key: cord-303381-xvzhb7ix authors: tsuruta, yuya; shibutani, shusaku t.; watanabe, rie; iwata, hiroyuki title: the requirement of environmental acidification for ibaraki virus infection to host cells date: 2015-08-28 journal: j vet med sci doi: 10.1292/jvms.15-0222 sha: doc_id: 303381 cord_uid: xvzhb7ix the effect of environmental acidification on ibaraki virus (ibav) infection was tested using endosomal inhibitory chemicals and low ph treatment. treatment of target cells with endosomal inhibitors significantly decreased the progeny virus production. ibav outer capsid proteins, vp5 and vp2, were removed from virion when purified ibav was exposed to low ph environment. further experiment showed that the exposure to low ph buffer facilitated ibav infection when the cellular endosomal pathway was impaired by bafilomycin a1. results obtained in this study suggest that acidic environment is essential to initiate ibav infection. ibaraki virus (ibav) is one of the strains of epizootic hemorrhagic disease virus (ehdv) serotype 2, a member of genus orbivirus in reoviridae family [11, 19] . it is known as a causative agent of ibaraki disease characterized by the paralysis of swallowing muscles. although the effective vaccine is widely used, the recent reemergence of ibaraki disease in combination with the co-circulation with other ehdv raised the alert level for the epidemic of ibaraki disease [13] . in addition, orbivirus family contains many viruses causing severe loss in the livestock industry. therefore, it is important to understand the detailed molecular mechanism of their infection. virus infection is initiated by the release of viral genome to the intracellular compartment of target cells. several mechanisms are known for the genome release to cytoplasm, and one of the most well known mechanisms is the direct fusion mechanism utilized by several enveloped viruses, such as human immunodeficiency virus-1, mouse hepatitis virus and paramyxoviruses [5, 6] . enveloped viruses are also known to use host endosomal pathway to facilitate their infection [12] . reovirus family does not have envelope, and its infection to host cells is believed to occur via host endosomal pathway [2, 12] . once attached to the cell surface, the host endosomal pathway incorporates particles. along with the endosome maturation, the dissociation of outermost capsid protein and proteolytic cleavage on viral protein will occur [3, 4] . especially, the proteolytic cleavage by endosomal proteases, cathepsin b and l, is an essential process for infection [1] . in other words, the expression level of those proteases affects the susceptibility of the host cells to virus infection. in addition to endosomal proteases, extracellular proteases also play an important role to promote certain reovirus infection in vivo. it was shown that tissue specific proteases contribute for the definition of target organ of rotavirus infection [10] . on the other hand, the critical condition to activate orbivirus infection remains unidentified. although it has been suggested that orbivirus also utilizes host endosomal pathway, no involvement of protease was reported [8] . from studies with enveloped viruses, it was shown that viruses utilize endosomal pathway in two different ways. some viruses, such as influenza virus, use low ph environment itself, and other viruses, such as severe acute respiratory syndrome virus, use endosomal proteases rather than low ph environment itself [12, 18] . to clarify which mechanism was used by orbivirus, we tested the effect of environmental ph for orbivirus infectivity using ibav. the utilization of endosomal pathway by ibav for infection was confirmed using endosome inhibitors. hmlu-1 (hamster lung) cells were infected with ibav in the presence of three different endosome inhibitors, bafilomycin a1 (baf a1, sigma, st. louis, mo, u.s.a.), chlorpromazine (cpz, abcam, cambridge, u.k.) and dynasore (wako, osaka, japan). those drugs inhibit clathrin dependent endocytosis in different manners. baf a1 is an antibiotic derived from streptomyces griseus and specifically inhibits vacuolar type h+ atpase [23] . cpz dislocates clathrin, and its adaptor protein from plasma membrane to cytosol [22] and dynasore inhibits gtpase activity of dynamin specifically [14] . all inhibitors work reversibly, and therefore, endosome pathway can restart once the drug was removed from the culture. hmlu-1 cells prepared in 6 well plate were treated with dulbecco's modified eagle's medium (dmem, wako) containing various concentrations of inhibitors for 30 min at 37°c. after cells were chilled on ice for 5 min, the media were removed, and cells were infected with ibav at moi=3 for 1 hr at 4°c. after a wash with pbs (−), cells were further incubated with dmem plus inhibitors for 30 min at 37°c, and the media were replaced with dmem containing 10% fetal bovine serum (10fdmem). cells were incubated for further 24 hr, and culture supernatant was collected. collected supernatant was subjected to plaque assay, and after staining with crystal violet solution (0.1% crystal violet in 10% buffered formalin and 20% methanol), the number of plaque was counted. to analyze the statistical significance of each inhibitor concentration group against mock treated group, statistical software r [16] was used to run dunnet's test [7] . figure 1a shows the number of infectious ibav in the supernatant of the cells treated with baf a1, cpz or dynasore. all inhibitors were shown to decrease virus titer when their concentration was elevated. the most significant decrease was observed when baf a1 concentration was higher than 6.25 nm (p=0.0013 for 6.25 nm against 0 nm). at the same time, the effect of those inhibitors to hmlu-1 viability was tested. hmlu-1 cells in 96 well multiwell plate were treated with media containing various concentrations of inhibitors for 1 hr without ibav infection. after 24 hr incubation, the number of viable cells was quantified using celltiter ® aqueous one solution reagent (promega, madison, wi, u.s.a.). as shown in fig. 1b , no inhibitors showed significant effect on viable cell numbers. these results indicated that ibav utilizes clathrin-dependent endosomal pathway for infection and coincided with the previous research on bluetongue virus entry [8] . to confirm the effect of low ph on virus infectivity, purified ibav was incubated in pbs (−) with several ph (ph=4, 7 or 9) for five min and infected to hmlu-1 at moi=0.01. we employed lower moi to protect cells from severe cpe at early time point and achieve multi cycle infection. dmem without fbs was used for control incubation, and the significance in the difference between control and each test group was analyzed by dunnet's test. as shown in fig. 2a , the promouse antisera raised against vp2 (anti-vp2) or purified ibav particle (anti-ibav) were used as first antibodies. duction of progeny virus was significantly suppressed when ibav was treated with pbs (ph4) compared to control treatment (p<0.001). other conditions did not give any impact on ibav infectivity (ph7 and ph9, p value was 0.993 and 0.906, respectively). the ph of pbs-ibav mix solution was confirmed with ph test paper after 5 min incubation (data not shown). many enveloped viruses lose its infectivity by low ph treatment, if they utilize endosomal acidification to trigger the activation of their protein that is responsible for infection [15, 17, 18] . the result obtained in this study was consistent with these observations suggesting that low ph treatment caused irreversible change on ibav particle that leads to the loss of infectivity. to understand the mechanism of this loss of infectivity, virus particle was collected by ultracentrifugation after low ph treatment. western blot analysis using anti-vp2 antisera and anti-ibav antisera was performed, and vp2, vp5 and vp7 were visualized (fig. 2b) . the approximate molecular weights of vp2, vp5 and vp7 are 115, 60 and 38 kda, respectively. in the case of btv, it was shown that vp2 and vp5 form outer capsid layer and play important roles for the initial step of infection [24] . vp2 is a protein reported as a sialic acid binding protein and implicated to play a role in the attachment to target cells [24] . vp5 is believed to work as a protein which disturbs the lipid bilayer of target cells [9] . as shown in the left panel of fig. 2b, vp2 was detected in the virus treated with pbs (ph7), whereas no vp2 was detected in the virus treated with pbs (ph4). similarly, less vp5 was detected in the virus treated with pbs (ph4) compared to the virus treated with pbs (ph7). the amount of vp7 seemed to be the same between pbs (ph4) and pbs (ph7), and hence, it was suggested that there are the same amounts of viral core in each sample. the result obtained above implicated that low ph treat-ment removes ibav outer capsid proteins from the particle and initiates its infection. figure 3b shows the viability of hmlu-1 after a series of treatment without virus infection, and no significant difference was observed (p=0.983, welch's t-test). results obtained in this approach confirmed the usage of clathrin-dependent endocytosis pathway by orbivirus and support the previous report [8] . in addition, it was proved that the exposure to low ph environment is the essential condition to initiate ibav infection. in the presence of baf a1, the acidification of late endosome, as well as its fusion to lysosome is impaired [20, 21] . although the cell surface viruses could be internalized during the post-treatment, they cannot be exposed to low ph environment in the presence of baf a1. however, if viruses were exposed to low ph at the cell surface before internalization, they acquired the infectivity and could start infection under the effect of baf a1. pbs (ph4) failed to enhance ibav infection under the influence of other two inhibitors. since those two inhibitors interrupt endosome pathway at the very beginning, no ibav could be incorporated during post-treatment. this might make both ibav groups to start infection at the same time and resulted in the same viral titer. since we could not deny an involvement of any proteases, additional research will be required to reveal the entire molecular mechanism of orbivirus infection. the importance of low ph environment for ibav activation that we proved in this study will help to design experimental approach for further studies. proteolytic disassembly is a critical determinant for reovirus oncolysis rotavirus cell entry intraluminal proteolytic activation plays an important role in replication of type 1 reovirus in the intestines of neonatal mice proteolytic digestion of reovirus in the intestinal lumens of neonatal mice the structural biology of type i viral membrane fusion virus entry: molecular mechanisms and biomedical applications a multiple comparison procedure for comparing several treatments with a control bluetongue virus entry into cells a capsid protein of nonenveloped bluetongue virus exhibits membrane fusion activity addition of exogenous protease facilitates reovirus infection in many restrictive cells the orbivirus genus. diversity, structure, replication and phylogenetic relationships ready, set, fuse! the coronavirus spike protein and acquisition of fusion competence reemergence of ibaraki disease in southern japan in 2013 dynasore, a cell-permeable inhibitor of dynamin fusion of enveloped viruses with cells and liposomes. activity and inactivation r: a language and environment for statistical computing acid-induced membrane fusion by the hemagglutinin protein and its role in influenza virus biology effects of low ph on influenza virus. activation and inactivation of the membrane fusion capacity of the hemagglutinin double-stranded rna of ibaraki virus inhibition of the vacuolar h(+)-atpase with bafilomycin reduces delivery of internalized molecules from mature multivesicular endosomes to lysosomes in hep-2 cells transport from late endosomes to lysosomes, but not sorting of integral membrane proteins in endosomes, depends on the vacuolar proton pump the use of inhibitors to study endocytic pathways of gene carriers: optimization and pitfalls metabolic products of microorganisms. 224. bafilomycins, a new group of macrolide antibiotics. production, isolation, chemical structure and biological activity bluetongue virus coat protein vp2 contains sialic acid-binding domains, and vp5 resembles enveloped virus fusion proteins acknowledgments. we thank members in the laboratory of veterinary hygiene and dr. brendan t. higgins for helpful comments and critical reading of the manuscript. this research was supported in part by research aid from yamaguchi university foundation and yamaguchi university fellowships for young researchers. key: cord-265740-wjdeps3h authors: radbel, jared; jagpal, sugeet; roy, jason; brooks, andrew; tischfield, jay; sheldon, michael; bixby, christian; witt, dana; gennaro, maria laura; horton, daniel b.; barrett, emily s.; carson, jeffrey l.; panettieri, reynold a.; blaser, martin j. title: detection of sars-cov-2 is comparable in clinical samples preserved in saline or viral transport media date: 2020-05-13 journal: j mol diagn doi: 10.1016/j.jmoldx.2020.04.209 sha: doc_id: 265740 cord_uid: wjdeps3h as the covid-19 pandemic sweeps across the world, the availability of viral transport media (vtm) has become severely limited, contributing to delays in diagnosis and rationing of diagnostic testing. given that sars-cov-2 viral rna has demonstrated stability, we posited that phosphate buffered saline (pbs) may be a viable transport medium, as an alternative to vtm), for clinical qpcr testing. we assessed the intraand inter-individual reliability of sars-cov-2 qpcr in clinical endotracheal secretion samples transported in vtm or pbs, evaluating the stability of the rt-qpcr signal for three viral targets (n gene, orf1ab, and s gene) when samples were stored in these media at room temperature for up to 18 hours. we report that using pbs as a transport medium has high intra-and inter-individual reliability, maintains viral stability, and is comparable to vtm in the detection of the three sars-cov-2 genes through 18 hours of storage. our study establishes pbs as a clinically useful medium that can be readily deployed for transporting and short-term preservation of specimens containing sars-cov-2. use of pbs as a transport medium has the potential to increase testing capacity for sars-cov-2, aiding more widespread screening and early diagnosis of covid-19. in december 2019, a novel coronavirus was recognized as causing a cluster of pneumonia cases in wuhan, china (1) . the infectious agent, an rna virus, was termed acute respiratory syndrome coronavirus 2 (sars-cov-2), and was identified as the cause of covid-19, a clinical syndrome manifested by an influenza-like illness that can progress to acute lung injury or acute respiratory distress syndrome (ards) with substantial mortality (2) . covid-19 has affected more than 3.1 million people causing more than 220,000 deaths worldwide (https://coronavirus.jhu.edu/map.html, last accessed april 29, 2020). sars-cov-2 detection using standard testing of upper airway secretions requires a nasopharyngeal (np) or oropharyngeal (op) swab that is transported to a clinical laboratory using viral transport media (vtm) (https://www.fda.gov/medical-devices/emergency-situations-medical-devices/faqs-diagnostic-testingsars-cov-2#offeringtests, last accessed april 29 2020). recently, op and saliva testing using vtm also were shown to be comparable to np swabs for detection of the virus (3, 4) . as the covid-19 pandemic has swept across the world, availability of vtm has become severely limited, impairing local and regional capacity for diagnosis. since sars-cov2 has capped rna with a 5′ gtp resembling host rna and that the virus sgrna manifests remarkable stability (5), we posited that qpcr detection of sars-cov-2 in specimens preserved in phosphate buffered saline (pbs), which is readily available, would be comparable to those in vtm. here, we report that sample preservation in pbs or vtm are comparably effective for the preservation of sars-cov-2 in endotracheal secretions. source materials. for transport media, we followed procedures outlined in standard references (5) (6) (7) (8) . we used phosphate buffered saline (pbs), a water-based salt solution containing disodium hydrogen phosphate, sodium chloride, potassium chloride and potassium dihydrogen phosphate, ph 7.2 (sigma aldrich, saint louis mo). viral transport media (vtm) was derived according to the centers for disease control and prevention (cdc) coronavirus outbreak response (https://www.cdc.gov/coronavirus/2019ncov/downloads/viral-transport-medium.pdf, last accessed april 29, 2020). in brief, a solution with hanks balanced salt solution (hbss), heat-inactivated fetal bovine serum (final concentration 2%), gentamicin 100µg /ml and amphotericin b 0.5 µg /ml was prepared and aliquoted into 2 ml screw top vials (6) . tubes then were stored at 4°c until use. experimental protocols: respiratory secretions from 16 confirmed covid-19 positive subjects were collected over a four-day period from an intensive care unit at robert wood johnson university hospital in new brunswick nj, according to a protocol approved by the rutgers irb (protocol # pro2020000800). all subjects were mechanically ventilated for acute hypoxemic respiratory failure due to confirmed covid-19 pneumonia. specimens were collected into a sterile container via closed circuit, in-line catheter suction of respiratory secretions from the endotracheal tube (et), as part of routine clinical care. swabs were then dropped into vials containing pbs or vtm and transported to the rucdr laboratory for analysis. to test intra-and inter-patient variation in efficacy of detecting sars-cov2 from et-derived samples, three experimental procedures were performed. eight samples from two subjects (four from each subject) were harvested at the same time and transported in either vtm or pbs. samples were processed immediately (at 0 hours) or after 2 hours at room temperature (rt). real time-qpcr (rt-qpcr) was performed on 1 sample from each transport medium at each incubation time (0 or 2 hours) and ct values for the sars-cov-2 nucleocapsid (n), open reading frame 1ab orf1ab, and spike protein (s) genes were compared; bacteriophage ms2 (ms2) spiked into the samples was used as a positive control. in a parallel experiment, the stability of detection of sars-cov2 sgrna by rt-qpcr in samples transported in pbs and vtm also was examined after remaining at room temperature for time points ranging from 0 to 18 hours. these experiments mimicked field conditions, in which specimens remain in transport at rt for periods up to 18 hours. twenty samples from each of two subjects were kept for 0, 2, 4, 6 h or overnight at rt in vtm or pbs to mimic these real-world transport condition. ct values were again compared for the rt-qpcrs for the three viral genes described above across the indicated time points. to examine inter-subject variance, we examined samples from an additional 12 patients whose et secretions were transported in either pbs or vtm; rt-qpcr was performed immediately on arrival in the lab for these 24 samples, and ct values were again compared between those transported in vtm or pbs. statistical analysis. pearson correlation was used to quantify association between repeated samples within subject, between transport media, and between the three genes. linear mixed effects models were used to address several of our research questions. these models were fitted separately for each of the 3 genes. the outcomes were the ct values, while the predictors included the transport media (vtm versus pbs) and hours in storage (0, 2h, 4h, 6h, or 18h). the models included a random intercept for each subject, to account for repeated observations (within-subject correlation). we used lme4 and ggplot2 packages in r for the linear mixed effects models and plots, respectively (https://www.rproject.org) (9) . analysis of repeated samples from the same subject. from two unique subjects, we obtained paired identical swab specimens that were transported in pbs and vtm. a total of 39 samples were analyzed, representing 10 for each subject in pbs and 9 in vtm, and the variation in ct plotted (figure 1) . as next, we used a linear mixed effects model (see methods) to compare the sensitivity of the two transport media in the timed samples, from 0 h to overnight (18h) storage. for all three genes, there were no significant differences, and in each case, the vtm values trended higher than for pbs. we next evaluated if across individual samples, the values obtained in testing one gene correlated with the results for the other genes. additionally, we tested whether the choice of transport medium made a difference. in total, for the 13 samples transported in vtm, the pair-wise correlations between the three genes ranged from 0.947 to 0.956. for the 14 samples transported in pbs, the correlations ranged from 0.963 to 0.991. thus, the results for the three genes were highly correlated independent of transport medium type. decay of the viral signal over time for specimens transported in the two media. we next considered whether there was decay in viral signal over time, and whether it differed according to the transport medium used. from the prior analyses, the coefficient of hours of storage was estimated to be negative in the models of all three genes tested (approximately -0.03 for each gene) and was not significantly different from zero (all p-values >0.5). these data indicate that storage at room temperature for up to 18 hours had little effect on the values detected in the rt-qpcrs for the three sars-cov-2 genes tested. however, these models did not include an interaction term for the transport medium used for storage and time, and there could be differences between the media. to assess this possibility, we performed the same analyses as above, but included interactions between hours of storage and transport medium used. for each of the three genes tested, both the main effect of time and the interaction between time and the transport medium were not statistically significant (all p-values >0.5) and estimates were close to 0. therefore, we did not detect decay in the viral signal or differential decay by transport medium over the time interval studied. during pandemics, molecular diagnostics are crucial to obtaining accurate and timely data to influence public health policy decisions in real time (10) . however, mounting demand for testing has caused a depletion of the viral transport media needed to perform sars-cov-2 pcr testing (11) . thus, in the midst of the sars-cov-2 pandemic, the fda has allowed laboratories to consider testing alternative transport media (https://www.fda.gov/medical-devices/emergency-situations-medical-devices/faqsdiagnostic-testing-sars-cov-2, last accessed april 29, 2020). our experiments using clinical samples demonstrate the efficacy of pbs as a transport medium and its applicability to clinically relevant conditions, such as overnight storage at room temperature. first, we determined that sars-cov-2 qpcr detection with pbs as a transport medium has high intra-patient reliability. next, using pbs for transport, we demonstrated strong inter-patient reliability of sars-cov-2 qpcr. we also found strong correlation of ct values from specimens transported in either pbs or vtm across multiple subjects with unknown viral loads. these results establish pbs as a dependable transport medium for use with clinical samples. our data are consistent with the recent demonstration that pbs is equivalent to vtm when each medium is spiked with known quantities of sars-cov-2 (12) . with little decay in signal over storage times up to 18h, pbs also has utility for laboratories that test for several sars-cov-2 genes that have different specimen processing times. testing can focus on any or all of the four sars-cov-2 structural proteins, including the spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins, or on any of their protein domains (13) . that results for all three viral genes tested were strongly correlated across samples from multiple subjects, support the robustness of the entire testing pathway, including transport. furthermore, delays in getting samples to the testing lab often occur in busy clinical settings (11) . as such, our findings that pbs acts as a stable storage medium with lack of significant viral decay for up to 18 h at room temperature prior to qpcr is advantageous. our study is limited in that we used tracheal secretions from mechanically ventilated patients, and we do not know the extent to which our results can be extended to np, op swabs and/or saliva testing. given the severity of illness in our subjects, they may have had higher viral loads than patients with milder disease in whom increased sensitivity of detection may be needed. however, the stability of the signal, with minimal changes in intensity for 18 hours, indicates the robustness of the methodology. the stability of sars-cov-2 in the environment (5) , which contributes to its widespread dissemination, may diminish the need for rapid transport of clinical specimens. the extent to which clinical laboratories can respond to the covid-19 pandemic is tied to the ability to develop and deploy proper diagnostic procedures (10) . early sars-cov-2 detection allows prompt treatment of infected patients and rapid implementation of control measures to limit viral transmission (14) . expanded testing capacity would also facilitate more widespread surveillance and containment of infectious transmission in communities, which could support policies to relax restrictions in work, travel, and social distancing. our study establishes pbs as a clinically useful transport medium with the potential to increase viral detection capacity, thus improving clinical care and surveillance efforts. the novel coronavirus originating in wuhan, china: challenges for global health governance return of the coronavirus: 2019-ncov. viruses, 2020, 12 pii: e135 consistent detection of 2019 novel coronavirus in saliva virological assessment of hospitalized patients with covid-2019 aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 concepts of clinical diagnostic virology transport of viral specimens centers for, disease control and prevention, & national institutes of, health. biosafety in microbiological and biomedical laboratories government printing office r: a language and environment for statistical computing. r foundation for statistical computing the role of community molecular diagnostics laboratories in the h1n1 pandemic sars-cov-2 testing evaluation of saline, phosphate buffered saline and minimum essential medium as potential alternatives to viral transport media for sars-cov-2 testing characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine diagnosing covid-19: the disease and tools for detection strom, and helmut zarbl. we also wish to thank marten/ups and dfyoung for in-kind logistics support.finally, we thank our courageous colleagues serving on the frontlines in the clinic. key: cord-259094-5qzbctan authors: xu, mei ling; kim, hyoung jin; chang, don yong; kim, hong-jin title: the effect of dietary intake of the acidic protein fraction of bovine colostrum on influenza a (h1n1) virus infection date: 2013-04-26 journal: j microbiol doi: 10.1007/s12275-013-2683-y sha: doc_id: 259094 cord_uid: 5qzbctan acidic protein levels in the milk decrease markedly as lactation progresses, suggesting that it is an important part of the colostrum. however, little attention has been paid to their biological function. in this study, we isolated the acidic protein fraction of bovine colostrum (afc, isoelectric point <5) by anion-exchange chromatography, and investigated the effect of its dietary intake on influenza a (h1n1) virus infection. 100% of mice infected with 1 ld(50) of the virus survived when administered afc for 14 days prior to infection, compared with 33% survival when administered phosphate buffered saline (pbs). moreover, consumption of afc reduced the weight loss associated with infection. we propose that dietary intake of afc has a prophylactic effect on influenza a virus infection. electronic supplementary material: supplementary material is available for this article at 10.1007/s12275-013-2683-y and is accessible for authorized users. breast milk delivers not only immunologic components that protect the infant against pathogen invasion, but also nutritional factors that promote normal organ development (wang and brand-miller, 2003) . colostrum is the natural food produced by female mammals during the first 24-72 h after giving birth (robison et al., 1988) . it contains concentrated nutrients, antibodies, cytokines, and growth factors (playford et al., 2000; jouan et al., 2006) , providing immediate immune protection to the newborns (asakuma et al., 2007; benson et al., 2012) . influenza is a single-stranded rna orthomyxovirus, subdivided into influenza a, b and c (gatherer, 2009) . the h1n1 and h3n2 subtypes of influenza a and influenza b are widely recognized as causes of seasonal flu (goldstein et al., 2011) . the virus can evade humoral immunity by antigenic drift, i.e. changes in the amino acid sequence of the surface hemag-glutinin and/or neuraminidase (webster et al., 1982; sandbulte et al., 2011) , which is why influenza can cause epidemics annually. although in most cases symptoms resolve within a week without the need for treatment, in high-risk groups influenza can lead to severe illness or death (world health organization, 2009 ). in the united states, the influenza-attributable mortality rate is estimated at 1.4-16.7 deaths per 100,000 cases, which translates to between 3,339 and 48,614 deaths annually (centers for disease control and prevention, 2010) . influenza is a serious public health and economic issue. in developed countries especially, epidemics can result in high levels of worker absenteeism and productivity losses (world health organization, 2009 ). therefore, much effort has been put into preventing infections. it is known that the oligosaccharide contents of bovine colostrum are different from those of human colostrum. 6sialyllactosamine and 3-sialyllactose are the most abundant in bovine colostrum while 3-sialyl-3-fucosyllactose and sialyllacto-n-tetraoses are in human colostrum (martin-sosa et al., 2003) . although there are differences in the oligosaccharide contents, both colostrums have been regarded to have biological functions to modulate diseases. previous reports have shown that human colostrum can modulate the immune system and enhance protection against pathogens (claud et al., 2003; wang and brand-miller, 2003) . dietary intake of bovine colostrum has also been suggested to prevent upper respiratory tract (urt) infections (brinkworth and buckley, 2003) . however, there has been no direct evidence that dietary bovine colostrum enhances protection against influenza infection. the amount of acidic proteins and sialic acid in milk is known to decrease significantly as lactation progresses (bezkorovainy, 1965; wang and brand-miller, 2003) , therefore, the acidic protein fraction is thought to have an important function in colostrum. in this study we isolated the acidic protein fraction of bovine colostrum (afc) and investigated how dietary intake of afc modulates the symptoms of influenza infection. bovine colostrum powder was provided by ildong foodis (korea). deae-sepharose cl-6b was purchased from ge healthcare (usa). a power pac 3000 power supply and electrophoresis kit (both from bio-rad, usa) were used for sds-polyacrylamide gel electrophoresis (sds-page, mini protean ® ii, bio-rad). the protein fraction separated by sds-page was visualized by silver staining (ge healthcare). the three mouse groups were given oseltamivir, pbs and afc orally for 14 days prior to infection with influenza a virus. after the virus challenge, the mice continued to receive oseltamivir, pbs and afc for 7, 3 and 3 days, respectively. they were then infected with 1 ld50 or 0.2 ld50 of the virus. survival rates and body weight changes were monitored for 14 days after infection. influenza a/pr/8/34 virus (h1n1) was kindly donated by prof. man-seong park (hallym university, korea). zoletil 50 (virbac, france) and rompun (bayer animal health, germany) were used to anesthetize the mice. phosphate buffered saline (pbs) and oseltamivir (roche, switzerland) were used as negative and positive controls in mouse experiments, respectively. a 40 g sample of bovine colostrum powder was dissolved in binding buffer (2,000 ml of pbs containing 0.025% tween 80, ph 7.0, final nacl concentration was adjusted to 0.13 m). this starting sample was dialyzed against 10 l of binding buffer at 4°c for 4 h. the dialysate was centrifuged for 10 min at 14,000×g, to obtain the supernatant (i.e. the loading sample). anion exchange chromatography was performed in batch mode. the loading sample was mixed with 40 ml deae-sepharose cl-6b resin equilibrated with binding buffer in a 2 l bottle, and the mixture was left at 4°c for 72 h. the unbound fraction was removed by centrifugation at 84×g for 5 min. the deae resin was centrifuged at 84×g for 5 min with 200 ml of the binding buffer. the afc fraction was isolated with elution buffer (to make elution buffer nacl was added to the binding buffer, final nacl concentration of elution buffer was adjusted to 0.652 m). the protein content of the starting sample, the loading sample and afc were analyzed as described below. the proteins in each sample were separated on 12% sds-page gels as described by laemmli (1970) and visualized by silver staining. protein concentration was determined by the bradford protein concentration assay (bio-rad). twodimensional electrophoresis (2de) of 800 μg protein samples was performed by genomine inc. (korea), and the separated proteins were visualized by coomassie blue staining. influenza a virus was propagated in 11-day old fertilized chicken eggs for 48 h at 37°c. egg allantoic fluid was clarified by centrifugation at 682×g for 10 min and filtered using a 0.22 μm syringe filter. the 50% lethal dose (ld50) of the virus was determined as described (reed and muench, 1938) . five-week-old female balb/c mice purchased from orient bio (korea) were divided into three groups (pbs, oseltamivir and afc) of five to six mice each. the dosing schedule for each group is shown in fig. 1 . the mice received pbs (200 μl/day) or afc (0.5 mg/kg/day, 0.5 mg indicates protein amount) orally for 14 days before infection. the protein concentration of afc was determined by bradford protein assay using commercial protein assay kit (bio-rad). they were anesthetized intraperitoneally with a 4:1 mixture of zoletil 50 and rompun and infected intranasally with 1 ld50 or 0.2 ld 50 of the influenza a (h1n1) virus. following infection, the pbs and afc groups continued to receive pbs (200 μl/day) and afc (0.5 mg/kg/day), respectively, for 3 days, and the oseltamivir group continued to receive oseltamivir (10 mg/kg/day) for 7 days. survival rates and changes in body weight were monitored for 14 days after infection. student's t-test was used to evaluate differences between the groups. p-values <0.05, or <0.01 in two-tailed tests, were considered statistically significant. as shown in fig. 2 , the starting sample and the loading sample were both found to contain three major protein bands with molecular masses of 52-76, 24-31, and 5-17 kda. there was no significant difference between the two samples in terms of protein composition. it has been suggested that bovine colostrum is composed of 50-75, 25-35, and 5-20 kda proteins (senda et al., 2011) , and our results were consistent with this finding. the 24-31 kda protein band was the most prominent in afc, the largest proportion of which was the 28 kda fraction (fig. 2) . we also compared the isoelectric points (pis) of the proteins in the starting sample and in afc (fig. 3) . most of the proteins in afc had pis <5 (fig. 3b) , while the pis of the proteins in the starting sample ranged from 4 to 10 (fig. 3a) , suggesting that afc contains mainly acidic proteins. whereas the survival rates in both the oseltamivir and afc groups were 100%, in the pbs group, only 33% of the animals challenged with 1 ld50 of the virus survived (fig. 4a ). at the same time, oral administration of afc significantly reduced body weight loss in the animals receiving 1 ld50 of the virus (fig. 4b) . we also compared the changes in body weight in the three groups infected with 0.2 ld 50 . as shown in fig. 5 , the mice in the oseltamivir and afc groups experienced no change in body weight, while significant weight loss was observed in the pbs group in days 4 to 7 after infection. these results demonstrate that dietary intake of afc can ameliorate the symptoms associated with influenza. picornaviruses, coronaviruses, adenoviruses, parainfluenza viruses and influenza viruses are the causal pathogens in the vast majority of urt infections (fendrick et al., 2003) . dietary supplementation with concentrated bovine colostrum proteins has been shown to reduce the incidence of symptoms of urt infection in adult men (brinkworth and buckley, 2003) . recently, patiroglu and kondolot also reported that oral administration of bovine colostrum lessened the severity of viral urt infections in iga-deficient children (patiroglu and kondolot, 2011) . based on these findings, we hypothesized that bovine colostrum would improve the symptoms associated with influenza virus. we performed a large number of animal experiments investigating the effect of total bovine colostrum, and found that while some experiments yielded significant reductions in influenza-related morbidity and mortality, the symptom-ameliorating effects of the same bovine colostrum products varied significantly (supplementary data fig. s1 ). therefore, the effect of total bovine colostrums on influenza virus infection in mice was significantly different from our expectation. on the other hand, afc showed promising effect for ameliorating symptoms caused by influenza infections (figs. 4 and 5) . previous studies implied that the intestinal conditions, i.e. bacterial flora and digestive enzymes involved in the absorption of colostrum components differed between human and animal subjects (petschow and talbott, 1994; johnson et al., 2007; kobayashi et al., 2012) . in other words, some components contained in bovine colostrum decrease the ameliorating effect or hinder the functions of novel compenents. we suggest that separations of novel components such as afc from the hindering components are thought to be critical in mouse experiment. we found that afc contains significantly higher level of proteins harboring α2,3-linked sialic acids than total bovine colostrums and acid protein fraction of mature bovine milk does (supplementary data fig. s2) , indicating that the anionexchange chromatography separates sialic acid rich fraction from the bovine colostrum. the sialic acid is known to inhibit influenza virus-mediated haemagglutination and infection (matrosovich and klenk, 2003) . in addition, α2,3linked sialic acid is a cell receptor for influenza a virus infection (glaser et al., 2005) . therefore, it is assumed that other components of afc such as free sialyl oligosaccharides may be beneficial on ameliorating the symptom caused by influenza virus infection. the composition and quality of pooled colostrum is known to depend on a number of factors, such as the health of the cows, the timing of colostrum collection, and the manufacturing process (kelly, 2003) . however, it is uncertain what amount of each component is required for its biological activity, and what specific components are biologically active in specific diseases, and this has hindered the standardization of bovine colostrums (struff and sprotte, 2007) . therefore, we believe that separating novel factors from colostrum, and collecting more data on the composition of colostrum and the activity of its components are critical for obtaining high-quality colostrum products. in this study, we provide the first evidence that orally supplemented afc is effective in reducing the symptoms associated with influenza a virus infection. further study of the mechanisms through which afc modulates these symptoms will provide new insight into why colostrum components are necessary for newborns. sialyl oligosaccharides of human colostrum: changes in concentration during the first three days of lactation a novel extract from bovine colostrum whey supports anti-bacterial and anti-viral innate immune functions in vitro and in vivo: i. enhanced immune activity in vitro translates to improved microbial clearance in animal infection models comparative study of the acid glycoproteins isolated from bovine serum, colostrum, and milk whey concentrated bovine colostrum protein supplementation reduces the incidence of selfreported symptoms of upper respiratory tract infection in adult males estimates of deaths associated with seasonal influenza -united states modulation of human intestinal epithelial cell il-8 secretion by human milk factors the economic burden of non-influenza-related viral respiratory tract infection in the united states the 2009 h1n1 influenza outbreak in its historical context a single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity predicting the epidemic sizes of influenza a/h1n1, a/h3n2, and b: a statistical method effects of feeding heat-treated colostrum on passive transfer of immune and nutritional parameters in neonatal dairy calves hormones in bovine milk and milk products: a survey bovine colostrums: a review of clinical uses species differences in tissue distribution and enzyme activities of arylacetamide deacetylase in human, rat, and mouse cleavage of structural proteins during the assembly of the head of bacteriophage t4 sialyloligosaccharides in human and bovine milk and in infant formulas: variations with the progression of lactation natural and synthetic sialic acid-containing inhibitors of influenza virus receptor binding the effect of bovine colostrum on viral upper respiratory tract infections in children with immunoglobulin a deficiency reduction in virus-neutralizing activity of a bovine colostrum immunoglobulin concentrate by gastric acid and digestive enzymes colostrum and milk-derived peptide growth factors for the treatment of gastrointestinal disorders a simple method of estimating fifty percent endpoints effects of passiveimmunity on growth and survival in the dairy heifer discordant antigenic drift of neuraminidase and hemagglutinin in h1n1 and h3n2 influenza viruses changes in the bovine whey proteome during the early lactation period bovine colostrum as a biologic in clinical medicine: a review. part i: biotechnological standards, pharmacodynamic and pharmacokinetic characteristics and principles of treatment the role and potential of sialic acid in human nutrition molecular mechanisms of variation in influenza viruses world health organization we thank prof. man-seong park (hallym university, korea) for providing influenza a virus. we thank ildong foodis (ildong foodis co. ltd., south korea) for providing colostrums. key: cord-267736-rya9w6sh authors: kang, xiaoping; li, yuchang; fan, li; lin, fang; wei, jingjing; zhu, xiaolei; hu, yi; li, jing; chang, guohui; zhu, qingyu; liu, hong; yang, yinhui title: development of an elisa-array for simultaneous detection of five encephalitis viruses date: 2012-02-27 journal: virol j doi: 10.1186/1743-422x-9-56 sha: doc_id: 267736 cord_uid: rya9w6sh japanese encephalitis virus(jev), tick-borne encephalitis virus(tbev), and eastern equine encephalitis virus (eeev) can cause symptoms of encephalitis. establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. currently, there are still no multiple antigen detection methods available clinically. an elisa-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. an elisa-array method for the simultaneous detection of five encephalitis viruses was developed in this study. seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the elisa-array. the elisa-array assay is based on a "sandwich" elisa format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. the developed elisa-array proved to have similar specificity and higher sensitivity compared with the conventional elisas. this method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. the results demonstrated that the developed elisa-array is sensitive and easy to use, which would have potential for clinical use. japanese encephalitis virus(jev), tick-borne encephalitis virus(tbev), eastern equine encephalitis virus (eeev), sindbis virus(sv), and dengue virus(dv) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [1] . establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. currently, elisa and ifa are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [2, 3] . there are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [4] [5] [6] . however, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [7] [8] [9] [10] . liew [11] validated one multiplex elisa for the detection of 9 antigens; anderson [12] used microarray elisa for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that elisa-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. however, the application of elisa-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. in this study, we developed an elisa-based array for the simultaneous detection of five encephalitis viruses. seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an elisa-array assay. the assay was validated using cultured viruses and inoculated chicken eggs with patient sera. the results demonstrated that this method combined the advantage of elisa and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus. monoclonal antibodies were prepared from hybridoma cell lines constructed by prof. zhu et al. purification was conducted by immunoaffinity chromatography on protein g affinity sepharose [13] . specific monoclonal antibodies (4d5 against jev, 2b5 against tbev, 1f1 against sv, 2b8 against serotype 2 dv, 4f9 against serotype 4 dv, 4e11 against eeev, and 2a10 against flavivirus) were selected for this study. all of the antibodies were raised according to standard procedures. using 4d5, 2b5, 1f1, 2b8, 4f9, and 4e11 as capture antibodies, detection antibodies (2a10, 1 f1, and 4e11) were coupled to biotin-nhs ester(pierce, germany) at 4°c for 3 h according to the manufacturer's instructions. unincorporated biotin was removed by desalt spin column (pierce). immunologic reactions were reported by streptavidin-hrp (cwbio, beijing, china) and super signal elisa femto maximum sensitive substrate. purified goat-anti mouse antibody was used as a positive control. jev and dv were cultured in c6/36 cells; sv, tbev, and eeev were cultured in bhk-21 cells. the culture of tbev and eeev was conducted in biosafety level 3 facility, however, jev, dv and sv were conducted in biosafety level 2 facility. viral titers were determined by the 50% tissue culture infectious dose (tcid 50 ) method. all the cultures were inactivated by 0.025% β-propionolactone at 4°c overnight, then 37°c for 1 h to decompose β-propionolactone. antibodies were spotted using a biodot machine (bd6000;california, usa) on elisa plates (30 nl/dot). the plates were blocked with 3% bsa-pbs in 37°c for 1 h, followed by washing 3 times with pbs containing 0.1% tween-20 for 2 min each. then, the plates were dried, sealed, and stored at 4°c before use [11] . when spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the elisa-array assay. the optimization was evaluated by dot morphology and signal intensity. the tested spotting buffers included 1 × phosphate buffer saline (pbs), pbs +20% glycerol, and 1 × pbs + 20% glycerol+0.004% triton-x100. a range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared. following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, hpr-labeled avidin, and substrate, followed by signal evaluation. antigen binding was performed in pbs(containing 0.1% tween-20 and 5% fcs) at 37°c for 2 h, followed by washing 3 times(1 × pbs containing 0.1% tween-20). incubation of elisa plates with biotinylated detecting antibody cocktails was performed in pbs (containing 0.1% tween-20 and 5% fcs) at 37°c for 2 h. after washing, specific binding of the detecting antibodies was reported by streptavidin-hrp and stained with super signal elisa femto maximum sensitive substrate (thermo scientific, rockford, usa) [11, 14, 15] . visualization of the plate was performed in ae 1000 cool ccd image analyzer(beijing bgi gbi biotech company., ltd, china). the signal intensity and background of each spot was read out and recorded with "monster"software. the positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2. the identical antibodies used in the elisa-array format were also tested in a conventional elisa format to determine the difference in sensitivity and specificity of the two methods. the conventional elisas were performed at the same time as the elisa-array assays to ensure similar reaction conditions. the conventional elisas were performed in an identical maner to the elisa-array, except that antibodies were coated at a concentration of 2 μg/ml in pbs (ph 7.4), and substrate tmb was used instead of super signal elisa femto maximum sensitive substrate [16, 17] . three serum samples were collected from patients with nervous system symptoms and histories of tick bites. the serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions (one for inactivation and one for rna extraction). the rna and inactivated samples were stored at -70°c before use. rna was extracted from the inoculated chicken eggs using a rneasy mini kit (qiagen inc., valencia, ca, usa) according to the manufacturer's instructions. all rna extraction procedures were conducted at bsl-3 facilities. the primers and probes were used as previously described [18] . the real-time rt-pcr was conducted with a quti-teck q-rt-pcr kit (qiagen inc,). the reaction consisted of 10 μl of 2 × reaction buffer (0.2 μl reverse transcription enzyme, and 250 nmol/l primers and probes). rna and deionized water were added to a final volume of 20 μl. pcr was performed with a lightcycler 2.0 (roche, switzerland) [19] . optimization of the elisa-array assay the spotted array layout is depicted in figure 1 and the efficacy of three different spotting buffers on the quality of the printed elisa-arrays were investigated by spot morphology observation and signal intensity comparison. the spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml (each was serially diluted 2-fold). the efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. figure 1 illustrates the array layout and figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2b5 by tbe virus culture detection. cross reaction detection was also conducted by applying jev, yf, and dv cultures. spot morphology observation (figures 2a, b , and 2c) demonstrated that spotting buffer containing pbs with 20% glycerol produced tailed spot morphology; buffers containing pbs alone and pbs with 20% glycerol +0.004% triton-x100 gave good spot morphology (round and full). buffers containing pbs with 20% glycerol and pbs with 20% glycerol+0.004% triton-x100 produced higher signal intensities than pbs alone. thus, pbs with 20% glycerol+0.004% triton-x100 was adopted as the optimized spotting buffer for subsequent experiments. simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. at this concentration, the signal intensity was higher and the cross-reaction did not appear (figure 2d ). consequently, spotting concentration optimization of other capture antibodies (4d5, 1f1, 4e11, and 2b8) demonstrated that 0.05 mg/ml was also suitable(data not shown). the optimized elisa array layout is shown in figure 3 , which was applied in the following experiments. successful detection of viral pathogens requires a test with high sensitivity and specificity. to evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. by testing serially-diluted viral cultures, including dv-2, dv-4, jev, tbe, sv, and eeev, the sensitivity of elisaarray and the identical conventional elisa were compared ( table 1 ). the detection limit of the two methods was compared and demonstrated. the cross-reactivity test was conducted using bhk-21 and vero cell lysate, yellow fever virus (yfv) cultures (5 × 10 5 tcid 50 /ml, west nile virus(wnv) cultures(2 × 10 6 tcid 50 /ml), and western equine encephalitis virus(1 × 10 7 tcid 50 /ml). the results demonstrated that neither the elisa-array nor traditional elisa displayed cross-reactivity. equal volumes of cultured tbev, jev, dv-2, dv-4, sv, and eeev were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. a cocktail of biotin conjugated antibody (2a10, 4e11, and 1f1) was used in all tests. the results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (figures 4 and 5) . chicken eggs inoculated with infected human serum were used for validation of the elisa-array assay. all samples showed high reaction signals with capture antibody 2b5, which was specific for tbev ( figure 6b ). the elisa-array assay suggested that the three patients were all infected with tbev. to verify the results tested by elisa-array, rna extracted from chicken eggs was applied to a real time-rt-pcr assay using primers and probes targeting tbev. the results were also positive (figure 6a) . the consensus detection results confirmed that the elisaarray assay was reliable. to be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] . multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. thus, an elisa-array, which combines the advantages of elisa and protein array, meets the above requirements. it has been reported that an elisa-array has been used in the diagnosis of cancer and auto-allergic disease [7, 12] ; however, no study has reported the detection of viral pathogens. in this study, we developed a multiplex elisa-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. the production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [14] . cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. first, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. then, the antibodies were screened by an elisa-array with a double-antibody sandwich elisa format. the antibodies which produced cross-reactivity and low-positive signals were excluded. finally, six antibodies were selected as capture antibodies. another monoclonal antibody, 2a10, which could specifically react with all viruses in the genus flavivirus was used for detecting antibody against dv, jev, and tbev. for the detection of eeev and sv, although the detecting and trapping antibodies were the same (1f1 and 4e11, respectively), the antibodies produced excellent positive signals. the epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. as one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. in the elisa-array assay, this problem exists as well. because of the limitation of available antibodies, this assay could only detect 5 pathogens. in the future, with increasing numbers of suitable antibodies, especially specific antibodies against flavivirus, this elisaarray might be able to test more pathogens and be of greater potential use. to make the assay more amenable to multiple virus detection, the assay protocol was optimized. in addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. heat inactivation was performed by heating the viral cultures at 56°c for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. thus, β-propiolactone treatment was chosen as the virus-inactivation method. a conventional elisa is a standard method in many diagnostic laboratories. we compared the elisa-array with a conventional elisa and confirmed that the advantage of the elisa-array was evident with comparable specificity and higher sensitivity than elisa. the time required for the elisa-array is significantly less than for conventional elisa (4 h vs. a minimum of 6 h, respectively). furthermore, less igg is required for printing than for coating elisa plates. coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of igg. for the elisa-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of igg. with the characteristics of ease of use, sensitivity, specificity, and accuracy, the elisa-array assay would be widely accepted for clinical use. direct broad-range detection of alphaviruses in mosquito extracts multiplexed protein measurement: technologies and applications of protein and antibody arrays multiplexed sandwich assays in microarray format detection of adamantane-resistant influenza on a microarray a novel magnetic bead bioassay platform using a microchip-based sensor for infectious disease diagnosis advances in viral disease diagnostic and molecular epidemicological technologies assessment of some tools for the characterization of the human osteoarthritic cartilage proteome high-throughput microarray-based enzyme-linked immunosorbent assay (elisa) development of a sensitive microarray immunoassay and comparison with standard enzyme-linked immunoassay for cytokine analysis multiplexed sandwich assays in microarray format validating a custom multiplex elisa against individual commercial immunoassays using clinical samples application of protein microarrays for multiplexed detection of antibodies to tumor antigens in breast cancer human neutralizing sars-cov specific fab molecules generated by phage display simultaneous multianalyte elisa performed on a microarray platform a multiplexed protein microarray for the simultaneous serodiagnosis of human immunodeficiency virus/hepatitis c virus infection and typing of whole blood a simple and rapid protein array based method for simultaneous detection of biowarfare agent microarray immunoassay for the detection of grapevine and tree fruit viruses development of a quantitative real-time rt-pcr assay with internal control for the laboratory detection of tick borne encephalitis virus (tbev) rna a duplex real-time reverse transcriptase polymerase chain reaction assay for detecting western equine and eastern equine encephalitis viruses high diagnostic accuracy of antigen microarray for sensitive detection of hepatitis c virus infection the role of biosensors in the detection of emerging infectious diseases submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this study was supported by the national natural science foundation of china (grant no. 81171663) and national key research special foundation of china (grant no. 2011zx10004-001). authors' contributions xk: designed the study, performed the laboratory testing, analyzed the test results, and co-wrote and edited the manuscript. h l and yl performed the virus cultures. lf performed laboratory testing. xc, fl, and gc performed real time-pcr. qz and yy organized the overall project and helped edit the manuscript. all of the authors read and approved the final manuscript. state key laboratory of pathogen and biosecurity, beijing institute of microbiology and epidemiology, beijing 100071, china the authors declare that they have no competing interests. key: cord-278802-bverdk5w authors: zhou, yefei; zhou, meixian; zhang, dunlin; zhang, honglin; zhang, liyang title: immune response of aa broilers to ibv h120 vaccine and sodium new houttuyfonate date: 2010-12-31 journal: research in veterinary science doi: 10.1016/j.rvsc.2010.04.010 sha: doc_id: 278802 cord_uid: bverdk5w abstract in this report, 120 healthy one-day-old aa broilers were divided into six groups. groups 1–4 received 100, 200, 400 and 800mg/l of sodium new houttuyfonate (snh) with ib vaccine h120 respectively. group 5 received pbs and h120 and group 6 il-2 and h120. the chickens were inoculated at 7 and 14days of age. on 0, 7, 14, 21, 28 and 35 post first vaccination, the dynamic changes of peripheral lymphocyte proliferation, cytokine assays and serum antibody titers were assayed respectively by mtt method, elisa and hemagglutination inhibition assay (hi). the results showed that sodium new houttuyfonate significantly raised ib antibody titer in the chickens and also markedly promoted lymphocyte proliferation. the serum levels of ifn-γ and il-4 in groups 1–4 were higher than those in groups 5 and 6. hence, the immunologic enhancement of snh was slightly superior to that of il-2 adjuvant. following challenge with ibv, chickens inoculated with snh showed fewer and less severe clinical signs, lower death rate and less kidney pathology, as compared to those of the control groups. it indicated that snh could enhance immune responses and increase protection against virulent ibv challenge in chickens. infectious diseases of animals, especially viral, often cause great losses in the domestic animal industries. infectious bronchitis (ib) is an acute and highly contagious respiratory disease of chickens, and is still a major health problem in the chicken industry. infectious bronchitis virus (ibv), an enveloped coronavirus containing an unsegmented, single-stranded, positive-sense rna genome, is one of the primary causes of respiratory disease in domestic fowl. infection with ibv reduces the performance of broilers and causes drops in egg production and egg quality (bijlenga et al., 2004; cavanagh and naqi, 2003; cavanagh, 2003) . there are problems with vaccines against infectious bronchitis applied in the field. live attenuated vaccine may spread to unvaccinated flocks (farsang et al., 2002) ; inactivated vaccine does not elicit an immune response against coronavirus (yang et al., 2004; stohlman et al., 1992) , and severe side effects and long-lasting local response may occurs after inoculation. clinical practice has indicated that the application of vaccine with adjuvant or immunopotentiator could improve efficacy. however, chemical adjuvants (e.g. aluminium hydroxide (al(oh) 3 ), aluminium phosphate (alpo 4 ), and mineral oil) commonly cause side effects, such as strong local reaction and carcinogenesis. sometimes a chemical adjuvant may fail to enhance the immunogenicity of a weakly antigenic vaccine (xie, 1995; gong and wu, 1996; sun, 1998) . therefore, there is a need to assess an immunopotentiator that is both safe and efficacious. sodium new houttuyfonate (snh, sodium lauroyl-a-hydroxyethyl sulfonate) is a novel antimicrobial medicine that has been recently developed (the chemical structure is shown in fig. 1) . previous studies showed that houttuyfonate homologues (hou-cn) had immunoregulatory activities and typical adjuvanticity, and could improve the immune ability of mice and inhibit the growth of staphylococcus aureus, bacillus subtilis and pneumococci (ye et al., 2006; wang et al., 2002 wang et al., , 2004 . it was reported that the immunoregulatory and antibacterial activity of snh were higher and stronger than that of sodium houttuyfonate (yuan et al., 2004a; ye et al., 2005; yang et al., 2007) . because of its potential pharmaceutical effects on health, the purpose of this study was to evaluate the immune enhancement properties of snh as a new type of immunopotentiator. snh was obtained from jangsu jichun pharmaceutical co., ltd. ib disease vaccine virus h120 strain (ha titer 2 â 10 8 ) was supplied by jiangsu provincial academy of agricultural science. after safety was tested by injecting the virus into 11-day-old spf chicken embryos, the virus and snh were mixed (v/v = 1:3), and four inocula, containing 100 mg/l (group 1), 200 mg/l (group 2), 400 mg/l (group 3) and 800 mg/l (group 4) of snh, were prepared. two additional inocula, pbs and h120 (group 5, negative control) and il-2 and h120 (group 6, positive control), were prepared. all vaccines contained the same antigen content. filtered (0.22 lm) rpmi 1640 media (gibco) supplemented with benzylpenicillin (100 iu/ml), streptomycin (100 iu/ml) and 10% fetal bovine serum (sijiqing, zhejiang province) was used for culturing the cells. the same formula, minus bovine serum, was used for washing and diluting the mitogen. cona (sigma), as the t-lymphocytes mitogen, was dissolved to 0.1 mg/ml in rpmi 1640 media without fetal bovine serum, which was added (10% of final concentration) after the cona solution was filtered (0.22 lm), and then stored at à20°c. the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (mtt, amresco co.) was dissolved to 5 mg/ml in calcium and magnesium-free (cmf) phosphate-buffered saline (pbs, ph7.4) and filtered (0.22 lm). lymphocyte separation media (ficoll-hypaque, q: 1.077 ± 0.002, rong-sheng biostix inc., shanghai) and mtt solution were stored at 4°c in the dark. dimethyl sulfoxide (dmso, zheng-xing institute of chemical engineering, suzhou) was ar. one hundred and twenty chickens were randomly divided into six groups. at seven days of age, the average maternal antibody titer was 3.5 log2 and the average body weight was 95 g. on 7 and 14 days of age, each chicken was injected intramuscularly with inocula according to group. five chickens were sampled randomly from each group for assay of lymphocyte proliferation by mtt method and 10 chickens from each group for assessment of serum hi antibody titer by the micro-method and cytokines by elisa kits at 0, 7, 14, 21 and 35 days post the primary immunization (dpi). all chickens were challenged with 100 eid of the ibv m41 strain (f12 passage) in 0.1 ml by the nasal-ocular route at 42 days of age, and the animals monitored for 14 days for clinical signs such as coughing, sneezing, ataxia, dyspnoea or death. 2.5. sample collection and assay 2.5.1. peripheral lymphocyte proliferation assay blood samples (10 ml per chicken and the birds were alternately used) were collected by cardiac puncture and transferred immediately into aseptic capped tubes with sodium heparin, then diluted with an equal volume of hank's solution and carefully layered on the surfaces of the lymphocyte separation media. after 20 min centrifugation at 800g, the white cloud-like lymphocyte band was collected and washed twice with rpmi 1640 media without fetal bovine serum. the resulting pellet was re-suspended to 5 â 10 6 /ml in rpmi 1640 media with fetal bovine serum and incubated in 96-well culture plates (nunclon), 80 ll/well, then another 20 ll/well of the cona (four wells) or rpmi 1640 with 10% fetal bovine serum (four wells) was added and each sample seeded eight wells. after 44 h of incubation at 37°c in a humid atmosphere of 5% co 2 (revco, co., usa), 20 ll/well of mtt was added, and the plates re-incubated for 4 h. the plates were centrifuged (10 min at 1000g, room temperature) and the supernatant carefully discarded. hundred microlitre/well of dmso was added and the plates shaken for 5 min to dissolve the formazan crystals. the absorbance of cell in each well was measured by microliter enzyme-linked immunosorbent assay reader (model dg-3022, east chinavacuum tube manufacturer) at a wave length of 570 nm and the stimulation index (si) was calculated. the stimulation index was determined from the formula: stimulation index (si) = experimental od/negative od (barta et al., 1992; maheswaran and thies, 1975) . to determine the immune activity of snh, the levels of ifn-c and il-4 in the vaccinated animals were examined. for the detection of ifn-c and il-4 production in the chickens, sera were obtained at 0, 7, 14, 21, 28 and 35 dpi and determined quantitatively using commercially available cytokine elisa kits (chicken ifn-c/il-4 kits, sensitivity: 1 pg/ml, adlitteram diagnostic laboratories (adl), usa) as per protocol. blood samples (3 ml per chick) were drawn from the main brachial vein into eppendorf tubes and allowed to clot at 37°c for 2 h. serum was separated by centrifugation and inactivated (56°c, 30 min) for use. briefly, two-fold serial dilutions of serum were made in a 96-well, v-shaped bottom microtiter plate containing 25 ll of pbs in all wells. 25 ll of ibv antigen (without purification, diluted to 4 ha units with pbs) was added to all the wells except the last row, which served as a control. serum dilutions ranged from 1:2 to 1:2048. the antigen-serum mixture was incubated for 10 min at 37°c. twenty five microliter of a 1% rooster erythrocyte suspension was added to each well and the plates were re-incubated for 30 min. a positive serum, a negative serum, erythrocytes, and antigen were also included as controls. the highest dilution of serum causing complete inhibition was considered the endpoint. the geometric mean titer was expressed as reciprocal log2 values of the highest dilution that displayed hi. all chickens were challenged with 100 eid of the ibv m41 strain in 0.3 ml by intramuscular injection (i.m.) at 49 days, and were examined daily for 2 weeks for clinical signs such as coughing, sneezing, ataxia, dyspnea or death. dead chicks were necropsied to confirm the death due to ibv infection. data are expressed as the mean ± s.d. duncan's multiple range test was used to determine the difference among every adjuvant and control groups. differences between means were considered significant at p < 0.05. the dynamic changes of stimulation index (si) value are listed in fig. 2 . on day 0 after primary vaccination, the values of groups 1-4 were larger than those of il-2 and pbs groups. at 7 dpi, the si values of 800 mg/l group were significantly higher than those for the pbs or il-2 groups (p < 0.05). at 14 dpi, the values for group 1 were higher than those for groups 2, 3 and 4; however, there was no significant difference between these groups. at 21, 28 and 35 dpi, the values of 200 mg/l group were significantly larger than those of il-2 or pbs groups (p < 0.05). at 21, 28 and 35 dpi, the values of 200 mg/l group were significantly larger than those of 100, 400 and 800 mg/l groups (p < 0.05). the elisa kits were employed to detect the production of ifn-c and il-4 in sera. the results showed that the mean concentration of ifn-c was higher in groups 1-4 compared to groups 5 and 6. the 800 mg/l group was significantly higher than the pbs or il-2 group at 7 and 14 dpi (p < 0.05), but there was no significant difference between the 100, 200 and 400 mg/l groups. at 21 and 28 dpi, the 200 mg/l group was significantly higher than the pbs or il-2 (p < 0.05), but there was no significant difference between other experimental groups (fig. 3a) . at 35 dpi, high levels of the experimental groups were observed comparing to the pbs or il-2, but there was no significant difference (p > 0.05). similarly, there was almost the same change in il-4 between groups 1-4 and group 6 after vaccination, but only at 7 and 14 dpi. including 800, 400 and 200 mg/l groups at 14, 21 and 28 dpi a significantly higher level of il-4 was observed compared to that in the pbs or il-2 groups (groups 5 and 6) (p < 0.05), whereas there was no significant difference between groups 1, 5 and 6 (p > 0.05) (fig. 3b) . the results indicated snh could promote both th1-type and th2-type cytokine responses, and there was a dose-dependant effect. the dynamic changes of hi antibody titer are listed in table 1 . on day 28 after primary vaccination, the antibody titers of groups 1-4 were higher than those of groups 5 and 6. at 14 and 21 dpi, group 4 was significant higher than the pbs or il-2 group (p < 0.05). except for groups 1 and 3, the titers of groups 2 and 4 at 21 dpi were significantly higher than those of groups 5 and 6 (p < 0.05). at 28 and 35 dpi, in comparison of other groups, the titer of group 2 was significantly larger (p < 0.05). chickens started to show clinical signs or die from viral infection on day 8 after challenge, but deaths occurred only in group 5. the chickens immunized with pbs were not protected and developed coughing, nasal discharge and dyspnea. the morbidity and mortality of the pbs immunized group 5 were 100% (10/10) and 80% (8/10), respectively, after challenge with ibv m41 stain (fig. 4) . chickens in groups 1-4 had lower morbidity, mortality and pathological changes when compared to those of the pbs group 5, but the difference was insignificant. groups 1-4 showed clinical signs such as depression, anorexia, a shrinking neck and fluffed down feathers, but no chicken died except for those in groups 1 and 6. this suggests that snh confers resistance against a virulent ibv challenge. the use of houttuyfonate as an adjuvant has been accompanied by side effects (e.g., anaphylactic shock, systemic anaphylaxis, dyspnea), and so its clinical use has been limited. recent pharmacological studies have shown that snh and houttuyfonate have similar pharmacological properties, with a variety of biological functions, in addition to antibacterial, diuretic, antitussive, but also play a strong role in immune regulation. in addition snh is safer and has fewer side effects than houttuyfonate (jiang et al., 2006; zhu et al., 2008a,b; yuan et al., 2004b) . however, its application in the poultry industry has seldom been reported. in this study, with chicken ibv h120 strain live attenuated vaccine in chickens, by measuring peripheral blood lymphocyte proliferation and serum cytokines and antibody titers, the humoral and cellular immunity induced with snh was researched. the results indicated snh could enhance immune function in a dose-dependent manner. the results show that snh played an important role in protecting against challenge by virulent virus. it is well known that in both mouse and human systems, the th1 and th2 paradigm has emerged as a critical concept governing cellular and humoral immune responses. ifn-c and il-4 are prototype th1 and th2 cytokines, respectively, whose antagonistic actions against each other have been widely reported (mosman and sad, 1996) . they reciprocally regulate not only th1 and th2 cell survival and subsequent differentiation, but also activation and differentiation of b cells as well as monocytes (constant and bottomly, 1997; snapper and paul, 1987; cao et al., 1989) . specific target molecules that are counter-regulated by ifn-c and il-4 play fig. 2 . dynamic change of lymphocyte proliferation responses in chicken (si value). an asterisk indicates the si value is significantly higher than the control group (p < 0.05). data were shown as mean ± s.d. error bars represent group standard deviations. arrows (;) indicate time of initial immunization and boost. important roles in mediating th1 or th2 immune responses. serum levels of the two cytokines increased, and this effect shows a certain degree of dose-effect relationship, especially obvious with the 200 mg/l dose. therefore it was important to determine the fig. 3 . serum concentrations (pg/ml) of ifn-c (a) and il-4 (b) in the serum. samples (n = 5) were examined by using commercially available chicken cytokine elisa kits. an asterisk indicates the concentration of cytokine is significant (p < 0.05). data were shown as mean ± s.d. error bars represent group standard deviations. arrows (;) indicate time of initial immunization and boost. table 1 the dynamic variation of antibody titers in chicken (log2). groups dpi 0 7 14 21 28 35 appropriate dose of snh to take advantage of the body's immune system. at the same time, t lymphocyte-mediated cellular immunity was also researched. the results show that snh can significantly enhance the immune function of chicken cells by promoting lymphocyte proliferation. so, snh improved cellular immunity in comparison with pbs or il-2. hi tests are mostly used to monitor antibody levels induced by ibv live attenuated vaccine. therefore, the hi titer was considered the key factor in the appraisal of the vaccine adjuvant in this study. although the maternal antibody ibv titers influence the results, the final result indicated no influence. the co-administration of antigen and snh increased hi titers, and the titers of the snh groups were significantly higher than those of the pbs or il groups, which confirmed that snh could improve antibody formation. thus, it qualifies as a new-type immunopotentiator. data within a column with different letters differ significantly (p < 0.05); the same letters means no significance. fig. 4. morbidity and mortality ratio of each group after challenging with ibv m41 strains optimum conditions for the chicken lymphocyte transformation test development and use of the h strain of avian infectious bronchitis virus from the netherlands as a vaccine: a review differential regulation of class ii mhc determinants on macrophages by ifn-c and il-4 severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus infectious bronchitis induction of th1 and th2 cd4+ t cell response: the alternative approaches molecular epizootiology infectious bronchitis virus in sweden indicating the involvement of a vaccine strain research actuality and tendency of immune adjuvant study of sodium houtluyfonate on acute toxicity development of a micro-culture system for stimulation of chicken peripheral blood lymphocytes with concanavalin a the expanding universe of t-cell subsets: th1 th2 and more interfern c and b cell stimulatory factor reciprocally regulate ig isotype production mouse hepatitis virus nucleocapsid protein specific cytotoxic t lymphocytes are ld restricted and specific for the carboxy terminus research headway of immunopotentiator. helongjiang ani. hus studies on adjuvanticity of sodium houttuyfonate and its mechanism effects of sodium houttuyfonate on phosphorylation of camk ii, creb and erk 1/2 and expression of c-fos in macrophages progress in the study of grave epidemic diseases immune prevention and cure of domestic animals and poultry a dna vaccine induces sars coronavirus neutralization and protective immunity in mice chemiluminescence determination of sodium new houttuyfonate in pharmaceutical preparations based on tween80 rhodamine b system effect of the surface activity on the antibacterial activity of octadecanoyl acetal sodium sulfite series interaction between houttuyfonate homologues and erythrocyte plasma membrane of rabbit in vitro relativity of surfactivity and antibacterial motive of houttuynine analogies effects of houttuyfonate homologues on immunity of mouse effects of sodium houtluyfonate on chicken cellar immune response effects of sodium new houtluyfonate on chicken immune response this work was supported by grants from the jiangsu province natural science foundation (08kjd180011), nanjing xiaozhuang college key program (2008nxy10). key: cord-002583-cgcf7mgj authors: zhuo, xun-hui; sun, hong-chao; huang, bin; yu, hai-jie; shan, ying; du, ai-fang title: evaluation of potential anti-toxoplasmosis efficiency of combined traditional herbs in a mouse model date: 2017-06-01 journal: journal of zhejiang university-science b doi: 10.1631/jzus.b1600316 sha: doc_id: 2583 cord_uid: cgcf7mgj toxoplasma gondii is a worldwide spread protozoan and is able to infect almost all warm-blood animals. no effective drugs are available clinically on toxoplasmosis. chinese traditional herbal medicines have provided remedies for many health problems. there exists a possibility that chinese herbs may provide protection against t. gondii. this work aims to assess the protective efficacy of combined chinese herbs against t. gondii. we screened five herbal medicines that have different pharmacological effects and combined them into a prescription according to the traditional chinese medicine compatibility principle. the drug potential and protective efficacy were evaluated through a mouse model by determining the survival time, the parasite load in blood and tissues, the change of cell proportions in blood and histological detection. the results showed that the survival time of mice in the 500 mg chinese herbs group and sulfadiazine group was significantly longer than that of the pbs control group. also the parasite load in blood and tissues of 500 mg chinese herbs and sulfadiazine groups was significantly lower than that of pbs group at 7 days post infection (dpi), which was in accordance with the result of histological detection. monocyte and neutrophil of infected mice were remarkably increased while lymphocyte was dramatically decreased compared to that of blank group at 7 dpi. the results demonstrated that the 500 mg dosage of our chinese herbs could slow down the replication of t. gondii and prolong the survival time of mice and could be considered as possible candidate drug against toxoplasmosis. toxoplasmosis caused by the intracellular protozoan toxoplasma gondii is a ubiquitous worldwide parasitic zoonotic disease (innes, 2010a) . it is usually asymptomatic in immune-competent individuals but may occasionally lead to severe ocular and neurological disorders (furtado et al., 2013) . when it comes to immune-compromised and congenitally infected individuals, toxoplasmosis can result in lethal systemic disease and eventually death (shen et al., 2016) . currently, the control of t. gondii mainly depends on chemotherapy, such as the combination of sulfadiazine and pyrimethamine, but these drugs have serious side effects (petersen and schmidt, 2003) . even though many antigens including rhoptry, surface, and dense granule excreted-secreted antigens were evaluated as potential vaccines (kur et al., 2009) , there is no licensed vaccine against t. gondii infection clinically making the situation even worse (jongert et al., 2009; innes, 2010b) . therefore, new efficient drugs and safe protective therapies are needed. artemisinin isolated from a chinese traditional medicine artemisia annua is now included in standard treatment worldwide for plasmodium falciparum malaria. subsequent to that, interest in chinese traditional medicine has risen to be considered for providing therapeutic reagents for many infectious diseases. the radix clematidis extract is reported to be a potent inhibitor of matrix metalloproteinase-9 (mmp-9) and notably blocks the nuclear factor-κb (nf-κb) pathway in breast carcinoma cells (noh et al., 2011). na-bangchang and karbwang (2014) found that a chinese traditional medicine atractylodes possesses various pharmacological effects with anticancer, anti-inflammatory and antimicrobial activity, and is now a potential chemotherapeutic for cholangiocarcinoma found in southeast asia. artemisia anomala has been widely and in long-term used for the treatment of inflammatory diseases in china. recent findings suggested that it manifests its antiinflammatory effects through inhibiting the expression of inducible nitric oxide synthase (inos) (tan et al., 2014) . glycyrrhizin, extracted from liquorice, was reported to have antiviral activity through inhibiting replication of the pathogen (cinatl et al., 2003) . throughout history, chinese traditional herbal medicines have provided many remedies for many health problems. the use of most herbal medicines dates back almost 2000 years in shennong's materia medica. however, limited information is available on their pharmacological activity since the use of herbal medicine has been based mainly on empirical treatment. as a consequence, there exists a possibility that many herbal medicines may have unknown effects on different health problems. in this study, we screened five herbal medicines that have different pharmacological effects and combined them into a prescription according to the traditional chinese medicine compatibility principle. we evaluated the protective efficacy provided by the different dosages of this prescription against the challenge of rh strain of t. gondii in a mouse model. female icr mice (6-8 weeks old) were kept in an animal experimentation laboratory under standard conditions according to the guidelines of the regulation for the administration of affairs concerning experimental animals of the people's republic of china. these experiments on mice were approved by zhejiang university experimental animal ethics committee (no. zju201308-1-10-072). tachyzoites of t. gondii rh strain were maintained in icr mice by intraperitoneal (i.p.) serial passage at regular 72 h intervals. the parasites were washed three times by phosphate-buffered saline (pbs) and centrifuged at 1000g for 5 min as previously described (mack and mcleod, 1984) . five kinds of chinese herbal medicines were purchased from huqingyu tang pharmaceutical co., ltd., hangzhou, china. according to the traditional chinese medicine compatibility principle, they were combined as follows: r. clematidis (10 g), fructus ulmi macrocarpae (8 g), atractylodes (15 g), a. anomala (10 g), and glycyrrhizae (6 g). on the basis of the traditional chinese medicine decoction method, herbal drugs were boiled for 3 h in water (1 l) followed by concentrating through a rotary evaporator into 2 g/ml of the whole drugs. sulfadiazine as a positive control drug was purchased from novartis (beijing, china). a total of 140 female mice were randomly divided into seven groups (a to g). mice in groups b-g were i.p. infected with 1×10 3 tachyzoites of t. gondii rh strain. different medical administrations were carried out after infection with t. gondii rh strain as follows: group a, blank control; group b, pbs control; group c, mice were treated with 10 mg/d of sulfadiazine through oral gavage; mice in groups d-g were treated with chinese herbs through oral gavage in single dosages of 300, 400, 500, and 600 mg/d, respectively, according to the chinese pharmacopoeia (national pharmacopoeia committee, 2010). the animals were observed daily for mortality. a total of three mice in each group were euthanized at 3 and 7 days post infection (dpi), when whole blood was collected for a blood routine test; liver, spleen, and lung tissues were collected for quantitative realtime polymerase chain reaction (pcr) and histological section assay. the presence of t. gondii dna was investigated by real-time pcr based on the sag1 gene as described previously (yu et al., 2013) . the genomic dna of blood, liver, spleen, and lung tissues was extracted using a universal genomic dna extraction kit (takara, china) according to the manufacturer's instructions. the forward and reverse primer sequences used in this study were 5'-ctgatgtcg ttcttgcgatgtggc-3' and 5'-gtgaagtggt tctccgtcggtgt-3', respectively, designed by the primer express software (pe applied biosystem), which could amplify a 128-bp fragment under predetermined conditions. sybr ® green was then used to detect fluorescence using the iq5™ real-time pcr system (bio-rad). sterile water and genomic dna of t. gondii tachyzoites were included as negative and positive controls in each run. in addition, a plasmid pmd-sag1 was constructed based on pmd18-t vector using the same primers. to establish standard curves, plasmid pmd-sag1 was tenfold serially diluted ranging from 2×10 4 to 2×10 9 copies per milliliter tested in each independent experiment for t. gondii quantification. each sample had three parallel wells and was detected three times. at least 30 µl of anticoagulated blood samples collected from mice of different groups at 3 and 7 dpi were applied to an idexx procyte dx ® hematology analyzer. each blood sample was read three times. the data were analyzed using the software spss 16.0 for windows (spss inc., chicago, il, usa) and statistical difference was accepted at the level of p<0.05. liver, spleen, and lung tissue specimens from different groups were first fixed in 10% (v/v) buffered formalin and then processed to paraffin for embedding. tissue sections were obtained at approximately 5-µm thickness and stained with haematoxylin and eosin (h & e) stains as described previously (fischer et al., 2008) . sections were examined under light microscopy. in order to determine whether these combined chinese herbs could provide effective protection against t. gondii rh infection, mortality was recorded daily following the t. gondii challenge until all mice were dead and survival curves of all groups were generated as shown in fig. 1 . the survival time of mice in the sulfadiazine group ((13.5±2.0) d) and 500 mg chinese herb group ((9.8±0.69) d) were significantly longer than that in the pbs control group (6.9±0.18 d) (p<0.05). the average survival time of mice in the 300, 400, and 600 mg chinese herb groups was all under 8 d which was slightly longer than that of pbs group, but the difference was not statistically significant (p>0.05). blood and tissue samples were collected from different experimental groups at 3 and 7 dpi with t. gondii, and then dna was extracted as the template for sag1-based real-time pcr to evaluate the parasite loads and dna copy number in t. gondiiinfected mice (fig. 2) . every blood and tissue dna sample was tested in triplicate and no amplification products were observed in wells from the blank control group. results from the blood are shown in fig. 2a . parasite loads in all groups at 7 dpi were survival rate of mice treated with sulfadiazine and different dosages of chinese herbs after challenge with 1×10 3 tachyzoites of t. gondii rh strain. mice treated with pbs were included as negative control and mice infected with no t. gondii were included as blank control. survival rate was monitored daily until all infected-mice died (n=10 mice per group) almost double that at 3 dpi, but no significant difference was observed among these groups. results of spleen, lung, and liver tissues presented a similar pattern in that parasite loads were all largely increased from 3 to 7 dpi, and mice of the pbs control group had statistically significantly higher parasite load compared with sulfadiazine and 500 mg chinese herb groups (p<0.05). however, the parasite loads of mice in 300, 400, and 600 mg chinese herb groups were all fewer than those in the pbs control group but not at a statistically significant level. blood samples were collected from different groups at 3 and 7 dpi directly into a 1-ml tube containing 0.3 mg dipotassium ethylenediamine tetraacetic acid (k 2 edta) and analyzed by an idexx procyte dx ® hematology analyzer. no significant increases were observed in eosinophil among the different t. gongdii-infected groups (fig. 3a) . the proportion of monocyte of infected mice was remarkably increased compared to that of the blank group at 7 dpi ( fig. 3b ; p<0.05). however, lymphocytes of the infected groups were dramatically decreased compared with the blank control group (fig. 3c) and those of the sulfadiazine and 500 mg chinese herb groups were significantly low compared to the other four infected groups (p<0.05). as shown in fig. 3d , neutrophil in the infected groups was increased more than double compared to the blank control group at 7 dpi (p<0.05), and the sulfadiazine and 500 mg chinese herb groups also increased significantly higher among these groups. blood (a), spleen (b), lung (c), and liver (d) tachyzoite burdens at 3 and 7 d after challenge. the dna copies of t. gondii (per milliliter blood or per gram tissue) in blood and tissues of mice after challenge with 1×10 3 tachyzoites of t. gondii rh strain per mouse. the counting of dna copies of t. gondii has been done in triplicate for each tissue and for three mice from each group. values are expressed as mean±sd. columns with different letters present statistical difference (p<0.05) pathological studies were performed through histological sections of liver, lung, and spleen tissues which were collected at 7 dpi from all groups. the livers of sulfadiazine and 500 mg chinese herbs-treated mice displayed few changes compared to normal mice (figs. 4c and 4f), but the livers of the pbs group mice showed a clear cellular separation, which revealed its morphologically incomplete and hepatocellular dysfunction (fig. 4b) , and the livers of 300, 400, and 600 mg chinese herbs-treated mice showed slightly more cellular separation . the lungs of all t. gondii-infected mice represented interstitial pneumonia at different degrees with much thicker alveolar walls compared to that of the blank control group (figs. 4i-4n) , and more severe cellular damage was observed in 300 and 600 mg chinese herbs-treated mice and the pbs control group. the spleen of the pbs group was extensively necrotic with few lymphoid cells and plenty of scattered cells observed. lymphoid follicles were also absent (fig. 4p) . the spleens of other groups also had evidence of necrosis and degeneration with small scattered nodules of dead lymphoid cells (figs. 4q-4u), but they were not as deteriorated as that of the pbs group shown in fig. 4p . t. gondii can infect almost all warm-blooded animals, but no available drugs can eliminate this pathogen from its host without any clinical side effects. the discovery of artemisinin, isolated from a. annua, which can provide effective treatment worldwide against p. falciparum malaria, raises the the results showed the percentage of cells in blood at 3 and 7 d after challenge with 1×10 3 tachyzoites of t. gondii rh strain per mouse. eosinophil (a), monocyte (b), lymphocyte (c), and neutrophil (d) cell percentages were analyzed by idexx procyte dx ® hematology analyzer at 3 and 7 dpi. blood samples were collected from three mice at each group at 3 and 7 dpi, respectively. values are expressed as mean±sd. columns with different letters present statistical difference (p<0.05) question as to whether chinese traditional medicines could afford protection against t. gondii. therefore, five herbal medicines that have different pharmacological effects were screened and combined into a prescription according to the traditional chinese medicine compatibility principle and then the drug potential and protective efficacy were evaluated through a mouse model. molecular methods, particularly real-time pcr, have been treated as sensitive and reliable tools for determining the parasite loads of t. gondii (bell and ranford-cartwright, 2002; li et al., 2012) . hence, we applied an sag1-based real-time pcr method to determine the parasite loads in blood and tissues. we found t. gondii dna could be detected in blood and tissues from all infected groups at 3 dpi with a relatively high level and was almost all doubled from 3 to 7 dpi while no significant difference was observed among these groups. the level of the parasite load of the liver of sulfadiazine and 500 mg chinese herbs-treated groups was lower than that of the pbs control group (p< 0.05). we also found the livers of sulfadiazine and 500 mg chinese herbs-treated mice displayed few changes while a clear cellular separation could be observed in the liver of pbs group mice that presented a morphologically incomplete and hepatocellular dysfunction. the lungs of sulfadiazine and chinese herbs-treated mice possessed a significantly lower parasite load than that of the pbs control group (p<0.05) and the histological result verified this from the morphological perspective. the lungs of all t. gondii-infected mice displayed interstitial pneumonia which had much thicker alveolar walls compared to those of the blank control group. however, the situations of the pbs group, the 300 and 600 mg chinese herbs-treated groups were the worst such that no complete pulmonary alveoli structure could be observed. this revealed the fact that the lungs barely functioned and resulted in rapid death at 7 dpi as shown in the survival curve. as for the spleen, the parasite in sulfadiazine and 500 mg chinese herbstreated groups was at a significantly lower level than that in the pbs group at 7 dpi (p<0.05). the spleens of all infected mice showed necrosis and degeneration. lymphoid follicles of the pbs group were barely observed, and those of sulfadiazine and 500 mg chinese herbs-treated groups also had evidence of necrosis and degeneration with small scattered nodules of dead lymphoid cells. these results were in accordance with the survival curve that the mice of sulfadiazine and 500 mg chinese herb groups lived much longer than the controls. mice treated with 500 mg chinese herbs ((9.8±0.69) d) showed a pathological examinations were based on h & e-stained 5 µm-thick longitudinal sections of liver, lung, and spleen tissues in each group at 7 dpi. liver (a-g), lung (h-n), and spleen (o-u) were collected from seven groups. scale bars represent 20 µm (a-g) and 50 µm (h-u) significantly longer survival time (p<0.05) than the pbs-treated mice. in addition, the average survival time of 500 mg chinese herbs-treated mice presented a similar level as many studies on dna vaccines; the average survival time of rrop18-immunized icr mice is 11.5 d (qu et al., 2013) and zheng et al. (2013) found that after being immunized with rrop5+ rsag1, mice had prolonged survival time of 12.1 d. we assume that t. gondii was able to move to most tissues of mice through blood based on the fact that dna copies of t. gondii had been detected in blood, liver, spleen, and lung. actually, dadimoghaddam et al. (2014) found t. gondii moved to various tissues within 24 h after i.p. injection and the largest number of parasites was observed in the heart, kidney, and liver. the movement trend of t. gondii and parasite load in tissues have been used mainly to evaluate the vaccine effect, anti-parasite and drug disease severity (bell and ranford-cartwright, 2002; romand et al., 2004) . the results gathered here suggested that these combined chinese herbs could afford efficient protection against acute t. gondii infection. machado et al. (2014) demonstrated that the increased monocytes and lymphocytes could be treated as a relevant hematological biomarker of acute retinochoroidal lesions caused by t. gondii. thus we used an idexx procyte dx ® hematology analyzer to determine the changes of cell proportions in blood at 3 and 7 dpi. we found that there was no significant difference among the t. gondii infected groups on eosinophil at 3 or 7 dpi. however, monocytes and neutrophils of infected mice were remarkably increased while lymphocytes were dramatically decreased compared to those of the blank control group at 7 dpi. on the other hand, the significantly lower lymphocyte and slighter lower monocyte of sulfadiazine and 500 mg chinese herbs-treated mice than those of other chinese herbs-treated mice suggests that effective drugs may decrease the cell proportions to hinder the cellular transmission of t. gondii. there are no differences between normal mice and infected mice at 3 dpi. we found t. gondii was capable of escaping the immune system in blood when combining the results of parasite load in blood at 3 dpi. in accordance with the results of machado et al. (2014) , the monocytes were increased drastically at 7 dpi. the recruitment of monocytes is essential in restricting the replication of the t. gondii murine model (mordue and sibley, 2003; robben et al., 2005) . on the other hand, monocytes as well as neutrophils and eosinophils are strong candidates for transport of t. gondii in blood in a way known as a "trojan horse" (lachenmaier et al., 2011) , so the activity of monocyte must be carefully controlled. however, the trend of lymphocytes was opposite to that of machado et al. (2014) . the percentage of lymphocytes was decreased in infected mice mainly because icr mice were sensitive to t. gondii and failed to eliminate the parasite, which was in accordance with the result that all infected mice eventually died. in summary, mice treated with chinese herbal medicines had been showed to elicit partial protection against acute t. gondii infection. moreover, our study indicated that 500 mg chinese herbs could afford more efficient protection than the other three dosages. the results also suggested that our 500 mg chinese herbs could slow down the replication of t. gondii and prolong the survival time of mice. these results demonstrated this combination of chinese herbs could be considered as possible candidate drugs against toxoplasmosis and also that the therapies of traditional chinese herbal medicine can be developed for anti-toxoplasma in the future. real-time quantitative pcr in parasitology glycyrrhizin, an active component of liquorice roots, and replication of sars-associated coronavirus tissue tropism and parasite burden of toxoplasma gondii rh strain in experimentally infected mice. asian pac hematoxylin and eosin staining of tissue and cell sections ocular toxoplasmosis i: parasitology, epidemiology and public health a brief history and overview of toxoplasma gondii vaccination against toxoplasma gondii: an increasing priority for collaborative research? vaccines against toxoplasma gondii: challenges and opportunities current status of toxoplasmosis vaccine development intracellular transport of toxoplasma gondii through the blood-brain barrier development and application of reverse transcription loop-mediated isothermal amplification for detecting live shewanella putrefaciens in preserved fish sample biomarker analysis revealed distinct profiles of innate and adaptive immunity in infants with ocular lesions of congenital toxoplasmosis new micromethod to study the effect of antimicrobial agents on toxoplasma gondii: comparison of sulfadoxine and sulfadiazine individually and in combination with pyrimethamine and study of clindamycin, metronidazole, and cyclosporin a a novel population of gr-1 + -activated macrophages induced during acute toxoplasmosis traditional herbal medicine for the control of tropical diseases pharmacopoeia of people republic of china radix clematidis extract inhibits tpa-induced mmp-9 expression by suppressing nf-κb activation in mcf-7 human breast cancer cells sulfadiazine and pyrimethamine in the postnatal treatment of congenital toxoplasmosis: what are the options? enhancement of protective immune response to recombinant toxoplasma gondii rop18 antigen by ginsenoside re recruitment of gr-1 + monocytes is essential for control of acute toxoplasmosis usefulness of quantitative polymerase chain reaction in amniotic fluid as early prognostic marker of fetal infection with toxoplasma gondii seroprevalence of toxoplasma gondii infection among hiv/aids patients in eastern china ethyl acetate extract of artemisia anomala s. moore displays potent anti-inflammatory effect evaluation of a real-time pcr assay based on the single-copy sag1 gene for the detection of toxoplasma gondii the virulence-related rhoptry protein 5 (rop5) of toxoplasma gondii is a novel vaccine candidate against toxoplasmosis in mice xun-hui zhuo, hong-chao sun, bin huang, hai-jie yu, ying shan, and ai-fang du declare that they have no conflict of interest.all institutional and national guidelines for the care and use of laboratory animals were followed. key: cord-275635-d50bxe7c authors: yuan, xiaomin; lin, huixing; fan, hongjie title: efficacy and immunogenicity of recombinant swinepox virus expressing the a epitope of the tgev s protein date: 2015-07-31 journal: vaccine doi: 10.1016/j.vaccine.2015.06.057 sha: doc_id: 275635 cord_uid: d50bxe7c to explore the possibility of developing a vaccine against transmissible gastroenteritis virus (tgev) infection, a recombinant swinepox virus (rspv-sa) expressing a tgev protective antigen has been constructed. immune responses and protection efficacy of the vaccination vector were assessed in both mice and pig models. an indirect elisa assay suggested that when mice were vaccinated with rspv-sa, the level of igg against tgev was enhanced dramatically. the cytokine assays were employed and the results indicated that both the th1-type and th2-type cytokine levels raised after vaccination with rspv-sa in mice models. results from the passive immunity protection test of new born piglets demonstrated that the recombinant live-vector vaccine, rspv-sa, could 100% protect piglets from the spv infection, and there was no significant clinical symptom in the rspv-sa treatment group during this experiment. the data suggest that the novel recombinant swinepox virus is a potential vaccine against tgev infection. transmissible gastroenteritis virus (tgev) is a member of coronaviridae, which is the etiological agent of transmissible gastroenteritis. although the virus is capable of infecting swine of all ages, suckling piglets are the most susceptible and have a mortality rate up to 100% [1] . tgev is a pleomorphic enveloped virus containing a positive-stranded rna genome and four structural proteins: the spike (s) protein, the integral membrane (m) protein, the minor envelope (e) protein, and the nucleocapsid (n) protein. among which, the spike (s) protein, one of the key structural membrane proteins of coronaviruses, is an attractive target for generating neutralizing antibodies against the virus due to the critical role it plays in the host cell invasion [2, 3] . precisely, the s protein mediates the attachment of virus particles to targets via binding of itself to the specific receptors. at the n terminus of the s protein, there are four antigenic sites, a, b, c, and d, which have been shown to be involved in the stimulation of neutralizing antibodies (fig. 1 ) [4] . previous studies have determined that the a site (which is fully dependent on glycosylation for proper folding) is predominantly responsible for stimulating neutralizing host antibodies [5] [6] [7] [8] [9] [10] . spv is a natural mild attenuated virus and has been widely applied as a vaccine. given that poxvirus-vectors can prevent a great deal of important diseases in both humans and animals it is not surprising that many of these vectors been licensed and used extensively [11] [12] [13] . additionally, spv is a safe vaccine vector as there is no risk of cross-species infection [14] . therefore, both for biological and clinical practicality, spv is regarded as an appropriate and promising veterinary vaccine for swine, owing to its ability to effectively express foreign genes, its large packaging capacity for recombinant dna, its low cost of delivery and its specific host restriction [15] . the potential value of spv as a live vector vaccine is being studied extensively. because spv is able to pack large amounts of recombinant dna and to induce appropriate immune responses in vivo, it is a promising candidate for the development of a recombinant vaccine [16, 17] . as of yet, pigs are the only known hosts of swinepox virus and therefore may be useful in developing a safe vaccine for clinical application [18, 19] . in this study, we constructed a recombinant swinepox virus expressing s-a (a epitope of the s protein) of tgev and characterized recombinant virus replication and expression of the s protein in pk-15 cells. we further investigated the potential of this approach for use in the vaccination of pigs against tge. type culture collection. swine transmissible gastroenteritis virus (tgev, china strain, shxb) was purchased from the jiangsu academy of agricultural sciences, and the titer was determined as 1 × 10 8 pfu/ml st cell. tgev convalescent positive serum was purchased from the jiangsu academy of agricultural sciences, and the neutralizing antibodies were used at a dilution of 1:10,000. the pusz11 swinepox virus vector was generated previously [20] . two primers, sa1 (5 -gcgtcgacatgggtcttggtatga-agcgtag-3 ) and sa2 (5 -cgggatcctta tagcgtcctgt-tagtttgtc-3 ) were used to amplify the s-a gene (516 bp, km980444) from the tgev genome, which was inserted into the pusz11 plasmid to construct pusz11-sa subsequently. the recombinant swinepox virus, rspv-sa, was generated by homologous recombination of wtspv with pusz11-s-a as previously described [21] . pcr and indirect immunofluorescence were employed to analyze the s-a gene expression and the expression of s protein. the replication capacity and genetic stability of rspv-sa were also evaluated by. the generation and screening of the recombinant swinepox virus assays were performed as described previously [20] . a subconfluent culture of pk-15 cells was infected with wtspv (0.02 moi) for 2 h, and subsequently transfected with 10 g of the pusz11-sa plasmid using exfect tm transfection reagent (vazyme biotech co., ltd). after 72 h, pk-15 cells were harvested and lysed by five rounds of freezing and thawing. subsequently, the lysate was used to infect pk-15 cells grown in a 12-well plate for further purification of recombinant viruses. 