infection was independent of hs (suppl. fig. s6 ). studies of pseudovirus were then extended to authentic sars-cov-2 virus infection using strain usa-wa1/2020. infection of vero e6 cells was monitored by double staining of the cells with antibodies against the sars-cov-2 nucleocapsid (n) and s proteins ( heparin inhibition (maroon and blue symbols). to rule out that the treatments caused a decrease in ace2 expression or a reduction in cell viability, vero cells were treated with heparin lyases and 100 µg/ml ufh, and ace2 expression was measured by western blotting and cell viability by celltiter-blue® (suppl. fig. s7a -b) . no effect on ace2 expression or cell viability was observed. these findings further emphasize the potential for using unfractionated heparin or other non-anticoagulant heparinoids to prevent viral attachment. j o u r n a l p r e -p r o o f 15 these findings were then extended to hep3b cells and mutants altered in hs biosynthesis using a viral plaque assay. virus was added to wildtype, ndst1 -/and hs6st1/2 -/cells for 2 hr, the virus was removed, and after 2 days incubation a serial dilution of the conditioned culture medium was added to monolayers of vero e6 cells. the number of plaques were then quantitated by staining and visualization. as a control, culture medium from infected vero e6 cells was tested, which showed robust viral titers. hep3b cells also supported viral replication, but to a lesser extent than vero cells. inactivation of ndst1 in hep3b cells abolished virus production, whereas inactivation of hs6st1/2 -/reduced infection more mildly, ~3-fold (fig. 7d) . hsase and ufh reduced infection more than 5-fold, but it had no effect on cell viability (suppl. in this report, we provide compelling evidence that hs is a necessary host attachment factor that promotes sars-cov-2 infection of various target cells. the receptor binding domain of the sars-cov-2 s protein binds to heparin/hs, most likely through a docking site composed of positively charged amino acid residues aligned in a subdomain of the rbd that is separate from the site involved in ace2 binding (fig. 1) . competition studies, enzymatic removal of hs, and genetic studies confirm that the s protein, whether presented as a recombinant protein (figs. 2-j o u r n a l p r e -p r o o f 16 5), in a pseudovirus (fig. 6) , or in authentic sars-cov-2 virions (fig. 7) , binds to cell surface hs in a cooperative manner with ace2 receptors. mechanistically, binding of heparin/hs to spike trimers enhances binding to ace2, likely increasing multivalent interactions with the target cell. this data provides crucial insights into the pathogenic mechanism of sars-cov-2 infection and suggests hs-spike protein complexes as a novel therapeutic target to prevent infection. the glycocalyx is the first point of contact for all pathogens that infect animal cells, and thus it is not surprising that many viruses exploit glycans, such as hs, as attachment factors. for example, the initial interaction of herpes simplex virus with cells involves binding to hs chains on one or more hs proteoglycans (shieh et al., 1992; wudunn and spear, 1989 ) through the interactions with the viral glycoproteins gb and gc. viral entry requires the interaction of a specific structure in hs with a third viral glycoprotein, gd (shukla et al., 1999) , working in concert with membrane proteins related to tnf/ngf receptors (montgomery et al., 1996) . similarly, the human immunodeficiency virus binds to hs by way of the v3 loop of the viral glycoprotein gp120 (roderiquez et al., 1995) , but infection requires the chemokine receptor ccr5 (deng et al., 1996; dragic et al., 1996) . other coronaviruses also utilize hs, for example nl63 (hcov-nl63) binds hs via the viral s protein in addition to ace2 (lang et al., 2011; milewska et al., 2018; milewska et al., 2014; naskalska et al., 2019) . in these examples, initial tethering of virions to the host cell plasma membrane appears to be mediated by hs, but infection requires transfer to a proteinaceous receptor. the data presented here shows that sars-cov-2 requires hs in addition to ace2. we imagine a model in which cell surface hs acts as a "collector" of the virus and a mediator of the rbd-ace2 interaction, making viral infection more efficient. hs varies in structure across cell types and tissues, as well as with gender and age (de agostini et al., 2008; feyzi et al., 1998; ledin et al., 2004; vongchan et al., 2005; warda et al., 2006; wei et al., 2011) . variation in competition by hs from different tissues supports this conclusion and raises the possibility that hs contributes to the tissue tropism and j o u r n a l p r e -p r o o f 17 the susceptibility of different patient populations, in addition to levels of expression of ace2 . coronaviruses can utilize a diverse set of glycoconjugates as attachment factors. human coronavirus oc43 (hcov-oc43) and bovine coronavirus (bcov) bind to 5-n-acetyl-9-oacetylneuraminic acid (hulswit et al., 2019; tortorici et al., 2019) , middle east respiratory syndrome virus (mers-cov) binds 5-n-acetyl-neuraminic acid (park et al., 2019) , and guinea fowl coronavirus binds biantennary di-n-acetyllactosamine or sialic acid capped glycans (bouwman et al., 2019) . whether sars-cov-2 s protein binds to sialic acid remains unclear. mapping the binding site for sialic acids in other coronavirus s proteins has proved elusive, but modeling studies suggest a location distinct from the hs binding site shown in fig. 1 (park et al., 2019; tortorici et al., 2019) . the s protein in murine coronavirus contains both a hemagglutinin domain for binding and an esterase domain that cleaves sialic acids that aids in the liberation of bound virions (rinninger et al., 2006; smits et al., 2005) . whether sars-cov-2 s protein, another viral envelope protein, or a host protein contributes to hs-degrading activity to aid in the release of newly made virions is unknown. the repertoire of proteins in organisms that bind to hs make up the so called "hs interactome" and consists of a variety of different hs-binding proteins (hsbps) (xu and esko, 2014) . unlike lectins that have a common fold that helps define the glycan binding site, hsbps do not exhibit a conserved motif that allows accurate predictions of binding sites based on primary sequence. instead, the capacity to bind heparin appears to have emerged through convergent evolution by juxtaposition of several positively charged amino acid residues arranged to accommodate the negatively charged sulfate and carboxyl groups present in the polysaccharide, and hydrophobic and h-bonding interactions stabilize the association. the rbd domains from the sars-cov-1 and sars-cov-2 s proteins are highly similar in structure (fig. 1g ), but the electropositive surface in sars-cov-1 s rbd is not as pronounced in sars-cov-2 s rbd (fig. 1h ). in accordance with this observation, recombinant rbd protein from sars-j o u r n a l p r e -p r o o f 18 cov-2 showed significantly higher binding to heparin-bsa, compared to rbd from sars-cov-1 (fig. 2b) . a priori we predicted that the evolution of the hs binding site in the sars-cov-2 s protein might have occurred by the addition of arginine and lysine residues to its ancestor, sars-cov-1. instead, we observed that four of the six predicted positively charged residues that make up the heparin-binding site are present in sars-cov-1 as well as most of the other amino acid residues predicted to interact with heparin ( fig. 1) . sars-cov-1 has been shown to interact with cellular hs in addition to its entry receptors ace2 and transmembrane protease, serine 2 (tmprss2) (lang et al., 2011) . our analysis suggests that the putative heparinbinding site in sars-cov-2 s may mediate an enhanced interaction with heparin compared to sars-cov-1, and that this change evolved through as few as two amino acid substitutions, thr444lys and glu354asn. further studies are underway to define the amino acid residues in the combining site for heparin/hs to test this hypothesis. the ability of heparin and hs to compete for binding of the sars-cov-2 s protein to cell surface hs and the inhibitory activity of heparin towards infection of pseudovirus and authentic sars-cov-2 illustrates the therapeutic potential of agents that target the virus-hs interaction to control infection and transmission of sars-cov-2. there is precedent for targeting proteinglycan interactions as therapeutic agents. for example, tamiflu targets influenza neuraminidase, thus reducing viral transmission, and sialylated human milk oligosaccharides can block sialic acid-dependent rotavirus attachment and subsequent infection in infants (hester et al., 2013; von itzstein, 2007) . covid-19 patients typically suffer from thrombotic complications ranging from vascular micro-thromboses, venous thromboembolic disease and stroke and often receive unfractionated heparin or low molecular weight heparin (thachil, 2020) . the findings presented here and elsewhere suggest that both of these agents can block viral infection (courtney mycroft-west, 2020; kim et al., 2020; liu et al., 2020; mycroft-west et al., 2020; tandon et al., 2020; wu et al., 2020) . effective anticoagulation is achieved with plasma levels of heparin of 0.3-0.7 units/ml. this concentration is equivalent to 1.6-4 µg/ml heparin (assuming that the activity of ufh is 180 units/mg). although this is sufficient to block spike protein binding to cells (fig. 4) , it would not be expected to prevent viral infection, but it should attenuate infection depending on the viral load (fig. 7) . the anticoagulant activity of heparin, which is typically absent in hs, is not critical for its antiviral activity based on the observation that mst derived heparin and split-glycol heparin is nearly as potent as therapeutic heparin ( figs. 4 and 6) . additional studies are needed to address the potential overlap in the dose response profiles for heparin as an anticoagulant and antiviral agent and the utility of nonanticoagulant heparins. antibodies directed to heparan sulfate or the binding site in the rbd might also prove useful for attenuating infection. in conclusion, this work revealed hs as a novel attachment factor for sars-cov-2 and suggests the possibility of using hs mimetics, hs degrading lyases, and metabolic inhibitors of hs biosynthesis for the development of therapy to combat covid-19. further information and request for resources should be directed to the lead contact, thomas mandel clausen (tmandelclausen@health.ucsd.edu) all developed sars-cov-2 expression plasmids produced in this study can be made available upon request to the lead contact. j o u r n a l p r e -p r o o f 28 this study did not generate any unique datasets or code. cell lines nci-h1299, a549, hep3b, a375 and vero e6 cells were from the american type culture collection (atcc). nci-h1299 and a549 cells were grown in rpmi medium, whereas the other lines were grown in dmem. hep3b cells carrying mutations in hs biosynthetic enzymes were previously derived from the parent hep3b line as described (anower et al., 2019) . all cell media were supplemented with 10% (v/v) fbs, 100 iu/ml of penicillin and 100 µg/ml of streptomycin sulfate, and the cells were grown under an atmosphere of 5% co 2 and 95% air. cells were passaged at ~80% confluence and seeded as explained for the individual assays. protein was produced in expicho or hek293-6e cells that were acquired from thermo fisher and grown according to the manufacturer's specifications. human bronchial epithelial cells were acquired from lonza. they were cultured in pneumacult-ex plus medium or to pneumacult-ali medium according to the manufacturer's instructions (stemcell technologies). specific details on the culture methods are described in the methods section. the collection of human tissue in this study abided by the helsinki principles and the an electrostatic potential map of the sars-cov-2 spike protein rbd domain was generated from a crystal structure (pdb:6m17) and visualized using pymol (version 2.0.6 by schrödinger). a dp4 fully sulfated heparin fragment was docked to the sars-cov-2 spike protein rbd using the cluspro protein docking server (https://cluspro.org/login.php) (kozakov et al., 2013; kozakov et al., 2017; vajda et al., 2017) . heparin-protein contacts and energy contributions were evaluated using the molecular operating environment (moe) software (chemical computing group). recombinant sars-cov-2 spike protein, encoding residues 1-1138 (wuhan-hu-1; genbank: mn908947.3) with proline substitutions at amino acids positions 986 and 987, a "gsas" substitution at the furin cleavage site (amino acids 682-682), twinstreptag and his 8x , was produced in expicho cells by transfection of 6 x10 6 cells/ml at 37 ºc with 0.8 µg/ml of plasmid dna using the expicho expression system transfection kit in expicho expression medium (thermofisher). one day later the cells were refed, then incubated at 32 ºc for 11 days. the conditioned medium was mixed with complete edta-free protease inhibitor (roche). samples of the recombinant trimeric spike protein ectodomain were diluted to 0.03 mg/ml in 1x tbs ph 7.4. carbon coated copper mesh grids were glow discharged and 3 µl of the diluted sample was placed on a grid for 30 sec then blotted off. uniform stain was achieved by depositing 3 µl of uranyl formate (2%) on the grid for 55 sec and then blotted off. grids were transferred to a thermo fisher morgagni operating at 80 kv. images at 56,000 magnification j o u r n a l p r e -p r o o f 31 were acquired using a megaview 2k camera via the radius software. a dataset of 138 micrographs at 52,000x magnification and -1.5 µm defocus was collected on a fei tecnai spirit (120kev) with a fei eagle 4k by 4k ccd camera. the pixel size was 2.06 å per pixel and the dose was 25 e − /å 2 . the leginon (suloway et al., 2005) software was used to automate the data collection and the raw micrographs were stored in the appion (lander et al., 2009) database. particles on the micrographs were picked using dogpicker , stack with a box size of 200 pixels, and 2d classified with relion 3.0 (scheres, 2012) . secreted human ace2 was transiently produced in suspension hek293-6e cells. a plasmid encoding residues 1−615 of ace2 with a c-terminal hrv-3c protease cleavage site, a twinstreptag and an his 8x tag was a gift from jason s. mclellan, university of texas at austin. briefly, 100 ml of hek293-6e cells were seeded at a cell density of 0.5 × 10 6 cells/ml 24 hr before transfection with polyethyleneimine (pei). for transfection, 100 µg of the ace2 plasmid and 300 µg of pei (1:3 ratio) were incubated for 15 min at room temperature. transfected cells were cultured for 48 hr and fed with 100 ml fresh media for additional 48 hr before harvest. secreted ace2 were purified from culture medium by ni-nta affinity chromatography (qiagen). filtered media was mixed 3:1 (v/v) in 4x binding buffer (100 mm tris-hcl, ph 8,0, 1,2 m nacl) and loaded on to a self-packed column, pre-equilibrated with washing buffer (25 mm tris-hcl, ph 8, 0.3 m nacl, 20 mm imidazole). bound protein was washed with buffer and eluted with 0.2 m imidazole in washing buffer. the protein containing fractions were identified by sds-page. j o u r n a l p r e -p r o o f sars-cov-2 spike protein in dpbs was applied to a 1-ml hitrap heparin-sepharose column (ge healthcare). the column was washed with 5 ml of dpbs and bound protein was eluted with a gradient of nacl from 150 mm to 1 m in dpbs. for binding studies, recombinant spike protein and ace2 was conjugated with ez-link tm sulfo-nhs-biotin (1:3 molar ratio; thermo fisher) in dulbecco's pbs at room temperature for 30 min. glycine (0.1 m) was added to quench the reaction and the buffer was exchanged for pbs using a zeba spin column (thermo fisher). heparin ( and incubated with s protein (100 nm). ace2 binding was measured to bound spike protein as described above. mixtures of stabilized (mut7) spike protein, 6x molar excess soluble ace2 ectodomain, with or without 9x molar excess an icosasaccharide (dp20) fragment derived from heparin were incubated at 4°c for 15 min or 1 hr. samples were diluted to 0.02 mg/ml with respect to spike protein in 1x pbs ph 7.4. carbon coated copper mesh grids were glow discharged at 20 ma for 30 s and 3 µl sample was applied for 20 s and blotted off. grids were washed five times in 10 µl 1x tbs ph 7.4 for 15 sec then stained and blotted twice with 3 µl 2% uranyl formate for 15 sec. grids were imaged with an fei tecnai spirit (120 kev) or fei tecnai f20 (200 kev) with an fei eagle ccd (4k) camera. data were collected on the fei tecnai f20 at 62,000x magnification, -1.5 µm defocus with a pixel size of 1.77 å per pixel. these datasets employed a box size of 256 and comprised 167 to 331 micrographs. data were collected on the fei tecnai spirit as described above. data collection on both microscopes was automated through leginon (suloway et al., 2005) . stored in the appion (lander et al., 2009 ) database, and particles were picked with dog picker . particles were 2d classified with relion 3.0 j o u r n a l p r e -p r o o f 34 (scheres, 2012) . trimeric 2d classes were selected for iterative 3d classification with relion 3.0. classifications were performed until 3d classes demonstrated ace2 occupancy throughout the relevant threshold-level of the spike protein as visualized using chimerax (goddard et al., 2018) . particle counts of final 3d classes were obtained with relion 3.0 (scheres, 2012) and the percentages of particles bound to 0, 1, 2, or 3 ace2 were calculated and visualized in graphpad prism 8. cells at 50-80% confluence were lifted with pbs containing 10 mm edta (gibco) and fresh human tissue was washed in pbs, frozen, and lyophilized. the dried tissue was crushed into a fine powder, weighed, resuspended in pbs containing 1 mg/ml pronase with 90% ethanol (esko, 1993) . for hs quantification and disaccharide analysis, purified hs was digested with a mixture of heparin lyases i-iii (2 mu each) for 2 hr at 37 °c in 40 mm ammonium acetate buffer containing the ace2 expression plasmid (addgene, plasmid #1786) (li et al., 2003) qpcr mrna was extracted from the cells using trizol (invitrogen) and chloroform and purified using the rneasy kit (qiagen). cdna was synthesized from the mrna using random primers and the superscript iii first-strand synthesis system (invitrogen). sybr green master mix (applied biosystems) was used for qpcr following the manufacturer's instructions, and the expression of tbp was used to normalize the expression of ace2 between the samples. the qpcr primers used were as follows: ace2 (human) forward: 5' -cgaagccgaagacctgttcta -3' and reverse: 5' -gggcaagtgtggactgttcc -3'; and tbp (human) forward: 5' -aacttcgcttccgctggccc -3' and reverse: 5' -gaggggaggccaagccctga -3'. to generate the cas9 lentiviral expression plasmid, 2.5 x 10 6 hek293t cells were seeded to a 10-cm diameter plate in dmem supplemented with 10% fbs. the following day, the cells j o u r n a l p r e -p r o o f 38 were co-transfected with the pspax2 packaging plasmid (addgene, plasmid #12260), pmd2.g envelope plasmid (addgene, plasmid #12259), and lenti-cas9 plasmid (addgene, plasmid #52962) (sanjana et al., 2014) in dmem supplemented with fugene6 (30µl in 600µl dmem). media containing the lentivirus was collected and used to infect a549 wt and a375 wt cells, which were subsequently cultured with 5 µg/ml and 2 µg/ml blasticidin, respectively, to select for stably transduced cells. a single guide rna (sgrna) targeting ace2 (5'-tggatacatttgggcaagtg -3') and one targeting b4galt7 (5'-tgacctgctccctctcaacg-3') was cloned into the lentiguide-puro plasmid (addgene plasmid #52963) following published procedure (sanjana et al., 2014) . the lentiviral sgrna construct was generated in hek293t cells, using the same protocol as for the cas9 expression plasmid, and used to infect a549-cas9 and a375-cas9 cells to generate crispr knockout mutant cell lines. after infection, the cells were cultured with 2 µg/ml puromycin to select for cells with stably integrated lentivirus. after 7 d, the cells were serially diluted into 96-well plates. single colonies where expanded and dna was extracted using the dneasy blood and tissue dna isolation kit (qiagen). proper editing was verified by sequencing (genewiz inc.) and gene analysis using the online ice tool from synthego (suppl. fig. 5 ). vesicular stomatitis virus (vsv) pseudotyped with spike proteins of sars-cov-2 were generated according to a published protocol (whitt, 2010) . briefly, hek293t, transfected to express full length sars-cov-2 spike proteins, were inoculated with vsv-g pseudotyped ∆gluciferase or gfp vsv (kerafast, ma). after 2 hr at 37°c, the inoculum was removed and cells were refed with dmem supplemented with 10% fbs, 50 u/ml penicillin, 50 µg/ml streptomycin, and vsv-g antibody (i1, mouse hybridoma supernatant from crl-2700; atcc). pseudotyped particles were collected 20 hr post-inoculation, centrifuged at 1,320 × g to remove cell debris and stored at −80°c until use. briefly, 100 µl of luciferin lysis solution was added to the cells and incubated for 5 min at room temperature. the solution was transferred to a black 96-well plate and luminescence was detected using an enspire multimodal plate reader (perkin elmer). data analysis and statistical analysis was performed in prism 8. fluor 594 labeling kits (invitrogen), respectively. zombie uv™ was used to gate for live cells in the analysis. cells were then analyzed using an ma900 cell sorter (sony). for 4 days. fresh medium, 100 µl in the apical chamber and 500 µl in the basal chamber, was added daily. at day 7, the medium in the apical chambers was removed, and the basal chambers were changed every 2-3 days with apical washes with pbs every week for 28 days. the apical side of the hbec ali culture was gently washed three times with 200 µl of phosphate buffered saline without divalent cations (pbs-/-). heparinase was added to the apical side for half an hour prior to infection. an moi of 0.5 of authentic sars-cov-2 live virus (usa-wa1/2020 (bei resources, #nr-52281)) in 100 µl total volume of pbs was added to the apical chamber with either dmso, heparinase (2.5mu/ml heparin lyase ii, and 5mu/ml heparin lyase iii (ibex)) or 100ug/ml of unfractionated heparin. cells were incubated at 37c and 5% co2 for 4 hours. unbound virus was removed, the apical surface was washed and the compounds were re-added to the apical chamber. cells were incubated for another 20 hours at 37c and 5% co2. after inoculation, cells were washed once with pbs-/-and 100 µl tryple (thermofisher) was added to the apical chamber then incubated for 10 min in the incubator. cells were gently pipetted up and down and transferred into a sterile 15 ml conical tube containing neutralizing medium of dmem + 3% fbs. tryple was added again for 3 rounds of 10 minutes for a total of 30 min to clear transwell membrane. cells were spun down and resuspended in pbs with zombie uv viability dye for 15 min in room temp. cells were washed once with facs buffer then fixed in 4% pfa for 30 min at room temp. pfa was washed off and cells were resuspended in pbs. zombie uv™ was used to gate for live cells in the analysis. infection was analyzed by flow cytometry as explained above. cell viability was assessed using the celltiter-blue® assay (promega). briefly, vero cells were seeded into a 96 well plate. the cells were treated with hsase mix (2.5 mu/ml hsase ii, and 5 mu/ml hsase iii; ibex) or 100 µg/ml ufh for 16 hrs. the viability of the cells using celltiter-blue® was measured according to the manufacturers protocol. briefly, the j o u r n a l p r e -p r o o f 42 celltiter-blue® reagent was added directly to the cell culture and the cells were incubated overnight. fluorescence was read at excitation 560nm and emission 590nm, using an enspire multimodal plate reader (perkin elmer). data analysis was performed in prism. the human bronchial epithelial cells were grown at an air-liquid interface as explained above. cell viability after treatment with hsase mix (2.5 mu/ml hsase ii, and 5 mu/ml hsase iii; ibex) or 100 µg/ml ufh for 16 hrs was measured by adding celltiter-blue® reagent directly to the transwell inserts and developed as explained above. all statistical analyses were performed in prism 8 (graphpad). all experiments were performed in triplicate and repeated