1.5 ml of medium with 1% lmp agarose (dingguo, beijing, china) was added to each well and incubation was continued for five days until plaques became visible under a light microscope. after 1-2 days, a second overlay medium containing x-gal was added. the plaques were resuspended in 0.3 ml of medium with 2% fbs. plaque isolation was repeated for 5-6 rounds until all plaques in a given well were stained blue. the recombinant spv bearing s-a of tgev was designated as rspv-sa. the rspv-sa genomic dna from the pk-15 cells infected with rspv-sa was extracted by sds-protease k-phenol. we utilized wtspv genomic dna from pk15 cells infected with wtspv as a negative control. pcr was performed for 5 min at 94 • c; followed by 32 cycles of 1 min at 94 • c, 1 min at 50 • c, and 1 min at 72 • c. amplifications were performed with dna polymerase (promega, shanghai, china) using primers sa1 (5 -gcgtcgacatgggtcttggtatgaagcgtag-3 ) and sa2 (5 -cgggatccttatagcgtcctgttagtttgtc-3 ). indirect immunofluorescence assays (ifa) were performed as described previously [21] . pk-15 cells were grown on a 24-well plate and infected with the wtspv and rspv-sa at 1 × 10 8 pfu/ml per well. pbs-treated cells were used as a negative control. at 60 h post-infection, cells were washed three times in pbst and fixed with cold methanol for 10 min at −20 • c. cells were then washed three times with pbst and blocked by pbst with 10% bsa. preparations were incubated for 1 h at 37 • c with tgev convalescent positive serum (1:1000 in dilution buffer, pbst with 1% bsa). after three washes with pbst, cells were treated with the rhodamineconjugated secondary antibody (staphylococcal protein a-rhod, boshide, wuhan, china) at a 1:5000 dilution (diluted in pbs) for 30 min at 37 • c. after a final wash with pbs, all wells were examined by fluorescence microscopy (zeiss, germany). nine six-week-old balb/c mice were randomly divided into three groups (3 mice per group), and immunized three times at 0, 14, and 28 days with rspv-sa (1 × 10 8 pfu/ml in 0.2 ml of pbs) or wtspv (1 × 10 8 pfu/ml in 0.2 ml of pbs), the control group injected with pbs. eight one-month-old swine (large white) were randomly divided into four groups (2 pigs per group) and were immunized twice at 0 and 28 days with infectious rspv-sa (1 × 10 8 pfu/ml in 2 ml of pbs), inactivated-tgev (1 × 10 8 pfu/ml in 2 ml of pbs), wtspv (1 × 10 8 pfu/ml in 2 ml of pbs) or pbs, each time via three routes: oral, nasal, and intraperitoneal. serum was collected 14 days after the last immunization. twelve one-day-old pigs were randomly divided into four groups for passive immunization experiments (3 pigs per group). high titers of antibodies were collected from piglets following the first immunization. mice and swine serum were incubated at 56 • c 30 min to complement inactivated. all experimental protocols involving mice or swine were approved by the laboratory animal monitoring committee of jiangsu province. pk-15 cell monolayers were infected with wtspv and rspv-sa (moi of 5) and incubated for 72 h at 37 • c. extracts, representing approximately 1 × 10 5 cells, were electrophoresed through an sds-12% polyacrylamide gel and the separated proteins were transferred onto a pvdf membrane. after a 2 h transfer, the membrane was blocked with 5% skim milk in phosphate buffered saline with 0.05% tween-20 (pbst) overnight at 4 • c. the membrane was incubated with swine convalescent serum (1:1000 dilution) containing tgev for 2 h at 37 • c and washed three times with pbst. immunodetection was performed with staphy-lococcal protein a-hrp at 37 • c. following the secondary antibody probing, the membrane was washed four times with pbst. the membrane was then developed with 3,3 -diaminoben-zidine substrate until optimal color development was observed. serum was collected from mice and pigs, and detected the tgev-specific antibodies by indirect elisa. the purified tgev was resuspended in 100 l pbs (ph 7.2), and used the best titer of virus for coating 96-well plates, which was determined by titration. samples were then incubated overnight at 4 • c. this incubation was followed by three pbst washes, and blocking with 5% skim milk (in pbst) at 37 • c for 2 h. serum samples were serially diluted and incubated at 37 • c for 1 h. the samples were set up at the same time and divided into three groups: the tgev positive serum, the negative control serum (spv positive serum) and the blank control (without serum). after three pbst washes, horseradish peroxidase (hrp)-conjugated goat anti-spa igg (1:10,000 diluted in pbst, signalway antibody) was added to each test well. the plates were then incubated at room temperature in the dark for 30 min and then washed three times with pbst. the tmb microwell peroxidase substrate system (tiangen) was used to develop the reaction. samples were developed for 20 min and the reaction terminated with 2.0 m sulphuric acid. all assays were performed in duplicate. a microplate reader (bio-rad) was used to measure the reaction product at an absorbance of 450 nm [22] . evaluation of cellular immunity was performed by detecting levels of ifn-␥ and il-4. three mice were sacrificed at 30 days after the first inoculation with wtspv (1 × 10 8 pfu/ml in 0.2 ml), pbs or rspv-sa (1 × 10 8 pfu/ml in 0.2 ml). the mouse spleen was removed aseptically. splenocytes were isolated, counted, and diluted to a density of 2 × 10 7 cells/100 l. the evenly separated cells were aliquot into 96-well plates (100 l/well). then, 100 l/well of dmem with 200 tcid 50 /100 l tgev was added to each well. after a 60 h incubation, the supernatants were collected and the mrna of ifn-␥ and il-4 were probed by rt-qpcr relative to ␤-actin as described previously [23, 24] . an assay for neutralizing antibodies was performed as described previously [18, 19] . to explore whether mice or swine generated tgev neutralizing antibodies, serum from the pbs, wtspv, inactivated-tgev and rspv-sa treated mice and pig were collected at 0, 14, 21, 35, 42 days post-primary immunization (1:100-1:12,800 dilution in a 100 l volume). and sera were mixed with equal volume of 100 tcid 50 /ml tgev and incubated at 37 • c. after 1.5 h incubation, we utilized sera treated viruses to infect st cells in 96-well plates and overlaid cells with agar at 37 • c in a 5% co 2 atmosphere. cells were monitored daily for three days to detect tgev-specific cpe. the virulent tgev strain shxb (1 × 10 8 pfu/ml in 1 ml) was mixed with 3 ml of the porcine antiserum induced by recombinants rspv-sa, inactivated tgev or pbs, incubated at 37 • c for 60 min, and administered using a gastric tube to 2-day-old swine born from tgev-seronegative sow. inoculated animals were fed three times per day with formula for newborns that contained 3 ml of the antiserum. at three days following viral infection, small intestine tissue was collected from newborn pigs sectioned for histology. microstructure characteristics were analyzed under the microscope (olympus, cx21fs1, philippines) [25] . all data were analyzed using one-way anova and values of p < 0.05 were considered significant. to analyse the recombinant virus and confirm the presence of the sa gene, two specific primers were designed to amplify the inserted sa gene. the gene encoding the neutralizing antigen epitopes of tgev is a 0.5 kb fragment and the specific fragments was detected in spv-sa as shown in fig. 2a . the recombinant spv-sa was also confirmed by observing blue foci in plaque assays. rspv-sa and wtspv were determined to be approximately 1 × 10 8 pfu/ml in 1 ml for both. as shown in fig. 2b , the ifa demonstrated that the expression of the a epitope of the s protein was present in infected cells. western blot analysis showed a specific band of target protein with a size of 19 kda in the cell lysates infected with rspv-sa (fig. 2e) . the 19 kda molecular weight is consistent with the predicted size of the sa protein of tgev. according to these data, we suggested that the a epitope of the s protein was expressed efficiently by the rspv-sa virus (fig. 2) . to monitor the s-specific antibody titers of mice vaccinated with rspv-sa, an elisa was used. after an initial boost 7 days postvaccination, the s-a-specific antibody titers increased gradually in mice (fig. 3a) . the antibody titers in rspv-sa-immunized mice were higher at all-time points post-vaccination (p < 0.01, n = 5) compared to wtspv or pbs-treated mice. the p/n value of the positive group was greater than 2.1. persistent high levels of neutralizing antibodies (fig. 4a) were detected in the rspv-sa group with a mean titer of 1:1000 at 42 days post-inoculation. in mice vaccinated with wtspv and rspv-sa at 30 days postinoculation (fig. 3b) , the qpcr results showed a distinct variation of il-4 and ifn-␥ between wtspv and rspv-sa treatment groups. the relative quantity normalized by beta-actin of il-4 mrna in rspv-sa was 1.47 times higher compared to wtspv. the ifn-␥ mrna levels increased 1.45 fold in rspv-sa when compared to wtspv. the concentrations of il-4 and ifn-␥ in rspv-sa-vaccinated mice were significantly higher than the control groups. these results indicate that rspv-sa induces th1-type and th2-type cytokine responses during cellular immunity. during the vaccination procedure, several poxes of 2 mmϕ could be observed around the injection position at five days post-inoculation in both rspv-sa and wtspv treatment groups, respectively, which was not observed in the inactivated-tgev and pbs treatment groups. this symptom could be disappeared spontaneously in the following 5 days. no pigs developed further symptoms such as fever or severe inflammation, and the spiritual condition and appetite of both treatment groups was considered as good. these two group pigs recovered in 14 days. all group pigs maintained rectal temperatures of 38.7-39.3 • c. from this experiment, we reveal that vaccination with rspv-sa and wtspv is well tolerated by pigs. rspv-sa induced a moderate level of tgev-specific igg as shown in fig. 5 . persistent high levels of tgev-specific neutralizing antibodies are shown in fig. 4b . the second boost led to of the levels of tgev-specific neutralizing antibodies. persistent high levels of neutralizing antibodies were detected in the rspv-sa group at a mean titer of 1:1400 in swine (p < 0.05, n = 5). all newborn piglets in different groups were fed with the mixture of tgev and the corresponding sera from pigs vaccinated with pbs, wtspv, inactivated-tgev, or rspv-sa. both pbs and wtspv treatment groups developed a very severe diarrhea symptom, significantly losing of weight and appetite, and the mortality rate was up to 100% in 3 days. meanwhile, in the inactivated-tgev and rspv-sa treatment groups, there was no obvious clinical symptoms and 0% morality rate observed (fig. 6 . and table 1 ). after the sacrificing of all the piglets, the small intestine tissue from different groups were used for the pathological examination. histological samples from pbs and wtspv treatment groups showed prominent histopathological changes in the small intestine characterized by serious fracture of the small intestine mucosa, epithelial expansion, vacuolar degeneration, necrosis, shedding, and lamina propria congestive edema and hemorrhage. and such pathological changes were not observed during the examination of the samples from inactivated-tgev and rspv-sa treatment groups. all the results indicate that rspv-sa inoculation provides complete protection passive immunity against tgev challenge in pigs (fig. 7) . in the present study, we have engineered the swinepox virus to express the a epitope of the tgev s protein. our data showed that this recombinant virus was not only able to induce a strong immune response against the a antigen of tgev in mice and pigs, but also had safety degree and protection efficacy against the virulent homologous tgev infection in pigs. the traditional way to protect swine from tgev infection is to immunize pregnant sows with inactivated or attenuated vaccine, resulting in the production of secretory iga in the colostrum. despite the protection that secretory iga offers piglets, this method does not provide protection after the cessation of breastfeeding. furthermore, inactivated or attenuated viral vaccines still maintain the ability of developing the viral toxicity [26] . currently, there are various potential vaccines against tgev but many present certain challenges, such as difficulties in the vaccination process, high production cost, low immunogenicity, biological risk and carcinogenicity. the results from our experiments indicate that the rspv-sa vaccine is a very promising candidate in the safety and immunogenicity respect. however, it should be noticed that, as an alive viral vector vaccine, it is possible that the spv per se would interfere the vaccination. to evaluate this possibility, 2000 randomly sampled pig sera were tested via the agarose gel diffusion assay, and the results showed the spv positive rate was around 1%. moreover, there was no report of spv epidemic in these recent years. thus, together with gel diffusion assay, the interfering from spv per se could be considered as minimum. for the first time, we generated a recombinant spv that expresses the neutralizing epitopes (a epitope in protein s) of tgev, and we verified that the s-a was expressed efficiently in our system. the antigen induced neutralizing antibodies against tgev in st cells, and potentiated strong th1-type and th2-type cytokine responses in our mouse model. these results suggest that rspv-sa induces humoral and cellular immune responses effectively and efficiently. given the th1 cell secretion of il-2, ifn-␥, ifn-␣, and tfn-␤, it is apparent that th1 cells play an important role in antiintracellular pathogenic infection. because th2 cells in our study secreted il-4, il-5, il-6 and il-10, it is likely that these cells were able to effectively stimulate b cell proliferation and hence, igg and ige antibody production (relevant to humoral immunity). we defined ifn-␥ and il-4 to be representative of the th1-type and th2-type cytokine secretory capacity, respectively. our results indicate that in our hands, both th1-type and th2-type cytokine levels increased dramatically. due to the immature immune system, the maternal antibodies from breast milk account for the immune defensive ability of piglets. based on the feasibility and relevant published papers [25] , we decided to mimic the breast milk from female pig by mixing the anti-serum generated by vaccinating the 30 days old piglets with spv-sa and the normal cow milk, and use it for passive immunity protection test on the 1days old piglets. in the study, the recombinant spv vectors were capable of protecting neonatal piglets against mortality and severe disease after a challenge with virus. the rspv-sa vector induced high titers of antibodies in swine. however it is worth noting that rspv-sa may enhance the anti-tgev antibody titer in swine as well as the anti-spv antibody, which will likely affect immune efficiency. with respect to clinical value, it is imperative to study this multivalent vaccine. to summarize, we first report that spv can be used as a live vector vaccine when it expresses the s-a protein of tgev. we determined that not only b-cells, but also t-cells were induced successfully. thus, rspv-sa provides thorough protection against virulent tgev challenge in swine. lastly, our data indicate that rspv-sa is a promising vaccine to prevent tgev infection. transmissible gastroenteritis virus infection: a vanishing specter coronaviruses: structure and genome expression antigenic structure of transmissible gastroenteritis virus. ii. domains in the peplomer glycoprotein bacterial expression of antigenic sites a and d in the spike protein of transmissible gastroenteritis virus and evaluation of their inhibitory effects on viral infection evaluation on the efficacy and immunogenicity of recombinant dna plasmids expressing spike genes from porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus construction, safety and immunogenicity analysis of attenuated salmonella typhimurium harbouring tgev dna vaccine localization of antigenic sites of the e2 glycoprotein of transmissible gastroenteritis coronavirus expression of swine transmissible gastroenteritis virus envelope antigens on the surface of infected cells: epitopes externally exposed clinical evaluation of transmissible gastroenteritis virus vaccines and vaccination procedures for inducing lactogenic immunity in sows a monoclonal antibody against transmissible gastroenteritis virus generated via immunization of a dna plasmid bearing tgev s1 gene 25 years and counting: centers for disease control and prevention identifies opportunities and challenges for diabetes prevention and control development of a recombinant vaccinia-rabies vaccine for oral vaccination of foxes against rabies recombinant fowlpox virus inducing protective immunity in non-avian species recombinant swinepox virus expressing betagalactosidase: investigation of viral host range and gene expression levels in cell culture construction of recombinant swinepox viruses and expression of the classical swine fever virus e2 protein replication and expression of a swinepox virus vector delivering feline leukemia virus gag and env to cell lines of swine and feline origin feline b7.1 and b7.2 proteins produced from swinepox virus vectors are natively processed and biologically active: potential for use as nonchemical adjuvants first insights into the protective effects of a recombinant swinepox virus expressing truncated mrp of streptococcus suis type 2 in mice immune responses and protection efficacy of a recombinant swinepox virus expressing ha1 against swine h3n2 influenza virus in mice and pigs a novel vaccine against streptococcus equi ssp. zooepidemicus infections: the recombinant swinepox virus expressing m-like protein joint production of prime/boost pairs of fowlpox virus and modified vaccinia ankara recombinants carrying the same transgene recombinant adenovirus encoding the ha gene from swine h3n2 influenza virus partially protects mice from challenge with heterologous virus: a/hk/1/68 (h3n2) co-expressing gp5 and m proteins under different promoters in recombinant modified vaccinia virus ankara (rmva)-based vaccine vector enhanced the humoral and cellular immune responses of porcine reproductive and respiratory syndrome virus (prrsv) parvovirus evades interferondependent viral control in primary mouse embryonic fibroblasts induction of antibodies protecting against transmissible gastroenteritis coronavirus (tgev) by recombinant adenovirus expressing tgev spike protein oral immunization of mice with recombinant lactococcus lactis expressing porcine transmissible gastroenteritis virus spike glycoprotein the authors of this paper have no financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. key: cord-338108-3rn6fwx3 authors: jin, yi; zhang, yuewei; wan, chunyan; wang, hongjun; hou, lingyu; chang, jianyu; fan, kai; xie, xiangming title: immunomodulatory activity and protective effects of polysaccharide from eupatorium adenophorum leaf extract on highly pathogenic h5n1 influenza infection date: 2013-09-18 journal: evid based complement alternat med doi: 10.1155/2013/194976 sha: doc_id: 338108 cord_uid: 3rn6fwx3 the development of novel broad-spectrum, antiviral agents against h5n1 infection is urgently needed. in this study, we evaluated the immunomodulatory activities and protective effect of eupatorium adenophorum polysaccharide (eap) against the highly pathogenic h5n1 subtype influenza virus. eap treatment significantly increased the production of il-6, tnf-α, and ifn-γ both in vivo and in vitro as measured by qpcr and elisa. in a mouse infection model, intranasal administration of eap at a dose of 25 mg/kg body weight prior to h5n1 viral challenge efficiently inhibited viral replication, decreased lung lesions, and increased survival rate. we further evaluated the innate immune recognition of eap, as this process is regulated primarily dectin-1 and mannose receptor (mr). these results indicate that eap may have immunomodulatory properties and a potential prophylactic effect against h5n1 influenza infection. our investigation suggests an alternative strategy for the development of novel antiinfluenza agents and benefits of e. adenophorum products. highly pathogenic h5n1 subtype influenza virus can be transmitted directly from poultry to human and cause acute respiratory infections. pandemic influenza virus h5n1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity [1, 2] . the symptoms in animals or humans infected with h5n1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome (sars) [3, 4] . the world health organization reported 622 human cases of highly pathogenic h5n1 influenza virus infection, including 371 deaths (a mortality rate >50%), from 2003 to 2013 (http://www.who.int/ influenza/human animal interface/h5n1 cumulative table archives/en/index.html). currently, the most effective preventive measure against the influenza virus is vaccination. several antiinfluenza medications have been widely used, including zanamivir (relenza) and oseltamivir (tamiflu). unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation [5, 6] . therefore, the development of novel antiinfluenza agents against the h5n1 subtype is very important. the invasive plant eupatorium adenophorum, native to central america, has a strong ability to adapt to different environments all over the world. this plant first invaded southern yunnan province (china) in the 1940s from burma and vietnam, and quickly spread across southwestern china throughout the 1950s [7, 8] . over the past 50 years, e. adenophorum has seriously impacted the ecological environment in china's middle subtropical zones, including yunnan, guizhou, sichuan, and guangxi provinces, by encroaching farmlands, pasture fields, and forests [7] . manual, chemical, or biological control of e. adenophorum has hindered its comprehensive development and utilization for economic benefit. many bioactive components isolated from e. adenophorum have shown antimicrobial activity and immunomodulating 2 evidence-based complementary and alternative medicine properties [9] . in a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated [10] . however, there have been few reports addressing the bioactivity of e. adenophorum polysaccharide (eap). the immunomodulating properties and therapeutic potential of a large number of botanical polysaccharides have been reported [11] . several polysaccharides from cordyceps militaris, portulaca oleracea, gracilaria lemaneiformis, gyrodinium impudium, and panax ginseng have been described as efficacious antiinfluenza agents against h1n1 and h3n2 strains [12] [13] [14] [15] . in recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of h5n1 vaccines in animal infection models [16, 17] . however, to our knowledge there have not been any reports regarding the treatment with eap against highly pathogenic h5n1 influenza. in the present study, we investigated the potential effect of eap against h5n1 influenza infection in a mouse model. immune enhancement effects and the innate immune recognition of eap were also evaluated. our results suggest the anti-h5n1 effects of eap offer an alternative strategy for developing antiinfluenza agents and the utilization of e. adenophorum products. virus. the h5n1 influenza virus (a/bar-headed goose/ qinghai/1/2010) used in this study was isolated from qinghai lake in may 2010. this isolate is highly pathogenic in poultry, mouse, and madin-darby canine kidney (mdck) cells. the virus was propagated in mdck cells at 37 ∘ c for 48 h, and the viral supernatant was harvested, aliquoted, and stored at −80 ∘ c. viral titers were determined by plaque assay as described previously [18] . animal and cells. 8-10-week-old female balb/c mice were obtained from vital river laboratories (beijing, china), and the original breeding pairs were purchased from charles river (beijing, china). mice were raised in independent ventilated cages (ivc) and received pathogen-free food and water. animal treatments were governed by the regulations of experimental animals of beijing authority, and approved by the animal ethics committee of the china agriculture university. the mouse leukemic monocyte macrophage raw 264.7 cell line, human lung adenocarcinoma epithelial a549 cell line, and madin-darby canine kidney (mdck) cell lines were provided by the cell resource center of peking union medical college. the cells were cultured and maintained according to the supplier's recommendations. yunnan province, china. the leaves were sliced and dried in shade. 100 g dried materials were powdered in a mixer and then filtered with 40 meshes. leaf powder was extracted by ultrasonic treatment with 1000 ml of distilled water for 45 min. the supernatant was collected and the precipitate resuspended in 1000 ml of distilled water and again extracted by ultrasonic treatment for 30 min. the resulting supernatant was combined with that obtained from the first ultrasonic treatment. the final aqueous fraction was evaporated to dryness in a rotary evaporator. the residue obtained was dissolved in distilled water and kept frozen at 4 ∘ c. the extract was centrifuged at 3000 g/min for 25 min and concentrated under 80 ∘ c for 8 h to prepare polysaccharide. the supernatant was then deproteinized using the sevag method, and dialyzed against water for 48 h. the final liquid was mixed with three-fold volume of 95% ethanol (v/v) and centrifuged at 3000 g/min for 10 min. the precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ c to obtained the crude polysaccharide (yield = 1.2%). eap content was determined by the phenol-h 2 so 4 method [19] . vitro. 2.5 ml a549 and raw 264.7 cells (4 × 10 5 /ml) per well were plated in 6-well plates and cultured at 37 ∘ c under 5% co 2 for 24 h. media was removed and 2.5 ml culture medium containing different concentrations of eap (50, 100, 200 g/ml) was added to each well. controls were treated with phosphate-buffered saline (pbs). cells were collected 36 h after treatment for rna extraction and quantitative polymerase chain reaction (qpcr). assay. mice were administrated eap at a dose of 5, 10, 25, or 50 mg/kg body weight, intranasally once daily for 5 days before the challenge. control mice were administered pbs using the same schedule. influenza virus stocks were diluted in pbs. mice were anesthetized with zotile (virbac, france) intramuscularly at 15 mg/kg (body weight) and then infected intranasally with 120 plaqueforming units (pfu) of h5n1 influenza virus in 50 l. the lung tissue of five mice per group was collected on day 0 before challenge for qpcr and elisa. lung tissue from another five mice on day 3 postinfection was collected for plaque assay and qpcr. ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. plaque assay. mdck cells were cultured in dmem (hyclone laboratories, logan, ut, usa) containing 10% fbs (hyclone laboratories), 100 u/ml penicillin, and 100 g/ml streptomycin (invitrogen, san diego, ca, usa). lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/ml of trypsin tpck (sigma-aldrich, st. louis, mo, usa). plates were incubated at 37 ∘ c, 5% co 2 for 60-72 h and stained with 1% crystal violet. total rna from 1 × 10 6 cells or 10 mg lung tissue were prepared by trizol (invitrogen) according to the manufacturer's instructions. dnaseitreated rna (0.2 g) was reverse transcribed into cdna using random primers. the expression of the hemagglutinin (ha) gene of h5n1 influenza virus was detected by qpcr using the power sybr green pcr master mix kit (applied biosystems, foster city, ca, usa). the following primers agg cac ca-3 5 -ctc ctt aat gtc acg cac gat ttc-3 h il-6 5 -cct tcg gtc cag ttg cct tct-3 5 -cca gtg cct ctt tgc tgc ttt c-3 h ifn were used: forward primer, 5 -cgc agt att cag aag aag caagac-3 ; and reverse primer, 5 -tcc ata agg ata gac cag cta cca-3 . the reaction was run on an abi 7500 thermal cycler with an initial denaturation step at 95 ∘ c for 10 min, followed by 40 cycles of 95 ∘ c for 15 s, 56 ∘ c for 30 s, and 72 ∘ c for 40 s. the copy number of the ha gene was calculated by 7500 software v2.0 (applied biosystems) using an ha-containing plasmid of known concentration as a standard. relative qpcr was performed for other eight genes: hactin, h il-6, h ifn-, and htnf-for a549 cells; mactin, mtlr-2, mtlr-4, mdectin-1, mmr, mil-6, mifn-, and mtnf-for raw264.7 cells. the sequences of primers were shown in table 1 . the reaction was run with 95 ∘ c for 10 min, followed by 40 cycles of denaturation at 95 ∘ c for 15 sec, annealing at 52 ∘ c for 30 s, and extension at 72 ∘ c for 40 s. the fold change in gene expression was normalized to controls (naive mice) by 2 −δδct using -actin as an internal standard [20] . 2.8. elisa. il-6, tnf-, and ifn-levels in lung were tested with elisa kits (boster, wuhan, china) according to the manufacturer's protocol. one gram of lung tissue from each mouse was ground in 1 ml pbs and centrifuged for 20 min at 5000 rpm. the supernatants were collected and diluted 10fold for elisa. 2.10. statistical analysis. the statistical analysis was performed using one-way anovas with spss 12.0 (spss taiwan corp., taiwan), and < 0.05 was considered significant. many botanical polysaccharides exhibit an immunomodulatory effect [11] . to determine the immunomodulatory properties of eap, we investigated the potential effect of the polysaccharides on a549 and raw264.7 cells. cells were treated with various concentrations of eap (50, 100, 200 g/ml) for 36 h. the mrna levels of il-6, tnf-, and ifn-were detected by qpcr. figure 1 shows the immunomodulatory activities of eap in vitro. various concentrations of eap triggered a strong secretion of il-6, tnf-, and ifn-in a dosedependent manner both in a549 cells (figures 1(a)-1(c) ) and raw264.7 cells (figures 1(d) -1(f)) compared with the pbs treatment group. to test whether eap could protect h5n1 infected mice, mice were treated with eap at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 pfu. ten mice per group were monitored for 14 days for the survival rate. as shown in figure 2 (a), all mice receiving pbs died at day 11. mice administrated 25 mg/kg eap had a survival rate of 50% at day 14, which was significantly higher than those receiving pbs (by log rank analysis). eap treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant. this result suggests that the protective effect of eap against h5n1 infection requires a moderate dose. eap treatment also alleviated weight loss in infected mice (figure 2(b) ). to determine the viral load in the lung of the infected mice, plaque assays and qpcr were performed. the pulmonary viral titers in the eap (25 mg/kg) group were significantly lower than the titers in the mice that received pbs at day 3 postinfection (figures 2(c) and 2(d) ). these data clearly indicate that intranasal administration of eap controls h5n1 viral replication and improves survival rates in a mouse model. the protective effect of eap against h5n1 virus is likely due to its immunomodulatory properties. to detect il-6, tnf-, and ifn-expression, lungs of five mice per group were collected at day 0 before infection and tested by qpcr and elisa. the mrna levels in the eap group (25 mg/kg) were significantly higher than those in the pbs control (naive mice) (figures 3(a)-3(c) ). soluble cytokine levels at day 0 were measured by elisa, and results were consistent with the qpcr results, even though ifn-production in the eap group was not significantly higher than that of the pbs group ( = 0.0599) (figures 3(g)-3(i) ). these results suggest that eap increases the il-6, tnf-, and ifn-production. il-6, tnf-, and ifn-expression at day 3 postinfection was determined by qpcr. in contrast, tnf-mrna levels following eap (25 mg/kg) treatment were significantly lower than those in the pbs group (figure 3(e) ), while il-6 and ifn-expression were only slightly lower (not significant) (figures 3(d) and 3(f) ). these results may be explained by a higher viral load, and the more severe inflammatory response in pbs treated mice. excessive inflammation can cause severe lung lesions during h5n1 influenza infection. to evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. the lungs of pbs treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls (figures 4(c) and 4(d) ). the lungs of eap (25 mg/kg) treated mice exhibited milder lesions than those receiving pbs, characterized by signs of bronchopneumonia with interstitial edema, and inflammatory cell infiltration around small blood vessels (figures 4(a) and 4(b) ). viral loads and inflammatory cytokine production in the lung were correlated; suggesting that eap treatment reduces lung lesions in h5n1 infected mice. polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as tnf-, il-6, il-8, and il-12 [11] . this immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors (prrs). to determine which receptor contributes directly to the innate immune recognition of eap, toll-like receptor 2 (tlr2), tlr4, dectin-1, and mannose receptor (mr) were examined by qpcr both in vivo and in vitro. mice were treated with eap at a dose of 25 mg/kg body weight intranasally once daily for 5 days, with control mice receiving pbs. lung total rna was prepared for qpcr. the expression of dectin-1 and mr in eap treated mice was significantly elevated compared with controls, while expression of tlr2 and tlr4 were slightly higher, but not statistically significant (figure 5(a) ). in vitro assay showed similar trends. as shown in figure 5 (b), raw264.7 cells were treated with 200 g/ml epa for 36 h before qpcr. dectin-1 and mr levels were significantly higher, while expression of tlr2 and tlr4 did not change. these data suggest that eap recognition occurred mainly via the dectin-1 and mr pathway. in this study, we evaluated the immunomodulatory activities and protective effect of eap against h5n1 influenza infection in a mouse model. to our knowledge, these findings are the first to show the anti-h5n1 effect of eap. intranasal administration of eap prior to h5n1 viral challenge improved survival rates of infected mice with a corresponding reduction of pulmonary viral load. the anti-h5n1 effect was very likely due to the innate immune recognition of eap and the secretion of innate immune mediators (il-6, tnfand ifn-) before infection. furthermore, the effect of eap on prr expression (including tlr2, tlr4, dectin-1, and mr) was determined both in vivo and in vitro. these results suggest that the innate immune recognition of eap was dependent upon the activation of the dectin-1 and mr pathways. our data demonstrate the feasibility of using eap as a novel immunomodulatory agent against influenza infection. unfortunately, the sugar composition of eap has not been characterized. the emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza [6, 21, 22] . thus, development of novel broad-spectrum antiinfluenza strategies is urgently needed. most botanical polysaccharides are ideal candidates for novel immunomodulatory agents due to their nontoxic properties and fewer side effects compared with bacterially derived polysaccharides. a number of polysaccharides isolated from plant and fungi exhibit effective antiviral benefits against influenza a virus (including h1n1 and h3n2 subtypes) [12] [13] [14] [15] . the use of polysaccharides as immunomodulatory agent in anti-h5n1 studies is rare. in this paper, our data show the immunomodulatory activities of eap both in vivo and in vitro. eap treatment elevated the production of il-6, tnf-, and ifnand provides a survival advantage in h5n1 infected mice. the survival rate following eap pretreatment (25 mg/kg body weight) was significantly higher than in mice receiving pbs (50% to 0%). in previous reports, high levels of proinflammatory cytokines and chemokines (including tnf-, il-6 and ifn-) were detected during h5n1 infection [23, 24] . this "cytokine storm" leads to the severe respiratory symptoms and host immune injury. thus, h5n1-induced cytokine storms are hypothesized to be the main cause of mortality, and the use of anti-inflammatory agents may therefore provide a therapeutic effect [25, 26] . however, it is unclear whether the lack of proinflammatory cytokines (such as tnfand il-6) facilitates viral clearance. interestingly, knockout 8 evidence-based complementary and alternative medicine mice deficient in tnf-, tnf-receptor, il-6, mip-1 , and il-1r or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following h5n1 influenza infection [27, 28] . interestingly, prophylactic treatment of tlr3 agonist polyiclc, which strongly upregulates cytokine production, provides protection against h1n1 and h5n1 infections [29, 30] . these conflicting studies may be explained in that the inflammatory response helps clear the virus, while aggravating host pathological damage. elevated production of cytokines, such as il-6, tnf-, and ifnare very important for viral clearance in the early stage of infection by activating the innate immune system. once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. in this case, receiving an immunomodulator alone cannot help animal to survive [25] . this likely explains why immunomodulator treatment prior to viral infection results in a better survival rate [26, 30] . in our study, treatment of eap shortly after infection or 24 h postinfection did not provide a survival advantage (data not show). the antiinfluenza properties of il-6, tnf-, and ifnhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. il-6 plays an important role in protecting against influenza a virus as it is required for viral clearance and essential for animal survival [31] . tnf-has been reported to exert a defensive effect against influenza infection in vitro [32] . ifn-treatment in the early stages of influenza infection improves the survival rate in mouse models [33] . in addition, high levels of ifn-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo [12] . in this report, intranasal administration of eap before h5n1 challenge elevates expression of il-6, tnf-, and ifncompared with mice receiving pbs. the high levels of these mediators contribute to the viral clearance and antiviral response. pulmonary viral titers following eap treatment were lower at day 3 postinfection. in contrast, il-6 and ifn-mrna levels were slightly lower, while tnf-production was significantly lower than that of pbs group. regarding the excessive inflammation induced by h5n1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response. therefore, the timing of eap treatment as a prophylactic agent is very important. the immunomodulatory activities of botanical polysaccharides are thought to be mediated by several prrs [11] . in this study, we examined the mrna levels of tlr2, tlr4, dectin-1, and mr after eap treatment. eap was found to upregulate dectin-1 and mr mrna expressions significantly both in vivo and in vitro. our hypothesis is that the innate immune recognition of eap is driven mainly via a dectin-1 and mr dependent pathway. binding to these receptors, eap may activate complex intracellular signaling pathways, and increase cytokine production, leading to an antiviral response. thus, the protection against h5n1 by eap treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. in conclusion, our study demonstrates that eap leaf extract is a prophylactic and immune enhancement agent against h5n1 influenza virus infection. treatment with eap effectively inhibits h5n1 viral replication and improves animal survival. this approach offers an alternative strategy for antiinfluenza immunomodulatory agent development, and benefits the utilization of e. adenophorum products. establishment of multiple sublineages of h5n1 influenza virus in asia: implications for pandemic control h5n1 influenza: a protean pandemic threat acute respiratory distress syndrome induced by avian influenza a (h5n1) virus in mice the comparative pathology of severe acute 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influenza viruses in mice proinflammatory cytokine responses induced by influenza a (h5n1) viruses in primary human alveolar and bronchial epithelial cells delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza a/h5n1 virus inhibition of highly pathogenic avian h5n1 influenza virus propagation by rna oligonucleotides targeting the pb2 gene in combination with celecoxib inhibition of the cytokine response does not protect against lethal h5n1 influenza infection a rationale for using steroids in the treatment of severe cases of h5n1 avian influenza activation of toll-like receptor signaling pathway for protection against influenza virus infection prophylactic, therapeutic and immune enhancement effect of liposome-encapsulated polyiclc on highly pathogenic h5n1 influenza infection essential role of il-6 in protection against h1n1 influenza virus by promoting neutrophil survival in the lung tumor necrosis factor alpha exerts powerful anti-influenza virus effects in lung epithelial cells ifn-treatment at early stages of influenza virus infection protects mice from death in a nk cell-dependent manner yi jin and yuewei zhang contributed equally to this work. key: cord-273035-sewfb3q8 authors: kang, xixiong; xu, yang; wu, xiaoyi; liang, yong; wang, chen; guo, junhua; wang, yajie; chen, maohua; wu, da; wang, youchun; bi, shengli; qiu, yan; lu, peng; cheng, jing; xiao, bai; hu, liangping; gao, xing; liu, jingzhong; wang, yiping; song, yingzhao; zhang, liqun; suo, fengshuang; chen, tongyan; huang, zeyu; zhao, yunzhuan; lu, hong; pan, chunqin; tang, hong title: proteomic fingerprints for potential application to early diagnosis of severe acute respiratory syndrome date: 2005-01-01 journal: clin chem doi: 10.1373/clinchem.2004.032458 sha: doc_id: 273035 cord_uid: sewfb3q8 background: definitive early-stage diagnosis of severe acute respiratory syndrome (sars) is important despite the number of laboratory tests that have been developed to complement clinical features and epidemiologic data in case definition. pathologic changes in response to viral infection might be reflected in proteomic patterns in sera of sars patients. methods: we developed a mass spectrometric decision tree classification algorithm using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. serum samples were grouped into acute sars (n = 74; <7 days after onset of fever) and non-sars [n = 1067; fever and influenza a (n = 203), pneumonia (n = 176); lung cancer (n = 29); and healthy controls (n = 659)] cohorts. diluted samples were applied to wcx-2 proteinchip arrays (ciphergen), and the bound proteins were assessed on a proteinchip reader (model pbs ii). bioinformatic calculations were performed with biomarker wizard software 3.1.1 (ciphergen). results: the discriminatory classifier with a panel of four biomarkers determined in the training set could precisely detect 36 of 37 (sensitivity, 97.3%) acute sars and 987 of 993 (specificity, 99.4%) non-sars samples. more importantly, this classifier accurately distinguished acute sars from fever and influenza with 100% specificity (187 of 187). conclusions: this method is suitable for preliminary assessment of sars and could potentially serve as a useful tool for early diagnosis. causative agent for sars (7, 8 ) . rapid progress has also been made in the determination of its genome sequences (9 -11 ) and the molecular evolution of the coronavirus (12 ) . identification of angiotensin-converting enzyme 2 as the viral receptor provided further information toward deciphering its molecular mechanisms of infection (13 ) . despite such advances in virologic studies, early diagnosis of sars has been based primarily on the clinical definitions released by who and cdc (14, 15 ) , which can be confusing or contradictory (16 ) . available serologic tests cannot guarantee an early diagnosis (17 ) , and pcrbased molecular detection of the viral rna suffers from unsatisfactory sensitivity and specificity (3, (17) (18) (19) . in the last year, failure to develop diagnostic tests for sars, especially in the acute phase, severely impacted specific prevention and treatment measures for sars. there is a need to establish a reliable diagnostic methodology for sars-cov, in particular, to distinguish the similar clinical manifestations of sars and other respiratory tract infections. this urgency is reinforced by the first sars case not linked to laboratory contamination, which occurred in guangdong, china this year (20 ) . proteomic analysis has provided a unique tool for the identification of diagnostic biomarkers, evaluation of disease progression, and drug development (21, 22 ) . surfaceenhanced laser desorption/ionization time-of-flight mass spectrometry (seldi-tof ms) enables rapid, reproducible protein/peptide profiling of multiple disease-specific biomarkers directly from crude samples (e.g., tissue cell lysates or body fluids) (23, 24 ) . small amounts of sample can be applied directly to a biochip coated with specific chemical matrices (e.g., hydrophobic, cationic, or anionic) or specific biochemical materials such as dna fragments or purified proteins. the bound proteins/peptides can then be analyzed by ms to obtain the protein fingerprints, or even amino acid sequence determinants, when interfaced to a mass spectrometric microsequencing device. analogous to the proteomic detection of various cancers (25, 26 ) , we used a weakly cationic proteinchip (wcx2 chip surface) to retrospectively analyze sars sera to determine whether there are distinct and reproducible protein fingerprints potentially applicable to the diagnosis of sars. we established a decision tree algorithm consisting of four unique biomarkers for acute sars in the training set and subsequently validated the accuracy of this classifier by use of a completely blinded test set. more than 2000 serum specimens from suspected/probable sars patients admitted to 38 major hospitals in the beijing area between april 14 and june 5, 2003, were eligible for inclusion. the serum procurement, data management, and blood collection protocols were approved by the beijing sars-control working group and were in accordance with who biosafety guidelines (27 ) . among the retrospective samples, only 74 were selected from probable patients whose blood samples were collected with onset of fever within 7 days at the time of admission (acute sars patients; table 1 ). probable cases were based on the eligibility criteria set forth by who (15 ) . these cases had also radiographic evidence of infiltrates consistent with pneumonia or respiratory distress syndrome on chest x-ray. the paired convalescent serum samples from the sars cohort tested positive for igm seroconversion by the ifa method (beijing genomics institute), and four samples also tested positive in a dna array test using nasopharyngeal samples. the 1067 non-sars control sethe patients and serum samples were then divided into two groups: one for the "training" set and the other for the blinded "test" set (tables 1 and 2 ). sars and non-sars control sera were all stored at ϫ80°c in 30-l aliquots. before each round of mass spectrometric assays, we routinely performed quality control of serum samples by the appearance and peak intensity of m/z 6635.09 (fig. 3a ). because the peak intensity of m/z 6635.09 remained relatively constant among spectra from different assays and different instruments, it was also used for normalization between each round of analyses. three different chip chemistries (hydrophobic, anionic, and cationic) were first evaluated to determine which affinity chemistry gave the best serum profiles in terms of the number and resolution of proteins. the weakly cationic exchange chip (wcx) gave the best results with mass spectra from 0 to 200 kda. the wcx chips in an 8-well bioprocessor format (ciphergen) were chosen to allow a larger volume of serum for the chip array. the bioprocessor was pretreated with 150 l of 100 mmol/l sodium acetate (ph 4) on a platform shaker at 250 rpm for 5 min. the excess sodium acetate was removed by inverting the bioprocessor on a paper towel. this process was repeated twice. the serum samples were thawed on ice in a biosafety level ii cabinet, and 20 l of each sample was mixed with 30 l of u9 buffer (9 mol/l urea, 10 g/l chaps in phosphate-buffered saline) in a 1.5-ml eppendorf tube and vortex-mixed at 4°c for 20 min. we then added 100 l of u1 buffer [u9 buffer diluted by ninefold (100 ml of u9 buffer plus 800 ml of tris-hcl) with 50 mmol/l tris-hcl (ph 7)] to the serum/urea mixture, vortex-mixed it for 10 min, and stopped the reaction by addition of 600 l of sodium acetate on ice. we applied 50 l of the serum/urea sample to each well, and the bioprocessor was sealed and shaken on a platform shaker at 250 rpm for 30 min. the excess serum/urea solution was discarded, and the bioprocessor was washed three times with 100 mmol/l sodium acetate as described above. the chips were removed from the bioprocessor, washed twice with deionized water, and air-dried. subsequently 0.5 l of eam sinapinic acid saturated in 500 ml/l acetonitrile-5 g/l trifluoroacetic acid was added to each well. after air-drying, the sinapinic acid application was repeated. chips were then placed in the protein biological system ii (pbs ii) mass spectrometer reader (ciphergen), and tof spectra were generated by an average of 104 laser shots collected in the positive mode. the settings for low-energy readings were set with a high mass of 50 kda and were optimized from 3 to 15 kda at a laser intensity of 200, detector sensitivity of 8, and a focus by optimization center. high-energy readings were set with a high mass of 200 kda and were optimized from 10 to 50 kda at a laser intensity of 230 and a detector sensitivity of 9. mass accuracy was calibrated externally by use of the all-in-one peptide molecular mass calibrator (ciphergen). sera from a healthy control were individually applied to seven bait surfaces of eight wcx2 chips and run during 3-day intervals for analysis of within-run reproducibility. in parallel, 40 samples (10 from sars patients, 10 from patients with fever, 10 from patients with pneumonia, and 10 from health controls) were applied in duplicate to a single chip and run on two different instruments (pbs ii and pbs iic; ciphergen) for between-run analysis of instrument drift. to avoid the possibility that placement or run order of samples would affect assay accuracy, samples were loaded on chips in a rotational fashion. in in the pericardium (n ϭ 1), upper right clavicle (n ϭ 1), lymph nodes (n ϭ 1), liver (n ϭ 1), and brain (n ϭ 1); accompanying hydrothorax was also observed in nine patients. brief, sample 1 was spotted on the 8-well directional chip (wells a to h) in duplicate in wells a and b and then in wells g and h of the second chip. samples 2, 3, and 4 were loaded on chips in the same rotation order. we also randomized the order of chip placement in the spectrometer to minimize bias from run order. spectra were collected for each sample and analyzed independently using the classification algorithm established in the training step. the peak at m/z 6635.09 in the quality-control serum was adjusted to have an intensity of 40 -60 for both the pbs ii and pbs iic. the peak intensity of m/z 6635.09 in the quality-control serum was used to normalize instrument resolution between the pbs ii and pbs iic. we normalized spectra using total ion current with an identical normalization coefficient and a low mass cutoff ͻ2000 da. if the factor was ͻ0.3 or ͼ2.9 after normalization to total ion current for the peak at m/z 3939, repeated runs would be performed. no outlier was rejected in the test. the "root" biomarker, m/z 3939, yielded the lowest and similar p value in both the pbs ii and pbs iic. peak detection was performed with biomarker wizard software 3.1.1 (ciphergen). the m/z ratios between 2000 and 20 000 were selected for analysis because this range contained the majority of the resolved protein and peptides. the m/z range between 0 and 2000 was eliminated from analysis to avoid interference from adducts, artifacts of the energy-absorbing molecules, and other possible chemical contaminants. peak detection involved baseline subtraction, mass normalization using a common calibrant peak (m/z 6635.09), and normalization to the total ion current intensity with a minimum m/z of 2000, using an external normalization coefficient of 0.2 (normalization factor for individual spectrum ϭ 0.2/average ion current for each spectrum) for spectra obtained at different times or locations. the settings used for autodetect peaks to cluster in the first pass were a signal-to-noise ratio of 5 and a minimum peak threshold of 5% of all spectra. the peak clusters were completed by second-pass peak detection using a signal-to-noise ratio of 2 and 0.3% of mass for the cluster window. an average of 99 peaks was detected in each spectrum. the mass range from 20 to 200 kda was analyzed in parallel. analytical procedure data analysis.the data analysis process used in this study involved three stages: (a) peak detection and alignment; (b) selection of peaks with the highest discriminatory power; and (c) data analysis using a decision tree algorithm. a random sampling (acute sars, fever, pneumonia, lung cancer, and healthy) with two strata (acute sars and non-sars) was used to separate the entire data set into training and test data sets. the training data set consisted of seldi spectra from 37 acute sars and 74 non-sars serum samples. the validity and accuracy of the classification algorithm were then challenged with a blinded test data set consisting of 37 acute sars and 993 non-sars samples. decision tree classification. construction of the decision tree classification algorithm was performed as described previously (26 ) with modifications based on the biomarker patterns software (ciphergen). classification trees were split into two branches or nodes, using one rule at a time. we set target the variable level at 2 and the minimum value at 0, and the decision was made based on the presence or absence and the intensity of one peak, using the gini or twoing method, favoring even splits from 0.00 to 2.00 and varied by 0.2 each time, and with v-fold cross-validation from 6 to 12 changed by 2 for the growth of 88 trees. the lowest cost tree (value ϭ 0.068; gini ϭ 2.0; v-fold ϭ 10) was selected for the final test. to identify the serum biomarkers that could distinguish sars from non-sars samples, we used a training set of specimens (37 sars acute and 74 controls; tables 1 and 2) and constructed the decision tree classification algorithm using 10 989 peaks [99 peaks ϫ (37 ϩ 74) spectra] of statistical significance identified in the low energy readings (see materials and methods). the classification algorithm used four peaks between 3 and 12 kda (m/z 3939.08, 4137.71, 8136.64, and 11 514.2) and generated five terminal nodes (fig. 1) . these discriminatory peaks efficiently split sars specimens into terminal nodes 3 and 5 and non-sars samples into terminal nodes 1, 2, and 4. each mass peak showed a mean intensity ratio of sars vs non-sars ͼ3 and a p value close to 0 (table 3) . notably, the protein or peptide with masses at 3939.08, 8136.64, and 11 514.2 da was up-regulated in patients with acute sars, whereas that of a mass at 4137.71 da was down-regulated compared with healthy controls or patients with respiratory tract infections. a representative spectrum of a sars specimen aligned with that of a healthy control ( fig. 2a) showed the four fingerprints in node 3 required for pattern recognition in the classifier. the unique presence of the root biomarker, m/z 3939.08, is demonstrated in the alignment of representative spectra of samples from patients with acute sars (1, 3, 5, and 7 days after the onset of fever; from terminal node 5) and those from healthy controls and patients with fever and influenza or pneumonia (fig. 2b) . this decision algorithm correctly classified 37 of 37 (100%) of the acute sars samples and 72 of 74 (97.3%) of the non-sars controls in the training set ( table 3) . the above classifier used only those masses in the low-energy readings (m/z ͻ50 000). to exhaust all meaningful serum biomarkers, we expanded the analysis of the same training samples in the high-energy setting (m/z combine two energy settings for analysis, we reasoned that the decision tree generated with only low-energy readings (fig. 1) would be more sensitive (100%) and more convenient for a clinical application. to determine the reproducibility of seldi spectra, mass location, and intensity from array to array on a single chip (intraassay) and between instruments (interassay), we first spotted the serum from a healthy control on seven baits in a single chip and collected seven independent spectra over a time span of 21 days (fig. 3a) . we then selected seven proteins in the range of 3-10 kda (m/z 4089.59, 5334.17, 5631.18, 5901.49, 6625.63, 7762.24 , and 7966.63; black arrows in fig. 3a ) to calculate the intraassay cv. these peaks were selected because they were in the proximity of the four biomarkers with comparable current intensities. the interassay experiments were similar except that sera from healthy controls and from patients with high fever, pneumonia, and sars were applied to a single chip, and the independent spectra were collected from two different instruments (pbs ii and pbs iic; fig. 3 , b and c). the mean intra-and interassay cvs for peak location were 0.02% and 0.03%, respectively. we considered masses with accuracies within 0.1% between spectra to be the same. the mean intra-and interassay cvs for the normalized intensity were 15% and 20%, respectively. cv calculations using lower intensity peaks (fig. 3a, gray arrowheads) , on the other hand, yielded results similar to those obtained with the seven high-intensity peaks (peak location, intra-and interassay cvs both 0.03%; peak intensity, intraassay cv ϭ 17% and interassay cv ϭ 18%). analysis of spectra from the completely blinded test set (37 acute sars and 993 controls; tables 1 and 2) accurately classified 36 of 37 (97.3%) sars specimens and accurately classified 987 of 993 (99.4%) of the controls as non-sars (table 3 ). more important was that the classification algorithm successfully distinguished acute sars from fever and influenza, with a sensitivity and specificity reaching 97.3% (36 of 37) and 100% (187 of 187; 60 of 60 with influenza), respectively. interestingly, when we tested the classifier using an additional control population of 40 samples from patients in the beijing area with measles after july 16, 2003 , who had no history of close contact with sars patients and had not visited those hospitals treating sars patients, the classifier had a specificity of 100% (95% confidence interval, 89 -100%; data not shown). several laboratory tests, based on either viral rna (3, 17, 19 ) or serology (6, 17 ) , have been developed to complement clinical characteristics and epidemiologic data in the identification of sars, but early detection of sars with sufficiently high sensitivity and specificity has not been achieved. the identification of proteins/peptides of pathophysifig. 3 . intra-and interassay reproducibility. (a), example of intraassay reproducibility of mass spectra and tree decision classification. serum from an unaffected healthy control was individually applied to seven bait surfaces on eight chips, and seven randomly selected peaks (arrows) in each spectrum over a course of 27 days were used as surrogate markers for calculation of cv. the reproducibility of seldi spectra, mass location, and intensity from spectrum to spectrum was determined accordingly. ologic significance (phenomic fingerprints) in crude biological and clinical samples by seldi-tof ms has been demonstrated in various cancer studies (28 ) . using a similar profiling strategy, we have established a classification algorithm that delineates probable sars patients as early as day 1 after self-described onset of symptoms from healthy individuals and from patients with respiratory tract infections in the training set (sensitivity ϭ 100%; specificity ϭ 97.3%). when applied to the blinded test set, this discriminatory profiling method precisely classified 97.3% of patients with acute sars and 99.4% of non-sars patients. more strikingly, our classifier was able to discriminate sars-cov infection from bacterial (mycoplasma, tuberculosis) and other local (influenza) or systemic (measles) viral infections of the respiratory tract with a specificity reaching 100%. this was attributable to the inclusion of corresponding inflammatory control samples in the training set and optimization of the classification algorithm. the biomarkers identified in the acute phase of sars seemed to remain throughout the convalescent phase of the disease because when we applied the identical tree classification to samples from patients in whom onset of fever had been ͼ2, 3, 4, and ͼ5 weeks previously, we could detect sars with sensitivities and specificities reaching 89.2% and 91.8%, 86.0% and 91.8%, 93.1% and 91.8%, and 79.5% and 91.8%, respectively (data not shown). one intriguing observation was that sars patients clustering in terminal node 3 all demonstrated moderate clinical features, whereas those in node 5 were severe cases. we are investigating the correlation between this proteomic pattern and the pathology of sars. these results represent, to the best of our knowledge, the most accurate laboratory technique for early detection of sars: pcr-based assays have a maximum sensitivity of 80% when used to test nasopharyngeal aspirates or plasma specimens (29, 30 ) . the proteomic method described here also has advantages over pcr-based assays in that it does not require bsl-3 containment and it can detect sars in serum samples. this is a critical alternative to pcr-based tests, which are challenged by low viral loads in nasopharyngeal aspirates and throat swab specimens in the acute phase of sars. instead of traditional chromatographic fractionation of samples, we directly spotted the crude serum on the wcx chips. by doing this we avoided the unnecessarily biased depletion of thousands of proteins and/or peptides associated with human serum albumin before ms analysis. processing of samples and generation of the diagnostic mass spectra by our method required only a small amount of serum (20 l vs several milliliters needed for pcr methods) and took ͻ3 h. high-throughput proteomic screening for sars in a 96-well format is also feasible. we adhered to the who case definition and eligibility criteria for sars and avoided using samples from non-sars controls from hospitals where sars patients had been admitted because these persons might have a history of close contact with sars patients or had been inside those sars hospitals. we further emphasized this point by sampling control sera from a nonepidemic region of the country. although the possibility might exist that the difference in serum fingerprints would reflect differences among sars and non-sars hospitals, the fact that all sars cases from 38 different hospitals fit into the single classification algorithm would likely rule out such a concern. more importantly, severe and mild cases of sars from different hospitals, which had been completely randomized in the experimental analysis, fell into distinct nodes of the tree classification, strongly indicating that the biomarkers we have identified were specific to sars and not the sites at which blood samples were collected. we further minimized the potential sampling bias by simultaneously using four biomarkers instead of one (e.g., m/z 3939.08), which nevertheless could sufficiently delineate sars from non-sars (sensitivity ϭ 93.7%; specificity ϭ 91.8%; data not shown). all sars and non-sars samples were from patients with the same ethnic background. sars and non-sars control sera collected at different times were all freshly aliquoted and properly stored at ϫ80°c. the differential protein pattern as the discriminator between sars and non-sars is independent of protein identities. the origins and full identities of the discriminating biomarkers are under investigation. to know their identities for the purpose of differential diagnosis is not absolutely required, as shown by numerous studies showing diagnosis of cancers by seldi methods. however, to characterize these peaks would certainly help in understanding the biological roles of these peptide/proteins and could potentially lead to the discovery of more direct diagnostic tools and novel therapeutic targets for sars-cov. cumulative number of reported probable cases of sars epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome identification of severe acute respiratory syndrome in canada coronavirus as a possible cause of severe acute respiratory syndrome newly discovered coronavirus as the primary cause of severe acute respiratory syndrome koch's postulates fulfilled for sars virus characterization of a novel coronavirus associated with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus comparative full-length genome sequence analysis of 14 sars coronavirus isolates and common mutations associated with putative origins of infection sars-beginning to understand a new virus angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus updated interim u.s. case definition of severe acute respiratory syndrome (sars) case definitions for surveillance of severe acute respiratory syndrome (sars) clinical presentations and outcome of severe acute respiratory syndrome in children clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study quantitative analysis and prognostic implication of sars coronavirus rna in the plasma and serum of patients with severe acute respiratory syndrome rapid diagnosis of a coronavirus associated with severe acute respiratory syndrome (sars) laboratory confirmation of a sars case in southern china disease proteomics biomedical informatics for proteomics clinical proteomics translating benchside promise to bedside reality seldi proteinchip ms: a platform for biomarker discovery and cancer diagnosis use of proteomic patterns in serum to identify ovarian cancer serum protein fingerprinting coupled with a pattern-matching algorithm distinguishes prostate cancer from benign prostate hyperplasia and healthy men who biosafety guidelines for handling of sars specimens proteomic applications for the early detection of cancer detection of sars coronavirus in plasma by real-time rt-pcr crouching tiger, hidden dragon: the laboratory diagnosis of severe acute respiratory syndrome key: cord-274396-l611eisi authors: park, su-jin; yu, kwang-min; kim, young-il; kim, se-mi; kim, eun-ha; kim, seong-gyu; kim, eun ji; casel, mark anthony b.; rollon, rare; jang, seung-gyu; lee, min-hyeok; chang, jae-hyung; song, min-suk; jeong, hye won; choi, younho; chen, weiqiang; shin, woo-jin; jung, jae u.; choi, young ki title: antiviral efficacies of fda-approved drugs against sars-cov-2 infection in ferrets date: 2020-05-22 journal: mbio doi: 10.1128/mbio.01114-20 sha: doc_id: 274396 cord_uid: l611eisi due to the urgent need of a therapeutic treatment for coronavirus (cov) disease 2019 (covid-19) patients, a number of fda-approved/repurposed drugs have been suggested as antiviral candidates at clinics, without sufficient information. furthermore, there have been extensive debates over antiviral candidates for their effectiveness and safety against severe acute respiratory syndrome cov 2 (sars-cov-2), suggesting that rapid preclinical animal studies are required to identify potential antiviral candidates for human trials. to this end, the antiviral efficacies of lopinavir-ritonavir, hydroxychloroquine sulfate, and emtricitabine-tenofovir for sars-cov-2 infection were assessed in the ferret infection model. while the lopinavir-ritonavir-, hydroxychloroquine sulfate-, or emtricitabine-tenofovir-treated group exhibited lower overall clinical scores than the phosphate-buffered saline (pbs)-treated control group, the virus titers in nasal washes, stool specimens, and respiratory tissues were similar between all three antiviral-candidate-treated groups and the pbs-treated control group. only the emtricitabine-tenofovir-treated group showed lower virus titers in nasal washes at 8 days postinfection (dpi) than the pbs-treated control group. to further explore the effect of immune suppression on viral infection and clinical outcome, ferrets were treated with azathioprine, an immunosuppressive drug. compared to the pbs-treated control group, azathioprine-immunosuppressed ferrets exhibited a longer period of clinical illness, higher virus titers in nasal turbinate, delayed virus clearance, and significantly lower serum neutralization (sn) antibody titers. taken together, all antiviral drugs tested marginally reduced the overall clinical scores of infected ferrets but did not significantly affect in vivo virus titers. despite the potential discrepancy of drug efficacies between animals and humans, these preclinical ferret data should be highly informative to future therapeutic treatment of covid-19 patients. low sn titers, resulting in a prolonged infection. as several fda-approved or repurposed drugs are being tested as antiviral candidates at clinics without sufficient information, rapid preclinical animal studies should proceed to identify therapeutic drug candidates with strong antiviral potential and high safety prior to a human efficacy trial. keywords covid-19, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), antiviral therapeutics, immunosuppression, serum neutralization, ferrets i n december of 2019, a novel coronavirus (cov) disease , identified in wuhan, hubei province, china, in patients with pneumonia, was found to be caused by a previously unknown betacoronavirus. the outbreak rapidly spread to other provinces in mainland china, and despite great efforts, the epidemic continued to spread from china to europe, north america, and other asian countries. the world health organization (who) announced that the severe acute respiratory syndrome cov 2 (sars-cov-2) epidemic was a public health emergency of international concern on 30 january 2020, and on 11 march 2020, the who director general characterized covid-19 as a pandemic (http://www.euro.who.int/en/health-topics/health -emergencies/coronavirus-covid-19/news/news/2020/01/2019-ncov-outbreak-is-an -emergency-of-international-concern; https://www.who.int/dg/speeches/detail/who -director-general-s-opening-remarks-at-the-media-briefing-on-covid-19---11-march -2020). currently, the number of people diagnosed with sars-cov-2 infection is increasing by approximately 10,000 cases a day. as of 17 april 2020, approximately 2,074,529 cases had been diagnosed as covid-19 and 139,378 deaths had occurred (1) . further, recent studies reported the detection of sars-cov-2 rna in various clinical specimens, suggesting other transmission routes for this virus besides respiratory secretions (2) (3) (4) . unfortunately, to date, no vaccines or antiviral drugs have been approved for the treatment of sars-cov-2 infection by regulatory agencies. researchers are fervently working to develop vaccines specifically for this virus, as well as potential treatments for covid-19. although many clinical trials are ongoing, there are no specific therapeutic agents approved for covid-19. developing sars-cov-2-specific antiviral drugs from scratch could take years; drugs that have already been approved by the u.s. food and drug administration (fda) have the potential to reach patients more quickly. during the sars outbreak in 2003, screening of approved drugs identified lopinavir-ritonavir, a human immunodeficiency virus type 1 (hiv-1) aspartate protease inhibitor, as effective against sars-cov replication (5) . the antiviral activity of lopinavir-ritonavir against middle east respiratory syndrome coronavirus (mers-cov) both in vitro (6) and in an animal model (7) has been reported, and case reports suggest that the combination of lopinavir-ritonavir with ribavirin and interferon alpha results in virologic clearance and survival (8, 9) . chloroquine (cq), a widely used antimalarial with immunomodulatory effects (10), was found in a recent study to inhibit the growth of sars-cov-2 in vitro (11) . however, this finding has not been strongly supported by clinical studies of approximately 100 sars-cov-2-infected patients (12, 13) . a derivative of chloroquine, hydroxychloroquine (hcq) sulfate, was first synthesized in 1946 by adding a hydroxyl group to cq, resulting in a compound found to be much less toxic than cq in an animal study (14) . in autoimmune diseases, hcq sulfate works by reducing inflammation (15) . however, recent reports have also shown heart risk concerns with the use of cq and hcq sulfate for covid-19 treatment. emtricitabine-tenofovir (truvada) is a prescription medicine for hiv approved by the u.s. fda for preexposure prophylaxis to reduce the risk of hiv infection in adults and adolescents. as a nucleotide analogue, it is reported that the active triphosphate form of this tenofovir diphosphate inhibits activity for rna-dependent rna polymerase (rdrp) of hiv and hepatitis b virus (hbv) (16, 17) . still, even these existing drugs will need rigorous testing for efficacy and safety and ultimately ramped-up production before they can be deployed widely against covid-19. generally, immunocompromised patients are more susceptible to bacterial, fungal, viral, and parasitic infections than healthy persons due to their inability to mount successful immune responses. this can be caused by impairment or weakening of the immune system by a number of conditions, including diseases (e.g., diabetes or hiv infection), malnutrition, and the use of certain medications. it has become apparent that sars-cov-2 infection also affects immunocompromised individuals more severely. a majority of covid-19 patients who were clinically diagnosed are older than ϳ60 years and have underlying complications, including heart disease, diabetes, hypertension, or cancer, indicating that age and reduced immune activity are the critical risk factors or determinants for covid-19 morbidity and mortality. we have recently established a ferret model for sars-cov-2 infection and transmission that highly recapitulates aspects of the human infection (18) . elevated body temperatures and virus replication were readily detected in sars-cov-2-infected ferrets. sars-cov-2-infected ferrets shed the virus through nasal washes and in saliva, urine, and fecal specimens. sars-cov-2 was transmitted readily to naive direct-contact ferrets but less efficiently to naive indirect-contact ferrets (18) . further, acute bronchiolitis was observed in infected lungs. in this report, we evaluated the efficacy of oral administration of lopinavir-ritonavir, hcq sulfate, and emtricitabine-tenofovir for sars-cov-2 infection in ferret infection models. we also treated ferrets with azathioprine, an immunosuppressive drug, and evaluated the replication kinetics of sars-cov-2. while most drug treatments reduced clinical symptoms (cs), none of them led to a significant reduction of in vivo virus titers in ferrets. thus, a drug candidate study in a robust preclinical animal model should greatly facilitate testing the efficacies and safety of therapeutic treatments for covid-19 patients. clinical features of sars-cov-2-inoculated ferrets treated with antivirals. in order to determine the antiviral efficacies of lopinavir-ritonavir, hydroxychloroquine (hcq) sulfate, or emtricitabine-tenofovir for treatment of sars-cov-2 infection, sars-cov-2 antibody-free ferrets (10/group) were inoculated with 10 5.8 50% tissue culture infective doses (tcid 50 )/ml of an nmc-ncov02 strain through the intranasal (i.n.) route ( fig. 1 ). at 1 day postinfection (dpi) with sars-cov-2, ferrets were administered lopinavir (24 mg/kg of body weight)-ritonavir (6 mg/kg), hydroxychloroquine sulfate (12.5 mg/kg), or emtricitabine (6 mg/kg)-tenofovir (9 mg/kg) daily via oral gavage for 14 days (fig. 1 ). in addition, to test the effect of immunosuppression on viral infection and clinical outcome, a group (n ϭ 10) of ferrets was also treated with phosphatebuffered saline (pbs) (as a control) or azathioprine, an immunosuppressive drug (10 mg/kg), for 7 days prior to sars-cov-2 infection (fig. 1) . while all groups of sars-cov-2-infected ferrets showed elevated temperatures at 4 to 6 dpi, the lopinavirritonavir-or emtricitabine-tenofovir-treated group exhibited mild fever compared with the pbs-treated group ( fig. 2a) . as with the pbs-treated group, the hydroxychloroquine sulfate-or azathioprine-treated group showed ϳ7% body weight loss at 8 dpi, while the lopinavir-ritonavir-or emtricitabine-tenofovir-treated group showed a ͻ4% change in body weight on average (fig. 2b ). to compare clinical features of sars-cov-2 infection following treatment with each drug, we developed an arbitrary scoring method to generate clinical symptom values based on 20-min observations of cough, rhinorrhea, and reduced activity and compared these cs values among the ferret groups as described in table 1 . the average cs value of pbs-treated control ferrets was 3.5 at 2 dpi, remained relatively high (ͼ5) for 3 to 6 dpi, and returned to normal (less than 1) by 11 dpi (table 1 ). the lopinavir-ritonavir-treated ferrets showed the reduced overall cs values (less than 5) that peaked at 4 to 5 dpi and resolved by 10 dpi (table 1) . the hcq sulfate-treated ferrets also showed reduced cs values at 6 to 8 dpi compared with those of the pbs-treated group, but overall clinical symptoms were similar to those of the pbs-treated control group (table 1) . although the emtricitabine-tenofovir-treated ferrets initially demonstrated clinical symptoms similar to those of the pbstreated control group, their overall cs values were relatively low at 3 to 8 dpi, and clinical symptoms were ultimately resolved by 9 dpi. finally, the azathioprine-treated immunosuppressed ferrets also showed cs values similar to those of the pbs-treated control group, but this group's clinical symptoms lasted slightly longer than those of the pbs-treated control group (table 1) . these results collectively showed that the lopinavir-ritonavir-, hcq sulfate-, or emtricitabine-tenofovir-treated group exhibited lower overall clinical scores than the pbs-treated control group. the overall clinical symptoms of immunosuppressed ferrets were similar but persisted slightly longer than those of pbs-treated control ferrets. comparisons of virus titers and shedding periods in antiviral-drug-treated ferrets. to evaluate the antiviral activity of each drug against sars-cov-2, we measured infectious virus titers in nasal washes from drug-treated ferrets (fig. 2c ). sars-cov-2 was isolated from all infected ferrets regardless of drug treatment from 2 dpi to 6 dpi, with similar virus titers (2.75 to 3.2 log 10 tcid 50 /ml). at 8 dpi, the emtricitabinetenofovir-treated ferrets exhibited reduced virus titers compared with those of the pbs-treated control group. although infectious virus was not detected in ferrets of the pbs-or antiviral-drug-treated groups at 10 dpi, three of four azathioprine-treated ferrets were positive for virus even at 10 dpi (fig. 2c) , suggesting delayed virus clearance in the upper respiratory tracts of immunocompromised ferrets. because gastrointestinal involvement has been documented in coronavirus infections of animals and humans (19) , we also collected fecal specimens and performed quantitative real-time pcr (qrt-pcr) to determine whether any of the drug treatments affected sars-cov-2 shedding in the gastrointestinal system (fig. 2d) . the results showed that the viral rna was present in fecal specimens of all groups from 2 to 8 dpi, with peak viral rna copy numbers observed at 4 to 6 dpi. however, there was no statistical difference in viral rna copy numbers among the groups during the experimental period. by 10 dpi, viral rna copy numbers declined in all drug-treated ferrets. to further evaluate virus titers in tissues, three ferrets from each group were euthanized at 4 and 8 dpi, and virus titers were measured in nasal turbinate and lungs. at 4 dpi, all groups of ferrets showed high virus titers of more than 3.0 log 10 tcid 50 /g in nasal turbinate tissues, and the virus was also isolated from their lung tissues (fig. 3) . at 8 dpi, while all nasal turbinate tissues were positive for virus, the azathioprinetreated group showed a much higher virus titer (ϳ3.0 log 10 tcid 50 /g) than those of the other groups (fig. 3a) . at 8 dpi, the azathioprine-treated group still had detectable virus titers in their lung tissues, whereas the rest of groups were negative for the virus (fig. 3b) . to compare the serum neutralization (sn) antibody titers among drug-treated groups, blood was collected from each group of ferrets at 10, 14, and 21 dpi. at 10 dpi, the pbs-treated control and drug-treated groups demonstrated sn titers greater than 40 (fig. 4) . the antiviral-treated groups showed sn titers similar to those of the pbs-treated control group until 14 dpi, but they exhibited lower sn titers at 21 dpi than the pbs-treated control group (fig. 4) . it is noteworthy that the azathioprine-treated immunosuppression group showed geometric mean sn titers of 33.6 and 47.5 at 10 and 21 dpi, respectively, suggesting the continuously reduced sn antibody response of the immunosuppressed group. in this study, we evaluated the antiviral efficacies of three fda-approved drug candidates against sars-cov-2 infection using a ferret infection model which has previously proven to be highly susceptible to sars-cov-2 infection (18, 20) . although several clinical trials continue to evaluate these drug candidates, most of the enrolled patient populations are considered heterogeneous with regard to the duration and severity of illness at enrollment. further, given the rapid spread of covid-19 around the efficacies of fda-approved drugs against sars-cov-2 ® world, there are relatively higher mortality rates in some regions and in certain age groups. therefore, the use of highly susceptible animal models and controlled experimental settings should be an effective approach prior to human clinical trials to evaluate the in vivo antiviral effects of potential therapeutics. ferrets treated with antivirals showed relatively reduced overall clinical scores compared with those of the pbs-treated control group, which displayed high overall clinical scores. of the three drugs, the emtricitabine-tenofovir-treated group showed a noticeable reduction in overall clinical scores (յ4) and a shorter duration (8 dpi) of clinical symptoms. although attenuation of the overall clinical scores was observed in lopinavir-ritonavir-and hcq-treated groups at some time points, their clinical durations were comparable with those of the pbs-treated control group. these results suggest that treatment with emtricitabine-tenofovir, a nucleotide analogue that inhibits rnadependent rna polymerase activity, may be the most likely candidate to reduce clinical symptoms, including the cough and morbidity of sars-cov-2-infected hosts. while the lopinavir-ritonavir protease inhibitor and a cq/hcq sulfate autophagy inhibitor have been shown to be effective against sars-cov, mers-cov, or sars-cov-2 in in vitro culture (5), several reports have already described no benefit for clinical improvement of covid-19 patients. furthermore, hqc has been reported to be associated with a number of side effects, including a heart rhythm problem, severely low blood pressure, and muscle or nerve damage. thus, these existing fda-approved drugs still need rigorous testing for efficacy and safety in an animal model prior to clinical trials of covid-19 patients. since the emergence of sars-cov-2 in china in december 2019, the number of cases has rapidly increased as the disease has spread globally. the increase in the number of cases is alarming and specially compounded by the possibility of viral transmission from asymptomatic individuals. several studies indicate that asymptomatic patients can transmit the virus to persons in close contact (21, 22) . therefore, although clinical symptoms were attenuated in ferret groups treated with antiviral candidates, we also evaluated virus titers in respiratory and gastrointestinal tracts using nasal washes and stool samples, respectively, from sars-cov-2-infected ferrets. regardless of antiviral candidate treatment, sars-cov-2 was detected at more than 2 log 10 tcid 50 /ml until 6 dpi, and there was no statistical difference between the pbs-treated control group and the antiviral-drug-treated groups. however, the emtricitabine-tenofovir-treated group showed relatively low virus tiers and shortened periods of virus shedding in nasal wash specimens compared with those of the other groups. currently, the role of sn antibody in the pathogenesis and disease clearance of sars-cov-2 is unclear. wu et al. (23) recently reported that the sn antibody levels in covid-19 patients were variable, depending on the immune status of the patient, and that about 30% of patients failed to develop high sn titers after sars-cov-2 infection, although the disease durations of these patients were comparable to those of others. this suggests that the sn antibody titer may be closely associated with immune activity rather than with the virus titer and disease duration in patients. interestingly, we found that the antiviral-treated groups showed lower sn antibody titers than the pbs-treated control group. it is possible that as the antiviral treatment reduced the overall clinical symptoms of sars-cov-2-infected ferrets, it might evoke weak immune responses and thereby lead to reduced neutralizing antibody responses. nevertheless, further immunological studies are needed to understand the detailed mechanisms of the low sn antibody titers in antiviral-treated ferrets. efficacies of fda-approved drugs against sars-cov-2 ® while covid-19 is typically characterized by respiratory symptoms, gastrointestinal symptoms have been reported in some cases. moreover, there is evidence of viral rna in the stools of sars-cov-2 patients. emtricitabine-tenofovir has reportedly shown antiviral efficacy in the gastrointestinal tract as well as in the respiratory tract (24) . however, qrt-pcr analysis of stools revealed no statistical difference in virus titers in stools among the pbs-treated control and the antiviral-treated groups, suggesting that none of the tested antiviral candidates significantly diminished gastrointestinal sars-cov-2 replication in infected ferrets. in conclusion, although there may be some discrepancies in drug efficacy between animals and humans, these results of a preclinical ferret infection study should aid in the selection of antiviral treatments of covid-19 patients. this also suggests that a robust preclinical animal model for sars-cov-2 infection is valuable in order to identify antiviral drugs for future human efficacy trials. a sars-cov-2 strain, nmc-ncov02, was propagated in vero cells in dulbecco's modified eagle medium (dmem; gibco, grand island, ny) supplemented with 1% penicillin-streptomycin (gibco) and tpck (tosylsulfonyl phenylalanyl chloromethyl ketone)-treated trypsin (0.5 g/ml; worthington biochemical, lakewood, nj) in a 37°c incubator supplemented with 5% co 2 for 72 h. propagated virus was stored at ϫ80°c as the working virus stock for animal studies. the 50% tissue culture infective dose (tcid 50 ) was determined through fixation and crystal violet staining. immunosuppression and antiviral-drug candidate treatments. ten ferrets were treated orally with azathioprine (10 mg/kg) (celltrion) daily for 7 days prior to sars-cov-2 infection, and treatment continued until 14 days postinfection (dpi) to reduce the immune response. as a control, pbs in the same volume was administered to 10 ferrets. to confirm the immunosuppressed status of ferrets, blood samples were collected from azathioprine-treated ferrets, and the percentage of lymphocytes was assessed at 7 and 4 days preinfection and at 7, 14, and 21 dpi. the reduction in lymphocyte numbers in azathioprine-treated ferrets compared with lymphocyte numbers in the pbs control group was confirmed (see fig. s1 in the supplemental material). for treatment with candidate antiviral drugs, groups of ferrets (10/group) were administered lopinavir (16 mg/kg)-ritonavir (4 mg/kg) (abbott), hydroxychloroquine sulfate (25 mg/kg) (elyson), or emtricitabine (6 mg/kg)-tenofovir (7.35 mg/kg) (gilead) daily via oral gavage starting at 1 dpi of sars-cov-2 infection and continuing until 14 dpi. experimental infection of ferrets. groups of 10-to 12-month-old female ferrets (10/group), seronegative for sars-cov-1 and sars-cov-2, were intranasally inoculated with 10 5.8 tcid 50 /ml of nmc-ncov02 under anesthesia. the body weights and temperatures of infected ferrets were monitored every other day until 14 dpi. nasal washes and stool specimens were collected every other day from the inoculated ferrets. blood was collected at 10, 14, and 21 dpi to measure the serum neutralization titer. three ferrets per group were euthanized at 4 and 8 dpi, and nasal turbinate and lungs were collected to measure tissue virus titers and examine lung histopathology. virus titers in nasal washes and tissues were determined by 50% tcid 50 assessment in vero cells, while the virus titers in stool specimens were measured with quantitative real-time pcr (qrt-pcr). briefly, total rna was extracted using trizol reagent (thermo fisher scientific) or an rneasy kit (qiagen), and cdnas were generated with a sars-cov-2specific primer by reverse transcription using quantitect reverse transcription (qiagen). qrt-pcrs were performed using a sybr green supermix (bio-rad) and a cfx96 touch real-time pcr detection system (bio-rad) with a spike gene-based, sars-cov-2-specific primer set as previously described (18) , and virus rna copy numbers were calculated as a ratio with respect to the standard control. clinical scoring methods of sars-cov-2-infected ferrets. the ferrets were monitored daily over a 14-day period for temperature change, weight loss, clinical symptom, and movement and activity change. briefly, the frequency of cough and rhinorrhea was assessed in each group of ferrets and scored on the basis of the following criteria: no evidence of cough (score, 0), occasional cough (score, 1), and frequent cough (score, 2) and no nasal rattling or sneezing (score, 0), moderate nasal discharge on external nares (score, 1), and severe nasal discharge on external nares (score, 2). a change in a ferret's activity was assessed and scored on the basis of the following criteria: normal movement and activity (score, 0), mildly reduced movement and activity (score, 1), and considerably reduced movement and activity (score, 2) for at least 20 min. neutralizing assay. sera were collected from each group of ferrets to detect the serum neutralization titer. heat-inactivated 10-fold-diluted serum samples were serially diluted by 2-fold. an equal volume of sars-cov-2 at 100 tcid 50 was added to all diluted samples. the mixture of serum and virus was incubated at 37°c for 1 h and then added to vero cells in a 96-well tissue culture plate for 90 min. the mixture of serum and virus was then removed, followed by two washes with cold pbs. fresh medium was added to infected cells, and cells were incubated at 37°c in 5% co 2 for 4 days. supernatants were removed, fixed with a 10% formalin solution, and stained with crystal violet to determine the titer. statistical analysis. to assess significant differences in values for weight loss, temperature, viral titers, and serum neutralization titers, statistical analyses were done. asterisks indicate the statistical significance between pbs-administered and treated ferrets determined by two-way analysis of variance (anova) and a subsequent dunnett test (*, p ͻ 0.05; **, p ͻ 0.001; and ***, p ͻ 0.0001). all statistical analyses were performed using graphpad prism version 8.20 for windows (graphpad software, la jolla, ca). park et al. who. 2020. coronavirus disease 2019 (covid-19) situation report 88. who detection of sars-cov-2 in different types of clinical specimens viral load of sars-cov-2 in clinical samples temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov-2 replication in vitro screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture treatment with lopinavir/ritonavir or interferon-␤1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset combination therapy with lopinavir/ritonavir, ribavirin and interferon-␣ of middle east respiratory syndrome virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen antimalarial drug chloroquine counteracts activation of indoleamine (2,3)-dioxygenase activity in human pbmc hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro hydroxychloroquine for sars-cov-2 infection: how did we get here? connecticut hospitals using anti-malaria drug chloroquine 'out of desperation' to treat covid-19 animal toxicity and pharmacokinetics of hydroxychloroquine sulfate hydroxychloroquine for autoimmune diseases advances in nucleotide antiviral development from scientific discovery to clinical applications: tenofovir disoproxil fumarate for hepatitis b tenofovir alafenamide (taf) as the successor of tenofovir disoproxil fumarate (tdf) infection and rapid transmission of sars-cov-2 in ferrets enteric involvement of severe acute respiratory syndromeassociated coronavirus infection susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2 transmission of efficacies of fda-approved drugs against sars-cov-2 ® 2019-ncov infection from an asymptomatic contact in germany a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient cohort and their implications drug-susceptible hiv-1 infection despite intermittent fixed-dose combination tenofovir/emtricitabine as prophylaxis is associated with low-level viremia, delayed seroconversion, and an attenuated clinical course all animal experiments were approved by the medical research institute, a member of the laboratory animal research center of chungbuk national university (larc) (approval number cbnur-1352-20), and were conducted in strict accordance with and adherence to relevant policies regarding animal handling as mandated under the guidelines for animal use and care of the korea centers for disease control (kcdc). the handling of virus was performed in an enhanced biosafety level 3 (bsl3) containment laboratory as approved by the korea centers for disease control and prevention (protocol kcdc-14-3-07). supplemental material is available online only. key: cord-276732-u2d1z4ip authors: mauri, tommaso; zambelli, vanessa; cappuzzello, claudia; bellani, giacomo; dander, erica; sironi, marina; castiglioni, vittoria; doni, andrea; mantovani, alberto; biondi, andrea; garlanda, cecilia; d’amico, giovanna; pesenti, antonio title: intraperitoneal adoptive transfer of mesenchymal stem cells enhances recovery from acid aspiration acute lung injury in mice date: 2017-03-06 journal: intensive care med exp doi: 10.1186/s40635-017-0126-5 sha: doc_id: 276732 cord_uid: u2d1z4ip background: mesenchymal stem cells (mscs) might act as fine-tuners of inflammation during acute lung injury. we assessed the effects of adoptive transfer of mscs in acid aspiration acute lung injury and explored the role of long pentraxin ptx3. methods: we conducted a prospective experimental interventional study on wild-type (wt) and ptx3-deficient (ptx3(−/−)) mice. acute lung injury was induced in wt and ptx3(−/−) mice by instillation of hydrochloric acid into the right bronchus. one hour later, animals received intraperitoneal sterile phosphate-buffered saline (pbs), wt-mscs (1 × 10(6)) or ptx3(−/−)-mscs (1 × 10(6)). twenty-four hours after injury, we measured the effects of treatments on arterial blood gases, wet/dry lung weight (w/d), ct scan analysis of lung collapse, neutrophils, tnfα and cxcl1 in bronchoalveolar lavage, and plasma ptx3. d-dimer was assayed in 1 week and oh-proline in 2 weeks to track the fibrotic evolution. results: in 24 h, in comparison to pbs, wt-mscs improved oxygenation and reduced w/d and alveolar collapse. these effects were associated with decreased concentrations of alveolar neutrophils and cytokines. wt-mscs increased d-dimer concentration and decreased oh-proline levels, too. treatment with ptx3(−/−)-mscs ameliorated oxygenation, w/d, and alveolar tnfα, though to a lesser extent than wt-mscs. ptx3(−/−)-mscs did not improve lung collapse, neutrophil count, cxcl1, d-dimer, and oh-proline concentrations. the protective effects of wt-mscs were dampened by lack of endogenous ptx3, too. conclusions: in acid aspiration acute lung injury, mscs improve pulmonary function and limit fibrosis by fine-tuning inflammation. the role of ptx3 in determining mscs’ effects might merit further scrutiny. electronic supplementary material: the online version of this article (doi:10.1186/s40635-017-0126-5) contains supplementary material, which is available to authorized users. the incidence of the acute respiratory distress syndrome (ards) is elevated, and mortality in recent studies still reaches 50% for the most severe form [1] [2] [3] [4] [5] . moreover, many ards survivors develop long-term lung fibrosis, reduced respiratory function, and poor quality of life [1] . at onset, ards is characterized by severe hypoxemia and lung edema, caused by dysregulated inflammation [1, 2] . overstimulation of leukocytes, cytokine storm, and altered tissue repair are key contributors to ards severity, mortality, and long-term morbidity [3] . however, we still lack effective pharmacological therapies that fine-tune these mechanisms [4] . mesenchymal stem cells (mscs) are multi-potent cells derived from adult tissues [6] . mscs secrete multiple molecules, including anti-inflammatory cytokines, growth factors, and anti-microbial peptides, and appear as fine-tuners of host inflammation [6] . previous studies showed that mscs administration in animal models of acute lung injury increased the ability of the host to eliminate the agent, regulate neutrophil recruitment, and reverse altered lung permeability, without additional injury [7] [8] [9] [10] . in addition, intraperitoneal (i.p.) route for the administration of mscs was recently described [11] . to our knowledge, the effects of i.p. mscs have never been assessed in experimental acid aspiration acute lung injury [12] ; moreover, the effects of mscs on the fibrotic long-term evolution of acute lung injury [13] have not been described, and key molecular determinants of mscs' effects are not fully understood. in the present study, we tested in a mouse model of acid aspiration acute lung injury the effects of i.p. mscs on the early acute inflammatory reaction and on the long-term fibrotic evolution [5, 12] . moreover, we explored the role of pentraxin 3 (ptx3) in mediating mscs' effects. ptx3 is an acute-phase inflammatory mediator produced by different cell types [3, 14] that exerts protective effects in experimental acute lung injury, closely resembling those of mscs [15] . previous studies indicated that mscs produce, store, and secrete ptx3 when activated [16, 17] . the research group of dr. g. d' amico generated ptx3-deficient mscs (ptx3 −/− -mscs) [18] , which showed a significant defect in promoting tissue repair in a mice model of wound healing compared to wild-type mscs (wt-mscs) [18] . in analogy, we investigated whether ptx3 deficiency in the mscs and/or at the endogenous level might impact the ability of mscs to promote short-and longterm recovery from acid aspiration acute lung injury. the hypothesis of this study was that early treatment with mscs in a murine model of acid-induced lung injury might exert short-and long-term beneficial effects by modulation of the inflammatory response and that lack of ptx3 in mscs might reduce their efficacy. procedures involving animals and their care were conducted in conformity with the institutional guidelines complying with national and international laws and policies. the experimental protocol was submitted to the italian ministry of health and approved by the animal care unit of the university of milan-bicocca, monza, italy. mscs were isolated from female c57bl/6 wt mice and from ptx3 −/− mice by previously described procedures [18] . cryopreserved aliquots of mscs were thawed 5-7 days before the experiments, seeded at 1000-2000 cells/cm 2 , and cultured at 37°c in a 5%-co 2 atmosphere. on early morning, mscs were dethatched by trypsin and used fresh for all the experiments performed that same day. fresh mscs at passages 5 to 7 were used for the present study. recent studies showed that ptx3 −/− -mscs were similar to wt-mscs in their ability to grow spontaneously, undergo mesengenic differentiation, and express common mscs' markers [18] . as already published, ptx3 −/− -mscs drastically decreased the mitogen-induced proliferation of lymphocyte in a dose-dependent manner similarly to wt-mscs [19] . moreover, ptx3 −/− -mscs did not store or release ptx3 while they tended to produce higher levels of tumor necrosis factor-stimulated gene 6 (tsg-6) [19] compared to wt-mscs (additional file 1: figure s1 ). acid aspiration acute lung injury was induced in wt-and ptx3 −/− -mice as previously described [12] . briefly, after intubation, 1.5 ml/kg of 0.1 m hydrochloric acid was instilled into the right lung, and after 10 min, the animals were extubated and placed in an oxygenated chamber. one hour later (to reproduce possible real life clinical timing), the mice received i.p. injection of sterile phosphate-buffered saline (pbs) or 1 × 10 6 wt-mscs or 1 × 10 6 ptx3 −/− -mscs (all in equal volume of 200 μl). figure 1 shows the experimental design of the study in wt mice. the following measures were performed in all wt mice: (a) twenty-four hours after hcl instillation, the mice were sacrificed and the following analysis were performed (detailed methods are described in additional files): -arterial blood gas analysis for gas exchange -wet-to-dry ratio as index of edema -micro-ct scan to measure change over time in non-aerated lung tissue expressed as percentage of the whole lung tissue, with more negative values representing larger decrease of alveolar collapse; -histopathology examination performed according to previous study [12] evaluating alveolar serofibrinous exudate and alveolar hemorrhage -bronchoalveolar lavage for differential cell count, total protein content (with bicinchoninic acid method) and keratinocyte chemoattractant (cxcl1, previously named kc), and tumor necrosis factor-α (tnf-α) were assayed by elisa -blood withdrawal for ptx3 levels measurement in plasma (elisa assay) (b)in 1 week from lung injury d-dimer (marker of fibrinolysis) [20] and matrix metalloproteinase 13 (mmp13), an enzyme that participates in collagen degradation [21] , were detected by elisa and by western blot in lungs lysate, respectively. (c) two weeks after acid-induced lung injury, the fibrotic evolution was evaluated [22] . in particular, we performed as follows: in ptx3 −/− -mice, instead, we measured only oxygenation and wet-to-dry lung weight ratio in 24 h and oh-pro content in 2 weeks. blinded researchers performed each analysis. data are expressed as mean ± standard deviation if normally distributed and as median (interquartile range) when non-normally distributed. one-way analysis of variance (anova) or kruskal-wallis and dunnett's or dunn's post hoc tests vs. pbs group were used to assess differences between treatment effects in wt mice, as appropriate. differences in physiologic variables measured in the right vs. left lung were assessed by t test or mann-whitney u test, as appropriate. p < 0.05 was considered statistically significant. detailed methods can be found in the additional file 1 of this article. mesenchymal stem cells enhance short-and long-term recovery from experimental acid aspiration acute lung injury in 24 h, i.p. administration of wt-mscs 1 h after induction of acid aspiration acute lung injury significantly improved arterial oxygenation and decreased the alveolar-arterial oxygen gradient in wt-mice in comparison to pbs (p < 0.05 and p = 0.001, respectively) ( fig. 2a and b) , without modification of paco 2 and even in presence of slightly worse ph values (additional file 1: table s1 ). early improvement in oxygenation yielded by wt-mscs was likely obtained by reduction of lung edema: in fact, the lungs' wetto-dry ratio in 24 h was decreased by wt-mscs in comparison to pbs (p < 0.05) (fig. 2e) . similarly, micro-ct scan analysis showed that the extent of lung collapse significantly decreased between 1 and 24 h in wt mice treated by wt-mscs (p = 0.01), likely indicating decreased superimposed weight from reduced lung edema (table 1 and fig. 3 ), but not in those treated by pbs. histology performed in 24 h showed decreased disruption of lung structures in mice treated by wt-mscs in comparison to pbs (table 1) , even though this difference did not reach statistical significance. bal total protein concentrations were left unchanged by wt-mscs treatment (fig. 2f) . mice treated by wt-mscs, indeed, showed significant reduction of total cell count in bal fluid in 24 h and substantial dampening of neutrophil recruitment into the alveoli (p < 0.05 for both; fig. 2c , d) in comparison to pbs. accordingly, levels of proinflammatory cytokines (i.e., cxcl1 and tnf-α) in bal fluid were significantly reduced by wt-mscs (p < 0.05 and p < 0.01, respectively), but not by pbs (table 1) . interestingly, circulating ptx3 was reduced in wt-mice treated by wt-mscs (albeit non-significantly) and not in wt-mice treated by ptx3-deficient mscs (table 1) . in this study, we showed that treatment by i.p. wt-mscs administered 1 h after acid aspiration attenuated the evolution of fibrosis, as demonstrated by lower collagen deposition (oh-pro assay) in 2 weeks (fig. 4a ) in comparison to mice treated by pbs. in 1 week, d-dimer concentration was significantly increased in the lungs of mice treated with wt-mscs (p < 0.001, fig. 4b ) in comparison to pbs, suggesting that dampening of long-term fibrotic evolution might have followed both reduced inflammation and enhanced fibrinolysis by wt-mscs in the days after injury. in an effort to evaluate whether i.p. wt-mscs migrate systemically in mice with acid aspiration acute lung injury, we performed western blot analysis to detect gfp + wt-mscs presence in the lungs, spleen, liver, and peritoneal lavage in 24 h. additional file 1: figure s3 shows actual blots with no apparent signal of gfp + wt-mscs presence in the lungs, spleen, and liver as opposed to positive controls. in the peritoneal lavage, instead, wt-mscs were still present in 24 h but by lower intensity, probably because, as previously shown [23] , they formed aggregates and adhered to the peritoneal cavity walls. figure s1 ). in 24 h, treatment by ptx3 −/− -mscs ameliorated oxygenation only to a lesser extent than wt-mscs (fig. 2) . reduced short-term effects on oxygenation in comparison to wt-mscs were paralleled by less effective reduction of wet-to-dry lung weight ratio by ptx3 −/− -mscs (fig. 2a, b , e) and the absence of effects of ptx3deficient cells on radiological signs of regional lung collapse and edema (table 1) . histology found reduction of lung injury, albeit non-significant (table 1 ). in summary, ptx3 −/− -mscs seemed less effective than wt-mscs in limiting formation of lung edema in 24 h after acid aspiration. at variance from wt-mscs, treatment with ptx3 −/− -mscs did not reduce total cell and neutrophil count (fig. 2) as well as cxcl1 levels in the alveolar space (table 1) . thus, the more limited effectiveness of ptx3 −/− -mscs in enhancing lung recovery after acid aspiration acute lung injury might have been related to ineffective reduction of the acute inflammatory processes. moreover, ptx3 −/− -mscs could not modulate fibrinolysis in the days following injury nor impact the long-term fibrotic evolution, as demonstrated by unchanged levels of d-dimer and oh-proline in comparison to pbs (fig. 4a, b) . however, wt-and ptx3 −/− -mscs did not seem to modulate activity of mmp13 in 1 week (additional file 1: figure s2 ) to impact remodeling and fibrosis. effects of study treatments on ptx3 knockout mice with acid aspiration acute lung injury extent of lung injury was similar between wt and ptx3 −/− mice (t test in 24 h in wtmice + pbs vs. ptx3 −/− -mice + pbs: pao 2 , p = 0.151; wet-to-dry lung weight, p = 0.099). when administered to ptx3 −/− -mice: wt-mscs improved lung function and reduced fibrosis, but the difference with pbs was non-significant (additional file 1: table s3 ); ptx3 −/− -mscs induced a further non-significant reduction of the alveolararterial gradient and of wet-to-dry lung weight ratio, while pao 2 and fibrosis worsened in comparison to wt-mscs (additional file 1: table s3 ). thus, endogenous ptx3 might collaborate in the protective effects of wt-mscs from fibrosis, while it might limit their effectiveness in reducing lung edema. more results are provided in the additional files of this article. study's main findings can be summarized as follows: wt-mscs dampen short-and long-term sequelae of acid aspiration acute lung injury in mice in terms of improved oxygenation, reduced edema causing lung collapse, and reduced fibrotic evolution, likely by fine-tuning the acute inflammatory reaction and the subsequent fibrinolysis and tissue repair process; moreover, lack of ptx3 gene in mscs and in the injured host might reduce the beneficial effects of mscs. in the present study, we administered i.p. wt-mscs 1 h after intratracheal instillation of hydrochloric acid, potentially reproducing real-life treatment of ards caused by aspiration of gastric contents [1, 12] , one of the major direct causes of ards [24, 25] with a mortality rate around 35-40% and significant long-term fibrosis [1] . in 24 h from injury, we could show multiple short-term beneficial effects of wt-mscs: as previously shown [7, 26] , mscs seemed to reduce the early inflammatory reaction in the lungs and to avoid excessive response and additional damage. in our study, indeed, mscs dampened leukocyte trafficking through the alveolar-epithelial barrier as well as their activation and release of primary inflammatory cytokines. in turn, as testified by oxygenation, wet-to-dry and ct scan data, this led to decreased accumulation and/or improved clearance of lung edema and inflammatory cells in the alveolar and thirdspace compartments and to attenuated extent of alveolar collapse. however, histology did not improve after wt-mscs administration, maybe due to insufficient numerosity; similarly, protein content in bal was not reduced by mscs, but this could have followed direct extravasation after acid-induced physical disruption of the alveolar-epithelial integrity. in our model, both lungs showed physiologic alterations, thus indicating that the left lung could completely compensate for the ventilation needs of the animals (additional file 1: table s2 ) [27] . in 2 weeks from acute lung injury onset, we also showed decreased long-term collagen deposition in the lungs associated with treatment by wt-mscs. moreover, the long-term reduction of fibrosis was preceded by increased fibrinolysis in 1 week. our data, in keeping with recent literature [7, 9, 20] , seem to suggest that the beneficial effects of mscs on the fibrotic evolution of acute lung injury might include reduction of the acute-phase inflammatory reaction and reduced fibrosis in 2 weeks. moreover, decreased respiratory effort during the early phases induced by improved gas exchange could have reduced interstitial lung edema [28] and the risk of additional ventilationinduced lung injury (vili) and fibrosis [13] . in summary, it would be tempting to say that ours and the previous data indicate that mscs might be regarded as personalized modular therapies limiting short-and long-term acute lung injury severity by finetuning inflammation and tissue remodeling. however, to date, whether these hypotheses hold true and will translate in improved mortality and long-term quality of life in human ards remains to be determined. ptx3 is a marker of severity in human ards [29] , and experimental models showed that ptx3 is as key determinant of the evolution, morbidity, and mortality of acute lung injury [30, 31] . a recent study by cappuzzello and colleagues showed that while wt-mscs improved tissue repair in experimental wound healing, ptx3 −/− -mscs could not [18] . similarly, we showed that the early dampening of leukocyte migration and release of pro-inflammatory cytokines in the injured lungs by wt-mscs could not be replicated when ptx3 −/− -mscs were adopted. this likely led to poorer effects on oxygenation and wet/dry ratios and no improvement in the ct scan analysis of lung collapse as well as no decrease in inflammatory cells and acute-phase primary cytokines in the bal. the positive effect of ptx3 −/− -mscs treatment on the tnf-α levels may depend on the anti-inflammatory role of tsg6 [32, 33] , which is highly expressed in ptx3 −/− -msc (additional file 1: figure s1 ). previous studies in a mice model of bilateral acid aspiration lung injury showed that interaction between ptx3 and pselectin is crucial for regulation of leukocyte recruitment with consequences on cytokine production and lung injury [23] , and similar mechanisms might underlie lack of lung protection by ptx3 −/− -mscs. moreover, we described that long-term fibrinolysis and subsequent fibrotic evolution could not be prevented by ptx3 −/− -mscs, possibly suggesting ptx3-mediated enhancement of lung tissue repair by wt-mscs [18, 20] . on the other hand, our data indicate that the beneficial effects exerted by wt-mscs are associated with a reduction in plasma ptx3, as if improvement of lung injury preceded modification of endogenous ptx3 production. however, lack of endogenous ptx3 seemed to reduce wt-mscs effects (additional file 1: table s3 ), maybe by impairment of local cell-to-cell interaction. our results do not generate a clear hypothesis on the role of ptx3 as molecular determinant of the lung protection exerted by mscs, and further studies are warranted, maybe exploring other etiologies and time-points. in our study, we could not detect presence of wt-mscs in the liver, spleen, or lungs in 24 h, while in keeping with previous findings [34] , a signal was still present in peritoneal lavage (additional file 1: figure s3 ). on the other hand, since levels of circulating ptx3 were lower and lung fibrinolysis was increased after administration of wt-mscs, we might speculate possible migration and direct effect of mscs at the site of injury but this cannot be concluded with any confidence. in summary, our data are not definitive to elucidate whether i.p. wt-mscs act through paracrine vs. direct mechanisms. this study suffers by a number of relevant limitations: as most of the measures required sacrifice of the animals (e.g., bal), we could not assess in the same animal all the effects at different time-points but each effect was assessed in a subset of animals receiving the same injury and therapy, which might have introduced some heterogeneity. we only examined three time-points (i.e., 24 h and 1 and 2 weeks), which might have prevented us from description of other effects of wt-mscs or ptx3 −/− -mscs in acid aspiration acute lung injury. apart from resources limitation, our choice was based on previous observations on the time-course of the studied animal model [12] . we described reduced effectiveness of wt-mscs induced by lack of ptx3 only in a murine model of non-infective acid aspiration lung injury and translation of these findings to other etiologies (e.g., infective pulmonary ards) and/or to the clinical setting warrants extreme caution. while we could determine significant effects of ptx3 presence in mscs in 1 week to modulate fibrosis, the downstream effects of ptx3 presence in mscs during the early acute phase (e.g., modulation of leukocyte recruitment by binding with p-selectin) remains to be elucidated. we did not evaluate the alteration of the alveolar-capillary permeability from a more molecular point of view (such as expression of tight or adherent junction proteins), but only by measuring the lung edema following such alterations (wet-to-dry ratio and the ct scan analysis). besides the standard histological analysis, we did not perform more quantitative approach using stereological assessment of the tissue injury, and this might have limited our possibilities to describe more significant differences. this is a preliminary study: the small number of mice used and lack of other administration routes could have reduced significant differences. the experiments on ptx3 −/− mice were subsequent and separate from those on wt mice. finally, volume of fluid instilled i.p. (i.e., 200 μl) might have induced cardiovascular impairment favoring pulmonary edema. the results presented here suggest that i.p. adoptive transfer of mscs enhances shortand long-term lung recovery when cells are administered 1 h after onset of acute lung injury. ptx3, an acute-phase inflammatory mediator, might play a role in the lung protection exerted by mscs, in particular against fibrosis, but this needs further clarification. studies on the molecular mechanisms of actions of mscs as well as on the risks associated with their administration should still proceed in parallel with ongoing translational studies [35] . in particular, ptx3 genetic polymorphisms have been associated with risk of microbial infections [36] [37] [38] , and it will be important to assess whether ptx3 polymorphisms are associated with outcome in ards and in clinical trials aimed to assess the potential of mscs. the acute respiratory distress syndrome soluble receptor for advanced glycation end-products 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microenvironment sets the humoral pattern recognition molecule ptx3 in a tissue repair mode phenotypic overlap between mmp-13 and the plasminogen activation system during wound healing in mice inhibition of pulmonary fibrosis by the chemokine ip-10/cxcl10 intraperitoneally infused human mesenchymal stem cells form aggregates with mouse immune cells and attach to peritoneal organs aspiration-induced lung injury epidemiology, patterns of care, and mortality for patients with acute respiratory distress syndrome in intensive care units in 50 countries therapeutic effects of human mesenchymal stem cell-derived microvesicles in severe pneumonia in mice unilateral acid aspiration augments the effects of ventilator lung injury in the contralateral lung spontaneous effort during mechanical ventilation: maximal injury with less positive endexpiratory pressure elevated plasma and alveolar levels of soluble receptor for advanced glycation endproducts are associated with severity of lung dysfunction in ards patients protective effects of long pentraxin ptx3 on lung injury in a severe acute respiratory syndrome model in mice long pentraxin ptx3 deficiency worsens lps-induced acute lung injury anti-inflammatory protein tsg-6 secreted by activated mscs attenuates zymosan-induced mouse peritonitis by decreasing tlr2/nf-κb signaling in resident macrophages intravenous hmscs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein tsg-6 regulation of leukocyte recruitment by the long pentraxin ptx3 mesenchymal stem (stromal) cells for treatment of ards: a phase 1 clinical trial genetic variations affect the risk of pseudomonas aeruginosa airway colonization in cystic fibrosis patients genetic ptx3 deficiency and aspergillosis in stem-cell transplantation implementation of a pan-genomic approach to investigate holobiont-infecting microbe interaction: a case report of a leukemic patient with invasive mucormycosis we are thankful to all the personnel working in the university of milan-bicocca, san gerardo hospital and humanitas institute for the continuous support and collaboration. the datasets supporting the conclusions of this article are included within the article (and its additional file). authors' contributions tm, vz, cg, gd, and ap made substantial contributions to the conception and design of the work. all authors performed the data acquisition, analysis, and interpretation for the work. tm, vz, cc, am, ab, cg, gd, and ap drafted the work and revised it critically for important intellectual content. the final approval of the version submitted for publication was carried out by all authors. tm, vz, and ap established the accountability for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. all authors read and approved the final manuscript. key: cord-259364-8zoav7b0 authors: xiao, yire; hu, qingqing; jiao, luoying; cui, xiping; wu, panpan; he, pan; xia, nana; lv, rui; liang, yuxin; zhao, suqing title: production of anti-trichophyton rubrum egg yolk immunoglobulin and its therapeutic potential for treating dermatophytosis date: 2019-09-09 journal: microb pathog doi: 10.1016/j.micpath.2019.103741 sha: doc_id: 259364 cord_uid: 8zoav7b0 the aim of this study was to estimate the therapeutic potential of specific egg yolk immunoglobulin (igy) on dermatophytosis caused by trichophyton rubrum. the igy was produced by immunizing hens with cell wall proteins of t. rubrum, extracted from eggs by peg precipitation and then purified by ammonium sulfate precipitation. the cross-reactivity (cr) with other fungi, growth inhibition on t. rubrum in vitro and therapeutic effect on t. rubrum infection in balb/c mice of the specific igy were then evaluated. antit. rubrum cell wall proteins igy (anti-trcwp igy) presented a certain degree of cross-reactivity with different fungi. in the in vitro and in vivo activity researches, anti-trcwp igy showed a significant dose-dependent growth inhibitory effect on t. rubrum in vitro and a significant dose-dependent therapeutic effect on t. rubrum infection in balb/c mice. dermatophytosis is a global health problem with high morbidity [1, 2] . according to the world health organization (who), dermatophytes affect about 25% of the world population. it is estimated that about 30%-70% of the infected adults are asymptomatic, and the incidence of the infection increases with age [3] . dermatophytes have been classified into three genera, trichophyton, microsporum and,epidermophyton. t. rubrum is particularly responsible for the major infections of dermatophytes [4, 5] , which is a kind of filamentous fungus that may cause tinea corporis, tinea cruris, and tinea pedis [6] . at present, most popular treatment options for dermatophytosis are topical use of antifungal chemicals such as amphotericin b, fluconazole, itraconazole, voriconazole, posaconazole, isavuconazole and terbinafine [7, 8] , oral antifungal drugs such as triazoles, griseofulvin, and terbinafine for systemic therapy of severe cases [9] . because of the fungal resistance problem of the antifungal chemicals, the research of their suitable alternatives is continuing [10] . human's regular interactions with fungi rarely result in diseases in persons with normal immunity. however, those with immune impairment are at high risk of fungal diseases. the recognition that impaired immunity is central to the pathogenesis of many fungal diseases and that antifungal agents are often ineffective in the setting of immune defects, provides a strong rationale for the development of immunebased therapies, or immunotherapy, for fungal diseases [11] . igy is produced by hens to provide their offspring with an effective humoral immunity against the common avian pathogens until full maturation of their own immune system. effective protection against salmonella enteritidis, salmonella e. typhimurium, campylobacter jejuni, escherichia coli etec, murine and bovine rotavirus, and bovine coronavirus infections in mice, pig, and calves has been obtained with the use of passively-administered egg yolk-derived antibodies [12] . ample data have demonstrated the protection of igy against different kinds of bacteria, virus, and parasites there are also reports about anti-fungi igys [13, 14] , reminding us that it may also be effective against t. rubrum. to explore the therapeutic potential of igy on dermatophytosis caused by t. rubrum, we immunized the hens with cell wall proteins of t. rubrum. the anti-t. rubrum igy was then extracted from eggs by peg precipitation and purified by ammonium sulfate precipitation. the purity, titer, and specificity of the igy were tested to evaluate the igy production process. a series of researches about cross-reactivity (cr) with other fungi, growth inhibition on t. rubrum in vitro and therapeutic effect on t. rubrum infection in balb/c mice of the igy have been carried out to assess the selectivity and therapeutic potential of the igy. t. rubrum (nbrc5467, provided by dr. song-chao yin from the third affiliated hospital of sun yat-sen university) was cultured at 28°c for 6 days in sabouraud agar medium. the t. rubrum spores were washed with sterile phosphate-buffered saline (pbs, ph7.4, 0.01 m), and then inoculated to the liquid sabouraud medium, shakingly cultured at 28°c, 160 rpm for 5 days. after that, t. rubrum hyphae were harvested by centrifugation, washed 3 times with sterile pbs (ph7.4, 0.01 m) and then freeze-dried. the cell wall proteins were extractedby referring to a reported method [15] . one g of hyphae was ground thoroughly in liquid nitrogen with a mortar and pestle into powder, dissolved with 20 ml pbs (ph7.4, 0.01 m); 200 μl 10 mm pmsf (beyotime) was added into the solution. the solution was put in an ice-bath and the cells were broken with an ultrasonic cell breakup instrument (2 s × 300 times, interval 5s). the precipitate was collected by centrifugation (4°c, 6000 g, 10 min) and washed five times with ice-cold water. after that a cold 0.1 m naoh solution was added to the precipitate and stirred at 4°c for 24hr. the supernatant was harvested by centrifugation(4°c, 12000g, 20min), and ph was adjusted to 7.0 with cold 0.1 m hcl solution. the solution containing cell wall proteins was dialyzed with pbs (ph7.4, 0.01 m) for 72hr and diluted to 1 mg/ml with pbs (ph7.4, 0.01 m). the concentration of the cell wall proteins was measured by bca assay. the protein components of the cell wall proteins were analyzed by sds-page (12%) method (bio-rad, usa). the cell wall proteins diluted to 1 mg/ml was mixed thoroughly with an equal amount of freund's adjuvant (sigma chemicals, st louis, mo, usa) until the emulsion was stable. 4 healthy 20-week-old roman laying hens housed at an experimental animal center were immunized and boosted via an intramuscular route (i.m. 0.5ml/hen, musculus pectoralis, left and right side) with the antigen emulsified in freund's complete adjuvant (for the primary immunization) or freund's incomplete adjuvant (for the subsequent booster injections). the birds were given four booster injections at two-week intervals. eggs were collected daily from 20d after the first immunization and stored at 4°c. eggs collected from unimmunized hens were used for the negative control [16] . isolation and purification of igy were carried out according to reported studies [17] . eggs were disinfected with 75% alcohol; the egg yolks were then separated from the whites with an egg yolk separator. the collected egg yolks were dissolved with a triple volume of pbs (ph7.4, 0.01 m) containing 3.5% peg6000 (w/v) and stirred for 40 min; the mixture was centrifuged at 4°c, 12000g for 20 min. the supernatant was collected, peg6000 was added to adjust the final polymer concentration to 12% (w/v), and the mixture was stirred for 40 min. the precipitate containing the crude igy was harvested by centrifugation at 4°c, 12000g for 20 min. the extracted crude igy was dissolved into the original volume of yolk in pbs (ph7.4, 0.01 m); the saturated ammonium sulfate solution was added to the equal volume and the mixture was stirred overnight at 4°c. the precipitate was collected by centrifugation (4°c, 12000g for 20 min) and washed with a 1:3 saturated ammonium sulfate solution. the precipitate was dissolved and dialyzed against pbs (ph7.4, 0.01 m) and then freeze-dried, the igy powder obtained was stored at −20°c. sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) was performed to determine the purity of the igy. a 10% polyacrylamide gel was used (bio-rad laboratories, usa). the analysis was conducted under reduced conditions. the sample(1 mg/ml) was mixed with sample buffer (5 × ) and held for 5-8 min at 100°c. ten micro liters of the sample was loaded into each well. pre-stained protein standard (fermentas, lithuania) was used as a molecular weight marker. the protein bands were visualized with coomassie brilliant blue r250 (sino pharma shanghai chemical reagent company). the gel was analyzed by bio-rad image analysis software. to confirm the specificity of the anti-trcwp igy, western blotting was conducted according to a previously published method [18] . the extracted t. rubrum cell well protein was subjected to sds-page with a 12% polyacrylamide gel. after electrophoresis, the gel was equilibrated in the transfer buffer for 15-30 min and the proteins were then electrically transferred onto a pvdf membrane (millipore, usa) for 3 h at 350 ma, 4°c. the membrane was stirred in freshly prepared ponceau solution (beyotime) for 1 min and washed with distilled water, and then cut into 0.5 cm strips, which were blocked with 5% fat-free milk for 2 h at room temperature and washed with tris buffer solution(0.02 m, ph7.6) containing 0.05% tween 20 (tbs-t). the strips were then incubated overnight at 4°c with a 1:100 dilution of anti-trcwp igy (1 mg/ml). igy from non-immunized hens was used as a negative control. after incubation, the strips were washed 3 times with tbs-t and incubated with hrp conjugated rabbit anti-chicken igy (promega, usa) diluted 1:10000. following 1 h of incubation at room temperature, the strips were washed three more times. after washing, ecl solution (millipore, usa) was added to the strips in a dark room with red light as the light source. the strips were photographed and developed. the titer of the specific igy was determined by indirect elisa as described previously by ref. [19] . ninety-six-well elisa plate was coated with t. rubrum whole-cell solution (1.2 × 10 8 cfu/ml in ph7.4, 0.01 m pbs) at 37°c for 2 h and 4°c overnight. the plate was washed 3 times with ph7.4, 0.01 m pbs containing 0.05% tween 20 (pbst). blocking was performed by adding 200 μl of 3.3% (w/v) fat-free milk, incubated at 37°c for 1 h. after washed 3 times with pbst, samples (100μl/well) were added to each well and incubated at 37°c for 1 h. the igy (1 mg/ml) was 2-fold serial diluted in pbs (ph7.4, 0.01 m). the igy from non-immunized hens was used as a negative control. the plate was washed 3 times again and 100μl/well of hrp affiliate rabbit anti-chicken igy (promega, usa; 1:5000) was added and the plate was incubated at 37°c for 1 h. after being washed 3 times, 100 μl of tmb substrate solution (huamei biological engineering co., ltd.) was added to each well for 15 min at 37°c. after that, the reaction was stopped by the addition of 50 μl of 2 m h 2 so 4 to each well. the optical densities of the wells were determined at 405 nm with a plate reader (bio-rad, usa). when od sample /od negative > 2.1 and od negative > 0.1, the maximum dilution multiple of the sample was determined as the igy titer. to estimate the cross-reactivity (cr) of anti-trcwp igy against other fungi, indirect elisas were performed by the use of antigens from different fungi according to the method described previously. antigens for elisa test with anti-trcwp igy. igy from non-immunized hens was used as a negative control. the cross-reactivity rate was calculated as follows: in the formula, dr f was the titer of igy against other fungi and dr t was the titer of igy against t. rubrum. the inhibitory effect of specific igy on t. rubrum growth in vitro was detected according to the m38-a2 method by the clinical and laboratory standards institute (clsi) [21] . t. rubrum was cultured at 28°c for 7 days in oats agar medium (30 g oats and 20 g agar in 1l water). the t. rubrum spores were washed with sterile pbs (ph7.4, 0.01 m) and diluted to 1.0 × 10 4 cfu/ml with rpmi1640. the specific igy was dissolved in sterile pbs (ph7.4, 0.01 m), diluted to 40 mg/ml, 20 mg/ml, 10 mg/ml, 5 mg/ml, 2.5 mg/ml, 1.25 mg/ml and 0.625 mg/ml, and then sterilized by filtration before use. 100 μl of cell suspension and 100 μl of different concentrations of igy solution were added into one well of the polystyrene plate and incubated at 28°c for 72 h, each igy concentration being triplicate. cell suspension plus pbs (ph7.4, 0.01 m) was used as a blank control and igy from non-immunized hens diluted with rpmi1640 was used as a negative control. the absorbencies of the mix solutions were then measured at 660 nm (bio-rad, usa). the growth-inhibitory curve of the specific igy on t. rubrum was graphed by using the concentration of igy as abscissa while od660 of the mix solution as ordinate. the mouse model infected with t. rubrum was built fby referring to a reported method [22] . experiments were carried out by following an accepted ethical protocol according to the guidelines of the guangdong university of technology for animal experiments(gdut/sbps/ 2017060501). the number of mice was reduced to an absolute minimum. animal suffering was limited to the experiment need and the sacrifice was done with carbon dioxide asphyxiation. balb/c mice (8 weeks old, purchased from medical experimental animal center of guangdong province) were maintained at a 12:12 h light/dark cycle with 18~26°c and 30-60% relative humidity, and provided ad-lib with food pellets and water. the animals were immunosuppressed with a daily intramuscular injection of triamcinolone acetonide injection (nanjing jindun animal pharmaceutical co., ltd. 0.3 ml per mouse) for 3 days. the back of each animal was depilated with 8% na 2 s solution, and the hairless skin was slightly rubbed with a piece of sterilized 1200 mesh sandpaper. the damaged skin (2 cm × 2 cm area) was inoculated with 200 μl t. rubrum conidia suspension (1.0 × 10 6 conidia/ml). after the inoculation, each animal was i.p. injected with dexamethasone sodium phosphate injection (shanghai shengguang animal health products co., ltd., 0.4 ml per mouse) daily for 3 days. after the successful fungal infection, 20 animals were divided randomly into 5 groups and treated with various drugs by smearing the drugs on the infected skin daily for 14 days. three groups were treated with 50 μl anti-trcwp igy at concentrations of 5 mg/ml, 10 mg/ml, and 20 mg/ml. the model control (mc) group was treated with 50 μl sterile pbs (ph7.4, 0.01 m). the positive control group was treated with 1 mg/ml fluconazole (nanjing zhuopu biotechnology co., ltd.) solution. the efficacy of specific igy was evaluated by clinical lesion score and histopathological examination of skin tissues. for clinical evaluation, changes in redness, ulcerative scaling, and hair loss at the inoculation site were visually examined and recorded by use of a lesion score. the cured, improved and aggravated were scored as 0, 1and 2 respectively. the skin lesion score of mice was the sum of the three scoring items. animals were sacrificed for skin biopsy 24 h after the final drug administration. infected skin tissues were excised, fixed in neutral buffered formalin, embedded in paraffin, and stained with hematoxylin/eosin (he) for histopathological examination. the experimental data were expressed as mean ± sd. statistical differences between mean values were evaluated by the program spss for windows, v. 17.0 (spss inc.; chicago, il, usa). differences with p < 0.05 were considered significant and those with p < 0.01 were considered highly significant. the cwp of t. rubrum was extracted from t. rubrum hyphae. as shown in fig. 1 , sds-page displayed that the cwp had about eight bands ranging from 35kd to 130 kd. igy was extracted from the egg yolk through the two-step peg precipitation method and purified with saturated ammonium sulfate. as shown in fig. 2 , sds-page revealed that the igy preparation was pure and dissociated into two protein bands with molecular weights of 68kd and 27kd, which was consistent with other reports (da silva and tambourgi 2010). calculated with the protein content test results, the yield of specific igy (freeze-dried powder) extracted and purified from egg yolkwas 8.7-9.1 mg/ml with a purity of more than 90%. as shown in fig. 3 , through immunoblotting, extracted igy could be bound to the second antibody. the specific igywas found specificallybound to related antigen protein, while a negative igy was not. eggs were collected 20d after the first immunization; elisa was performed to assess the immune response of the purified igy. the titer of specific igy began to increase on the 20th day after the first immunization, as shown in fig. 4 , and reached the highest level on the 40th day after the first immunization. the highest titer of specific igy could reach 1:16 000.and was maintained for 30 days before declining (fig. 4) . cross-reactivity (cr) of anti-t. rubrum igy with other trichophyton strain or other fungi were tested by elisa by using antigens from different fungi. as shown in fig. 5 , specific igy showed a certain degree of cross-reactivity with different fungi. the most significant cross-reactivity was with t. mentagrophytes, a trichophyton strain (fig. 5) . as shown in the growth inhibitory curve (fig. 6) , compared with the control igy, specific igy showed a significant dose-dependent inhibitory effect on cell growth of t. rubrum. skin lesion scores after 14 days of treatment of 5 experimental groups were shown in fig. 7 . in comparison with the model control (mc) group treated with sterile pbs (ph7.4, 0.01 m), scores of specific igy groups decreased significantly and the treatment effect increased following the increase of dosages(p < 0.05,p < 0.05,p < 0.01) (fig. 7) . skin biopsies of infected areas of the experimental groups were subjected to histological analysis. in comparison with the normal untreated controls (fig. 8a) , the mc group showed fungal elements, damage of epithelium, dermis granulomas, and infiltration of inflammatory cells into the dermis, indicating the successful infection (fig. 8b) . the fluconazole group showed no obvious inflammation or tissue destruction (fig. 8c) . fewer inflammatory cells, weakened epithelium damage and hyperplastic tissue of cuticle were observed in the 5 mg/ml, 10 mg/ml, and 20 mg/ml specific igy groups (fig. 8d , e, and 8f). dermis granulomas decreased when the concentration of specific igy increases (fig. 8) . (color should be used in print). some studies have proved that specific igys were effective to bovine mastitis caused by e. coli and staphylococcus aureus [23] [24] [25] . in those studies, inactivated strains were used as antigens to immunize hens. the studies showed that s. aureus specific igys could inhibit the growth of s. aureus and the internalization of s. aureus by bovine mammary epithelial cells, and could decrease the bacterial count in milk and cure experimental mastitis (83.3%) and clinical mastitis (50%). igys were observed ineffective to lyse gram-positive bacteria, especially the encapsulated strain, but presented some inhibitory effect under growth conditions. e. coli specific igys could inhibit the growth of e. coli bybeing bound with it in a dose-dependent manner and enhance phagocytosis of e. coli by milk macrophages and polymorphonuclear neutrophil leukocytes. there were also other studies about antifungal igys such as anti-c. albicans igys [14, 26, 27] . one of the studies evaluated in vitro and in vivo effectiveness of igy against c. albicans by using proteins from sonicated cells of c. albicans as antigen to immunized hens. in the study, anti-c. albicans igy significantly reduced the adherence capacity of c. albicans to human pharynx carcinoma cells. oral administration of anti-c. albicans igy by mice significantly reduced the number of c. albicans and the scores of tongue lesions; anti-c. albicans igy also reduced colonization of c. albicans in mouse organs, indicating that anti-c. albicans igy had a protective effect against the oral candidiasis of experimentally infected mice and reduced the dissemination of c. albicans [26] . in this study, we investigated the growth inhibition activity of anti-trcwp igy to t. rubrum and the protective effect of anti-trcwp igy against dermatophytosis of experimentally infected mice. we immunized the hens with cell wall proteins extracted from t. rubrum. the cell wall was considered to be the major part of the deposition of fungal immune response products. many studies have shown that cell wall proteins have good immunogenicity and can induce specific antibodies [28] [29] [30] . in our study, the cell wall proteins showed good immunogenicity, highest titer of anti-trcwp igy reaching 1:16000. the anti-trcwp igy expressed significant inhibitory effect on t. rubrum in a dose-dependent manner compared with igy from unimmunized hens,and significantly reduced lesion scores of mice experimentally infected with t. rubrum. moreover, the anti-trcwp igy reduced infiltration of inflammatory cells, epithelium damage and hyperplastic tissue of cuticle. cell wall proteins of pathogenic fungi are crucial for growth, virulence, and pathogenicity as well as important in mediating the hostfungus interaction. the cell wall provides both adhesive properties critical for the invasion of host tissue and protection against the host defense mechanisms [31] . the first step of dermatophytes to infect the host is to inoculate the germinating fungus into the skin, which adheres to the cuticle. the occlusion and impregnation of the cuticle accelerate the adhesion process. the fungal spores sprout after being adhered to the cuticle for a long time; the mycelia continue to spread, especially in the lower layer of the cuticle [32] . anti-trcwp igy may be directly bound to the proteins on the fungal cell wall, inhibit the proliferation of t. rubrum, reduce the adherence capacity of t. rubrum to host cells and prevent further invasion of the fungal hypha to the host. anti-trcwp igy showed some degree of cross-reactivity with other fungi, most significantly with t. mentagrophytes, which indicated that the cell wall protein components of t. rubrum and t. mentagrophytes were quite similar. the anti-trcwp igy may also be effective to other fungal pathogens of dermatophytosis. this result was similar to another study. in that study, anti-e. coli o111 igy showed strong growth inhibition activity to both e. coli o111 and five other e. coli strains isolated from mastitis cows [23] . owing to the effect on pathogenic microorganisms, igys have been widely added into foods to prevent microbial infection of animals or aquatic organisms and widely added to cosmetics to resist bacteria and viruses to protect skin. they have also been added to mouthwash and toothpaste to prevent oral infection, to yogurt or food to prevent and treat helicobacter pylori and gastrointestinal virus infection. anti-trcwp igy can be used in a detergent to prevent dermatophytosis, and can be used in combination with antifungal chemicals to improve the therapeutic effect of dermatophytosis and reduce the generation of drug resistance. in conclusion, we presented evidence for the activity of anti-trcwp igy against t. rubrum. anti-trcwp igy showed a significant dosefig. 7 . skin lesion scores in t. rubrum-infected balb/c mice. after being infected, the mice were treated with pbs (model control), 5 mg/ml, 10 mg/ml, or 20 mg/ml specific igy or 1 mg/ml fluconazole (positive control). changes in redness, ulcerative scaling, and hair loss at the inoculation site were visually examined and recorded by using a lesion score. the cured, improved and aggravated were scored as 0, 1and 2 respectively. the skin lesion score of mice was the sum of the three scoring items. the data were presented as means ± sd (n = 4).*p < 0.05, **p < 0.01 in comparison with mc group. dependent growth inhibitory effect on t. rubrum in vitro and a significant dose-dependent treatment effect on t. rubrum infection in balb/c mice. these results suggested that anti-trcwp igy could be used as preventive immunotherapy against t. rubrum. the authors have no conflict of interest to declare. analysis of the dermatophyte species isolated in the british isles between 1980 and 2005 and review of worldwide dermatophyte trends over the last three decades assessment of dermatophytosis treatment studies: interpreting the data dermatophytes: 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characterization of specific egg yolk immunoglobulin (igy) against mastitis-causing escherichia coli characterization of chicken egg yolk immunoglobulins (igys) specific for the most prevalent capsular serotypes of mastitis-causing staphylococcus aureus efficacy of specific egg fig. 8. histopathological examination of skin sections from t. rubrum-infected balb/c mice. (a) normal group. (b) model control group. (c) fluconazole group. (d-f) 5 mg/ml, 10 mg/ml yolk immunoglobulin (igy) to bovine mastitis caused by staphylococcus aureus in vitro and in vivo effectiveness of egg yolk antibody against candida albicans (anti-ca igy) use of candida-specific chicken egg yolk antibodies to inhibit the adhering of candida to denture base materials: prevention of denture stomatitis a method for identification of dermatophyte antigens in situ by an immunoperoxidase technique in light and electron microscopy fluorescence microscopy in dermatology vaccination procedures and the infectivity of dermatophyte lesions the vaccines and antibodies associated with als3p for treatment of candida albicans infections arthroconidia as vectors of dermatophytosis we thank dr. songchao yin (the third affiliated hospital of sun yat-sen university) for providing fungi strain and technical guidance.this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. supplementary data to this article can be found online at https:// doi.org/10.1016/j.micpath.2019.103741. key: cord-013265-qrfi6e5c authors: isono, toshihito; domon, hisanori; nagai, kosuke; maekawa, tomoki; tamura, hikaru; hiyoshi, takumi; yanagihara, katsunori; kunitomo, eiji; takenaka, shoji; noiri, yuichiro; terao, yutaka title: treatment of severe pneumonia by hinokitiol in a murine antimicrobial-resistant pneumococcal pneumonia model date: 2020-10-15 journal: plos one doi: 10.1371/journal.pone.0240329 sha: doc_id: 13265 cord_uid: qrfi6e5c streptococcus pneumoniae is often isolated from patients with community-acquired pneumonia. antibiotics are the primary line of treatment for pneumococcal pneumonia; however, rising antimicrobial resistance is becoming more prevalent. hinokitiol, which is isolated from trees in the cypress family, has been demonstrated to exert antibacterial activity against s. pneumoniae in vitro regardless of antimicrobial resistance. in this study, the efficacy of hinokitiol was investigated in a mouse pneumonia model. male 8-week-old balb/c mice were intratracheally infected with s. pneumoniae strains d39 (antimicrobial susceptible) and nu4471 (macrolide resistant). after 1 h, hinokitiol was injected via the tracheal route. hinokitiol significantly decreased the number of s. pneumoniae in the bronchoalveolar lavage fluid (balf) and the concentration of pneumococcal dna in the serum, regardless of whether bacteria were resistant or susceptible to macrolides. in addition, hinokitiol decreased the infiltration of neutrophils in the lungs, as well as the concentration of inflammatory cytokines in the balf and serum. repeated hinokitiol injection at 18 h intervals showed downward trend in the number of s. pneumoniae in the balf and the concentration of s. pneumoniae dna in the serum with the number of hinokitiol administrations. these findings suggest that hinokitiol reduced bacterial load and suppressed excessive host immune response in the pneumonia mouse model. accordingly, hinokitiol warrants further exploration as a potential candidate for the treatment of pneumococcal pneumonia. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 pneumonia represents a major threat in a globalized society as it spreads easily by droplets. there are very few therapeutic agents that can tackle infection by drug-resistant bacteria or emerging viruses [1] . furthermore, self-organ damage owing to excessive host immunity can also occur in severe pneumonia [2] . therefore, treatment of severe pneumonia requires both elimination of drug-resistant microorganisms and suppression of hyperimmunity that damage the host lungs. hinokitiol, a tropolone-related compound has been isolated from chamaecyparis obtusa and thuja plicata donn ex d. don, which are trees in the cypress family, as β-thujaplicin [3, 4] . previous studies have shown antitumor, anti-inflammatory, antioxidant, and antibacterial activities of hinokitiol [5] [6] [7] [8] [9] . we recently showed that hinokitiol was effective against streptococcus pneumoniae in vitro, regardless of antimicrobial resistance [10] . at present, no studies have examined the effect of hinokitiol on pneumococcal infection in vivo. s. pneumoniae is a gram-positive diplococcus resident in the human upper respiratory tract [11] . this bacterium causes not only pneumonia but also otitis media, sinusitis, bronchitis, and empyema [12] , as well as invasive pneumococcal diseases, including meningitis, bacteremia, and septicemia [13] . the primary treatment of pneumococcal diseases relies on the use of effective antibiotics. in the early 2000s, macrolides were strongly recommended for pneumococcal pneumonia [14] [15] [16] ; however, their widespread use has augmented the risk of infection with macrolide-resistant s. pneumoniae [17, 18] . our previous study showed that more than 80% of s. pneumoniae isolated in municipal hospitals were non-susceptible to macrolides [19] . in addition to the appropriate prescription and consumption of antibiotics, new therapeutic agents need to be developed. in this study, we investigated the effect of hinokitiol on bacterial loads and analyzed the suitable administration route of hinokitiol using a murine pneumococcal infection model. furthermore, we also examined the effect of hinokitiol on inflammation in a severe pneumonia murine model. antimicrobial-susceptible s. pneumoniae strain d39 (serotype 2) was purchased from the national collection of type cultures (salisbury, uk). macrolide-resistant s. pneumoniae clinical strain nu4471 (serotype 19, azithromycin minimum inhibitory concentration (mic) � 1 mg/ml) was isolated from patients with respiratory tract infections [20] . all strains were grown in tryptic soy broth (becton dickinson, franklin lakes, nj, usa) at 37˚c as described previously [21] . male 8-10-week-old balb/c mice were obtained from nihon clea tokyo, japan. mice were maintained under standard conditions in accordance with our institutional guidelines. all animal experiments were approved by the institutional animal care and use committee of niigata university (sa00223). hinokitiol was purchased from fujifilm wako pure chemical industries (osaka, japan) and solubilized in phosphate-buffered saline (pbs) containing 10% ethanol. thiazolyl blue tetrazolium bromide (mtt) was purchased from sigma-aldrich (st. louis, mo, usa). the anesthetic mixture consisted of medetomidine hydrochloride (domitol; meiji seika pharma co., ltd., tokyo, japan), midazolam (dormicum; astellas pharma inc., tokyo, japan), and butorphanol (vetorphale; meiji seika pharma co., ltd.) mixed at 0.15, 2.0, and 2.5 mg/kg body weight with normal saline (otsuka pharmaceutical factory, inc. tokyo, japan) [22] . balb/c mice were injected intraperitoneally once a day with 0, 10, 20, 30, 50, and 100 mg/kg hinokitiol for 7 days. body weight was monitored every day. the change in body weight was considered an indicator of systemic toxicity of hinokitiol. the to evaluate the toxicity of intratracheally administered hinokitiol, mice were injected 500 μg/ml hinokitiol in 50 μl pbs. human alveolar epithelial cell line a549 (atcc ccl-185) was obtained from riken cell bank (ibaraki, japan). cells were seeded in a 96-well plate (becton dickinson) and grown to 90% confluence in dulbecco's modified eagle medium (fujifilm wako pure chemical industries) supplemented with 10% fetal bovine serum (japan bio serum, hiroshima, japan) and 100 u/ml penicillin and 100 μg/ml streptomycin (both fujifilm wako pure chemical industries) at 37˚c in 5% co 2 . a549 cells were treated with various concentrations (10-1000 μg/ml) of hinokitiol or 0.5% triton x-100 for 3 h. mtt assays were performed to determine cell viability as described previously [23] . mice (n = 8 per group) were anesthetized with a mixture of medetomidine hydrochloride, midazolam, and butorphanol and were intratracheally infected with s. pneumoniae strain d39 or strain nu4471 [1.0 × 10 9 colony forming units (cfu) in 50 μl pbs] using the microsprayer aerosolizer (penn-century inc., philadelphia, pa, usa). uninfected control mice (n = 5 per group) were intratracheally injected with 50 μl pbs (uninfected-pbs group) or 500 μg/ml hinokitiol (uninfected-hinokitiol group). at 1 h after infection, all infected mice were anesthetized again and 500 μg/ml hinokitiol (infected-hinokitiol group) was administered intratracheally to infected mice, while control mice were administered 50 μl pbs (infected-pbs group). to evaluate the efficacy of repeated hinokitiol administration in a pneumonia mouse model, all mice were intratracheally infected with s. pneumoniae strain nu4471 (3.0 × 10 8 cfu/mouse). after 1 h, 50 μl pbs was injected into the respiratory tract (untreated group; n = 5), followed by intratracheal injection of pbs every 24 h. in the hinokitiol injection group, all mice were initially intratracheally administered with hinokitiol 1 h after infection. the single administration group (n = 5) was injected with pbs after initial hinokitiol administration at 24 h intervals. in the double administration group (n = 5), the second administration of hinokitiol was performed at 24 h after infection, and pbs was administrated after 48 h from infection. in the three-dose group (n = 5), hinokitiol was intratracheally administered every 24 h after the first administration. all mice were sacrificed 72 h after from pneumococcal infection. at 18 h after infection, balf and serum samples were collected from five mice. lung tissue samples were collected from three mice. balf samples were plated onto blood-agar plates and cultured aerobically to count cfu. to determine the concentration of pneumococcal dna in murine serum, dna was isolated from 50 μl of serum obtained from the infected mice using qiaamp spin columns (qiagen, hilden, germany). then, absolute quantification by realtime pcr was performed using the steponeplus real-time pcr system via the sybr green detection protocol (thermo fisher scientific, waltham, ma, usa). the primers used for realtime pcr targeted a fragment of the pneumolysin-encoding gene of s. pneumoniae [24] . the forward primer sequence was 5 0 -agcgatagctttctccaagtgg-3 0 and the reverse primer sequence was 5 0 -cttagccaacaaatcgtttaccg-3 0 . for pathological examinations, lung tissue samples were fixed with 4% paraformaldehyde (fujifilm wako pure chemical industries). paraffin-embedded sections were made by biopathology institute co., ltd (oita, japan). lung sections were stained with hematoxylin and eosin (sakura finetek japan co., ltd., tokyo, japan). they were observed under a biorevo bz-9000 microscope (keyence, osaka, japan). to detect neutrophil elastase (ne) in lung tissue, paraffin-embedded sections were treated with rabbit anti-mouse ne antibody (abcam, cambridge, uk) in blocking solution (thermo fisher scientific). after overnight incubation at 4˚c, the secondary alexafluor 488-conjugated goat anti-rabbit igg antibody (thermo fisher scientific) in blocking buffer was added, followed by a 2 h incubation in the dark. the samples were then washed with pbs and mounted with nuclear staining mounting medium (prolong tm diamond antifade mountant with dapi; thermo fisher scientific). finally, samples were observed with a confocal laser-scanning microscope (carl zeiss, jena, germany). the number of neutrophils in balf was counted by flow cytometry. prior to staining cells in balf, their surfaces were blocked with anti-cd16/32 antibody (thermo fisher scientific) at 4˚c for 5 min [25] . then, cells were stained with anti-cd11b-allophycocyanin and ly-6gphycoerythrin antibody (thermo fisher scientific) in pbs containing 1% bovine serum albumin (sigma-aldrich) at 4˚c for 40 min in the dark [26] . samples were washed and fixed with 4% paraformaldehyde in pbs. flow cytometry analysis was performed on a novocyte flow cytometer with novoexpress software (acea biosciences, san diego, ca, usa) [26] . the cells were gated by their forward and side scatter. neutrophils were identified by the expression of both ly-6g and cd11b [26] . ne activity in balf was determined using the specific substrate n-methoxysuccinyl ala-ala-pro-val p-nitroanilide (merck millipore, billerica, ma, usa) as described previously [27] . briefly, samples were incubated in 0.1 m tris-hcl (ph 8.0) containing 0.5 m nacl and 1 mm substrate at 37˚c for 24 h, after which absorbance at 405 nm was measured. for cytokine and chemokine measurements, balf samples were centrifuged at 2000 × g at 4˚c for 15 min. then, enzyme-linked immunosorbent assay was performed to determine the concentrations of (i) c-x-c motif chemokine ligand l (cxcl-1; proteintech group, chicago, il, usa), (ii) interleukin (il)-1β, il-6, and tumor necrosis factor (tnf) (biolegend inc., san diego, ca, usa) in balf and serum according to the manufacturers' instructions. data were analyzed statistically by analysis of variance with tukey's multiple-comparison test. where appropriate (comparison of two groups only), unpaired t-tests were conducted. all statistical analyses were performed using graphpad prism software version 8.03 (graphpad software, inc., la jolla, ca, usa). values of p < 0.05 were considered significant. we first evaluated the systemic toxicity of hinokitiol. balb/c mice were intraperitoneally injected with hinokitiol once a day for 7 days as specified in the supplementary information. as shown in s1 fig, administration of �20 mg/kg hinokitiol significantly decreased body weight 3 days after the first administration (p < 0.05). hence, we lowered the maximum hinokitiol concentration for systemic administration in mice to 10 mg/kg. we used this dosage to test whether intraperitoneal administration of hinokitiol ameliorated pneumonia in a mouse intratracheal pneumococcal infection model. there was no significant decrease on pneumococcal cfu in the bronchoalveolar lavage fluid (balf) (s2 fig; p > 0 .05) and little effect on the transcriptional levels of various cytokine genes, such as cxcl1, il1b, il6, and tnf, in lung tissue (s3 fig; p > 0.05 ). the negative results from systemic administration of hinokitiol prompted us to test whether intratracheal administration of hinokitiol represented a better route for therapeutic intervention in pneumococcal pneumonia. evaluation of hinokitiol cytotoxicity against a549 alveolar epithelial cells in vitro revealed no significant decrease in viability except at 1000 μg/ml, whereby cell viability was reduced by 54% (fig 1a; p < 0.05). furthermore, compared with pbs injection, intratracheal hinokitiol administration caused negligible morphological changes in lung tissues in mice (fig 1b) . our previous in vitro study showed that 0.3 μg/ml hinokitiol exerted antibacterial effect against s. pneumoniae regardless of antimicrobial resistance [10] . here, compared with pbs injection, intratracheal administration of 500 μg/ml hinokitiol in a pneumococcal pneumonia mouse model decreased inflammatory cell migration and prevented destruction of alveolar tissue (fig 2) . in addition, intratracheal administration of hinokitiol significantly decreased cfu in balf of both antimicrobial-susceptible strain d39 and macrolide-resistant strain nu4471 (fig 3a; p < 0.05). because the severity of pneumococcal pneumonia is related to bacteremic pneumonia [28] , next, we investigated whether hinokitiol treatment inhibited s. pneumoniae. fig 3b shows that compared with pbs injection in mice, hinokitiol administration significantly decreased s. pneumoniae serum dna concentration (p < 0.05). previous studies have shown that excessive activation of neutrophils causes the release of ne, which contributes to severe lung injury [28] . therefore, controlling the excessive accumulation of neutrophils may be an essential therapeutic strategy to combat severe pneumonia [28, 29] . fig 4c) . similarly, ne activity in balf was significantly decreased in the hinokitiol administrated group which infected with pneumococcus compared to pbs injected group (fig 4d; p < 0.05). excessive production of inflammatory cytokines or chemokines is a hallmark of tissue injury and severe pneumonia [30, 31] . the concentration of cxcl-1, il-1β, il-6, and tnf was significantly lower in the balf from the infected-hinokitiol group compared with that from the infected-pbs group (fig 5; p < 0.05) . serum il-6 and tnf levels are associated with mortality of patients with pneumonia [32] . our previous study revealed that pneumococcal bacteremia induced an increase in serum cytokine levels [33] . here, the concentration of cxcl-1, il-1β, and il-6, but not tnf, was significantly decreased in the serum of the infected-hinokitiol group compared with that in the infected-pbs group (fig 6; p < 0.05) . meanwhile the chemokine and cytokines concentrations in balf and serum of uninfected control groups were either below (il-6) or just above the limit of detection (cxcl-1, il-1β and tnf). taken together, these findings suggested that the antibacterial action of hinokitiol in the lungs of pneumonia mice reduced the production of cytokines or chemokines in balf and serum. the above results suggested that intratracheal hinokitiol administration was effective in inhibiting s. pneumoniae in mouse lung. however, in clinical settings, antimicrobial agents are administrated several times for the treatment of pneumococcal pneumonia, and thus, we next sought to investigate whether repeated hinokitiol injection decreased the bacterial load in (fig 7; p < 0.05) . meanwhile, no significant differences were observed in balf and serum bacterial loads at 72 hours post-infection in the group between the untreated group and the groups which administrated hinokitiol once or twice. these results suggest that repeated administration of hinokitiol is required for the effective treatment of pneumococcal pneumonia. the present study showed that intratracheal administration of hinokitiol decreased the bacterial load in a mouse pneumonia model regardless of macrolide resistance of s. pneumoniae. as a result, the production of inflammatory cytokines and release of ne also decreased, eventually preventing the disruption of lung tissue. these results suggest that the antibacterial activity of hinokitiol has potential as a treatment against pneumococcal pneumonia. an important aspect when selecting a drug is to evaluate and compare its therapeutic index [34] . although hinokitiol exhibits antibacterial effect against various pathogenic organisms [10] , dosages above 12.7 and 14.8 mg/kg/day in male and female rats, respectively, have been shown to produce systemic side effects such as chronic toxicity [35] . based on our observations and these values, intraperitoneal injection of 10 mg/kg hinokitiol would be the maximum concentration for systemic administration in mice. however, this amount did not decrease bacterial load in our mouse pneumonia model, suggesting that this concentration is inadequate to achieve mic against s. pneumoniae in the lungs. topical administration of antibacterial agents has been successfully evaluated in the clinic for the prevention as well as therapeutic treatment of invasive pulmonary infections. our in vitro findings indicate that hinokitiol exhibited little cytotoxicity against pulmonary alveolar epithelial cells. aminoglycoside formulations for inhalation were developed to maximize drug delivery to the respiratory tract and minimize toxicity [36] [37] [38] . opting for an alternative route such as intratracheal administration of hinokitiol achieved the mic against s. pneumoniae, reducing disruption of lung tissue and suppressing bacteremia without systemic side effects. intratracheal administration demonstrated the efficacy of hinokitiol in a mouse pneumonia model; however, this may not necessarily indicate successful and complete treatment of pneumonia. clinically, treatment of infectious diseases requires thorough bactericidal effect until the infectious inflammation is resolved [39, 40] . therefore, a repeated dose of antibiotics is often prescribed to maintain an adequate concentration of the drug at the site of infection [41] [42] [43] . our repeated administration experiment showed that although single or two-time hinokitiol administration did not significantly lower the bacterial load 4 days after infection, repeating the procedure three times successfully decreased the bacterial load in the mouse pneumonia model. this finding suggests that hinokitiol administrated intratracheally does not exhibit antibacterial activity for an extended period and repeated administration may be required to treat pneumococcal pneumonia. effective use of hinokitiol will demand further research to optimize the administration interval and treatment period. hinokitiol intratracheal injection: neutrophil elastase (ne: green), dapi (blue). magnification: 40×; scale bar: 50 μm. (d) ne activity in balf of mice infected with either antimicrobial-susceptible s. pneumoniae strain d39 or macrolide-resistant strain nu4471 and then treated with pbs or hinokitiol. data represents the mean ± sem (n = 5) and was evaluated using one-way analysis of variance with tukey's multiple-comparisons test. � significantly different from the infected control group at p < 0.05. nd: not-detected, ns: not-significant; � p < 0.05. https://doi.org/10.1371/journal.pone.0240329.g004 antimicrobial resistance is a global problem [44] ; one way to deal with it is by developing alternative antimicrobials [45] , including from plant essential oils [46] . hinokitiol has been shown to inhibit the growth of antimicrobial-resistant organisms in vitro, such as methicillinresistant staphylococcus aureus and multidrug-resistant s. pneumoniae [10, 27, 47] . in this study, intratracheal administration of hinokitiol suppressed bacterial pneumonia induced by macrolide-resistant pneumococci. thus, hinokitiol could represent a new weapon against the growing class of macrolide-resistant s. pneumoniae. neutrophils are rapidly recruited to the lungs of pneumonia patients [48] . previous studies have shown that s. pneumoniae induced the release of ne from neutrophils, leading to the disruption of alveolar epithelial cells [28] . moreover, several animal studies have reported that the inhibition of ne activity suppressed lung injury in pneumococcal pneumonia [49, 50] . in this study, intratracheal hinokitiol administration reduced neutrophil infiltration and elastase release in the lungs of pneumonia mice, consequently diminishing lung injury and pneumococcal dna detection in serum. excessive production of inflammatory mediators induces a severe systemic response in pneumonia [51] . although antibacterial treatment is an important approach against pneumonia, regulating excessive inflammation is equally essential [52, 53] . hinokitiol was previously shown to reduce the transcription of inflammatory cytokines from epithelial cells in vitro [6, 54] . in this study, intratracheal administration of hinokitiol decreased the concentrations of cytokines and chemokines in balf and serum. these findings suggest that the anti-inflammatory property of hinokitiol, in addition to its antibacterial activity, might ameliorate the severity of pneumonia by downregulating the production of inflammatory mediators. certain limitaitions are noted in the current study. first of all, the administration of hinokitiol 1 h after bacterial inoculation is not representative of the clinical scenario, where antibiotics and other therapies are often initiated days after infection onset. additionally, most mouse experiments were only evaluated at a single time point in this study as obtaining balf and lung tissues requires the mice to be sacrificed. finally, the study does not include any conventional antibiotic comparators. the results of multiple doses indicate the limit of the antibacterial effect of hinokitiol and are expected to play a role as an adjunct to other antibacterial drugs. therefore, further research is needed to apply hinokitiol to the treatment of pneumonia, including hinokitiol alone as well as in combination with existing antibacterial drugs and hinokitiol. in conclusion, the present study showed that intratracheal hinokitiol administration suppresses acute pneumococcal pneumonia in vivo, regardless of whether bacteria are susceptible to macrolides. in case of severe pneumonia caused by antimicrobial-resistant pneumococci, a systemic dose of existing antimicrobials alone may not be successful [55, 56] . adjunctive use of hinokitiol may facilitate the treatment of pneumococcal pneumonia and reduce the use of existing antimicrobials. supporting information s1 fig. changes in body weight in mice intraperitoneally injected with hinokitiol. male 8-week-old balb/c mice were intraperitoneally injected once a day with 0, 10, 20, 30, 50, and 100 mg/ kg hinokitiol in pbs containing 10% ethanol buffer for 7 days. body weight was monitored every day. data represent the mean ± sem (n = 4) and were analyzed using two-way anova with dunnett's multiple comparison test; � p < 0.05. (tif) male 8-week-old balb/c mice were intratracheally infected with s. pneumoniae strain d39 (1.0 × 10 9 cfu in 50 μl pbs) and, 1 h after infection, they (n = 5) were intraperitoneally injected with hinokitiol (10 mg/kg, n = 5) or pbs (containing 10% ethanol, n = 5). after 18 h, balf samples were plated on blood-agar plates and cultured aerobically to count cfu. data represent the mean ± sem (n = 5) and were analyzed using student's t-test; � p < 0.05. (tif) s3 fig. hinokitiol does not affect the transcription of chemokine and cytokine genes in mice. real-time pcr was performed to quantify the transcription of cxcl1, il1b, il6, and tnf in mouse lung tissue. total rna was extracted from mouse lung tissue using tri reagent (molecular research center, inc., cincinnati, oh, usa), and quality was assessed spectrophotometrically at 260 and 280 nm. the rna was reverse-transcribed using superscript vilo master mix (thermo fisher scientific, waltham, ma, usa) and the cdna was quantified using the steponeplus real-time pcr system according to the manufacturer's protocol. values were normalized to those of gapdh mrna and are presented as fold change relative to the mrna transcript levels of the control group. data represent the mean ± sem (n = 5) and were analyzed using student's t-test; � p < 0.05. (tif) all mice were intratracheally infected with s. pneumoniae strain nu4471 (3.0 × 10 8 cfu/mouse). pbs injection or hinokitiol (500 μg/ml in pbs) injection via the tracheal route was started after 1 h from infection. pbs or hinokitiol injection into the air tract was performed at 24 h intervals. all mice were sacrificed and samples were collected after 72 h from infection. 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polymorphonuclear leukocyte effects of specific neutrophil elastase inhibitor, sivelestat sodium hydrate, in murine model of severe pneumococcal pneumonia inhibition of neutrophil elastase reduces lung injury and bacterial count in hamsters proinflammatory and anti-inflammatory cytokines as mediators in the pathogenesis of septic shock impact of macrolide therapy on mortality for patients with severe sepsis due to pneumonia addition of a macrolide to a beta-lactam-based empirical antibiotic regimen is associated with lower in-hospital mortality for patients with bacteremic pneumococcal pneumonia anti-inflammatory effects of hinokitiol on human corneal epithelial cells: an in vitro study a review of clinical failures associated with macrolide-resistant streptococcus pneumoniae resistance among streptococcus pneumoniae: implications for drug selection we would like to thank editage (www.editage.com) for english language editing. key: cord-011106-h20vbmbo authors: takeda, yohei; okuyama, yuko; nakano, hiroto; yaoita, yasunori; machida, koich; ogawa, haruko; imai, kunitoshi title: antiviral activities of hibiscus sabdariffa l. tea extract against human influenza a virus rely largely on acidic ph but partially on a low-ph-independent mechanism date: 2019-10-16 journal: food environ virol doi: 10.1007/s12560-019-09408-x sha: doc_id: 11106 cord_uid: h20vbmbo influenza a virus (iav) infection is perennially one of the leading causes of death worldwide. effective therapy and vaccination are needed to control viral expansion. however, current anti-iav drugs risk inducing drug-resistant virus emergence. although intranasal administration of whole inactivated virus vaccine can induce efficient protective immunity, formalin and β-propiolactone are the currently used and harmful inactivating agents. here, we analyzed the antiviral activity of hibiscus (hibiscus sabdariffa l.) tea extract against human iav and evaluated its potential as a novel anti-iav drug and a safe inactivating agent for whole inactivated vaccine. the in vitro study revealed that the ph of hibiscus tea extract is acidic, and its rapid and potent antiviral activity relied largely on the acidic ph. furthermore, the mouse study showed that the acidic extract was not effective for either therapeutic or vaccination purposes. however, hibiscus tea extract and protocatechuic acid, one of the major components of the extract, showed not only potent acid-dependent antiviral activity but also weak low-ph-independent activity. the low-ph-independent activity did not affect the conformation of immunodominant hemagglutinin protein. although this low-ph-independent activity is very limited, it may be suitable for the application to medication and vaccination because this activity is not affected by the neutral blood environment and does not lose antigenicity of hemagglutinin. further study of the low-ph-independent antiviral mechanism and attempts to enhance the antiviral activity may establish a novel anti-iav therapy and vaccination strategy. outbreaks of seasonal influenza caused by influenza a virus (iav) or b virus occur all over the world and lead to approximately 3 to 5 million severe cases and 290,000 to 650,000 deaths every year (world health organization 2018). moreover, novel iav strains have emerged repeatedly 1 3 in the past, leading to a large number of deaths by pandemics in the twentieth century (frederick 1958; saunders and krewski 2016; world health organization 2009) . to combat iav, there is a relentless need to develop more effective antiviral therapies and vaccine strategies. the calyces of hibiscus sabdariffa l. is an ingredient in hibiscus tea. hibiscus tea contains abundant bioactive compounds including anthocyanins, polyphenols, organic acids, and flavonoids (da-costa-rocha et al. 2014) . earlier reports have shown various biological and pharmacological activities of hibiscus tea and hibiscus tea-derived compounds like anti-inflammatory, anti-oxidant, anti-cholesterol, anti-hypertensive, anti-bacterial, and antiviral activities (d'souza et al. 2016; da-costa-rocha et al. 2014; hassan et al. 2017; joshi et al. 2015) . we previously reported the antiviral activity of hibiscus tea extract against h5 subtype avian influenza viruses (baatartsogt et al. 2016) . hibiscus tea extract inactivated the avian influenza viruses, showing activity in 10 min. based on these results, we hypothesized that these potent and rapid antiviral activities could be used to develop a novel anti-iav drug and a safe inactivating agent for the whole inactivated vaccine. a number of anti-iav drugs have been in practice or under development (li et al. 2015; noshi et al. 2018) . although these drugs elicit the therapeutic effect, viral strains resistant against these drugs tend to appear after prolonged use (hurt 2014; li et al. 2015) , and some of these drugs also exhibit adverse effects. rna polymerase inhibitor favipiravir showed teratogenicity and embryotoxicity in animal experiments. therefore, its clinical use is strictly restricted in japan (nagata et al. 2015) . in addition, the possibility of insufficient existing drug supply is a concern during pandemics. finding novel anti-iav substances would be a promising approach for treatment to deal with these problems. vaccination is also important to control iav expansion. one of the effective vaccination strategies is intranasal (i.n.) administration of whole inactivated vaccine. whole inactivated vaccine can prime potent protective immunity because it preserves virus particle integrity (soema et al. 2015) . in addition, i.n. vaccination can induce systemic igg production and mucosal iga production, the latter of which is indispensable to prevent viral infection establishment (rose et al. 2012; tamura et al. 2016) . formalin and β-propiolactone (β-pl) are widely used as inactivating agents for whole-virus vaccines (delrue et al. 2012) . however, removal of these substances is essential due to their chemical toxicity, making the procedure time consuming and expensive. finding a safe anti-iav compound that can be used as a novel inactivating agent for a whole-virus vaccine may solve critical problems. here, we attempted to clarify the mechanism of antiviral activity against h1n1 human iav strain a/puerto rico/8/1934 (pr8 virus) and identify the antiviral compounds present in hibiscus tea extract. we focused on three compounds including protocatechuic acid (pca), ferulic acid, and (2s,3r)-tetrahydro-3-hydroxy-5-oxo-2,3-furandicarboxylic acid (hibiscus acid). pca and ferulic acid showed antiviral activity against several virus species (hassan et al. 2017; joshi et al. 2015) . hibiscus acid is one of the major organic acids in hibiscus calyces (da-costa-rocha et al. 2014 ). in addition, we assessed hibiscus tea extract's potential as a candidate for novel anti-iav drug and as an inactivating agent for whole-virus vaccines. pr8 virus (atcc® catalog no. vr-95™) was purchased from atcc (manassas, va), and propagated in the allantoic fluid of 10-day-old embryonated chicken eggs. madin-darby canine kidney (mdck) cells were kindly provided by dr. h. nagano (hokkaido institute of public health, sapporo, japan). for passaging, mdck cells were cultured in dulbecco's modified eagle's minimal essential medium (dmem) (nissui pharmaceutical co., ltd., tokyo, japan) supplemented with 10% fetal bovine serum, 20 μm l-glutamine (wako pure chemical industries, ltd., osaka, japan), 0.15% nahco 3 (wako pure chemical industries, ltd.), 2 µg/ml amphotericin b (bristol-myers squibb co., new york, ny), and 100 µg/ml kanamycin (meiji seika pharma co., ltd., tokyo, japan). after viral inoculation, mdck cells were cultured in viral growth medium composed of dmem supplemented with 0.2% bovine serum albumin (wako pure chemical industries, ltd.), 0.01% glucose (wako pure chemical industries, ltd.), 0.15% nahco 3 , 2.5 mm hepes (wako pure chemical industries, ltd.), and 0.0006% trypsin (wako pure chemical industries, ltd.). ten to 13-week-old female balb/c mice were purchased from clea japan inc. (tokyo, japan) or generated in our laboratory. all animal experiments were approved by the institutional animal care and use committee of obihiro university of agriculture and veterinary medicine and performed in compliance with institutional guidelines. pca was purchased from tokyo chemical industry co., ltd. (tokyo, japan). ferulic acid was purchased from cayman chemical co. (an arbor, mi). formalin and β-pl were purchased from wako pure chemical industries, ltd. hibiscus tea (roselle) was purchased from nakazen corp. (nanjo, japan). to prepare acidic crude hibiscus tea extract (hib[crude]), 2 g of hibiscus tea powder was boiled in 100 ml ultrapure water until the volume reached 50 ml. to isolate the small molecular weight (< 3 kda) fraction (frhibis), hib[crude] was fractionated using vivaspin® 6 (3000 mwco pes) (sartorius ag., göttingen, germany) (baatartsogt et al. 2016 ). hibiscus acid was isolated as previously described (ibnusaud et al. 2002; ibrahim et al. 2000) . in brief, hibiscus tea powder was soaked in a sufficient volume of ultrapure water at 24 °c for 24 h, and this process was repeated several times. the combined extract was concentrated under reduced pressure to obtain a viscous syrup. methanol was added to the syrup to precipitate insoluble materials. the precipitate was removed by filtering, and the filtrate was concentrated to obtain a second viscous syrup that was extracted several times with acetone. acetone was removed under reduced pressure, and the extract was further extracted several times with ether. the ether was removed under reduced pressure, and a crude acid was obtained which was recrystallized (ether + chloroform) to obtain pure hibiscus acid (molecular weight: 190.1, 0.1 g, 1%) in the form of colorless crystal. the structure and purity of isolated hibiscus acid was confirmed using previously reported spectroscopic and other physical analyses (ibnusaud et al. 2002; ibrahim et al. 2000) . hibiscus acid-na was synthesized as previously described (ibnusaud et al. 2002; ibrahim et al. 2000) . in brief, saturated aqueous sodium bicarbonate was added dropwise into an aqueous hibiscus acid solution (0.1 g, 0.53 mmol, in 1 ml water) at 0 °c until the ph of the solution was neutral. after removing water under reduced pressure, the residue was triturated and washed using dry acetone. the product was finally dried under reduced pressure to obtain a colorless solid (molecular weight: 234.1, 0.1 g, 81%). 