as indicated in the figure legends. data was analyzed statistically using unpaired t-tests when two groups were being compared or by one-way anova without post-hoc correction for multiple comparisons. ic 50 values and confidence intervals were determined using non-linear regression using the inhibitor vs. response least squares fit algorithm. the error bars in the figures refer to mean plus standard deviation (sd) values. the specific statistical tests used are listed in the figure legends and in the methods section. experiments were evaluated by statistical significance according to the following scheme; ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001. after 48 hr, cell culture supernatants were collected and stored at -80°c. virus titers were determined by plaque assays on vero e6 monolayers greiner bio-one, #662160) and rocked for 1 hr at room temperature. the cells were subsequently overlaid with mem containing 1% cellulose the plaques were visualized by fixation of the cells with a mixture of 10% formaldehyde and 2% methanol (v/v in water) for 2 hr. the monolayer was washed once with pbs and stained with 0.1% crystal violet (millipore sigma # v5265) prepared in 20% ethanol the pennsylvania state university, following the guidelines approved by the institutional biosafety committees. human bronchial epithelial cell air-liquid interface generation and infection human bronchial epithelial cells (hbecs, lonza) were cultured in t75 flasks in plus medium according to manufacturer instructions (stemcell technologies) to generate air-liquid interface (ali) cultures, hbecs were plated on collagen i-coated 24 well transwell inserts with a 0.4-micron pore size (costar, corning) at 5x10 4 cells/ml. cells were maintained for 3-4 days in pneumacult-ex plus medium until confluence, then changed to pneumacult-ali medium triglyceride-rich lipoprotein binding and uptake by heparan sulfate proteoglycan receptors in a crispr/cas9 library of hep3b mutants remdesivir for the treatment of covid-19 -preliminary report guinea fowl coronavirus diversity has phenotypic consequences for glycan and tissue binding heparan sulfate proteoglycans and viral attachment: true receptors or adaptation bias? viruses 11 undersulfated and glycol-split heparins endowed with antiangiogenic activity the 2019 coronavirus (sars-cov-2) surface protein (spike) s1 receptor binding domain undergoes conformational change upon heparin binding identification of a major co-receptor for primary isolates of hiv-1 hiv-1 entry into cd4+ cells is mediated by the chemokine receptor cc-ckr-5 special considerations for proteoglycans and glycosaminoglycans and their purification order out of chaos: assembly of ligand binding sites in heparan sulfate age-dependent modulation of heparan sulfate structure and function bioengineering murine mastocytoma cells to produce anticoagulant heparin ucsf chimerax: meeting modern challenges in visualization and analysis structural analysis of urinary glycosaminoglycans from healthy human subjects human milk oligosaccharides inhibit rotavirus infectivity in vitro and in acutely infected piglets human coronaviruses oc43 and hku1 bind to 9-o-acetylated sialic acids via a conserved receptor-binding site in spike protein domain a loss of bcl-6-expressing t follicular helper cells and germinal centers in covid-19 stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis initial step of virus entry: virion binding to cell-surface glycans how good is automated protein docking? the cluspro web server for protein-protein docking appion: an integrated, database-driven pipeline to facilitate em image processing inhibition of sars pseudovirus cell entry by lactoferrin binding to heparan sulfate proteoglycans evolutionary differences in glycosaminoglycan fine structure detected by quantitative glycan reductive isotope labeling heparan sulfate structure in mice with genetically modified heparan sulfate production assessing ace2 expression patterns in lung tissues in the pathogenesis of covid-19 angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus proteoglycans and sulfated glycosaminoglycans sars-cov-2 spike protein binds heparan sulfate in a length-and sequence-dependent manner entry of human coronavirus nl63 into the cell human coronavirus nl63 utilizes heparan sulfate proteoglycans for attachment to target cells stable heparin-producing cell lines derived from the furth murine mastocytoma herpes simplex virus-1 entry into cells mediated by a novel member of the tnf/ngf receptor family heparin inhibits cellular invasion by sars-cov-2: structural dependence of the interaction of the surface protein (spike) s1 receptor binding domain with heparin membrane protein of human coronavirus nl63 is responsible for interaction with the adhesion receptor structures of mers-cov spike glycoprotein in complex with sialoside attachment receptors localisation and distribution of o-acetylated n-acetylneuraminic acids, the endogenous substrates of the hemagglutinin-esterases of murine coronaviruses, in mouse tissue mediation of human immunodeficiency virus type 1 binding by interaction of cell surface heparan sulfate proteoglycans with the v3 region of envelope gp120-gp41 improved vectors and genome-wide libraries for crispr screening relion: implementation of a bayesian approach to cryo-em structure determination cell surface receptors for herpes simplex virus are heparan sulfate proteoglycans a novel role for 3-o-sulfated heparan sulfate in herpes simplex virus 1 entry nidovirus sialate-o-acetylesterases: evolution and substrate specificity of coronaviral and toroviral receptor-destroying enzymes the sweet spot: defining virus-sialic acid interactions automated molecular microscopy: the new leginon system effective inhibition of sars-cov-2 entry by heparin and enoxaparin derivatives. biorxiv the versatile heparin in covid-19 structural basis for human coronavirus attachment to sialic acid receptors the war against influenza: discovery and development of sialidase inhibitors structural characterization of human liver heparan sulfate dog picker and tiltpicker: software tools to facilitate particle selection in single particle electron microscopy function, and antigenicity of the sars-cov-2 spike glycoprotein isolation and characterization of heparan sulfate from various murine tissues site-specific glycan analysis of the sars-cov-2 spike a comprehensive compositional analysis of heparin/heparan sulfate-derived disaccharides from human serum generation of vsv pseudotypes using recombinant deltag-vsv for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines cryo-em structure of the 2019-ncov spike in the prefusion conformation vaccines and therapies in development for sars-cov-2 infections initial interaction of herpes simplex virus with cells is binding to heparan sulfate demystifying heparan sulfate-protein interactions structural basis for the recognition of sars-cov-2 by full-length human ace2 cov-2 spike protein interacts with heparan sulfate and ace2 through the rbd • heparan sulfate promotes spike-ace2 interaction • sars-cov-2 infection is co-dependent on heparan sulfate and ace2 • heparin and non-anticoagulant derivatives block sars-cov-2 binding and infection in brief provide evidence that heparin sulfate is a necessary co-factor for sars-cov-2 infection. they show that heparin sulfate interacts with the receptor binding domain of the sars-cov-2 spike glycoprotein we thank scott selleck (the pennsylvania state university), eugene yeo (uc san diego), john guatelli (uc san diego), mark fuster (uc san diego) and stephen schoenberger (la jolla institute for immunology) for many helpful discussions, and annamaria naggi and giangiacomo torri from the ronzoni institute for generously providing split-glycol heparin. this key: cord-103940-a2cqw8kg authors: shi, yuejun; shi, jiale; sun, limeng; tan, yubei; wang, gang; guo, fenglin; hu, guangli; fu, yanan; fu, zhen f.; xiao, shaobo; peng, guiqing title: insight into vaccine development for alpha-coronaviruses based on structural and immunological analyses of spike proteins date: 2020-06-09 journal: biorxiv doi: 10.1101/2020.06.09.141580 sha: doc_id: 103940 cord_uid: a2cqw8kg coronaviruses that infect humans belong to the alpha-coronavirus (including hcov-229e) and beta-coronavirus (including sars-cov and sars-cov-2) genera. in particular, sars-cov-2 is currently a major threat to public health worldwide. however, no commercial vaccines against the coronaviruses that can infect humans are available. the spike (s) homotrimers bind to their receptors through the receptor-binding domain (rbd), which is believed to be a major target to block viral entry. in this study, we selected alpha-coronavirus (hcov-229e) and beta-coronavirus (sars-cov and sars-cov-2) as models. their rbds were observed to adopt two different conformational states (lying or standing). then, structural and immunological analyses were used to explore differences in the immune response with rbds among these coronaviruses. our results showed that more rbd-specific antibodies were induced by the s trimer with the rbd in the “standing” state (sars-cov and sars-cov-2) than the s trimer with the rbd in the “lying” state (hcov-229e), and the affinity between the rbd-specific antibodies and s trimer was also higher in the sars-cov and sars-cov-2. in addition, we found that the ability of the hcov-229e rbd to induce neutralizing antibodies was much lower and the intact and stable s1 subunit was essential for producing efficient neutralizing antibodies against hcov-229e. importantly, our results reveal different vaccine strategies for coronaviruses, and s-trimer is better than rbd as a target for vaccine development in alpha-coronavirus. our findings will provide important implications for future development of coronavirus vaccines. importance outbreak of coronaviruses, especially sars-cov-2, poses a serious threat to global public health. development of vaccines to prevent the coronaviruses that can infect humans has always been a top priority. coronavirus spike (s) protein is considered as a major target for vaccine development. currently, structural studies have shown that alpha-coronavirus (hcov-229e) and beta-coronavirus (sars-cov and sars-cov-2) rbds are in lying and standing state, respectively. here, we tested the ability of s-trimer and rbd to induce neutralizing antibodies among these coronaviruses. our results showed that beta-covs rbds are in a standing state, and their s proteins can induce more neutralizing antibodies targeting rbd. however, hcov-229e rbd is in a lying state, and its s protein induces a low level of neutralizing antibody targeting rbd. our results indicate that alpha-coronavirus is more conducive to escape host immune recognition, and also provide novel ideas for the development of vaccines targeting s protein. hcov-nl63) and beta-covs (hcov-oc43 and hcov-hku1) are well adapted to 68 humans and widely circulate in the human population, with most infections causing 69 mild disease in immunocompetent adults (3, 5, 6). in addition, sars-cov, 70 sars-cov-2 and mers-cov belong to beta-cov and are highly pathogenic (7-9). as the primary glycoprotein on the surface of the viral envelope, the spike (s) 80 glycoprotein is the major target of neutralizing antibodies (nabs) elicited by natural 81 infection and key antigens in experimental vaccine candidates. the s protein contains 82 two subunits responsible for receptor binding (s1 subunit) and membrane fusion (s2 83 subunit) (11). in particular, the s1 subunit of the prefusion s protein is structurally (17, 18) . the s1 subunits of beta-and gamma-cov strains utilize the cross-subunit 103 packing mode, reducing the conformational conflict of the rbd in a standing state 104 (13, 19, 20, 24). in contrast, alpha-and delta-cov strains both utilize an intrasubunit 105 packing mode, and the s1-ctd is limited by the conformational conflict with 106 surrounding domains (12, 14, 16-18, 21, 24) . hence, the s1-rbd in the s trimer was 107 captured in two different states among different coronaviruses. in the beta-covs 108 (sars-cov, sars-cov-2 and mers-cov), the s1-rbd adopts a "standing" state, 109 which is believed to be a prerequisite for receptor binding and rbm-specific antibody 110 6 binding (13, 19, 20) . nevertheless, the s1-rbds of alpha-covs all adopt "lying" state, 111 which is considered more conducive to evading antibody recognition (12, 14, 16, 21 mers-cov. among them, the s protein or rbd was the major targets (45) (46) (47) . compared with beta-covs, relatively few studies have investigated two 123 alpha-hcovs: hcov-229e and hcov-nl63. however, their s1 subunit structure and 124 receptor recognition pattern, especially the structure of the rbd and its state in the s 125 trimer, differ substantially from those of beta-covs, suggesting different s protein 126 immune responses between alpha-and beta-covs. importantly, considering the low 127 homology between different coronavirus genera, related research on alpha-covs can 128 not only help to elucidate the differences between s proteins that adopt different rbd 129 states but can also facilitate the development of coronavirus vaccines. in this study, 130 we selected sars-cov, sars-cov-2, and hcov-229e as models, which adopt the 131 two rbd states, and evaluated and compared immune responses to the s trimers and 132 7 rbds of these coronaviruses through immunological and bioinformatics approaches. 