10 5 50% tissue culture infective dose (tcid 50 )/ml of pr8 virus propagated in allantoic fluid was mixed with an equal amount of phosphate-buffered saline (pbs), hib[crude], frhibis, pca, ferulic acid, hibiscus acid, or hibiscus acid-na. the effective concentrations of pca, ferulic acid, hibiscus acid, and hibiscus acid-na in the mixture were 2.5 mg/ml, 1 mg/ml, 2.5 mg/ml, and 2.5 mg/ml, respectively. ph of the mixture was adjusted within a range of acidic to around neutral by adding hcl (wako pure chemical industries, ltd.) or naoh (wako pure chemical industries, ltd.). ph of the mixture was determined by measuring the ph of a virus-free mixture as a substitute for the virus-containing mixture. the mixture was placed at 26 °c for 10 min, 24 h, or 48 h. after each reaction time, the mixture was inoculated into mdck cells and a tenfold serial dilution was performed. three days after inoculation, the viral titer was evaluated by observing the cytopathic effect (cpe) on mdck cells. we calculated log 10 tcid 50 /ml using the behrens-kärber method (kärber 1931) . pr8 virus propagated in allantoic fluid was mixed with an equal amount of neutral and acidic ph pbs, hib[crude], frhibis, or pca. the effective concentration of pca in the mixture was 2.5 mg/ml. the mixture was placed at 26 °c for 10 min or 48 h. a twofold serial dilution was performed after each reaction time. an ha test was conducted using 0.5% chicken blood cells (nippon bio-test laboratories inc., asaka, japan) according to the who manual on animal influenza diagnosis and surveillance (who manual, world health organization 2002). the ha test was also conducted with formalin-, β-pl-, or acidic hib[crude]-inactivated purified pr8 virus. mice were intranasally given 10 1 tcid 50 /50 μl pr8 virus propagated in allantoic fluid. eight hours after the viral inoculation, 200 μl of neutral pbs or acidic hib[crude] was administered once orally (p.o.) (day 0). from day 1 to 4, pbs or hib[crude] was administered p.o. twice a day. each group had 5 mice. the change in the body weight of each mouse was monitored daily. mice were euthanized when the body weight decreased to 75% or 2 weeks after the viral inoculation. to determine the lung viral titer, mice were euthanized at days 3 and 5 and lungs were harvested from the euthanized mice. the lungs were homogenized in 20% (w/v) pbs supplemented with 5 μg/ml amphotericin b, 1 mg/ml kanamycin, and 100 μg/ml gentamicin (msd k.k., tokyo, japan). the lung homogenate supernatant was added to mdck cells, and a tenfold serial dilution was performed. three days after inoculation, the viral titer (log 10 tcid 50 /g tissue) was evaluated. to prepare formalin-inactivated whole-virus vaccine, 1 mg/ml of purified pr8 virus was mixed with 0.1% formalin and kept at 4 °c for 48 h. after viral inactivation, 0.6% na 2 s 2 o 4 (wako pure chemical industries, ltd.) was added and the mixture was placed at 4 °c overnight. similarly, 1 3 to prepare β-pl-inactivated whole-virus vaccine, 1 mg/ ml of purified pr8 virus was mixed with 0.1% β-pl and placed at 4 °c for 48 h. afterward, formalin and β-pl were removed using membrane dialysis with spectra/por® (8,000 mwco) (spectrum laboratories inc., los angeles, ca). the concentration of formalin-or β-pl-inactivated pr8 virus was measured using the micro bca protein assay kit (pierce, rockford, il) and adjusted to 300 μg/ ml. to prepare acidic hib[crude]-inactivated whole-virus vaccine, 600 μg/ml of purified pr8 virus was mixed with an equal amount of acidic hib[crude] and placed at 26 °c for 10 min. the inactivated virus was directly used to vaccinate mice. viral inactivation was confirmed based on the absence of viral infectivity by egg inoculation followed by an ha test. 50 μl pbs, formalin-, β-pl-, or acidic hib[crude]-inactivated pr8 virus vaccine was intranasally administered in mice (first vaccination) under light anesthesia with isolflurane (intervet k.k., tokyo, japan). the second vaccination was performed 2 weeks after the first vaccination. there were 5 mice in each group. six weeks after the second vaccination, the mice were intranasally inoculated with 10 2 tcid 50 /50 μl of pr8 virus propagated in allantoic fluid (day 0). changes in body weight were monitored daily after pr8 viral inoculation. the mice were euthanized when the body weight decreased 75%. all surviving mice were euthanized on day 14. blood samples were collected on day 0 (pre-inoculation) and day 14 (post-inoculation) to evaluate anti-pr8 virus antibody (ab) titer using the hemagglutination inhibition (hi) test. the obtained serum samples were treated with receptordestroying enzyme (denka seiken co., ltd., japan) to remove non-specific ha inhibitors. the serum was mixed with chicken blood cells (the final concentration was 10%), and the mixture was rotated at room temperature for 1 h to remove any non-specific ha factors. the supernatant was collected after centrifugation, and the hi test was conducted in the presence of pr8 viral antigen according to the who manual. the serum anti-pr8 virus ab titer was determined as log 2 hi titer. since hib[crude] is acidic, we assessed whether the anti-pr8 virus activity of hib[crude] depends on its acidic ph. pr8 virus was mixed with non-ph-adjusted and phadjusted pbs or hib[crude] (ph of the mixture ranged from 3.0 to 7.2), and the titer of the treated virus was determined. the ph of the mixture containing non-phadjusted hib[crude] was around 3.0. after a 10-min reaction, hib[crude] and pbs of ph ≤ 4.0 strongly inactivated the pr8 virus, but the effect of hib[crude] at ph 4.0 seemed to be slightly higher than pbs at ph 4.0. on the other hand, both pbs and hib[crude] of ph ≥ 5.0 did not inactivate pr8 virus. after a 24-h reaction, pr8 virus was inactivated using both pbs and hib[crude] at ph ≤ 5. on the other hand, the viral titer of hib[crude]-treated pr8 virus was 10 1 -times lower than pbs-treated pr8 virus under the conditions of ph ≥ 6.0 (fig. 1a ). the differences were statistically significant (p < 0.05 or 0.01). we also evaluated viral titers of acidic and neutral (ph-adjusted) frhibis-treated pr8 virus. the ph of the mixture containing non-ph-adjusted frhibis was around 3.0. acidic frhibis inactivated pr8 virus after a 10-min reaction, but neutral frhibis did not. after a 48-h reaction, the viral titer of neutral frhibis-treated pr8 virus was consistent with neutral hib[crude]-treated pr8 virus and 10 2 -times lower (p < 0.01) than pbs-treated pr8 virus (fig. 1b) . these results show that frhibis has two different anti-pr8 virus activities: one is a rapid and potent activity largely dependent on acid, and the other is a slow and weak low-ph-independent activity. next, we evaluated the anti-pr8 virus activity of pca, ferulic acid, and hibiscus acid. these compounds are acidic in solution. pr8 virus was treated with pbs or each of these solutions, and the titer of the virus was measured. both acidic (non-ph-adjusted) and neutral (ph-adjusted) solutions were tested to correlate between acidic ph of the solutions and the anti-pr8 virus activity. the estimated ph values of the mixtures containing acidic pca, ferulic acid, and hibiscus acid were approximately 5.0, 6.5, and 3.0, respectively. the mixtures containing acidic pbs (ph 5.0, 6.5, and 3.0) were also tested as acidic solution controls. after a 10-min reaction, the titers of acidic and neutral pca-treated pr8 virus were 1 3 similar to a pbs-treated pr8 virus. acidic pbs and pca completely inactivated pr8 virus after a 48-h reaction. on the other hand, the viral titer of pca-treated pr8 virus was 10 1.5 -times lower (p < 0.01) than pbs-treated pr8 virus under neutral conditions (fig. 2a) . neither a 10-min nor 48-h reaction in acidic or neutral conditions showed anti-pr8 viral activity for ferulic acid (fig. 2b) . finally, anti-pr8 viral activity of hibiscus acid was analyzed. hibiscus acid-na is a disodium salt of hibiscus acid, and its ph was approximately neutral. acidic hibiscus acid inactivated pr8 virus after a 10-min or 48-h reaction, but neutral hibiscus acid and hibiscus acid-na did not do so (fig. 2c) . these results show that pca has weak low-ph-independent anti-pr8 virus activity. next, we assessed whether acidic and neutral hibiscus tea extracts affect the ha activity of pr8 virus. pr8 virus was mixed with acidic or neutral pbs, hib[crude], or frhibis. ha titer of pr8 virus was measured after a 10-min or 48-h reaction. acidic pbs-, hib[crude]-, and frhibis-treated pr8 virus lost ha activity in 10 min. it was confirmed earlier that the acidic hib[crude] did not directly affect chicken blood cell agglutination (data not shown). after a 48-h reaction, the ha titer of neutral hib[crude]-and frhibis-treated pr8 virus did not decrease compared to pbs-treated pr8 virus (fig. 3a) . ha titers of acidic and neutral pca-treated pr8 virus were also measured. acidic pbs-and pca-treated pr8 virus lost ha activity after a 48-h reaction. on the other hand, the ha titer of neutral pca-treated pr8 virus did not decrease compared to pbs-treated pr8 virus (fig. 3b) . these results show that neutral hib[crude], frhibis, and pca does not affect viral ha activity. next, we assessed the therapeutic efficacy of p.o. administration of acidic hibiscus tea extract in pr8 virus-infected mice. neutral pbs or acidic hib[crude] was administered p.o. to mice twice a day for the first 4 days after pr8 virus inoculation (fig. 4a) . a significant loss in body weight was observed in all treated mice (fig. 4b) . all pbs-treated mice and 80% (4 out of 5) hib[crude]-treated mice died within 8 days post-infection. although only one hib[crude]-administered mouse survived and recovered from the disease, there was no significant difference in the survival rate between the pbs-and hib[crude]-treated groups (fig. 4c) . the viral titer in the lungs was determined on days 3 and 5, and it was found to be similar to levels in both the pbs and hib[crude] groups (fig. 4d) . these results show that p.o. administration of acidic hib[crude] does not exert a therapeutic effect in pr8 virus-infected mice. next, we assessed the efficacy of acidic hibiscus tea extract as an inactivating agent for whole virus. the pr8 virus inactivated by formalin, β-pl, or acidic hib[crude] was intranasally administered to mice twice before administering a lethal dose of pr8 virus (fig. 5a) . non-vaccinated mice receiving pbs served as a control group. non-vaccinated mice rapidly lost body weight after the viral inoculation, and all mice in the group died by day 7. on the other hand, the body weight in the formalin-and β-pl-treated groups did not change, and all the mice survived until day 14. the mice lost body weight in the hib[crude]-treated group, but the degree of weight loss was ameliorated compared to the non-vaccinated group. the vaccine efficacy was much lower in the hib[crude]-treated group, but the survival rate was significantly improved (fig. 5b, c) . the hi-active ab titer in serum was also measured pre-inoculation on day 0 and postinoculation on day 14. prior to inoculation, the hi titers were below the detection limit in all non-vaccinated mice, but the titers were in the range of 2 9 to 2 12 in the formalin and . the ph of each aqueous solution was adjusted to acidic or approximately neutral. the ph shown was an approximate value with a range ± 0.3. the mixture was placed at 26 °c for 10 min or 48 h and mixed with an equal volume of chicken blood cells. ha titer of the mixture was evaluated after a 30-min reaction. error bars indicate mean ± sd; n = 3 to 6 per group. student's t test with bonferroni's test (a), and student's t test (b) were performed to analyze statistical significance. the results represent more than two independent experiments 1 3 β-pl groups. in the hib[crude] group, the titers were less than 2 5 in all mice and below the detection limit in two mice (fig. 5d) . although the hi titers were low in the hib[crude] group, the hi-positive mice survived until day 14 (data not shown). post-inoculation hi titer did not differ from the preinoculation titer in the formalin and β-pl groups. in the hib[crude] group, the hi titer increased to the same level as the formalin and β-pl groups (fig. 5d) . these results indicate that formalin-and β-pl-inactivated vaccine completely prevented viral infection, but acidic hib[crude]-inactivated vaccine did not prevent infection. the viral particle structure integrity in the whole inactivated vaccine relates to protective activity against iav infection. we assessed whether the conformation of hemagglutinin (hain) was preserved in each vaccine using the ha assay. ha activity of iav is lost by a change in hain conformation (sriwilaijaroen and suzuki 2012) . intact (untreated) or inactivated pr8 virus was mixed with chicken blood cells and the ha titer was measured. the ha titer of acidic hib[crude]-treated pr8 virus was below the detection limit, but the ha titer of formalin-and β-pl-treated pr8 virus was similar to the intact pr8 virus (fig. 5e) . these results show that formalin and β-pl treatments but not acidic hib[crude] preserve hain conformation. earlier studies indicated that the extract derived from hibiscus sabdariffa l. calyces has antiviral activity against the h5 subtype of highly pathogenic and low-pathogenic avian influenza viruses, herpes simplex virus-2, feline calicivirus, murine norovirus-1, hepatitis a virus, and aichi virus (baatartsogt et al. 2016; d'souza et al. 2016; hassan et al. 2017; joshi et al. 2015) . moreover, the leaves of hibiscus sabdariffa l. also showed anti-measles virus activity (sunday et al. 2010 ). however, antiviral activity mechanism against these viruses is still unclear. since many acidic compounds in hibiscus calyces contribute to the low ph, we focused on correlating anti-pr8 viral activity and the acidic ph of hibiscus tea components. we found that not only hib[crude] but also pbs of ph ≤ 4.0 potently inactivated pr8 virus in a short period of time (10 min) (fig. 1) . this finding indicates that the anti-pr8 virus activity of hibiscus tea extract is largely dependent on acid. however, we could not deny a possibility that there are unknown antiviral compounds which show its activity only in the acidic condition. this unknown mechanism might partially contribute to the viral inactivation. we further analyzed the anti-pr8 virus activity of pca, ferulic acid, and hibiscus acid, which are all components of hibiscus tea extract. in the previous reports, pca showed antiviral activity against feline calicivirus, murine norovirus-1, and herpes simplex virus-2 (hassan et al. 2017; joshi et al. 2015) . ferulic acid showed antiviral activity against feline calicivirus and murine norovirus-1 (joshi et al. 2015) . here, acidic pca and hibiscus acid showed anti-pr8 virus activity. non-ph-adjusted ferulic acid solution with a relatively higher ph (ph 6.5) did not inactivate pr8 virus. when the iav particle is exposed to acidic ph conditions, viral hain changes its conformation and irreversibly loses binding affinity for sialic acid (quan et al. 2011; sriwilaijaroen and suzuki 2012) . here, all pr8 virus particles treated with acidic pbs-, (fig. 3) . it is also known that low-ph treatment of iav induces m1 protein layer loss and coagulation of ribonucleoprotein complexes (fontana et al. 2012 ). these comprehensive structural changes due to acid could have resulted in rapid and potent viral inactivation from acidic hibiscus component treatment. many iav strains are susceptible to acid, but the degree of sensitivity varies between strains (puri et al. 1990; ruigrok et al. 1984) . some highly pathogenic strains of avian h5 influenza virus like h5n1 strain isolated from thailand were resistant to ph 3.0 (treated for 10 min at room temperature) treatment (wanaratana et al. 2010) . although acidic hib[crude] and frhibis inactivated highly pathogenic and low-pathogenic avian h5 influenza viruses in 10 min in our previous study (baatartsogt et al. 2016) , we have yet to determine whether all potent and rapid antiviral activity against these h5 viral strains is completely acid dependent. here, frhibis showed potent acid-dependent antiviral activity and weak low-ph-independent antiviral activity against pr8 virus (fig. 1) . the neutral pca also showed anti-pr8 virus activity (fig. 2) . the anti-pr8 virus activity by both neutral frhibis and pca did not affect hain activity (fig. 3) . since pca accounts for 24% of hibiscus sabdariffa water extract (da-costa-rocha et al. 2014), a major part of the anti-pr8 virus activities of neutral hibiscus extract may be due to pca. in addition, frhibis also contains abundant bioactive compounds such as phenolic compounds (da-costa-rocha et al. 2014) . several studies reported that polyphenol-rich plant extracts and plant-derived phenolic compounds have anti-iav activities (droebner et al. 2007; kim et al. 2013; yamada et al. 2009 ). these phenolic compounds in frhibis may also contribute to antiviral activity. an attempt to identify the mechanism of low-ph-independent anti-pr8 virus activity of pca and to search for other low-ph-independent anti-pr8 virus constituents is currently ongoing. we assessed the therapeutic efficacy of p.o. administration of acidic hib[crude] in pr8 virus-infected mice. however, no therapeutic effect was observed (fig. 4) . in the case of p.o. administration, hib[crude] is first absorbed from the digestive tract and distributed into systemic blood. subsequently, it reaches the respiratory organs where iav resides. the neutralized hib[crude] in the blood loses potent anti-iav activity due to acid, and the low-ph-independent antiviral activity is inadequate to inactivate virus in vivo. the low content, weak antiviral activity, and short biological half-life of low-ph-independent anti-iav compounds in hib[crude] could contribute to therapeutic failure. to achieve a successful p.o. administration therapy using hibiscus tea components such as pca, it would be necessary to chemically modify them to extend their biological half-life and enhance antiviral activity or use them in combination with a carrier to retain them in blood. in iav vaccination, i.n. vaccine administration induces effective anti-iav immunity. we previously showed that i.n. co-administration of innate immune receptor adjuvants with split vaccine efficiently induced both systemic and mucosal anti-iav immunity (takaki et al. , 2017 takeda et al. 2018 ). on the other hand, whole inactivated vaccine comprises viral structural components with immune priming activity like immunostimulatory viral rna. we assessed the utility of acidic hib[crude] as a safe inactivating agent for whole-virus vaccine. however, unlike the viruses inactivated by formalin and β-pl, the viruses inactivated by acidic hib[crude] failed to induce efficient protective immunity (fig. 5) . as shown in other reports (pawar et al. 2015) , formalin-and β-pl-treated virus but not acidic hib[crude] showed intact ha activity (fig. 5e ). hain is an immunodominant protein (kaminski and lee 2011; liu et al. 2018; sautto et al. 2018) . the acidic ph of hib[crude] could cause the loss of antigenicity by inducing a change in hain conformation. these results show that the acidic hibiscus components cannot be used as inactivating agents for vaccine preparation. on the other hand, low-ph-independent activity of frhibis and pca which does not affect hain conformation is suitable for vaccine application. however, these weak virucidal effects have not yet been sufficient for use as an inactivating agent. further study of the low-ph-independent anti-iav mechanism and enhancement of its antiviral activity may contribute to iav vaccine strategy development. in conclusion, we showed the rapid and potent anti-pr8 virus activity of 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molecular basis of the structure and function of h1 hemagglutinin of influenza virus antiviral effect of hibiscus sabdariffa and celosia argentea on measles virus toll-like receptor 3 in nasal cd103 + dendritic cells is involved in immunoglobulin a production cgamp promotes germinal center formation and production of iga in nasal-associated lymphoid tissue vaccine adjuvant arnax promotes mucosal iga production in influenza ha vaccination. biochemical and biophysical research communications intranasal inactivated influenza vaccines: a reasonable approach to improve the efficacy of influenza vaccine? the inactivation of avian influenza virus subtype h5n1 isolated from chickens in thailand by chemical and physical treatments pdf?seque nce=1&isall owed=y world health organization mechanism of the antiviral effect of hydroxytyrosol on influenza virus appears to involve morphological change of the virus publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations conflict of interest the authors declare that they have no conflict of interest. key: cord-309742-fd1qmr87 authors: slepushkin, vladimir a.; staber, patrick d.; wang, guoshun; mccray, paul b.; davidson, beverly l. title: infection of human airway epithelia with h1n1, h2n2, and h3n2 influenza a virus strains date: 2016-12-14 journal: mol ther doi: 10.1006/mthe.2001.0277 sha: doc_id: 309742 cord_uid: fd1qmr87 three subtypes of influenza a virus cause human disease: h1n1, h2n2, and h3n2. although all result in respiratory illness, little is known about how these subtypes infect differentiated airway epithelia. therefore, we assayed a/pr/8/34 (h1n1), a/japan/305/57 (h2n2), and x31 (h3n2) influenza virus strains for binding and infection on fully differentiated primary cultures of airway epithelia isolated from human bronchus, grown on semiporous filters at an air–liquid interface. in this model system, viral infectivity was highest when virus was applied to the apical versus the basolateral surface; japan was most infectious, followed by pr8. the x31 strain showed very low levels of infectivity. confocal microscopy and fluorescence-resonance energy transfer studies indicated that japan virus could enter and fuse with cellular membranes, while infection with x31 virions was greatly inhibited. japan virus could also productively infect human trachea explant tissues. these data show that influenza viruses with saα2,3gal binding specificity, like japan, productively infect differentiated human airway epithelia from the apical surface. these data are important to consider in the development of pseudotyped recombinant viral vectors for gene transfer to human airway epithelia for gene therapy. influenza viruses comprise some of the most common pathogens of the human upper and lower respiratory tract (1) . the virus lipid envelope surrounds a segmented rna genome, with the envelope protein, hemagglutinin (ha), responsible for binding and fusion to host cells (1) . another viral surface glycoprotein, neuraminidase, is important in both budding and entry (1) . humans are infected by influenza a virus subtypes with the classification of h1n1, h2n2, and h3n2. the ha proteins bind to sialic acid-bearing receptors on host cells. specificity for sialic acid residues terminating in ␣2,6-galactose ␤1,4-n-acetyl glucosamine (sa␣2,6gal) or sa␣2,3gal is determined by ha protein sequence (2) (3) (4) (5) (6) . earlier studies showed that many human isolates preferentially bind and agglutinate red blood cells bearing oligosaccharides terminating in sa␣2,6gal while the avian strains often exhibit specificity for erythrocytes bearing sa␣2,3gal receptors (3, 4, 7) . in subtypes cultured in the laboratory, the host cell used to culture the virus can direct ha-coding sequence alteration (5, 8 -10) and alter sialic acid-binding specificity. cells in the human trachea express both types of oligosaccharides (11) . using specific lectins it was shown that ciliated cells express sa␣2,6gal moieties on their surface, while mucus-secreting goblet cells stain positive for sa␣2,3gal. the ability of influenza to infect differentiated airway cells expressing one, both, or neither oligosaccharide is not currently known. however, an earlier report showed that an h3 human virus strain with sa␣2,6gal specificity could bind to formaldehyde-fixed human respiratory epithelium (12) . experiments that examined influenza virus-host cell interactions, using dog renal epithelia (13) (14) (15) , showed that virus binding and budding occur preferentially at the apical surface of polarized cells. binding to cultured airway cells or tissues has also been examined. prior reports demonstrated that an h1n1 strain could bind and propagate in dissociated airway epithelia cultured from fetal lung tissue explants (16) . the strain pr8 was also shown to productively infect airway epithelial cells grown as dissociated cells on plastic dishes (17) . thus these experiments together with earlier studies (11) do not directly address the polarity of infection or budding of influenza strains on differentiated respiratory epithelia. a culture system which closely resembles the airway epithelium both morphologically and physiologically has been described (18 -21) . in this model, airway epithelia cells are isolated from human trachea or bronchus and the cells dissociated. the cells are grown on filter inserts and cultured at the air-liquid interface. in approximately 2 weeks, the culture is multilayered and polarized, with basal cells, intermediate cells, and columnar cells present. the most apical layer contains both ciliated and nonciliated cells. this model has been used to demonstrate the polarity of infection of recombinant retroviruses (21) , recombinant adenoviruses (22, 23) , and recombinant adeno-associated viruses (24) . we examined the polarity of influenza virus binding, fusion, and budding in human airway epithelia cultures. the h3 strain, x31, with sa␣2,6gal specificity; a/japan/ 305/57, an h2n2 influenza strain with sa␣2,3gal specificity; and a strain with mixed specificity (a/pr8, h1n1) were tested. our results demonstrate that influenza a strains bind to the apical surface of the epithelia. for h2n2 and h1n1 this results in a productive infection. we also demonstrate entry and fusion of the h2n2 virus with cellular membranes and show that x31 virions can bind but do not productively infect differentiated airway epithelia. primary culture of human airway epithelia. primary cultures of human airway epithelia were prepared from bronchi by enzymatic dispersion according to established methods (18, 22) . briefly, epithelial cells were disassociated and seeded onto collagen-coated, semipermeable membranes with a 0.4-m pore size (millicell-ha; surface area, 0.6 cm 2 ; millipore corp., bedford, ma). millicell inserts were placed into 24-well plastic cell culture cluster (costar, cambridge, ma). twenty-four hours after seeding, the mucosal medium was removed and the cells were allowed to grow at the air-liquid interface as reported previously (21) . the culture medium, which consisted of a 1:1 mixture of dulbecco's modified eagle medium and ham's f-12 with 2% ultroser g (sepracor, inc., marlborough, ma), 100 u of penicillin/ml, and 100 g of streptomycin/ml, was added to the wells to feed cells from the basolateral surface. only well-differentiated cultures ͼ2 weeks old were used in these studies, with approximately 4 ϫ 10 5 cells/millicell. the presence of tight junctions was confirmed by transepithelial resistance measurements (resistance ͼ500 ⍀ ⅐ cm 2 ). influenza viruses. influenza virus preparations were purchased from spafas laboratory (storrs, ct). preparations were provided as concentrated and partially sucrose-gradient-purified preparations. three strains of influenza a virus were used in this study: a/pr/8/34 (h1n1) (pr8), a/japan/ 305/57 (h2n2) (japan), and x31 (h3n2). the latter strain is a reassortment with surface glycoprotein genes from a/aichi/2/68 (h3n2) and internal genes from a/pr/8/34 (25) . the total protein concentration of each virus preparation was 2 mg/ml. viruses were titrated by serial dilutions on mdck cells in the presence of 2 g/ml tpck-treated trypsin (sigma, st. louis, mo) (26) . infected wells were determined by the presence of hemagglutination activity in the culture medium. the tissue culture infectious dose 50% (tcid 50 ) was calculated using previously described methods (27) . for measurement of the kinetics of influenza virus production from infected airway cells in one infectious cycle, 100 l of the virus preparation was diluted in dulbecco's pbs (life technologies, inc., grand island, ny) and applied to the apical surface of the epithelia (m.o.i. of 0.1). after incubation for 30 min at 37°c the cells were washed three times with pbs and incubation at 37°c was continued for various time periods with 100 l of pbs left on the surface of epithelia. release of the progeny virus into pbs or basolateral cell culture medium was assayed by hemagglutination with human donor erythrocytes (7) . there was no hemagglutination activity in the third pbs wash. to infect airway epithelia with different influenza virus strains from the basolateral side, the millicell culture insert was turned over and virus preparations diluted in pbs were applied in a 100-l volume to the bottom of the membrane (m.o.i. ϭ 0.1). after incubation for 30 min at 37°c, the suspension was removed and the membranes were washed three times with pbs. the millicell insert was returned to its regular upright position with 100 l of pbs added to the apical side. after incubation for 16 h at 37°c, virus hemagglutination titer was assayed in both the apical pbs and the basolateral cell culture medium. for testing viral infectivity during a multiple cycle infection, 100 l of the virus preparations was added to the apical side of the airway epithelia at an m.o.i. of 0.1 and incubated for 30 min at 37°c. the viral suspension was then removed and the cells were washed three times with pbs. epithelia were incubated at 37°c for 18 h to allow for initial virus production. to assay for virus release, 100 l of pbs was added to the mucosal surface and cells were incubated at 37°c for 1 h. to test the effects of protease on influenza virus ha, tpck-treated trypsin was added to the pbs (final concentration of 2 g/ml) in some experiments. after incubation, surface liquid was collected and assayed by hemagglutination for the presence of the virus. the cells were incubated further at 37°c without medium on the apical side. these steps were repeated every 24 h for 4 days. after 96 h of incubation the cells were lysed in laemmli sample buffer (28) , and viral proteins were analyzed by immunoblot assay. metabolic labeling of virus proteins. airway epithelia were infected with different influenza virus strains from the apical side, at an m.o.i. of 0.1, for 2 h at 37°c. after the initial infection epithelia were incubated in methionine-and cysteine-free cell culture medium (life technologies, inc.) for 8 h at 37°c to accommodate the initial steps of the virus replication cycle. [ 35 s]methionine (nen life science products, boston, ma) was then added to the basolateral culture medium (50 ci/ml final) and incubation continued for 10 h at 37°c. cell-free virus was collected from the apical side and cells were lysed in laemmli sample buffer. analysis of radioactively labeled proteins was done by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) with subsequent autoradiography. antiviral polyclonal antibodies. polyclonal antibodies against japan and x31 strains were generated by inoculation of sheep with the corresponding partially purified formalin-inactivated virus by elmira biologicals (iowa city, ia). the antibodies were purified from serum using a combination of ammonium sulfate precipitation and deaecellulose chromatography (29) . for immunostaining, purified antibodies were labeled with either texas red or fluorescein isothiocyanate (fitc) using fluoreporter protein labeling kits (molecular probes, inc., eugene, or). reactions were performed according to the manufacturer's recommendations. immunoblot assay. following page, proteins were transferred to nitrocellulose membranes (immobilon-nc; millipore corp.) using a semidry transfer cell (trans-blot sd; bio-rad laboratories, hercules, ca), 0.5 h at 50 ma. membranes were blocked with 5% fat-free dry milk, 2 h at room temperature, followed by overnight incubation at 4°c with anti-japan sheep serum diluted 1:5000 in 0.1% tween 20/pbs solution. specific virus proteins were stained with peroxidase-conjugated affinipure donkey antisheep igg (jackson immunoresearch laboratories, inc., west grove, pa), used at 1:20,000 dilution for 2 h at room temperature. the bands were visualized on x-ray film after development with the ecl kit (amersham pharmacia biotech uk ltd., little chalfont, england). staining of membranes with preimmune serum was negative. influenza virus interactions with airway epithelia. partially purified preparations of influenza virus were labeled with n-(lissamine rhodamine b sulfonyl) diacyl phosphatidylethanolamine (rh-pe) (avanti polar lipids, inc., alabaster, al) by passive insertion into the virus envelope according to earlier described methods (30) . cellular membranes were labeled with n-octadecyl-nј-(5-(fluoresceinyl))thiourea (f18; molecular probes) by addition of the dye into the basolateral culture medium at a final concentration of 10 m and incubation for 2 h at 37°c. f18-labeled airway epithelia were infected with rh-pe-labeled influenza virus by adding 100 l of virus suspension to the apical surface (m.o.i. ϭ 5). virus was allowed to internalize for 30 min at 37°c. cells were then washed three times with pbs and fixed with 2% paraformaldehyde for 30 min at room temperature. cells were then washed two times with pbs and the millicell inserts removed and mounted onto slides for microscopy. cells were scanned using a mrc confocal microscope (bio-rad). images were acquired using two photomultipliers, set for a double-labeling protocol (fluorescein and pe-rhodamine), and analyzed using cut plane program software, allowing for xy and xz axis stacking of sections. for fluorescence-resonance energy transfer (fret) assays cells were scanned using customized settings with the excitation wavelength set for fluorescein only. images were acquired using the same double-labeling protocol as above. at this excitation wavelength, detection of red indicates fret. airway epithelia were cooled on ice and influenza virus preparations diluted 10-fold in pbs were added to the apical side (m.o.i. of 1). the virus was allowed to adsorb for 30 min at 4°c. virus was then removed, cells were washed three times with cold pbs, and 37°c-warm fitc-dextran solution (3000 mw, anionic, lysine fixable; molecular probes) 0.5 mg/ml in pbs was added. cells were incubated at 37°c for 5 min, washed three times with pbs, and fixed with 2% paraformaldehyde for 30 min at room temperature. cells were counterstained with texas red-labeled lectin from maackia amurensis (sigma), specific for sialic acid. infection of tracheal explants. blocks of human trachea, approximately 0.1 cm 2 in size, were placed in airway culture medium in a 24-well plastic cluster dish. explants were infected with the influenza virus preparations diluted to the same concentration as for evaluation of fluid-phase endocytosis (above). virus was incubated for 2 h at 37°c. excess virus was then removed by four washings with pbs, with the last wash tested for virus by hemagglutination assay. infection of airway epithelia was allowed to proceed overnight at 37°c in the airway culture medium, and the release of the progeny virus was assayed by hemagglutination. twenty-four hours after infection explants were washed two times with pbs and fixed with 2% paraformaldehyde. fixed tissues were cryoprotected and embedded and 15-m sections obtained for staining. the sections were stained with fitc-or texas red-labeled sheep igg against japan or x31 strains. photomicrographs were acquired using a leica dm rbe fluorescence microscope equipped with a sony digital camera and captured with adobe photoshop software. we first compared the abilities of x31 (h3n2), japan (h2n2), and pr8 (h1n1) strains to infect cultures of primary human airway epithelia. this model culture system, in which human airway epithelia are cultured at the airliquid interface (18, 22) , has been invaluable in testing how other viruses can or cannot access intracellular compartments when applied to the mucosal surface (21, 22, 31) . the titers of the strains tested were determined in mdck cells, and sa-gal specificities were confirmed on erythrocytes. titers and sa-gal specificities are listed in table 1 . the viruses were applied to the apical side of the airway epithelia at m.o.i. equal to 0.1. the amount of the virus released to the apical surface fluid was measured at different time points by hemagglutination. the results of a representative experiment are shown in fig. 1 . in all cases, virus production in airway epithelia was highest for the japan strain. the number of virus progeny produced by pr8 was considerably lower than that of japan, but higher than that of x31. for japan, virus release started 10 h after virus inoculation and continued to rise throughout the 18-h time course. the virus production was somewhat delayed compared to the mdck cell model, in which maximal virus production was reached in approximately 10 h (32). however, the kinetics of virus production observed in our experiments were very similar to previous reports for human airway cell lines infected with the h1n1 influenza a strain (16) . there was no drop in transepithelial resistance measurements throughout the study (data not shown), indicating that the epithelia remained intact. our first experiment tested the ability of influenza a viruses to productively infect airway epithelia when applied to the mucosal surface. we next compared the amount of virus produced after basolateral or apical application. the virus released into the basolateral or apical compartments was measured by hemagglutination. apical infection of the airway epithelia produced 8-to 16fold more virus than basolateral infections (fig. 2) . when japan strain was allowed to infect from the top of the cultures, approximately 2-fold more virions were generated than when pr8 was applied and approximately 100fold more viruses than following x31 infection. in similar studies, we found no cross-release of the virus (data not shown). cleavage of hemagglutinin is an important, and often a host-limiting, step of influenza virus infection. in most cell culture systems used for replication of influenza virus the addition of exogenous trypsin is required for multiple cell infection. we asked whether a multiple-cycle infection could proceed in airway cells without the addition of proteases. to perform this experiment, airway epithelia were infected with x31, pr8, or japan strains and then washed with pbs with or without addition of trypsin. virus production was monitored by hemagglutination. protease treatments and assays were repeated on days 2, 3, and 4 after infection. it was found that the difference in tcid 50 between japan virus-infected filters treated and untreated with trypsin did not exceed 1 log (fig. 3a) . infectivity of x31 strain under either condition was very low (data not shown), precluding our ability to test for cleaved ha. immunoblot assays were done to confirm that japan virus hemagglutinin was cleaved in the absence of exogenously added proteases (fig. 3b) . japan strain hemagglutinin precursor (ha0) in the absence of trypsin can be seen in fig. 3b (lane ϫt) . the addition of trypsin facilitated more complete digestion of the hemagglutinin protein (fig. 3b, lane ϩt) , but as noted this additional cleavage did not dramatically increase virus production. the x31 strain is a reassortment virus generated for vaccination purposes (33) . prior work showed that h3n2 strains similar to x31 could productively infect nasal polyps grown as organ cultures (34) . also, other work showed effective binding of x31 to formaldehyde-fixed tracheal tissues. to determine if the block in x31 infection was after entry or after binding, both metabolic labeling and binding assays were performed. cultures of airway epithelia were infected with x31 or japan strains and metabolically labeled with side were analyzed by sds-page. the data in fig. 4a show accumulation of influenza nucleoprotein (np) in apical washings from japan-infected cells (fig. 4a , lane js) but not x31-infected cells (fig. 4a, lane xs) . the authenticity of viral np protein was confirmed using an immunoblot with anti-japan antiserum (fig. 4b) . this result indicates that the hindrance to x31 infection occurs at early stages of the infectious cycle. japan and x31 strain infection was monitored by confocal microscopy after application of fluorescently labeled virions to the cultures (m.o.i. ϭ 5) to determine if the x31 strain was capable of entry into human airway epithelia. to distinguish between viruses and cells, the rhodaminelabeled lipid probe rh-pe was introduced into the viral envelope, and cellular membranes were labeled with f18 (fluorescein lipid probe). thirty minutes after application of virus, cells were washed, fixed, and observed by confocal microscopy. the photomicrographs in fig. 5 show an en face view (figs. 5a and 5b) or x-z stacked series (figs. 5c and 5d) from the acquired images of influenza virusinfected airway epithelia. japan virions were noted both apically and internally (fig. 5c ). in contrast, the x31 strain is localized to the apical surface only (fig. 5d) . we next tested for evidence of fusion using the fret assay. the assay was designed to test for fret between the fluorescein label in cellular membranes and the rhodamine fluorophore in the viral envelopes. this process can take place only if the distance between the two dyes is in the range of 10 to 100 å (35), as can be accomplished during membrane fusion processes. figures 5e and 5f are representative of the level of fret occurring in the presence of excitation of fluorescein only. fret, detected as excitation of the ph-re label, was observed throughout the thickness of the culture after application of labeled japan strain (fig. 5e ). for cultures to which rh-pe-labeled x31 was applied, fret was rarely detected (fig. 5f ). fluid-phase endocytosis is minimal in fully differentiated airway epithelia (36) . to investigate if application of influenza virus would increase endocytosis, fitc-labeled dextran, as a fluid-phase marker, was applied to the cells after their infection with japan or x31 strains. after incubation with virus for 5 min at 37°c (m.o.i. ϭ 1), cells were washed and intracellular fluorescence was observed using confocal microscopy. the results of this experiment show that endocytosis in the cells infected with japan strain was significantly enhanced, probably as a result of virus uptake (fig. 6a) . in contrast, fitc-dextran uptake in the cells infected with x31 was very low (fig. 6b) and was indistinguishable from that of control, uninfected cells (not shown). to confirm the ability of japan to productively infect human airway cells, experiments were done on freshly explanted human trachea. after overnight infection with japan strain, culture fluid was collected and assayed by hemagglutination for the presence of the virus. the japan strain produced a ha titer of 1:8. in similar studies with pr8 and x31, the titers were 1:8 and undetectable, respectively. thus qualitatively, in both the cell culture and the explant models, japan and pr8 influenza strains were capable of infection, while x31 was not. the infection of the tracheal epithelium by japan virus was also confirmed by immunostaining (fig. 7) . notably, only the apical surface was positive for influenza proteins. the detection of virus proteins was replication-dependent, as staining of control tracheal explants, fixed immediately after adsorption of the viruses, was negative (data not shown). we compared the infectious activity of three influenza a strains in differentiated primary human airway epithelia grown at an air-liquid interface. each of the viruses tested was representative of a different ha subtype. x31 (h3n2) has binding specificity for sa␣2,6gal, japan (h2n2) binds preferentially to sa␣2,3gal, and pr8 (h1n1) is able to utilize both types of oligosaccharides (6) . we confirmed the sa-gal specificity (3, 7) of our viruses in hemagglutination inhibition tests with normal horse serum (table 1) . based on studies of virus binding to fixed sections of human trachea (12) , and human respiratory tissue (34), we initially hypothesized that application of viruses with sa␣2,6gal specificity would be most likely to result in a productive infection in human airway epithelia. however, our data show that influenza strains with preferential binding to sa␣2,3gal receptors productively infect human airway epithelia. this was recently confirmed by clinical observations; avian influenza viruses isolated from infected patients in hong kong were found to be sa␣2,3gal specific (37) . prior studies using specific lectin staining showed that tracheal epithelia express sialyloligosaccharides with both sa␣2,3gal and sa␣2,6gal linkages (11, 12) . in earlier work by baum and paulson (11) , goblet cells and their corresponding mucus droplets stained with lectins specific for sa␣2,3gal, while ciliated cells stained predominantly with lectins specific for sa␣2,6gal. these data supported studies done by liu (38) , showing binding of dye-labeled human isolates with sa␣2,6gal specificity to ciliated epithelia of the ferret trachea. similar studies, using paraformaldehyde-fixed preparations of human trachea, confirmed binding of viruses with sa␣2,6gal specificity to ciliated cells (11) . interestingly, the authors of this study also noted labeled virus with sa␣2,6gal specificity situated over goblet cells, but attributed this binding to tissue preparation artifact. in our experiments binding and infection were tested using viable cells, with clear evidence of sa␣2,3gal-specific strain binding and fusion with the apical membranes of polarized airway epithelia. like japan, x31 could bind to the apical surface of airway epithelia. however, it could not enter the cells; there was minimal evidence of membrane fusion within the cells as was found with the japan strain, and there was little to no virus produced after 18 h of infection. the block in infection was not due to defective virus, since the titer in mdck cells was similar to those of the other viruses tested. also, we did not find an overall block in infection by other h3n2 strains with sa␣2,6gal specificity, since other h3n2 human isolates could productively infect human airway epithelia from the apical surface (39) . one possibility for the inability of the x31 strain to infect airway epithelia could be mutations within ha that do not alter binding specificity, but do inhibit interaction with a secondary receptor or entry of the virus into cells. sequencing of the ha rna encoding the ha1 subunit from x31 and a/aichi/2/68 virions revealed only a conservative mutation: ile 182 in aichi to val in x31 (data not shown). however, we cannot discount that mutations in ha2 or other proteins were responsible for the low infectious activity of x31 in airway epithelia. a second possibility may relate to the susceptibility of japan or x31 ha to inactivation (40) . work by korte and colleagues demonstrated that x31 ha is more sensitive to inactivation after preincubation in low-ph buffer (ph 5.0 -5.2) than japan virus. those studies showed that a 10-min preincubation of x31 at ph 5.0 reduced the fusogenic capacity of the virus by 70%. japan was more stable under these conditions. when similar experiments were done using pr8, the rate and extent of inactivation were greater than for x31. however, we noted that pr8 was more infections than x31 in airway epithelia, suggesting that inactivation of x31 ha may not be the sole determinant for its inability to infect airway epithelia in this study. finally, the observed differences between the parent strains (pr8 and a/aichi) and their reassortant (x31) could be due to altered interactions between surface glycoproteins derived from the h3n2 strain and the membrane proteins (m1 and m2) originating from h1n1 virus. how the requirements for these interactions would differ between kidney cells and airway epithelia is unclear at this time. infection of epithelial cells by most viruses is polarized in terms of entry and release of virus particles (reviewed in (41) ). we noted previously that entry of moloney murine retrovirus vectors with amphotropic or xenotropic envelopes into human airway epithelia was restricted to the basolateral surface (21) . in contrast, influenza virus infection resulted from binding to the apical plasma membrane ((32) and this study), similar to coronavirus (42, 43) and measles virus (44) . researchers investigating viral vectors for application to the treatment of cystic fibrosis have shown that one of the major limitations to infection of airway epithelia from the apical side is receptor inaccessibility (21) (22) (23) (24) . recently, methods have been developed to transiently disrupt the tight junctions to allow envelope viruses access to all cell layers of the epithelia (45) . an alternative method to target retroviral vectors to airway epithelia from the mucosal side may be to pseudotype them with envelopes shown to allow for productive infection when applied to the apical surface. our data suggest that japan, but not x31, could be a candidate envelope protein for such an approach. for consideration of gene therapy for cf or other airway diseases, it would be advantageous to use an envelope not currently circulating in the human population (like japan). this would preclude problems of existing neutralizing antibodies. such envelopes, coupled with lentivirus vectors, which we previously showed to result in longterm expression (46) , may be ideal. in conclusion, we confirm the previously reported polarity of influenza virus binding and budding, but using a model of well-differentiated airway epithelia cultured at an air-liquid interface. our data revealed interesting differences between x31 infectivity in mdck vs airway epithelia and suggest that the block in x31 infection was due to inhibition of infection after binding to the cell surface. we demonstrate that japan virus can bind and fig. 6. fluid-phase endocytosis in human airway epithelia infected with japan or x31 strain of influenza virus. influenza virus preparations were added to airway epithelia in the cold, followed by addition of a 37°c solution of fitc-dextran to detect fluid-phase endocytosis. after a 5-min incubation cells were fixed, stained with texas red-labeled lectin to label the apical surface, and observed by confocal microscopy. the photomicrographs are a stacked x-z series. background levels of endocytosis (not shown) in the absence of added virus were uniformly low as previously published (36) . cryosection of trachea explant infected with japan virus. trachea explants were left uninfected or were infected with the japan influenza strain as described under materials and methods. cryosections (15 m) were stained with fitc-labeled antibodies against japan virus. the photomicrograph shown is representative of the level of anti-japan immunoreactivity seen in three independent experiments using blocks of trachea from different donor lungs. sections from control tissues, or mock-infected tissues, were negative (not shown). the arrow points to the apical side of tracheal epithelium. nuclei were detected using dapi staining. fuse with apical cellular membranes, increase fluid-phase endocytosis, and replicate in viable cultures 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infection of cultured primary human airway cells conformational intermediates and fusion activity of influenza virus hemagglutinin virus infection of polarized epithelial cells advances in virus research mhv-a59 enters polarized murine epithelial cells through the apical surface but is released basolaterally human coronavirus 229e infects polarized airway epithelia from the apical surface entry and release of measles virus are polarized in epithelial cells increasing epithelial junction permeability enhances gene transfer to airway epithelia in vivo feline immunodeficiency virus vectors persistently transduce nondividing airway epithelia and correct the cystic fibrosis defect key: cord-275779-ocbygkyb authors: ye, zi-wei; yuan, shuofeng; poon, kwok-man; wen, lei; yang, dong; sun, zehua; li, cun; hu, meng; shuai, huiping; zhou, jie; zhang, mei-yun; zheng, bo-jian; chu, hin; yuen, kwok-yung title: antibody-dependent cell-mediated cytotoxicity epitopes on the hemagglutinin head region of pandemic h1n1 influenza virus play detrimental roles in h1n1-infected mice date: 2017-03-21 journal: front immunol doi: 10.3389/fimmu.2017.00317 sha: doc_id: 275779 cord_uid: ocbygkyb engaging the antibody-dependent cell-mediated cytotoxicity (adcc) for killing of virus-infected cells and secretion of antiviral cytokines and chemokines was incorporated as one of the important features in the design of universal influenza vaccines. however, investigation of the adcc epitopes on the highly immunogenic influenza hemagglutinin (ha) head region has been rarely reported. in this study, we determined the adcc and antiviral activities of two putative adcc epitopes, designated e1 and e2, on the ha head of a pandemic h1n1 influenza virus in vitro and in a lethal mouse model. our data demonstrated that sera from the e1-vaccinated mice could induce high adcc activities. importantly, the induction of adcc response modestly decreased viral load in the lungs of h1n1-infected mice. however, the elevated adcc significantly increased mouse alveolar damage and mortality than that of the pbs-vaccinated group (p < 0.0001). the phenotype was potentially due to an exaggerated inflammatory cell infiltration triggered by adcc, as an upregulated release of cytotoxic granules (perforin) was observed in the lung tissue of e1-vaccinated mice after h1n1 influenza virus challenge. overall, our data suggested that adcc elicited by certain domains of ha head region might have a detrimental rather than protective effect during influenza virus infection. thus, future design of universal influenza vaccine shall strike a balance between the induction of protective immunity and potential side effects of adcc. introduction influenza viruses, as one of the major zoonotic pathogens, have continuously caused global health concerns due to their high propensity for unpredictable genetic mutation in major surface antigens, hemagglutinin (ha), and neuramindase. antivirals and vaccines are vital in combating the threat of influenza epidemics and pandemics. however, the increasing usage of licensed antivirals has resulted in the global emergence of amantadine-and/or oseltamivir(1) . on the other hand, seasonal influenza vaccines have to be updated annually due to the continuous antigenic drift and shift (2) . otherwise, the mismatch between vaccinated formulations and that of natural selection would considerably limit the effectiveness of influenza vaccines. neutralizing antibodies have traditionally been thought to provide protection against influenza viruses. nevertheless, the effectiveness induced by such vaccines is limited by the emergence of mutant viruses that are resistant to antibodymediated neutralization (3) . in this regard, the quest for universal influenza vaccines has fueled the interest in broadly reactive antibodies in addition to direct virus neutralizations. antibody-dependent cell-mediated cytotoxicity (adcc) uses effector arms of both innate and adaptive immune responses to eliminate target cells. natural killer (nk) cells, upon triggered by specific adcc antibodies, mediate the clearance of virusinfected cells (4) . the nk cell cd16 receptor recognizes the fc portion of igg1 antibodies that in turn bind to antigens on virus-infected cells (5) . this interaction induces degranulation of nk cells to release perforin/granzymes as well as secretion of antiviral cytokines such as interferon-γ (ifn-γ) and tumor necrosis factor-α (tnf-α) (5) . since adcc could invoke protective immune response against infections from a broad array of viruses, the adcc antibody response was incorporated as one of the most important features of potential universal vaccine candidates by the world health organization. notably, multiple components of influenza viruses are known to induce adcc, including the viral surface proteins such as ha (6) and m2 ectodomain (7, 8) , as well as the internal proteins including np and m1 (9) . the glycoprotein ha consists of two functional domains, the immunodominant globular head domain and the more conserved stalk domain (10) . conventionally, neutralizing antibody response to influenza virus is dominated by antibodies that target the head region, which block the virus receptor-binding site. compared with the bulky globular ha head, the ha stem region is far less immunogenic, and antibodies directed to this domain occur at a relatively low frequency. however, a rare class of neutralizing antibodies that target a conserved site in the ha stem was reported recently, which might shed new light for the development of universal influenza vaccines (6) . we have previously identified two putative adcc epitopes that mapped to the ha head of a pandemic h1n1 influenza virus (11) . both epitopes, designated e1 and e2, revealed by epitope mapping of convalescent-phase plasma igg antibodies from six h1n1-infected human subjects, are dominant and highly conserved (11) . in this study, we further dissected the function of the two adcc epitopes in vitro and in a lethal mouse model. surprisingly, our results demonstrated that the adcc response elicited by the e1 epitope triggered a detrimental rather than protective effect against influenza virus infection. while the antiviral efficacy provided by the stalk-specific adcc antibodies has been confirmed (12) , our data raised concerns on the side effect of certain ha head epitopes in devising a universal influenza vaccine. in this regard, our study suggested that a delicate balance between protective immunity and over induction of adcc should be maintained, which should be an important consideration in evaluating vaccine safety. the la4 cell line, which was derived from mouse lung adenoma, was maintained in dmem/f-12 medium (gibco) supplemented with 20% heat-inactivated fetal bovine serum (fbs), 50 u/ml penicillin, and 50 μg/ml streptomycin (p/s). peripheral blood mononuclear cells (pbmcs) were prepared by ficoll-paque separation (13) of heparinized whole blood obtained from healthy balb/c mice (6-8 weeks old). to prepare the adcc target cells, la4 cells were transfected with an ha expression plasmid that based on the cdna fragment of influenza virus strain a/hong kong/415742/2009(h1n1)pdm09. specifically, the full-length ha fragment was cloned into a mammalian expression vector peak10 plasmid containing a mouse igg1 fc gene (ch2 + ch3) (14) . the pandemic h1n1 influenza virus strain a/hong kong/415742/2009(h1n1)pdm09 was used for in vitro virus infection; while its mouse-adapted version, a/hong kong/415742md/2009 (h1n1)pdm09 was propagated in embryonated hens' eggs and utilized for in vivo experiment (15) . the viruses were stored in −80°c in aliquot and titrated by standard plaque assay. all experiments with live viruses were conducted using biosafety level 2 facilities as described previously (16) . balb/c female mice, 6-8 weeks old, were kept in biosafety level 2 housing and given access to standard pellet feed and water ad libitum. all experimental protocols followed the standard operating procedures of the biosafety level 2 animal facilities and were approved by the animal ethics committee in the university of hong kong (17) . vaccinations were carried out to immunize the mice with e1 or e2 or ha epitopes by dna priming and protein boost. pbs was used as a negative control. the specified vaccination scheme was listed in table 1 . to prepare the dna plasmids, either of the e1 or e2 fragment (11) was cloned into the mammalian expression vector peak10 as described for the ha plasmid construction. priming of the mice by electroporation. to prepare protein for vaccination, recombinant ha, e1, and e2 fusion proteins were expressed in freestyle 293ft™ transient expression system (invitrogen) and purified by protein a affinity (ge healthcare). subsequently, proteins were dialyzed and concentrated with vivaspin 20 centrifugal concentrator (ge healthcare), followed by protein boosting through intramuscular route. each mouse received 25 μg protein at each protein boosting. sera were obtained at day 68 postimmununization before virus challenge. antibody titers raised against e1, e2, and ha in mouse sera were evaluated by elisa as previously described with some modifications (18) . mouse sera collected from the pbs-treated group were taken as a background control. immunized mice (15 mice/group) were inoculated with five 50% lethal dose (ld50) of mouse-adapted pandemic h1n1 influenza virus by intranasal route, i.e., 1,000 pfu/mouse. animal survival and body weight were monitored daily for 14 days after virus challenge. a body weight loss of 30% was set as the humane endpoint. three mice per group were randomly selected and euthanized on day 3 and 5 post-challenge, respectively. mouse lungs were collected from the euthanized mice. half of the lung tissues were harvested for virus titration by rt-qpcr methods (19) , while the other half were immediately fixed in 10% of pbs buffered formaldehyde for histopathological analyses as described previously (20) . slides were examined in a blinded manner and scored with a semiquantitative system according to the relative degree of inflammation and tissue damage (21) (22) (23) (24) . inflammation was scored as follows: 0, no inflammation; 1, perivascular cuff of inflammatory cells; 2, mild inflammation (extending throughout 25% of the lung); 3, moderate inflammation (25-50% of the lung); 4, severe inflammation involving over one half of the lung. antibody-dependent cell-mediated cytotoxicity activity, reflected by the rate of cell death, was measured by a flow cytometry-based assay that described previously with some modifications (11) . generally, the pkh-67 dye (sigma) was utilized to label the target cells, i.e., ha-transfected la-4 cells; while 7-aminoactinomycin d (7-aad; invitrogen) was used to stain the dead cells that mediated by adcc activity. experimentally, pkh-67-labeled target cells and unlabeled effector cells (i.e., pbmcs) were prepared in rpmi 1640 medium (gibco) containing 10% fbs and 1% p/s with a cell density of 10 6 and 10 8 cells/ml, respectively. subsequently, 50 μl of target cells were dispensed into a round-bottom 96-well plate, followed by addition of 1 μl of mouse serum. mouse serum concentration in each group was normalized before addition according to their titers determined by elisa. one hour after incubation, 50 μl of effector cells were added to incubate with the target cells. three hours later, another 1 μl of 7-aad was added to each well before performing the flow cytometry. cell death was determined with a facsaria iii flow cytometer using the bd facs software (bd biosciences). percent cell death was calculated by software analysis of four identifiable cell populations: live effector cells (no dye), dead effector cells (7-aad positive), live target cells (pkh-67 positive), and dead target cells (pkh-67 and 7-aad double positive). assay controls used to define cell populations included target cells alone (background cell death) and target cells with 1 μl triton x-100 added (maximum cell death). percent adcc was calculated as [(percent experimental lysis − percent spontaneous lysis)/(percent maximum lysis − percent spontaneous lysis)] × 100%, in which percent spontaneous lysis refers to the percent lysis of infected cells with effectors but in the absence of serum, while percent maximum lysis refers to the percent lysis of infected cells with effectors in the presence of 1% triton x-100. experiments were performed in triplicate and repeated twice for confirmation. immunostaining was performed as previously described to visualize perforin expression in mouse lung tissues (25) . rat anti-mouse perforin (abcam ab16074) and goat anti-rat alexa 594 were used as primary and secondary antibodies, respectively. images were acquired with a carl zeiss lsm 780 system. table s1 in supplementary material. statistical comparison was performed by student's t-test using graphpad prism 6. differences were considered statistically significant when p < 0.05. results adcc responses are enhanced by the sera of e1-vaccinated mice in our previous study, we mapped two putative adcc epitopes, e1 and e2, on the ha head region. by depleting e1 and/or e2 from clinical plasma igg antibodies, the adcc activity dropped significantly, which suggested that the two epitopes played potential roles in eliciting adcc (11) . in this study, we sought to confirm the function of these putative epitopes in the induction of adcc activity using a mouse model. immunization of mice gave raise to igg titers against the e1, e2, or ha protein, as quantified by elisa (figure 1) . a prime/boost immunization strategy was adopted, and mice that immunized with pbs or ha were included as a negative or a positive control, respectively ( table 1 ). our results indicated that serum samples from mice vaccinated with e1 ( figure 1a ) or e2 ( figure 1b) both exhibited strong dilution-dependent antibody responses, reaching a titer of more than 1:5000. additionally, using ha as the coating antigen for elisa, we demonstrated that e1 and e2 sera could bind fulllength ha at a comparable efficiency ( figure s1 in supplementary material). taken together, our data suggested that the vaccination was successful and the resultant serum samples could be utilized for further investigations. next, adcc activities in serum samples from e1-, e2-, or ha-immunized mice were evaluated by flow cytometry-based adcc assays. to this end, the ha-transfected la4 cells were labeled with the cell-membrane marker pkh67 and utilized as target cells for adcc-specific antibody binding. subsequently, the vaccinated mouse serum was added to bridge the interaction between target cells and pbmc effector cells (figure 2) . the presence of adcc-mediating antibody was determined by analyzing the cytotoxicity rate within the cell mixture that contained the target cells, serum, and effector cells, in which the dead target cell population was revealed by the cell death marker, 7aad (figure 3) . as shown in figures 3a-g , sera from the e1-vaccinated mice consistently triggered the highest 7aad + rate among all evaluated groups, suggesting that a higher percentage of cell lysis was induced in the e1 group in comparison to the other groups. the percentage of cytotoxicity was normalized using the formula reported by srivastava et al., with spontaneous and maximum cell cytotoxicity rate taken into consideration (11) . quantitatively, sera from the e1-vaccinated mice elicited a significantly increased (p < 0.05) adcc activity in comparison with the pbs-vaccinated group (figure 3h) . of note, though sera from the e2-vaccinated mice triggered a subtle increase in adcc activity, the difference was not statistically significant ( figure 3h) . intriguingly, albeit ha contained the e1 epitope, sera from the ha-vaccinated group did not induce a significantly elevated cytotoxicity response in comparison to that of the pbs control group (figure 3h ). since e1 was capable of inducing adcc activities, we hypothesized that e1-vaccinated mice could potentially be protected by the elicited adcc activity after influenza virus challenge. to this end, we inoculated the vaccinated mice with pandemic h1n1 influenza virus in a lethal mouse model ( figure 4a) . as shown in figure 4b , mice in the ha-vaccinated group, as a positive control, demonstrated a substantial reduction of viral load on both day 3 and day 5 post-inoculation in comparison to the pbs-vaccinated group. importantly, we detected an approximately one log decrease of viral load in the mouse lungs of the e1-vaccinated group in comparison to that of the pbs-vaccinated group on day 5 post-inoculation, while no significant difference could be observed between the two groups on day 3 post-virus challenge (figure 4b ). in addition, the viral load in the lungs of the e1-vaccinated mice was notably lower on day 5 when compared with that of day 3, suggesting that the adcc effect was triggered between day 3 and 5 postinoculation ( figure 4b) . figure 2 | schematic diagram of the antibody-dependent cell-mediated cytotoxicity (adcc) assay. adcc activity was determined by a flow cytometrybased assay using two fluorescent dyes. pkh-67, a membrane-labeling dye, was used to specifically identify the ha-transfected target cells. 7aad was excluded by viable cells but could penetrate the cell membrane of dead or dying cells and intercalate into double-stranded dna. briefly, 50 μl of pkh-67-labeled target cells (10 6 cells/ml) was dispensed into a round-bottom 96-well plate, followed by addition of e1/e2/ha/pbs sera and effector/pbmc cells. following a 3-h incubation, 7-aad was added. cell death was determined on a facsaria iii flow cytometer using bd facs diva software (bd biosciences). percent cell death was analyzed by the flowjo software. in parallel, we measured the survival rate and body weight changes of the mice. as shown in figure 4c , all mice from the ha-vaccinated group survived the course of infection while all mice received pbs-treatment died, indicating that the virus challenge was successful. unexpectedly, our results demonstrated that the mice in the e1-vaccinated group succumbed to influenza virus infection at a significantly earlier time (p < 0.0001) postinoculation when compared with that of the pbs-vaccinated control group (figure 4c ). in line with the survival rate, mice from the e1-vaccinated group suffered from an apparently accelerated weight lost starting on day 3 post-inoculation in comparison to mice from the pbs-and e2-vaccinated groups, although the difference did not reach statistical significance ( figure 4d) . next, we carried out histopathological examinations on the lung sections of the virus-infected mice. using uninfected mouse lung tissues as a control (figures 5i,j) , our observation showed that on both day 3 and day 5, mice from the ha-vaccinated group (figures 5g,h) exhibited ameliorated alveolar morphology changes when compared with those from the e1 (figures 5c,d) , e2 (figures 5e,f) , and pbs (figures 5a,b) groups. importantly, the lung pathological scores of mice from the ha-vaccinated group on both day 3 and day 5 were significantly lower than those of the pbs-treated mice (figures 5k,l) ; while mice from the e1-vaccinated group suffered from a significantly more dramatic interstitial inflammatory infiltration than that of the pbs-treated mice on day 5 ( figure 5l ). this result indicated that the detrimental lung damage of e1-vaccinated mice, possibly triggered by adcc, might account for the reduced viral load in lungs as well as the earlier drop in survival. to address whether the severe lung damage in the e1-vaccinated group could be attributed to the adcc effect, we performed immunofluorescence staining to visualize the expression level of perforin among different mouse groups. perforin is released by activated nk cells and is known as a marker of adcc activation (28) . as quantitated in figure 6m , the e1-vaccinated mice (figures 6d-f) demonstrated the highest perforin expression level in the lung sections amongst the other three groups on day 5 post infection (figures 6a-c,g-l) . however, the mrna expression level of perforin was not significantly different across all evaluated groups ( figure 6n) . binding of fc receptor (fcr) on effector cells triggers the secretion of cytotoxic granules as well as antiviral cytokines and chemokines, such as ifn-γ and tnf-α (4). to investigate if elevated expression of proinflammatory cytokines might contribute to the lung damage, we measured the mrna expression level of representative cytokines including tnf-α (figure 7a) , il-1β (figure 7b) , and ifn-γ (figure 7c ) in the mouse lungs samples. our results showed that the gene expression of all three proinflammatory cytokines were significantly enhanced in the e1-vaccinated group when compared with those of the pbstreated group (figure 7) , which were in line with the perforin protein expression pattern in figure 6 . together, our data suggested that the e1 epitope from the ha head domain mediated unfavorable adcc that resulted in a more severe lung damage and exacerbated mouse mortality despite a successful control of the h1n1 influenza viral load. antibody-dependent cell-mediated cytotoxicity, as a bridge of the innate and adaptive immunity, plays important roles in humoral and cellular immune response (4, 9) . since adcc antibodies are known to recognize a wide range of viral proteins that lead to the lysis of virus-infected cells, a better understanding on the adcc mechanism during influenza virus infections will facilitate the development of universal influenza vaccines with broader protections (4, 9, 29) . the conserved viral proteins or domains, such as m2 extracellular domain and ha stem domain, have been widely studied as potential targets of domain-based universal influenza vaccines. jegerlehner and colleagues have demonstrated that the protective role of m2 adcc-mediating antibodies was dependent on fcr (7, 8) . dilillo et al. provided further support that the influenza-specific adcc antibody, though elicited by the ha stem, also required fcrs interaction for protection against lethal influenza infection (6) . collectively, both studies highlighted the functional importance of fcr during adcc-mediated virus clearance. on the other side, unexpected cases have been reported that young adults without prior exposure to the 1968 h3n2 virus produced robust adcc-mediating antibody response upon infection of this virus strain. some individuals even possessed cross-reactive adcc-mediating antibodies toward avian h5n1 and h7n7 strains in the absence of prior exposure (30) . however, the mechanism of such antibody responses remains unclear to date. in this study, we investigated the adcc effect of the two putative ha head epitopes in vitro and in vivo. our data demonstrated that e1-induced adcc activity against h1n1 influenza virus infection in vitro (figure 3) . unexpectedly, although e1 vaccination decreased the viral load in h1n1-infected mice (figure 4b) , it induced exacerbated lung damage ( figure 5 ) and a higher level of nk activity (figure 6 ) that accelerated mouse death ( figure 4c ). nk cells, which offer the first line of defense against virus infection, have been widely considered to be beneficial to the host during viral infections. however, a recent report by zhou et al. revealed that adoptive transfer of nk cells from influenza virus-infected lungs, but not uninfected lung, resulted in a more rapid weight loss and increased mortality of virus-infected mice (31) . this finding was in line with our observation that e1-induced adcc exhibited deleterious impact to promote mortality during influenza virus infection. most healthy donors have a persistently low level of crossreactive adcc-mediating antibodies, while these cross-reactive antibodies are found in individuals in the absence of detectable neutralization (4, 9) . in our previous study, both e1 and e2 epitopes were identified as putative regions that could induce adcc activity. the depletion of such antibodies in human plasma significantly decreased the adcc effect. however, for certain samples, it appeared that more diluted plasma exhibited higher adcc activity than less diluted plasma, and the use of igg antibodies at a low concentration led to a higher adcc activity than the use of igg antibodies at a high concentration (11) . to date, there is no conclusive study on the correlation between antibody concentration and adcc activity, neither was the optimal concentration of adcc antibodies that could protect against virus infection elucidated. in this context, we demonstrated here that an overwhelming production of adcc antibodies in the absence of neutralization might not play a protective role against influenza virus infection. indeed, multiple factors such as saturation of antibodies or interference from non-adcc antibodies may contribute to the induction of adcc (4, 11) . in this case, the threshold level of protective adcc-mediating antibodies should be investigated in further studies. various adcc assays that mainly differ in the choice of effector cells and measurement of adcc activity have been reported (4, 9) . for example, some studies used ha-transfected or virus-infected a549 cells as target cells, which were susceptible results are presented as bar charts with mean values ± sd. differences between groups were compared using the t test. *p < 0.05 and **p < 0.01 as compared to the pbs-immunized group. survival rate (c) and body weight (d) of the mice (9 mice per group) vaccinated with pbs (red), e1 (blue), e2 (green), and ha (purple) were monitored for 14 days. the body weight values are shown as means ± sd for the mice that were alive at each time point (***p < 0.0001). to nk cell-mediated adcc after incubating with the sera from healthy donors or clinical blood samples (6, 32, 33) . in our case, we isolated pbmcs from healthy mice as effector cells and measured the death rate of target cells in the presence of vaccinated mouse sera (figure 2) . at the same time, utilization of flow cytometry for quantitation of cell cytotoxicity provided an efficient and precise way to assess the adcc responses (figure 3) . importantly, the h1n1-infected la4 cells showed a low background of cell death in the absence of antibodies (figure 3c) , which suggested la4 as an ideal cell line for the mouse-specific adcc assay. collectively, the established in vitro adcc assay, together with the balc/c mouse model, might be applied for the evaluation of other influenza-specific adcc epitopes. experimental mouse models are an invaluable scientific resource that allow comprehensive investigation of key biological questions in vivo and provide an essential platform in the study of many human diseases. it has been widely acknowledged that the mouse and human antibody repertoire share a general similarity (34) (35) (36) . however, differences in germline antibody repertoire exist between species, and the number of mature naïve b cells from mice is smaller than that from humans (37) . both variations may contribute to the dissimilarities in the antibodies elicited by the e1-containing fragments in humans compared to those in mice. due to the diversity of the b cell antigen receptor repertoire between the mouse and human model, the antibodies bind to the same fragments in distinct host models might potentially have slightly different epitopes. alternatively, there may be fundamental differences between murine and human in terms of the regulation in nk cell cytotoxic granule secretion (38) . surprisingly, distinct expression patterns of perforin were detected between protein ( figure 6m ) and mrna (figure 6n ) levels. the discrepancy might be explained by a previous finding that resting murine nk cells are "pre-armed" with high amounts of perforin mrna, which can be rapidly translated into protein upon activation in vitro and in vivo (38). this mechanism of murine nk cells facilitates a better control of perforin expression, allowing a rapid production of effector proteins without the need of de novo gene transcription (38). upon activation, adcc effector cells produce various cytokines such as tnf-α, il-1β, and ifn-γ. further, cytokines may represent one of the arming pathways that stimulate the translation of perforin (38). notably, human nk cell is minimally cytotoxic at rest, expresses little perforin protein, and becomes potently cytotoxic only after cytokine activation (39, 40) . our result showed that tnfα, il-1β, and ifn-γ were significantly elevated in e1-vaccinated group when compared with those of the pbs-treated group (figure 7) . in this regard, the upregulation of these cytokines may activate the cytotoxic perforin to cause the detrimental damage in mouse lungs. in our previous study, e1 and e2 epitopes were located on the ha head ( figure s2 in supplementary material), both of which are conserved in h1n1 strains from 2009 (2009_h1n1) (11) . to date, however, the role of ha head during influenza virus infection and adcc activation has not been fully delineated. in a recent study, dilillo and colleagues reported that neutralizing antibodies targeting the ha stem but not the ha head were capable of conferring influenza-specific adcc (6) . they proposed that only the anti-stem antibodies could bind in a correct conformation to ligate fcrs, which was based on the observation that a strain-specific anti-ha head antibody (py102) was unable to mediate fcγr binding and to protect mice. on the other hand, ha head-induced adcc activities were reported by a number of other groups (41) (42) (43) , which was in agreement with our findings. the discrepancy between these observations might be due to the sequential and structural variations among subtypes/strains of influenza virus used. interestingly, although both e1 and e2 epitopes are located on the ha head, serum from the ha-vaccinated group did not trigger a significantly elevated cytotoxicity response (figure 3h) , implying that additional regulators exist to control the adcc activity. in summary, we provided in vitro and in vivo evidence to verify the effect of the ha head epitope e1-mediated adcc. importantly, our data suggested that e1-mediated adcc alone caused detrimental effect during influenza virus infection, which raised concerns on using this conserved non-neutralizing region of the ha head in future designs of a universal influenza vaccine. in fact, the "headless ha" has been recommended in several vaccine designs that aimed to make use of adcc antibodies (44) (45) (46) (47) . our data provided further evidence in support of this "headless ha" vaccine design strategy. emerging influenza antiviral resistance threats influenza vaccine -outmaneuvering antigenic shift and drift variable influenza vaccine 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and human immunology transitional b cells: how well are the checkpoints for specificity understood? differences in mouse and human nonmemory b cell pools acquisition of murine nk cell cytotoxicity requires the translation of a pre-existing pool of granzyme b and perforin mrnas the biology of human natural killer-cell subsets differential expression of human granzymes a, b, and k in natural killer cells and during cd8+ t cell differentiation in peripheral blood influenza virus a(h1n1)2009 antibody-dependent cellular cytotoxicity in young children prior to the h1n1 pandemic antibody-dependent cell-mediated cytotoxicity to hemagglutinin of influenza a viruses after influenza vaccination in humans cross-reactive influenza-specific antibody-dependent cellular cytotoxicity antibodies in the absence of neutralizing antibodies the quest for a universal flu vaccine: headless ha 2.0 preparation of influenza virus subviral particles lacking the ha1 subunit of hemagglutinin: unmasking of cross-reactive ha2 determinants vaccination with soluble headless hemagglutinin protects mice from challenge with divergent influenza viruses stalking influenza by vaccination with pre-fusion headless ha ministem key: cord-335310-61wibso4 authors: chen, hui-wen; huang, chen-yu; lin, shu-yi; fang, zih-syun; hsu, chen-hsuan; lin, jung-chen; chen, yuan-i; yao, bing-yu; hu, che-ming j. title: synthetic virus-like particles prepared via protein corona formation enable effective vaccination in an avian model of coronavirus infection date: 2016-08-15 journal: biomaterials doi: 10.1016/j.biomaterials.2016.08.018 sha: doc_id: 335310 cord_uid: 61wibso4 the ongoing battle against current and rising viral infectious threats has prompted increasing effort in the development of vaccine technology. a major thrust in vaccine research focuses on developing formulations with virus-like features towards enhancing antigen presentation and immune processing. herein, a facile approach to formulate synthetic virus-like particles (svlps) is demonstrated by exploiting the phenomenon of protein corona formation induced by the high-energy surfaces of synthetic nanoparticles. using an avian coronavirus spike protein as a model antigen, svlps were prepared by incubating 100 nm gold nanoparticles in a solution containing an optimized concentration of viral proteins. following removal of free proteins, antigen-laden particles were recovered and showed morphological semblance to natural viral particles under nanoparticle tracking analysis and transmission electron microscopy. as compared to inoculation with free proteins, vaccination with the svlps showed enhanced lymphatic antigen delivery, stronger antibody titers, increased splenic t-cell response, and reduced infection-associated symptoms in an avian model of coronavirus infection. comparison to a commercial whole inactivated virus vaccine also showed evidence of superior antiviral protection by the svlps. the study demonstrates a simple yet robust method in bridging viral antigens with synthetic nanoparticles for improved vaccine application; it has practical implications in the management of human viral infections as well as in animal agriculture. vaccine is historically the most effective countermeasure against infectious threats, as agents resembling pathogens are administered to mount an immune response against specific targets. amidst continuing and emerging viral threats, vaccine technology continues to advance with the aim of effectively promoting antiviral immune responses, and a major development effort lies in retaining or integrating virus-like features in vaccine formulations for improved immune processing. several morphological and antigenic characteristics of viral particles have been demonstrated to promote immune potentiation. for example, particles at the nanoscale have been shown to have better lymphatic transport as compared to smaller subunit antigens [1, 2] . in addition, the display of multiple antigens on a single particle facilitates more effective antigen presentation to immune cells [1] . as compared to traditional vaccine formulations, vaccines preserving virus-like features have shown superior capability in eliciting immune responses [3e5] . these results and observations have also prompted material scientists to apply synthetic nanomaterials towards mimicking viral features for vaccine development [6e9] . given their high radii of curvature, synthetic nanoparticles frequently possess high surface energies that induce adsorption of biomolecules in a phenomenon known as protein corona formation. in protein-rich media, strong nanoparticle/protein association occurs spontaneously as a means to passivate surface energies, and the resulting particles are encased in a protein layer that dictates the particles' interactions with the environment [10, 11] . while protein corona formation is gaining increasing scientific interest owing to its implications in biomedical applications [10, 12, 13] , we herein demonstrate harnessing this phenomenon can be beneficial towards mimicking viral features for vaccine applications. we show that synthetic virus-like particles (svlps) with close semblance to native virions in physicochemical properties and antigen display can be facilely prepared through spontaneous antigen-particle association in optimized incubation conditions. using 100 nm gold nanoparticles (aunp), a biologically inert material commonly used for biomedical research [14e16] , and a spike glycoprotein derived from an avian infectious bronchitis virus (ibv), a single-stranded positive-sense rna virus that belongs to the family coronaviridae [17] , we controlled the incubation condition to prepare spike glycoprotein-laden svlps (fig. 1) . the morphological features and antigen display by the svlps were compared to native ibv viral particles using nanoparticle tracking analysis and immunogold staining. in addition, vaccination potency between the svlps and free spike glycoproteins was compared in an avian model of coronavirus infection. a commercial whole inactivated virus (wiv) formulation that is the current standard vaccine for ibv management was examined in parallel. coronaviruses are a major viral family of which the most publicized examples include the pathogens behind severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) [18] . in animals, ibv is a prime example of coronavirus that infects the respiratory and urogenital tracts of chickens, posing a serious economic threat as one of the most important pathogens in the poultry industry. the ibv spike glycoprotein, which forms the large, pental-shaped spikes on the surface of the virion, is chosen as the antigen candidate as it is implicated as a determinant of virus pathogenicity. among coronaviruses, spike glycoproteins possess a variety of biological functions, including triggering cell attachment, inducing cell-cell fusion, and binding to cellular receptors [19, 20] . as spike glycoproteins are the primary targets in ongoing vaccine development efforts for coronavirus vaccinations, the present study has broad implications across both human and animal disease management [21, 22] . s. frugiperda sf9 (atcc crl-1711) insect cells were cultured in grace's insect cell medium (invitrogen, carlsbad, ca) and supplemented with 10% fbs (thermo fisher, rockford, il) and 1% p/ s/a antibiotics (biological industries, beit-haemek, israel) at 27 c. 100 nm gold nanoparticle (aunp) solution was purchased from sigma-aldrich (st. louis, mo). avian coronavirus ibv strain 2575/98 was propagated in 10-dayold specific-pathogen-free (spf) chicken embryos via the allantoic route as previously described [23] . the virus titers of ibvs were determined with the method of reed and muench [24] in spf chicken embryos and expressed as 50% embryo infectious dose (eid 50 ) [25] . the virus-containing allantoic fluid was concentrated and purified using sucrose gradient solution as previously described to derive the native virions [23] . full spike (s) protein of avian coronavirus ibv was cloned and expressed using the bac-to-bac baculovirus expression system (invitrogen). briefly, a recombinant plasmid was constructed by inserting full spike protein gene of ibv strain 2575/98 (accession no. dq646405) [26] into the pfastbac-1 vector using the following primer set: ibv-s-bamhi-f: 5 0 -ttggg atccg atgtt ggtga agtca c-3 0 ; ibv-s-sali-f: 5 0 -cttgt cgaca ttaaa cagac ttttt aggt-3 0 . the recombinant pfastbac-1 shuttle vector was then transposed to the bacmid in e. coli strain dh10bac, and recombinant bacmid was purified using the hipure plasmid midiprep kit (invitrogen). sf9 cells were used for transfection with the recombinant bacmid, and recombinant baculoviruses were then harvested in the supernatant and designated rbac-2575s. recombinant spike proteins (r2575s) were harvested from sf9 cells infected with rbac-2575s (multiplicity of infection ¼ 1). sf9 cells were washed and lysed with the i-per insect cell protein extraction reagent (thermo fisher). recombinant proteins were purified using the glycoprotein isolation kit, cona (thermo fisher) according to the manufacturer's instructions. after purification, r2575s protein was stored in 10% sucrose at à20 c. citrate-buffered 100 nm gold nanoparticles were washed repeatedly in water to remove the citrate stabilizer, and the resulting pellet was resuspended in 10% sucrose. protein solutions ranging in concentrations between 100 mg/ml to 3 mg/ml of purified spike proteins were then mixed with 1 â 10 11 /ml of gold nanoparticles (determined by nanoparticle tracking analysis) in 10% sucrose. the mixtures were bath sonicated for 1 min followed by incubation in an ice bath for 30 min. the nanoparticles were then removed from unbound spike proteins via centrifugation at 1500g for 3 min. following 3 centrifugal washes with 10% sucrose, pelleted nanoparticles were mixed with 1x pbs and sonicated in a bath sonicator for 30 s. dispersible, stabilized svlps were retrieved and their protein content was quantified using a bca protein assay (thermo fisher) with 25 ml of 1 â 10 11 particles/ml following the manufacturer's protocol. visualization of unstable nanoparticles and colloidally stable svlps was performed using a 200 kv high resolution transmission electron microscope (fei tecnai tf20). particle stability was assessed by monitoring the size of svlps for 7 days. particle size, polydispersity index (pdi), and concentrations were measured by nanoparticle tracking analysis using nanosight ns-500 (malvern, uk) at a concentration of 1 â 10 8 particles/ml based on the manufacturer's instructions. particle size and zeta potential were also measured by dynamic light scattering using zetasizer nano zs at a concentration of 1 â 10 10 particles/ml (malvern, uk) based on the manufacturer's instructions. antigen display was examined using freshly prepared svlps. antigen retention was examined by mixing svlps in protein-poor (pbs) or in protein-rich (10% bsa) conditions for varying periods of time. at 0, 3, 10, and 24 h marks, svlps were pelleted from their respective solutions. the particles were then processed using a previously published protocol with sds-page loading buffer for protein removal and quantification [27] . ibv spike proteins eluted from the svlp were analyzed in 6% discontinuous sds-page under non-reducing condition. protein gel was then transferred onto a 0.45 mm nitrocellulose membrane (bio-rad). after transfer, the membrane was soaked in blocking buffer (5% skim milk in pbs) at room temperature for 1 h and probed with anti-s monoclonal antibody (mab) for another 1 h. after three washes, the membrane was incubated with peroxidase-conjugated goat anti-mouse igg (h þ l) (jackson immunoresearch laboratories, west grove, pa) in blocking buffer at room temperature for 1 h. after three washes, the protein blots were detected with either tmb membrane peroxidase substrate (kpl) or enhanced chemiluminescence (ecl) substrate (pierce). band intensities were analyzed via imaging analysis using imagej. presence of ibv spike proteins on the svlps was further verified by immunogold staining, and purified ibv 2575/98 virions were used as a control. briefly, 3 ml of svlp or virion samples were deposited onto a glow-discharged carbon-coated grid for 2 min. the virion sample was fixed with 4% paraformaldehyde for 5 min. after 3 washes with pbs, the samples were blocked with 1% bsa for 15 min. the samples were then incubated with anti-s mab for 1 h. after pbs washes, the samples were incubated with 6 nm goldconjugated goat anti-mouse igg (jackson immunoresearch laboratories) for another 1 h. after pbs washes, native virions were further stained with 1% uranyl acetate for 15 s. all experiments were performed at room temperate. particles were visualized under a 200 kv high resolution transmission electron microscope (fei tecnai tf20). the care and use of animals were approved by the institute animal care and use committee, national taiwan university (approval no. ntu-102-el-89). all animal experiments were carried out in accordance with the approved guidelines. 8-week old balb/c mice were injected with 50 ml of pbs, free protein formulation, or svlps containing 2 mg of viral antigens via the intrafootpad route. after 24 h, the mice were sacrificed and the popliteal lymph nodes were harvested (n ¼ 6). cryosections (6 mm) were made and fixed for 10 min in acetone, followed by 8 min in 1% paraformaldehyde. sections were blocked by 5% normal goat serum (invitrogen) in pbs for 10 min and stained with anti-s mab for 4 h at room temperature. after washes, sections were further incubated with fitc-conjugated anti-mouse igg (jackson immu-noresearch laboratories) for 1 h at room temperature. nuclei were counterstained with dapi (invitrogen). fluorescence signal was observed under a fluorescence microscope (leica dmi8), and quantified via imaging analysis using imagej. 8-week old balb/c mice were injected intramuscularly in the thigh with 100 ml of formulations containing pbs, free protein, or svlps (10 mg of viral antigens) mixed with the complete freund's adjuvant. mice blood was collected on day 14 and 21 for antibody titer quantification (n ¼ 4e5 per group). three-week-old spf chickens were obtained from jd-spf biotech (miaoli, taiwan). chickens were randomly divided into four different experimental groups (n ¼ 4e6 per group) receiving pbs, free protein (r2575s), whole inactivated virus (wiv) vaccine (merial laboratories, lyon, france), or svlps. briefly, free protein or svlps (10 mg of viral antigen in 100 ml) were emulsified with the complete freund's adjuvant and administered via an intramuscular route. the commercially available wiv vaccine (oily-adjuvanted) was administered to chickens according to the manufacturer's recommendation (0.3 ml per chick). chicken sera and tears were collected on day 0 (before immunization), 14, and 21 post-immunization. all chickens were intranasally challenged with ibv 2575/98 live virus (10 6 eid 50 ) on day 21, and were observed for disease signs for 7 days. chickens were sacrificed on day 28. for serum iga and igg virus-specific elisa, 100 ng of purified ibv 2575/98 virions was diluted with coating buffer (15 mm na 2 co 3 and 35 mm nahco 3 , ph 9.6) and coated onto flatbottomed microtiter plates (nunc) at room temperature overnight. the wells were washed with pbst (0.1% tween 80 in pbs) three times and blocked with blocking reagent (5% skim milk in pbst) at 37 c for 1 h. after washes, 100 ml of chicken serum was added and incubated at room temperature for 1 h. following three washes, 100 ml of peroxidase-conjugated goat anti-chicken igy (h þ l) or iga (jackson immunoresearch) in blocking buffer was added into each well and incubated at room temperature for 1 h. after three washes, 100 ml of sureblue reserve tmb microwell peroxidase substrate (kpl) was added to each well and incubated in the dark at room temperature for 10 min. the reaction was stopped by adding 100 ml of tmb stop solution (kpl). the od was measured at 450 nm using an automated plate reader (thermo fisher). for total tear iga quantification, elisa was performed with chicken iga elisa kit (ab157691, abcam) according to the manufacturer's protocol. on day 28 post immunization, chicken spleens were minced and passed through a 70-mm cell strainer (corning) to obtain single-cell suspensions. red blood cells (rbcs) were lysed using an rbc lysis buffer (ebiosciences), and cells were resuspended in rpmi 1640 medium (gibco, grand island, ny) containing 10% fbs. viable cells were determined by trypan blue staining. 10 6 splenocytes were plated in 96-well u-bottom plates (corning), and were stimulated with 1 mg of purified ibv 2575/98 virions in the presence of brefeldin a (golgiplug, bd biosciences) for 6 h at 37 c. for the quantification of cytokine expression, the stimulated splenocytes were lysed, and total rna was isolated by trizol (invitrogen) according to the manufacturer's manual. real-time rt-pcr was performed using iscript (bio-rad) and iq sybr green supermix kit (bio-rad) with previously described primers for chicken ifn-g and gapdh [28] . melting curve analysis following real-time pcr was conducted to verify the specificity for each primer set. all obtained ct values were normalized to gapdh. the relative expression of chicken ifn-g (fold change of naive control) was determined by a 2 àddct method [29] . disease signs of chickens were recorded on a daily basis after virus challenge. the clinical score index of ibv infection was interpreted according to a previously described method [30] . the clinical signs were evaluated as: 0 ¼ no clinical signs; 1 ¼ lacrimation, slight shaking, watering feces or tracheal rales; 2 ¼ lacrimation, presence of nasal exudate, depression, water feces, apparent sneezing or cough; 3 ¼ high degree of lacrimation, nasal exudate, and severe watery feces; 4 ¼ death. after necropsy, gross lesions at the tracheas and kidneys were recorded. chicken kidneys were further harvested and homogenized in tryptose phosphate broth (bd biosciences). viral load in kidneys was assessed by quantitative rt-pcr described below. 2.11. viral rna quantification rna in chicken kidneys was extracted using trizol (invitrogen) according to the manufacturer's manual. for viral load assessment, quantitative rt-pcr was performed with iscript (bio-rad) and iq sybr green supermix kit (bio-rad) using previously described primer sets that target the s protein gene of ibv (rc2u and rc3l) [31] and chicken 28s rrna [32] . quantitative rt-pcr experiments were performed in duplicates. data was expressed as arbitrary units. data was analyzed by anova followed by dunnett's multiple comparison tests using graphpad prism (graphpad software, san diego, ca). p values smaller than 0.05 were considered significant. following aunp incubation in solutions of different protein concentrations, the resulting nanoparticles were pelleted from free proteins and re-dispersed through sonication in pbs. consistent with previous studies on nanoparticle/protein interactions [33] , it was observed that higher protein concentrations yielded particles with increased colloidal stability as evidenced by the disappearance of a discernable pellet and a purple solution characteristic of aunp suspensions ( fig. 2a) . svlps prepared from the 3 mg/ml protein suspension were readily dispersible and manifest as distinct, nonclustered nanoparticles under transmission electron microscopy (fig. 2b) , indicating passivation of the high particle surface energy upon sufficient protein coating. in contrast, particle preparations with lower protein content (1000 mg/ml) yielded clustered aunps. to further characterize svlps, we assessed aunps, svlps, and native ibv virions (fig. 2c) using nanoparticle tracking analysis, which examines particle samples on a particle-by-particle basis via tracking of scattered laser light from individual particles [34] . between aunps and svlps, we observed an overall reduction in the light scattering intensity. given that aunps are known to scatter light at an extraordinary efficiency, the intensity reduction in svlps can be attributed to successful protein coating, which restricts light passage to the aunp surfaces. likewise, native virions have the lowest light scattering under the analysis as they are comprised entirely of organic materials. the result demonstrates the feasibility of studying the evolution of nanoparticle protein corona formation using nanoparticle tracking analysis, which reveals changes in light scattering and size simultaneously. upon examining the size distributions of the different particles, svlps showed a broader distribution as compared to the sharply distributed 100 nm aunps. protein corona formation increased the nanoparticle size from 100.6 nm (pdi ¼ 0.012) to 139.2 nm (pdi ¼ 0.073) and increased the zeta potential from à23.2 mv to à16.7 mv (fig. 2c,d) . in comparison to native ibv virions, which have an average diameter of 147.3 nm (pdi ¼ 0.081) and a zeta potential of à16.6 mv, the svlps are similar in overall physicochemical properties. examination of particle stability showed that the svlps remained stable in pbs over a 7-day period with its size ranging from 136.7 nm (pdi ¼ 0.071) to 140.2 nm (pdi ¼ 0.091) (fig. 2e) . analysis of antigen display with freshly prepared svlps showed that 1 â 10 11 aunps retained 23.5 ± 2.2 mg of spike proteins, corresponding to approximately 900 ibv spike proteins per particle. western blotting using analysis revealed a sharp protein band of approximately 160 kda (fig. 2f) , which is characteristic of the viral antigen [17] . transmission electron microscopy and immunogold staining further highlight the similarity between svlps and native ibv virions. it was observed that immunogold clustered around the svlps, mirroring the staining pattern on the native virions (fig. 2g) . these observations demonstrate the close semblance between the svlps and native virions regarding their physicochemical properties and antigen display. examination of antigen retention in protein-poor (1x pbs) and protein-rich (10% bsa in 1x pbs) conditions also shed light on the characteristics of the protein corona around the svlps. in pbs, particle-bound antigen level remained steady over a span of 24 h, yielding similar ibv spike protein band intensities across the different incubation samples (fig. 2h) . a rapid drop-off in particlebound spike protein was observed upon incubation in 10% bsa. immediate retrieval of svlps from the bsa solution resulted in~65% reduction in the spike protein level, and at the 24 h mark,~25% of the initial antigen remained on the svlps. this observation suggests the formation of two distinctive corona layers distinguishable by their interaction dynamics with surrounding biomolecules, reflecting the presence of both a reversible "soft corona" and an irreversible "hard corona" that have been frequently observed in prior nanoparticle studies [35e37]. the results indicate that approximately 200e250 ibv spike proteins are stably bound to each svlps. these proteins are expected to remain in the particulate form in complex biological environments upon in vivo administration. to examine antigen delivery and lymphatic transport by the svlps as compared to free spike proteins, svlp formulation was administered to mice through a footpad injection. popliteal lymph nodes, which are the draining lymph nodes by the footpads, were subsequently collected and sectioned for immunofluorescence assay. ibv spike protein-specific immunofluorescence staining showed a significantly enhanced antigen delivery by the svlps as compared to the free protein formulation, resulting in an increased number of fluorescent punctates (green) in the lymph node sections (fig. 3a) . imaging analysis on multiple lymph node sections showed that the svlp formulation increased lymphatic delivery by approximately 6 fold (fig. 3b) . the observation of increased delivery attests to the strong protein/particle binding in the "hard corona" layer as the particle carrier is capable of facilitating antigen transport in vivo. the enhanced lymph node localization of the svlps is consistent with prior observations on nanoparticles and virus-like particles [2] . owing to their nanoscale morphology and physicochemical properties, these nanoparticles are known to facilitate free lymphatic drainage via convective transport [38, 39] as well as cell-mediated lymphatic delivery via increased cellular uptake [2] . immunogenicity of the svlps was also examined following intramuscular inoculation in mice. anti-ibv igg serum titers were compared between mice vaccinated with svlps and with free ibv spike proteins (fig. 3c) , and it was observed that the svlps elicited significantly higher igg levels, demonstrating improved vaccination potency over the free protein formulation. the improved immunogenicity can be explained in part by the enhanced antigen delivery to the lymph node, where a high number of antigen presenting cells reside. in addition, the particulate nature of the svlps likely also favors other immune activation mechanisms, such as improved cellular uptake, enhanced complement activation [38] and presentation by follicular dendritic cells [40] . these nanoparticle-specific immunological features make the svlps a promising vaccine candidate for disease management. to evaluate the svlps' effectiveness against viral infections, we vaccinated spf chickens with free ibv spike proteins or svlps (10 mg of total viral antigens) via the intramuscular route. as an additional reference, a commercial wiv vaccine for ibv was administered based on the manufacturer's suggested dosage. following vaccination, blood and tear were collected for analysis and a live ibv challenge was performed (fig. 4a) . elisa analysis showed that the svlps were superior in generating both igg and iga titers as compared to the free protein formulation and the wiv vaccine (fig. 4b,c) . the total iga in the tears of the vaccinated chickens were also quantified. despite that intramuscular vaccination is generally known to be non-ideal for promoting mucosal immunity [41] , elevation of tear iga level was observed for all three vaccine formulations (fig. 4d) . it is expected that mucosal vaccination in future studies may further increase tear iga levels and better highlight the differences among the formulations in eliciting mucosal immunity. besides humoral immunity, cellular immunity, a major component of effective antiviral immune responses [42] , was analyzed using splenocytes extracted on day 28. the svlp sample showed a significant increase in the ifn-g mrna level as compared to the control, free protein, and the wiv vaccine samples (fig. 4e) , demonstrating superior promotion of antigen-specific cellular immunity. we further examined the effect of the different vaccinations in protecting against a viral challenge. clinical scores evaluated based on stamina, posture, and voice show that the svlp group had the lowest overall symptoms, on par with animals vaccinated with the wiv formulation (fig. 5a,b) . in comparison, vaccination with the free protein formulation was less effective and highly variable in moderating the disease symptoms. on day 28, necropsies were performed to examine the tracheas and kidneys, which are characteristic sites for infections by ibv [43] . as indicated in the gross lesion photos, the best antiviral protection was observed in the svlp-immunized group, whereas organs from the free protein group and the wiv vaccine group showed observable mucus secretion and petechiae in tracheas (fig. 5d, upper panel, arrowed) and swollen lesions and hemorrhages in kidneys (fig. 5d, lower panel, arrowed) . the prophylactic effect of the svlp vaccination was further demonstrated by examining the viral load in kidneys. analysis by quantitative rt-pcr showed that immunization with svlps more consistently reduced the viral content, resulting in the lowest relative viral mrna expression across the animal samples (fig. 5c) . the results further corroborate the protective effect by the svlp vaccination, which enhanced both humoral and cellular immunity for increased protection against the viral challenge. coronavirus spike proteins are the primary antigenic signatures on coronaviruses as they contribute to the characteristic crown-like morphology underlining this virus family. as these proteins comprise the outermost layer of coronaviruses, the spike proteins have a pivotal role in viral pathogenesis and are recognized as the primary target for vaccine preparations [44] . present vaccination strategies for coronaviruses include recombinant viruses and viruslike particles, and there is a continuing effort in developing new strategies for improving vaccine potency and safety [22] . to the best of our knowledge, incorporating coronavirus spike protein with synthetic nanoparticles has not been previously explored. by exploiting the high surface energies of synthetic nanoparticles, spontaneous assembly of svlps covered with ibv spike proteins were demonstrated. the strong particle/antigen association resulted in virus-sized particulates displaying ibv spike proteins, and the svlps elicited strong immune protection against a live ibv challenge. the enhanced immunopotentiation by the particle carrier is consistent with previous studies and echoes the curious observation that gold nanoparticles not only promote humoral but also cellular immune responses upon association with antigens [14, 15] . as the increased cellular immune response suggests that the nanoparticles may play a role beyond a passive antigen carrier, future studies examining the impact of nanomaterials and nanoparticle surface energies on immunological interactions are warranted. it should be noted that the phenomenon of protein corona formation is an evolving field of study in which scientists continue to examine nanomaterials in biological medium with increasing complexity [45e47]. subtle changes on the environment and on nanoparticle properties can have dramatic and unpredictable impact on the overall corona identity with significant biological implications. to demonstrate a practical utility for the protein corona phenomenon, the present study adopts a reductionist approach in examining protein-particle interactions. aunps are incubated in a highly controlled condition with proteins of a singular species to form svlps with virus-mimetic features, and the dynamics of such association are expected to vary with different biomolecules and nanomaterials [48] . in general, inorganic nanoparticles promote stronger protein adsorption as compared to organic nanoparticles as inorganic nanoparticles tend to have higher surface energies. decreasing particle size also tends to increase biomolecule interactions as it increases radii of curvature of nanoparticle surfaces. other forces, such as electrostatic interactions, van der waals forces and covalent interactions all play intertwining roles in governing the nano-bio interface, and factors including nanoparticle functionalizations, buffer conditions, and biomolecule species have significant impact on the corona formation [48] . nonetheless, in a controlled and optimized condition, the phenomenon may be exploited to facilely prepare formulations with defined characteristics and favorable biological performance. the present work takes advantage of this spontaneous interaction between nanomaterials and biomolecules towards improving vaccine development. this strategy may find practical applications in disease management against coronaviruses as well as other infectious threats. in summary, we demonstrate by incubating viral antigens with synthetic nanoparticles in optimized conditions, spontaneous formation of protein corona induces the assembly of virus-like nanostructures with viral antigens encasing the particulate core. results from the present study validate the successful preparation of svlps via nanoparticles' innate tendency to induce protein coating. in comparison to typical virus-like particle preparations, the present strategy offers practical advantages owing to its simple and facile process. amidst the growing health threats of coronavirus infections as well as the ongoing economic impact of ibv infections, virus-like particles are garnering increasing scientific interest as vaccine candidates owing to their improved efficacy in comparison to subunit antigens [49, 50] . in the present study, vaccination with the svlps resulted in enhanced humoral and cellular immune responses, improving protection against an avian model of coronavirus infection as compared to 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reversible versus irreversible binding of transferrin to polystyrene nanoparticles: soft and hard corona the evolution of the protein corona around nanoparticles: a test study exploiting lymphatic transport and complement activation in nanoparticle vaccines a sensitive in vivo model for quantifying interstitial convective transport of injected macromolecules and nanoparticles follicular dendritic cells: dynamic antigen libraries what role does the route of immunization play in the generation of protective immunity against mucosal pathogens? contributions of humoral and cellular immunity to vaccine-induced protection in humans coronavirus avian infectious bronchitis virus the spike protein of sars-covea target for vaccine and therapeutic development effects of the presence or absence of a protein corona on silica nanoparticle uptake and impact on cells protein corona fingerprinting predicts the cellular interaction of gold and silver nanoparticles understanding and controlling the interaction of nanomaterials with proteins in a physiological environment understanding biophysicochemical interactions at the nano-bio interface mers-cov viruslike particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques assembly and immunogenicity of coronavirus-like particles carrying infectious bronchitis virus m and s proteins the study was supported by the ministry of science and technology (103-2321-b-002-066, 104-2321-b-002-023, 105-2321 -b-001-055) and national taiwan university (104r7320). key: cord-009800-tajkpgkj authors: orellana, mhica v.; perry, mary jane title: an immunoprobe to measure rubisco concentrations and maximal photosynthetic rates of individual phytoplankton cells date: 2003-12-22 journal: limnol oceanogr doi: 10.4319/lo.1992.37.3.0478 sha: doc_id: 9800 cord_uid: tajkpgkj the cross‐reactivity of an immunological probe to the key photosynthetic enzyme rubisco (ribulose‐1,5‐bisphosphate carboxylase/oxygenase) was characterized as part of a larger effort to determine maximal photosynthetic rates of individual phytoplankton cells. polyclonal antiserum was produced against purified rubisco from the marine diatom chaetoceros gracilis. the results of western immunoblotting demonstrated that the antiserum reacted positively with rubisco from 38 species of algae and higher plants and failed to react with only three species of dinoflagellates and one prochlorophyte species. however, the binding affinity or the strength of the cross‐reaction for the polyclonal antiserum with purified rubisco varied among species. the antiserum was then affinity purified against spinach rubisco and its binding affinity for purified rubisco determined by elisa. two taxonomic groupings resulted: one with high‐binding affinity (these species included chrysophytes, bacillariophytes, prymnesiophytes, and chlorophytes) and the other with low‐binding affinity (dinophytes and cyanophytes). rubisco concentration per cell and light‐saturated rates of photosynthesis were highly correlated for cultures of the diatom thalassiosira weissflogii. these results indicate that affinity‐purified antiserum can be rigorously characterized for use in quantifying rubisco concentration and for assessing the maximal photosynthetic potential of individual phytoplankton cells. assessing the magnitude and variance of primary production in the marine environment is a major goal of biological oceanography. variability in rates of photosynthesis have been observed on all spatial scales from that of the individual phytoplankton cell to the global ocean (watt 197 1; platt and sathyendranath 1988 ) and on all temporal scales from picoseconds to geological epochs (kolber et al. 1988 ; barnola et al. 1987) . understanding the causes of variability in primary production is important if we are to predict nonlinear responses of marine food webs to environmental perturbations or to develop realistic models of the biogeochemical flux of carbon in the ocean. achievement of that goal requires --acknowledgments we thank r. a. cattolico for the prochloron sp. lysate and access to her laboratory facilities, g. kenny for heterotrophic specimens, e. duffield for macroalgal samples, m. c. talbot for the photosynthetic measurements, and the following for advice: s. newman (rubisco purification), b. watson (affinity purification), r. horner (systematic classification), r. davis, and c. roesler (computer programing studies of photosynthesis on a diversity of spatial and temporal scales. photosynthetic c fixation has been measured by nah'"co, incorporation (steemann nielsen 1952) for 40 yr. as generally applied, the 14c method determines c assimilation rates for "bulk" phytoplankton in mixed-species assemblages but gives little or no information about the variability in photosynthetic activity at the scale of the individual species or cell. statistical information on the underlying variability at the level of the individual phytoplankton cell is necessary to resolve differences in primary production and loss at the next highest scale (i.e. bulk assemblages). sampling at the scale of the individual cell provides the most basic information for interpreting variance in primary productivity among bulk assemblages (huston et al. 1988) . a limited number of oceanographic studies of photosynthetic or growth rates of individual phytoplankton cells or species have been carried out with autoradiography (iturriaga and marra 1988) , 14c labeling of individual cells (rivkin 1990) , and cell division patterns (weiler and eppley 1979) . although the results of these studies demonstratc significant interspecific differences in rates, relatively few cells can be processed because single-cell methods are extremely labor intensive and, hence, limited in their general applicability to field studies of primary productivity. however, with the application of flow cytometry to oceanography, it is possible to analyze autofluorescent and light-scattering properties for thousands of individual cells in a very short time (e.g. minutes). by coupling flow cytometry with immunological probes for taxonomic identification (shapiro et al. 1989) or quantification of specific cellular components (orellana et al. 1988) , it is possible to address questions relating to the variability in photosynthetic rates and physiological processes among individual cells or taxonomic groups in phytoplankton assemblages. the specific goal of the present work was to develop a method to determine maximal photosynthetic potential for individual phytoplankton cells. to do so, we developed an immunological probe against the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco). the reasons for selecting this key enzyme of the calvin-benson cycle are as follows. rubisco is responsible for net coz assimilation. under conditions which favor carboxylase activity as opposed to oxygenase activity (e.g. high ratios of co2 to o,), maximal light-saturated rates of photosynthesis are proportional to rubisco concentrations (bjorkman 198 1; rivkin 1990 ). the enzyme is ubiquitous in all photoautotrophic plants and is also found in various photosynthetic bacteria. the large subunit of rubisco contains the active catalytic site and is highly conserved (miziorko and lorimer 1983) . because it is a soluble protein and a major constituent of phytoplankton cells, rubisco can be easily detected in individual cells with immunofluorescent probes. in order to use an immunoprobe to quantitatively measure rubisco concentration within individual cells in a mixed-species assemblage, it is imperative to determine: first, whether the probe is truly specific to only the large subunit of rubisco; second, whether the probe binds to the rubisco large subunit of all phytoplankton species with equal affinity; and third, whether immunologically determined concentrations of rubisco are well correlated with maximal photosynthetic rates. in this paper, we demonstrate that these criteria were satisfied. the results indicate that an affinity-purified anti-rubisco probe can be used to quantify rubisco concentrations and maximal photosynthetic potential of individual phytoplankton cells in mixed-species assemblages. cell culture-cultures of 38 species of algae were grown at 20°c; the other two algal species, gonyaulax tamarensis and peridinium balticum, were grown at 15" and 28°c. most species were grown in the light under a 12 : 12 l/d regime; g. tamarensis and porphyra yezoensis were grown under regimes with 18 : 6 and 16 : 8 l/d. all diatom cultures and cultures of seven additional species (synechoccocus bacillaris, dunaliella tertiolecta, dunaliella salina, tetraselmis suecica, cryptomonas profunda, isochrysis galbana, and ochromonas dank&) were grown in modified-imr media (perry et al. 198 1) at 300 pmol photons m-2 s-l. chlamydomonas moewusii, porphyridium purpureum, and cyanophora paradoxa were grown in 3 n+ medium (hoham 1971) at 300, 220, and 100 pmol photons m-2 s-l. anabaena cylindrica was grown in ccap media (george 1976) at 100 pmol photons m-2 s-l and monodus sp. in asp2 (hedberg 1983 ) at 300 pmol photons m-2 s-l. the remaining chlorophytes, dinophytes, and phaeophytes were grown in modified f/2 medium (waaland and watson 1980) at 150 or 300 ,umol photons m-2 s-l. a lysate of prochloron sp. was obtained from r. a. cattolico. rubisco from spinacea oleracea was purified from partially purified rubisco (sigma). the two heterotrophic species were obtained from g. kenny. whole-cell zysates-cells in the logarithmic growth phase were harvested by centrifugation. they were resuspended in 15-40 ml of rubisco buffer (25 mm tris-hcl, ph 8.1, 10 mm mgcl,, 10 mm kci, 1 mm dtt (dithiothreitol), 0.5 mm egta [ethyleneglycol-bis(b-amino-ethyl ether)-n,n'tetraacetic acid], 0.4 mm pmsf (phenylmethylsulfonylfluoride), 0.7 pg ml-l leu-peptin, 0.5 pg ml-l pepstatin}, and broken at 1,15 5 kg m-2 with a french pressure cell. rubisco purification -purified enzyme was prepared according to newman and cattolico (1987a) . whole-cell lysates were centrifuged at 15,000 x g for 15 min, the supernatant was adjusted to 55% (v/v) (nh,),so,. the resultant precipitate was collected by centrifugation at 18,000 x g for 15 min and resuspended in 2 ml of rubisco buffer. the resuspension was loaded onto a 10-40% linear sucrose gradient prepared in rubisco buffer and centrifuged at 184,000 x g for 140 min. absorbance was monitored at 280 nm for each fraction. rubisco identity was confirmed by a 14clabeling activity assay (newman and cattolico 1987a) ; fractions containing rubisco were pooled and stored at -40°c. polyclonal antiserum production -rubisco holoenzyme was purified from the diatom chaetoceros gracilis and the antiserum produced in a new zealand rabbit according to vaitukaitis (198 1) . preimmune s&urn was obtained before initiation of the injection series. the antiserum was prepared according to hurn and chantler (1980) and stored at -40°c. acffinity purijication of polyclonal antiserum-spinach rubisco (sigma r-8000) was coupled to a cyanogen bromide-activated sepharose column (sigma c 9 142) and crosslinked with 1% glutaraldehyde in pbs (10 mm na,hpo,, ph 7.2, 0.15 m nacl) for 1 h. the antiserum was loaded onto the column and the column washed with pbs. antibodies that cross-reacted with domains on spinach rubisco were linked to the spinach-sepharose column, while antibodies that did not recognize domains on spinach rubisco were eluted and discarded. the reactive antibodies were eluted with 100 mm glycine, ph 3.2; these affinity-purified monospecific antibodies were washed with pbs and stored at -40°c. rubisco samples were diluted 1 : 2 in sample buffer containing 0.5 m tris hcl, ph 6.8, 10% (v/v) glycerol, 2% (w/v) sds, and 5% (v/v) 2-/3-mercaptoethanol. the samples were heated for 4 min at 95°c. discontinuous sds-page gel electrophoresis was carried out according to laemmli (1970) with 25 ma at room temperature. the gels were stained with 0.1% coomassie brilliant blue r-250 in 4oyo methanol (v/v) with acetic acid (10% v/v), destained, and scanned with a laser densitometer (helena) for estimation of protein in the gel bands. western blots-the separated proteins on the gels were electrophoretically transferred to nitrocellulose membrane overnight according to towbin et al. (1979) . after electrotransfer, the membrane was blocked with 2% bsa (bovine serum albumin) in tbs (tris-buffered saline: 20 mm tris-hcl, ph 7.5, 500 mm nacl), washed in tbs, and incubated in primary (anti-rubisco) antiserum diluted to 1 : 1,000 with 2% bsa in ttbs (0.05% tween 20 in. tbs) for 2 h. the membrane was washed in ttbs and incubated in secondary antibody (biorad goat anti-rabbit igg conjugated with horseradish peroxidase). the immunoprecipitate was visualized by incubating the membrane in substrate solution [0.6 mg ml-' 4-chloro-1-naphthol in methanol, 8 ml of pbs, and 0.1% h202 (v/v)]. the reaction was terminated with deionized water. dot blot-a series of concentrations from 5 to 1,000 ng of purified rubisco was loaded onto nitrocellulose membrane according to moeremans et al. (1984) . the immunobinding and detection were carried out as described for the western immunoblotting. elisa assay-purified rubisco, in concentrations ranging from 24 to 500 ng, was diluted in 0.1 m carbonate buffer (0.1 m nahco,, ph 9.6, 10 mm mgc12) and immobilized in triplicate wells of costar no. 3590 microtiter plates by passive attachment during overnight incubation at 4°c according to catt and millard (1988) with modifications. the plates were washed in pbs, incubated in a blocking buffer (2% bsa in pbs), rewashed in pbs-t (pbs with 0.05% tween 20$, and incubated with affinity-purified antiserum, diluted to 1 : 100 in pbs-t with 2% bsa, for exactly 2 h at room temperature. the plates were washed in pbs-t and incubated with secondary antibody (biorad goat antirabbit igg conjugated with alkaline phosphatase, diluted 1: 3,000 in 2% bsa in pbs-t) for 2 h. the plates were washed and incubated in substrate solution (4-nitrophenyl phosphate in 0.1 m diethanolamine buffer, ph 9.8, 1 mm mgc12). the reaction was terminated with naoh (13 mm), and optical density at 405 nm was measured with an elisa plate reader (cambridge technology). the linear portions of the hyperbolic elisa-binding curves were analyzed by analysis of variance within species and analysis of homogeneity of slope regressions among species. immunofluorescent staining-the procedure consists of three steps: fixation, permeabilization, and rubisco immunofluorescent staining. cells were harvested in a refrigerated centrifuge at 500 x g and 4°c for 10 min and subsequently fixed in freshly prepared 2% paraformaldehyde in pbs ph 8.0 at 4°c for 10 min. cells were permeabilized in 0.5% dms0 (dimethylsulfoxide) in pbs for 3-5 min on ice. tests for potential leakage of rubisco from the cells during fixation and permeabilization were carried out by dot blot immunoassay (moeremans et al. 1984 ). because paraformaldehyde cross-links this soluble enzyme and prevents its leakage from the cell (ohba et al. 1979) , no rubisco was found in the supernatant. although paraformaldehyde changes enzyme secondary structure, no loss of antigenicity for rubisco was observed. the cells were stained by first incubating them on ice with affinity-purified rubisco antiserum diluted (1 : 100) in pbs with 2% bsa. after 2 h, the cells were washed with 15 ml of pbs and then incubated with a goat antirabbit secondary antibody conjugated with fitc (fluorescein isothiocyanate) at a 1 : 300 dilution for 1 h. the cells were washed three times for 5 min in 15 ml of pbs. three controls were run at the same time on fixed and permeabilized cells to evaluate nonspecific staining: cells without either primary or secondary antisera, cells incubated in preimmune serum and secondary antiserum, and cells incubated in secondary antiserum alone. the fixation and staining procedures were evaluated qualitatively by epifluorescence microscopic observations. flow cytometric analysis-the fluorescence of individual cells was quantified with a coulter epics profile flow cytometer with an argon laser tuned to 48 8 nm and operated at 15 mw. the epics profile was calibrated with "immuno-check" fluorospheres (coulter corp.). short-pass (530 nm) and long-pass (630 nm) filters were used to detect the green and red fluorescence from fitc and chl a, respectively. forward-angle scatter data also were collected to test cell integrity. fluorescence and light scatter data were recorded on a 3-decade log,, scale, stored as one-or two-parameter histograms, and transformed to a linear scale for analysis. the magnitude of rubisco immunostaining was calculated as the difference between the green fluorescence of the primary plus secondary immunostained cells and that of the control cells stained with only secondary antiserum. photosynthetic measurements -photosynthetic rate measurements were determined for cultures of the diatom thalassiosira weissflogii grown on a 15 : 9 l/d cycle at 5,50, 100, 150, or 300 hmol photons m-2 s-l at 20°c. i rra d iance was measured with a quantum scalar irradiance meter (biospherical qsl-100). cultures were adapted to their growth irradiance for 3 weeks before initiation of experiments. they were maintained at low cell densities in logarithmic growth by diluting the cultures every other day with fresh media to avoid self-shading and to ensure nonlimiting nutrient concentrations. specific growth rates (d-l) were determined from cell counts. chl a was measured fluorometrically (lorenzen 1966) . photosynthetic rates were measured by nah14c0, incorporation in p vs. z (photosynthesis vs. irradiance) incubators with 48 irradiances, ranging from 3.7 to 1,525 pmol photons m-2 s-l (talbot et al. 1985) . one-milliliter aliquots were incubated for 0.5 h at 20°c with 0.06 &i 14c ml-l; total activity was determined with pea (phenethylamine; iverson et al. 1976 ). the incubation was terminated by adding 250 ~1 of 6 n hcl; samples were shaken for 1 h to remove excess 14c02. the p vs. i data were fit to a nonlinear exponential equation to derive the photosynthetic parameter p,,, (light-saturated photosynthetic rate). polyclonal antiserum -cross-reactivity of the anti-rubisco polyclonal antiserum with rubisco from 44 species of autotrophs (representing 14 groups of microalgae, macroalgae, and one higher plant) was analyzed by western blot analysis. in addition, wholecell lysates from two heterotrophic species were analyzed. most species were analyzed a minimum of 2 times. almost all the species cross-reacted with the probe, except for the one species of prochlorophyte, the two heterotrophic protozoans, and three species of dinoflagellates. a fourth dinoflagellate, g. tamarensis, exhibited positive crossreactivity against its purified rubisco but failed to cross-react when its whole-cell lysate was tested. in almost all cases where cross-reactivity was observed only one crossreacting band, representing the large subunit of the rubisco holoenzyme, was found in whole-cell lysates. the results from these western blots are summarized in table 1 and demonstrate specificity of the antiserum for the rubisco large subunit as well as a pattern of broad interspecific crossreactivity. however, western blots are strictly a qualitative indicator of the presence or absence of antibody-antigen crossreactivity and are not a quantitative index of the strength of the cross-reaction. when the more quantitative dot blot assays were used with purified rubisco from different species, a high degree of variability in the binding affinity with the polyclonal antiserum was evident (fig. 1) . as a consequence of this interspecific heterogeneity in the strength of the antibody-antigen binding, it was necessary to affinity purify the polyclonal antiserum. afinity-purijed antiserum -for wholecell lysates, the rubisco large subunit was the only band evident in the sds-page gel (fig. 2, left) which cross-reacted with the antiserum in the western blot (fig. 2, right) , demonstrating the high specificity of the antiserum for the rubisco large subunit. the affinity-purified probe cross-reacted with the rubisco large subunit (fig. 2, right) , confirming that no unexpected alteration occurred in probe specificity due to affinity purification. the binding capacity or strength of the antibody-antigen interaction between purified rubisco and the affinity-purified antiserum was quantified by elisa assay. the results of the assay were highly reproducible, with an average se of 0.192 for all points on the curves. variability between abcdefg fig. 1 . dot blot analysis to measure cross-reaction between polyclonal rubisco antiserum and nanogram concentrations of purified rubisco from: a-olisthodiscus luteus; b-isochrysis galbana; c-chaetoceros gracilis; d-phaeodactylum tricornutum; e-porphyra yezoensis; f-tetraselmis suecica; g-chlamydomonas moewusii. assays run on different days was low; results of two independent experiments with purified rubisco from i. galbana are shown in fig. 3 . no statistically significant difference was found between the slopes for the two experiments (p 5 0.001). when purified rubisco from 11 species representing different systematic groups was analyzed by elisa, two distinct groupings were evident (table 2) . group 1 had a high binding affinity for the affinity-purified antiserum and included species representing the chrysophytes, prymnesiophytes, bacillariophytes, chlorophytes, and rhodophytes. it should be noted that the binding affinity for the affinity-purified antiserum to rubisco, purified 0 100 200 300 400 500 fsochrysls r"bl.?.ccl ("0) fig. 3 . elisa assays using affinity-purified ru-b~sco antiserum and purified rub&a from zsochrysrs galbana. gd is optical density. each assay was nm in triplicate; open and closed symbols represent assays run at different times. from the immunizing species c. gracilis was greater than for the other species in group 1 and that rhodophyte species had a lower affinity. the binding affinity in group 2 was much lower; the second group of species included the cyanophyte synechoccocus bacillaris and the dinophytes glenodinium sp., gymnodinium simplex, and amphidinium carterat?. rub&o concentration and the light-saturated rate ofphotosynthesis-the growth rate of t. weissfrogii ranged from 0.09 d-l at 5 pm01 photons mm2 ski to 1.06 d-' at 300 firno photons m-2 sk'; however, growth was not saturated at the highest irradiance. the concentration of chl n cell-l decreased by a factor of 2.9 as growth irradiance increased from the lowest to the highest irradiance, while the cellular absorption crosssection estimated from flow cytometric chl a fluorescence cell-l according to perry and porter (1989) changed by a factorof 1.9 over the growth irradiances examined. light-saturated rates of photosynthesis per cell increased by a factor of 4.4 as a function of increasing growth irradiance. figure 4 shows an example of t. weissfrogii cells grown at 25 ~mol photons mm2 ski that were immunostained with the anti-rub&o probe. the cells were analyzed flow cytometrically and displayed on a 3-decade log,, scale. the control cells were fixed, permeabilized, and stained with only the second antibody. they exhibited very low background green fluorescence relative to the rub&co-stained cells. the geometric mean in relative fluorescence units, converted to linear units from log,, units, was 3 for the control cells while the geometric mean for the immunostained cells was 172.5. the background fluorescence of the control cells included a component due to native green autofluorescence (shapiro 198 8 ) plus a component due to nonspecific staining by the secondary goat antirabbit antibody. in general the total fluorescence of the control cells was low, averaging between 3 and 8% of the signal for the rubisco-stained cells. figure 5 shows the regression (r2 = 0.90) of the concentration of rubisco per cell determined by immunofluorescence against p,,, per cell. probe characterization -if an immunological probe is to be used to quantify rubis-experimental d 100 260 fltc green fluorescence p,ax (pg c cell"h") incorporation measurements @= -11.6 1 + 1.34x; r2 = 0.90). co concentration in individual phytoplankton cells in a mixed-species assemblage, it must meet the following criteria: cross-react with only the rubisco large subunit; 'bind quantitatively to the rubisco large subunit; and have equal affinity for the rubisco large subunit from all species of marine phytoplankton. the affinity-purified monospecific polyclonal antiserum reported here meets these criteria for group 1 species. one reason that it was possible to select for a species-independent rubisco probe is that the large subunit protein is highly conserved in an evolutionary context (miziorko and lorimer 19 8 3) . immunological cross-reactivity (table l) , hybrid reconstitution (andrews and lorimer 1987) , heterologous hybridization (shively et al. 1986) , and amino acid sequences (miziorko and lorimer 1983) all suggest that the large subunit of higher plants, green algae, and cyanobacteria is conservative, particularly at the active sites. taxa-specific differences have been reported for other anti-rubisco antisera (plumley et al. 1986; newman and cattolico 1987b); it should be noted that these polyclonal antisera were not affinity purified to conservative sites. in contrast, in the present study the antiserum was affinity purified to rubisco from a distant species in an effort to select only antibodies to conservative sites. the data in table 1 show that for 44 species tested with our c. gracilis polyclonal anti-rubisco antiserum, 38 exhibited positive cross-reactivity. the species that did not. cross-react included three species of dinoflagellates (a. carterae, gonyaulux catenella, and heterocapsa niei), the one species of prochlorophyte, and the two nonphotosynthetic heterotrophic protozoans. the lack of cross-reactivity with the dinoflagellates is puzzling. it may reflect a low total concentration of rubisco in the whole-cell lysate, since positive cross-reactivity was detected in a western blot of purified rubisco from g. tamarensis but not in the whole-cell lysate (table 1) . alternatively, the lack of cross-reactivity could reflect a minimal number of common domains on the large subunit of these dinoflagellate species or degradation of the enzyme during lysate preparation. coprecipitation of rubisco with the membrane fraction during centrif-ugation is also possible, as it has been previously observed that extraction in certain buffers, such as those containing mg2+, can result in association of rubisco with membranes in some species (mcneil and walker 1981) . the results of the western blot analyses with whole-cell lysates demonstrated a high degree of cross-reactivity across species groups (table l) , as might be predicted from the conservative structure of the rubisco large subunit. ihowever, the dot blot analysis with purified rubisco revealed high variability in the binding affinity of the polyclonal antiserum with the large subunit of different species (fig. 1) . the source of this heterogeneity is probably the presence of a diversity of antibody molecules in the polyclonal antiserum, with each type of antibody having a different affinity or strength of interaction to the different antigenic domains on the rubisco large subunit (atassi 1984) . although the active site of the large subunit of rubisco is highly conserved, some of the antibodies could have been produced against nonconservative sites. such heterogeneity in the binding affinity would prevent the use of rubisco probes to quantify rubisco concentrations in single cells present in a mixed-species assemblage. this problem was addressed by affinity purification of the polyclonal antiserum against rubisco from spinach, a species evolutionarily distant from c. gracilis. the reasoning is that only a subset of antibodies, i.e. those that react with the most conservative sites on the protein, are selected by binding to the affinity-purification column, while the antibodies that do not recognize domains on spinach rubisco are eluted and eliminated. the antibodies that do react with domains on spinach rubisco should be monospecific, and therefore, should theoretically cross-react with the same sites on all phytoplankton species and with the same binding affinity. these concepts were tested with purified rubisco. the results presented in fig. 2 indicate that the arrtiserum was monospecific and bound only to the rubisco large subunit. the results presented in table 2 indicate that for our affinity-purified antiserum the concept of uniform binding affinity applied only to group 1 species; the implication is that our affinity-purified antiserum can be used only to quantify rubisco in species of chrysophytes, bacillariophytes, prymnesiophytes, and chlorophytes (group 1). although the rhodophyte species in group 1 had a slightly lower binding affinity, this deviation is not critical to our application, because this group is not phytoplanktonic. a significantly lower binding affinity was observed for group 2, which consisted of dinophyte and cynophyte species. the explanation for this pattern, particularly for the dinophytes, is not obvious; however, a comparison of their rubisco structure might yield interesting evolutionary patterns. the oceanographic implications of the characterization and standardization of the rubisco antiserum is that the affinity-purified probe can be used in mixed-species assemblages to quantify rubisco concentrations in individual phytoplankton cells, particularly for mixed-species assemblages dominated by chrysophytes, bacillariophytes, prymnesiophytes, and chlorophytes. because the probe has a much lower binding affinity for dinophytes and cyanophytes, rubisco cannot be measured in these groups and it is essential to identify individuals belonging to these groups. oceanic coccoid cyanobacteria can be distinguished flow cytometrically by their phycoerythrin fluorescence and characteristic low forwardscatter signal (olson et al. 1988; perry and porter 1989) . dinoflagellates are more problematic; however, cell-surface antibody probes may prove to be sufficient for discriminating dinoflagellates by flow cytometry. the technology for image and fluorescence analysis of immobilized cells is rapidly improving and may also provide a solution for dinoflagellate recognition. this study demonstrates the importance of immunoprobe standardization. although rubisco has been previously quantified with heterologous antisera by a variety of different methods (radioimmunoassay, radial immunodiffusion, rocket immunoelectrophoresis, and dot blots), the strength of the antibody-antigen interaction or affinity of the antiserum for rubisco purified from different species has not, to our knowledge, been quantified previously. equal binding affinity of the antiserum with different species has been assumed. although this may be true when rubisco from closely related species is being measured, the results of the immunoprobe standardization demonstrate that the assumption is incorrect when rubisco is measured in an evolutionarily different group of species. the crossreactivity patterns reported by plumley et al. (1986) and newman and cattolico (1987b) suggest that not all the antibodies in their polyclonal mixtures were against conservative sites, similar to our polyclonal mixture prior to affinity purification. their findings further confirm the need for rigorous characterization of antisera used in mixed-species applications. monoclonal antibodies to the active site of rubisco may provide a more universal solution. rubisco concentration and the light-saturated rate of photosynthesis-a strong relationship between maximal extractable rubisco activity and maximal capacity for photosynthesis has been observed in higher plants (bjorkman 198 1) and in microalgae (rivkin 1990) . total in vitro rubisco activity has been correlated with maximal light-saturated rates of photosynthesis in the diatom phaedactylum tricornutum at low growth irradiance (beardall and morris 1976) , the diatom c. gracilis grown under nitrogen stress (m. j. perry unpubl.), the green alga scenedesmus obliques (senger and fleischhaker 19 7 8) , the dinoflagellate pyrocystis noctiluca (rivkin et al. 1982) , and several other individual species isolated from natural populations (rivkin 1990 ). in studies of marine phytoplankton assemblages, total rubisco activity has also been correlated with light-saturated rates of photosynthesis (glover and morris 1979; smith et al. 1983 ). however, a number of the past observations of the relationship between p max and rubisco concentration, as determined by in vitro assay activity, have been conflicting. for example, glover and morris (1979) and smith et al. (1983) found that rubisco activity would not account for 100% of the photosynthetic potential, as a consequence either of partial recovery of enzyme activity or differing physiological responses of individual species within a mixed-species assemblage. this observa-tion led glover and morris to conclude that rubisco activity was a poor indicator of the light-saturated rates of photosynthesis. the relationship in the past between in vitro rubisco activity and p,,,, has 'been controversial. first, the early in vitro enzymatic assays resulted in unnaturally low rates of co, fixation because the importance of the sequence of substrate addition on rubisco activation was not appreciated until the mid-1970s (lorimer 1979) . second, the importance of light activation of calvin cycle enzymes and the role of specific inhibitors on in vivo photosynthetic rate and in vitro rubisco activity was not fully recognized until more recently (ogren et al. 1986 ). third, in vivo net co2 fixation varied greatly in response to changes in the ratio of coz to o2 because of the bifunctional nature of rubisco as both a carboxylase and an oxygenase (jordan and ogren 198 1) . the enormous variation in the ratio between terrestrial and marine ecosystems suggests caution in directly extrapolating results from one system to another. relatively few immunological measurements of both rubisco concentration and photosynthetic rates have been made for microalgae. the results that are available for chlorophyte species show good agreement between rubisco concentration and p inaxsukenik et al. (1987) measured the rubisco large subunit in the marine alga dunaliella tertiolecta with a radioimmunoassay. they reported that p,,, per cell was independent of growth irradiance between 80 and 2,000 pmol photons me2 s-l. the cellular concentration of the large subunit of rubisco, measured both immunologically and by in vitro activity assays, exhibited the same pattern as p,,,. when the freshwater alga tetraedron minimum was grown at 50 and 500 pmol photons m--2 s-l, the immunologically determined rubisco concentration per cell was constant while p max per cell varied only 25% between the high-and low-light adapted cultures (fisher et al. 1989 ). oceanographic applications-the ability to measure rubisco protein and predict maximal photosynthetic rates in individual cells presents many opportunities; its widespread application will depend on the uni-versality of this relationship in phytoplankton. this approach will provide statistical information on the variability in primary product ion potential among individual phytoplankton cells within mixed-species assemblages. to achieve the main goal of determining the in situ photosynthetic rate of individual cells in the ocean, we need three other parameters in addition to p,,, per cell: underwater irradiance, absorption cross-section of the individual cell, and the functional form of the photosynthesis vs. absorption relationship. spectral underwater irradiance can be readily obtained, while perry and porter ( 1989) have demonstrated a strong correlation between single-cell chl a fluorescence determined flow cytometritally and the whole-cell absorption crosssection. theoretical and empirical advances on the functional relationship between photosynthesis and absorption are continuing; by coupling these advances with information on p,,, per cell, in situ irradiance, and single-cell absorption it will be possible to model in situ production for individual cells. by combining this approach with taxonspecific probes (shapiro et al. 1989) , it should be possible to assess the contribution by specific taxa to primary productivity. rub&o: structure and prospects of improvement the biochemistry of plants. v. 10. academic immune recognition of proteins vostok ice core provides 160,000-year record of atmospheric co the concept of light intensity adaptation in marine phytoplankton: some experiments with phaeoductylum tri-corm&m responses to different quantum flux densities the measurement of ribulose-1,5bisphosphate carboxylase/ oxygenasc concentrations in the leaves of potato plants by enzyme linked immunosorption assays changes in the levels of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) in tetraedron minimum (chlorophyta) during light and shade adaptation culture centre of algae and protozoa list of strains photosynthetic carboxylating enzymes in marine phytoplankton investigation of the chloroplast genome in two marine algae, codium fragile and monodus sp laboratory and field studies on snow algae of the pacific northwest production of reagent antibodies new computer models unify ecological theory temporal and spatial variability of chroococcoid cyanobacteria synechococcus spp. specific growth rates and their contribution to primary production in the sargasso sea loss of radiocarbon in direct use of aquasol for liquid scintillation counting of solutions containing 14c-nahco 198 1. species variation in the specificity of ribulose biphosphate carboxylase/oxygenase effects of growth irradiance and nitrogen limitation of photosynthetic energy conversion in photosystem 2 cleavage of structural proteins during the assembly of the head of bacteriophage t4 a method for the continuous measurement of in vivo chlorophyll concentration. deep-sea res evidence for the existence of discrete activator and substrate sites for co, on ribulose-1 $bisphosphate carboxylase 198 1. the effect of magnesium and other ions on the distribution of ribulose-1,5-bisphosphate in chloroplast extracts ribulose-1,5-bisphosphate carboxylase-oxygenase sensitive visualization of antigen-antibody reactions in dot and blot immune overlay assays with immunogold and immunogold/silver staining structural, functional and evolutionary analysis of ribulose-1,5-bisphosphate carboxylase from the . chromophytic alga olisthodiscus luteus progress in photosynthesis research the regulation of rubisco activity reaction of formaldehyde with calf thymus nucleohistone analysis of synechococcus pigment types in the sea using single and dual beam cytometry probes for assessing single-cell primary production: antibodies against ribulose-1,5-bisphosphate carboxylase (rubpcase) and peridinin/ chlorophyll a protein (pcp) determination of the cross-section absorption coefficient of individual phytoplankton cells by analytical flow cytometry oceanic primary production: estimation by remote sensing at local and regional scales ribulose biphosphate carboxylase from three chlorophyll c-containing algae: physical and immunological characterizations lightshide adaptation by the oceanic dinoflagellate pvrocystis noctiluca and p. fusiformis adaptation of the photosynthetic apparatus of scenedesmus obliques to strong and weak light conditions differences in pigments, photosynthetic capacity, quantum yield and dark reactions practical flow cytometry immunochemical characterization of ultraplankton species molecular evolution of the large subunit of ribulose-1,5-bisphosphate/oxygenase rubisco. fems (fed photoadaptation of carboxylating enzymes and photosynthesis during spring bloom the use of radioactive carbon (c4) for measuring organic production in the sea light-saturated photosynthesis limitation by electron transport or carbon fixation? photosynthesis vs. light intensity measurements: a miniaturized incubator electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications i98 1. production of antisera with small doses of immunogen: multiple intradermal injections isolation of a cell-fusion hormone from gr@hsia pacifica kylin, a red alga measuring the primary production rates of individual phytoplankton species in natural mixed populations. deep-sea res temporal patterns of division in the dinoflagellate genus ceratium and its application to the determination of growth rate key: cord-002013-rb9xdzro authors: zheng, xuexing; wong, gary; zhao, yongkun; wang, hualei; he, shihua; bi, yuhai; chen, weijin; jin, hongli; gai, weiwei; chu, di; cao, zengguo; wang, chong; fan, quanshui; chi, hang; gao, yuwei; wang, tiecheng; feng, na; yan, feihu; huang, geng; zheng, ying; li, nan; li, yuetao; qian, jun; zou, yong; kobinger, gary; gao, george fu; qiu, xiangguo; yang, songtao; xia, xianzhu title: treatment with hyperimmune equine immunoglobulin or immunoglobulin fragments completely protects rodents from ebola virus infection date: 2016-04-12 journal: sci rep doi: 10.1038/srep24179 sha: doc_id: 2013 cord_uid: rb9xdzro recent successes with monoclonal antibody cocktails zmapp(tm) and mil77 against ebola virus (ebov) infections have reignited interest in antibody-based therapeutics. since the production process for monoclonal antibodies can be prolonged and costly, alternative treatments should be investigated. we produced purified equine antisera from horses hyperimmunized with ebov virus-like particles, and tested the post-exposure efficacy of the antisera in a mouse model of infection. balb/c mice were given up to 2 mg of purified equine antisera per animal, at 30 minutes, 1 or 2 days post-infection (dpi), in which all animals survived. to decrease the possibility of serum sickness, the equine antisera was digested with pepsin to generate f(ab′)(2) fragments, with in vitro neutralizing activity comparable to whole immunoglobulin. full protection was achieved with when treatment was initiated at 1 dpi, but the suboptimal protection observed with the 30 minute and 2 dpi groups demonstrate that in addition to virus neutralization, other fc-dependent antibody mechanisms may also contribute to survival. guinea pigs given 20 mg of antisera or f(ab′)(2) at or starting at 1 or 2 dpi were also fully protected from ebov infection. these results justify future efficacy studies for purified equine products in nhps. of these reasons means that there are high personal risks involved with working on ebov in a laboratory setting, and as such it is classified as a biosafety level 4 (bsl-4) agent. in the spring of 2014, a new ebov variant emerged in the west african nation of guinea 4 , an area in which the virus had not been previously reported. the outbreak soon spread to neighbouring sierra leone and liberia. occasionally, cases have been exported into other countries through travel; with countries located in africa, europe and north america all having recorded ebov cases imported by travel, or repatriation of infected citizens. as of mid-december 2015, there are over 11,000 fatalities and 28,000 infections 5 , the largest evd outbreak in history. although the outbreak is now largely under control with no reported cases since the week of november 29 th , 2015 5 , the virus had shown itself at the peak of the outbreak to be extremely resistant to traditional containment methods designed to curb ebov transmission. passive immunotherapy with sera of animal origin has been used for over 120 years to treat bacterial and viral infections, envenomations and drug intoxications. in 2012, a report demonstrated that the passive transfer of igg from nonhuman primate (nhp) survivors of ebov disease to naive nhps was sufficient to confer post-exposure protection against ebov challenge in all animals 6 . building on these findings, cocktails of monoclonal antibodies (mabs) raised against the ebov glycoprotein (gp) were soon shown afterwards to be effective in the treatment of ebov disease 7, 8 . this culminated in the development of zmapp tm , a combination of three mabs produced in genetically modified tobacco plants, which were shown to reverse advanced ebov disease in experimentally-infected nhps 9 , and may have provided a survival benefit when administered to ebov-infected patients 10 . a second antibody cocktail (mil-77), which was based on zmapp tm and produced in modified cho cells, were later shown to have similar efficacy to zmapp tm in nhps 11 . therefore, passive immunotherapy is an extremely promising approach to control ebov disease. due to the ease of management, high antibody yield and low risk of human contamination by virus or adventitious agents, horses are the most commonly used animal species in the production of hyperimmune sera. immunization itself is standardized and performed under optimal conditions for both personnel and animals. passive immunotherapy is still commonly used in countries that are still resource-poor medically, and treatment with immune globulin against rabies virus 12 and clostridium tetani 13 infections remains frequent in africa, asia and latin america. the lower manufacturing costs of hyperimmune equine antisera therefore represents an attractive alternate avenue of treatment, especially to developing and third-world countries, compared to the more costly production process of ebov gp-specific mabs. to investigate further, we first prepared purified anti-ebov antisera via the immunization of horses with ebov enveloped virus-like particles (evlp), which consists of ebov viral protein 40 (vp40) and gp. the equine antisera was then tested in vitro using a neutralization assay with recombinant ebov expressing egfp (ebov-egfp), as well as in vivo against a lethal challenge with mouse-adapted ebov (ma-ebov) in immunocompetent balb/c mice. to investigate whether ebov infections can be controlled by virus neutralization alone, and to prevent the possible induction of serum sickness in humans that would be administered antisera, the post-exposure efficacy of f(ab′ ) 2 (immunoglobulin treated with pepsin to remove the fc regions of the antibody) were also investigated side-by-side with equine antisera in all experiments as a potential alternate treatment. guinea pigs then were used to confirm the efficacy results from mouse studies, due to their status as a higher phylogenic species which more closely models hallmarks of evd in humans. additionally, prior to efficacy studies both the equine antisera and f(ab′ ) 2 had been evaluated for safety in the peking union medical college center for new drug safety evaluation, chinese academy of medical sciences, which is certified by the food and drug agency of the people's republic of china. both equine-derived products were found to meet safety standards for clinical use in china. immunization of horses and production of equine antibody products. the horses were immunized with evlp produced from the infection of sf9 cells with rbv-vp40-gp. the filamentous evlp were observed under an electron microscope (supplementary figure 1 ) and confirmed to be morphologically similar to ebov. immunoblotting of rbv-vp40-gp infected sf9 cell lysates demonstrated that the evlp contained ebov gp and vp40 (supplementary figure 2) . three horses were immunized intramuscularly (im) with 7 injections of evlp over 11 weeks and the hyperimmune sera were collected from each animal at specified timepoints ( fig. 1a) to determine the serum titers by neutralization assay against a recombinant hiv-1 virus pseudotyped with ebov gp. a pseudotyped-virus was used for these studies to confirm the in vitro efficacy of the antisera preparations under biosafety level 2 (bsl-2) conditions, before subsequent studies in the bsl-4 laboratory. neutralizing serum titers were detectable after the 5th immunization at week 6, and increased until the 7th immunization at week 11 (fig. 1b) . the serum neutralizing antibody titers of two horses were 1:40,960 after seven immunizations, and was 1:20,480 for the third animal. the equine antisera (purified by ammonium sulphate-based precipitation) was then digested with pepsin at a concentration of 5-10 u/ml for 45 to 90 min and purified to generate f(ab′ ) 2 fragments. the purity of the antisera and f(ab′ ) 2 preparations, as determined by sds-page followed by thin layer chromatography, was determined to be 95% and 83%, respectively (fig. 2) . approximately 2000 ml of purified antisera could be obtained from each horse during each collection, with the stock concentration between 20-25 mg/ml. up to 3-5 collections can be performed on each horse, therefore each hyperimmunized horse yields between 120 to 250 g of purified antisera. in vitro characterization of equine antisera and f(ab') 2 . the equine antisera and f(ab′ ) 2 products were first characterized against a laboratory generated ebov-egfp for their potential to neutralize ebov. equine antisera and f(ab′ ) 2 were found to possess levels of similar neutralizing activity, with the half effective maximal concentration (ec 50 ) of the products to be 1.07 μg/ml and 2.12 μg/ml, respectively (fig. 3) . moreover, the neutralization activities of both the equine antisera and f(ab′ ) 2 were complete at a concentration of 12.5 μg/ml, scientific reports | 6:24179 | doi: 10.1038/srep24179 indicating that the two products were indistinguishable from each other in terms of neutralization at high concentrations. ebov-specific igg in the equine antisera preparations were also determined by elisa. serial 2-fold the neutralizing activities of equine antisera and f(ab′ ) 2 against ebov-egfp were compared over different concentrations (x-axis). total fluorescence from infected veroe6 cells at 3 dpi were shown as a percentage of the fluorescence observed with the pbs control, which is set at 100% (y-axis). samples were processed in triplicate, and error bars indicate standard error. data shown in this figure are representative of three independent neutralization studies with f(ab′ ) 2 or antisera. dilutions of stock antisera with a concentration of 18-20 mg/ml were assayed in triplicate over three independent experiments, and the titer was determined to be between 10,240 and 20,480 endpoint dilutions. were first assessed in guinea pigs, and found to be 8-12 hours and 3-4 days, respectively (supplementary table 1 ). the protective efficacy of equine antisera and f(ab′ ) 2 were assessed over three experiments in balb/c mice. the first experiment was to compare the efficacy of antisera and f(ab′ ) 2 treatments side-by-side. groups of 8 mice were given intraperitoneal (ip) injections of f(ab′ ) 2 at 200 μg per dose, twice daily for 3 days (b.i.d. × 3d), starting at either 1 or 2 days post-infection (dpi) with a uniformly lethal dose of ma-ebov. for comparison, groups of 8 mice were administered an ip injection of equine igg at 200 μg per dose (q.d. × 1d), at either 1 or 2 dpi. control mice were given an equal volume of pbs in place of the treatment (q.d. × 1d) at 1 dpi. pbs was used instead of non-specific immunoglobulin or f(ab′ ) 2 , because previous studies involving passive transfer of non-specific antisera in mice and nhps did not result in protection 14, 15 , and thus survival due to the non-specific effects of serum proteins is considered unlikely. control mice lost approximately 23% of its body weight over the course of the experiment and none survived, with a mean time to death (mtd) of 6.4 ± 0.5 dpi. five of eight mice given f(ab′ ) 2 starting at 1 dpi survived (p-value < 0.0001, compared with pbs group), with an average weight loss of 18.7% and a mtd of 8.3 ± 0.6 dpi; however, none of the mice given f(ab′ ) 2 starting at 2 dpi survived the challenge (p-value = 0.9777, compared with pbs group), with an average weight loss of 19.0% and a mtd of 5.8 ± 1.8 dpi (fig. 4a,b) . five of eight mice given antisera at 1 dpi survived (p-value < 0.0001, compared with pbs group), with an average weight loss of 14.6% and a mtd of 8.3 ± 0.6 dpi (fig. 4c ). three of eight mice given antisera at 2 dpi survived (p-value = 0.0446, compared with pbs group), with an average weight loss of 12.3% and a mtd of 6.6 ± 0.6 dpi (fig. 4d ). comparing groups with equal treatment times, there was no statistical difference between f(ab′ ) 2 at 1 or 2 dpi (p-value 1.0000 and 0.0613, respectively). however, given that multiple administrations of f(ab′ ) 2 were required to achieve similar protection levels demonstrated by a single injection of antisera, the results suggest that equine antisera is a superior product to f(ab′ ) 2 in terms of efficacy, possibly due to a longer in vivo half-life of equine antiseras. since f(ab′ ) 2 appear to be promising early in ma-ebov infection, the dosage of this treatment was increased for a second experiment. groups of 9-10 mice were given ip injections of f(ab′ ) 2 at 1 or 2 mg per dose, twice daily for 3 days (b.i.d. × 3d), starting at either 30 minutes or 1 dpi with ma-ebov. control mice were treated with pbs (q.d. × 1d) at 1 dpi. control mice lost approximately 17.3% of its body weight over the course of the experiment and none survived, with a mtd of 6.3 ± 1.0 dpi. partial survival was observed when treatment began 30 minutes after challenge. four of nine mice in the 1 mg group survived (p-value < 0.0001, compared with pbs group), with an average weight loss of 6.8% and a mtd of 11.6 ± 1.5 dpi, whereas four of ten mice in the 2 mg group survived (p-value < 0.0001, compared with pbs group), with an average weight loss of 6.2% and an mtd of 12.5 ± 1.4 dpi (fig. 5a,b) . in contrast, all mice survived if treatment was initiated at 1 dpi, with negligible weight loss (less than 5%) observed in both the 1 and 2 mg groups (p-value < 0.0001, compared with pbs group), indicating that protection was complete. these results indicate that f(ab′ ) 2 can contribute to protection from ma-ebov, but only within a certain timeframe after challenge. the efficacy of equine antisera at higher doses was then investigated in a third experiment. groups of 10 mice were given ip injections of antisera at 2 mg per dose (q.d. × 1d), at 30 minutes, 1 or 2 dpi with ma-ebov. control mice were treated with pbs (q.d. × 1d) at 1 dpi. control mice lost approximately 17.3% of its body weight over the course of the experiment and none survived, with a mtd of 6.3 ± 1.0 dpi. all mice treated with antisera at 30 minutes and at 1 dpi survived with negligible weight loss (p-value < 0.0001, compared with pbs group). nine of ten mice treated with antisera at 2 dpi also survived the challenge (p-value < 0.0001, compared with pbs group), with the lone non-survivor dying at 7 dpi and the average peak weight loss determined to be 7.9% within this treatment group (fig. 6a,b) . these results again indicate that equine antisera, compared to f(ab′ ) 2 is able to offer a greater contribution to protection from ma-ebov in the mouse model due to the need for fewer administrations to achieve a similar level of protection. guinea pigs were then used to confirm the protective effects from antisera and f(ab′ ) 2 in a higher phylogenic species for ebov challenge, since these animals better mimic hallmarks of human ebov infections, such as coagulation abnormalities 16 . groups of 6 animals were given a single ip injection of antisera (q.d. × 1d) starting at 1 or 2 dpi with ga-ebov, or twice-daily ip injections of f(ab′ ) 2 for three days (b.i.d. × 3d), starting at 1 or 2 dpi. each injection dose contained 20 mg antisera or f(ab′ ) 2 . a group of five control animals were given a single injection of pbs at 1 dpi. control guinea pigs lost approximately 17.9% of its body weight over the course of the experiment and none survived, with a mtd of 8.2 ± 0.6 dpi. all guinea pigs that were given antisera at 1 or 2 dpi survived (p-values = 0.0011 for both groups, compared with pbs group), with no clinical signs of disease or significant weight loss (fig. 7a,b) . in addition, animals that were given f(ab′ ) 2 starting at 1 or 2 dpi survived ga-ebov challenge (p-values = 0.0011 for both groups, compared with pbs group) without signs of disease or significant weight loss (fig. 7a,b) . the disparity between the efficacy of f(ab′ ) 2 between mice and guinea pigs may be attributed to a much higher dosage of f(ab′ ) 2 administered per weight: each f(ab′ ) 2 dose for guinea pigs was 100× higher than those given to mice, but the guinea pigs used in this experiment were only 10-20 times bigger than the mice by weight. the lack of bsl-4 laboratories globally, trained personnel as well as the rigors of working under high biocontainment conditions have severely hampered basic research with ebov, leading to a dearth of vaccines and therapeutics for use in humans. these weaknesses were exposed with the 2014-15 ebov crises, and highlight the value and need for basic and translational science that occurs prior to an impending threat. in an effort to save lives, several experimental candidate treatments that had been tested for efficacy in mice or nhps, in addition to approved or nearly-approved drugs developed against unrelated pathogens, were expedited for use in humans under compassionate circumstances, with varying degrees of success 17 . of these, antibody-based treatments appear to hold the most promise in the clinic and thus should be further investigated for use in humans. passive immunotherapy against ebov had been tried in the past. towards the end of the 1995 ebov outbreak in kikwit, zaïre (presently democratic republic of the congo), the passive transfer of whole blood from ebov survivors to patients resulted in the survival of seven out of eight recipients 18 . however, safety concerns with blood transfusions, such as the spreading of blood-borne diseases, allergic reactions and concerns of inefficacy 19 mean that this approach is controversial and will likely not be used unless a better alternative was unavailable. in the 1990s, russian investigators had prepared hyperimmunized antisera from horses vaccinated with inactivated ebov, which were shown to be effective when administered at a dose of 1 mg/kg to baboons, with 100% survival (2 survivors out of 2) when given 2 hours before challenge, and 67% (2 survivors out of 3) when given immediately after challenge 20 , although baboons are known to be more resistant to ebov infection compared to other nhp species 21 . the survival benefits of the equine antisera were limited to a delay in the onset of viremia clinical symptoms when tested in cynomolgus macaques immediately after challenge, at a dose of 60 mg for an animal weighing between 5-6 kg 22 . comparing to successful passive immunotherapy studies with purified igg from nhps (80 mg/kg), as well as zmapp and mil-77 (50 mg/kg), the dosage for the cynomolgus macaque experiment was likely too low at the time. use of equine antisera as an emergency post-exposure treatment against ebov has been approved in russia, and several investigators that had been accidentally exposed to the virus have been administered this treatment, although it is not clear if they had actually been infected 23 . the results of this study show that both equine antisera and f(ab′ ) 2 preparations, which can be rapidly produced in large quantities at a lower cost compared to mabs, are effective in the post-exposure treatment of ma-ebov challenged mice and ga-ebov infected guinea pigs. f(ab′ ) 2 was developed in this study due to past documented issues with antisera administrations, in which equine botulinum toxin and anti-snake venom antisera was shown to produce serum sickness at high doses 24 . however, mice that were given a single dose of antisera demonstrated more complete protection over a greater period of time compared to multiple injections of f(ab′ ) 2 . this suggests that f(ab′ ) 2 has a shorter half-life compared to igg (which has been confirmed in this study), and/or that virus neutralization plays a partial role in survival from ebov. the observation that administering f(ab′ ) 2 at 1 dpi is more efficacious than when the same treatment was given at 30 minutes post-exposure (fig. 5a ) was also observed in past studies with therapeutic ebov gp-specific monoclonal antibodies 25, 26 , and suggests that virus neutralization may play a bigger role in protection at a later timepoint after ebov challenge. with regards to neutralization, past reports have shown that while elevated antisera levels in nhps vaccinated against ebov correlated with survival, levels of specific neutralizing antibodies did not 27 . it is a possible explanation as to why mab kz52, which was originally selected for its ability to neutralize ebov 28 , failed to protect nhps when given at a dose of 50 mg/kg starting 1 day before challenge 29 , and suggests that in the future, the screening of potentially efficacious antibodies against ebov should not be based solely on the ability of the antibody to neutralize virus. fc-dependent antibody functions, which include antibody dependent cellular cytotoxicity, complement-dependent cytotoxicity, and neonatal fragment crystallizable receptor (fcr)-mediated cross-presentation, likely play a role in protection. for instance, studies in mice against yellow fever virus and west nile virus infections have shown that the protective mechanisms of monoclonal antibodies are dependent on fcr 30, 31 . furthermore, complement component c1q has been previously shown in influenza virus and west nile virus infections to directly enhance the neutralizing activities of antibodies 32, 33 . these mechanisms are not mutually exclusive, but determining their relative importance of each proposed mechanism to efficacy and survival will yield further insight into the specific mechanisms in which antibodies help protect against ebov disease. furthermore, the findings of this study justify the testing of equine igg in a higher phylogenic species for ebov, such as nhps, and may result in the development of a safe and economical method for the production of an effective ebov therapeutic. ethics statement. mice animals. balb/c mice and hartley guinea pigs were purchased from a commercial supplier (charles river). the animals were kept in sterile, autoclaved cages and provided sterilized food and water ad libitum. animals were also provided red, plastic shelters inside the cages as an added source of stimulation. horses were purchased from a commercial supplier (red hill military horse farm) and provided food and water ad libitum. the sequences of ebov gp and vp40, with lengths of 2,031 and 981 bp, respectively, were derived from genbank (zaire ebolavirus strain zaire 1995, complete genome; genbank accession no. ay354458). the gp and vp40 genes were cloned into the puc-57 vector, named puc-gp and puc-vp40, and then inserted into pfastbac dual vector (life technologies) under the polyhedrin promoter and p10 promoter respectively, resulting in plasmid pfastbac dual-vp40-gp. the purified plasmid was transformed into dh10 ™ bac e. coli (life technologies) for transposition into a bacmid. a cellfection ® ii reagent (life technologies) was used according to manufacturer instructions to generate recombinant baculoviruses co-expressing ebola vp40 and gp (rbv-vp40-gp). evlp production and inspection. to produce evlp, sf9 cells were infected with rbv-vp40-gp at a multiplicity of infection (moi) of 1, and incubated at 27 °c for 4 days. culture supernatants were harvested and centrifuged at 2000 × g for 30 min to remove cells, and then pelleted by ultracentrifugation at 30,000 × g for 60 min at 4 °c. the pellets were re-suspended in pbs and purified through a 10-30-50% discontinuous sucrose gradient at 25,000 × g for 90 min at 4 °c. the evlp band obtained between 30% and 50% density range was collected, washed, and re-suspended in pbs 34 . for immunoblotting, evlp and control sample (cell culture supernatant) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing conditions, transferred onto a polyvinylidene fluoride (pvdf) membrane (whatman, kent, uk) and then probed with mouse anti-ebov gp polyclonal antibody (prokaryotic expression gp protein immunized mice) or mouse anti-ebov vp40 monoclonal antibody (ab1918, abcam) at a dilution of 1:200 overnight at 4 °c. the sample was then incubated with horseradish peroxidase (hrp)-conjugated goat anti-mouse secondary antibody at a dilution of 1:4000 (millipore, boston, ma, usa) for 60 min at 37 °c. the pvdf membrane was colored with a chemiluminescence solution (pierce biotechnology, rockford, il, usa). for electron microscopy, evlp were applied onto a carbon-coated formvar grid, which was immediately stained with 1% phosphotungstic acid and then observed by a transmission electron microscope. horse immunizations. horses (n = 3) were vaccinated intramuscularly (subcutaneous multi-point injection) with either 3 or 5 mg of evlps emulsified in freund's complete adjuvant/freund's incomplete adjuvant at weeks 0, 2, 4, 5, 6, 7 and 9 (7 times). blood samples were collected from the jugular vein at 1 week prior to the first immunization and 1 week after each immunization, and the sera were stored at − 80 °c until further analysis. fragmentation and purification of equine antibody products. the equine antisera and f(ab′ ) 2 were produced at changchun institute of biological products co., ltd., using the large-scale, current good manufacturing practice compatible equine antiserum manufacturing platform. equine antisera and f(ab′ ) 2 preparations were characterized according to guidelines set forth by chinese pharmacopoeia (2010 edition), including appearance, color, visible foreign matter, ph value, f(ab′ ) 2 and immunoglobulin content, and sterility. for equine antisera, the blood was taken from the jugular vein of immunized horses, and the sera were separated 2 h later. the horse sera were diluted 8-fold with pbs, centrifuged at 12,000 rpm for 30 min, and supernatants were subjected to filtration through a 0.45 μm filter. the antisera were purified by ammonium sulphate precipitation 35 , followed by salt column, and then stored in 0.9% nacl solution. for f(ab′ ) 2 : horse sera were diluted 3-fold and the ph was adjusted to 3.2 using 1 m hcl, and then the antisera were subjected to shock digestion at 37 °c for 30 min with pepsin, and 0.4 m naoh was added to terminate digestion. the digestion products were subjected to salt column purification, followed by protein a column. the flow-through fluid was harvested and subjected to ultrafiltration to remove the pepsin and small molecular proteins, and the f(ab′ ) 2 was stored in 0.9% nacl solution. sds-page and thin layer chromatography. the purified equine antisera and f(ab′ ) 2 samples were mixed with non-reducing (without β -mercaptoethanol) protein sample buffer, heated at 95 °c for 5 min, and then subjected to sds-page (12% gel, staining for 3 h and destaining for 2 h); and then the target fractions in the gel were examined by thin layer chromatography scanner (cs-9301, shimadzu), (transmission, zigzag scan, dual wavelength, swing width: 8 mm, delta y: 0.1 mm) to determine the purity of equine antisera and f(ab′ ) 2 . elisa. ebov gp expressed by e. coli bl-21 was diluted with 0.1 m bicarbonate buffer ph 9.6 to a final concentration of 10 μg/ml, and coated on 96-well elisa plates overnight at 4 o c (100 μl/well). after blocking with angiotensin converting enzyme (ace) buffer (bio-rad, california, usa) at 37 °c for 2 h, the plates were incubated with 100 μl of serial 2-fold dilutions of stock equine antisera in triplicate at 37 °c for 1.5 h. after washing, 100 μl of hrp-conjugated goat anti-horse igg (comwin biotech co., ltd. beijing, china) diluted 1:20000 in pbs-0.5% tween was added to the wells and incubated at 37 °c for 50 min. after washing, 100 μl of substrate 3′,5,5′ -tetramethylbenzidine (tmb, sigma) was added and incubated at room temperature for 30 min. the reaction was stopped by adding 50 μl of 0.5 m h 2 so 4 , and the absorbance was measured at 450 nm. a dilution was considered positive if the absorbance reading was at least twice that of the negative control (pbs) at the same dilution. the igg endpoint titer was calculated as the highest dilution still showing a positive result. the assay was performed independently three times. half-life studies in guinea pigs. group of 4 guinea pigs were administered an ip injection of 1 ml purified equine antisera (neutralization titer 1:20480) or f(ab′ ) 2 (neutralization titer 1:20480). blood samples were collected at 0, 1, 4, 8, 12, 24, 48, 72, 96, 120, 144 h post-injection, and sera were used for determination of neutralization titer against a recombinant hiv-1 virus pseudotyped with ebov gp. the time range post-infection in which the titer decreases by 50% is considered the half-life. each sample was performed in triplicate. determining the protective efficacy of equine antisera. balb/c mice, 4-6 week old, female and weighing between 15-19 g, were randomly assigned into groups of 8-10 mice. all animals were challenged intraperitoneally (ip) with a dose of 1000 × ld 50 mouse-adapted ebov (ma-ebov, usamriid/balb/c-lab/ cod/1976/mayinga-ma-p3) in 200 μl dmem. the treatment was given ip (q.d. × 1d) at 1 or 2 days post-infection (dpi) with 0.2 mg equine anti-ebov antisera per mouse, or in a subsequent experiment, given ip at 30 minutes, 1 or 2 dpi with 2 mg equine anti-ebov antisera per mouse. the control group was given the same volume of pbs as mock treatment. all animals were monitored for signs of disease, survival and weight change for 16 days, and survival was monitored for 12 additional days. female strain hartley guinea pigs, 4-6 week old and weighing between 200 and 300 g, were randomly assigned into groups of 6 animals. all animals were challenged ip with 1000 × ld 50 guinea pig-adapted ebov (ga-ebov, vector/c.porcellus-lab/cod/1976/ mayinga-gpa-p7) in 1 ml dmem. the treatment was given ip (q.d. × 1d) at 1 or 2 dpi with 20 mg equine anti-ebov igg per animal. a control group of 5 guinea pigs were given the same volume of pbs as mock treatment. all animals were monitored for signs of disease, survival and weight change for 15 days, and survival was monitored for 13 additional days. determining the protective efficacy of f(ab′) 2 . balb/c mice, 4-6 week old, female and weighing between 15-19 g, were randomly assigned into groups of 8-10 mice. all mice were challenged ip with a dose of 1000 × ld50 ma-ebov in 200 μl dmem. the treatment was given ip at 1 or 2 dpi with 200 μg f(ab′ ) 2 per mouse, or in a subsequent experiment, given ip at 30 minutes, 1 or 2 dpi with either 1 mg or 2 mg of f(ab′ ) 2 per mouse. the treatment was given via ip twice a day for 3 days (b.i.d. × 3d). the control group was given the same volume of pbs as a mock treatment. all animals were monitored for signs of disease, survival and weight change for 16 days, and survival was monitored for 12 additional days. female strain hartley guinea pigs, 4-6 week old, were randomly assigned into groups of 6 animals. all animals were challenged ip with 1000 × ld 50 guinea pig-adapted ebov in 1 ml dmem. the treatment was given ip at 1 or 2 dpi with 20 mg f(ab′ ) 2 per animal. the treatment was given via ip twice a day for 3 days (b.i.d. × 3d). a control group of 5 guinea pigs were given the same volume of pbs as mock treatment. all animals were monitored for signs of disease, survival and weight change for 15 days, and survival was monitored for 13 additional days. pseudotyped virus neutralization assay. titers of equine antisera and f(ab′ ) 2 from horses was tested in an neutralization assay in huh-7 cells against recombinant hiv-1 virus pseudotyped with ebov gp. the method for generating pseudotyped viruses was described in a previous publication 36 . briefly, pseudotyped virus containing supernatants were incubated either with serially diluted horse sera at 37°c for 1 h, before addition to pre-plated target cells in 96-well culture plates (density of 10 4 cells/well). cells were re-fed fresh medium 4 h after addition, and followed by lysing cells at 48 h using cell lysis buffer (promega) and transferring the lysates into 96-well luminometer plates. luciferase substrate (promega) was added to the plates, and the relative luciferase activity was determined. the inhibition of pseudotyped was presented as % inhibition. the highest serum dilution giving over 50% reduction of luciferase activity was regarded as the neutralizing antibody titer. ebov-egfp neutralization assay. serial two-fold dilutions of f(ab′ ) 2 or antisera (between 12.5 to 0.097656 μg/ml) were incubated with 100 plaque forming units (pfu) of ebov expressing egfp (ebov-egfp, nml/h.sapiens-lab/cod/1976/mayinga-egfp-p3) at 37 °c for 1 h, transferred to vero e6 cells and incubated at 37 °c for 1 h, and then replaced with dmem supplemented with 2% fetal bovine serum. control wells contained pbs instead of f(ab′ ) 2 or antisera, and all samples were repeated in triplicate. the plates were fixed with 10% phosphate buffered formalin at 72 h after infection, and scored for the intensity of egfp using the biotek synergy ht microplate reader. the results were expressed as a percentage of the fluorescence reading with the control (which is set at 100%), and fitted to a 4-parameter logistic curve (graphpad). statistical analysis. the p-values for rodent studies were determined using the log-rank (mantel-cox) test. calculated values of less than 0.05 were considered statistically significant. all in vivo studies were performed once. outbreaks chronology: ebola virus disease clinical management of filovirus-infected patients organization of patient care during the ebola hemorrhagic fever epidemic in kikwit, democratic republic of the congo emergence of zaire ebola virus disease in guinea ebola situation report -16 postexposure antibody prophylaxis protects nonhuman primates from filovirus disease delayed treatment of ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques successful treatment of ebola virus-infected cynomolgus macaques with monoclonal antibodies reversion of advanced ebola virus disease in nonhuman primates with zmapp clinical care of two patients with ebola virus disease in the united states two-mab cocktail protects macaques against the makona variant of ebola virus use of a reduced (4-dose) vaccine schedule for postexposure prophylaxis to prevent human rabies passive transfer of antibodies protects immunocompetent and imunodeficient mice against lethal ebola virus infection without complete inhibition of viral replication postexposure antibody prophylaxis protects nonhuman primates from filovirus disease animal models for ebola and marburg virus infections backs against the wall: novel and existing strategies used during the 2014-2015 ebola virus outbreak treatment of ebola hemorrhagic fever with blood transfusions from convalescent patients. international scientific and technical committee from whole blood to component therapy: the economic, supply/demand need for implementation of component therapy in sub-saharan africa disease modeling for ebola and marburg viruses passive immunization of ebola virus-infected cynomolgus monkeys with immunoglobulin from hyperimmune horses defense against filoviruses used as biological weapons hypersensitivity reactions associated with botulinal antitoxin epitopes involved in antibody-mediated protection from ebola virus ebola gp-specific monoclonal antibodies protect mice and guinea pigs from lethal ebola virus infection. plos neglected tropical diseases 6, e1575 immune parameters correlate with protection against ebola virus infection in rodents and nonhuman primates ebola virus can be effectively neutralized by antibody produced in natural human infection neutralizing antibody fails to impact the course of ebola virus infection in monkeys the fc portion of antibody to yellow fever virus ns1 is a determinant of protection against yf encephalitis in mice antibodies against west nile virus nonstructural protein ns1 prevent lethal infection through fc gamma receptor-dependent and -independent mechanisms natural antibody and complement mediate neutralization of influenza virus in the absence of prior immunity complement protein c1q reduces the stoichiometric threshold for antibody-mediated neutralization of west nile virus ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies a quantitative immunochemical measure of the primary interaction between i bsa and antibody structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the s protein of middle east respiratory syndrome coronavirus x.z., g.w., y.z., h.w., s.h., y.b., x.q., s.y. and x.x. performed the experiments and analyzed the results. x.z., g.w., g.k., g.f.g., x.q., s.y. and x.x. wrote the manuscript. w.c., h.j., w.g., d.c., z.c., c.w., q.f., h.c., y.g., t.w., n.f., f.y., g.h., y.z., n.l., y.l. and j.q. prepared the horse antisera. g.f.g., x.q., s.y. and x.x. jointly supervised the work. all authors reviewed the manuscript. key: cord-305648-majanu8l authors: schountz, tony; calisher, charles h.; richens, tiffany r.; rich, audrey a.; doty, jeffrey b.; hughes, mark t.; beaty, barry j. title: rapid field immunoassay for detecting antibody to sin nombre virus in deer mice date: 2007-10-17 journal: emerg infect dis doi: 10.3201/eid1310.070383 sha: doc_id: 305648 cord_uid: majanu8l we developed a 1-hour field enzyme immunoassay (eia) for detecting antibody to sin nombre virus in deer mice (peromyscus maniculatus). the assay specificity and sensitivity were comparable to those of a standard eia. this test will permit identification of rodents with antibody to this and perhaps other hantaviruses. horseradish peroxidase enzyme-linked immunosorbent assay (pageia) to detect antibodies to snv in deer mice (7) . the test can be completed in ≈1 hour under relatively primitive fi eld conditions. the assay has advantages over more laborious assays used for similar purposes and, because it is mammal-specifi c rather than species-specifi c, we expect this assay will be applicable to serologic tests of mammals of many other species. a fragment of the s segment (nt 43-394) encoding part of the nuclecapsid was cloned into pet21b with a c-terminal his tag to produce a 15-kda truncated antigen (8) for use in the assay. deer mice were trapped near fort lewis, colorado, and blood was collected as previously described (9) ; whole blood was diluted in (1:100) 1 ml of phosphate-buffered saline (pbs) in 96 deep-well plates (p-dw-11-c, axygen, union city, ca, usa) at time of collection to expedite sample loading. the remainder of the blood was frozen on dry ice and returned to the laboratory for additional testing. wells of 96-well polyvinyl chloride plates (falcon 353912, bd biosciences, san jose, ca, usa) were coated with 100 μl of 2 μg/ml recombinant nucleocapsid in pbs and blocked (0.25% gelatin in pbs) a week in advance. wells were washed in the fi eld 3× with 200 μl of pbs (ph 7.0) by using an 8-channel pipettor, and blood in pbs was added from the deep well plate; positive and negative (1:100) controls (diluted in pbs) were included. plates then were incubated at ambient temperature (range ≈23°c-29°c) for 30 min. after 3 more washes with pbs/0.5% tween-20, 100 μl of pretitrated staphylococcal protein-a/streptococcal protein-g horseradish peroxidase conjugate (pierce biotechnology, inc., rockford, il, usa) diluted 1:1,000 in pbs was added for 30 min. plates again were washed 3× with pbs-tween-20, and 100 μl of activated abts substrate was added to each well. after 15 min of incubation at ambient temperature, wells were scored by using a 0-4+ system, with 0 indicating no reaction (i.e., clear, no color) and 4+ representing the strongest signal (i.e., dark green color). samples deemed 1+, 2+, 3+, or 4+ were considered positive (very weak, weak, strong, very strong, respectively). samples were retested under laboratory conditions with pageia and standard centers for disease control and prevention (cdc) enzyme immunoassay (eia) (5) . blood samples from 222 deer mice were collected during 3 trapping sessions in the summer of 2006, and 39 samples were scored as positive in the fi eld by pageia; 183 were negative by the fi eld pageia, repeat laboratory pageia, and the standard eia in the laboratory. one sample (ha-2564) was scored negative by fi eld and laboratory pageia, but (low) positive (optical density [od] of 0.327) by conventional eia (table) . of the 39 samples that were scored positive in the fi eld, 5 discrepancies between these and laboratory tests were found (table) . one sample (ts-0830-7) scored as 1+ in the fi eld was determined to be negative on subsequent laboratory testing by both pageia and conventional eia. the other 4 samples (hb-2628, ha-2609, ha-2616, hb-2710) were scored as positive by fi eld and laboratory pageia but negative by conventional eia. in the fi eld, each of these samples was scored as 1+ or 2+ and had ods of 0.331-0.664 by laboratory pageia. however, ods ranged from 0.076 to 0.228 by conventional eia. †field scores were based upon visual inspection without instrumentation, with 0 as negative, 1+ as very weak, 2+ as weak, 3+ as strong, and 4+ as very strong, relative to positive and negative control samples. ‡field-collected samples were retested by pageia under laboratory conditions and the od reported here. the instrument was blanked on the negative control sample. §od >0.200 above the negative control was considered positive. ¶for laboratory eia, od was recorded for diluted (1:100) blood with both snv antigen (numerator) and control antigen (denominator). #a sample tested with snv antigen and having an od >0.300 was considered positive if the od of that sample with the control antigen was <0.150. in regard to sample hbv-2717, the od of the laboratory eia with antigen was 0.293, very near the acceptable minimum, and the background was 0.027, which is very low; this sample was considered provisionally positive. the pageia results were similar to results of conventional eia, with a specifi city of 82.9% (184 negatives/222 total rodents) versus 84.7% (188/222) for conventional eia. the sensitivity of the pageia was 97.1% (34 positive by pageia/35 positive by conventional eia). we have modifi ed an existing serologic assay so that it is suitable for use in the fi eld. the assay relies on a staphylococcal protein-a and streptococcal protein-g horseradish peroxidase conjugate (10) . each protein has the capacity to bind to the fc portions of antibodies, including immunoglobulin m (igm) and iga for protein a (11, 12) , but has highest affi nity for igg subclasses of many mammalian species. all samples scored 3+ or 4+ were also positive in laboratory tests when results were read by using a spectrophotometer. thus, we are confi dent that such samples in the fi eld will indicate seropositive animals. because we are suggesting that this assay be used for identifying seropositive rodents and not for determining seroprevalence (although it appears to be adequate for those studies as well) and to be conservative, we considered only samples that appeared dark green (3+ and 4+) in the fi eld assay to be positive with relative certainty. to minimize the complexity of the pageia under fi eld conditions, we did not use a negative control antigen to assess nonspecifi c reactivities of serum samples. use of this test will allow deer mice with antibody to snv to be identifi ed. deer mice are the population most likely to be naturally infected with that virus, and those rodents can be retained for further testing and for studies of tissues, live cells, and body fl uids to be used for subsequent laboratory investigations, such as for determining cellular immunologic responses, viremia levels, viruria levels, and virus shedding in excreta and secreta. additional limitations of the pageia are similar to those of other serologic tests. pageia can detect only seropositivity, which is not necessarily indicative of current infection or of current shedding of virus. it also binds only with high affi nity to igg; thus, it is not useful for discriminating other immunoglobulin classes, such as igm, the presence of which usually indicates recent infection. because of the broad mammalian species specifi cities of a protein-a and protein-g conjugate, the rapid pageia likely can be used to test for antibodies to other antigens in other mammals. lee et al. (7) characterized the reactivities of protein a and protein g with igg from rodents of several species. they found that serum specimens from both sigmodontine rodent species (deer mice and hispid cotton rats, sigmodon hispidus) they tested were recognized by protein-a and/or protein-g conjugates. similar laboratorybased pageias have also been used to detect antibody to antigens of agents causing other infectious diseases, including severe acute respiratory syndrome coronavirus-like viruses and nipah virus in bats (13) (14) (15) . hantaviruses: a global disease problem serologic and genetic identifi cation of peromyscus maniculatus as the primary rodent reservoir for a new hantavirus in the southwestern united states experimental infection model for sin nombre hantavirus in the deer mouse (peromyscus maniculatus) persistent sin nombre virus infection in the deer mouse (peromyscus maniculatus) model: sites of replication and strand-specifi c expression utilization of autopsy rna for the synthesis of the nucleocapsid antigen of a newly recognized virus associated with hantavirus pulmonary syndrome rapid and specifi c detection of sin nombre virus antibodies in patients with hantavirus pulmonary syndrome by a strip immunoblot assay suitable for fi eld diagnosis detection of antibody for the serodiagnosis of hantavirus infection in different rodent species purifi cation and characterization of the sin nombre virus nucleocapsid protein expressed in escherichia coli natural history of sin nombre virus in western colorado antibodies: a laboratory manual differential binding of iga proteins of different subclasses and allotypes to staphylococcal protein a crystal structure of a staphylococcus aureus protein a domain complexed with the fab fragment of a human igm antibody: structural basis for recognition of b-cell receptors and superantigen activity severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats fruit bats as reservoirs of ebola virus nipah virus in lyle's fl ying foxes we thank brian hjelle for helpful discussions on the manuscript.funding was provided by nih contract ai25489 (to t.s., c.h.c., and b.j.b.) and grant ai054461 (to t.s.). c.h.c. was also partially funded by cdc, contract us3/ccu 813420-09. beta beta beta biological honor society and the university of northern colorado also provided support (to a.a.r. and t.s., respectively).dr schountz is an assistant professor of microbiology in the school of biological sciences at the university of northern colorado. his research interest is the immunologic basis of persistence of zoonotic agents. key: cord-001732-4eyn7pjq authors: riede, o; seifert, k; oswald, d; endmann, a; hock, c; winkler, a; salguero, f j; schroff, m; croft, s l; juhls, c title: preclinical safety and tolerability of a repeatedly administered human leishmaniasis dna vaccine date: 2015-04-30 journal: gene ther doi: 10.1038/gt.2015.35 sha: doc_id: 1732 cord_uid: 4eyn7pjq the leishmaniases are a complex of vector-borne diseases caused by protozoan parasites of the genus leishmania. leishdnavax is a multi-antigen, t-cell epitope-enriched dna vaccine candidate against human leishmaniasis. the vaccine candidate has been proven immunogenic and showed prophylactic efficacy in preclinical studies. here, we describe the safety testing of leishdnavax in naive mice and rats, complemented by the demonstration of tolerability in leishmania-infected mice. biodistribution and persistence were examined following single and repeated intradermal (i.d.) administration to rats. dna vectors were distributed systemically but did not accumulate upon repeated injections. although vector dna was cleared from most other tissues within 60 days after the last injection, it persisted in skin at the site of injection and in draining lymph nodes. evaluation of single-dose and repeated-dose toxicity of the vaccine candidate after i.d. administration to naive, non-infected mice did not reveal any safety concerns. leishdnavax was also well tolerated in leishmania-infected mice. taken together, our results substantiate a favorable safety profile of leishdnavax in both naive and infected animals and thus, support the initiation of clinical trials for both preventive and therapeutic applications of the vaccine. the leishmaniases are a complex of diseases caused by protozoan parasites of the genus leishmania with up to 1.6 million cases reported worldwide annually 1 and~350 million people at risk to develop leishmaniasis. 