133 we also investigated the mechanism through which the hcov-229e s trimer 134 produced effective nabs. finally, we provide possible vaccine strategies for alphato address this issue, we performed b-cell epitope predictions for the s trimers 152 and rbds of alpha-cov (hcov-229e) and beta-covs (sars-cov and 153 sars-cov-2). the predicted positive residues (the corresponding spatial epitope and 154 8 linear epitope) are displayed on the structural surface ( fig. 2a, 2c and 2e) , and the 155 distribution of positive residues on the rbd is summarized in table 1 . a total of 51 156 and 26 amino acid residues located on the rbd were predicted to be conformational 157 epitopes for sars-cov and sars-cov-2, respectively. of these, 47 and 25 residues 158 were located in the sars-cov rbm subdomain and in the sars-cov-2 rbm 159 subdomain, respectively. the linear b-cell epitope prediction results were similar in 160 sars-cov and sars-cov-2. however, in hcov-229e, only 3 residues located in 161 the rbm subdomain were predicted to be conformational epitopes, and 9 residues 162 were predicted to be linear epitopes. the same results also appeared in the 163 hcov-229e s trimer: fewer positive residues were located in the rbd than in the 164 sars-cov or sars-cov-2 rbm subdomain ( fig. 2a, 2c and sars-cov-2-immunized mice had a good neutralizing ability ( fig. 3i and 3j) . 205 for hcov-229e, the s trimer serum had a comparable neutralizing ability to that of 206 sars-cov or sars-cov-2, but the rbd serum had no detectable neutralizing 207 ability (fig. 3k) . our experimental results indicate that the lying state of the rbd in 208 the hcov-229e s-trimer induces the production of very few antibodies targeting the 209 rbd, but the s-trimer still produces strong neutralizing antibody levels. in this study, we found that more rbd-specific antibodies were induced by the 211 s trimer with the rbd in the standing state than the s trimer with the rbd in the 212 lying state, and the affinity between rbd-specific antibodies and the s trimer was 213 also higher in the standing state. however, we also found that fewer nabs were 214 induced by the rbd of hcov-229e than by the rbds of sars-cov or 215 sars-cov-2. in terms of hcov-229e, the distribution of the potential residues in the 216 rbm was lower than that of sars-cov or sars-cov-2, which may have been 217 caused by different rbm patterns and exposure degrees. when we compared the 218 reported nab epitopes of sars-cov and alpha-cov tgev with our results (47), 219 they were basically consistent. therefore, we believe that this finding illustrates the 220 11 inherent difference between the rbds of alpha-and beta-cov. 221 the intact and stable s1 subunit of hcov-229e is a prerequisite for the 222 production of effective nabs 223 our experimental results showed that hcov-229e s-trimer can induce strong 224 nab levels, while the rbd alone is less immunogenic. next, we will explore which 225 functional domains of the s-trimer are involved in the generation of nabs. to clarify 226 this issue, we immunized mice with the hcov-229e s trimer (10 µg), s1 (10 µg), 227 ntd (10 µg), rbd (10 µg) and ntd+rbd (5 µg+ 5 µg). meanwhile, to better 228 confirm our results, the hcov-229e strain vr740 was used for the neutralizing assay. 229 the results indicated that the s trimer serum had the best neutralizing ability, followed 230 by the s1 and ntd+rbd sera, while the ntd and rbd sera alone had no detectable 231 neutralizing effects (fig. 4a) . the results indicate that the s1 region in the s-trimer 232 should be the key region for nabs induction. to further verify the importance of the 233 complete s1 structure in the s-trimer, we designed two s trimer mutants, namely, an 234 ntd-deficient s trimer and an s65c/t472c s trimer, the s1 subunit integrity or 235 stability of which was destroyed ( fig. 4c and 4f ). mutant proteins disrupt the 236 conformational conflicts that limit rbd standing, significantly improving their ability 237 to bind hapn ( fig. 4d and 4g) . however, an incomplete or unstable s1 238 conformation significantly reduces the level of nabs induced by the s-trimer (fig. 4e 239 and 4k). taken together, these results showed that the intact and stable s1 subunit of 240 hcov-229e is a prerequisite for the production of effective nabs. furthermore, our experimental results show that rbd has a higher ability to bind 242 12 to the receptor hapn (fig. 4b) , which indicates that the characteristics of rbd itself 243 may lead to the generation of less neutralizing antibodies. furthermore, we screened 244 monoclonal antibodies using s-trimer, and the results showed that few antibodies 245 targeting s1-rbd (fig. 5a) . to further determine the ability of rbd to induce 246 antibodies itself, we screened monoclonal antibodies targeting the s1 region and 247 found that the proportion of antibodies targeting rbd was approximately 20% (fig. 248 5b ). since the s1 protein is expressed in a monomeric form, rbd is not restricted by 257 we compared the structures of s trimers and rbds among alpha-coronaviruses 258 (figs. 1b and 6a) . we also predicted the potential b-cell epitopes for their rbds 259 ( fig. 6a; table1) . in alpha-cov, the s-trimer had a closed s1 subunit with three 260 "lying" rbds (fig. 1b) . moreover, the rbds consist of a standard β-sandwich fold 261 core and three short discontinuous loops in the same spatial region (12, 14, 16, 21, 26, 262 27, 48) (fig. 6a) . meanwhile, we performed a structural conservative analysis and the 263 results showed that the rbd structures of hcov-nl63, pedv, and fipv are most 264 13 similar to hcov-229e, with rsmd values of 1.9, 2.0, and 2.2, respectively (fig. 6b) . 265 in addition, the distribution of potential b-cell epitopes in the rbds of alpha-covs 266 was also similar to that of hcov-229e (fig. 6a and 6c; table1) . based on the above 267 data, inherent differences exist in the rbds between alpha-and beta-covs (figs. 2 268 and 6a). however, the alpha-and beta-covs show high similarity in their rbds and 269 similar potential immune characteristics within their respective genera (figs. 2, 3, 6a 270 and 6b). accordingly, in alpha-covs such as hcov-229e, subunit vaccines should 271 prioritize the s-trimer rather than the rbd. in beta-covs such as sars-cov and 272 sars-cov-2, the s trimer and rbd are both good candidates for subunit vaccines 273 (fig. 7) . 274 in summary, we systematically analyzed the conformational states and igg (1:5,000 diluted in pbst with 1% bsa (w/v), boster) was used for detection. signal reading was carried out in the same manner. hbs buffer was used as a mock then the plates were reacted with the hybridoma culture supernatants at 37℃ for 1h. hrp-conjugated goat anti-mouse igg (1:5,000 diluted in pbst with 1% bsa (w/v), boster) was used for detection. signal reading was carried out in the manner 368 described above. hybridoma culturing medium was used as a mock control. ratification vote on 446 taxonomic proposals to the international committee on taxonomy of viruses origin and evolution of pathogenic coronaviruses genetic recombination, and pathogenesis of coronaviruses clinical 456 features of patients infected with 2019 novel coronavirus in wuhan genomic analysis of human coronaviruses oc43 (hcov-oc43s) circulating in france from 460 2001 to 2013 reveals a high intra-specific diversity with new recombinant 461 genotypes coronavirus as a possible cause of severe acute respiratory syndrome anonymous. 2020. the species severe acute respiratory syndrome-related 470 coronavirus: classifying 2019-ncov and naming it sars-cov-2 structure, function, and evolution of coronavirus spike proteins cryo-em analysis of a feline coronavirus 479 spike protein reveals a unique structure and camouflaging glycans cryo-em structure of the 2019-ncov spike in the 483 prefusion conformation the 3.1-angstrom cryo-electron microscopy 485 structure of the porcine epidemic diarrhea virus spike protein in the prefusion 486 structural basis for human 489 coronavirus attachment to sialic acid receptors the human coronavirus hcov-229e s-protein glycan shield and fusion activation of a 495 deltacoronavirus spike glycoprotein fine-tuned for enteric infections cryo-electron microscopy structure of porcine deltacoronavirus spike 499 protein in the prefusion state cryo-em structures of mers-cov and sars-cov spike 502 glycoproteins reveal the dynamic receptor binding domains cryo-electron 505 microscopy structures of the sars-cov spike glycoprotein reveal a antigenic and immunogenic 597 characterization of recombinant baculovirus-expressed severe acute respiratory 598 syndrome coronavirus spike protein: implication for vaccine design recombinant receptor binding domain protein induces 602 partial protective immunity in rhesus macaques against middle east 603 respiratory syndrome coronavirus challenge immunogenicity and structures of a rationally designed prefusion mers-cov 608 spike antigen structural bases of coronavirus attachment to host aminopeptidase n and its 611 inhibition by neutralizing antibodies the x-ray crystal structure of human 613 aminopeptidase n reveals a novel dimer and the basis for peptide processing comparison of coronaviruses a 619 sequence homology and bioinformatic approach can predict candidate 620 targets for immune responses to sars-cov-2 clustal w and clustal x version 2.0 s1: receptor-binding subunit; s2: 628 membrane fusion subunit; ntd: n-terminal domain; rbd: receptor-binding domain 629 (magenta). (b) overall structure comparison of coronavirus s trimers structure-based b-cell epitope predictions of beta-cov (sars-cov and 637 sars-cov-2) and alpha-cov (hcov-229e). (a, c and e) the predicted b cell 638 30 epitopes of sars-cov, sars-cov-2 and hcov-229e are shown. the linear (red 639 cartoon) and conformational (yellow sphere) b cell epitopes were bepipred 2.0 or discotope 2.0 and labeled onto the corresponding structure by 641 and f) the complex structures of the rbds of sars-cov sars-cov-2 and hcov-229e with the receptors (hace2 and hapn) are shown. the 643 interface area of each complex and the surface area of each rbd were calculated via 644 the rbm region of the rbd and the receptors (hace2 and hapn) are 645 shown in red and cyan immunological analysis of beta-cov (sars-cov and sars-cov-2) and hcov-229e). (a and b) cross-reactivity of the sars-cov s trimer and 648 mice sera of sars-cov s trimer (red) 649 and sars-cov rbd (blue) were 10-fold serially diluted (starting with 500-fold 650 dilution) and reacted with the s trimer (a) or rbd (b), respectively cross-reactivity of the sars-cov-2 s trimer and rbd-specific sera is determined by 652 mice sera of sars-cov-2 s trimer (magenta) and sars-cov-2 rbd (slate) were 2-fold diluted and reacted with sars-cov-2 s trimer (c) and rbd (d) -reactivity of the hcov-229e s trimer and rbd-specific sera is determined 655 by elisa. mice sera of hcov-229e s trimer (orange) and hcov-229e rbd the antibody titers of sera from mice immunized with 10 μg of the hcov-229e rbd 658 (brown) and 50 μg of the hcov-229e rbd (purple) all data above are presented as the dilution that remained positive. (i, j and k) the neutralization 662 assay of mouse sera from the spike trimer and rbd against sars-cov, sars-cov-2 663 and hcov-229e pseudoviruses is determined. the data are presented as the mean 664 reciprocal ic 90 titer. the limit of detection for the assay depends on the initial dilution 665 and is represented by the intact and stable s1 subunit of hcov-229e is a prerequisite for the 667 production of effective nabs. (a) the neutralization abilities of mouse sera from the 668 b) determination of the affinity of ntd and rbd with the receptor hapn. 670 (c) structural model of hcov-229e-s-△ntd. magenta: rbd; green: sd1; cyan: 671 sd2. (d) dose-dependent binding of hcov-229e-s-△ntd and hapn. (e) the 672 neutralization ability of mouse sera from hcov-229e-s-△ntd was measured via 673 pseudovirus neutralization assay magenta: rbd; blue: 675 ntd; green: sd1; cyan: sd2. (g) dose-dependent binding of h) the neutralization ability of mouse sera 677 from hcov-229e-s-s65c/t472c was measured via pseudovirus neutralization assay the limit of detection for the assay depends on the initial dilution and is 680 represented by dotted lines，a reciprocal ic 90 titer of 10 was assigned. besides monoclonal antibody epitope mapping of the hcov-229e spike protein monoclonal antibody (mab) epitope regions in the hcov-229e spike protein (a) and 684 s1 domain (b). supernatants of positive hybridomas were reacted with the data are presented as the od 450 686 (bottom). mabs and their epitope regions are indicated below the schematic of the 687 b cell epitope analysis of the rbd regions of alpha-coronavirus spike proteins. 689 (a) structures of the rbds from alpha-covs (hcov-229e the linear (red cartoon) and conformational 691 (yellow sphere) b cell epitopes were predicted by bepipred 2.0 or discotope 2.0 and 692 labeled onto the corresponding rbd structure by pymol. (b) structural comparison 693 of the rbds from alpha-covs. (c) sequence alignment of the rbds from 694 alpha-covs. the rbm or putative rbm region is shown in cyan foundation (program no. 2662017py028). the authors declare no competing interests. fig. 7 potential vaccine strategies for alpha-and beta-covs. the model showed that 698 the rbds of the alpha-cov s trimers are in a lying state. in this state, the s protein 699 cannot bind to the receptor, but meanwhile, this state is also conducive to escaping the 700 immune response target the rbd, and the rbds of the alpha-covs also induces 701 fewer nabs; thus, their s-trimers can be an effective potential subunit vaccine. in key: cord-133453-23rfdkuw authors: chen, jiahui; gao, kaifu; wang, rui; wei, guowei title: prediction and mitigation of mutation threats to covid-19 vaccines and antibody therapies date: 2020-10-13 journal: nan doi: nan sha: doc_id: 133453 cord_uid: 23rfdkuw antibody therapeutics and vaccines are among our last resort to end the raging covid-19 pandemic.they, however, are prone to over 1,800 mutations uncovered by a mutation tracker. it is urgent to understand how vaccines and antibodies in the development would be impacted by mutations. in this work, we first study the mechanism, frequency, and ratio of mutations on the spike (s) protein, which is the common target of most covid-19 vaccines and antibody therapies. additionally, we build a library of antibody structures and analyze their 2d and 3d characteristics. moreover, we predict the mutation-induced binding free energy (bfe) changes for the complexes of s protein and antibodies or ace2. by integrating genetics, biophysics, deep learning, and algebraic topology, we deduce that some of the mutations such as m153i, s254f, and s255f may weaken the binding of s protein and antibodies, and potentially disrupt the efficacy and reliability of antibody therapies and vaccines in the development. we provide a strategy to prioritize the selection of mutations for designing vaccines or antibody cocktails. the expeditious spread of coronavirus disease 2019 pandemic caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has led to 34,667,658 confirmed cases and 1,030,040 fatalities as of september 30, 2020. in the 21st century, three major outbreaks of deadly pneumonia are caused by βcoronaviruses: sars-cov (2002) , middle east respiratory syndrome coronavirus (mers-cov) (2012), and sars-cov-2 (2019) [1] . similar to sars-cov and mers-cov, sars-cov-2 causes respiratory infections, and the transmission of viruses occurs among family members or in healthcare settings at the early stages of the outbreak. however, sars-cov-2 has an unprecedentedly high infection rate compared to sars-cov and mers-cov [2] . considering the high infection rate, high prevalence rate, long incubation period [2] , asymptomatic transmission [3, 4] , and potential seasonal pattern [5] of covid-19, the development of specific antiviral drugs, antibody therapies, and effective vaccines is of paramount importance. traditional drug discovery takes more than ten years, on average, to bring a new drug on the market [6]. however, developing potent sars-cov-2 specified antibodies and vaccines is a relatively more efficient and less timeconsuming strategy to combat covid-19 for the ongoing pandemic [7] . antibody therapies and vaccines depend on the host immune system. recently studies have been working on the host-pathogen interaction, host immune responses, and the pathogen immune evasion strategies [8] [9] [10] [11] [12] [13] , which provide insight into understanding the mechanism of antibody therapies and vaccine development. the immune system is a host defense system that protects the host from pathogenic microbes, eliminates toxic or allergenic substances, and responds to an invading pathogen [14] . it has innate immune system and adaptive immune system as two major subsystems. the innate system provides an immediate but non-specific response, whereas the adaptive immune system provides a highly specific and effective immune response. once the pathogen breaches the first physical barriers, such as epithelial cell layers, secreted mucus layer, mucous membranes, the innate system will be triggered to identify pathogens by pattern recognition receptors (prrs), which is expressed on dendritic cells, macrophages, or neutrophils [15] . specifically, pprs identify pathogen-associated molecular patterns (pamps) located on pathogens and then activate complex signaling pathways that introduce inflammatory responses mediated by various cytokines and chemokines, which promote the eradication of the pathogen [16, 17] . notably, the transmission of sars-cov-2 even occurs in asymptomatic infected individuals, which may delay the early response of the innate immune response [8] . another important line of host defense is the adaptive immune system. b lymphocytes (b cells) and t lymphocytes (t cells) are special types of leukocytes that are the acknowledged cellular pillars of the adaptive immune system [18] . two major subtypes of t cells are involved in the cell-mediated immune response: killer t cells (cd8+ t cells) and helper t cells (cd4+ cells). the killer t cells eradicate cells invaded by pathogens with the help of major histocompatibility complex (mhc) class i. mhc class i molecules are expressed on the surface of all nucleated cells [19] . the nucleated cells will firstly degrade foreign proteins via antigen processing when viruses infect them. then, the peptide fragments will be presented by mhc class i, which will activate killer t cells to eliminate these infected cells by releasing cytotoxins [20] . similarly, helper t cells cooperate with mhc class ii, a type of mhc molecules that are constitutively expressed on antigen-presenting cells, such as macrophages, dendritic cells, monocytes, and b cells [21] . helper t cells express t cell receptors (tcr) to recognize antigen bound to mhc class ii molecules. however, helper t cells do not have cytotoxic activity. therefore, they can not kill infected cells directly. instead, the activated helper t cells will release cytokines to enhance the microbicidal function of macrophages and the activity of killer t cells [22] . notably, an unbalanced response can result in a "cytokine storm," which is the main cause of the fatality of covid-19 patients [23] . correspondingly, a b cell involves in humoral immune response and identifies pathogens by binding to foreign antigens with its b cell receptors (bcrs) located on its surface. the antigens that are recognized by antibodies will be degraded to petites in b cells and displayed by mhc class ii molecules. as mentioned above, helper t cells can recognize the signal provided by mhc class ii and upregulate the expression of cd40 ligand, which provides extra stimulation signals to activate antibody-producing b cells [24] , rendering millions of copies of antibodies (ab) that recognize the specific antigen. additionally, when the antigen first enters the body, the t cells and b cells will be activated, and some of them will be differentiated to long-lived memory cells, such as memory t cells and memory b cells. these long-lived memory cells will play a role in quickly and specifically recognizing and eliminating a specific antigen that encountered the host and initiated a corresponding immune response in the future [25] . the vaccination mechanism is to stimulate the primary immune response of the human body, which will activate t cells and b cells to generate the antibodies and long-lived memory cells that prevent infectious diseases, which is one of the most effective and economical means for combating with covid-19 at this stage. as mentioned above, secreted by b cells of the adaptive immune system, antibodies can recognize and bind to specific antigens. conventional antibodies (immunoglobulins) are y-shaped molecules that have two light chains and two heavy chains [26] . each light chain is connected to the heavy chain via a disulfide bond, and heavy chains are connected through two disulfide bonds in the mid-region known as the hinge region. each light and heavy chain contain two distinct regions: constant regions (stem of the y) and variable regions ("arms" of the y) [27] . an antibody binds the antigenic determinant (also called epitope) through the variable regions in the tips of heavy and light chains. there is an enormous amount of diversity in the variable regions. therefore, different antibodies can recognize many different types of antigenic epitopes. to be specific, there are three complementarity determining regions (cdrs) that are arranged non-consecutively in the tips of each variable region. cdrs generate most of the diversities between antibodies, which determine the specificity of individuals of antibodies. in addition to conventional antibodies, camelids also produce heavy-chain-only antibodies (hcabs). hcabs, also referred to as nanobodies, or vhhs, contain a single variable domain (vhh) that makes up the equivalent antigen-binding fragment (fab) of conventional immunoglobulin g (igg) antibodies [28] . this single variable domain typically can acquire affinity and specificity for antigens comparable to conventional antibodies. nanobodies can easily be constructed into multivalent formats and have higher thermal stability and chemostability than most antibodies do [29] . another advantage of nanobodies is that they are less susceptible to steric hindrances than large conventional antibodies [30] . considering the broad specificity of antibodies, seeking potential antibody therapies has become one of the most feasible strategies to fight against sars-cov-2. in general, antibody therapy is a form of immunotherapy that uses monoclonal antibodies (mab) to target pathogenic proteins. the binding of antibody and pathogenic antigen can facilitate either immune response, direct neutralization, radioactive treatment, the release of toxic agents, or cytokine steam inhibition (aka immune checkpoint therapy). the sars-cov-2 entry of a human cell facilitated by the process of a series of interactions between its spike (s) protein and the host receptor angiotensin-converting enzyme 2 (ace2), primed by host transmembrane protease, serine 2 (tmprss2) [31] . as such, most covid-19 antibody therapeutic developments focus on the sars-cov-2 spike protein antibodies that were initially generated from patient immune response and t-cell pathway inhibitors that block t-cell responses. a large number of antibody therapeutic drugs are in clinical trials. 