2 it is estimated that the most severe form, visceral leishmaniasis (vl), causes up to 40 000 deaths per year. 1 measures to control transmission of the parasite were of limited success to date. despite advances in treatment of vl over the last decade, drug toxicity and heat stability, difficult routes of administration and variation in drug efficacy between endemic areas remain issues to be fully solved. a preventive and therapeutic leishmaniasis vaccine prospectively represents the most cost-effective and successful measure to control the leishmaniases worldwide. 3 immunity to the intracellular parasite leishmania is associated with t-cell-mediated immune responses. 4 dna vaccines are particularly able to deliver antigens into the major histocompatibility complex class i processing pathway, thereby inducing cd8 cytotoxic t-cell immune responses, 5, 6 necessary for clearance of leishmania. in addition, cd4 t cells and b cells are activated by dna vaccines. hence, this vaccination technology is a promising approach for developing a leishmaniasis vaccine. 7 an appropriate preclinical safety profile of a vaccine candidate has to be established prior to initiation of clinical phase 1 studies. the us food and drug administration (fda) and the world health organization recommend the evaluation of distribution and persistence of dna vaccines as well as their local reactogenicity and systemic toxicity. 8, 9 results of corresponding studies suggest that dna vaccines are safe, 10, 11 which has been confirmed in several clinical trials. 12 however, immunogenicity of dna vaccines was often modest in humans, necessitating better delivery methods and improved vaccine antigen expression. 12 in addition, safety concerns prompted avoidance of antibiotic resistance genes and other selection markers for plasmid propagation. 13 state-of-the-art plasmids are consequently more efficacious, safer and smaller than classical plasmids. 14 minimalistic immunogenically defined gene expression (midge) vectors represent one of the most rigorous concepts for improvement. 15 midge vectors are small linear double-stranded dna (dsdna) molecules, solely containing sequences required for their function in vivo and no bacterial plasmid backbone sequences as they have been shown to negatively impact transgene expression and immunogenicity. 16, 17 midge-th1 vectors are midge vectors with a short peptide (pkkkrkvedpyc) covalently attached, enhancing the immune responses to encoded antigens. 18, 19 recently, preclinical data on biodistribution and toxicity of midge and midge-th1 vectors have been published, 20, 21 indicating an excellent safety profile after a single administration. leishdnavax is a mixture of five midge-th1 vectors each encoding a different leishmania antigen: kmp11, cpa, cpb, p74 (elongation factor 1-alpha) or tsa. 22 antigen selection and sequence design followed a novel rational approach. in a series of animal experiments it was demonstrated that leishdnavax is immunogenic and effective against challenge with leishmania donovani, the causative agent of vl. 22 the vaccine is aimed to be administered to humans in both preventive and therapeutic settings. here, we present comprehensive preclinical safety and tolerability data for leishdnavax. a repeated-dose toxicity study in naive mice and a biodistribution and persistence study in rats were performed. moreover, the effect of leishdnavax administration on parasite burden and standard tolerability parameters in mouse models of vl were evaluated. in all studies, potential cumulative effects of repeated dosing of leishdnavax were addressed to allow for a better risk-assessment prior to multi-dose clinical trials. leishdnavax is systemically distributed after intradermal administration biodistribution of the vaccine was assessed in rats after intradermal (i.d.) injection of a 120 μg dose. total dna was extracted from tissue samples taken 24 h post injection and midge-th1 vector dna quantified using a quantitative pcr (qpcr) assay. vector dna was found in a varying number of animals per group and in all tissues except for bone marrow (figure 1a ). the vector copy numbers per μg total dna ranged from 4.04 × 10 3 (geometric mean, ovaries, 4/5 animals positive) to 2.55 × 10 9 (geometric mean, skin of injection site, all animals positive). comparable distribution pattern after single or repeated administration next, we tested whether repeated administration alters the distribution pattern or causes accumulation of vector dna in tissues. one hundred and twenty micrograms of leishdnavax were administered to rats i.d. at the same site four times at weekly intervals. vector dna was quantified in total dna extracted from tissue samples collected 24 h after the last injection. distribution patterns and amounts of vector dna were comparable 24 h after single and repeated injection (figures 1a and b) . copy numbers per μg total dna ranged from 4.63 × 10 3 (geometric mean, lung tissue, 5/10 animals positive) to 4.50 × 10 7 (geometric mean, skin of injection site, all animals positive). midge-th1 vectors persist at injection site and in draining lymph nodes persistence of midge-th1 vectors was examined 14 days and 60 days post treatment. fourteen days after four injections leishdnavax was cleared from the majority of organs (figure 1c ). high vector copy numbers per μg total dna were found in skin of injection site (all animals positive, geometric mean: 3.45 × 10 6 copies) and in inguinal lymph nodes (7/10 animals positive, geometric mean: 2.88 × 10 4 copies). sixty days after four injections, midge-th1 vectors were cleared from most tissues but persisted in inguinal lymph nodes (5/10 animals positive, geometric mean: 1.44 × 10 4 copies) and skin of injection site (all animals positive, geometric mean: 6.93 × 10 5 copies) (figure 1d ). to assess toxicity of leishdnavax in naive mice, sterile phosphate-buffered saline (pbs, placebo) and ascending doses of the vaccine were injected either once or five times in weekly intervals (table 1) . leishdnavax was well tolerated at all doses tested. no animal died during the in-life phase, and no change in behavior or external appearance of the animals was noted. comparing placebo-treated to leishdnavax-treated animals, no differences were observed in body weight, food and drinking water consumption, ophthalmological and auditory examinations, urinalysis, organ weights and the neurological screening. no local intolerance reactions were noted after single or repeated vaccine dosing. minor deviations in single animals/groups for few blood parameters (large unstained cells, albumin/globulin ratio, αamylase) were classified as not vaccine-related. macroscopic post mortem examination of organs revealed an enlarged spleen in a single male animal 1 day after single administration of 100 μg leishdnavax, which possibly indicated the activation of the lymphatic system. in all animals the histological findings were within the normal range of variation. no leishdnavax-related morphological lesions were found. no signs of auto-immune reactions were observed, and there were no elevated levels of serum antibodies against dsdna detected. biological activity of the vaccine lot was proven by induction of antigen-specific serum immunoglobulin g (igg) in mice of satellite groups (table 1) . a dose-dependent increase of leishdnavaxspecific igg levels was observed (figure 2 ), in line with data from efficacy studies as previously reported. 22 rats receiving the vaccine during the biodistribution study did not exhibit any vaccine-related local or systemic signs of toxicity, and also necropsy did not reveal any vaccine-related toxicity. leishdnavax is well tolerated in l. donovani-infected mice tolerability of leishdnavax and effects on visceral parasite burden were evaluated in balb/c and c57bl/6 mice. groups of mice infected with l. donovani were injected with one, two or three doses of 100 μg leishdnavax or pbs in weekly intervals. parasite burden was evaluated seven days and 29 days after the last injection. no significant difference in hepatic or splenic parasite burden was found between groups receiving equal numbers of injections of pbs or leishdnavax. in both treatment groups infection with l. donovani followed the known kinetic 23 with distinct organ-specific growth patterns in livers and spleens of infected mice. figure 3 shows the parasite burden as determined in the balb/c and one c57bl/6 experiment. no significant difference in weights between pbs and leishdnavax-treated groups of mice was observed. serum samples from mice, which had received three injections of pbs or leishdnavax, were subjected to analyses of standard biochemical parameters; alanine transaminase, aspartate aminotransferase, urea and creatinine. no significant differences between pbs and leishdnavax-treated groups were found for these parameters in balb/c mice. significant differences were found in levels of creatinine in c57bl/6 mice in two separate experiments. however, neither the time point nor the direction of difference were consistent between experiments ( table 2) . histological analysis of livers and spleens in repeated experiments and the kidney, heart and lung in a single experiment (c57bl/6 mice) revealed similar lesions in all animals (pbs and leishdnavax treated) in terms of location and severity. lesions were consistent with infection and included severe granulomatous hepatitis and splenitis with intralesional parasites, granulomatous/interstitial pneumonia and interstitial nephritis and glomerulonephritis. biodistribution and persistence of dna vaccines are studied to estimate the duration of antigen expression and the risk of vector integration into host genomic dna. 8, 9 we examined the biodistribution of leishdnavax after a single and, in contrast to previously published results, 20,21 also after a series of four injections at weekly intervals. this condensed application scheme is relevant for clinical approaches with: (i) a limited number of injections over a longer period of time as accepted for prophylactic vaccinations and (ii) vaccination regimes requiring more injections in shorter intervals, for example, therapeutic vaccinations. in line with recommendations of the us fda, 8 organ and tissue samples were taken at several time points following injections and vector dna was quantified using quantitative pcr. twenty-four hour after single or repeated injection, midge-th1 vector dna was detected in almost all organs and tissues examined, suggesting that it was distributed systemically, most likely via the lymphatic system and the blood stream. a similar distribution pattern has been described for midge-th1 vectors per day of sampling, five male and five female animals per group were killed. b satellite group to assess biologic activity of the vaccine lot and induction of antibodies against dsdna. figure 2 . immunogenicity of ascending leishdnavax doses in mice. groups of 10 balb/c mice (5 male, 5 female) were immunized i.d. at the tail base five times in weekly intervals with either pbs or leishdnavax (10, 50 or 100 μg per dose). fourteen days after the last immunization leishdnavax-specific antibodies (total igg) in sera were quantified by elisa using plates coated with a mixture of recombinant leishdnavax antigens. ***p ⩽ 0.001 (student's t-test); n.s.: not significant. encoding hepatitis b surface antigen, 21 indicating that distribution of midge-th1 vectors is independent of the encoded protein as previously reported for plasmid dna vectors. 11 systemic distribution at early time points has also been shown for naked plasmid dna upon i.m. and i.d administration. 24, 25 in accordance with other reports, 11, 26, 27 the highest vector amounts were detected in samples taken from the site of injection. consistently, lymph nodes draining the site of injection contained the second highest copy numbers, probably due to skin immune cells taking up dna at the site of injection and migrating to the lymph nodes. at day 14 after the fourth injection, vectors were cleared from nearly all organs except from skin and inguinal lymph nodes. whereas the amount of vector dna in skin samples was~10-fold lower compared with 24 h after the last injection, vector copy numbers in inguinal lymph node samples were reduced 100-fold. this finding is in line with reports on i.d.-administered plasmid dna vaccines, [27] [28] [29] suggesting that plasmids and midge-th1 vectors ( 21 and this report) follow a similar pattern of distribution and persistence. midge-th1 vector dna was not cleared from skin and inguinal lymph nodes within 60 days after four injections. although this 20 this short time to overall clearance was probably related to wider dispersion of dna upon multiple jet injection, the lower dna concentration (five jet injections of 10 μg dna at 1 mg ml − 1 versus one needle injection of 120 μg dna at 4 mg ml − 1 ), as well as the rapid entry into the blood circuit due to the high vascularization and low retention rate of macromolecules in tumor tissue as compared with normal skin tissue. though only a side-by-side comparison would be conclusive, we propose that these parameters strongly affected the time to overall clearance than the dna dose. in fact, the rats in our study received up to 0.6 μg leishdnavax dna per gram body weight, thus a lower relative dna dose than that administered to mice of the referenced study (~1.6 μg tumor necrosis factor alpha midge dna per gram body weight). 20 a persistence of at least 60 days as observed in our study suggests that vector dna had entered long-living cells residing in the skin. whether or not this vector dna is still functioning cannot be concluded from our results, as the quantitative pcr method targeted a short consensus sequence only. notably, midge-th1 vector dna did not accumulate in any tissue after repeated injections. at 60 days after one injection 21 or after four injections of leishdnavax, comparable amounts of midge-th1 vector dna were found, indicating that repeated injections do not enhance persistence. therefore, future evaluations of biodistribution and persistence of midge-th1 vectors in similar settings can be based on a single injection. long-term persistence of vector dna in tissues can be related to integration into host genomic dna and thus, theoretically bears tumorigenic potential. however, published data show that the integration rate of plasmid dna vectors does not exceed the rate of spontaneous mutation events within the host genome. 26, 27, 30 notably, for linear dna vectors with a structure similar to midge vectors a very low level of integration has been described. 31 furthermore and in contrast to closed circular plasmid dna molecules, integration of covalently closed linear dna constructs rather leads to disruption of chromosomes followed by apoptosis of affected cells, hence minimizing the risk of replicating unwanted genetic rearrangements. 32 on the basis of these published results, the risk related to potential integration of midge-th1 vectors into host genomic dna can be considered as low, though it was not assessed in this work. in a repeated-dose toxicity study in naive mice, we tested three ascending doses of leishdnavax. the highest dose of 100 μg corresponds to a human dose of~300 mg dna on a mg kg − 1 body weight base. doses in published clinical trials ranged from 0.1 mg 33 to 4 mg 34 of plasmid dna, so our study design includes a high safety margin. to further extend the safety margin, the vaccine was administered in a condensed schedule of five vaccinations (one more than the estimated maximum number for clinical application) in weekly intervals. importantly, a no observed adverse effect level, that is, the highest dose without significant toxicity, was not reached with the doses tested. no local intolerance reactions related to the vaccine were observed. the results established in naive mice are corroborated by safety data obtained from the biodistribution study. rats received 120 μg leishdnavax, corresponding to a human dose of~24 mg (based on mg kg − 1 body weight). there were no vaccine-related toxic effects observed either. our results further confirm the findings of other investigators reporting excellent preclinical safety profiles of dna vectors. 10, 11, 20, 26, 27, 30 in leishmania-infected and diseased individuals, tolerability of the vaccine may be different than in healthy, naive vaccinees. moreover, owing to the ability of leishmania amastigotes to exploit host igg as virulence factor, 35 there is the theoretical risk of vaccine-related immune-enhancement of infection and pathogenesis as discussed for other infectious diseases. 36, 37 hence demonstration of absence of disease exacerbation was included into the development program of leishdnavax. we evaluated the tolerability of leishdnavax in two different mouse strains, chosen for their inherent differences in cytokine responses to infection with l. donovani. 38 these experiments demonstrate that leishdnavax had no effect on the kinetics of the parasite burden with no vaccine-related enhancement of infection at any of the time points investigated. the kinetic of infection corresponded to the well-documented pattern in mice with a rapid increase in hepatic parasite burden, followed by a decline in parasite numbers and clearance and a slower increase in parasite numbers in the spleen with the establishment of chronic infections. 23 results for all other standard parameters were similar for treated and nontreated mice, demonstrating a good tolerability of the vaccine candidate in infected animals. in summary, we have shown here that leishdnavax, a novel dna vaccine candidate against leishmaniasis is safe and well tolerated in both naive and leishmania-infected mice. after repeated i.d. injections, the vaccine was rapidly cleared from most organs and tissues of rats and persisted in the skin of injection site and its draining lymph nodes only. we conclude that leishdnavax has a favorable safety profile that supports the initiation of human clinical trials. leishdnavax is an equimass mixture of five midge-th1 vectors, dissolved in sterile pbs. each vector encodes one leishmania antigen: kmp11, cpa, cpb, p74 or tsa, respectively. their dna sequences and expression cassettes of midge-th1 vectors have been described previously. 22 midge-th1 vectors were synthesized in a simple and standardized procedure. 18 forty wistar rats (20 per sex, janvier labs, saint-berthevin, france), 8 weeks of age, were allocated to single-or four-dose study groups with 10 animals each (five per sex). animals received needle injections of 120 μg leishdnavax per injection (~5.3 × 10 13 midge-th1 vector molecules, injection volume 30 μl) i.d. at the dorsal base of tail, either once or four times in weekly intervals. animals injected with a single dose were killed 24 h after administration. four-dose groups were either killed 24 h, 14 days or 60 days after the fourth injection. clinical signs were recorded before, 30 min and 4 h after treatment, thereafter once daily. twenty-four hours after injection, a modified irwin test 39 was performed on the single-dose group and on a non-treated control group to assess acute neurological toxicity of the vaccine. throughout the study, mortality was evaluated once daily and body weight once weekly. at killing, animals were anesthetized deeply by isoflurane inhalation and blood was collected from the plexus ophthalmicus. gross pathology was performed and samples of the following tissues were taken: blood, thigh bone marrow, brain, heart, kidneys, liver (lobus quadratus), lymph nodes (lnn. axillares, inguinales, mesenteriales), lung, thigh muscle, ovaries, skin of injection site, spleen and testes. to avoid cross-contamination with vector dna single-use scalpels were applied for each sample. all other equipment was thoroughly decontaminated after each dissection. samples were immediately frozen in liquid nitrogen and stored at − 80°c until processing. quantitative analyses of dna vector amounts were performed at imgm laboratories (martinsried, germany). total dna was extracted from safety and tolerability of a leishmaniasis dna vaccine o riede et al ~20 mg tissue or up to 280 μl blood, respectively, using the dneasy blood&tissue kit (qiagen, hilden, germany) and stored at − 20°c. a taqman-based quantitative realtime-pcr assay was established and characterized using a standard curve of leishdnavax ranging from 10 vector copies per reaction to 1.05 × 10 11 vector copies per reaction with 200 ng genomic dna from the livers of naive animals as background. measurements were carried out with 200 ng of extracted total dna in 384 well plates with a reaction volume of 20 μl on an ab7900ht instrument (life technologies, carlsbad, ca, usa). primers (eurofins genomics, ebersberg, germany) and hydrolysis mgb probe (life technologies) were designed to detect a consensus sequence present on each midge-th1 vector. the forward primer (5′-gtcgtttagtgaaccgtcagatca-3′) anneals to the cmv promoter region, the hydrolysis mgb probe (5′-fam-tttattgcggtagtttatca-nfq-3′) and the reverse primer (5′-gcacg actgcgttagcaatttaa-3′) anneal to the intron. limit of detection and lower limit of quantification of the assay were determined at 525 copies of midge-th1 vector per reaction (that is, 2625 copies per μg total dna). all samples of extracted dna were measured in triplicates. midge-th1 vector copy numbers were calculated according to the leishdnavax standard curve running in parallel on each plate and expressed as copy number per μg total dna. acceptability criteria of measurements included standard curve coefficient of correlation ⩾ 0.95 and detected copy numbers of no-template-controls below the limit of detection. in addition, the s.d. of the quantification cycle values of at least two replicate measurements had to be below 0.5, otherwise the measurement was repeated. assessment of vaccine toxicity. the study was performed in accordance with glp regulations principles and german animal welfare regulations at lpt laboratory of pharmacology and toxicology, hamburg, germany with prior approval by lpt's institutional animal care and use commissary (study no. 26814) and by the competent authority (behörde für gesundheit und verbraucherschutz, amt für verbraucherschutz lebensmittelsicherheit und veterinärwesen, billstraße 80, 20539 hamburg, germany, v 11307-591-00.33). one hundred and forty balb/c mice (70 male, 70 female) were randomized and allocated to study groups. animals were housed individually. at first treatment mice were 44-49 days (males) or 60-65 days (females) of age with a body weight of 18.2-22.5 g (males) or 18.0-20.4 g (females). ten animals each (five per sex) received one i.d. needle injection (volume: 25 μl) into the dorsal tail base with a dose of 10, 50 or 100 μg leishdnavax or the placebo (pbs). they were killed 24 h post injection for evaluation of acute toxic reactions. thirty animals each receiving placebo and 100 μg dose, or 20 animals each receiving 10 μg and 50 μg dose (injection volume: 25 μl) were injected by needle i.d. into the dorsal tail base five times in weekly intervals. animals were killed after a recovery period of 24 h, 14 days or 61 days (placebo and 100 μg dose group only) following the fifth injection (table 1) . animals were observed for clinical signs including injection site reactogenicity before and 30 min and 4 h after each dosing and daily between injections. mortality was evaluated twice daily throughout the study. body weight of animals was recorded at day of group allocation, on the day of first dosing and three times a week thereafter. food consumption was determined weekly and water consumption was recorded daily. neurological screening 40 and assessment of grip strength 41 was conducted on all animals 24 h after first dosing. ophthalmological and auditory examinations were performed pre-dose, at the end of the treatment period and at the end of the recovery period. for urinalysis, mice were placed in funnel cages in groups of five per sex and received 30 ml tap water per kg body weight by gavage. subsequently, urine was collected for 6 h and the following parameters were measured: volume, weight, ph, specific gravity, osmolality, protein, glucose, bilirubin, urobilinogen, ketones, hemoglobin, nitrite, epithelial cells, leukocytes, erythrocytes, organisms, further constituents, crystalluria. at killing, blood was taken for clinical biochemistry (albumin, globulin, bilirubin, cholesterol, triglycerides, creatinine, glucose, protein, urea, calcium, alanine aminotransferase, alkaline phosphatase, α-amylase, aspartate aminotransferase, creatine kinase, glutamate-dehydrogenase, γ-glutamyl-transferase, lactate dehydrogenase) and hematology (hemoglobin content, erythrocytes, leukocytes, differential blood count, reticulocytes, platelets, mean platelet volume, red cell distribution width, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration). gross pathological examination was conducted, organ weights were taken and samples of the following organs and tissues processed for histological examination: adrenal gland, aorta abdominalis, bone (os femoris with joint), bone marrow, brain, cecum, coagulating gland with seminal vesicle, epididymis, eye with optical nerve and harderian gland, exorbital lacrimal gland, gall bladder, heart, skin and subcutaneous tissue of injection site, large intestine, small intestine, kidney and ureter, liver, lung, lymph nodes (mandibular, mesenteric), mammary gland, muscle, sciatic nerve, esophagus, ovary, pancreas, pituitary gland, prostate, salivary glands, spinal cord, spleen, sternum, stomach, testicle, thymus, thyroid, tongue, trachea, urinary bladder, uterus and vagina. histological examination was performed on samples of all animals with the highest expected burden, that is, after receiving five injections of each 100 μg leishdnavax and 24 h of recovery, and the respective placebo group as control. moreover, the immunologically relevant organs thymus, spleen and lymph nodes as well as the skin at the injection site of all animals of all groups were examined histologically. in general, tissue samples were fixed in 7% buffered formalin (eyes in davidson´s solution), embedded in paraffin, sections prepared and routinely stained with haematoxylin-eosin. samples from mice receiving five times a dose of 10 or 50 μg, respectively, were scheduled for histologically examination only in case of vaccine-related findings in the group receiving five times 100 μg, or if gross pathology revealed vaccine-related changes. as there were no respective findings, these samples were not histologically examined. biologic activity of the vaccine lot and assessment of antibodies against dsdna. in all, 100 balb/c mice (50 male, 50 female) were randomized and allocated to satellite study groups of the toxicity study resembling the repeated-dose treatment schedule (table 1) . at first treatment, animals were 44-49 days (males) or 60-65 days (females) of age with a body weight of 17-19.3 g (males) or 17-18.4 (females) . at study termination, blood samples were taken and serum obtained for enzyme-linked immunosorbent assay to test for antibodies against vaccine antigens and for radioimmunoassay to test for antibodies against dsdna. these analyses were performed under non-glp regulations conditions. leishdnavax-specific antibodies were detected performing an enzyme-linked immunosorbent assay as previously described 22 with minor modifications. plates (nunc maxisorp, thermo scientific, roskilde, denmark) were coated per well with 100 μl of 5 μg ml − 1 antigen-mix (kmp11, cpa, cpb, p74, tsa 1:1:1:1:1) in pbs (fisher scientific, paisley, uk). recombinant proteins were obtained from proteogenix, oberhausbergen, france (cpa, cpb, p74, tsa) or were kindly provided by professor c jaffe (kmp11). antigen mix was assembled at lpt. plates were incubated overnight at 4°c, then washed three times with wash buffer (0.05% (v/v) tween in pbs) and subsequently blocked with assay diluent (5% (w/v) bovine serum albumin (sigma aldrich, st louis, usa) in pbs) for 1 h at room temperature. plates were washed three times with wash buffer and 100 μl per well of 1:50 diluted serum samples were added. the plates were incubated for 2 h at room temperature and subsequently washed five times with wash buffer. hundred micolitre per well of anti-mouse igg-hrp (conc.: 11.3 mg ml − 1 ), (sigma aldrich) were added at a 1:5000 dilution and plates were incubated for 2 h at room temperature. after washing five times developer solution containing 3,3',5,5'-tetramethylbenzidine (sigma aldrich) was added and the reaction stopped by adding acid solution when the color developed. absorbance was measured at 450 nm. to assess the induction of an immune response against dsdna a radioimmunoassay was performed at ibl international gmbh (hamburg, germany) using a kit for detection of anti-dsdna antibodies (ibl international). in brief, 25 μl of serum sample was mixed with 150 μl of 125 i-labeled dsdna tracer and incubated at 37°c for 60 min. subsequently, 1 ml of cold ammonium sulfate solution was added to the sample followed by vortexing. tubes were centrifuged (15 min at 1500 g) and supernatant was removed. radioactivity was counted using a gamma counter and the dsdna binding capacity was calculated according to a standard curve. under specific pathogen-free conditions in individually ventilated cages and exposed to 12 h light-12 h dark cycles. standard rodent diet (rm no 1 expanded) and de-ionized water were supplied ad libitum. l. donovani (strain mhom/et/67/hu3) was maintained in rag-1 (b6) ko mice and amastigotes harvested from spleens of infected animals 440 days after infection. infection and treatment schedule. female balb/c and c57bl/6 mice (6-8 weeks of age at the start of experiments) were infected by injecting 2 × 10 7 freshly harvested l. donovani amastigotes intravenously into a tail vein as described. 42 following infection, animals were sorted into groups of three to five mice per cage and two cages assigned to each treatment group. groups of mice received either one, two or three injections of leishdnavax containing 20 μg dna/antigen (corresponding to 100 μg total dna) or pbs, administered in volumes of 25 μl i.d. at the base of the tail using 29g single-use needles (bd microfine plus insulin syringes). the first dose was administered 7 days after infection and repeat doses in 7-day intervals. groups of mice were killed 7 days after having received the last dose of leishdnavax or pbs. in a further experiment in c57bl/6 mice additional groups were killed 29 days after the last of a total of three doses of leishdnavax or pbs. determination of efficacy and tolerability parameters. mouse weights were recorded prior to the first dose of treatments administered and in weekly intervals thereafter. the injection site was monitored following administration of treatments and animals were observed daily by trained staff for the whole duration of the experiments. mice were humanely killed by exsanguination under terminal anesthesia and blood collected by cardiac puncture. livers and spleens were removed and their weight recorded. tissue impression smears were prepared, fixed in 100% methanol and stained in 10% giemsa. parasite burden was determined microscopically and leishman-donovan units calculated by the formula number of parasites per host cell nucleus × organ weight in mg, as described previously. 43 serum was harvested from blood stored overnight at 4°c by centrifugation at 1500 g, 4°c for 15 min and stored at − 80°c. biochemical analysis of standard serum parameters was carried out by laboklin gmbh&co.kg (bad kissingen, germany). for histology, organs were fixed in 10% neutral buffered formalin, embedded in paraffin and routinely stained with hematoxylin and eosin. histological data were evaluated in blinded fashion. data from the repeated-dose toxicity study in naive mice were analyzed using student's t-test for numerical functional tests (p ⩽ 0.01), dunnet's multiple t-test for body weight, food consumption, hematology, clinical biochemistry and organ weights (p ⩽ 0.01) and fisher's exact test for histology (p ⩽ 0.05), respectively. toxicity data of the biodistribution study were evaluated as follows: parameters of the irwin test were analyzed by mann-whitney u-test (p ⩽ 0.05) and normality of body weight data by shapiro-wilks test. in case of normality, an analysis of variance was performed with post hoc dunnett's test for multiple comparisons; otherwise, a non-parametric kruskal-wallis test with post hoc dunnett's test was employed (p ⩽ 0.05). igg levels were statistically analyzed using graphpad prism 5 (graphpad software inc., la jolla, usa). normality of data was tested with shapiro-wilk test prior to applying either two-tailed student's t-test or mann-whitney test to analyze differences between means of two groups (p ⩽ 0.05). to evaluate tolerability of the vaccine in infected mice, comparisons between three or more groups were made by one-way analysis of variance followed by bonferroni's multiple comparison's test with comparison between pbs and vaccine treated groups. comparisons between two groups were made by an unpaired t-test assuming gaussian distribution (graphpad prism 6). a p-value of o0.05 was considered statistically significant. or, do, ae and cj are employees and ms is chairman of the executive board of mologen ag. ch is employee of imgm laboratories gmbh. aw is employee of lpt gmbh & co. kg. mologen ag owns a patent for the midge-th1 vector (pct/ de02/03798p74). these affiliations do not alter the authors' adherence to gene therapy policies on sharing data and materials. fjs declares no conflict of interest. leishmaniasis worldwide and global estimates of its incidence report of a meeting of the who expert committee on the control of leishmaniases case study for a vaccine against leishmaniasis leishmaniasis: complexity at the host-pathogen interface heterologous protection against influenza by injection of dna encoding a viral protein induction of antigen-specific cytotoxic t lymphocytes in humans by a malaria dna vaccine dna vaccines: a rational design against parasitic diseases us food and drug administration. guidance for industry. considerations for plasmid dna vaccines for infectious disease indications who annex 1: guidelines for assuring the quality and non-clinical safety evaluation of dna vaccines toxicological safety evaluation of dna plasmid vaccines against hiv-1, ebola, severe acute respiratory syndrome, or west nile virus is similar despite differing plasmid backbones or gene-inserts biodistribution of dna plasmid vaccines against hiv-1, ebola, severe acute respiratory syndrome, or west nile virus is similar, without integration, despite differing plasmid backbones or gene inserts clinical applications of dna vaccines: current progress cpmp/bwp/3088/99: note for guidance on the quality, preclinical and clinical aspects of gene transfer medicinal products marker-free plasmids for biotechnological applicationsimplications and perspectives dna immunisation with minimalistic expression constructs silencing of episomal transgene expression by plasmid bacterial dna elements in vivo minicircle dna is superior to plasmid dna in eliciting antigen-specific cd8+ t-cell responses priming of immune responses to hepatitis b surface antigen with minimal dna expression constructs modified with a nuclear localization signal peptide effect of different nuclear localization sequences on the immune responses induced by a midge vector encoding bovine herpesvirus-1 glycoprotein d intratumoral dispersion, retention, systemic biodistribution, and clearance of a small-size tumor necrosis factor-alpha-expressing midge vector after nonviral in vivo jetinjection gene transfer combination of midge-th1 dna vaccines with the cationic lipid saint-18: studies on formulation, biodistribution and vector clearance modular multiantigen t cell epitope-enriched dna vaccine against human leishmaniasis differential regulation of the immune response in the spleen 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dna ministrings: highly safe and effective gene delivery vectors phase 1 clinical trials of the safety and immunogenicity of adjuvanted plasmid dna vaccines encoding influenza a virus h5 hemagglutinin phase 1 safety and immunogenicity evaluation of advax, a multigenic, dna-based clade c/b' hiv-1 candidate vaccine intrinsic antibodydependent enhancement of microbial infection in macrophages: disease regulation by immune complexes after a tick bite in a tick-borne encephalitis virus endemic area: current positions about post-exposure treatment traitors of the immune system-enhancing antibodies in hiv infection: their possible implication in hiv vaccine development the capacity to produce ifn-gamma rather than the presence of interleukin-4 determines the resistance and the degree of susceptibility to leishmania donovani infection in mice comprehensive observational assessment: ia. a systematic, quantitative procedure for assessing the behavioral and physiologic state of the mouse a neuromuscular screen for use in industrial toxicology a method for the routine assessment of fore-and hindlimb grip strength of rats and mice n-(2-hydroxypropyl)methacrylamide-amphotericin b (hpma-amb) copolymer conjugates as antileishmanial agents regulation of leishmania populations within the host. i. the variable course of leishmania donovani infections in mice the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in the credit line; if the material is not included under the creative commons license, users will need to obtain permission from the license holder to reproduce the material we thank the leishdnavax consortium for helpful discussions, mologen ag's production and qc department for manufacturing the vaccine, shantanabha das for providing the leishmania antigen elisa protocol and charles jaffe for providing recombinant kmp11. this work was funded by the european commission as part of the 7th framework programme (#223189). key: cord-001065-j4hvyyoi authors: boncristiani, humberto f.; evans, jay d.; chen, yanping; pettis, jeff; murphy, charles; lopez, dawn l.; simone-finstrom, michael; strand, micheline; tarpy, david r.; rueppell, olav title: in vitro infection of pupae with israeli acute paralysis virus suggests disturbance of transcriptional homeostasis in honey bees (apis mellifera) date: 2013-09-05 journal: plos one doi: 10.1371/journal.pone.0073429 sha: doc_id: 1065 cord_uid: j4hvyyoi the ongoing decline of honey bee health worldwide is a serious economic and ecological concern. one major contributor to the decline are pathogens, including several honey bee viruses. however, information is limited on the biology of bee viruses and molecular interactions with their hosts. an experimental protocol to test these systems was developed, using injections of israeli acute paralysis virus (iapv) into honey bee pupae reared ex-situ under laboratory conditions. the infected pupae developed pronounced but variable patterns of disease. symptoms varied from complete cessation of development with no visual evidence of disease to rapid darkening of a part or the entire body. considerable differences in iapv titer dynamics were observed, suggesting significant variation in resistance to iapv among and possibly within honey bee colonies. thus, selective breeding for virus resistance should be possible. gene expression analyses of three separate experiments suggest iapv disruption of transcriptional homeostasis of several fundamental cellular functions, including an up-regulation of the ribosomal biogenesis pathway. these results provide first insights into the mechanisms of iapv pathogenicity. they mirror a transcriptional survey of honey bees afflicted with colony collapse disorder and thus support the hypothesis that viruses play a critical role in declining honey bee health. over the last years, the declining health of the honey bee (apis mellifera) and other pollinators has caused concern all over the world. particularly over the last six years, honey bee health has shown alarming rates of deterioration [1, 2, 3] , questioning the sustainability of our food production system. there are many possible threats to honey bees health, including pesticides, malnutrition, management stress, and pathogens [3, 4, 5, 6, 7] . numerous studies suggest that novel or emerging pathogens play a role in honey bee health declines [1, 2, 3, 7, 8, 9] . however, insufficient knowledge on honey bee pathogens compromises our ability to assess their importance and to develop control measures. this is particularly true for honey bee viruses although their importance for honey bee losses has become evident in recent years [6, 7, 8, 10, 11, 12] . specifically in combination with the ectoparasitic bee mite varroa destructor [13, 14, 15 ] that serves as a vector, several viruses appear to become more virulent [16, 17, 18] . viruses may cause covert infections [19] and were considered mostly harmless until varroa mites were introduced to a. mellifera populations almost 30 years ago [20, 21, 22] . the increased virulence of viruses has been confirmed experimentally by direct inoculation of bees with viruses [23, 24, 25, 26, 27] , opening an important research field to explore. approximately twenty honey bee viruses have been described so far [4, 16, 28, 29] , affecting the morphology, physiology, and behavior of bees. most belong to the families dicistroviridae [30] and iflaviridae in the order picornavirales. viruses in these families have a positive sense rna genome, covered by an icosahedral, pseudo t = 3 structure symmetry capsid (around 30 nm) that is responsible for rna protection, host specificity, and tissue infection. picornaviruses are well known for their capacity to shut off the translational system of their host cells, by cleavage of translation factors leading to a decrease in cap-dependent host translation, a conserved replication strategy among all members studied to date [31, 32] . picornavirus infection also triggers hostimmune responses (i.e., pkr) that result in decreased capdependent (host) translation. picornaviruses circumvent this immune response by encoding an internal ribosomal entry site (ires) which is recognized and translated by the host machinery (non-canonical translation) [30, 32] . over time, the accumulation of produced virus particles and repression of the synthesis of essential cell components lead to cell death in most cases [32] . little is known about the specific biology of the viruses in these families that infect honey bees, although they contain important bee pathogens, such as deformed wing virus (dwv) and israeli acute paralysis virus (iapv). iapv has previously been associated with the unusual honey bee disappearance syndrome called colony collapse disorder (ccd) [8] and is frequently seen in many honey bee pathogen surveys [6, 7] . despite the importance of iapv and the feasibility to work with iapv in the laboratory [25, 33] , little is known about iapv's interactions with its host and resulting pathogenesis. in general, the lack of adequate tools for honey bee virus research has hampered our understanding of basic biology of the relevant viruses and little is known about the molecular bases of honey bee viruses replication and pathogenesis [18, 28] . therefore, many assumptions regarding their replication are made based on other picornaviruses (e.g., cricket paralysis virus [34, 35] and human poliovirus [32] ), highlighting the need of specific, mechanistic studies on honey bee viruses [36] . focusing on iapv, we report here the development of an inoculation method of invitro reared honey bee worker pupae that provides the basis for mechanistic, in-depth studies of honey bee viruses. we report acute but variable disease symptoms, compare viral replication among pupae of two colonies and patrilines within these colonies, and report on measures of gene expression in response to viral infection that indicate major disruption of cellular homeostasis. initially, approximately 20 adult bees from a heavily iapvinfected colony were frozen in liquid nitrogen, ground to a fine powder, and homogenized in 10 ml extraction buffer (0.1 m potassium phosphate buffer, ph 7.5, 0.2% diethyldithiocarbamate, 1/5 volume of diethyl ether). emulsification ensued by adding 5 ml carbon tetrachloride and centrifuging at 10,000 rpm for at 4uc for 10 minutes (rotor: sorvall rc-5b) and collecting the supernatant. the supernatant containing viruses was filtered through a 0.2micron filter (milex-gs, millipore, #slgs033ss) to remove small tissue debris, fungi, and bacteria. the filtrate was then centrifuged at 30,000 rpm (61,740 rcf) in a beckman lb-70m ultracentrifuge with a 70.1/ti rotor for six hours at 4uc to pellet picornavirus particles. the pellet was resuspended in 0.2 ml of pbs and centrifuged against a cscl gradient (0.44 g/ml) at 52,000 rpm (185,000 rfc) overnight (beckman lb-70m ultracentrifuge, 70.1/ti rotor). the fractions containing virus particles were dialyzed using ''slide-a-lyzer dialysis cassettes'' against cold (10uc) 0.2 ml of pbs overnight. rt-pcr was conducted to test for the presence of acute bee paralysis virus (abpv), israel acute paralysis virus (iapv), kashmir bee virus (kbv), sacbrood virus (sbv), chronic bee paralysis virus (cbpv), black queen cell virus (bqcv), and deformed wing virus (dwv). iapv, and small amounts of abpv, dwv, and bqcv were detected in the purified viral solution (positive amplification with pcr primers). viral quantification was performed by absolute quantification using the standard curve method as described previously [37] [38] . 5.0 ml of viral solution was examined for the presence of virus particles and their phenomenological characterization by electron microscopy. virus particles were negatively stained with 2% uranyl acetate on a formvar-coated ni grid and viewed in a hitachi h-7000 electron microscope at 150,000x to 200,000x. iapv replicates readily in pupae [25] . therefore, white-eyed pupae were inoculated for virus propagation, using 1.0 ml of the viral suspension per pupa. after 4 days of incubation, with disease symptoms apparent (figure 1 ), viruses were purified using the approach outlined above. qrt-pcr showed ,10 5 more iapv genomes than the second most detected virus, dwv after a single round of virus injection pupal amplification, and isolation. this procedure was repeated twice using pupae from very strong hives to further reduce contaminating viruses and increase the amounts of iapv. the high concentration of iapv over other honey bee viruses in these purifications allowed us to strongly dilute the inoculum, decreasing the chances of cross inoculation with another virus. in the experiments described below, we injected 10 4 iapv genome equivalents keeping the probability of cross contamination at negligible levels. pupae were individually homogenized and submitted to total rna extraction, using trizolh (invitrogen, carlsbad ca) following the manufacturer's protocol. the resultant rna pellets were resuspended in diethyl pyrocarbonate-treated water in the presence of rnase inhibitor (invitrogen, carlsbad ca) and treated with dnase i (invitrogen, carlsbad ca) to remove any contaminating dna. the resulting rna was quantified on a nanodrop spectrophotometer (thermo scientific, wilmington, de). first-strand cdna was then synthesized by incubating 2mg of total rna per sample in a 96-well plate with master mix containing 50 u superscript ii (invitrogen, carlsbad, ca), 2 nmol dntp mix, 2 nmol poly(dt)18, and 0.1 nmol poly (dt) (12) (13) (14) (15) (16) (17) (18) at 42uc for 50 minutes followed by 15 minutes at 70uc as described previously [39] . the cdna was diluted 1:5 with molecular-grade water. the primers used in this study were validated for relative quantification of the target genes and are commonly used in honey bees [39, 40, 41] . reactions to amplify the cdna products were conducted in 96-well plates using the applied biosystems step one real time pcr machine (applied biosystems, carlsbad, california). one microliter of diluted cdna from each sample was used as a template for rt-qpcr reactions using sybr green tm (applied biosystems, carlsbad, california) following the manufacturer's protocols. the reactions were conducted under a fixed thermal protocol consisting of 3 minutes at 95uc, followed by 40 cycles of a three-step protocol of 95uc for 20 sec, 60uc for 30 sec, 72uc for 1 minute. fluorescence measurements were taken at each cycle during the last 72uc step. this procedure was followed by a melt-curve dissociation analysis to confirm the specificity of the reactions. based on the results of the preliminary experiments 1 and 2 (preliminary experiments s1), a more extensive iapv inoculation experiment was designed to study the time line of infection and associated gene expression patterns, and to assess bees for variability in iapv susceptibility. one microliter of the inoculant (pbs as control or virus solution containing 10 4 genome equivalents of iapv) was injected using a nanojet tm syringe pump (chemix, usa) with an infusion flow rate of 0.1ml/sec, following manufacturer's parameters. the needle was inserted in the lateral abdomen between the fourth and fifth tergite of young, white-eye honey bee pupae ( figure 2a ). two strong, iapv-free hives were selected from the uncg research apiary, representing two distinct sources of bees for the experiment. from each hive, 200 white-eye pupae were collected for each of the following treatment groups: without inoculation (w/o), pbs inoculated (pbs), and iapv inoculated (iapv). from each treatment group and hive, 50 bees were frozen at 0 h, 5 h, 20 h and 48 h after inoculation and a subset of these samples was individually analyzed for viral titers and gene expression patterns. the first time point directly after inoculation was used as a control of the initial states of the bees in the experimental and control groups. the time point of five hours post-infection was chosen to measure the virus impact before completion of the replication cycle, based on the assumption that iapv follows the picornavirus family average timing for a replication cycle, of 7-12 hours [32, 34, 35] . any gene expression changes at this time point represent the bees' response to inoculation without complications from virus-related tissue damage. the time point of 20 h postinfection was considered representative of events after one complete cycle of virus replication, and the 48 h time point represents the established diseased state, characterized by visual symptoms. based on the results of the preliminary experiments (preliminary experiments s1), we tested the effect of iapv injection on gene expression of six commonly used reference genes that have been reported to be constantly expressed across different experimental conditions [4, 7, 41] . we studied the transcription of actin, ribosomal 28s rna, ribosomal 18s rna, ribosomal protein rps5, mgst1, and histone h2a, under iapv infection. histone h2a is not common used in honey bees, but it was added to our experiment because its expression is constitutive and cell-cycle independent, and it is commonly used on other models [42] . the sequences of utilized h2a primers are: 59-aaaggaaattacg-cagaacga-39 (h2a forward) and 59-cggctaaatattc-cataacgg-39 (h2a reverse). in addition, the titers of iapv and dwv were quantified in these samples. one hind leg was removed from each pupa and stored at -20uc before dna extraction to determine the subfamily (patriline) for each individual. dna was extracted from each leg using a standard chelex 100h method [43] . briefly, each sample was incubated for 60 min at 55uc, 15 min at 99uc, 1 min at 37uc, and 15 min at 99uc in 150 ml of a 5% chelex 100h solution with 5 ml 0.35 mg/ml proteinase k. subfamily identification for each sample was determined using microsatellite alleles following previously described methods [44] . this genomic paternity analysis was conducted using two multiplex pcr reactions (plex 1 and plex 2) with 10 ml reaction volumes containing approximately 100 ng sample dna, 16pcr buffer (takarah without mgcl 2 ), 1 mg/ml bsa, 1.5 u taq polymerase, 300 mm dntps, and either 1.5 mm (plex 1) or 1.1 mm (plex 2) mgcl 2 . following [45] , primer sets in plex 1 included 2.0-2.5 pmol am061, am052, am010, and am553, and primer sets in plex 2 included 2.5-3.5 pm am043, am098, am125, and am059. all reactions were performed using a thermoh px2 thermocycler with 7 min at 95uc followed by 30 cycles of 30 sec at 95uc, 30 sec at 55uc (plex 1) or 54uc (plex 2), and 30 sec at 72uc, then a final 10 min at 72uc. the pcr products were run on an abi 3730h dna analyzer at the genomic sciences laboratory at ncsu. data was acquired with genemapper 4.0 (abi) to score microsatellite fragment sizes. loci with poor amplification were excluded from analyses and only samples for which more than half of the loci could be scored were used for analysis. the data were analyzed with the computer package colony 1.2 to assign subfamily membership to each sample [46] . all experiments revealed that the absolute quantities (ct values) of the standard reference genes were affected by the iapv treatment (see results) and that they did not fulfill the criterion of expression stability. therefore, a larger set of potential reference genes was evaluated in the main experiment (raw data s1). however in the absence of an internal control, the transcript level of these genes and iapv could not be normalized by the customary dct or ddct methods [47, 48] . instead, transcripts were evaluated by ct values, based on the assumption that the amount of template after quantification and appropriate dilution did not differ systematically among treatment groups. to ensure consistency, a fixed fluorescence threshold for each gene and experiment was determined manually to avoid inter rt-qpcr runs inconsistencies. tests of technical error indicated a high replicability for several genes, with variation between replicate ct values of 1.3% on average (minimum: 0.002%, maximum: 2.6%). all statistical analyses of this study were done using the r stats package, version 2.15.0, http://www.r-project.org/ or with spss 20.0 (ibm). heat maps were generated by heatmap.plus r package version 1.3. patterns of gene expression were analyzed with parametric linear models, using time and treatment as fixed effects. bonferroni and scheffe's post-hoc tests were performed and did not differ in their results. in the experiment, the virus titers of inoculated individuals were compared among colonies and patrilines within colonies. patrilines that were represented by only one individual were omitted from the patrline but not the colony analysis. thus, separate anovas were used instead of one nested anova. attempts to isolate pure iapv directly from naturally infected adult bees were unsuccessful due to co-infection of the bees with other honey bee viruses. pcr tests resulted repeatedly in positive amplification of multiple viruses, such as bqcv, abpv, and dwv. co-infection seems to be the rule rather than the exception and it is generally rare to find bees infected with a single virus [49] . however, our artificial inoculation of pupae led to selective increases of iapv, relative to the other viruses. after three rounds of pupae inoculation and subsequent virus purification, the amount of iapv was at least 10 5 genome copies higher than all other common honey bee viruses found in our initial inoculum (bqcv, abpv, dwv). after serial dilutions, iapv was the only virus that could be detected by pcr. electron microscopy analysis of this sample showed uniform viral particles around 27 nm ( figure 2b ), consistent with picornavirus particles. sequence analysis verified these particles to be iapv. the site for injection of the virus inoculum into the honey bee was chosen based on the ability of the pupae to complete development to become an adult after sham injections. when the junction between the last abdominal sternites (figure 2a ) was selected more than 95% of bees were able to complete development after pbs inoculation. this region is very soft, enabling smooth penetration of the needle with little physical damage to the pupae. in addition, varroa destructor nymphs were often observed in this same region when pupae were prepared for inoculations, suggesting that this area is an attractive feeding site. in the standardization process both controls, without inoculation (w/o) and pbs buffer injected bees (pbs), developed normally ( figure 1a) , culminating in full development after 144 hours ( figure 1c ). iapv-inoculated bees showed strong but variable symptomatology over time ( figures 1a and 1b) , inhibited metamorphosis, and ultimately death. symptoms ranged from a complete cessation of development with no visual evidence of disease ( figure 1b-1) , rapid darkening of body parts ( figure 1b-3 and 1b-4) , simultaneous darkening and hindered development ( figure 1b-5) , to apparently normal development ( figure 1b-2) with eventual sudden death. iapv titer increased in all inoculated bees but no correlation between symptomatology and virus titer determined by rt-qpcr at the end of the experiment was observed. the experiment investigated iapv infections in pupae from two unrelated colonies to compare these two colonies and patrilines within the colonies. rt-qpcr analyses showed no initial evidence of iapv infection in either experimental colony and even the initial inoculum was below our detection limit (figure 3) . a twofactorial anova indicated that the two colonies differed significantly in the build-up of virus titers (f colony (1,104) = 5.3, p = 0.023; f time (3,104) = 69.7, p,0.001; f interaction (3,104) = 8.0, p,0.001). specifically, a significant difference between the colonies was found at 20 hours (f colony (1,42) = 39.2, p,0.001; figure 3 ). post-hoc tests also revealed a significant difference among all time points, except between 0 and 5 hours. within colonies, some patriline differences were suggestive ( figure s1 ) but not significant after bonferroni correction (colony 1: symptomatic differences were also observed between the two colonies (table 1) . generally, pupae from colony 1 showed some evidence of developmental completion, as evidenced by the presence of brown eye pigmentation and darkened abdomens. pupae from colony 2 showed higher development debilitation: few individuals developed eye pigmentation or darkened abdomens. no correlations between virus titer and symptomatology were found. three-factorial anova revealed a significant treatment effect on the expression on all six genes ( table 2 ). in general, gene expression also differed among time points but the differences for actin were not significant. in contrast, the two colonies only differed in actin expression (table 2) . post-hoc tests of main treatment effects showed significantly higher gene expression in the iapv-inoculated bees compared to the two control groups for actin, 28s rrna, and mgst1. conversely, 18s rrna was significantly less expressed in iapv-inoculated bees than in both control groups. for histone h2a, significantly lower expression was found in the untreated group than in the pbs and virus injected group, and all treatment groups differed significantly in the rps5 expression in the following order: ''control group'',''pbs-injected'',''iapv-injected''. post-hoc test results for time effects were more complex (results s1). the anova models also revealed many significant interaction terms (results s1), indicating time-specific and colony-specific treatment effects (figure 4) . the entire anova model explained most of the gene expression variation for rps5 (65.8%), followed by actin (62.1%), mgst (40.7%), h2a (38.0%), 18s rrna (35.0%), and 28s rrna (18.9%). the additionally performed ancovas revealed that all associations between iapv and transcripts were also significant, independently of treatment or timing ( table 2) . no correlations between gene expression and symptomatology were found. honey bee viruses play an important role in the recent declines in honey bee health [8, 9, 16, 17, 50, 51] but very little is known about how virus infections damage honey bees. the developed study model is a crucial step to much-needed mechanistic studies of honey bee viruses. on the one hand, honey bee pupae do not require feeding and can be easily maintained under laboratory conditions until full development into adult. they are highly relevant in the pathology of several viruses [16] . on the other hand, iapv has proven an excellent choice because its preferential replication in pupae [25] enables the production of inoculum that is virtually free of contaminating viruses. in addition, iapv is relevant for bee health [8] but it is not ubiquitous in the bee population, which makes it possible to set up experiments with iapv-free bees. our experiment showed significant differences in iapv replication between the two studied colonies, and also suggested patrilineal variation, although small sample sizes per patriline precluded significance after correction for multiple testing. environmental factors are not to be disregarded and can include colony propolis [52] and pesticide [53] levels, or larval nutrition [54] . colony 01 showed a more abrupt increase in virus titers, while iapv increased more gradually in colony 02. however, the relative resistance of bees from colony 02 only delayed iapv build-up and the iapv titers were invariably high in pupae after 48 hours. the identification of genotypic variation in virus susceptibility would improve the prospect for selective breeding to improve honey bee health. in any case, our study demonstrates significant heterogeneity in virus amplification and gene responses (see below), highlighting the importance of standardization in honey bee health studies. distinct symptomology patterns were observed between the two colonies (table 01) . it is not clear whether virus-induced tissue damage and necrosis or melanization as part of the immune response is responsible for the observed darkening. melanization is key component of insect immune response and is active in antiviral immunity [55, 56, 57] . melanization would be predicted to correlate with resistance to iapv, contrary to our observations of the darkening of larvae. therefore, necrosis or other forms of cell death are a more likely explanation for the tissue darkening [58] . quantifying gene expression responses to iapv infection in the honey bee pupa according to standard protocols was complicated because iapv infection significantly affected all investigated reference genes in all experiments (preliminary experiments s1 and figure 4 ), precluding their meaningful incorporation into dct or ddct analyses [48] . even though relative quantification has considerable advantages and is used almost universally, it depends on appropriate references [59] , which were not available for our study. therefore, we relied on absolute quantification after standardizing the amount of rna in each experiment. the ct values were converted to another measure of absolute quantification (copy number) for iapv by comparison to a standard curve. variation in cdna synthesis or other inequalities among samples might have contributed some experimental error. however, it is highly unlikely that technical errors are responsible for the observed significant differences among our experimental treatments, particularly given the consistency among our three separate experiments. thus, we conclude that ct values are most appropriate for our study and absolute quantification is necessary in studies of the transcriptional response to virus infections in honey bees. caution needs to be exerted in general when interpreting relative gene expression patterns with respect to virus infections in honey bees and other organisms [60, 61] . the investigation of multiple reference genes confirmed the earlier conclusions that basic cellular pathways were significantly being affected by iapv infection (preliminary experiments s1). interestingly, the transcription of many genes in the pbs-injected bees was intermediate between the negative control and the iapv group, demonstrating an effect of wounding itself. overall, the expression of all genes was affected by time, although for actin this effect was non-significant in the full factorial model. in contrast, actin was the only gene that exhibited an overall expression difference between the two colonies. furthermore, all genes were significantly associated with iapv titers, independently of the treatment effects. in sum, all analyzed genes failed to fulfill the criteria for a reliable reference gene and instead indicated a profound disruption of fundamental cellular processes by iapv. in addition to treatment effects, the expression of the putative reference genes also changed over time or was dependent on genotype. this transcriptional instability of putative reference genes might present a general disadvantage of the pupa as study system because the ongoing metamorphosis presumably affects numerous genes, independently of any treatment effects [62] . the biological interpretation of the main effects of host colony, time, and treatment is complicated by numerous significant interaction effects observed. for actin all interactions among the three factors were significant and for the 28s rrna no significant interactions were observed. the other four genes all showed a significant 3-way interaction and one or two 2-way interactions. interactions between time and treatment are not surprising for any transcript because the treatment effects only appear at the later stages of the experiment. however, interactions between colony and treatment confirm the finding that source colony significantly affects the interaction between iapv and its host. bees of the more resistant colony 02 showed a down-regulation of the 18s rrna by iapv injection. in contrast, the transcript was increased by iapv injection in bees from the more susceptible colony 01. similarly, for most other transcripts, the strongest up-regulation by iapv occurred after 20 hours in colony 01 but after 48 hours in colony 02. further experiments are needed to determine the causal relationships among host genotype and environment, gene expression patterns, and iapv replication. the observed gene expression patterns could be due to viral manipulation of the cells to increase virus replication or present cell compensatory responses to iapv infection. typically, picornaviruses express a protease that cleaves the scaffold eif4g initiator factor. this process inhibits the 59 cap mediated translation of cellular peptides and redirects the cell translational machinery to viral mrnas that depend on internal ribosomal entry sites (ires)-mediated translation [30, 31] . the proteasemediated shut-down of cellular translation is widespread [63, 64] and homologs of the protease gene have been identified in all members of the dicistroviruses so far [30] . however, direct evidence for a translational inhibition that increases transcriptional activities via feedback loops is so far missing for all honey bee viruses and insect picornaviruses in general. rps5 is a key component for ires recognition in the dicistrovirus family [65, 66, 67] . the up-regulation of this gene benefits virus replication directly, suggesting that rps5's strong and consistent upregulation may be directly induced by iapv. however, the widespread transcriptional response to iapv also suggests that the cell may respond to the lack of certain cell components by increasing their transcription. the up-regulation of actin, mgst1, and the histone h2a in most experimental groups suggests a far-reaching, although variable, response in a range of basic cellular processes in addition to a disturbance of the ribosomal biogenesis pathway discussed below. more research is needed to understand these processes and it variability among environments and genotypes. the three components of the ribosomal biogenesis pathway studied exhibited different responses to iapv injection. while 28s rrna, and rps5 transcripts were invariably increased after iapv replication (20 and 48 hours post-injection), 18s rrna transcripts were decreased in colony 02 at both time points and were only increased in the more susceptible colony 01 at the first time point. the reason for these disparities is unclear, particularly because the 18s rrna and 28s rrna transcripts are derived from a polycistronic precursor mrna [68] . however, the regulated balance between small and large ribosomal subunits [69] is controlled by independent maturation pathways [70] and iapv presumably affects these pathways differently. the differences between colonies may indicate that the more resistant individuals may have either resisted transcriptional manipulation by iapv or dedicated more cellular resources to immediate immune functions instead of ribosomal biogenesis. consistent with this interpretation, gene expression patterns converged between the two colonies at the later time point. ribosome biogenesis is a highly complex and energetically costly pathway that is essential for all eukaryotic cells [70] . it is highly regulated and integrated with other cell functions, such as p53 signaling, and deregulation of ribosomal biosynthesis has been associated with oncogenesis [71] and apoptosis [72] . apoptosis is a widespread cellular response to virus infection [73] and could explain some of the observed differences in iapv symptomology. on the other hand, viruses can also directly interfere with the ribosomal biogenesis pathway by either up-or down-regulation [74, 75] . in any case, our result of a disturbance of the ribosomal biogenesis corroborates an important microarray survey of transcripts in the honey bee intestine that has linked picornaviruses and 28s rrna transcript abundance to colony collapse disorder [9] . the injection of pbs served as an experimental control to account for the effect of wounding during iapv inoculation. for all genes, the observed transcription patterns of the pbs-injected bees were intermediate between the iapv-injected and the negative control group. this observation may suggest that a similar disruption of basic cellular functions occurs in response to wounding and cellular trauma, resulting in profound changes at the transcriptome level [76] . however, our data show also an increase of dwv titers in the pbs-injected pupae over time and relative to both other treatment groups. increased dwv titers in response to wounding have been observed before [77] . the increase of another picorna-like virus may have triggered responses in the pbs-injected bees that were similar to the iapv injection, supporting a similar gene expression pattern observed between pbs group and iapv injected bees compared to the negative control groups. in contrast to the pbs-injected bees, the iapv-injected bees did not show an increase in dwv titers, suggesting that iapv or cellular responses to iapv interfere with dwv replication [78] . in summary, this study introduces an important model system to advance mechanistic studies on virus-host interactions in insects. it is particularly valuable to study honey bee viruses and their role in compromising honey bee health. the results demonstrate significant variability and indicate sources for this variability. the transcriptional analyses show profound, correlated perturbations of basic cellular functions and call into question the use of typical reference genes in this system. the investigated responses to iapv inoculation in honey bees seem typical for picornavirus infections and provide a first step towards understanding the basic biology of this important honey bee virus. more detailed studies need to follow to manipulate virus and host and assess host responses to iapv at the systemic level. figure s1 patriline differences of iapv titers within colonies suggest a genetic basis for virus resistance in honey bees. preliminary experiments s1 two experimental inoculations of a limited number of bees with iapv suggested that viral infection causes broad-scale alterations of transcript patterns, including immune and reference genes. raw data s1 raw ct values for all qpcrs run in the main experiment. (pdf) results s1 the expression of genes evaluated by full, 3factorial anovas indicated that developmental time is a significant factor, that was as important as treatment but for most genes interacted with treatment. 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translational response in fission yeast ribosome biogenesis: of knobs and rna processing changes in ribosome biogenesis may induce cancer by down-regulating the cell tumor suppressor potential ribosomal stress induces l11-and p53-dependent apoptosis in mouse pluripotent stem cells apoptosis in viral pathogenesis genome-wide expression profiling shows transcriptional reprogramming in fusarium graminearum by fusarium graminearum virus 1-dk21 infection cardiovirus 2a protein associates with 40s but not 80s ribosome subunits during infection epigenetic reprogramming during wound healing: loss of polycomb-mediated silencing may enable upregulation of repair genes expression levels of immune-genes in developing workers of apis mellifera in response to reproductive timing and infestation level by the parasitic mite varroa destructor competition-colonization dynamics in an rna virus we would like to thank the usda-beltsville bee research laboratory members: dr. miguel corona, for the histone h2a primer design and mr. andy ulsamer and ms. michele hamilton for their technical support. in addition, we would like to thank all members of the uncg social insect group for their suggestions and support. key: cord-001017-4qfhltg4 authors: chatterjee, dhriti; biswas, kaushiki; nag, soma; ramachandra, s. g.; das sarma, jayasri title: microglia play a major role in direct viral-induced demyelination date: 2013-06-20 journal: clin dev immunol doi: 10.1155/2013/510396 sha: doc_id: 1017 cord_uid: 4qfhltg4 microglia are the resident macrophage-like populations in the central nervous system (cns). microglia remain quiescent, unable to perform effector and antigen presentation (apc) functions until activated by injury or infection, and have been suggested to represent the first line of defence for the cns. previous studies demonstrated that microglia can be persistently infected by neurotropic mouse hepatitis virus (mhv) which causes meningoencephalitis, myelitis with subsequent axonal loss, and demyelination and serve as a virus-induced model of human neurological disease multiple sclerosis (ms). current studies revealed that mhv infection is associated with the pronounced activation of microglia during acute inflammation, as evidenced by characteristic changes in cellular morphology and increased expression of microglia-specific proteins, iba1 (ionized calcium-binding adaptor molecule 1), which is a macrophage/microglia-specific novel calcium-binding protein and involved in membrane ruffling and phagocytosis. during chronic inflammation (day 30 postinfection), microglia were still present within areas of demyelination. experiments performed in ex vivo spinal cord slice culture and in vitro neonatal microglial culture confirmed direct microglial infection. our results suggest that mhv can directly infect and activate microglia during acute inflammation, which in turn during chronic inflammation stage causes phagocytosis of myelin sheath leading to chronic inflammatory demyelination. microglia are specialized macrophages of the cns that constitute 5-20% of total glial cells in rodents, depending on the specific neuroanatomical region of the cns. microglia are distinguished from neuron as well as glial cells, such as astrocytes and oligodendrocytes, by their origin, morphology, gene expression pattern, and function. while neuron and conventional glial cells are neuroectodermal in origin, microglia are of haematopoietic origin and act as primary responding cells for pathogen infection and injury like monocytes/macrophages in peripheral tissues. microglia exhibit several features that distinguish them from other populations of macrophages, such as their "ramified" branches that emerge from the cell body and communicate with surrounding neurons and other glial cells. microglia can rapidly respond to infectious and traumatic stimuli and adopt a "phagocytotic" nature. activated microglia are known to produce many proinflammatory mediators including cytokines, chemokines, reactive oxygen species (ros), and nitric oxide which mainly contribute to the clearance of pathogens or infections. however, prolonged or unwarranted microglial cell activation may result in pathological forms of inflammation which can lead to several neuroinflammatory conditions of the nervous system. microglia-mediated innate immune response in the cns is now considered to be potentially one of the major pathogenic factors in a number of cns neuroinflammatory diseases that lack 2 clinical and developmental immunology the prominent leukocytic infiltrates of adaptive immune responses [1] . neuroinflammation is associated with many neurodegenerative diseases, including alzheimer's disease (ad), parkinson's disease (pd), amyotrophic lateral sclerosis (als), and multiple sclerosis (ms) [2] . while ad, pd, and als are commonly known to be neurodegenerative disease with underlying neuroinflammatory mechanisms, ms is one of the major chronic inflammatory cns diseases in humans with heterogeneous (chronic/remitting) clinical presentations and course [3, 4] . ms is believed to be an autoimmune inflammatory demyelinating disease in which exposure of genetically predisposed people to environmental factors triggers a breakdown in t-cell tolerance to myelin antigens. demyelination is a complex process, and while the precise mechanisms of this pathology are unclear, inflammatory demyelination is thought to be the result of adaptive immune-mediated responses to myelin antigens in the myelin sheaths of axons and/or in the myelin-forming oligodendrocytes. most studies have focused on the pathogenic role of myelin-specific cd4 + t cells because of the relatively strong association of susceptibility to ms with major histocompatibility complex (mhc) class ii alleles [5, 6] . there is also increasing recognition of the potential importance of cd8 + t cells in the pathogenesis of demyelination [7, 8] . however, the contribution of innate immune cells in mediating ms pathogenesis has been recently gained attention, as several studies demonstrated the role of various innate immune cells in mediating ms pathogenesis, in particular, the potential anti-inflammatory or proinflammatory function of microglial cells along with its physical interaction with myelin [9] [10] [11] . for long time, microglia were known to be present in the chronic inflammatory demyelinating plaque to remove myelin from the dead sick neuron in ms patients but the emerging recognition of microglia as cns resident immune cells and their role in cns health and diseases stimulated substantial efforts to redefine the role and function of microglia in the regulatory mechanisms of demyelination. ms is best studied in some experimental models such as experimental autoimmune encephalitis (eae), theiler's murine encephalomyelitis (tmev), and mouse hepatitis virus-(mhv-) induced neuroinflammation. virtually, all types of adaptive immune response have been proposed to play important roles in the pathogenesis of eae [4, 12] , tmev [13] , and a neurotropic strain of mouse hepatitis virus (mhv); mhv-jhm [14, 15] , mimicking the pathogenesis of the ms. upon intracranial (i.c.) infection of neurotropic mhvs, acute meningoencephalitis (with or without hepatitis) is the major pathologic process (see supplementary figure 1 available online at http://dx.doi.org/10.1155/2013/510396) [16] . natural and genetically constructed recombinant mhv strains (generated by targeted rna recombination) with differential pathological properties were used in several studies to understand the mechanisms of demyelination and concomitant axonal loss [17] [18] [19] [20] . the outcome and degree of mhv-induced disease are dependent on several factors, including the age and strain of the mouse, the strain of mhv, and the route of virus inoculation. even very closely related strains of mhv differ in pathogenic properties. some strains of mhv are purely hepatotropic (e.g., mhv-2) [21] ; some are primarily neurotropic (e.g., jhm, mhv-4, an isolate of jhm) [15, 22] ; while others (e.g., mhv-a59 and mhv3) [16, 23] are both hepatotropic and neurotropic. viral titer reaches its peak at days 3 and 5 postinfection (p.i.) [21] . infectious virus is cleared within the first 10-14 days; however, at this time mice begin to develop demyelination, either clinical or accompanied by chronic hind limb paralysis [16, 24] . both mhv-jhm and mhv-a59 cause inflammatory demyelination in the brain and spinal cord whereas mhv3 only causes vasculitis [23, 25] . it was formerly believed that in primary mhv-induced demyelination neuronal axons remain relatively preserved. recently, it has been shown that axonal damage is, in large part, immune mediated in mhvinfected mice and occurs concomitantly with demyelination. concurrent axonal loss and demyelination have recently also been observed with s protein recombinant demyelinating strain-infected mouse spinal cord [20] . evidence from highly neurovirulent jhm strains of mhv suggests that mhv-induced demyelination is primarily immune mediated [26, 27] . clearance of infectious virus is mediated by both cytolytic and cytokine-mediated mechanisms and microglia, and t cells modulate pathologic changes. demyelination can be prevented in jhminfected lymphocyte-deficient (rag −/− ) mice [28] . however, demyelination will occur upon transfer of splenocytes from immunocompetent mice to rag −/− mice [28] . it has also been shown by depletion and transfer studies in the jhm model that cd8 + t cells can induce demyelination. these studies suggest that an intact adaptive immune system is required to promote demyelination in jhm-mhv infection. contrary to these findings, demyelination, induced by mhv-a59, has been shown to develop in adult immunocompromised mice lacking b and t cells [29] . it has also been demonstrated that the depletion of cd4 + or cd8 + t cells after the acute stage of infection does not reduce demyelination [28, 30] . indeed, mhv-a59 or its isogenic spike protein (hostattachment protein) recombinant strain, rsa59 [19, 20, 31] , induces a ms-like disease in mice mediated by microglia, along with a small population of t cells. the mechanism of demyelination is at least, in part, due to macrophagemediated myelin stripping, with some direct axonal injury as well as without involving the conventional t cells. in our current studies, we have used rsa59 infection in vivo, in vitro, and ex vivo as a model to understand whether mhv can directly infect cns resident microglia and the mechanism of microglial activation in the induction of chronic demyelination. use of animals and all experimental procedures were reviewed and approved by the institutional animal care and use committee at the indian institute of science education and research kolkata and indian institute of science, bangalore india. animal protocols adhered to the guidelines of the cpcsea, india. rsa59 an isogenic recombinant demyelinating strain of mhv-a59, where the spike gene (encodes virus host-attachment protein), was exchanged by mhv-a59 spike gene only in the background of mhv-a59 gene by targeted rna recombination as described in our previous studies [18, 19] . this recombinant strain also expresses enhanced green fluorescence protein (egfp) [32] for easy detection of viral particle by egfp fluorescence. to engineer the targeted recombinant strains, molecularly cloned vector pmh54 [33, 34] , which contains the entire 3 end of the genome from mhv-a59, was used for construction of the recombinant viruses. rsa59 and rsmhv2 are isogenic except the spike protein. rsa59 strain is expressing the mhv-a59 spike in the mhv-a59 background, whereas rsmhv2 strain is expressing the mhv-2 spike in the mhv-a59 background [18] . furthermore, egfp gene was inserted into the mhv genome in place of the nonessential gene 4 in both rsa59 and rsmhv2 [19] . in order to replace gene 4 with the egfp gene, pmh54 was modified by the introduction of a sali site 42 nucleotides downstream of the intergenic sequence for gene 4a and a noti site 102 bp upstream of the stop codon for gene 4b, using the quick change site-directed mutagenesis kit (stratagene, la jolla, ca, usa). (these are coding-silent nucleotide changes.) the coding sequence of egfp was cleaved from the pegfp-n1 vector (clontech, palo alto, ca, usa) using sali and noti and inserted in the place of the sali/noti fragment of pmh54. the resulting plasmid contains 760 bp of non-mhv sequence, including the 722-bp egfp open-reading frame, replacing the entire gene 4a and the rest 213 bp of gene 4b. previous studies reported with jhm strain revealed that the interruption of the orf 4 did not alter the neurovirulence neither in vivo nor the replication in vitro [35] . targeted recombination was used to select mhv isolates with stable and efficient expression of the gene encoding egfp to facilitate the in vivo detection of virus in the mouse cns as well as to trace the viral entry and spread in tissue culture. the viruses replicated with similar kinetics as wild-type virus both in tissue culture and in the mouse cns. they caused similar encephalitis and demyelination in animals as the wildtype virus or their recombinant strains; however, they were somewhat attenuated in virulence [19] . four-week-old, ten mhv-free, c57bl/6 (b6) mice (jackson laboratory, obtained from iisc, bangalore, india) were inoculated intracranially with 50% ld 50 dose of rsa59 strain (20,000 pfu) as described previously [19, 32] . mice were monitored daily for signs of disease. three mock-infected controls were inoculated similarly but with an uninfected cell lysate at a comparable dilution. three mice were sacrificed in between days 5, 6, or 7 (period for peak of inflammation postinfection for routine paraffinbased histopathological analysis), and the other three were used for frozen sections. the rest of the infected mice were sacrificed at day 30 postinfection for routine paraffin-based histopathological analysis. cervical, thoracic, and lumbar regions of spinal cord were successively processed, and 4 quadrants (dorsal/posterior column, anterior column, and two anterior horns) from two separate sections of each spinal cord level were examined. histopathology. at 5, 6, or 7 and 30 days postinfection, brain and spinal cord tissues were harvested from both mock infected and rsa59-infected mice. for routine paraffin sectioning, brain and spinal cord tissues were postfixed in 4% pfa for overnight. fixed tissues were processed and 5 micron thin sections were prepared for routine cns pathology, whereas frozen sections tissues were postfixed with 4% pfa for 4−6 hours and then transferred in 4% sucrose solution for 4-6 hours and in 20% sucrose solution for 16-24 hours and mounted in cryomatrix (thermo shandon). ten micron thin sections were prepared for frozen tissue immunofluorescence. the paraffin-embedded tissue sections were stained with hematoxylin and eosin (h&e) to determine the inflammation, whereas luxol fast blue (lfb) staining was used to detect the loss of myelin sheath. all slides are coded and read in blind manner. analysis. serial sections from brain and spinal cord were stained by the avidin-biotinimmunoperoxidase technique (vector laboratories) using 3,3-diaminobenzidine as substrate and a 1 : 100 dilution of anti-iba1 (wako, richmond, va, usa), 1 : 100 dilution of anti-cd45 (lca; leukocyte common antigen, ly-5, bd pharmingen), anti-iba1 (wako, richmond, va, usa), or cd3 (dako; carpinteria, ca, usa), and 1 : 20 dilution of monoclonal antibody directed against the nucleocapsid protein (n) of mhv-jhm (monoclonal antibody clone 1-16-1 (kindly provided by julian leibowitz)) as primary antibodies. control slides from mock-infected mice were incubated in parallel. cryosections from the spinal cord tissues were washed with pbs at room temperature in a humidified chamber, incubated for 10 min at room temperature with 1 mg/ml nabh 4 in pbs to reduce autofluorescence, washed, incubated for 1 h at room temperature with 1 m glycine in pbs to reduce nonspecific cross-linking, and then washed subsequently with pbs, pbs with 0.5% triton x-100 (tx), and pbs with tx and 2% goat serum (gs). the sections were incubated overnight at 4 ∘ c with a 1 : 100 dilution of a rabbit anti-iba1 antibody diluted in pbs with tx and gs, washed, and then incubated with a secondary antiserum diluted into pbs with gs for 2 hrs at 37 ∘ c. all incubations were carried out in a humidified chamber. viral antigen was detected by egfp in a fluorescein isothiocyanate channel [19] . control slides were incubated in parallel with preimmune rabbit sera, and sections from mock-infected mice were incubated with secondary antibodies only. tissue sections were sequentially washed with pbs plus tx and with pbs and mounted and visualized by ix-81 fluorescence microscopy with a 40x uplanapo objective, with the iris diaphragm partially closed to limit the contribution of out-of-plane fluorescence, and with filter packs suitable for green fluorescence and red fluorescence. images were acquired with a hamamatsu orca-1 charge-coupled device camera and image-pro image analysis software (media cybernetics, silver spring, md, usa). chronic inflammatory stage of rsa59 infection. to further characterize the presence of microglia in the chronic demyelinating plaque at the ultrastructural level, mice were anesthetized, perfused with 4% pfa, and spinal cords from mock-infected and rsa59-infected were harvested and fixed overnight in 2% glutaraldehyde as described earlier [20] . samples for transmission electron microscopy (tem) were postfixed with 1% osmium tetroxide, dehydrated, and flatembedded in poly-bed 812 epoxy resin (polysciences). half micrometer thick sections were cut from the lesional epicenter, stained with toluidine blue, and examined by light microscopy. ultrathin tem sections (600å) were cut from representative foci of demyelination from the toluidine bluestained semithin sections and mounted on 200 mesh copper grids, stained with uranyl acetate and bismuth subnitrate, and viewed under a jeol jem 1010. culture. four-week-old, mhv-free, c57 bl/6 were perfused transcardially with sterile pbs. spinal cord was harvested and washed with pbs containing 1% penicillin/streptomycin (pen/strep). the spinal cord was then embedded in 2% agarose mould, and 200 micron thick crosssections were prepared by vibratome (leica vibrating blade microtome; vt1200s). the slices were washed twice with pbs containing 1% pen/strep. the slices were then transferred to a 24-well plate with one slice in each well. 500 l of dmem containing 10% fbs, 1% pen/strep, and 1% l-glutamine were added in each well and incubated overnight with 5% co 2 . after 24 hrs of explantation, slices were infected with rsa59 at 20,000 pfu (half of the ld 50 dose) in low serum (2%) containing medium for 1 hr and then washed with pbs to remove the unbound viruses, and 10% serum containing medium were added to the infected culture and the cultures were maintained for 72 hrs. at 24 hrs, 48 hrs, and 72 hrs of postinfection, slices were processed for immunostaining with anti-iba1 antibody, and egfp fluorescence was used to detect viral antigen. briefly, at different times postinfection slices were washed gently with pbs and fixed with 4% pfa for 2 hours. postfixed slices were washed with pbs, permeabilized with 0.5% triton x-100 for 5 mins, and blocked with 1% goat serum for 1 hr at rt followed by overnight incubation with anti-iba1 antibody. for better staining, next day antibody solution was replaced with fresh antibody and incubated at 4 ∘ c for additional 16-20 hrs. slices were washed to remove any unbound antibody and then labelled with tritc conjugated goat anti-rabbit igg for 16 hrs. labelled slices were then washed to remove any unbound fluorescent tagged antibody and then mounted in vectashield (vector laboratories, ca, usa with dapi and observed in zeiss confocal microscope (lsm710). images were acquired and processed by using zen2010 software (carl zeiss). brain. primary cultures of mixed glia from day 0 to day 3 newborn mice were prepared as described previously [36] . briefly, following the removal of meninges, brain tissues were minced and incubated in a rocking water bath at 37 ∘ c for 30 min in hanks balanced salt solution (hbss, gibco) in the presence of 300 g/ml of dnasei (sigma) and 0.25% trypsin (sigma). enzyme-digested-dissociated cells were triturated with 0.25% of fetal calf serum (fcs), followed by a wash and centrifugation (300 ×g for 10 min). the pellet was resuspended in hbss, passed through a 70 micron nylon mesh, followed by a second wash and centrifugation (300 ×g for 10 min). following dilutions with astrocytespecific medium (dulbecco's essential medium containing 1% penicillin-streptomycin, 0.2 mm l-glutamine, and 10% fcs), cells were plated and allowed to adhere for 1 day in a humidified co 2 incubator at 37 ∘ c. after 24 hrs, any nonadherent cells were removed and fresh astrocyte-specific medium was added. adherent cells were maintained in astrocyte-specific medium for 10 days. culture. after establishment of the mixed glia culture, feeding was stopped for 10 days to allow for significant microglial growth on top of the astrocyte monolayer. the microglia population peaked at 12-14 days in these cultures. to remove any cells adherent to the astrocyte monolayer, microgliaenriched cultures were thoroughly agitated in an orbital incubator shaker (200 rpm for 40 min at 37 ∘ c). immediately following agitation, all cells suspended in the culture medium were collected and centrifuged at 300 ×g for 5 min at 4 ∘ c. the cell pellet was resuspended and diluted with fresh astrocytespecific medium bringing the cells to a final concentration of 8 × 10 5 cells/ml; 1 ml was added to each well of a two-well cc2-treated chamber slide (specifically made for primary cell culture; nunc) or 2 ml/well of a six-well plate. after 30 min, any non-adherent cells were discarded and adherent cells were maintained in fresh astrocyte-specific medium until infected with a medium change every 3-4 days. to examine different cell types in a given culture, primary antibodies directed against cell-specific antigens were used to determine the presence and/or purity of each of the major glial cell types as described previously. microglia were labelled with biotinylated anti-mouse cd11b (chemicon, diluted 1 : 100 in f-12 nutrient medium) followed by cy3streptavidin (jackson immunoresearch, diluted 1 : 200 in f-12). astrocytes were labelled with polyclonal rabbit antiglial fibrillary acidic protein (anti-gfap; dako) followed by either goat anti-rabbit alexa488 (molecular probes), cy2, or fitc (jackson immunoresearch) secondary antibodies. before processing for double-label immunofluorescence microscopy, cells were washed in f-12 nutrient medium. cells were incubated with primary antibodies to the surface clinical and developmental immunology 5 markers cd11b at room temperature followed by three 2 min washes with f-12. cells were then incubated with fluorescently coupled secondary antibodies for 30 min followed by three washes with pbs containing ca ++ /mg ++ . surfacelabelled cells were fixed for 10 min in 4% paraformaldehyde followed by pbs washes, permeabilized with pbs/tx (pbs with ca ++ /mg ++ , 0.5% triton-x) for 5 min, and successively washed with pbs/tx/gs (pbs with ca ++ /mg ++ , 0.5% triton-x, 2.0% normal goat serum) three times for 5 min each. cells were incubated for 30 min with the astrocytic marker gfap, washed three times with pbs/tx, labelled with an appropriate secondary antibody, and stained with dapi (1 : 500 diluted in pbs without ca ++ /mg ++ from 5 g/ml stock solutions) for 5 min. cells were then washed, mounted using vectashield (vector laboratories), and visualized by fluorescence microscopy (olympus i x-80) with a 20 planapo objective (1.0 numerical aperture). images were acquired with a hamamatsu orca ccd camera and data were analyzed by using image-pro software. on day 2 after seeding, neonatal microglial cultures were infected at a multiplicity of infection (moi) of 2 : 1 with rsa59 or mock-infected with noninfected cell lysate. after allowing viral adsorption for 1 hr, cells were washed and placed in fresh media without virus. at 6, 12, and 24 hrs after infection, cultures were examined by microscopy for egfp fluorescence. 2.14. rsa59 growth curve. confluent monolayers of l2 cells were infected with undiluted and 1 : 2 diluted culture supernatant collected from the in vitro infected microglia and incubated for 1 hr at 37 ∘ c. following adsorption, the cells were washed with tris-buffer saline 3 times and then fed with dmem with 20% fbs mixed with 0.4% agarose for overlaying. 48 hours postinfection, culture was subjected for plaque count [32] . to confirm the rsa59-induced cns inflammation, brain and spinal cord sections from day 7 (peak of inflammation) and day 30 (peak of demyelination) postinfected mice were stained with h&e or lfb and examined. rsa59-induced meningitis (supplementary figure 1(a) ), and encephalomyelitis (accumulation of inflammatory cell and perivascular cuffing) ( supplementary figures 1(b) and 1(c) ) were observed as shown previously [18, 19] (supplementary figure 1 ; these data are partly published but for the ready information compiled in one figure. ). to characterize inflammatory cell types, brain sections from day 7 postinfection were stained immunohistochemically with anti-cd45 (leukocyte common antigen (lca)), anti-cd11b and/or anti-iba1 (macrophage/microglial marker), or anti-cd3 (pan t-cell marker) (data not shown). the majority of inflammatory cells in rsa59-infected brains were immunoreactive for both lca (supplementary figure 1(d) ) and cd11b (supplementary figure 1(e) ) and iba1 (supplementary figure 1(f) ). some cd3-stained infiltrating t cells were also found (data not shown), although nonspecific background staining of neurons with available anti-cd3 antibodies made quantification difficult. no cd4-and cd19positive cells but few cd8-positive cells were observed in the inflamed brain and spinal cord sections in rsa59-infected mice (data not shown). demyelination was observed by lfb staining as early as day 7 as examined (supplementary figure 1(h) ) and it reaches its peak at day 30 postinfection (supplementary figure 1(i) ) as observed earlier [20, 31] . lfb-stained spinal cord section showed no myelin loss (supplementary figure 1(g) ). together, the data indicate that rsa59 causes meningoencephalitis and demyelination. cns inflammation consists of a mixed population of inflammatory cells, predominantly macrophages/microglia as well as a smaller population of t lymphocytes as shown previously [17, 20, 37] . infection during acute inflammation. previously, it has been demonstrated that neurotropic strains of mhv can directly infect different neural cell types [16, 19, 38, 39] but there is no evidence whether neurotropic strain can directly infect microglia or only acquire activity indirectly due to the infection of other neural cell types. in order to determine the tropism of rsa59 in cns resident microglia, fourweek-old, mhv-free, c57bl/6 (b6) mice (jackson laboratory) were inoculated intracranially with rsa59. mice were sacrificed at the peak of inflammation (day 6), and the spinal cord sections were prepared for cryostat sectioning. since rsa59 expresses egfp, viral antigen was viewed directly by fluorescence microscopy. identification of cns resident microglia was performed by using iba1 as a specific marker for microglia/macrophages [40] . while iba1 immunofluorescence was observed in both gray and white matter, double fluorescence/immunofluorescence demonstrated dual labelling of egfp (viral antigen) positive iba1 positive microglia/macrophages were present only in the white matter of rsa59 infected mice (figure 1 ). in the white matter, all the microglia (iba1-positive) were not infected as shown by arrowheads (figures 1(e) and 1(f) ). in the control mock infected spinal cord section, no double fluorescent labelled cells were observed as expected (data not shown). previous studies demonstrated that with time of postinfection viral antigen spread from gray matter to white matter [19] in the infected mice. this phenomenon is more prevalent in the spinal cord of infected mice as gray and white matter is clearly separated from white matter. immunostained section demonstrated that the viral antigen is localized both in gray and white matter at day 7 postinfection (figure 2(a) ). at day 30 viral antigen is below the detection level, more specifically after day 10 postinfection (as observed) viral antigen is below the detection limit (data not shown) as discussed previously [18, 19] . to determine whether microglia also follow the trajectory of the viral spread at days 7 and 30 postinfection, debris in demyelinating plaque. previously microglial accumulation was observed in the demyelinating plaque of rsa59 with an emphasis on the stripping of the myelin sheath [20] . to reemphasize on the accumulation of microglia in the demyelination plaque during chronic phase of the inflammation at ultrastructural level, semithin sections were cut at 1 micron intervals from five infected mice at day 30 post infection. semithin sections were stained with toluidine blue. control mock-infected mouse spinal cord was used to evaluate for background fixation and/or postfixation artefacts ( supplementary figure 2(a) ). rsa59-infected spinal cords showed significant myelin loss and accumulation of phagocytotic microglia within plaques as observed earlier ( supplementary figures 2(b) and 2(c)). representative foci of demyelination were selected from semithin sections, and 600å ultrathin sections from poly-bed embedded blocks were processed for tem. high-resolution tem images show accumulation of large number of microglia with no basement membrane which is the characteristic features of microglia/macrophages (supplementary figure 2(e) ). multiple vacuoles with myelin fragments were seen within the cytoplasm of the microglia in the plaque (supplementary figure 2(f) ). no such microglial accumulation was observed in the control mock infected mice at high-resolution tem images (supple figure 2(d) ). cells. in vivo colocalization of iba1 with egfp-(viral antigen) positive cells demonstrated that rsa59 can directly infect microglia but that does not confirm that infected microglia were resident microglia because in intracranial (ic) inoculation blood brain barrier can be disrupted and blood monocytes/macrophages can migrate and acquire infection. (figures 3(g) and 3 (k)) which demonstrated that cns resident microglia can directly acquire infection and become activated (by morphological analysis as number of processes increased and enlarged). arrowheads in figures 3(b) , 3(c), 3(d), 3(f), 3(g), 3(h), 3(j), 3(k), and 3(l) showed that some of the resident microglia did not get infection. control noninfected explant cultures were also immunolabeled with anti-iba1 antibody (figures 3(a) , 3(e), and 3(i)) as microglia in vitro in culture system behave like activated macrophages due to perturbation of the culture system. figures 3(d) , 3(h), and 3(l) show a merged image of egfp (viral antigen; green), iba1 (microglia; red), and dapi (nucleus; blue) and demonstrate the presence of viral antigen in the cell cytoplasm of microglia. due to the thickness of the slices, clarity of the images was slightly compromised. rsa59 infection in ex vivo explant cultures demonstrated that in the absence of peripheral immune cells cns resident microglia can directly be infected. syncytia. in order to determine whether rsa59 can infect microglial cells in vitro in absence of any neural cells, 2-dayold neonatal microglial cultures were infected at a multiplicity of infection (moi) of 2 : 1 with rsa59 or mock infected with noninfected cell lysate. microglia harvested in the cell suspension by the conventional shake-off method as described earlier [36] were 99 ± 0.5% positive for cd11b staining (figure 4(a) ). very few gfap (astrocyte marker) positive cells were observed in the isolated microglia culture (data not shown). at 0, 6, 12, and 24 hrs after infection, cultures were examined by microscopy for egfp fluorescence. at 0 and 6 hrs, no fluorescence was observed in the infected culture but at 12 hrs bright fluorescence started to appear denoting the presence of viral antigen in the microglia. at 12 hrs postinfection, infected microglia demonstrated stressed morphology and started to fuse with the neighbouring cells, and at 24 hrs postinfection, most of the infected cells were involved into large syncytia formation (figure 4(c) ) which is a characteristic of some enveloped rna viruses and more specifically characteristic of mhv-a59 (parental strain of rsa59), an enveloped demyelinating strain of mhv [41] . nucleus of the syncytia was very obvious as shown in figure 4 staining. in vitro experiment demonstrated that rsa59 can infect primary microglia in isolated culture and can also induce syncytia in primary microglia. to demonstrate that the virus is replicating in the microglia, culture supernatant of infected microglia was assessed by routine plaque assay [32] . routine plaque assay found very few plaques which were below the detection limit. but there, discrete syncytia was observed in the infected plates which denoted that the titer could be 30-50 pfu/ml. to understand the cellular mechanism of demyelination of neurotropic strain of mhv, prior studies in our laboratory have analyzed the detailed pathogenesis of recombinant mhv strain, rsa59 (demyelinating strain (dm)) and compared it with rsmhv2 (nondemyelinating strain (ndm)) that is isogenic except for the spike gene that encodes the virus-host-attachment spike glycoprotein [18, 20] . both strains are capable of causing hepatitis, encephalitis, and meningitis. however, the two strains differ in their ability to induce subsequent demyelination and axonal loss [20] . seven days post infection, rsa59 produces demyelination that is best observed in the spinal cord at day 30 postinfection (peak of inflammation). in contrast, rsmhv2 does not produce demyelination and only rarely demonstrates axonopathic changes in spinal cord white matter [20] . the inability of rsmhv2 to induce demyelination is due in part to a lack of transport of viral antigen (and the subsequent inflammatory reaction) to the white matter. furthermore, in vivo and in vitro experiments demonstrate deficits in the ability of rsmhv2 to spread between neurons when compared to interneuronal spread by rsa59 [20] . rsa59-induced demyelination occurs in the setting of both axonal degeneration and macrophage mediated myelin stripping along intact axons [20] . while spike glycoprotein mediates spread of viral antigen to white matter through axonal transport, specific mechanisms leading to subsequent demyelination are not known. one plausible explanation is that mhv spreads intra-axonally within gray matter and when it reaches the white matter, viral particles may need to spread directly into oligodendrocytes, astrocyte, and microglia, using the spike protein, and can induce local oligodendroglial dystrophy and inflammation. viral antigen in white matter axons may be sufficient to trigger an inflammatory response via microglial activation. infected and activated microglia due to its intrinsic nature of chemotaxis can recruit more microglia to the site of inflammation and strip myelin from the damaged axon and successively cause demyelination. our current in vivo studies support this hypothesis that rsa59 can infect cns resident microglia. the migration and activation of numerous microglia to the white matter during acute inflammation and the retention of microglia in the chronic inflammatory plaque reinforce the hypothesis that cns resident microglia can be recruited to the region of local cns injury. ultrastructural morphology of microglia containing multiple vacuoles with myelin fragments in the cytoplasm in the demyelinating plaque further substantiate that cns resident activated microglia can mediate myelin stripping and can successively mediate demyelination. as rsa59 spread intra-axonally, no colocalization was observed within the gray matter cns resident microglia. if haematogenous propagation of peripheral monocytes/macrophages occurred to the cns, one would expect more widespread distribution of activated microglia throughout the spinal cord which may not discriminate gray/white matter track. furthermore, the delay in complete development of demyelination following partial resolution of encephalitis (up to 30 days after peak inflammation) documented in previous studies would not be expected [16, 18, 31] . moreover, ex vivo colocalization of egfp-positive cells with microglia confirmed that rsa59 can directly infect cns resident microglia in absence of peripheral immune cells. in vitro infections of neonatal microglia demonstrate that rsa59 not only infects, but microglia can also forms syncytia which suggests that microglia supports rsa59 infection via cell-to-cell contact. current combined in vivo, in vitro, and ex vivo explants culture studies established that the recruitment of microglia occurred from the cns resident microglial pool rather than peripheral monocyte/macrophages. our current studies are focused on the understanding of the innate immune mechanism of cns resident microglia activation and maturation to perform phagocytotic activity. affymetrix microarray analyses for mrna expression have revealed that expression of inflammatory mediators by mhv infected microglia, including chemokine and inflammatory cytokines. mhv infection of the mouse spinal cord was also associated with increased expression of genes involved in ifn signalling compared to mock-infected controls in the cns. during chronic infection (day 30 postinfection), microglia are still present within areas of demyelination and microgliaassociated inflammatory mediators are still produced which indicates that microglia are still active. our results suggest that putative activated microglia and inflammatory mediators contribute to a local cns microenvironment that eventually regulates viral replication and ifn-gamma production during acute phase of infection. sequentially, ifn-can activate microglia by promoting phagolysosomes maturation and activation (engulfment of the myelin sheath) leading to demyelination. affymetrix microarray data warrants further confirmation. viral infection in the cns is classically recognized as inflammatory in nature, with meningeal perivascular and parenchymal infiltrates of peripheral leukocytes but rsa59 infection could be an exception where inflammation can proceed with cns resident glial activation without involving the peripheral immune responses like rabies virus infection [42] , hiv infection [43] , and prion diseases [44, 45] . in this perspective, it is tempting to speculate that the underlying mechanism of chronic myelin loss in ms could be a combination of persistence of myelin-related autoimmunogens that has escaped self-tolerance with persistence of activated cns resident microglia which can mediate demyelination by phagocytised myelin. microglia are known for their innate immune function for long time but the role of microglia in chronic inflammation opens a new episode in the field of glial biology of neuroinflammatory diseases. the concept of chronic inflammation as opposed to acute inflammation is more relevant in the context of understanding other cns diseases, more specifically neurodegenerative diseases like alzheimer's disease, amyotrophic lateral sclerosis, parkinson's disease, and huntington's disease. these neurodegenerative diseases lack the prominent infiltrates of mononuclear cells but the underlying mechanism of inflammation could be through activation of cns resident microglia. activation of cns resident microglia in the context of chronic neuroinflammation as one of the underlying mechanism of neurodegeneration warrants further study. microglia as the prime components of an intrinsic cns resident immune system become a major focus in cellular neuroimmunology and, therefore, in neuroinflammation. it has been known for long time that in absence of conventional t cells microglia play a major role in neurotropic mhv-induced demyelination but the mechanism of infection and route of infection were not very clearly known for long time. our current microglial tropism studies revealed that rsa59, an isogenic demyelinating strain of mhv, can infect and activate cns resident microglia, and microglia can help to mediate demyelination by engulfing myelin debris. rsa59-induced neuroinflammatory models are helpful in understanding direct cns cellular injury and demyelination that does not require an intact adaptive immune system. understanding the role of direct cns resident microglial infection and activation will shed some light on the pathogenesis of cns inflammatory disease, not only infectious diseases but also chronic cns disorders. the vision of cnsresident-microglia-driven neuroinflammatory responses in rsa59 with neuropathological consequences has extended the avenue to explore the contribution of microglia in chronic neuroinflammatory cns diseases. microglia and neuroinflammation: a pathological perspective mechanisms underlying inflammation in neurodegeneration multiple sclerosis immunology of multiple sclerosis a full genome search in multiple sclerosis a complete genomic screen for multiple sclerosis underscores a role for the major histocompatibility complex a pathogenic role for myelin-specific cd8 + t cells in a model for multiple sclerosis autoreactive cd8 + tcell responses to human myelin protein-derived peptides role of the innate immune system in the pathogenesis of multiple sclerosis t cells-innate immune lymphocytes? multiple sclerosis: a complicated picture of autoimmunity myelin-specific cd8 t cells in the pathogenesis of experimental allergic encephalitis and multiple sclerosis persistent infection with theiler's virus leads to cns autoimmunity via epitope spreading pathogenesis of mouse hepatitis virus-induced demyelination chronic central nervous system demyelination in mice after jhm virus infection experimental demyelination produced by the a59 strain of mouse hepatitis virus a mechanism of virus-induced demyelination demyelination determinants map to the spike glycoprotein gene of coronavirus mouse hepatitis virus demyelinating and nondemyelinating strains of mouse hepatitis virus differ in their neural cell tropism mechanisms of primary axonal damage in a viral model of multiple sclerosis mouse hepatitis virus type-2 infection in mice: an experimental model system of acute meningitis and hepatitis pathogenesis of virusinduced demyelination selective tropism of a neurotropic coronavirus for ependymal cells, neurons, and meningeal cells limbic encephalitis after inhalation of a murine coronavirus ependymitis, leukoencephalitis, hydrocephalus, and thrombotic vasculitis following chronic infection by mouse hepatitis virus 3 (mhv 3) bystander cd8 t cell-mediated demyelination after viral infection of the central nervous system demyelination induced by murine hepatitis virus jhm strian (mhv-4) is immunologically mediated cd4 and cd8 t cells have redundant but not identical roles in virusinduced demyelination neither b cells nor t cells are required for cns demyelination in mice clinical and developmental immunology persistently infected with mhv-a59 cd4 + and cd8 + t cells are not major effectors of mouse hepatitis virus a59-induced demyelinating disease experimental optic neuritis induced by a demyelinating strain of mouse hepatitis virus enhanced green fluorescent protein expression may be used to monitor murine coronavirus spread in vitro and in the mouse central nervous system the internal open reading frame within the nucleocapsid gene of mouse hepatitis virus encodes a structural protein that is not essential for viral replication analysis of a recombinant mouse hepatitis virus expressing a foreign gene reveals a novel aspect of coronavirus transcription inactivation of expression of gene 4 of mouse hepatitis virus strain jhm does not affect virulence in the murine cns magnetic cell sorting: a fast and effective method of concurrent isolation of high purity viable astrocytes and microglia from neonatal mouse brain tissue macrophagemediated optic neuritis induced by retrograde axonal transport of spike gene recombinant mouse hepatitis virus selective localization of wild type and mutant mouse hepatitis virus (jhm strain) antigens in cns tissue by fluorescence, light and electron microscopy viral infections and demyelinating diseases neurofibromatosis-1 heterozygosity increases microglia in a spatially and temporally restricted pattern relevant to mouse optic glioma formation and growth syncytia formation induced by coronavirus infection is associated with fragmentation and rearrangement of the golgi apparatus rabies virusinduced activation of mitogen-activated protein kinase and nf-b signaling pathways regulates expression of cxc and cc chemokine ligands in microglia microglia in human immunodeficiency virusassociated neurodegeneration neuroinflammation in alzheimer's disease and prion disease atypical inflammation in the central nervous system in prion disease key: cord-277054-eq4obbte authors: kaur, manpreet; rai, anant; bhatnagar, rakesh title: rabies dna vaccine: no impact of mhc class i and class ii targeting sequences on immune response and protection against lethal challenge date: 2009-03-26 journal: vaccine doi: 10.1016/j.vaccine.2009.01.128 sha: doc_id: 277054 cord_uid: eq4obbte rabies is progressive fatal encephalitis. who estimates 55,000 rabies deaths and more than 10 million pep every year world-wide. a variety of cell-culture derived vaccines are available for prophylaxis against rabies. however, their high cost restricts their usage in developing countries, where such cases are most often encountered. this is driving the quest for newer vaccine formulations; dna vaccines being most promising amongst them. here, we explored strategies of antigen trafficking to various cellular compartments aiming at improving both humoral and cellular immunity. these strategies include use of signal sequences namely tissue plasminogen activator (tpa), ubiquitin (uq) and lysosomal-associated membrane protein-1 (lamp-1). tpa, lamp-1 and their combination were aimed at enhancing the cd4(+) t cell and antibody response. in contrast, the uq tag was utilized for enhancing cd8(+) response. the potency of modified dna vaccines assessed by total antibody response, antibody isotypes, cytokine profile, neutralizing antibody titer and protection conferred against in vivo challenge; was enhanced in comparison to native unmodified vaccine, but the response elicited did not pertain to the type of target sequence and the directed arm of immunity. interestingly, the dna vaccines that had been designed to generate different type of immune responses yielded in effect similar response. in conclusion, our data indicate that the directing target sequence is not the exclusive deciding factor for type and extent of immune response elicited and emphasizes on the antigen dependence of immune enhancement strategies. rabies, progressive fatal encephalitis [1] is caused by rabies virus of genus lyssavirus. majority of rabies cases reported from developed countries involve wild animals like raccoons, skunks, bats, and foxes. however, it is a major health concern in developing countries which account for more than 99% of all human deaths from rabies [2] . exposure to rabid dogs is the cause of bulk (>99%) of human rabies deaths world-wide [3] . india has a particularly severe problem, with as many as 30,000 human deaths and 2 million people requiring post-exposure vaccination yearly. stray and community dogs cause vast majority of human cases. though potent and safe cell-culture derived inactivated vaccines are available, their efficacy may be compromised by disruption of cold chain storage, poor general health status of the subject, poor vaccination techniques. as a consequence, several approaches are currently being investigated experimentally; out of which dna vaccines appear to be particularly promising as they can induce persistent, cell-mediated and humoral immune responses to antigens isolated from a variety of viral, bacterial, and parasitic pathogens. besides their immunogenicity, dna vaccines offer several other practical advantages. dna vaccines being free from foreign proteins may not cause the various side reactions, which may be observed for conventional vaccines. in addition to the safety, they may have benefits of being inexpensive, overcome the need of time consuming procedures that are needed for purification of subunit proteins, are stable, can be stored and transported at room temperature. in animal models of human disease, dna vaccines have been shown to induce protective responses against hiv, influenza, bovine herpes virus, rabies, leishmaniasis, malaria, herpes simplex virus, and tuberculosis [4] [5] [6] . in addition, human clinical trials have established their safety and potency, further encouraging studies in this direction [6] [7] [8] [9] [10] . in this regard, various dna vaccination strategies have shown to provide protection against lethal rabies virus challenge. these strategies relied on the usage of adjuvants like cationic-lipids [11, 12] , intradermal injection using gene gun [13] [14] [15] , repeated dna vaccination [16, 17] or dna vaccine in association with a single dose of anti-rabies immune serum [18] for immune enhancement. prophylactic immunization was found to be effective in preventing canine rabies [17, 19, 20] . rabies dna vaccine has also been found to be highly efficient in large size mammals [21] . for post-exposure treatment, single dose of rabies dna vaccine was found to be as potent as 5 dose regimen of cell-culture vaccine in balb/c mice [22] . considering these studies, we attempted further improvement in humoral and cell-mediated immune response elicited by dna vaccination by antigen trafficking to various cellular compartments. efficient delivery of antigens to both mhc class i and class ii processing and presentation pathways is required for generating an ideal immune response comprising of both cd8 + and cd4 + (cellmediated) and antibody (humoral) immune response. accordingly, this study investigates strategies for targeting glycoprotein antigen to mhc class i and class ii pathways for improving its antigenicity, immunogenicity and protective efficacy. for targeting mhc class ii pathway, we utilized tissue plasminogen activator (tpa) and human lysosomal-associated membrane protein-1 (lamp-1) signal sequences. tpa-fused antigens are highly expressed secreted proteins with elevated uptake by antigenpresenting cells; and thus, bring about a more generalized activation of the immune system. they have been shown to induce significant humoral and cell-mediated responses [23] . lamp-1 is a type of transmembrane protein localized predominantly to lysosomes and late endosomes. antigen trafficking of lamp-1-fused antigens to the cellular site of mhc class ii processing and presentation pathway could enhance its presentation to mhc class ii restricted cd4 + t cells [24] and thus augment the humoral response. on the other hand, for directing the antigen to mhc class i, signal sequence of ubiquitin a-76 (uq) was employed. uq-conjugated antigens are trafficked through the proteasome, an organelle that generates short peptides for presentation via the mhc class i pathway [25] . such uq-conjugated antigens are expected to enhance the cellular immune response. uq-conjugated proteins have been shown to generally undergo rapid intracellular degradation and can elicit cytokine responses in the absence of specific antibody production [26] . thereby, we explored the potential of targeted dna vaccine encoding the glycoprotein antigen fused to tpa, lamp-1 or uq to elicit superior immune response in comparison to the unmodified (without target sequence) dna vaccine. baby hamster kidney (bhk)-21 cells; procured from national centre for cell science (nccs), pune, india were maintained in dulbecco's modified eagle's medium (dmem, sigma) supplemented with 10% of heat-inactivated fetal bovine serum (fbs, biological industries) and 100 u/ml penicillin (amersham) and 100 g/ml streptomycin (amersham), in a humidified 5% co 2 incubator at 37 • c. virus pitman-moore (pv-11) strain of rabies virus was propagated on bhk-21 cells. virus was purified, inactivated with beta-propionolactone (bpl) and used for in vitro re-stimulation assay. the challenge virus standard (cvs-11) strain was propagated and maintained in mice brain. it was titrated on bhk-21 cells to determine the optimal dose for rapid fluorescence focus inhibition test (rffit) to determine virus neutralization antibodies and for intracerebral rabies virus challenge to determine protection conferred. plasmid ptarget-rab-g [27] was used as the parental plasmid for construction of all the clones used in this study. the glycoprotein (1.57 kb) gene was amplified by pcr from ptarget.rabgp plasmid using sequence-specific primers (supplementary table 1 ) and cloned in eukaryotic plasmids bearing address tags; pdnavacc vectors (nature technology corporation, nebraska). the sequences of clones bearing the address tags, pgp-native, ptpa.gp.lamp-1 (genbank accession number eu715585), ptpa.gp (genbank accession number eu715586), puq.gp (genbank accession number eu715587), pgp.lamp-1 (genbank accession number eu715588) were confirmed by sequencing using abi prism, model 3730, version 3.0 (sequencing primers listed in supplementary table 1) and designated as represented in the fig. 1 . the respective constructs were processed for the purification of plasmid dna using the endofree plasmid isolation maxi kit (qiagen) according to the manufacturer's instructions. the purified plasmid dna (1-2 mg/ml) was dissolved in autoclaved milli quartz (mq) water and stored at −20 • c, until further use. the glycoprotein gene was similarly pcr amplified (see supplementary table 1 for primer sequences) using ptarget.rabgp as template and cloned in pqe30 expression vector (t5 expression system). rgp was expressed as a fusion protein with 6× histidine tag in e. coli sg (prep-4) strain and was purified on a ni 2+ -nta column to more than 95% homogeneity under native conditions. the rgp was dialyzed against 10 mm hepes overnight and stored in aliquots at −80 • c. the ability of vaccine constructs to express glycoprotein antigen was studied in vitro in a mammalian cell-culture system. briefly, bhk-21 cells were cultured and seeded at a density of 1 × 10 6 cells/ml in a 24-well tissue culture plate, a day prior to transfection. bhk-21 cells were subsequently transfected with 800 ng dna complexed with 2 l of lipofectamine (invitrogen) and 8 l of plus buffer (invitrogen). the analysis of expression and localization was carried out 40 h post-transfection. for assessing expression, flow cytometric analysis was carried out, in which bhk-21 cells were transfected with various dna vaccine combinations. transfected cells were fixed with 2% paraformaldehyde (pfa) and then permeabilized in 0.1% triton x-100 in pbs. the cells were then probed with mouse anti-rabies polyclonal sera, diluted in pbs containing 0.5% bsa; followed by staining with alexa fluor 488 labeled secondary antibody (molecular probes) diluted in pbs containing 0.5% bsa and analyzed for fluorescence using cell lab quanta tm sc mpl flow cytometer (beckman coulter inc.). control healthy bhk-21 cells were also stained. ten thousand cells per sample were analyzed using fl1 filter (525 nm) and percent green fluorescent cells were recorded using quanta sc mpl analysis software version 1.0 (beckman coulter inc.). for immunoblotting, total cell lysate was prepared. transfected cells were solubilized using radioimmunoprecipitation assay (ripa) buffer [50 mm tris(hydroxymethyl)aminomethane (tris), ph 7.4, 150 mm sodium chloride (nacl), 0.1% sodium dodecyl sulphate (sds), 1% triton x-100, 1% sodium deoxycholate, 1 mm phenyl methyl sulphonyl fluoride (pmsf)] supplemented with protease inhibitor cocktail (sigma). lysosomal fraction was extracted using lysosome enrichment kit for tissue and cultured cells (pierce) according to the manufacturer's protocol. the presence of lysosomes in different fractions was determined by analyzing the activity of ␤-hexosaminidase [28] . cell membrane protein fraction was prepared by qproteome membrane protein kit (qiagen) according to the manufacturer's protocol. the presence of cell membrane in the fractions was determined by the associated nadh oxidase activity [29] . culture supernatant proteins were precipitated by ice-cold acetone. solubilized proteins from the total cell lysate, lysosomes, membranes and cell-culture supernatants were subjected to 12% sds-page, blotted on to nitrocellulose membrane and probed with mouse anti-rabies polyclonal sera, followed by incubation with goat anti-mouse immunoglobulins conjugated with alkaline phosphatase (sigma) and visualized with nbt/bcip substrate (sigma). all animal experiments were conducted in compliance with the animal ethics committee. four to six weeks old female balb/c mice were used to verify the immunogenicity of the constructs. mice were purchased from nin, hyderabad; and maintained in pathogen free environment at the animal house facility. each group comprised of ten mice. mice were vaccinated intramuscularly (i.m.) with 100 g endotoxin-free plasmid dna in 200 l pbs/animal in the individual groups (dna vaccine or vector control), thrice at three-week intervals. control mice were immunized with only pbs. the mice from each group were bled at days, 20, 41 and 62; sera were prepared and stored at −80 • c. antigen-specific antibody (igg total) and isotypes (igg1, igg2a) levels were determined by elisa in the serum from immunized mice. recombinant glycoprotein, expressed in bacterial system (500 ng/well) in 100 l of 0.1 m pbs was coated overnight at 4 • c [30] . plates were then blocked with 2% bsa in pbs for 2 h at 37 • c followed by three washings with pbs-tween 20 (0.05%). this was followed by incubation with sera samples for 2 h at 37 • c and washing with pbst. secondary antibodies, anti-mouse igg or its isotypes conjugated with horseradish peroxidase; raised in sheep (santa-cruz) were incubated for 1 h at 37 • c. estimation of the enzymatic activity was carried out using tmb as the substrate. the reaction was stopped with 50 l of 1 m h 3 po 4 and the absorbance was measured at 450 nm, with 630 nm as the reference filter using microplate reader (bio rad). the antibody response generated in a group of vaccinated mice was represented as the geometric mean of the absorbance obtained by pooled serum samples of the animals; the reaction being carried out in triplicates. mouse sera were tested in vitro for the presence of virusneutralizing antibodies with rffit, as described previously [31] . briefly, sera from mice were heat inactivated at 56 • c for 30 min. 100 l of various sera dilutions were mixed with 100 l of the cvs-11 strain of rabies virus (containing 50 ffd 50 ) in 96-well tissue culture plate and incubated at 37 • c, 5% co 2 for 90 min. after the incubation period, bhk-21 cells (1 × 10 5 ) were added to each well and the plates were incubated for 40 h, following which they were fixed with chilled acetone and stained with fitc-conjugated antirabies monoclonal antibody (vmrd, usa) for 45 min. the wells were washed thrice with pbs, mounted in glycerol: pbs (1:1), and visualized under fluorescence microscope (nikon, japan). data were expressed as the neutralizing antibody titer that is the mean of the serum resulting in a 50% reduction in the number of the virus-infected cell foci in the presence of the test serum. rabies reference antiserum of known international units (iu/ml) of rabies virus-neutralizing antibody was included as positive control in the assay. splenic cells were prepared by grinding spleens between frosted slides. erythrocytes were lysed with 0.1 m ammonium chloride. remaining spleen cells were washed twice with dmem medium and then were suspended in complete dmem medium supplemented with 10% heat-inactivated fetal bovine serum and 10 −6 m 2-mercaptoethanol. viability was determined by trypan blue exclusion test. splenocytes were cultured in triplicate (1 × 10 6 cells/well) in a 24-well culture plate (costar), stimulated without antigen or with 5 g/ml of bpl inactivated pv-11 virus or concanavalin a (cona) (1 g/ml; sigma), and incubated at 37 • c under 5% co 2 and 95% humidity. supernatants were harvested after 24, 48 and 72 h and the levels of cytokines were determined. levels of il-2, il-4, il-12, and ifn-␥ were determined using bd opt eia tm kits according to manufacturer's protocol (pharmingen). briefly, 96-well microtiter elisa plate was coated with capture antibody of the respective cytokines and incubated overnight at 4 • c. plate was aspirated and washed thrice and blocked with 200 l subsequently, the protein samples were resolved on 12% sds-page under reducing conditions and transferred onto nitrocellulose membrane. presence of rabies glycoprotein was detected using mouse polyclonal anti-rabies hyperimmune sera followed by alkaline phosphatase-conjugated anti-mouse igg. the blot was developed using bcip/nbt as substrate. of 2% bsa for 2 h at 37 • c. after the incubation period, plate was aspirated and washed thrice and incubated with the harvested supernatants for 2 h at rt. the plate was then aspirated and washed five times; plate was incubated with detector (anti-mouse igg-hrp) for 1 h at rt. following this, plate was aspirated and washed 7 times and incubated with 100 l substrate solution for 30 min in dark at rt. reaction was stopped by adding 50 l stop solution to each well. the absorbance was read at 450 nm using a microplate reader (bio rad) within 30 min of stopping the reaction. the concentrations of cytokines in the culture supernatants were calculated using a linear regression equation obtained from the absorbance values of the standards provided by the manufacturer. each vaccine construct was tested in two independent experiments. for challenge, immunized mice were inoculated with 20 ld 50 of rabies virus cvs-11 strain intracerebrally 21 days postimmunization. the challenged mice were observed for 18 days for symptoms indicative of rabies virus infection. mice that developed complete bilateral hind leg paralysis, characteristic of the terminal stage of rabies, were euthanized for humanitarian reasons. upon challenge, pbs or vector vaccinated mice died within 6-13 days. surviving mice were kept and observed for an additional two to three weeks to ensure that they survived the infection. survivorship rates obtained with the different vaccine constructs were compared. the experimental data were analyzed by sigma plot 10.1 and were expressed as means ± standard deviations (s.d.). comparisons between individual data points were made using a student's t-test and levels of significance (p value) were determined. p value <0.05 was considered statistically significant. rv-g based dna vaccine constructs were made wherein the glycoprotein gene was fused to various signal sequences (fig. 1) to analyze the influence of signal sequences on immunogenicity and generation of rvna titers. the sequences of insert in native dna vaccine construct (pgp) and modified constructs bearing the target sequences, pt.gp.l, pt.gp, pu.gp, pgp.l were confirmed by sequencing. for assessing the expression of dna vaccine constructs, transiently transfected bhk-21 cells were subjected to flow cytometric analysis. the percentages of cells stained with the antibody are shown in the figure (fig. 2a) . the transfected cells were expressing the rabies glycoprotein as indicated by fluorescence recorded as 69.33%, 55.28%, 74.43%, 69.41 %, 74.85 % for pgp, pt.gp.l, pt.gp, pu.gp and pgp.l respectively; whereas the control cells revealed a low fluorescence signal (8.39%) (fig. 2a) . as the majority of cells showed expression of the rabies glycoprotein, it can be inferred that all the constructs were capable of expressing the protein efficiently in transfected cells. for assessing the localization of dna vaccine constructs, transiently transfected bhk-21 cells were subjected to subcellular fractionation and subsequently visualization by western blotting. for the same, total cell lysate, lysosomal fraction, membrane fraction and cell-culture supernatant of tranfected cells were resolved on sds-page followed by probing with hyperimmune polyclonal serum from mice immunized with rabies virus. prominent immunoreactive protein bands were observed on the blot corresponding to cell lysate of the all the dna vaccine constructs (fig. 2b, topmost panel) . there was no corresponding band in cell lysate from vector-transfected or mock-transfected bhk-21 cells (data not shown). the observed molecular weight of approximately 67 kda was consistent with the expected sizes of glycosylated glycoprotein. analysis of subcellular fraction revealed that the pgp.l construct encoded lysosomal form of glycoprotein (second panel). the pt.gp construct encoded secreted form of glycoprotein (third panel). further, dual tagged construct pt.gp.l expressed both secreted and lysosomal form of glycoprotein (second and third panels). pu.gp construct was exclusively expressed in cell associated form (topmost panel). the native construct encoded membrane associated glycoprotein (fourth panel). expression of glycoprotein in various subcellular fractions was comparable to that from cell lysate. to address the issue if these vaccines could induce efficient humoral response was assessed. groups of 10 balb/c mice were vaccinated intramuscularly with dna encoding either the unmodified or modified antigen. anti-glycoprotein antibody response was estimated by elisa with recombinant glycoprotein (expressed in bacterial system, unpublished data). all the mice sero-converted after priming, however, maximum titers were obtained after second booster both for unmodified as well as modified dna vaccine. for clarity, only the means and standard deviations for each group are shown (fig. 3) . all vaccine groups mounted antibody response higher than the unmodified vaccine. the highest antibody response was generated in the pgp.l immunized group (p value <0.005), closely followed by pt.gp.l. there was insignificant antibody response in vector and pbs immunized mice. trafficking of glycoprotein through different pathways may affect the type of immune response elicited against it. like pt.gp mediated trafficking of glycoprotein from cytoplasm to secretion pathway, which targets molecules through the endoplasmic reticulum and golgi; may lead to higher induction of th2 type of immune response. likewise, pgp.l mediated trafficking may drive the glycoprotein through trans-golgi network directly to 4 . the isotype profile of the rv-g-specific igg1 (black bars) and igg2a (gray bars) titers in mice immunized by different protocols. each group of mice (n = 10) was immunized respectively by dna, vector or pbs. mice were bled at three weeks after the last immunization and glycoprotein-specific igg1 and igg2a titers were detected by elisa. optical density was measured at 450 nm. data shown represent geometric mean titers and standard deviations for each group of animals. endosomes and then to lysosomes, again influencing the th2 type response. pt.gp.l may channelize the glycoprotein through either of the above pathways, to affect the th2 type of immune response. on the contrary, pu.gp is expected to enhance the proteolysis of conjugated glycoprotein mediated by the ubiquitin-proteasome pathway for enhancing the processing and presentation for th1 type of immune response. to examine such a possibility, serum from mice immunized with pgp, pt.gp, pt.gp.l, pu.gp and pgp.l was assayed by probing with isotype specific secondary antibodies. immunization with all the constructs led to an igg1-dominated response (fig. 4) indicating the th2 bias. thus, our results show that addition of signal sequence did not affect the isotype inclination following the immunization and there was convergence of the antibody response, in spite of differential targeting. further, we explored the possibility of enhancement in neutralizing antibodies against glycoprotein when modified antigens were employed for the immunization experiments. rabies virusneutralizing antibody (rvna) titers were assessed by rffit; three weeks post the last immunization corresponding to the time of lethal challenge. the rvna titer in all the groups of immunized mice was >0.5 iu/ml; the minimum titer against rabies as recommended by who. as shown in fig. 5 , the highest geometric mean rvna titer was observed for pgp.l (16 iu/ml, p value <0.005), followed by pt.gp.l and pu.gp with titer of 8 iu/ml. the neutralizing antibody potential of tpa tagged vaccine was found be the lowest, equivalent to the unmodified antigen based dna vaccine (4 iu/ml). in comparison, vector or pbs immunized group did not induce significant neutralizing antibodies. t helper cells (th1/th2) play an important role in eliciting both humoral and cellular responses via expansion of antigenstimulated b cells and cd8 + t cells or ctls respectively. the levels of some cytokines which may play key roles in the induction of protective immune responses against rabies virus were studied as parameters of polarization of immune response. th1 cytokines (il-2, il-12, and ifn-␥) and th2 cytokine (il-4) were measured from splenocytes from immunized mice by elisa at 24, 48 and 72 h after re-stimulation with inactivated pv-11 virus. il-2 production substantially increased on immunization with pgp.lamp-1; 28.03 pg/ml i.e., ∼14 fold higher as compared to the response from control (splenocytes from pbs immunized mice) was observed (p value <0.005). all the constructs exhibited significant il-4 and ifn-␥ production. there was no significant increase in the cytokine levels of mice immunized with vector or pbs. il-12 production also strongly increased in case of pgp.lamp-1, ∼35 fold superior than the control group (p value <0.005). the cytokine profile is summarized in fig. 6 . the ability of these dna vaccines to elicit protective responses in immunized mice was assessed by intracerebral challenge with 20 ld 50 of virulent rabies virus cvs strain. for controls, vector and pbs immunized mice were also challenged. the lethality of the challenge was confirmed by death of all the mice in the vector and pbs immunized group within 4-11 days post-challenge. groups of vaccinated mice that developed significant levels of virus-neutralizing antibodies also survived rabies virus challenge. the protection conferred by dna vaccines was found to be significant (p value <0.005). all modified antigen groups apart from pt.gp conferred higher protection than the unmodified dna vaccine, with 60% and 40% protection levels conferred respectively. surviving mice did not show any signs of rabies virus infection. kaplan-meier curves for survival of dna vaccine immunized mice are summarized in fig. 7 . a variety of cell-culture derived vaccines are available for prophylaxis against rabies [1] . however, the high cost of the vaccination therapy along with the risk of developing anaphylactic, neuroparalytic or encephalitic side reactions limit their therapeutic application. these facts indicate the need of more faithful candidate vaccines which must be capable of inducing strong immune response to protect from infection. more importantly, the candidate immunogen must be able to induce a strong th2 immunity as it has been established that th2; that is, the humoral immune response plays a predominant role in induction of protective immunity against rabies virus [32] [33] [34] . rabies virus glycoprotein is the main antigen responsible for inducing the production of rabies virus-neutralizing antibodies and for conferring immunity against lethal rabies infection. out of the various strategies being employed for enhancing the immunoprophylactic potential of vaccination strategy, dna vaccines have been the most promising. in an effort to develop an optimal dna vaccine against rabies virus, this study was aimed at evaluating the immune enhancement potential of different antigen targeting strategies to selectively improve responses mediated by cd8 + and cd4 + t lymphocytes and by antibodies, induced after intramuscular immunization with dna plasmids. addition of target sequences like tpa, lamp-1, uq have been employed for vaccination against various pathogens including sars coronavirus [35] ; dengue virus [36] ; orthopox virus [37]; influenza a [38] ; mycobacterium [39] . the signal sequences would target the heterologous protein to different sites targeting the model; for (i) high expression and secretion by fusing with tpa and a more generalized activation of the immune system for induction of significant humoral and cell-mediated responses [23] (ii) lysosomal degradation by fusing with lamp-1 and class ii presentation [40, 41] ; (iii) wider and enhanced immune response by fusion with tpa and lamp-1 and (iv) cytoplasmic degradation by the proteasome by fusing with ubiquitin and class i presentation [42] . classically, the transmembrane (tm) region is excised from the dna vaccine immunogen, such that it can be secreted into extracellular milieu. targeted dna vaccines based on immunogens with deleted tm have been successfully employed for vaccination against tumours [43] . nevertheless, wang et al. found that hemagglutinin (ha) proteins from different serotypes of influenza a virus elicits contrasting response to full length and truncated transmembrane forms [44] . further, rath et al. reported that tm domain along with a secretion signal of rv glycoprotein was required for eliciting highest level of neutralizing antibodies. they inferred that tm domain is critical for proper folding of protein otherwise the critical epitopes may get disrupted [30] . gupta et al. also reported that dna vaccine encoding rabies virus glycoprotein lacking transmembrane domain though enhances antibody response but does not confer protection [35] . therefore, we retained the tm domain in our dna vaccine constructs and utilized full gene for targeting strategies. thus, different plasmid dna constructs were made-pgp, the unmodified constructs and modified constructs including p.gp.l (n terminal tpa and c terminal lamp-1), pt.gp (n terminal tpa), pu.gp (n terminal uq), and pgp.l (c terminal lamp-1). transient transfection of bhk-21 cells with all the plasmid dna constructs revealed expression of rabies glycoprotein by flow cytometric analysis. majority of the cells were found to express glycoprotein as seen by the fluorescence monitored by cell sorter. thus, dna vaccine constructs were capable of efficiently expressing the glycoprotein. distribution of chimeras was analyzed by subcellular fractionation and immunoblotting. total cell lysate of transfected bhk-21 cells of all the constructs expressed glycoprotein at approximately 67 kda. the observed high molecular weight of rv-g expressed in bhk-21 cells could be due to the influence of host factors on glycosylation [45] . morimoto et al. showed both bhk and murine neuroblastoma (mna) cell lines, transformed with the same retroviral expression vector encoding rv-g cdna, show different patterns of glycosylation of the expressed rv-g [45] . rrv-g expressed by bhk cells was highly glycosylated and sialylated in comparison to mna expressed rrv-g, indicating that the glycosylation and sialylation of rv g is dependent on the cellular conditions in which rv-g is produced. analysis of subcellular fractions indicated that glycoprotein along with the targeting sequences was suitably recognized by mammalian cells and directed towards the respective pathway. from flow cytometric and immunoblotting analysis of transfected cells, it can be inferred that there was efficient recognition and expression of dna vaccine immunogens in the mammalian system. the signal sequences successfully directed the glycoprotein to respective cellular locations, with comparable levels of expression as of total cell lysate. vaccination of mice with all rv-g plasmid dna constructs led to the generation of anti-rv-g antibodies. all the modified vaccines elicited higher anti-rv-g antibody levels than the unmodified one. the highest antibody response was observed with pgp.lamp-1. the generation of rvna is the most important adaptive immune system response for conferring protection against rabies. therefore, to compare the utility of the rv-g plasmid dna constructs, rvna response elicited by each construct was determined by rffit. the neutralizing antibodies were more than 0.5 iu/ml, which is the minimum titer recommended by who. the highest rvna titer was elicited by pgp.lamp-1 which is also supported by an enhanced antibody response by elisa, in comparison to other rv-g constructs. the effectiveness of the constructs to induce th1/th2 type of immune response was indirectly evaluated by determining th1 (igg2a) and th2 (igg1) antibody isotypes. we found a strong igg1 response in all the dna constructs. even though igg2a antibodies were produced, the ratios of igg1/igg2a were consistently more than one, thus emphasizing on the th2 bias. presence of both types of immune responses may be due to the presence of more than one type of antigenic sites in the glycoprotein immunogen. it is worth noting that differential targeting for enhancing th1 and th2 responses yielded in effect a similar response. the increase in antibodies to dna vaccine may reflect an effect of the antigen on the t helper cell response needed to promote differentiation of naïve b cells into antibody secreting plasma cells. this was assessed by cytokine profiling of splenocytes immunized with signal sequence tagged glycoprotein based vaccine or only vector; upon in vitro stimulation with inactivated pv-11 virus. we found that all the cytokines analyzed could be detected from the splenocytes of dna vaccine immunized mice, with a pronounced enhancement in the level of il-2 and il-12 in the pgp.lamp-1 immunized group. for other cytokines, namely il-4 and ifn-␥, similar levels of cytokines were observed for all the four groups, with the level being several folds in comparison to the splenocytes from control group. antigen binding to the t cell receptor (tcr) stimulates the secretion of il-2, and the expression of il-2 receptors il-2r. the il-2/il-2r interaction then stimulates the growth, differentiation and survival of antigen-selected cytotoxic t cells via the activation of the expression of specific genes. il-2 facilitates production of immunoglobulins made by b cells and induces the differentiation and proliferation of natural killer cells. il-12, produced mainly by macrophages and dendritic cells, is quickly induced by viral infections or by vaccination stimuli. il-12 strengthens the non-specific immune responses by activating nk cells to produce ifn-␥ and in synergy with ifn-␥, drives the differentiation of cd4 + t cells into th1 cells, more adapted to the control of viral infections. various groups of immunized mice when challenged with cvs virus showed higher protection as compared to a vehicle control. high titers of rvna and protection conferred in dna vaccines might be due to the possibility that modified immunogens led to the expression of rv-g with appropriate folding and better accessibility of epitopes to immune system, critical for generating rvna titers. in spite of similar magnitude of immune response generated, protective efficacies against viral challenge varied. the unmodified and secreted forms of vaccines were found to be inferior in inducing protection against viral challenge. xiang et al. also reported that secreted form of vaccine did not confer significant protective immunity [46] . protection against rabies virus is mainly mediated by neutralizing antibodies [47] ; subtle differences in the conformation of the secreted protein, not readily detectable by conventional biochemical methods, might select a different repertoire of neutralizing antibodies with lower avidity to the full length g protein present on the surface of viral particles, thus being less able to prevent the spread of virus [48] . interestingly, pt.gp.l, pt.gp, pu.gp, pgp.l dna vaccine combinations designed to generate different types of immune responses yielded in effect similar data. a probable explanation for this could be that the tagged antigens evoke similar levels of immunity and act to enhance survival via the same primary protective mechanism. we observed that ubiquitination of antigen for mhc class i targeting also enhanced the igg1 antibody and cd4 + mediated cytokine response. thus, we infer that the peptides generated by proteasomal degradation could also be presented by mhc-ii. while, there is no specific information of how protein processing in transfected cells occurs in vivo, different mechanisms have been postulated. they include direct priming by somatic cells, direct priming by antigen-presenting cells, or cross priming of antigen-presenting cells. activation by cross priming appears to be the most probable immune mechanism which occurs following intramuscular vaccination that could be shared by the tpa, lamp-1 and uq vaccines [24, [49] [50] [51] [52] . cross priming may occur via exit of exogenous antigens from the endocytic compartments and its processing in the cytosol, recycling of mhc-i molecules through endosomal/lysosomal pathway and transfer of processed peptides to the endosomal compartments. it is well known that cd4 + t-cell stimulation can result from endocytosis of exogenous peptides or proteins followed by antigenic processing via mhc class ii pathways [53] . lamp-1 targeting of antigen has been reported to increase the number of immunogenic peptide epitopes that activate cd4 + t cells, thus inducing a broadened immune response in comparison to untargeted antigen [54] . recent studies have also demonstrated that exogenous proteins or peptides, possibly complexed to heat shock proteins, can be taken up by antigen processing cells, processed through the mhc class i pathway, and ultimately stimulate naïve cd8 cells [55, 56] . thus, via cross-priming mechanisms, secreted fusion proteins expressed from tpa plasmids, membrane bound fused proteins expressed from lamp-1 or peptides released from cells transfected with the uq constructs could induce both cd4 + and cd8 + t-cell populations. several researchers have applied targeting strategies and reported conflicting results with different antigens and different infectious systems. successful targeting was demonstrated for several pathogens including human papillomavirus [57] , influenza a [38] ; mycobacterium [39] ; but not for all the constructs tested against malaria [58] . thus, a tagged dna vaccine may represent an 'ideal' immunogen for generating protective immune response, nevertheless; the antigen dependence of immune strategies has to be considered for successful vaccination against any pathogen. further, optimization of immunization doses, routes, schedule, adjuvant supplementation and a greater understanding of the immune mechanisms responsible for producing protective immunity in response to dna vaccination should facilitate the creation of further improved rabies dna vaccination strategies. rabies re-examined world survey of rabies: no. 32 for the year 1996. geneva, world health organization rabies: a new look at an old disease genetic immunization: a new era in vaccines and immunotherapy first human trial of a dna based vaccine for treatment of human immunodeficiency virus type 1 infection: safety and host responses cellular cytotoxic response induced by dna vaccination in hiv-1-infected patients induction of antigen-specific cytotoxic t lymphocytes in humans by a malaria dna vaccine excellent safety and tolerability of the human immunodeficiency virus type 1 pga2/js2 plasmid dna priming vector vaccine in hiv type 1 uninfected adults phase i clinical trial safety of dna-and modified virus ankara-vectored human immunodeficiency virus type 1 (hiv-1) vaccines administered alone and in a prime-boost regime to healthy hiv-1-uninfected volunteers sustained protective rabies neutralizing antibody titers after administration of cationic lipid-formulated pdna vaccine dna vaccination of mice against rabies virus: effects of the route of administration and the adjuvant monophosphoryl lipid a (mpl r ) gene gun particle-mediated vaccination with plasmid dna confers protective immunity against rabies virus infection rabies vaccination: comparison of neutralizing antibody responses after priming and boosting with different combinations of dna, inactivated virus, or recombinant vaccinia virus vaccines rabies dna vaccination of non-human primates: post-exposure studies using gene gun methodology that accelerates induction of neutralizing antibody and enhances neutralizing antibody titers immunization of dogs and cats with a dna vaccine against rabies virus immunization of dogs with a dna vaccine induces protection against rabies virus post-exposure dna vaccination protects mice against rabies virus canine rabies dna vaccination: a single-dose intradermal injection into ear pinnae elicits elevated and persistent levels of neutralizing antibody field trials of a very potent rabies dna vaccine which induced long lasting virus neutralizing antibodies and protection in dogs in experimental conditions rabies dna vaccine in the horse: strategies to improve serological responses postexposure therapy in mice against experimental rabies: a single injection of dna vaccine is as effective as five injections of cell culture-derived vaccine immunogenicity of dna vaccines expressing tuberculosis proteins fused to tissue plasminogen activator signal sequences dna vaccine encoding human immunodeficiency virus-1 gag, targeted to the major histocompatibility complex ii compartment by lysosomal-associated membrane protein, elicits enhanced long-term memory response enhancing dna immunization dna vaccination against tuberculosis: expression of a ubiquitin-conjugated tuberculosis protein enhances antimycobacterial immunity development of rabies dna vaccine using a recombinant plasmid cortisone-evoked decrease of acid-galactosidase, glucuronidase, n-acetyl-glucosaminidase and arylsulphatase in the ileum of suckling rats breaking down the wall: fractionation of mycobacteria dna vaccine for rabies: relevance of the trans-membrane domain of the glycoprotein in generating an antibody response a rapid fluorescent focus inhibition test (rffit) for determining rabies virus-neutralizing antibody rabies virus glycoprotein. ii. biological and serological characterization oral immunization and protection of raccoons (procyon lotor) with a vaccinia-rabies glycoprotein recombinant virus vaccine experimental immunization of cats with a recombinant rabies-canine adenovirus vaccine elicits a long-lasting neutralizing antibody response against rabies sars coronavirus nucleocapsid immunodominant t-cell epitope cluster is common to both exogenous recombinant and endogenous dna-encoded immunogens dna vaccines against dengue virus based on the ns1 gene: the influence of different signal sequences on the protein expression and its correlation to the immune response elicited in mice targeting the vaccinia virus l1 protein to the cell surface enhances production of neutralizing antibodies immunization with plasmid dna encoding influenza a virus nucleoprotein fused to a tissue plasminogen activator signal sequence elicits strong immune responses and protection against h5n1 challenge in mice evaluation of the anti-tuberculosis activity generated by different multigene dna vaccine constructs the enhanced immune response to the hiv gp160/lamp chimeric gene product targeted to the lysosome membrane protein trafficking pathway hiv-1 p55gag encoded in the lysosome-associated membrane protein-1 as a dna plasmid vaccine chimera is highly expressed, traffics to the major histocompatibility class ii compartment, and elicits enhanced immune responses regulating protein degradation by ubiquitination dna vaccines encoding full-length or truncated neu induce protective immunity against neu-expressing mammary tumors hemagglutinin (ha) proteins from h1 and h3 serotypes of influenza a viruses require different antigen designs for the induction of optimal protective antibody responses as studied by codon-optimized ha dna vaccines comparison of rabies virus g proteins produced by cdna-transfected animal cells that display either inducible or constitutive expression of the gene immune responses to nucleic acid vaccines to rabies virus use of mouse anti-rabies monoclonal antibodies in post-exposure treatment of rabies antibodies i post exposure treatment of rabies in vivo priming by dna injection occurs predominantly by antigen transfer mhc-i class restricted ctl responses to exogenous antigens priming of cytotoxic t lymphocytes by dna vaccines: requirement for professional antigen presenting cells and evidence for antigen transfer from myocytes dna vaccines: immunology, application, and optimization alternative processing of endogenous or exogenous antigens extends the immunogenic, h-2 class-i restricted peptide repertoire capture and processing of exogenous antigens for presentation on mhc molecules characterization of mhc class ii-presented peptides generated from an antigen targeted to different endocytic compartments receptor-mediated uptake of antigen/heat shock protein complexes results in major histocompatibility complex class i antigen presentation via two distinct processing pathways multiple antigen-specific processing pathways for activating naive cd8 + t cells in vivo characterization of hpv-16 e6 dna vaccines employing intracellular targeting and intercellular spreading strategies targeting antigen to mhc class i and class ii antigen presentation pathways for malaria dna vaccines inactivated pv viral antigen and rabies reference antiserum were kindly provided by dr. v. srinivasan, indian immunological ltd. the authors acknowledge dr. shardul solanke (national biotechnology center, ivri, india) for transfection and anuj kumar sharma (school of biotechnology, jnu, india) for flow cytometry studies. special thanks are extended to dr. subhash chandra (cornell university, ny) for vital inputs in the study. this work was supported by department of biotechnology, government of india. manpreet kaur is recipient of senior research fellowship from csir, government of india. supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.vaccine.2009.01.128. key: cord-264267-weat0qs6 authors: kleine-weber, hannah; schroeder, simon; krüger, nadine; prokscha, alexander; naim, hassan y.; müller, marcel a.; drosten, christian; pöhlmann, stefan; hoffmann, markus title: polymorphisms in dipeptidyl peptidase 4 reduce host cell entry of middle east respiratory syndrome coronavirus date: 2020-01-21 journal: emerg microbes infect doi: 10.1080/22221751.2020.1713705 sha: doc_id: 264267 cord_uid: weat0qs6 middle east respiratory syndrome (mers) coronavirus (mers-cov) causes a severe respiratory disease in humans. the mers-cov spike (s) glycoprotein mediates viral entry into target cells. for this, mers-cov s engages the host cell protein dipeptidyl peptidase 4 (dpp4, cd26) and the interface between mers-cov s and dpp4 has been resolved on the atomic level. here, we asked whether naturally-occurring polymorphisms in dpp4, that alter amino acid residues required for mers-cov s binding, influence cellular entry of mers-cov. by screening of public databases, we identified fourteen such polymorphisms. introduction of the respective mutations into dpp4 revealed that all except one (δ346-348) were compatible with robust dpp4 expression. four polymorphisms (k267e, k267n, a291p and δ346-348) strongly reduced binding of mers-cov s to dpp4 and s protein-driven host cell entry, as determined using soluble s protein and s protein bearing rhabdoviral vectors, respectively. two polymorphisms (k267e and a291p) were analyzed in the context of authentic mers-cov and were found to attenuate viral replication. collectively, we identified naturally-occurring polymorphisms in dpp4 that negatively impact cellular entry of mers-cov and might thus modulate mers development in infected patients. middle east respiratory syndrome coronavirus (mers-cov) is an enveloped virus with a single-stranded rna genome of positive polarity. it belongs to the coronaviridae family (genus betacoronavirus), which is part of the order nidovirales. mers-cov was isolated in 2012 from the sputum of a 60 year old man suffering from acute pneumonia and renal failure in saudi arabia [1] . since its discovery, mers-cov has caused 2,442 human infections of which 842 (34.5%) had a fatal outcome (as of may, 2019) [2] . dromedary camels are reservoir hosts of mers-cov and display only common cold-like symptoms upon infection but constitute the main source of human infections. transmission to humans occurs via close contact to animals or contaminated animal products [3] [4] [5] [6] . human-to-human transmissions seem limited and were mainly observed in health care settings, leading to mers outbreaks in hospitals [7] [8] [9] [10] [11] [12] . finally, differences in the tissue specific expression of the cellular receptor for mers-cov, dpp4, were recently suggested to account for the differences in mers-cov transmission and disease induction in camels and humans, respectively [13, 14] . in order to infect a host (cell) and replicate, mers-cov has to deliver its genome into the cellular cytoplasm for gene translation and genome replication. this process is facilitated by the viral spike (s) glycoprotein, a type-i transmembrane protein embedded in the viral envelope. for host cell entry, the surface unit, s1, of mers-cov s binds to the cellular type-ii transmembrane protein dipeptidyl peptidase 4 (dpp4, cd26) [15] . the structure of the interface between dpp4 and mers-cov-s was resolved on the atomic level and fifteen residues in dpp4 were found to make direct contact with residues in the viral s protein [16] . upon dpp4 engagement, mers-cov s undergoes proteolytic activation through the cellular serine protease tmprss2 or the endosomal cysteine protease cathepsin l [17] [18] [19] , which allows the transmembrane unit, s2, of mers-cov s to fuse the viral membrane with cellular membranes. dpp4 is a prolyl oligopeptidase that is expressed in various tissues [20] and involved in multiple biological processes including t-cell activation [21] , control of the activity of growth factors, chemokines and bioactive peptides [22] [23] [24] , and regulation of the glucose metabolism [25] . mature dpp4 is embedded in the plasma membrane as a homodimer and each monomer consists of an n-terminal cytoplasmic domain, followed by a transmembrane domain and a large ectodomain, which can be further subdivided into a short stalk domain, a glycosylation-rich and a cysteine-rich region as well as the c-terminal catalytic domain (α/ β-hydrolase domain) [26] . polymorphisms in the dpp4 gene were implicated in several diseases and conditions, including diabetes [27, 28] and myocardial infarction [29] but their potential impact on mers-cov infection has not been analyzed. we asked whether naturally-occurring amino acid polymorphisms in dpp4 residues making contact with mers-cov s have an impact on mers-cov entry. we identified fourteen polymorphisms by screening public databases and introduced the respective mutations into a dpp4 expression plasmid. we identified four mutations that reduced mers-cov s binding to dpp4 and mers-cov s-driven host cell entry without affecting dpp4 expression at the cell surface. 293t cells were transfected with expression vectors for wt or mutant dpp4, or empty expression vector (negative control). at 16 h post transfection, the culture medium was replaced and the cells were further incubated for additional 32 h. then, the cells were washed with pbs and mixed with 2x sds-sample buffer (0.03 m tris-hcl, 10% glycerol, 2% sds, 0.2% bromophenol blue, 1 mm edta). cell lysis was achieved by incubating the samples for 10 min at room temperature followed by incubation at 96°c for an additional 10 min. the samples were further loaded on polyacrylamide gels and sds-page (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was performed. next, the proteins were transferred onto nitrocellulose membranes (hartenstein gmbh) by immunoblotting. the membranes were further blocked by incubation in pbs-t (pbs containing 0.5% tween 20 and 5% skim milk powder) for 30 min at room temperature. afterwards, the membranes were incubated overnight at 4°c with undiluted supernatant of a hybridoma cell line secreting anti-cmyc antibody 9e10 (for dpp4 detection) or pbs-t containing anti-ß-actin (actb) antibody (mouse, 1:1,000, sigma aldrich). following three washing intervals with pbs-t, the membranes were further incubated with pbs-t containing horseradish peroxidaseconjugated anti-mouse antibody (goat, 1:5,000, dianova) for 1 h at room temperature before an in house-prepared enhanced chemiluminescent solution (0.1 m tris-hcl [ph 8.6], 250 µg/ml luminol, 1 mg/ ml para-hydroxycoumaric acid, 0.3% h 2 o 2 ) was added and signals were recorded using the chemocam imaging system and the chemostar professional software (intas science imaging instruments gmbh). in order to quantify the signal intensity of the protein bands, the program imagej (fiji distribution) [30] was used. to account for differences in the total protein content of the samples and variations, we normalized the dpp4 signals against the respective signals of the loading control (actb). 293t (human kidney cells, dsmz no. acc 635), bhk-21 (hamster kidney cells, dsmz no. acc 61) and vero 76 (african green monkey kidney cells, kindly provided by andrea maisner, philipps-university marburg) were cultivated in dulbecco's modified eagle medium (pan-biotech) while caco-2 cells (human colorectal adenocarcinoma cells) were cultivated in minimum essential medium (thermofisher scientific). the media were supplemented with 10% fetal bovine serum (biochrom), 100 u/ml of penicillin and 0.1 mg/ml of streptomycin (pan-biotech). all cell lines were incubated at 37°c and 5% co2 in a humidified atmosphere. for subcultivation and seeding, cells were washed with phosphate-buffered saline (pbs) and detached by incubation with trypsin/ edta solution (pan-biotech) (bhk-21, vero 76 and caco-2) or by resuspending the cells in culture medium (293t). transfection of 293t and bhk-21 cells was carried out by calcium-phosphate precipitation or with the help of icafectin-441 (in-cell-art) or fugene hd (promega). all dpp4 mutants were generated based on a pcdna3.1/zeo(+)-based expression vector in which the coding sequence for human dpp4 (genbank: xm_005246371.3) containing an c-terminal cmyc epitope was inserted into via bamhi/ecori restriction sites. the following aa (amino acid) substitutions were introduced via overlap-extension pcr: k267e, k267n, q286k, t288i, t288s, a289v, a291p, a291v, r317k, y322h, i346t, i346v and k392n . in addition, a deletion mutant was generated that lacks aa residues 346-348 (δ346-348). information on dpp4 polymorphisms was retrieved from the ensembl database (https://www.ensembl.org/index.html) [31] and the single nucleotide polymorphism database (dbsnp) of the national center for biotechnology information (ncbi) (https://www.ncbi.nlm.nih.gov/snp) [32] , and is based on data provided by the gnomad database (genome aggregation database, https://gnomad. broadinstitute.org/), topmed program (trans-omics for precision medicine, https://www.nhlbiwgs.org/), exac consortium (exome aggregation consortium, http://exac.broadinstitute.org/) [33] and the 1000g project (1,000 genomes project, http://www. internationalgenome.org/) [34] (for detailed information see supplementary table 1) . we further utilized pcaggs-based expression vectors for vesicular stomatitis virus (vsv) glycoprotein (g), mers-cov s wildtype (wt) and mers-cov s (d510g) (the latter two either untagged or equipped with a c-terminal v5 epitope) that have been described elsewhere [35] [36] [37] . in addition, a previously described expression vector for angiotensin converting enzyme 2 was employed [38] . similar to the strategy used for the generation of dpp4 mutants, we employed the overlap-extension pcr technique to introduce a single mutation into the mers-cov s open reading frame, thus generating untagged and v5-tagged mers-cov s (d539n). soluble s comprising the s1 subdomain of mers-cov s (aa residues: 1-747) fused to a human igg fc tag was generated by inserting the pcr-amplified s1 sequences into the pcg1fc vector [39] (kindly provided by georg herrler, university of veterinary medicine hannover) making use of the bamhi/sali restriction sites. in addition, we generated an expression vector for the enhanced green fluorescent protein (egfp) by inserting the egfp coding sequence, which was pcr-amplified from the pegfp-c1 vector (clontech), into the pcaggs plasmid using the ecori/xhoi restriction sites. all pcr-amplified sequences were subjected to automated sequence analysis (microsynth seqlab) to verify their integrity. sequences of primers used for cloning of the different constructs are available upon request. analysis of dpp4 surface expression by immunofluorescence analysis bhk-21 cells were grown on coverslips and transfected with the different dpp4 constructs or empty expression vector using icafectin-441 (in-cell-art) at 24 h post seeding according to the manufacturer's instructions. after changing the culture medium at 4 h post transfection, the cells were incubated for additional 20 h. then, the culture medium was aspirated and the cells were washed with pbs, before they were fixed by incubated with pbs containing 4% paraformaldehyde (pbs/pfa) for 15 min at room temperature. subsequently, the cells were washed with 0.1 m glycine/ pbs solution followed by a washing step with pbs. next, the coverslips were incubated with anti-dpp4 antibody (mouse, diluted 1:200 in pbs containing 1% bovine serum albumin [pbs/bsa], abcam) for 1 h at 4°c. for this, the coverslip was put on a drop (20 µl) of antibody solution that was added on a sheet of parafilm inside a humidity chamber (a glass dish in which the parafilm was placed on wet paper tissue). thereafter, the cells were washed 3x with pbs before incubation with alexafluor568-conjugated antimouse antibody (goat, 1:1000, diluted in pbs/bsa, thermofisher scientific) for 30 min at 4°c was performed. subsequently, the cells were washed 3x with pbs. finally, the cells were incubated with dapi (4',6-diamidino-2-phenylindole, carl roth) and mounted in prolong gold antifade mountant (ther-mofisher scientific) before they were analyzed using a zeiss lsm800 (zeiss) confocal laser scanning microscope and the zen imaging software (zeiss). analysis of dpp4 surface expression by flow cytometry bhk-21 cells were transfected with expression vectors for wt or mutant dpp4, or empty expression vector (negative control). at 16 h post transfection, the culture medium was replaced and the cells were further incubated for additional 32 h. then, the cells were washed with pbs, resuspended in pbs/ bsa and pelleted by centrifugation (600 x g, 5 min, 4°c). after aspiration of the supernatant, the cells were resuspended in pbs/bsa containing anti-dpp4 antibody (mouse, diluted 1:100, abcam) and incubated for 1 h at 4°c. next, the cells were pelleted, washed with pbs/bsa, pelleted again, resuspended in pbs/bsa containing alexafluor488-conjugated anti-mouse antibody (donkey, diluted 1:500, thermo-fisher scientific) and incubated for 1 h at 4°c. subsequently, the cells were washed (as described above) and resuspended in pbs/pfa for 2 h at 4°c for fixation. finally, the cells were washed (as described above) and resuspended in pbs/bsa for flow cytometric analysis using an lsr ii flow cytometer and the facs diva software (both bd biosciences). additional data analysis was carried out using the fcs express 4 flow research software (de novo software). for quantification of dpp4 surface expression, the mean fluorescence intensity (mfi) value of the negative control was subtracted from all samples. for normalization of dpp4 surface expression, values obtained for cells expressing dpp4 wt were set as 100% and the relative surface expression of the respective dpp4 mutants was calculated accordingly. in order to generate soluble mers-cov s for binding studies, 293t cells were transfected with an expression vector for the s1 subunit of mers-cov s fused to the fc fragment of human immunoglobulin g (solmers-s1-fc). at 24 h post transfection, the culture medium was exchanged and the cells were further incubated for 24 h before culture supernatants were harvested and freed from cellular debris by centrifugation (4,700 x g, 10 min, 4°c). the clarified supernatants were loaded on vivaspin protein concentrator columns with a molecular weight cut-off of 30 kda (sartorius) and centrifuged at 4,700 x g at 4°c until the sample was 10-fold concentrated. for the binding studies with solmers-s1-fc, a similar protocol was followed as described for the analysis of dpp4 surface expression with the exceptions that sol-mers-s1-fc was used instead of the primary antibody (1:10 dilution in pbs/bsa) and that an alexafluor488conjugated anti-human antibody (goat, 1:500 dilution in pbs/bsa, thermofisher scientific) was employed as the secondary antibody. bhk-21 cells transfected with expression vectors for wt or mutant dpp4, ace2 or empty expression vector (both negative controls) were analyzed by flow cytometry for solmers-s1-fc binding using an lsr ii flow cytometer and the facs diva software (both bd biosciences). additional data analysis was carried out using the fcs express 4 flow research software (de novo software). for quantification of solmers-s1-fc binding, the mfi value obtained for cells transfected with empty expression vector was subtracted from all samples. further, binding of solmers-s1-fc to cells expressing dpp4 wt was set as 100% and the relative binding efficiencies to cells expressing the respective dpp4 mutants or ace2 were calculated accordingly. 293t cells (grown in 6-well plates) were cotransfected with expression plasmids coding for solmers-s1-fc and wt or mutant dpp4. cells transfected with empty expression vector instead of dpp4 or sol-mers-s1-fc (or both) served as controls. at 16 ,400 x g at 4°c, before 400 µl of the supernatant were mixed with 50 µl of protein a-sepharose (1 g protein a-sepharose [sigma-aldrich] in 4 ml pbs) while the residual 100 µl of the cell lysate were mixed with 100 µl 2x sds-sample buffer and incubated for 15 min at 96°c (these samples were later analyzed to confirm comparable total protein levels [via detection of actb] as well as comparable dpp4 and solmers-s1-fc levels before the co-immunoprecipitation [co-ip] step.). following incubation of the lysate/protein a-sepharose mixtures for 2 h at 4°c in an overhead shaker, the samples were centrifuged for 5 min at 16,400 x g at 4°c to pellet the protein a-sepharose/solmers-s1-fc/dpp4-complexes. after aspiration of the supernatant, 500 µl of np40 lysis buffer (without protease inhibitors) were added and the cells were mixed by vortexing, before being centrifuged again. this washing routine was repeated three times, before finally 50 µl of 2x sdssample buffer were added to the pelleted complexes and the samples were further incubated for 15 min at 96°c. thereafter, the samples were subjected to sds-page and western blot analysis (see above). detection of dpp4 (lysate and co-ip samples) and actb (lysate samples) was carried out as described above. solmers-s1-fc was detected (lysate and co-ip samples) by incubation with a peroxidase-conjugated anti-human antibody (goat, 1:5,000, dianova). signal intensities of the protein bands were quantified as described above. further, signals obtained for dpp4 were normalized against the respective signals for solmers-s1-fc in order to account for variations in transfection efficiency and sample processing. for the binding studies with soluble dpp4, a similar protocol was followed as described for the analysis of binding of solmers-s1-fc with the exceptions that a soluble dpp4 fused to the fc region of human igg (soldpp4-fc, acro biosystems) was used instead of solmers-s1-fc (1:200 dilution in pbs/bsa) and that an alexafluor488-conjugated anti-human antibody (goat, 1:500 dilution in pbs/bsa, thermofisher scientific) was employed as the secondary antibody. 