28 currently, most antibody therapy developments focus on the use of antibodies isolated from patient convalescent plasma to directly neutralize sars-cov-2 [32] [33] [34] , although there are efforts to alleviate cytokine storm. a more effective and economical means to fight against sars-cov-2 is vaccine [35] , which is the most anticipated approach for preventing the covid-19 pandemic. a vaccine is designed to stimulate effective host immune responses and provide active acquired immunity by exploiting the body's immune system, including the production of antibodies, which is made of an antigenic agent that resembles a disease-causing microorganism, or surface protein, or genetic material that is needed to generate the surface protein. for sars-cov-2, the first choice of surface proteins is the spike protein. there are four types of covid-19 vaccines, as shown in figure 1 . 1) virus vaccines use the virus itself, in a weakened or inactivated form. 2) viral-vector vaccines are designed to genetically engineer a weakened virus, such as measles or adenovirus, to produce coronavirus s proteins in the body. both replicating and non-replicating viral-vector vaccines are being studied now. 3) nucleic-acid vaccines use dna or mrna to produce sasr-cov-2 s proteins inside host cells to stimulate the immune response. 4) protein-based vaccines are designed to directly inject coronavirus proteins, such as s protein or membrane (m) protein, or their fragments, into the body. both protein subunits and viral-like particles (vlps) are under development for covid-19 [36] . among these technologies, nucleic-acid vaccines are safe and relatively easy to develop [36] . however, they have not been approved for any human usage before. however, the general population's safety concerns are the major factors that hinder the rapid approval of vaccines and antibody therapies. a major potential challenge is an antibody-dependent enhancement, in which the binding of a virus to suboptimal antibodies enhances its entry into host cells. all vaccine and antibody therapeutic developments are currently based on the reference viral genome reported on january 5, 2020 [37] . sars-cov-2 belongs to the coronaviridae family and the nidovirales order, which has been shown to have a genetic proofreading mechanism regulated by non-structure protein 14 (nsp14) in synergy with nsp12, i.e., rna-dependent rna polymerase (rdrp) [38, 39] . therefore, sars-cov-2 has a higher fidelity in its transcription and replication process than other single-stranded rna viruses, such as the flu virus and hiv. even though the s protein of sars-cov-2 has been undergoing many mutations, as reported in [40, 41] . as of september 30, a total of 1811 mutations on the s protein has been detected on 63556 complete sars-cov-2 genome sequences. therefore, it is of paramount importance to establish a reliable computational paradigm to predict and mitigate the impact of sars-cov-2 mutations on vaccines and antibody therapies. moreover, the efficacy of a given covid-19 vaccine depends on many factors, including sars-cov-2 biological properties associated with the vaccine, mutation impacts, vaccination schedule (dose and frequency), idiosyncratic response, assorted factors such as ethnicity, age, gender, or genetic predisposition. the effect of covid-19 vaccination also depends on the fraction of the population who accept vaccines. it is essentially unknown at this moment how these factors will unfold for covid-19 vaccines. it is no doubt that any preparation that leads to an improvement in the covid-19 vaccination effect will be of tremendous significance to human health and the world economy. therefore, in this work, we integrate genetic analysis and computational biophysics, including artificial intelligence (ai), as well as additional enhancement from advanced mathematics to predict and mitigate mutation threats to covid-19 vaccines and antibody therapies. we perform single nucleotide polymorphism (snp) calling [41, 42] to identify sars-cov-2 mutations. for mutations on the s protein, we analyze their mechanism [43] , frequency, ratio, and secondary structural traits. we construct a library of all existing antibody structures from the protein data bank (pdb) and analyze their two-dimensional (2d) and three-dimensional (3d) characteristics. we further predict the mutation-induced binding affinity changes of antibody and s protein complexes using a topology-based network tree (topnettree) [44] , which is a state-of-the-art model that integrates deep learning and algebraic topology [45] [46] [47] . after identifying mutations that are potentially disruptive to antibody and s protein interactions, we further infer their threats to vaccines based on antibody binding site analysis, mutation-induced disruptive free energy, and mutation occurrence frequency. we combine frequency and free energy change to prioritize mutation threats and guild the development of future vaccines and antibody therapies. as a fundamental biological process, mutagenesis changes the organism's genetic information and servers as a primary source for many kinds of cancer and heritable diseases, which is a driving force for evolution [48, 49] . generally speaking, virus mutations are introduced by natural selection, replication mechanism, cellular environment, polymerase fidelity, gene editing, random genetic drift, gene editing, recent epidemiology features, host immune responses, etc [50, 51] . notably, understanding how mutations have changed the sars-cov-2 structure, function, infectivity, activity, and virulence is of great importance for coming up with life-saving strategies in virus control, containment, prevention, and medication, especially in the antibodies and vaccines development. genome sequencing, snp calling, and phenotyping provide an efficient means to parse mutations from a large number of viral samples [40, 42] (see the supporting material (s1)). in this work, we retrieved over 60,000 complete sars-cov-2 genome sequences from the gisaid database [52] and created a real-time interactive sars-cov-2 mutation tracker( https://users.math.msu.edu/users/weig/sars-cov-2 mutation tracker.html) to report over 18,000 single mutations along with its mutation frequency on sars-cov-2 as of september 30. figure 2 is a screenshot of our online mutation tracker. it describes the distribution of mutations on the complete coding region of sars-cov-2. the y-axis shows the natural log frequency for each mutation at a specific position. a reader can download the detailed mutation snp information from our mutation tracker website. as mentioned before, the s protein has become the first choice for antibody and vaccine development. among 63,556 complete genome sequences, 1811 unique single mutations are detected on the s protein, and the h-index of s protein is 52 [40, 40] the number of unique mutations (n u ) is determined by counting the same type of mutations in different genome isolates only once, whereas the number of non-unique mutations (n nu , i.e., frequency) is calculated by counting the same type of mutations in different genome isolates repeatedly. table 1 lists the distribution of 12 snp types among unique and non-unique mutations on the s protein of sars-cov-2 worldwide. it can be seen that c>t and a>g are the two dominated snp types, which may be due to the innate host immune response via apobec and adar gene editing [43] . moreover, 133 non-degenerated mutations occurred on the s protein receptor-binding domain (rbd), which are relevant to the binding of sars-cov-2 s protein and most antibodies as well as ace2. additionally, 59 mutations occurred on the s protein domain (residue id: 14 to 226) are relevant to the binding of another antibody (4a8) and sars-cov-2 s protein. furthermore, since antibody cdrs are random coils, the complementary antigen-binding domains must involve random coils as well. table 2 lists the statistics of non-degenerate mutations on the secondary structures of sars-cov-2 s protein. here, the secondary structures are mostly extracted from the crystal structure of 7c2l [53] , and the missing residues are predicted by raptorx-property [54] . we can see that for both unique and non-unique cases, the average mutation rates on the random coils of the s protein have the highest values. particularly, the 23403a>g-(d614g) mutation on the random coils has the highest frequency of 39967. if we do not consider the 23403a>g-(d614g) mutations, then the unique and non-unique average rates on the random coils of s protein still have the highest values (0.98 and 10.83), indicating that mutations are more likely to occur on the random coils. consequently, the natural selection of mutations may tend to disrupt antibodies. table 2 : the statistics of non-degenerate mutations on the secondary structure of sars-cov-2 s protein. the unique and non-unique mutations are considered in the calculation. n u , n nu , ar u , ar nu represent the number of unique mutations, the number of nonunique mutations, the average rate of unique mutations, and the average rate of non-unique mutations on the secondary structure of s protein, respectively. here, the secondary structure is mostly extracted from the crystal structure of 7c2l, the missing residues are predicted by raptorx-property. we construct a sars-cov-2 antibody library of 28 3d antibody structures deposited in the pdb. among them, the binding sites of 27 antibodies are on the rbd of the s protein. while another antibody, 4a8 [53] , has a distinguished binding domain. additionally, mr17-k99y is a mutant of antibody mr17 [55] . we align 26 antibody structures, excluding mr17-k99y, with sars-cov-2 s protein in figure 3 . ace2 is included as a reference. clearly, except for antibody 4a8, all other 26 structures bind to the s protein rbd. it is interesting to note that 4a8 locates on a different domain. the pdb ids of these complexes can be found in figure 4 . [56] , cr3022 [61] , ey6a [62] , and 4a8, all the other 23 antibodies have their binding sites spatially clashing with that of ace2. notably, the paratope of h014 [67] does not overlap with that of ace2 directly, but in terms of 3d structures, their binding sites still overlap. this suggests that the bindings of 23 antibodies are in direct competition with that of ace2. theoretically, this direct competition reduces the viral infection rate. for such an antibody with strong binding ability, it will directly neutralize sars-cov-2 without the need of antibody-dependent cell cytotoxicity (adcc), antibody-dependent cellular phagocytosis (adcp), or other immune mechanisms. the paratopes of s309, cr3022, and ey6a on the rbd are away from that of ace2, leading to the absence of binding competition [62, 69, 70] . one study shows that the adcc and adcp mechanisms contribute to the viral control conducted by s309 in infected individuals [69] . for cr3022, one research indicates that it neutralizes the virus in a synergistic fashion [71] . for ey6a, the hypothesis is that the binding of ey6a could inhibit the glycosylation of ace2 [62] . a more radical example is 4a8 [53] , it binds to the n-terminal domain (ntd) of the s protein (figure 3 (h)), which is quite far from the rbd, it is speculated (i) the 3d structure of s protein rbd. the red, green, and blue represent for helix, sheet, and random coils of rbd, respectively. the darker color represents the higher mutation frequency on a specific residue. the antibodies are s309 (6m0j) [56] , cc12.1 (6xc2) [57] , cc12.1 and cr3022 (6xc3) [57] , cc12.3 (6xc4) [57] , cc12.3 and cr3022 (6xc7), c105 (6xcm) [58] , regn10933 and regn10987 (6xdg) [59] , cv30 (6xe1) [60] , fab 2-4 (6xey) [55] , cr3022 (6yla) [61] , h11-d4 (6yz5), cr3022 and h11-d4 (6z2m) [61] , h11-h4 (6zbp), ey6z and nanobody (6zcz) [62] , ey6z (6zer) [62] , p2b-2f6 (7bwj) [63] , bd23 (7byr) [64] , b38 (7bz5) [65] , cb6 (7c01) [66] , 4a8 (7c2l) [53] , sr4 (7c8v) [55] , b38 (7c8w), h014 (7cah) [67] , mr17-k99y (7can) [55] , bd-604 (7ch4), bd-629 (7ch5), bd-236 (7chb), bd-236 and bd-368-2 (7che), bd-604 and bd-368-2 (7chf), bd-368-2 (7chh), cova2-04 (7jmo) [68] , and cova2-39 (7jmp) [68] . that 4a8 may neutralize sars-cov-2 by restraining the conformational changes of the s protein, which is very important for the sars-cov-2 cell entry [53] . any antibody or drug that can inhibit serine protease tmprss2 priming of the s protein priming can effectively stop the viral cell entry [31] . figure 3 provides a visual illustration of antibody and ace2 competitions. it remains to know in the residue detail what has happened to these competitions. to better understand the antibody and s protein interactions, we study the residue contacts between antibodies and the s protein. we include the ace2 as a reference but excluding antibodies 4a8 and mr17-k99y. in figure 4 , the paratopes of 26 antibodies and ace2 were aligned on the s protein rbd 2d sequence, and their contact regions are highlighted. from the figure, one can see that, except for h014, s309, cr3022, and ey6a, all the other 22 antibodies have their antigenic epitopes overlapping with the ace2 rbd, especially on the residues from 486 to 505 of the sars-cov-2 rbd. therefore, these 22 antibodies competitively bind against ace2 as revealed in figure 3. the next question is whether there is any connection or similarity between the antibody paratopes in our library, particularly for those antibodies that share the same binding sites. to better understand this perspective, we carry out multiple sequence alignment (msa) to further study the similarity and difference among existing antibodies. many antibodies are very similar to each other and can be described in a few groups. the first group includes bd-629, cc12.3 [57] , cova2-04 [68] , cv30 [60] , cc12.1 [57] , b38 [65] , bd-236, bd-604, ey6a, and regn10933 [59] , as well as cb6 [66] . their identity scores to cb6 are 87. 39 therefore, multiple sequence alignment suggests that the paratopes of the antibodies bd-629, cb6, cova2-04, cv30, cc12.1, cc12.3, c105, bd-604, bd-236, and b38 are almost identical. similarly, the paratopes of the antibodies h11-h4, h11-d4, nb are highly consistent. so are the antibodies regn10987, cova2-39, and p2b-2f6. the above similarity indicates that the adaptive immune systems of individuals have a common way to generate antibodies. on the other hand, the existence of three distinct groups, as well as antibody 4a8 suggests the diversity in the immune response. note that we have also included ace2 in our msa as a reference but none of the existing antibodies is similar to ace2, because they were created from entirely different mechanisms. to investigate the influences of existing s protein mutations on the binding free energy (bfe) of s protein and antibodies, we consider 133 mutations occurred on the s protein rbd which are relevant to the binding of sars-cov-2 s protein and antibodies as well as ace2. additionally, 59 mutations occurred on the ntd of the s protein (residue id: 14 to 226) which are relevant to the binding of sars-cov-2 s protein and antibody 4a8 (pdb: 7c2l). we predict the free energy changes following existing mutations using our topnettree model [44] . the rbd mutations are computed which are in the distance of 10å to antibodies. our predictions are built from the x-ray crystal structure of sars-cov-2 s protein and ace2 (pdb 6m0j) [56] , and various antibodies (pdbs 6wps [69] , 6xc2 [57] , 6xc3 [57] , 6xc4 [57] , 6xc7, 6xcm [58] , 6xdg [59] , 6xe1 [60] , 6xey [72] , 6yla [61] , 6yz5, 6z2m, 6zbp, 6zcz [62] , 6zer [62] , 7bwj [63] , 7byr [64] , 7bz5 [65] , 7c01 [66] , 7c2l [53] , 7c8v [55] , 7c8w, 7cah [67] , 7can [55] , 7ch4, 7ch5, 7chb, 7che, 7chf, 7chh, 7jmo [68] , and 7jmp [68] ). the bfe change following mutation (∆∆g) is defined as the subtraction of the bfe of the mutant type from the bfe of the wild type, ∆∆g = ∆g w − ∆g m where ∆g w is the bfe of the wild type and ∆g m is the bfe of mutant type. therefore, a negative bfe change means that the mutation decreases affinities, making the protein-protein interaction less stable. we first present the bfe changes ∆∆g of sars-cov-2 s protein binding domain with antibody 4a8 in figure 5 , which is the only complex that is not on the rbd in our collections of s protein and antibody complexes. most mutations have small changes in their binding free energies, while some of them have large changes. notably, 25 out of 59 mutations on the binding domain have positive bfe changes, which means that the mutations increase affinities and would make protein-protein interactions more stable. however, the majority (58%) of mutations have negative bfe changes, including high-frequency mutations, m153i, s254f, and s255f. it is also noted that many mutations on the binding domain, such as g142d and k147n, have significant negative free energy changes. the mutations on the binding domain with negative binding affinities reveal that the binding of antibody 4a8 and s protein will be potentially disrupted. next, we study the bfe changes ∆∆g induced by 39 mutations on the sars-cov-2 s protein rbd for the antibody fab 2-4 (pdb: 6xey) in figure 6 . most mutations induce small changes in the thee binding free energies, while mutations, g485r and s494l, have large negative bfe changes. overall, 27 out of 39 mutations on the rbd lead to negative bfe changes, which means 69% of mutations will potentially weaken the binding between antibody fab 2-4 and s protein. particularly, mutation s477n on the rbd induces a negative bfe change with a high frequency of 3269. while some mutations leading to positive bfe changes, more mutations induce negative bfe changes with large magnitude. antibody fab 2-4 shares a similar binding domain with ace2 and thus is a potential candidate for the direct neutralization of sars-cov-2. however, bfe change predictions indicate that the mutations on s protein weaken the fab 2-4 binding with s protein and make it less competitive with ace2. in figure 7 , we illustrate antibody b38 (pdb: 7c8w), which shares the binding domain with ace2 as well. one can notice that only four mutations, r403s, f490s, l455f, and s494l, have the magnitude of bfe changes larger than 1 kcal/mol and all are negative bfe changes. the rest mutations have a small magnitude of changes. mutation v483a has a frequency of 31 and small positive bfe changes. interestingly, mutation s494l induces large bfe changes for antibodies b39 and fab 2-4. antibody b39 will reduce its competitiveness with ace2 if mutations r403s, f490s, l455f, and s494l become dominant. finally, we consider the bfe change predictions for antibody s309 and s protein complex, whose receptor binding motif (rbm) does not overlap with the rbm of ace2. the bfe changes induced by 30 mutations are predicted. among them, 11 changes are positive. similar to the aforementioned antibodies, most of the mutations lead to small changes in their binding affinity magnitude but three mutations, t345s, v395i, and k444n, induce large negative changes. the binding of antibody s309 might be disrupted, considering that a majority of mutations induce negative bfe changes with large magnitude. while antibodies play a variety of functions in the human immune system such as neutralization of infection, phagocytosis, antibody-dependent cellular cytotoxicity, etc., their binding with antigens is crucial for these functions. our analysis of bfe changes following mutations on s protein suggests that some antibodies will be less affected by mutations, which is important for developing vaccine and antibody therapies. the bfe change analysis of other antibodies is described in the supporting material (s3). in this section, we build a library of mutation-induced bfe changes for all mutations and all antibodies. in principle, we could create a library of all possible mutations for all antibodies, as we did for ace2 [73] . here, we limit our effort to all existing mutations. antibody 4a8 on the ntd has been discussed above. we consider antibodies on the rbd. based on our earlier analysis, three types of sars-cov-2 s protein secondary structural residues have f338l g339d e340k v341i y365h s366p s366f v367f n370s d405v v407i r408i r408t k417r k417n i418v n439k n440k l441i different mutation rates. among them, the random coils are major components of the rdb and the ntd, as shown in fig. 3 . therefore, mutations on the rbd are split into three categories based on their locations in secondary structures helix, sheet, and coil. in figure 9 , we present the bfe changes for ace2 and antibodies induced by mutations on helix residues of the s protein rbd. the frequency for each mutation is also presented. most mutations on helix residues lead to positive bfe changes (green squares), whereas some mutations induce negative bfe changes (pink squares). the n439k mutation having the largest frequency, 106, shows mild bfe changes on ace2 and antibodies. mutations k417n and y505c induce positive bfe changes on most of ace2 and antibodies. especially, antibodies c105 and bd-604 have larger bfe changes than ace2, which indicates that they are stronger competitive than ace2. antibody cb6 may be potentially a good therapeutic candidate as its bfe changes are positive following all mutations, but this needs to be confirmed by other mutations on the coil and sheet residues. in figure 10 , we present the bfe changes for ace2 and antibodies along with frequencies on mutations of sheet residues of the s protein rbd. the mutation r403s has a large variance of the bfe changes such that both positive and negative changes occurred on antibodies and ace2. clearly, antibodies bd23, bd38, cb6, mr17, and mr17-k99y lead to negative bfe changes on mutations of rbd sheet residues, which reduce their competitive binding ability with ace2 after mutations. as for mutations with high frequencies, the mutation r403k has negative changes on most antibodies, which poses a danger of disrupting the binding of antibodies and s protein. figure 11 presents the bfe changes for ace2 and antibodies along with the log of frequencies on each mutation of coil residues on the s protein rbd. overall, most mutations on coil residues lead to negative bfe changes. interestingly, cv30 has the most positive bfe changes following mutations, which can be a good candidate for potential therapy. for the high-frequency mutation s447n, the bfe changes are mild on ace2 and antibodies. however, mutation l455f induces negative bfe changes for all antibodies except for cova2-39, which is considered as a potentially dangerous mutation for antibody therapies. n354d n354s n354k r357k k378n t393p v395i r403k r403s l452m l452r y453f q493l s494p s494l y508h in statistics, most mutations (94 of 133) occur on residues whose secondary structures are coil, while 20 out of 133 mutations are on the helix, and 19 out of 133 mutations are on the sheet. here, 12 mutations on the random coils and 2 mutations on helix are not calculated due to the far distance to antibodies. moreover, residues on coil have more negative bfe changes (548 negative bfe changes vs. 534 positive bfe changes), while residues on the helix or sheet have more positive bfe changes (74 and 95 negative bfe changes vs. 94 and 123 positive bfe changes, respectively). lastly, the maximum bfe changes of the helix, sheet, and coils are 4.47 kcal/mol, 4.63 kcal/mol, and 4.52 kcal/mol, while the minimum bfe changes are -2.91 kcal/mol, -2.95 kcal/mol, and -3.33 kcal/mol, respectively. binding affinity changes (kcal/mol) figure 12 : illustration of sars-cov-2 mutation-induced maximal and minimal binding free energy changes for the complexes of s protein and 28 antibodies or ace2. here, the maximal change strengthens the binding while the minimal change weakens the binding for each complex. figure 12 indicates the bfe changes extreme values (maximal in blue and minimal in red) of the complexes of s protein ace2 or antibodies following mutations. many antibodies, such as cr3022 and cr3022 h11-d4, are not very sensitive to the current s protein mutations. however, some other antibodies, such as cv30, fab 2-4, and ey6z nb, can be dramatically affected by sars-cov-2 mutations. the increasing number of affected and dead individuals, the global spread situation, and the lack of prophylactics and therapeutics give rise to the urgent demand for the prevention of covid-19. vaccination is the most effective and economical means to prevent and control pandemics [35] . currently, 213 vaccines are in various clinical trial stages, as reported in an online covid-19 treatment and vaccine tracker ( https://covid-19tracker.milkeninstitute.org/#vaccines intro). broadly speaking, there are four types of coronavirus vaccines in progress: virus vaccines, viral-vector vaccines, nucleic-acid vaccines, and proteinbased vaccines, as shown in figure 1 . the first type of vaccine is the virus vaccine, which injects weakened or inactivate viruses to the human body. a virus is conventionally weakened by altering its genetic code to reduce its virulence and elicit a stronger immune response. a biotechnology company codagenix is currently working on a "codon optimization" technology to weaken viruses, and it has weakened virus vaccine is in progress [74] . unlike a weakened virus, the inactivated virus cannot replicate in the host cell. a virus is inactivated by heating or using chemicals, which induces neutralizing antibody titers and has been proven to have its safety [75] . at this stage, both sinopharm, which works with the beijing institute of biological products and wuhan institute of biological products, and sinovac, which works with institute butantan and bio farma is developing inactivate sars-cov-2 vaccines that are in phase iii clinical trials. the second type of vaccine is the viral-vector vaccine, which is genetically engineered so that it can produce coronavirus surface proteins in the human body without causing diseases. there are two subtypes of viral-vector vaccines: the non-replicating viral vector and the replicating viral vector. there are 4 non-replicating viral vector vaccines in phase iii trials. astrazeneca and the university of oxford, whose vaccine is in phase iii trials in many countries. it works by taking a chimpanzee virus and coating it with the s proteins of sars-cov-2. the chimp virus causes a harmless infection in humans, but the spike proteins will activate the immune system to recognize signs of a future sars-cov-2 invasion. notably, the booster shots can be needed to keep long-lasting immunity. moreover, at this stage, only one replicating viral-vector vaccine is in phase i. institut pasteur themis, in cooperating with the university of pittsburgh cvr and merck sharp & dohme is developing such a replicating viral vaccine, which tends to be safe and provoke a strong immune response [36] . the third type of vaccine is nucleic-acid vaccines, including two subtypes: dna-based vaccines and rna based vaccines. at least 40 teams are currently working on nucleic-acid vaccines since they are safe and easy to develop. the dna-based vaccine works by inserting genetically engineered blueprints of the viral gene into small dna molecules such as plasmids for injection. moreover, the electroporation technique is employed to create pores in membranes to increase dna uptake into cells. the injected dna will produce mrna by transcription with the help of the nucleus in human cells. such an mrna will translate viral proteins (mostly spike proteins), which are dutifully produced by cells in response to the genes, alarm the immune system, and should produce immunity. currently, there are four dna-based vaccines in phase ii. similar to dna-based vaccines, the rna-based vaccines provide immunity through the introduction of rna, which is encased in a lipid coat to ensure its entering into cells. two rna-based vaccines are in phase iii, and companies such as moderna, biontec, and pfizer are working on the advanced development of rna-based vaccines. the fourth type of vaccine is the protein-based vaccines, which aims to inject viral proteins directly to human bodies to trigger immune readiness. protein subunits vaccine is one of the subtypes of the proteinbased vaccine. more than 60 teams are working on vaccines with viral protein subunits, such as spike proteins and membrane (m) proteins. another subtype of the protein-based vaccine is the virus-like particle (vlp) vaccine. the vlp vaccines closely resemble viruses. however, they are not infectious since they do not contain viral genetic material. the non-replicating propriety provides a safer alternative to weakened virus vaccines, the hpv vaccine or newer flu vaccines are vlp vaccines. currently, 16 teams are working on the vlp vaccines for the future prevention of covid-19. since the structural basis of antibody cdrs, or paratope, is random coils, we hypothesize that cdrs favor antigenic random coils as complementary epitopes, i.e., antigenic determinants [76, 77] . figure 13 depicts the 3d structure of s protein, where the random coils are drawn with green strings, and the other secondary structure is described with the purple surface. it shows that the rbd and the ntd mostly consist of random coils. the rbd is the antigenic determinant of 27 structurally-known sars-cov-2 antibodies; meanwhile, the ntd is the binding domain of antibody 4a8, which confirms our hypothesis. figure 14 marks the secondary structure of the s protein. the red, blue, and green colors represent helix, sheet, and random coils of s protein. it can be seen that the s protein mostly consists of random coils, which means there are many other potential antigenic epitopes on the s protein for antibody cdrs. we believe that the emphasis of direct binding competition with ace2 in the past [62, 69, 70] has led to the neglecting of many important antibodies that do not bind to the rbd. therefore, we suggest that researchers pay more attention to antibodies that do not bind to the rbd. vaccine efficacy is an essential issue for the control of the covid-19 pandemic. s protein is one of the most popular surface proteins for the vaccine development. however, mutations accumulated on the s protein of sars-cov-2, which may reduce the vaccine efficacy. as we found in section 2, mutations are more likely to happen on the random coils of s protein, which may have a devastating effect on vaccines in the development. as shown in figure 12 , mutations could considerably weaken the binding between the s protein and antibodies and thus pose a direct threat to reduce the efficacy of vaccines. however, there are a few obstacles in determining the exact impacts of mutations to covid-19 vaccines. firstly, the four types of vaccine platforms can produce very different virus peptides, which will result in different immune responses, as well as antibodies. secondly, even for a given vaccine platform, the different peptides may be produced due to different immune responses caused by gender difference, age difference, race difference, etc. therefore, in this work, we proposed to understand the impact of sars-cov-2 mutations on covid-19 vaccines by the statistical analysis. by evaluating the binding affinity changes induced by 28 existing sars-cov-2 antibodies, as shown in figure 9 to figure 11 , we can notice that the k417n, y505c, f456l, and f486l mutations enhance the binding of almost all of the 28 antibodies. in contrast, the r403k, l455f, and p491r mutations have weakened the binding of almost all of the existing antibodies. moreover, mutation k378n enhances the binding of antibody ey6a, whereas mutation v395i weakens the binding of antibody s309. furthermore, it is noticed that many mutations such as k417n, q414r, and g585r considerably disrupt many antibodies and thus may bring a threat to future vaccines. figure 12 depicts the maximal and minimal binding free energy changes for s protein complexes and 28 antibodies or ace2. it can be seen that antibodies cr3022, cr3022 h11-d4, bd-629, bd-604, and bd-362-2 are not very sensitive to the current mutations on the s protein. however, other antibodies, such as cv30 and ey6a, are very sensitive to current mutations. in a nutshell, by setting up a sars-cov-2 antibody library with the statistical analysis based on the mutation-induced binding free energies changes, we can estimate the impacts of sars-cov-2 mutations on covid-19 vaccines, which will provide a way to infer how a specific mutation will pose a threat to vaccines. this approach works better when more antibody structures become available. another important factor in prioritization is mutation frequency. figures 9, 10, and 11 have provided frequency information from our snp calling. once a mutation is identified as a potential threat, it can be incorporated into the next generation of vaccines in a cocktail approach. in principle, all four types of vaccine platforms allow the accommodation of new viral strains. coronavirus disease 2019 (covid-19) pandemic has gone out of control globally. there is no specific medicine and effective treatment for this viral infection at this point. vaccination is widely anticipated to be the endgame for taming the viral rampant. another promising treatment that is relatively easy to develop is antibody therapies. however, both vaccines and antibody therapies are prone to more than 18,000 unique mutations recorded in the mutation tracker. we present a prediction of mutation threats to vaccines and antibody therapies. first, we identify existing mutations on the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) spike (s) protein, which is the man target for both vaccines and antibody therapies. we analyze the mechanism, frequency, and ratio of mutations along with the secondary structures of the s protein. additionally, we build a library of antibodies with structures available from the protein data bank (pdb) and analyze their two-dimensional (2d) and three-dimensional (3d) characteristics by employing computational biophysics. we further predict the mutation-induced binding free energy (bfe) changes of s protein and antibody complexes by a model called topnettree based on deep learning and algebraic topology. from these studies, we infer that some of s protein mutations may disrupt the binding of antibodies and s protein, which will further affect the efficacy and reliability of vaccines. to prioritize mutation threats, we also take into consideration of mutation occurrence frequency. the resulting algorithm indicates that some high-frequency mutations such as m153i, s254f, and s255f with negative bfe changes may potentially disrupt the efficacy and reliability of vaccines and antibody therapies currently in the development. our method can provide the efficient prioritization of mutations to guild the design of the next generation of vaccines and antibody therapies. supporting material is available for: s1 method; s2 multiple sequence alignments of the antibodies and pairwise identity scores; and s3 mutation-induced changes of binding free energies of antibody-sars-cov-2 spike protein complexes. p337s a344t a344s t345s r346k r346t a348s a348t s359n c361s c361s d364y a372v s373l f374l v382l p384s p384l t385a a411s g413r q414e q414k q414p q414r t415s d427y s438f k444r k444n v445i v445f v445a g446s g446v n450k l455f f456l r457k k458q k458n s459f s459y n460t i468f i468v i468t s469p t470n e471q e471g e471d i472v a475v g476s s477g s477t s477i s477n s477r t478a t478k t478i p479s p479l n481h n481d g482s v483f v483a e484k e484q e484d g485s g485r f486l f490l f490s p491r l518i h519q 4 3 genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding covid-19 vaccine development and a 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supported in part by nih grant gm126189, nsf grants dms-1721024, dms-1761320, and iis1900473, michigan economic development corporation, george mason university award pd45722, bristol-myers squibb, and pfizer. the authors thank the ibm tj watson research center, the covid-19 high performance computing consortium, nvidia, and msu hpcc for computational assistance. rw thanks dr. changchuan yin for useful discussion. the authors declare no competing interests.