293t cells transfected with expression vectors for wt or mutant (d510g and d539n) mers-cov s, or empty expression vector (negative control) were analyzed by flow cytometry for soldpp4-fc binding using an lsr ii flow cytometer and the facs diva software (both bd biosciences). additional data analysis was carried out using the fcs express 4 flow research software (de novo software). for quantification of soldpp4-fc binding, the mfi value obtained for cells transfected with empty expression vector was subtracted from all samples. further, binding of soldpp4-fc to cells expressing mers-cov s wt was set as 100% and the relative binding efficiencies to cells expressing the respective mers-cov s mutants were calculated accordingly. we employed a previously described protocol for the generation of vsv pseudotype particles (vsvpp) that is based on a replication-deficient vsv vector that lacks the genetic information for vsv-g but instead contains the genetic information for egfp and firefly luciferase (fluc) as reporters of transduction efficiency (vsv*δg-fluc, kindly provided by gert zimmer, institute of virology and immunology, mittelhäusern/switzerland) [37, 40] . in brief, 293t cells transfected with expression vectors for mers-cov s, vsv-g (positive control) or empty expression vector (negative control) were inoculated with vsv*δg-fluc for 1 h before being washed with pbs and further incubated for 16 h with culture medium that was supplemented with anti-vsv-g antibody (i1, mouse hybridoma supernatant from crl-2700; atcc) (except for cells expressing vsv-g). the produced vsvpp were inoculated onto bhk-21 cells expressing wt or mutant dpp4, or no dpp4 (empty expression vector, negative control) and incubated for 16-18 h before fluc activity in cell lysates was quantified as an indicator for transduction efficiency using the beetle-juice kit (pjk) and a plate luminometer (hidex) [41] . bhk-21 cells were transfected with expression vectors for wildtype or mutant dpp4 (k267e or a291p), or empty expression vector (negative control) using fugene hd (promega) according to the manufacturer's instructions. at 24 h posttransfection, the cells were infected with mers-cov (human betacoronavirus 2c emc/2012, mers-cov emc-2012, genbank accession number: jx869059) at a multiplicity of infection of 0.01 for 1 h. thereafter, the inoculum was removed and the cells were washed 3x with pbs before fresh medium was added and the first sample (time point 0 h postinfection) was taken. the cells were further incubated and additional samples were taken at 24 and 48 h postinfection. viral titers in the culture supernatant were analyzed by quantitative reversetranscriptase pcr, using the upe assay according to a published protocol [42] . in brief, viral rna was isolated from cell culture supernatant using the nucleospin rna virus kit (macherey-nagel), reversetranscribed into cdna using the superscript iii one step rt-pcr system (thermofisher scientific) and analyzed on a lightcycler 480 qpcr cycler platform (roche) with primers and conditions as specified for the upe assay [42] . in vitro-transcribed standard samples containing defined amounts of mers-cov fragments (10, 100, 1,000 and 10,000 copies) were included for absolute quantification as genome equivalents (ge). the dpp4 protein structure (4pv7) [43] and the structure of the complex formed by the mers-cov s receptor binding domain bound to dpp4 (4l72) [16] were retrieved from the research collaboratory for structural bioinformatics protein database (rscb pdb, https://www.rcsb.org/). structure visualization and colorization was performed using the yasara software (http://www.yasara.org/index.html) [44] and ucsf chimera version 1.14 (developed by the resource for biocomputing, visualization, and informatics at the university of california, san francisco) [45] . one-way or two-way analysis of variance (anova) with dunnett's posttest was used to test for statistical significance. only p values of 0.05 or lower were con identification of polymorphisms in dpp4 that alter amino acid residues which make contact with mers-cov s the binding interface between mers-cov s and the cellular receptor dpp4 was resolved by wang and colleagues using crystallography, revealing the interacting amino acid residues for each binding partner [16] : fourteen residues of mers-cov s (y499, n501, k502, l506, d510, r511, e513, d537 g538, d539, y540, r542, w553 and v555) interact with a total of fifteen residues in dpp4 (k267, f269, q286, t288, a289, a291, l294, i295, h298, r317, y322, r336, q344, i346 and k392) [16] , which are distributed over the glycosylation-rich domain and the cysteinerich domain ( figure 1a-c) . in order to identify polymorphic residues in dpp4 that contact mers-cov s, we screened public databases that provide information on polymorphic amino acid residues based on data derived from different bio projects (i.e. gnomad, topmed, exac, 1000g; more information is given in the materials and methods section). by this method we found that nine out of the fifteen dpp4 residues interacting with mers-cov s are polymorphic (k267, q286, t288, a289, a291, r317, y322, i346 and k392) ( figure 1c ). while five of these residues can be replaced by only a single different amino acid residue (q286[k], a289 . circles with sticks represent glycosylation sites, while small numbers indicate the amino acid residues. triangles below the domains highlight the positions of amino acid residues that directly interact with mers-cov s (grey triangles mark residues for which no polymorphism has been reported, while red triangles indicate polymorphic residues). (b) side (left) and top (right) view of homodimeric dpp4 (the dotted line indicates the border between the two monomers and the cellular plasma membrane is schematically depicted below the side view model of dpp4). the protein model was constructed on the published crystal structure (4pv7) deposited in rscb pdb and the binding interface with mers-cov s has been highlighted (green). (c) close-up on the dpp4 residues that directly interact with mers-cov s and for which no polymorphic (yellow) or polymorphic (red) residues have been reported. in addition, the specific residues in dpp4 (regular letters and numbers), including the respective polymorphic residues (letters in brackets), and the corresponding interacting residues in mers-cov s (italicized letters and numbers) are indicated. (d) frequency of polymorphic dpp4 residues in the human population. public databases (see supplementary table 1 and the materials and methods section for detailed information) were screened for the frequency of the polymorphic residues under study (y-axis). error bars indicate standard error of the mean (sem) and refer to polymorphic residues found in more than one database. table 1 ). finally, the frequency of these polymorphisms in the human population is low, ranging roughly from 1:19,000 (a289v) to 1:245,000 (t288i) ( figure 1d and supplementary table 1 ). we next introduced the polymorphisms in a dpp4 expression plasmid. western blot analysis and signal quantification revealed that all resulting dpp4 variants were robustly expressed and total expression levels were comparable (figure 2a-b) . as dpp4 needs to be transported to the plasma membrane to be engaged by mers-cov s for host cell entry, we next investigated whether the presence of the polymorphic dpp4 residues has an impact on dpp4 cell surface localization. for this, we performed flow cytometry and confocal laser scanning microscopy, using transfected bhk-21 cells and an antibody targeting the dpp4 ectodomain. we found that all dpp4 variants but one, a deletion variant lacking amino acid residues 346-348 (δ346-348), displayed comparable cell surface expression levels ( figure 3a-b) . we next investigated whether polymorphic dpp4 residues impact mers-cov host cell entry. for this, we made use of vesicular stomatitis virus (vsv) pseudotypes (vsvpp) bearing mers-cov s or vsv g, which does not bind to dpp4 and served as negative control [46] . as expected, vsvpp harboring vsv g were able to efficiently transduce bhk-21 target cells irrespective of dpp4 expression. in contrast, transduction of bhk-21 cells mediated by mers-cov s critically depended on ectopic expression of human dpp4, in accordance with published findings [47] ( figure 4) . notably, four dpp4 polymorphisms -k267e, k267n, a291p and δ346-348 -severely reduced mers-cov s-driven transduction compared to dpp4 wt (figure 4 ). in order to analyze whether the reduction in mers-cov s-driven host cell entry would translate into attenuated mers-cov replication, we next investigated two dpp4 polymorphisms (k267e and a291p) in the context of infection with authentic mers-cov. when followed over a period of two days post infection it was observed that mers-cov replication in bhk-21 cells expressing human dpp4 was significantly reduced when dpp4 contained either k267e or a291p ( figure 5 ). after the identification of dpp4 polymorphisms that reduce s-driven cellular entry of rhabdoviral vectors as well as mers-cov replication, we next sought to investigate whether the attenuating phenotype was due to reduced binding of mers-cov s to dpp4. for this, we used soluble mers-cov s, produced by fusing the s1 subunit, which contains the dpp4 binding domain, to the fc portion of human immunoglobulin g (solmers-s1-fc). co-immunoprecipitation analysis demonstrated that dpp4 variants harboring polymorphisms k267e, k267n or a291p, which were not compatible with efficient mers-cov s-driven host cell entry, displayed significantly reduced ability to interact with mers-cov s as indicated by weaker dpp4 signals upon protein a-sepharosemediated pull-down of dpp4/solmers-s1-fc (as compared to dpp4 wt, figure 6a-b) . notably, dpp4 variant δ346-348 could be as efficiently coimmunoprecipitated as dpp4 wt, indicating that its inefficient receptor function was solely due to its defect in proper surface transport. the findings obtained by co-ip analysis were confirmed by flow cytometry. it was revealed that polymorphisms that reduced mers-cov s-driven host cell entry (k267e, k267n, a291p and δ346-348) and spread of authentic mers-cov (k267e and a291p) also reduced mers-cov s binding to cells expressing dpp4 on the cell surface ( figure 6c ). in addition, polymorphism a289v, which decreased mers-cov s-driven transduction to a lesser extent than the aforementioned polymorphisms (figure 4) , also reduced mers-cov s binding to dpp4. thus, dpp4 polymorphisms k267e, k267n and a291p reduce mers-cov s-driven host cell entry and mers-cov infection by diminishing mers-cov s binding to dpp4. host cell entry of mers-cov critically depends on the interaction between the viral s protein and the cellular receptor dpp4. a link between obesity or underlying diseases like diabetes mellitus, which both can affect dpp4 expression levels [48] , and the risk of fatal outcome of mers-cov infection has been made [49] . moreover, alanine scanning mutagenesis identified dpp4 residues critical for mers-cov entry, including k267, l294, i295, r317 and r336 [50, 51] . however, the impact of natural-occurring variations on host cell entry of mers-cov has not been addressed so far. we identified dpp4 polymorphisms that reduce s protein-driven host cell entry and replication of authentic mers-cov by lowering the binding efficiency of mers-cov s to dpp4, suggesting that the dpp4 phenotype may impact the course of mers-cov infection. western blot analysis, flow cytometry and confocal laser scanning microscopy revealed that none of the polymorphisms studied, except deletion of amino acids 346-348, had a significant impact on total or cell surface expression of dpp4, at least in the context of dpp4 transfected cells. four polymorphisms located at three different sites in dpp4 (k267e, k267n, a291p and δ346-348) severely reduced s protein-driven host cell entry. as dpp4 δ346-348 was shown to be incompatible with robust cell surface transport but able to interact with mers-cov s in co-ip analysis, we conclude that the reduction in entry efficiency is solely due to insufficient dpp4 surface levels. in contrast, reduction of host cell entry by k267e, k267n and a291p could not be explained by reduced dpp4 expression and these polymorphisms were thus further investigated. mers-cov infection of bhk-21 cells transfected to express dpp4 wt and variants k267e or a291p revealed that k267e or a291p were not compatible with robust mers-cov replication. finally, co-ip analyses and binding studies with soluble mers-cov s showed that these dpp4 polymorphisms reduced s protein binding to dpp4. when looking at the crystal structure of the complex consisting of the mers-cov s receptor binding domain bound to dpp4, these observations do not come as a surprise. dpp4 residue k267 has been reported to contact mers-cov s residues g538 and d539, including a salt bridge interaction with d539 [16] . the exchange of k267 to either glutamate (e) . whole cell lysates (wcl) were prepared and analyzed for dpp4 expression by sds-page under non-reducing conditions and wb using a primary antibody targeting the c-terminal cmyc epitope and a peroxidase-conjugated secondary antibody. further, expression of beta-actin (actb) was analyzed as a loading control. shown are the expression data from a representative experiment. numbers at the left indicate the molecular weight in kilodalton (kda). (b) quantification of total dpp4 expression in wcl. after normalization of dpp4 band intensities with that of the corresponding actb bands. dpp4 wt expression was set as 100% and the relative expression of mutant dpp4 was calculated accordingly. presented are the combined data of three independent experiments with error bars indicating the sem. no statistical significance for differences in total expression between wt and mutant dpp4 was observed by one-way analysis of variance with dunnett's posttest (p > 0.05, not significant [ns]). . surface expressed dpp4 was stained by subsequent incubation of the non-permeabilized cells with a primary antibody that targets the dpp4 ectodomain and an alexafluor488-conjugated secondary antibody. fluorescent signals representing surface-expressed dpp4 were analyzed by flow cytometry and the mean fluorescence intensity (mfi) values for each sample were calculated. for normalization, the mfi value of the negative control was subtracted from all samples. further, surface expression of dpp4 wt was set as 100% and the relative surface expression of the dpp4 mutants was calculated accordingly. shown are the combined data of three experiments with error bars indicating the sem. statistical significance for differences in surface expression between wt and mutant dpp4 was tested by one-way analysis of variance with dunnett's posttest (p > 0.05, not significant; p ≤ 0.05, *). (b) dpp4 surface expression was further analyzed by immunofluorescence analysis. for this, dpp4 wt or dpp4 mutants were expressed in bhk-21 cells grown on coverslips (cells transfected with empty expression vector served as negative control). after fixation of the cells, surface expressed dpp4 was stained by subsequent incubation of non-permeabilized cells with a primary antibody that targets the dpp4 ectodomain and an alexafluor568-conjugated secondary antibody. in addition, cellular nuclei were stained with dapi. finally, images were taken using a confocal laser scanning microscope at a magnification of 80x. or asparagine (n) likely abolishes/decreases the interaction with mers-s due to the different biochemical properties of k267 (positively charged, basic) versus e267 (negatively charged, acidic) and n267 (not charged, acidic) (supplementary figure 1) . for dpp4 residue a291, which has been reported to contact the mers-cov s residue e513, no information on the type of interaction is available [16] . here, we speculate that the bulky and distorted side chain of proline (in comparison to the small side chain of alanine) abolishes/decreases interaction with mers-cov s residue e513 (supplementary figure 1) . in contrast to that, valine contains a small side chain and also has identical biochemical properties as alanine and thus might be efficiently contacted by e513 of mers-cov s, which is why we did not observe any impact of polymorphisms a291v on mers-cov s-driven entry and mers-cov s mers-cov s binding/interaction (supplementary figure 1 ). the observation that certain polymorphisms in dpp4 reduced mers-cov s binding and viral entry triggered the question whether residues in mers-cov s that are in direct contact with the respective dpp4 residues are also polymorphic. indeed, we obtained initial evidence to support such a concept. thus, we found that residue 539 in mers-cov s which contacts dpp4 residue 267 is polymorphic, with certain mers-cov variants harboring an asparagine instead of an . reduced mers-cov s-driven host cell entry is caused by inefficient s protein binding to dpp4 harboring polymorphic amino acid residues. in order to investigate whether reduced mers-cov s-driven host cell entry and mers-cov replication is due to inefficient mers-cov s binding to dpp4 harboring amino acid polymorphisms at the binding interface, we performed co-immunoprecipitation (co-ip) as well as binding experiments with a soluble s protein comprising the s1 subunit of mers-cov s fused to the fc region of human igg. (a) 293t cells were cotransfected with expression plasmids coding for soluble, fc-tagged mers-cov s1 (solmers-s1-fc) and the indicated dpp4 variant containing a c-terminal cmyc-tag. cells that were transfected only with empty expression vector alone, or empty expression vector instead of either solmers-s1-fc or dpp4 served as controls. at 48 h posttransduction, cells were lysed and incubated with protein a sepharose. next, samples were subjected to sds-page and western blot analysis. dpp4 levels were detected via antibodies specific for the cmyc-tag, whereas solmers-s1-fc was detected using a peroxidase-coupled anti-human antibody. similar results were obtained in three individual experiments. analysis of whole cell lysates (wcl) for expression of solmers-s1-fc, dpp4 and ß-actin confirmed comparable ß-actin levels in each sample and comparable expression levels for solmers-s1-fc and dpp4. (b) for quantification of mers-cov s/dpp4 interaction we first normalized the dpp4 signals against the respective solmers-s1-fc signals. then, mers-cov s/dpp4 interaction was set as 100% for wildtype (wt) dpp4 and the relative interaction efficiency for each dpp4 mutant was calculated accordingly. presented are the mean data from three independent experiments. error bars indicate the sem. statistical significance of differences in mers-cov s/dpp4 interaction between wt and mutant dpp4 was analyzed by one-way analysis of variance with dunnett's posttest (p > 0.05, ns; p ≤ 0.05, *; p ≤ 0.001, ***). (c) soluble mers-cov s1-fc was incubated with bhk-21 cells expressing wildtype (wt) or mutant dpp4, or cells transfected with empty expression vector or an ace2-expression plasmid (controls). to detect bound s protein, the cells were subsequently incubated with an alexafluor488-conjugated anti-human antibody directed against the fc-tag. fluorescent signals representing bound solmers-s1-fc were analyzed by flow cytometry and mfi values for each sample were calculated. for normalization, the mfi value of the negative control (empty expression vector) was subtracted from all samples. further, binding of sol-mers-s1-fc to cells expressing dpp4 wt was set as 100% and the relative binding to cells expressing the dpp4 mutants was calculated accordingly. shown are the combined data of five independent experiments with error bars indicating the sem. statistical significance of differences in solmers-s1-fc binding to cells expressing wt or mutant dpp4 was analyzed by one-way analysis of variance with dunnett's posttest (p > 0.05, ns; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***). aspartate residue at this position. d539n reduced entry into cells expressing relatively low amounts of dpp4 but had no effect on entry into cells expressing high amounts of dpp4 (supplementary figure 2) . moreover, and more interestingly, d539n slightly rescued mers-cov s-driven entry from the negative effect exerted by dpp4 polymorphism k267n (supplementary figure 2) . similarly, residue 510 in mers-cov s, which is known to interact with dpp4 residues 317 and 322, was found to be polymorphic, and previous studies demonstrated that polymorphism d510g reduced dpp4 binding but also increased resistance to neutralizing antibodies [37] . notably, d510g slightly increased entry via dpp4 harboring polymorphism y322h and allowed mers-cov s to use dpp4 with polymorphism r317k with the same efficiency as wt dpp4. it should be stated that none of these effects was statistically significant and that dpp4 and mers-cov s polymorphisms occur with low frequency. although it is unlikely that the dpp4 polymorphisms have emerged as a result of evolutionary pressure from mers-cov infections, our results suggest that certain existing dpp4 polymorphism(s) might foster the emergence of mers-cov variants with altered biological properties. the polymorphisms studied here occur with relatively low frequencies of one per ∼19,000 (a289v) to ∼245,000 (t288i) individuals. however, detailed information on the geographic distribution or incidence in certain ethnical groups is largely missing. thus, dpp4 polymorphisms could contribute to the perplexing absence of mers cases in africa, where the virus circulates in camels [52] [53] [54] [55] [56] [57] . however, recent evidence suggests that sequence variations between african and arabian mers-cov might be a factor [53, 57] . more importantly, it remains to be analyzed how frequent dpp4 polymorphisms that affect s protein binding occur in the middle east and 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reagents. we further thank a.-s. moldenhauer for excellent technical support. no potential conflict of interest was reported by the authors. this work was supported, including the efforts of stefan pöhlmann and christian drosten, by the bundesministerium für bildung und forschung [grant numbers 01ki1723d and 01ki1723a], network project rapid (risikobewertung bei präpandemischen respiratorischen infektionserkrankungen). the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. key: cord-351498-bmq6zcb0 authors: martínez-sernández, victoria; perteguer, maría j.; hernández-gonzález, ana; mezo, mercedes; gonzález-warleta, marta; orbegozo-medina, ricardo a.; romarís, fernanda; paniagua, esperanza; gárate, teresa; ubeira, florencio m. title: comparison of recombinant cathepsins l1, l2, and l5 as elisa targets for serodiagnosis of bovine and ovine fascioliasis date: 2018-03-21 journal: parasitol res doi: 10.1007/s00436-018-5809-7 sha: doc_id: 351498 cord_uid: bmq6zcb0 infections caused by fasciola hepatica are of great importance in the veterinary field, as they cause important economic losses to livestock producers. serodiagnostic methods, typically elisa (with either native or recombinant antigens), are often used for early diagnosis. the use of native antigens, as in the mm3-sero elisa (commercialized as bio k 211, bio-x diagnostics), continues to be beneficial in terms of sensitivity and specificity; however, there is interest in developing elisa tests based on recombinant antigens to avoid the need to culture parasites. of the antigens secreted by adult flukes, recombinant procathepsin l1 (rfhpcl1) is the most commonly tested in elisa to date. however, although adult flukes produce three different clades of cls (fhcl1, fhcl2, and fhcl5), to our knowledge, the diagnostic value of recombinant fhcl2 and fhcl5 has not yet been investigated. in the present study, we developed and tested three indirect elisas using rfhpcl1, rfhpcl2, and rfhpcl5 and evaluated their recognition by sera from sheep and cattle naturally infected with f. hepatica. although the overall antibody response to these three rfhpcls was similar, some animals displayed preferential recognition for particular rfhpcls. moreover, for cattle sera, the highest sensitivity was obtained using rfhpcl2 (97%), being equal for both rfhpcl1 and rfhpcl5 (87.9%), after adjusting cut-offs for maximum specificity. by contrast, for sheep sera, the sensitivity was 100% for the three rfhpcls. finally, the presence of truncated and/or partially unfolded molecules in antigen preparations is postulated as a possible source of cross-reactivity. fascioliasis (or fasciolosis) is recognized as one of the most important food-borne trematodiasis in the veterinary field because of the economic impact it has on livestock (mainly sheep and cattle) producers (schweizer et al. 2005; charlier et al. 2007; charlier et al. 2009; mezo et al. 2011 ). the genus fasciola includes two species, f. hepatica and f. gigantica, the former of which is distributed worldwide and the second of which is restricted to several regions of africa and asia ). the broad distribution of f. hepatica seems to be associated with its ability to adapt to new definitive hosts and with its capacity to infect different snail species (intermediate hosts) living in diverse habitats and under different environmental conditions (correa et al. 2010) . when humans or animals are infected by ingestion of the metacercariae (present in vegetables or contaminated water), the parasites excyst in the intestine, transverse the intestinal wall and the peritoneal cavity, and then migrate to the liver where they feed and grow for 8-12 weeks (andrews 1999) . finally, the parasites move into biliary ducts where they mature and begin producing eggs. nevertheless, sometimes a small proportion of parasites reaches ectopic locations (mas-section editor: xing-quan zhu * florencio m. ubeira fm.ubeira@usc.es coma et al. 2013 coma et al. , 2014 , thus preventing elimination of the eggs through the biliary ducts. considering the characteristics of the biological cycle of fasciola, diagnosis of infections by either f. hepatica or the more pathogenic f. gigantica (valero et al. 2016) has traditionally been carried out by microscopic observation of parasite eggs in fecal samples from infected hosts (mas-coma et al. 2014 ). however, these methods are tedious, have poor sensitivity, and depend to a greater or lesser degree on the experience of the examiner. moreover, fecal examination cannot detect acute infections when the parasites feed on liver parenchyma, before egg production has started (charlier et al. 2014; mas-coma et al. 2014 ). in the past two decades, considerable effort has been made to develop rational elisa methods for the diagnosis of human and animal infections caused by f. hepatica and f. gigantica in order to prevent the limitations inherent in microscopic examination of fecal samples (espino and dumenigo 2003) . such methods include the use of whole or purified natural antigens from fasciola as well as recombinant antigens, to detect anti-fasciola circulating antibodies (cornelissen et al. 1999; carnevale et al. 2001b; espinoza et al. 2007; mezo et al. 2007; gonzales santana et al. 2013; gottstein et al. 2014) . they also include elisa methods capable of detecting secreted antigens present in serum or fecal samples from infected humans and animals (espino and finlay 1994; abdel-rahman et al. 1998; mezo et al. 2004; ubeira et al., 2009; george et al., 2017) . because antibody titers may remain elevated for long periods of time after treatment of the infection, elisa methods for the detection of specific antibodies cannot distinguish between current or past infections. nevertheless, these elisas offer the advantage of being able to diagnose early prepatent (acute) infections (salimi-bejestani et al. 2007; mezo et al. 2010a,b; mas-coma et al. 2014) , and, for this reason, they are frequently used to screen herds of domestic animals in serum and mostly in milk samples (mezo et al. 2010b; duscher et al. 2011; mezo et al. 2011 ). most of these methods are indirect or capture elisas that include fasciola cathepsins l (cls) as antibody targets, as these antigens probably induce the highest proportion of anti-fasciola antibodies during natural infections. however, except for elisas based on the use of single recombinant cls (rcls) or recombinant procathepsins l (rpcls), typically l1 clade members, the suitability of each of the several cathepsins secreted by fasciola for detecting anti-fasciola antibodies by elisa has not previously been investigated. this aspect is relevant, as it has been reported that the pattern of cathepsins that are secreted by f. hepatica varies during its biological cycle, so that immature parasites begin producing fhcl3, fhcl4, and cathepsins b, but that expression is gradually replaced by fhcl1, fhcl2, and fhcl5 in adults (robinson et al. 2008; smooker et al. 2010; cwiklinski et al. 2015) . moreover, production of these fhcls by adult flukes has been reported to be imbalanced, with proportions of 69, 22 and 9% for fhcl1, fhcl2, and fhcl5, respectively ). it is therefore important to determine which of these molecules are most relevant from the point of view of their antigenicity and suitability as target antigens for serodiagnosis of human and animal infections by fasciola. in the present study, we developed and tested indirect elisas based on recombinant procathepsin l1 (rfhpcl1), rfhpcl2, or rfhpcl5, in order to investigate which of these targets are best recognized by sera from sheep and cattle naturally infected with f. hepatica. we also evaluated if any of these rfhpcls is advantageous over the use of native cls using as reference method of this study the mm3-sero elisa, in which the monoclonal antibody (mab) mm3 captures native fasciola cls (mezo et al. 2007 ). cattle a slaughterhouse that processes cattle from the whole region was visited fortnightly during a year. at each visit, 15 adult cows (over 2 years old; friesian breed, females) were selected at random, and their livers as well as stool and blood samples were collected and transported to the laboratory promptly. in total, samples from 100 naturally f. hepatica-infected and 200 f. hepatica-free adult cattle, as determined, respectively, by the presence or the absence of flukes in livers (gold standard), were collected. for recovering and counting all flukes, each liver was thoroughly examined. in the first step, we proceeded to the opening of the bile ducts and the gallbladder to obtain the most of intact adult flukes and then the livers were cut into slices (approximately 1 cm thick) which were manually squeezed to obtain the immature flukes inhabiting the parenchyma. this procedure prevented any possible problems of low sensitivity when liver inspection is not performed thoroughly, as has been described in previous studies (rapsch et al. 2006; charlier et al. 2008) . in addition to fluke counts, samples of feces from all animals were subjected to sedimentation (anderson et al. 1999) and flotation (maff 1971) procedures to concentrate eggs from fasciola or other parasites, and then they were microscopically examined. the remaining fecal material was frozen at − 25°c for subsequent further quantitation of fasciola antigens by the mm3-copro elisa (mezo et al. 2004; martínez-sernández et al. 2016) . as we prioritized testing simultaneously the sera with the four f. hepatica antigens in a single run, the sample size was limited to 33 f. hepatica-infected and 22 f. hepatica-free cows, which was optimal for the conditions of our laboratory. for the first group, the infected animals (n = 100) were stratified in three categories according to their parasite burden (low: 1-10 flukes, middle: 11-25 flukes, and high: ≥ 26 flukes) and animals from each group were selected by random sampling using epidat 3.1 software (consellería de sanidade, xunta de galicia, spain). to assure a better comparison of the performance of the different elisas evaluated in the present study, animals with low parasite burdens represented the 60% (n = 20) of the sample, while animals from the remaining categories constituted both the ≈ 20% (n = 6 and 7) of the sample. of the 200 f. hepatica-free cattle, 22 were selected by simple random sampling. serum and fecal samples used in the present study were obtained from the herd of a commercial farm suffering from fascioliasis (infected sheep) and from the fluke-free herd maintained at ingacal (non-infected sheep). the sheep in both herds were an autochthonous galician breed (braza gallega^). the sample size was limited by the availability of animals to conduct the study. in the first herd, the 15 sheep suffering chronic fascioliasis (using both data from coprology and mm3-copro elisa as gold standard) were sampled. in the second herd, samples were taken from another 15 sheep chosen completely at random. as for cattle samples, the feces from all sheep were examined for the presence of eggs from fasciola and other parasites. fecal aliquots were also frozen at − 25°c for further quantitation of fasciola antigen by the mm3-copro elisa (mezo et al. 2004; martínez-sernández et al. 2016 ). the f. hepatica excretory-secretory antigens (esas) used in the mm3-sero elisa (see below) were obtained as previously reported (mezo et al. 2003) . briefly, live adult flukes were collected from the bile ducts of naturally infected cows and washed, first in sterile saline solution containing antibiotics (penicillin/streptomycin) and glucose (2 g/l), at 38°c, and then in rpmi 1640 cell culture medium supplemented with 20 mm hepes, 0.3 g/l l-glutamine, 2 g/l sodium bicarbonate and antibiotics, at 38°c under 5% co 2 in air. the flukes were then transferred to 75-cm 2 tissue culture flasks and maintained in culture medium (3 ml/fluke) at 38°c under 5% co 2 in air. after 24 h incubation, the medium containing the secreted antigens was removed and centrifuged at 10,000g for 20 min at 4°c in the presence of protease inhibitors (sigmafast protease inhibitor tablets, sigma-aldrich, madrid, spain). the supernatant was then passed through a 0.45-μm pore filter disk, concentrated in an amicon 8050 ultrafiltration cell (amicon, inc., beverly, ma) equipped with a ym10 membrane (10 kda molecular weight cut-off), dialyzed against pbs, sterilized by filtration and, finally, stored at − 80°c until required. the protein concentration was measured using the micro bca protein assay kit (pierce; thermo fisher scientific, barcelona, spain). the recombinant ani s 1 allergen (rani s 1) (anadón et al. 2010; cuéllar et al. 2012) included in the trisakis 170 kit (lin et al. 2014) , was produced in e. coli and purified and refolded as previously described (anadón et al. 2010 ). hybridoma cells secreting mab mm3 were obtained as previously described (mezo et al. 2004; martínez-sernández et al. 2014) . the secreting hybridoma cells were grown intraperitoneally in pristan-primed balb/c mice, and the anti-f. hepatica igg1/ĸ antibodies were isolated from the ascitic fluid by affinity chromatography on a protein g column (hitrap protein g, ge healthcare, madrid, spain) according to the manufacturer's protocol. cloning of the genes encoding fhpcl1, fhpcl2, and fhpcl5 and expression of recombinant proteins messenger rna (mrna) was obtained from adult specimens of f. hepatica as previously reported . briefly, whole f. hepatica mrnas were obtained using the fast track mrna isolation kit (invitrogen, san diego, ca), according to the manufacturer's instructions, and the mrna concentrations were determined by spectrophotometry (nanodrop; thermo fisher scientific). a collection of cdna was prepared with one microgram of mrna using the marathon cdna amplification kit (clontech, palo alto, ca), according to the manufacturers' instructions. the double-strand cdnas were subsequently ligated to the marathon adaptors (ap1). the procedures used to clone and subclone fhpcl1 are reported elsewhere . the deduced amino acid sequence was annotated under genbank accession number cca61803.1. for expression of the corresponding protein, the fhpcl1 gene was directionally cloned into the pqe-30 expression vector (qiagen; qiagen iberia sl, madrid, spain) and further transformed into the e. coli strain m15 [prep4]. primers corresponding to sequences described by kofta et al. (2000) were synthesized to amplify the full cathepsin molecules: f-kofta (5′ atgtggttcttcgtattagc 3′) and rkofta (5′ ccaagtatttttaacaatccaata 3′). pcr reactions were carried out under conditions of low stringency, in order to amplify genes corresponding to different types of cathepsins. the cdna described above was used as template. the pcr products were cloned into the pgem-t vector (promega biotech ibérica sl, madrid, spain), and dna from recombinant plasmids was automatically sequenced using fluorescence-base labeling with the abi prism system (perkin elmer, langen, germany) and the universal primers d and sp6. a clone similar to the fhcl2 (gb|cca61803.1) was obtained. the fhpcl2 without the signal peptide was directionally cloned (sac i -sma i) into the pqe-30 expression vector (qiagen) using primers f-pcl2 (5′ tcgaatga cgatttgtg 3′) and r-pcl2 (5′ ttcacggaaatc 3′), and was then further transformed into the e. coli m15 [prep4] cells. the selected fhpcl2 sequence was annotated in genbank under accession number ky464953. generic primers, f-pcl5 (5′ tcaaatgacgattt g t g g c at c a at g g a a g 3 ′ ) and r-pcl5 (5′ tcacggaaattgtgccaccatcgggac 3′), were designed to directly clone the fhpcl5 gene using the cdna collection as template. considering that the fhpcl1, fhpcl2, and fhpcl5 sequences were almost identical at the amino terminal region and quite similar at the carboxy terminal region (both used to design the different sets of primers), and as fhpcl5 is the least abundant member of the fhcls expressed by adult worms ), numerous clones had to be sequenced to obtain a sequence compatible with the characteristics previously reported by norbury et al. (2012) for the cl5 clade. the selected fhpcl5 sequence was annotated in genbank under accession number ky392883. for protein expression, competent e. coli m15 [prep4] cells were transformed with the fhpcl5 gene directionally cloned (bam h i -sac i) into the pqe-30 expression vector (qiagen) with the primers f2-pcl5 (5′ tcaaatgacgatttgtgg 3′) and r2-pcl5 (5′ tcacggaaattgtgc 3′). expression, purification, and refolding of rfhpcl1, rfhpcl2, and rfhpcl5 recombinant fhpcls expression was induced by adding 1 mm iptg to the transformed e. coli cultures . once the expression was induced, cultures were maintained 4 h at 37°c. then, e. coli cells were harvested by centrifugation, and the insoluble recombinant proteins were purified with b-per reagent (thermo fisher scientific), solubilized and purified by affinity chromatography with his-select nickel affinity gel (sigma-aldrich) under denaturing conditions (8 m urea), as indicated by the supplier. the different rfhpcls were refolded as previously described ) with a few modifications. briefly, each elute from the affinity column was pretreated with 10 mm dtt for 1 h at room temperature (rt), and subsequently diluted at a ratio of 1/10 in tbs (50 mm tris, 150 mm nacl, ph 8) plus 10 mm cysteine, 1 mm cystine, and 1 mm edta. once dialyzed against tbs (ph 8), each rfhpcl was dialyzed against pbs and concentrated by membrane-filtration in an amicon-stirred ultrafiltration cell equipped with a filtron omega series membrane (10 kda nominal molecular weight limit; pall corporation, port washington, ny). finally, the protein concentration was determined with the micro bca protein assay kit, and the refolded recombinant proteins were stored at − 80°c until use. to investigate the optimal concentration of each rfhpcl for use as target in indirect elisa, we tested several concentrations of each antigen in the range of 1-10 μg/ml in pbs. polystyrene microtiter plates (greiner bio-one; soria-melguizo, madrid, spain) were coated with 100 μl/well of each rfhpcl dilution and incubated for 2 h at 37°c. the plates were then washed three times with pbs and blocked with 200 μl/well of 1.5% sodium caseinate in pbs for 1 h at rt. aliquots of 100 μl of an appropriate dilution of mab mm3 (1/2000) in pbs containing 0.05% tween 20 and 1% skimmed dry milk (pbs-t-sm) were then added to each well, and the plates were incubated for 30 min at rt with shaking at 750 rpm. the plates were then washed five times with pbs-t, and bound mm3 antibodies were detected after incubation with hrp-labeled goat anti-mouse igg secondary antibodies (bio-rad, madrid, spain) diluted 1/3000 in pbs-t-sm for 30 min at rt with orbital shaking at 750 rpm. the plates were then washed, as above, and incubated for 20 min at rt with 100 μl/well of the enzyme substrate opd (sigmafast opd, sigma-aldrich). finally, the optical density (od) was measured at 492 nm. recombinant procathepsin dilutions containing the same equivalent concentration (i.e., yielding the same od signal with mab mm3) were selected for use in an indirect elisa, as indicated below. indirect elisas with rfhpcl1, rfhpcl2, rfhpcl5, and rani s 1 the wells of elisa plates were coated with 100 μl of each rfhpcl (rfhpcl1, rfhpcl2, or rfhpcl5) or with the anisakis simplex allergen rani s 1, all at a concentration of 2.5 μg/ml in pbs, and incubated for 2 h at 37°c. the plates were then washed three times with pbs and blocked with 200 μl/well of 1.5% sodium caseinate in pbs for 1 h at rt. aliquots of 100 μl of sheep or cattle sera (from f. hepaticainfected and f. hepatica-free animals) diluted 1/100 in pbs-t-sm were added to each well of the plates in duplicate, and incubated for 30 min at rt with orbital shaking at 750 rpm. the plates were then washed five times with pbs-t, and bound igg antibodies were detected with either hrpconjugated mouse anti-sheep/goat igg monoclonal antibodies (1/30,000 in pbs-t-sm; sigma-aldrich), or hrp-conjugated sheep anti-bovine igg1 polyclonal antibodies (1/6000 in pbs-t-sm; bio-rad), and opd, as indicated above. the mm3-sero elisa was performed as previously described (mezo et al. 2007 ) but with some modifications. briefly, elisa plates were coated with purified mab mm3 (100 μl/well at 5 μg/ml), incubated on at 4°c, washed three times with pbs, and blocked with 200 μl/well of 1.5% sodium caseinate in pbs for 1 h at rt. aliquots of 100 μl of f. hepatica esas at 1 μg/ml in pbs or pbs only were added to each well in odd (ag+) and even (ag−) plate rows, respectively. the plates were incubated for 2 h at rt and then washed three times with pbs, before 100 μl of each serum sample (from sheep or cattle) diluted 1/100 in pbs-t-sm was added to each ag+ and ag− well in duplicate. the plates were then incubated for 30 min at rt with shaking at 750 rpm, washed five times with pbs-t, and specific sheep or bovine igg was detected as described above. the od value for each sample was calculated as od1-od2, where od1 is the value for the ag+ well, and od2 is the value for the ag− well. the significance of the differences in coproantigen values for fecal samples obtained from parasitized cattle classified according to their parasite burden (low, middle, high; see above) was determined by the kruskal-wallis test (nonparametric anova) and dunn's multiple comparison test. the analysis was conducted using the graphpad instat statistical package (graphpad software inc., san diego, ca). differences were considered significant at p < 0.05. pearson's correlation coefficients (r) were calculated to compare the data obtained analyzing samples from infected animals with the different indirect and capture elisas using originpro 7.5 (originlab corporation, northamptom, ca). the cut-off values for each elisa and species tested (sheep or cattle) were calculated from the od values obtained testing the sera from f. hepatica-free animals. two methods were used to calculate the cut-off values for the different indirect elisas. in method a, designed to guarantee maximum specificity (i.e., 100%; see martínez-sernández et al. 2016) , the cut-off values were calculated as the sum of the highest od value obtained on testing the negative sera plus 1sd. these cut-offs were used to obtain the sensitivities of each test, which were calculated using epidat 3.1 software as the number of true positives (correctly identified by each test), divided by the total number of infected animals. in method b, the cut-off values were obtained by receiver operating characteristic (roc) analysis. the roc curves were generated using the medcalc software (medcalc software, ostend, belgium) following the methodology proposed by delong et al. (1988) . the cut-off values defined by the youden index (i.e., that maximize the sum of sensitivity and specificity) were used to estimate the sensitivity and specificity of each test. finally, the cut-offs and the corresponding sensitivities of the mm3-sero elisa were determined considering method a only. comparison of the fhpcl1, fhpcl2, and fhpcl5 amino acid sequences the sequence alignments of the three fhpcls used in this study are shown in fig. 1 . the displayed sequences corresponded to the full-length propeptide (residues s 1 -r/ l 91 ) lacking the deduced signal peptide (residues 1-15). the alignments revealed a high percentage of sequence identity among the three fhpcls, although the percentage of identity was higher between fhpcl1 and fhpcl5 (87%), in comparison with the 84% calculated for fhpcl2 and fhpcl5, and the 78% for fhpcl1 and fhpcl2. assignation of the sequence gb|cca61803.1 to fhcl1 was previously reported . regarding the sequence gb|ky464953, we observed that it differs by three nucleotides, but only by one residue (l 52 s), from sequence gb|abq95351.1 (baspinar et al. unpublished results) and by seven nucleotides, which translate into five residue changes (l 52 s, f 86 y, n 110 d, p 149 t, l 152 f), from the sequence gb|aac47721.1 (dowd et al. 1997) , both of which were classified as fhcl2. interestingly, these three sequences totally coincide with the key residues y 69 , m 70 , a 135 , l 160 , t 161 , a 163 , and l 209 (mature protein numbering) reported by norbury et al. (2012) as being typical of the fhcl2 clade. the sequence gb|ky392883 was classified as fhcl5 because of its similarity to the sequence gb|aaf76330 reported by smooker et al. (2000) and classified as fhcl5 by norbury et al. (2012) . our sequence has four nucleotide changes, two of which translate into two amino acid substitutions (i 263 t and m 295 t), relative to gb|aaf76330. however, both sequences share the characteristic residues l 69 , m 70 , a 135 , l 160 , n 161 , g 163 , and l 209 reported by norbury et al. (2012) in the mature fhcl5. as indicated in the previous section, fasciolosis in cattle was determined by liver necropsy and further confirmation by fecal egg counting and coproantigen detection (mm3-copro elisa). the non-infected group tested negative in fluke, egg, and coproantigen determinations. coprology also revealed that most cows in both groups had intestinal nematodes from one or more genera of trichostrongylidae, trichuridae (trichuris spp.), and strongylidae. additionally, the farm records for individual cows revealed that most were routinely treated with albendazole during the dry period and vaccinated against infectious bovine rhinotracheitis and bovine respiratory disease (bovine respiratory syncytial virus, parainfluenza virus type 3, and mannheimia haemolytica). some were also vaccinated at the end of the gestation period against coronavirus, rotavirus, and e. coli. regarding sheep, f. hepatica eggs (ranging between 0.3 and 9 eggs per gram of feces) and coproantigens (fecal antigen concentration ranging from 7.1 to 200.5 ng/ml) were detected in all sheep from the infected herd but were absent in the fluke-free herd. as for cattle, infections by gastrointestinal nematodes were detected in most animals from both infected and non-infected herds. specifically, n e m a t o d es i n t h e f a m i l i e s tri c h o s t r on g y l i d a e , molineidae (nematodirus spp.), ancylostomatidae, strongylidae, and trichuridae (trichuris spp.) were identified. the fecal antigen concentrations, parasite load and other characteristics from the infected animals included in the study are shown in table 1 . recognition of rfhpcl1, rfhpcl2, and rfhpcl5 by sera from infected and non-infected cattle and sheep the antigens rfhpcl1, rfhpcl2, and rfhpcl5 were evaluated in indirect elisa testing sera from cows and sheep naturally infected with f. hepatica and from non-infected animals. as indicated in the previous section, the optimal concentration of the target antigens was determined in elisa plates by their recognition by mab mm3, which binds to an epitope that is present in the three fhcl clades. moreover, as the mm3 epitope is conformational in nature , their recognition by this mab in elisa strongly suggests correct folding of these recombinant molecules. the results presented in fig. 2a (fasciola-infected) and fig. 2b (fasciola-free) show the individual igg antibody response of the 33 and 22 cows, respectively, to the three recombinant cathepsins in indirect elisa, and to native cathepsins in capture elisa (mm3-sero). most of the sera from fasciolainfected cows produced od signals higher than 0.5 to the three rfhpcls although, considering the samples individually, the antibodies in some sera displayed a preference for certain rfhpcls. for example, this was the case with serum #5, which reacted preferentially with rfhpcl5 and rfhpcl1, and serum #15 which produced the highest od value with rfhpcl2. finally, only two serum samples (#14 and #29) produced low responses with ≤ 0.25 od (fig. 2a) . with respect to sera from fasciola-free cattle (fig. 2b) , unlike for the elisa mm3-sero, several serum samples produced high backgrounds in indirect elisa, with od values ≥ 0.1. the highest background values were obtained with rfhpcl1 (sera #10, #17, and #19) and with rfhpcl5 (sera #10, #15, and #21), although one sample (serum #22) also produced a considerable background (od > 0.2) with rfhpcl2. the antibody responses of sera from fasciola-infected and fasciola-free sheep are shown in fig. 3a , b. regarding infected sheep, the response to the three recombinant antigens follows the same pattern as indicated above for cattle, with good elisa signals and only one sample yielding an od signal diagnostic value of the indirect elisa with rfhpcl1, rfhpcl2, or rfhpcl5 versus the mm3-sero capture elisa for a better comparison of the signals obtained in indirect elisa with rfhpcl1, rfhpcl2, and rfhpcl5 with those of the classical mm3-sero elisa, we elaborated a graph grouping the response given by each individual serum in each of the four elisas tested (fig. 4a, b) . in these figures, the corresponding cut-offs, calculated as the maximum od value from negative sera plus one sd (method a) or obtained by roc analysis (method b) are represented, respectively, by horizontal dashed red and blue lines. the sera from infected cattle produced similar mean od signals (p > 0.05 for all comparisons), ranging from od = 0.177 to od = 2.398 for rfhpcl1-elisa, from od = 0.192 to od = 2.268 for rfhpcl2-elisa, from od = 0.186 to od = 2.673 for rfhpcl5-elisa, and from od = 0.254 to od = 2.238 for mm3-sero elisa (fig. 4a ). however, due to the big differences observed between the cut-off of the reference test mm3-sero elisa (od = 0.043) and the calculated cut-offs for the indirect elisas (od = 0.632, 0.310, and 0.597, respectively, with method a, and 0.265, 0.265, and 0.285, respectively, with method b) several positive sera were incorrectly classified by these latter. specifically, n = 4, n = 1, and n = 4 sera from infected cattle were misclassified by rfhpcl1, rfhpcl2, and rfhpcl5 elisas, respectively, considering the cut-off obtained by method a, while n = 2, n = 1, and n = 2 of these sera were incorrectly classified using the cut-off obtained by method b (roc analysis). these results yielded sensitivity values of 87.9, 97.0, and 87.9%, respectively, considering the former cut-offs (method a), and sensitivities of 93.9, 97.0, and 93.9%, respectively, considering the latter cut-offs (method b). although the sensitivity values of the rfhpcl1 and rfhpcl5 elisas obtained using cut-off values calculated with roc curves (method b) are apparently better than those obtained with method a, it is relevant to consider that this increase in sensitivity was done at expenses of lowering the specificity of the assays by about 5% (see table 2 ). with respect to infected sheep, the distribution of od signals obtained in indirect elisa with the three rfhpcls was similar to that obtained for infected cattle (the lowest od signal obtained with rfhpcl5 (od = 0.185) and the highest with rfhpcl2 (od = 2.260)). interestingly, as the cut-off values calculated with method a (rfhpcl1, od = 0.140; rfhpcl2, od = 0.114; rfhpcl5, od = 0.095; mm3-sero, age (range) 8.8 (4-12) 8.5 (9-12) 7.1 (4-10) 4.7 (3-8) a statistic differences (p < 0.05) with group harboring 1-10 flukes b statistic differences with group harboring 1-10 flukes (p < 0.01) and with group harboring 11-25 flukes (p < 0.05) c animals in production, not sacrified at the moment of sample collection od = 0.055) or the method b (rfhpcl1, od = 0.121; rfhpcl2, od = 0.100; rfhpcl5, od = 0.085) were much lower than for cattle, the three elisa variants and the mm3-sero elisa were capable of correctly classifying all sera from infected and non-infected sheep, i.e., with a sensitivity and specificity of 100% (table 2) . nevertheless, the comparison of the od signals obtained in any of the indirect elisas with those yielded by the mm3-sero capture elisa (either for cattle or sheep) revealed that the signal-tonoise ratio was much more favorable in the latter. finally, the r values obtained on comparing the four elisa methods for fasciola-infected cattle and sheep sera are shown in table 3 . the correlation was strongest between the data obtained with rfhpcl5 and rfhpcl1 elisas for both cow and sheep sera, while the lowest r value corresponded to the comparison between the od values of the tested cow sera in the mm3-sero and rfhpcl5 elisas. the relatively large proportion of sera from fasciola-free cattle with cross-reactive antibodies to one or more of the recombinant rfhpcls used in this study (see fig. 2b ) led us to investigate whether these cows also had cross-reactive antibodies to other recombinant or non-recombinant antigens. for this purpose, we evaluated the presence of igg antibodies reactive with a recombinant a. simplex allergen (rani s 1) expressed in e. coli and purified using the same procedure as for rfhpcls, or reactive with mouse igg1 (purified mab mm3 without f. hepatica antigens, which is used as control for each individual serum in the mm3-sero elisa). a relevant proportion of sera from cattle infected with f. hepatica had igg antibodies reactive with rani s 1, three of which produced od signals above 1.0 (see fig. 5a ). although the reactivity of sera from the fasciola-free population of cattle was much lower (fig. 5b) , these samples still showed a notable reactivity to rani s 1 (fig. 5b) . moreover, anti-mouse igg1 crossreactivity was also observed for a significant number of sera within the samples from fasciola-infected and fasciola-free cows (fig. 5e, f) . on the other hand, regarding serum samples from sheep, the population infected with f. hepatica did not recognize the rani s 1 (fig. 5c ) allergen, and only two serum samples from non-infected sheep produced od values > 0.2 (fig. 5d) . also, no reaction to mouse igg1 antibodies was observed when testing sheep sera from both populations (fig. 5g, h) . this is the first study that comparatively evaluates the performance of three rfhpcls (rfhpcl1, rfhpcl2, and rfhpcl5) as targets antigens in elisa for the serodiagnosis of fasciolosis in sheep and cattle. these molecules were selected since (i) they are produced by adult flukes and thus continue stimulating the immune system during the chronic phase of the illness, and (ii) fasciola cathepsins l1, l2, and l5 contain a common epitope recognized by mab mm3, which is the capture antibody in the mm3-sero elisa (mezo et al. 2007) . although rfhpcls from the l1 clade have already been successfully used to develop a very sensitive and specific lateral flow test for immunodiagnosis of human fascioliasis , as well as to design elisa tests for human (o'neill et al. 1999; carnevale et al. 2001a, b; gonzales santana et al. 2013; gottstein et al. 2014) and animal use (cornelissen et al. 2001; kuerpick et al. 2013; selemetas et al. 2014; bloemhoff et al. 2015) , the performance of rfhpcls from l2 and l5 clades as elisa targets has not previously been investigated. from a functional point of view, a single amino acid substitution may be sufficient to affect substrate specificity in cysteine proteases from f. hepatica (smooker et al. 2000) . the comparison of the new fhpcl2 (gb|ky464953) and fhpcl5 (gb|ky392883) sequences described in the present study with other reported cathepsins l2 (gb|abq95351.1; gb|aac47721.1) and l5 (gb|aaf76330) revealed some nucleotide and amino acid changes. however, as none of the amino acid changes were in the positions referred by norbury et al. (2012) as being typical of the fasciola l2 and l5 clades, the assignation of our recombinant molecules as fhpcl2 and fhpcl5 was probably correct. the alignment of the sequences of the three fhpcls used in this study showed a mean of amino acid changes of 17%, which, besides affecting substrate specificity, it may be enough to induce variations in the level of circulating antibodies produced in infected animals. the results showing preferences in procathepsin recognition in the population of sera from fasciola-infected animals, mainly cattle (see fig. 2a ), are consistent with this hypothesis. more importantly, our results indicated that indirect elisas based on rfhpcl1, rfhpcl2, and rfhpcl5 show differences in sensitivity, despite the fact that the target antigens were expressed and purified in the same way. specifically, considering the od values obtained with the different immunoassays, the cut-off values calculated to maximize specificity (method a), and the number of false negatives obtained for each rfhpcl, the rfhpcl2 might be a better target antigen than the other rfhpcls. however, the mm3-sero elisa had a better performance, as this method classified correctly all samples from sheep and cattle. there are at least two explanations for the superiority of the mm3-sero elisa over the elisas based on the use of individual rfhpcls: (i) mab mm3 is able to virtually capture all native cathepsin isoforms of the cl1, cl2, and cl5 clades from fasciola ; and this study), including those from the more pathogenic f. gigantica species valero et al. 2016) . this may explain the fact that od values obtained in mm3-sero elisa tended to be higher than in indirect elisas among infected animals with low anti-fasciola antibody levels (see fig. 2a ), and (ii) the mm3-sero elisa includes an individual control for each serum, which ensures that particular animals with anti-species cross-reactive antibodies or antibodies against other irrelevant proteins present in the elisa plate (see fig. 5e , f) can be easily detected, thus allowing this signal to be subtracted from that of the corresponding positive well. consequently, good od signals and low backgrounds (which translates into lower cut-offs) are normally achieved, yielding an excellent signalto-noise ratio, for both cattle and sheep sera. these results suggest that the use of a single recombinant cathepsin/ procathepsin as target antigen in elisas for serodiagnosis of fascioliasis may limit the sensitivity of the assay when testing sera from some species, particularly cattle. considering the above characteristics of the mm3-sero elisa, its highest sensitivity (see table 2 ) over the indirect elisas based on the use of a single f. hepatica cathepsin/ procathepsin l1, l2, or l5 is not surprising. however, considering that representatives of all mature fasciola cls are captured in mm3-sero, the high backgrounds obtained with the rfhpcl1, rfhpcl2, and rfhpcl5 antigens for fasciola-free cattle (see fig. 2b ) may appear unexpected at first sight. moreover, this phenomenon was not limited to the samples investigated in this study, nor to the expression of rpfhcls in e. coli. in agreement with this, a similar finding was recently reported in a study where the performance of an indirect elisa based on a rfhpcl1 expressed in the yeast pichia pastoris was compared with that of a commercial indirect elisa test containing a purified fraction from fasciola esas (f2 antigen; institute pourquier, montpellier, france) (kuerpick et al. 2013 ). cross-reactivity was also observed by cornelissen et al. (2001) who tested sera from sheep and cattle harboring other parasites, mainly nematodes, suggesting that the cross-reactivity may be due to common epitopes between recombinant fasciola cathepsins and antigens present in other parasites. however, as such cross-reactivity did not occur with the three tested rfhpcls (see fig. 2b ), this explanation seems unlikely. another possible source of cross-reactivity is that rfhpcls produced in e. coli were contaminated by bacterial proteins retained in the imac column during the affinity purification process. coelution of native e. coli proteins (e.g., proteins containing clusters of histidine residues) with recombinant proteins is a recognized feature, mainly when recombinant proteins are expressed at low level (robichon et al. 2011 ). however, this seems also unlikely for several reasons: (i) rfhpcls are produced as inclusion bodies, and contaminating proteins are removed during the washing procedures before denaturation and subsequent imac purification of rfhpcls; (ii) as indicated above, the reactivity of a particular serum does not occur with the three rfhpcls, despite the fact that all of them were produced and purified following the same procedure; and (iii) preincubation of the cross-reactive sera with a bacterial extract prior to the incubation in elisa plates, or the use of a rfhpcl1 expressed with a second tag in order to eliminate possible remaining contaminants in a capture elisa did not eliminate the observed backgrounds (data not shown). conversely, we hypothesize that the phenomenon may be related to a greater opportunity of cross-reactive antibodies to bind non-specific linear epitopes present in rpfhcl preparations than in native cls. while in native antigens, the availability of non-specific linear epitopes may be low due to the correct folding of the protein, such epitopes may be exposed for antibody-binding when truncated, partially folded, or unfolded molecules are present, as occurs frequently when recombinant proteins are overexpressed in bacterial or yeast cell systems (lilie et al. 1998; cornelissen et al. 2001 ). in addition, an unexpectedly high number of samples from fasciola-infected and non-infected cattle reacted with a recombinant form of the a. simplex ani s 1 allergen (fig. 5a, b) , an antigen that is present in a fish parasite never naturally in contact with cattle, or with other herbivorous animals (anadón et al. 2010 ). such allergen is structurally related with kunitz-type serine protease inhibitors (audicana and kennedy 2008) . thus, with respect to fasciola-infected cows, it could be hypothesized that cross-reactivity with rani s 1 is due to the presence of common epitopes between this allergen and the kunitz-type molecules secreted by fasciola (bozas et al. 1995; di maggio et al. 2016; smith et al. 2016) . nevertheless, the fact that the response was irregular among these cows, and that it also occurred, to a lesser extent, among fasciola-free cows, suggests again that cross-reactive antibodies may be induced against antigens not related with fasciola but which share some linear epitopes. considering the above argumentations and the sensitivity obtained in the indirect elisas tested, the usual purification conditions of recombinant proteins expressed in bacterial or yeast systems may render these antigens not suitable for use as target in elisas for serodiagnosis of fascioliasis in certain animal species. besides, there is also experimental data suggesting that cross-reactivity may not occur in the same way in different host species. in this sense, we observed that the od responses to rfhpcls were more homogeneous in infected sheep than in cattle (see figs. 3a and 2a, respectively), which was also reflected by the higher r values obtained for sheep sera relative to cattle sera, when comparing the od values obtained on testing the different rfhpcls (see table 3 ). moreover, interspecific differences in the generation of cross-reactive antibodies to fasciola procathepsins can also be deduced from a previous study in which no cross-reactivity was observed for human sera in a lateral flow test based on the same rfhpcl1 ) used in the present study. this reflects that some species may be more prone to induce cross-reactive antibodies than others or that the antigens to which humans are exposed have fewer common epitopes with fasciola cls than those to which ruminants are exposed. the limited number of positive and negative sera tested in this study may not be sufficient to obtain precise values of sensitivity and specificity for the three indirect elisas based on recombinant rfhpcls and to obtain cut-off values valid for use under field conditions. however, this did not prevent drawing conclusions about their performance with respect to the mm3-sero elisa, as they were tested simultaneously with the same sera. moreover, it did not condition the fact that recombinant cls purified by imac may be more prone to be recognized by cross-reactive antibodies than the corresponding natural antigens. finally, although liver necropsy and coproantigen detection may not be perfect gold standards, since some animals may only have migrating or ectopic flukes, considering the age of cattle in our study (4-12 years old), the probability of having a single contact within the previous weeks before slaughtering is minimal. in summary, our results show that rfhpcl2 may be more suitable than rfhpcl1 and rfhpcl5 as target antigen in indirect elisa for the serodiagnosis of fascioliasis in ruminants, particularly in cattle, as these molecules, unless purified to homogeneity and correctly folded, may be more frequently recognized by cross-reactive antibodies than the corresponding natural antigens. these results reflect that further research into methods of producing and purifying recombinant fasciola cathepsins is required to overcome the need to collect live adult parasites in abattoirs, or from experimental infections, to obtain natural antigens. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. blood and fecal samples were collected from naturally infected sheep and cattle by veterinarians from the bcentro de investigaciones agrarias de mabegondo^(ingacal, a coruña, spain). the samples were collected either during routine control and treatment of herds (sheep) or in the slaughterhouse (cattle). all procedures were carried out in strict accordance with spanish and eu legislation (law 32/2007, r.d. 53/2013, and council directive 2010/63/eu). evaluation of a diagnostic monoclonal antibody-based capture enzyme-linked immunosorbent assay for detection of a 26-to 28-kd fasciola hepatica coproantigen in cattle diagnosing human anisakiasis: recombinant ani s 1 and ani s 7 allergens versus the unicap 100 fluorescence enzyme immunoassay the sensitivity and specificity of two methods for detecting fasciola infections in cattle the life 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hepatica in cattle use of a pre-selected epitope of cathepsin-l1 in a highly specific peptide-based immunoassay for the diagnosis of fasciola hepatica infections in cattle early immunodiagnosis of fasciolosis in ruminants using recombinant fasciola hepatica cathepsin l-like protease bridging gaps in the molecular phylogeny of the lymnaeidae (gastropoda: pulmonata), vectors of fascioliasis ani s 1 and ani s 7 recombinant allergens are able to differentiate distinct anisakis simplex-associated allergic clinical disorders the fasciola hepatica genome: gene duplication and polymorphism reveals adaptation to the host environment and the capacity for rapid evolution comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach across intramammalian stages of the liver fluke fasciola hepatica: a proteomic study isolation of a cdna encoding fasciola hepatica cathepsin l2 and functional expression in saccharomyces cerevisiae fasciola hepatica -monitoring the milky way? the use of tank milk for liver fluke monitoring in dairy herds as base for treatment strategies international handbook of foodborne pathogens sandwich enzyme-linked immunosorbent assay for detection of excretory secretory antigens in humans with fascioliasis evaluation of fas2-elisa for the serological detection of fasciola hepatica infection in humans application of a coproantigen elisa as an indicator of efficacy against multiple life stages fasciola hepatica infections in sheep the diagnosis of human fascioliasis by enzyme-linked immunosorbent assay (elisa) using recombinant cathepsin l protease comparative assessment of elisas using recombinant saposin-like protein 2 and recombinant cathepsin l-1 from fasciola hepatica for the serodiagnosis of human fasciolosis successful dna immunisation of rats against fasciolosis evaluation of a recombinant cathepsin l1 elisa and comparison with the pourquier and es elisa for the detection of antibodies 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detection of fasciola hepatica coproantigens in sheep and cattle using a new monoclonal antibody (mm3) the use of mm3 monoclonal antibodies for the early immunodiagnosis of ovine fascioliasis kinetics of anti-fasciola igg antibodies in serum and milk from dairy cows during lactation, and in serum from calves after feeding colostrum from infected dams field evaluation of the mm3-sero elisa for detection of anti-fasciola igg antibodies in milk samples from individual cows and bulk milk tanks association between anti-f. hepatica antibody levels in milk and production losses in dairy cows molecular and immunological characterization of fasciola antigens recognized by the mm3 monoclonal antibody analysis of fasciola cathepsin l5 by s2 subsite substitutions and determination of the p1-p4 specificity reveals an unusual preference short report: immunodiagnosis of human fascioliasis using recombinant fasciola hepatica cathepsin l1 cysteine proteinase estimating the true prevalence of fasciola hepatica in cattle slaughtered in switzerland in the absence of an absolute diagnostic test engineering escherichia coli bl21(de3) derivative strains to minimize e. coli protein contamination after purification by immobilized metal affinity chromatography zoonotic helminth infections with particular emphasis on fasciolosis and other trematodiases proteomics and phylogenetic analysis of the cathepsin l protease family of the helminth pathogen fasciola hepatica: expansion of a repertoire of virulenceassociated factors an integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen fasciola hepatica: proteins associated with invasion and infection of the mammalian host evaluation of an enzyme-linked immunosorbent assay for detection of antibodies to fasciola hepatica in milk estimating the financial losses due to bovine fasciolosis in switzerland weather and soil type affect incidence of fasciolosis in dairy cow herds unexpected activity of a novel kunitz-type inhibitor: inhibition of cysteine proteases but not serine proteases a single amino acid substitution affects substrate specificity in cysteine proteinases from fasciola hepatica cathepsin b proteases of flukes: the key to facilitating parasite control? mm3-elisa detection of fasciola hepatica coproantigens in preserved human stool samples mm3-elisa evaluation of coproantigen release and serum antibody production in sheep experimentally infected with fasciola hepatica and f. gigantica higher physiopathogenicity by fasciola gigantica than by the genetically close f. hepatica: experimental long-term follow-up of biochemical markers the authors declare that they have no competing interests. key: cord-308461-4lhh3du0 authors: ueki, hiroshi; wang, i-hsuan; zhao, dongming; gunzer, matthias; kawaoka, yoshihiro title: multicolor two-photon imaging of in vivo cellular pathophysiology upon influenza virus infection using the two-photon impress date: 2020-01-29 journal: nat protoc doi: 10.1038/s41596-019-0275-y sha: doc_id: 308461 cord_uid: 4lhh3du0 in vivo two-photon imaging is a valuable technique for studies of viral pathogenesis and host responses to infection in vivo. in this protocol, we describe a methodology for analyzing influenza virus–infected lung in vivo by two-photon imaging microscopy. we describe the surgical procedure, how to stabilize the lung, and an approach to analyzing the data. further, we provide a database of fluorescent dyes, antibodies, and reporter mouse lines that can be used in combination with a reporter influenza virus (color-flu) for multicolor analysis. setup of this model typically takes ~30 min and enables the observation of influenza virus–infected lungs for >4 h during the acute phase of the inflammation and at least 1 h in the lethal phase. this imaging system, which we termed two-photon impress (imaging pathophysiology research system), is broadly applicable to analyses of other respiratory pathogens and reveals disease progression at the cellular level in vivo. in vivo two-photon imaging is an analytical approach that can be used to visualize cell dynamics and hemodynamics in organs or tissues of live animals. information in real time obtained by using this approach, such as changes in cell behavior and morphology, tissue localization, and blood flow, has revealed highly sophisticated and dynamic systems of living organisms. during in vivo imaging, the blood circulation in the tissue being observed is maintained; therefore, this technique is also effective for analyzing the migration and invasion of immune cells in the inflammatory environment. observations in physiological environments deepen our understanding of host response mechanisms under both steady-state and disease conditions. computed tomography, x-ray, and ivis spectrum (an in vivo imaging system) imaging methods have been used as non-invasive approaches; however, these techniques have low spatiotemporal resolution and have been able to estimate only the site of inflammation in an organ 1, 2 . therefore, it is impossible to observe cellular responses of the immune system using these approaches. by contrast, a two-photon excitation laser microscope, the light source of which is a near-infrared laser that produces low damage to cells but has long-reaching depth in tissue, enables us to capture the movement of cells in living animals at high resolution. two-photon imaging has been in use in biological science since the 1990s; it has progressed at a remarkable rate, and observation methods for various organs, including brain, liver, and lymph nodes, have been reported 3, 4 . in this protocol, we describe how to use it to image virus-infected lungs. we have previously demonstrated that this protocol works by using mice infected with mouse-adapted seasonal influenza virus (h1n1) or highly pathogenic avian influenza virus (h5n1) 5 . the lung, which is a respiratory organ, has contact with the outside environment and is an important organ for research on immunity to infectious diseases. in the seventeenth century, marcello malpighi discovered pulmonary capillaries and alveoli in the frog lung by using optical microscopy 6 ; now fluorescent reporter mice facilitate the study of disease models in conjunction with two-photon excitation microscopy (table 1) . however, a challenge encountered when imaging the lung is that it is constantly moving during respiration. the lung has been stabilized in several ways during in vivo observation by microscopy, including bronchus clamping, prolonged apnea, gluing, and suction 7, 8 ; however, it is difficult to reduce motion artifacts due to lung respiratory movement under physiological conditions and hence to obtain high-quality images. bronchus clamping can suppress respiratory motion artifacts of the lung lobe 9, 10 ; however, it is not suitable for long-term observation because it causes severe hypoxia. although prolonging apnea is less invasive [11] [12] [13] , it does not allow researchers sufficient time to observe the lung for image acquisition by two-photon excitation microscopy, and the quality of the images tends to deteriorate over time. gluing addresses the above limitations 14, 15 ; however, it can induce shear force injury and inflammation, which affect the interpretation of results. a suction window, which is currently the most commonly used stabilizing system during lung imaging, achieves moderate immobilization of the lung and high-quality images [16] [17] [18] [19] ; however, the observation period is limited to ≤12 h. ex vivo imaging of lungs and in vivo imaging of trachea have also been performed as complementary methods 8 . each of these methods has its advantages and disadvantages, and it is important to select and optimize the method best suited to the goal of the experiments and disease model. in vivo observation of lungs has been performed using various lung disease and experimental models, including bacterial infection, allergen inoculation, tumor metastasis, and lipopolysaccharide (lps)-induced sepsis (table 1) . however, for viral respiratory diseases, such as influenza, other than an observation in a methodology report 20 , only analyses of the trachea in vivo [21] [22] [23] and isolated lungs had been performed 24 , with no analysis of the lung in vivo, until our recent publication 5 (table 1) . unlike ex vivo methods, which involve isolated or sliced lungs, in vivo imaging using two-photon excitation microscopy of live animals enables researchers to observe hemodynamics, migration and extravasation of immune cells, as well as interactions among immune cells during influenza virus infection. however, it is technically demanding to perform two-photon excitation microscopy of live influenza virus-infected lung, which exhibits severe inflammation, requiring the development of highly sophisticated, less invasive instruments and surgical techniques. in addition, when observing animals infected with pathogenic viruses, specialized facilities and instruments are frequently required to avoid the spread of the virus. furthermore, because many types of immune cells infiltrate the infected lung in an inflammatory environment, it is necessary to distinguish the target immune cells from the infected cells by using fluorescent labels in the infected microenvironment. to detect multiple fluorescent signals excited simultaneously by a two-photon excitation laser, fluorochromes with different spectra and equal brightness must be selected; however, there is currently no comprehensive database of fluorescent reagents, fluorescent reporter viruses, and reporter mouse lines available for lung in vivo imaging. we therefore also provide a database of fluorescent dyes, antibodies, and reporter mouse lines that can be used in combination with a reporter influenza virus (color-flu) [25] [26] [27] for multicolor analysis under pathological conditions in this protocol. our system uses suction-based lung stabilization 16, 28 to improve an existing in vivo two-photon imaging system for influenza virus-infected lung as a model of an acute inflammatory respiratory disease 5 . we have successfully used c57bl/6 mice and transgenic mice of the c57bl/6 background (6-to 10-week-old males and females). by using our method, described in detail here, it is possible to visualize and analyze the behavior of immune cells and their interactions with infected cells during an influenza virus infection, which creates an acute inflammatory environment. a limitation of two-photon excitation microscopy is that the observation depth that can be achieved is a maximum of~70 μm. therefore, we cannot observe the bronchial region. this limitation is linked to the wavelength of the infrared laser and detector capability of the microscope. however, as laser technology develops, the observation depth achievable using this method will improve. in this protocol, we describe the application of this methodology to influenza virus infection of the lungs because this is what we have used it for previously. this protocol could be applied not only to studies of the early stages of inflammation due to infection or other causes, but also to analyses of tissue regeneration mechanisms in lungs that are in the process of recovering from infection or other protocol nature protocols injuries. the information provided will also be useful to those using two-photon imaging analysis for the evaluation of the effects of drugs and vaccines, as well as biological events in the lungs and other organs (e.g., liver, spleen) 5 . moreover, with minor modifications, the approach could be applied to analyses of other respiratory diseases, including other infectious models (e.g., severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers)), pulmonary fibrosis, and tumor metastasis. . dna fingerprinting showed that this cell line has the same origin as one obtained from atcc (cat. no. ccl-34, rrid:cvcl_0422) ! caution all viruses and infected animals should be handled in accordance with your institution's biosafety regulations. all work on highly pathogenic avian influenza viruses must be performed under biosafety level 3 (bsl3) regulations. accordingly, all our in vivo imaging studies were performed in the bsl3 facility at the university of tokyo (tokyo, japan), which is approved for such use by the ministry of agriculture, forestry, and fisheries of japan c critical the cells should be regularly checked to ensure that they are not contaminated with mycoplasma. • isoflurane (msd animal health) ! caution isoflurane is an anesthetic gas associated with adverse health outcomes. it should be used in a well-ventilated room or with another appropriate removal system. store it in a locked drawer at room temperature (18-25°c). • sevoflurane (maruishi pharmaceutical) ! caution sevoflurane is an anesthetic gas associated with adverse health outcomes. it should be used in a well-ventilated room or with another appropriate removal system. store it in a locked drawer at room temperature. c critical all reagents should be prepared under sterile conditions. fluorescent reagents should be protecting from light during the setup procedure because they are light sensitive. to prepare 10 mg/ml of tamoxifen solution in sunflower seed oil, dissolve 100 mg of tamoxifen in 1 ml of ethanol (99.5%) and add 9 ml of sunflower seed oil. after adding the ethanol and sunflower seed oil, mix well by vortexing and sonication. this solution can be stored in a refrigerator (2-8°c) for a week. ! caution tamoxifen powder should be handled in a hood. to avoid inhalation and contact with skin, wear rubber gloves and a surgical mask. prepare a solution at a concentration of 2 mg/ml in sterile 1× pbs or saline, make aliquots in 1.5-ml microtubes, and store them in a refrigerator (2-8°c) for up to 2 weeks. inject 50 μl (100 μg) of fluorescent dextran i.v. per mouse. qtracker 655 vascular labels immediately before use, add 5 μl of the stock solution to 95 μl of sterile 1× pbs or saline to make 100 μl total and inject 50 μl i.v. at a concentration of 0.1 μm. prepare a solution at a concentration of 1 × 10 8 beads/ml in sterile 1× pbs or saline, make aliquots of the solution in dark 1.5-ml microtubes, and store them in a refrigerator (2-8°c) for long periods (~3 months). immediately before use, mix well and inject 50 μl i.v. per mouse. qdot 655 wga immediately before use, add 5 μl of the stock solution to 95 μl of sterile 1× pbs or saline to make 100 μl total and i.v. inject 50 μl. prepare a solution at a concentration of 100 μm in sterile 1× pbs or saline, dispense the solution into dark 1.5-ml microtubes, and store them in a refrigerator (2-8°c) for up to 2 weeks. inject 50 μl of fluorescent dextran i.v. per mouse. divide the 5 mm dmso stock solution into dark 1.5-ml microtubes and store them at −20°c for up to 3 months. immediately before use, prepare a solution at a concentration of 50 μm in sterile 1× pbs or saline and i.v. inject 50 μl per mouse. prepare a solution at a concentration of 100 mm in sterile 1× pbs or saline, dispense the solution in dark 1.5-ml microtubes, and store them at −20°c for up to 3 months. immediately before use, prepare a solution at a concentration of 1 mm in sterile 1× pbs or saline and inject 50 μl i.v. per mouse. prepare a solution at a concentration of 10 mm in sterile 1× pbs or saline, make aliquots of the solution in dark 1.5-ml microtubes, and store them in a refrigerator (2-8°c) for up to 2 weeks. inject 50 μl of the solution i.v. per mouse. prepare a working solution according to the vendor's manual, dissolve pan caspase in vivo probe in 5 μl of dmso, and add 55 μl of 1× injection buffer (from the kit). inject 60 μl of the solution i.v. per mouse within 1 h of preparation. prepare a working solution according to the vendor's manual, dissolve 100 μl of pkh26pcl in 900 μl of ethanol and store at room temperature for up to 3 months. immediately before use, prepare a solution at a concentration of 10 μm in sterile dilution buffer (from the kit) and inject 50 μl intranasally per mouse. cellrox green, orange, and deep red immediately before use, add 50 μl of the stock solution to 450 μl of sterile 1× pbs or saline to make 500 μl total and inject 50 μl i.v. at a concentration of 250 μm. lysotracker blue, green, red, and deep red immediately before use, add 50 μl of the stock solution to 450 μl of sterile 1× pbs or saline to make 500 μl total and inject 50 μl i.v. at a concentration of 100 μm. mitotracker orange cmtmros, red cm-h2xros, and red fm immediately before use, dilute 50 μg of mitotracker in 1 ml of dmso and inject 50 μl i.v. at a concentration of 100 μm. c critical the mitotracker solution should be prepared fresh each time immediately before use. prepare the solution at a concentration of 10 mm in sterile 1× pbs or saline, make aliquots in dark 1.5-ml microtubes, and store them in a refrigerator (2-8°c) for up to 2 weeks. immediately before use, prepare a solution at a concentration of 10 μm in sterile 1× pbs or saline and inject 50 μl i.v. per mouse. prepare the solution at a concentration of 10 mm in dmso, make aliquots in dark 1.5-ml microtubes, and store them in a refrigerator (2-8°c) for up to 2 weeks. immediately before use, prepare a working solution at a concentration of 1 mm in sterile 1× pbs or saline and inject 50 μl i.v. per mouse. prepare each solution at a concentration of 1 mm in dmso, make aliquots in dark 1.5-ml microtubes, and store them in a refrigerator (2-8°c) for up to 1 week. immediately before use, prepare solutions at a concentration of 100 μm in sterile 1× pbs or saline and inject 50 μl i.v. per mouse. dilute fluorescent antibodies to a concentration of 1 µg per 10 μl with sterile 1× pbs or saline and inject 50 μl i.v. per mouse. ! caution it should be noted that antibody staining may affect the target cell behavior; for example, at a high dose (~200 μg), antibodies may neutralize cell activities and/or cause antibody-dependent cytotoxic activity [35] [36] [37] . in our studies, we use 5 µg of antibody for brightness screening because inoculation of fluorochrome-conjugated anti-ly-6g antibody at low doses (1-40 µg) into mice does not affect neutrophil recruitment 38 . the contribution of ly-6g, which is expressed predominantly on murine neutrophils, to recruitment during inflammation remains a matter of debate. it has been reported that low-dose antibody treatment inhibited ly-6g ligation and the recruitment of neutrophils to the site of inflammation 39 ; however, a more recent study indicated that ly-6g knockout did not affect either neutrophil differentiation or recruitment to the site of inflammation in catchup mice 32 . therefore, a low dose of anti-ly-6g antibody is used in our protocol. laser path adjustment system an overview of the laser path adjustment system is shown in fig. 1 . our two-photon excitation laser (chameleon vision ii) unit is placed on an anti-vibration table outside the bsl3 facility. the laser beam enters the bsl3 room, where the two-photon excitation scanning microscope is located, through a window (composed of a small glass window (wg12012-b) and a planar window (rs seal)) connecting the inside and the outside of the bsl3 facility (fig. 1c,d) . the laser path connecting the laser source unit and the two-photon excitation microscope is adjusted by automated laser beam alignment and the aligna 4d stabilization system is adjusted with two active mirrors. ! caution this system adjusts the laser path passing from the outside to the inside of the bsl3 facility for maintenance purposes, so there is no need for this setup unless you are using pathogens that require bsl3 containment. heat is generated when the laser source unit is running, so keep the temperature and humidity constant by using air conditioning equipment. ! caution the system should be operated only by users trained to deal with unenclosed high-power invisible beams and should be placed in an appropriate enclosure with interlocking doors. two-photon excitation laser scanning microscopy system for in vivo imaging of virus-infected mouse lungs in a bsl3 facility a schematic of the arrangement of the in vivo lung imaging system for virus-infected mouse is shown in fig. 2a, and layout examples are shown in fig. 2b -g. this in vivo lung imaging system is based on the upright microscope lsm 780 nlo system, which is equipped with four different lasers (excitation at 405, 488, 543, and 633 nm) for confocal imaging and a two-photon excitation laser (excitation at 630-1,050 nm). to be able to perform the surgical procedure on the mouse, we replaced the sample stage with a large, flat one (microscope stage for in vivo experiment) as shown in fig. 2b ,c. to efficiently excite multiple fluorescent proteins and fluorescent dyes simultaneously, the wavelength of the infrared laser should be set at 910 nm. all fluorescent spectra between the 410-and 695-nm wavelengths can be detected using a 20× water immersion lens, and we record signals in lambda image stacks (0.13 frames per s, 1,024 × 1,024 pixels) and acquire z-stack images with z-depths of 5 μm (total of 10-μm z-depth). we perform spectral separation of the acquired lambda stacks by using the linear unmixing function of the zen software. although the lsm 780 microscope system is controlled by a primary personal computer, we recommended adding >64 gb of ram for appropriate imaging analysis. we keep the mice on a heated stage on the sample stage and record their vital signs using a labox-1 pulse oximeter. to observe the lungs of the mice with a thoracotomy, we place the ventilator with an airway pressure monitor and anesthesia machine for rodents in appropriate positions on the stage. we installed high-efficiency particulate air (hepa) filters in the exhalation duct of the ventilation system (fig. 2b,d) , and the operator wore a positive pressure mask (versaflo faceshields) and a tyvek suit (fig. 2e-g) to avoid exposure to the viruses. ! caution the wavelength and power of the excitation laser should be adjusted appropriately according to the experimental conditions. increasing the laser power enhances target signals and enables detection of second-harmonic generation (shg), in which structures with repeating patterns lead to the formation of a signal. shg is a useful phenomenon for visualizing collagen fibers in the lung without staining; however, it should be noted that the autofluorescence of lung tissue is also enhanced under excessive excitation conditions ( supplementary fig. 1 ). when using this protocol, we did not perform experiments under which shg occurs, in order to minimize autofluorescence; it is better to adjust the laser power according to the experimental purpose. when the wavelength of the excitation laser is too short, the autofluorescence signal becomes very strong and it is difficult to observe properly. by contrast, when the laser wavelength is too long, it becomes difficult to obtain a signal because of the short excitation energy (supplementary fig. 2 ). ! caution although color separation of emission using a conventional optical band-pass filter is also available for this protocol, multispectral imaging is a useful approach for simultaneously analyzing multiple targets by eliminating tissue autofluorescence and identifying fluorescent labels with overlapping spectra 40, 41 . in vivo two-photon imaging is performed under conditions of single stimulation with a two-photon excitation laser; limitations exist regarding available fluorescent reagents/proteins for multiple labeling of target cells and lung architecture. therefore, we recommend using a multispectral approach to produce crosstalk-free images of fluorescence with overlapping spectra that cannot be separated by using band-pass filters. before starting experiments, it is necessary to collect spectral signatures of the emission signal of each fluorescent reagent and protein as reference spectra under the same excitation condition as will be used in the experiment. to observe the mouse lung using an upright microscope, it is necessary to prepare a thoracic suction window to immobilize the lung. in the bsl3 facility, animal experiments must be performed while wearing two or three layers of latex gloves; therefore, the thoracic suction window was designed for easy handling, even in the bsl3 facility, and to be minimally invasive for the infected animals (fig. 3a-c and supplementary fig. 3 ). to position a cover glass for each observation, flatten the upper surface of the thoracic suction window so that a commercially available cover glass will fit. this device is also designed to reduce concavity and convexity as much as possible so that blood containing virus cannot accumulate. connect the thoracic suction window to an aspirator through a waste tank and a suction regulator. to prevent the spread of virus-containing aerosols, install hepa filters between the waste tank and the suction regulator as shown in fig. 3d . starting up the imaging system equipment • timing 20-30 min 2 on the day of analysis, turn on the two-photon excitation laser and the aligna 4d control unit placed outside the bsl3 facility, and verify that they are working. c critical the aligna 4d control unit needs to be kept on. 3 wearing a tyvek suit, positive pressure mask, and gloves according to the guidelines for the bsl3 facility, enter the bsl3 facility where the imaging system is housed. ? troubleshooting 4 turn on the microscope controllers, confocal lasers, and the computer for the two-photon excitation microscope and the aligna 4d system. 5 launch the microscope control software zen and turn on the lasers, including the two-photon excitation laser. 6 launch the aligna 4d control software kangoo and adjust the laser path connecting the laser source unit and the microscope (supplementary fig. 4) . ? troubleshooting 7 wrap the hot plate with aluminum foil, turn it on, and keep it at 35°c. sterilize the surgical area and tools with 70% ethanol and place all instruments within easy reach. animal anesthesia • timing 2-3 min 8 turn on the gas anesthesia vaporizer and supply 5% isoflurane to a mouse anesthesia induction chamber. 9 anesthetize the influenza virus-infected mouse with 5% isoflurane in a mouse anesthesia induction chamber. subsequently, transfer the mouse to the hot plate while supplying 2% isoflurane via an anesthetic mask. ? troubleshooting 10 inject the chosen fluorescent dyes and antibodies via the retro-orbital plexus (as shown in supplementary video 1) using an insulin syringe. tables 4 and 5 show the brightness levels of antibodies and fluorescence of dyes, respectively, in vivo. ! caution when working with viruses in a bsl3 containment, it is not safe to use needles, so we avoid them as much as possible, which is a standard precaution in high-containment laboratories. in addition, in the bsl3 facility, animal experiments must be performed wearing two or three layers of latex gloves. tail-vein administration is a common method; however, it is not easy to perform these procedures with so many layers of gloves. use tweezers to hold down the mouse to make the administration route. when an infected animal is not used, an administration route can be created via the tail vein or the jugular vein. ? troubleshooting surgical procedure • timing 10-15 min c critical before experimenting with infected animals, practice the surgical procedures with euthanized animals. 11 place the mouse on its back and tape the anterior limbs with adhesive tape (fig. 4a) . 12 using straight scissors, cut the skin beneath the chin in the middle and expose the trachea (fig. 4b) . insert a tracheal cannula and intubate the mouse to facilitate mechanical ventilation with a ventilator (fig. 4c) . turn on the ventilator, ventilate the mouse at a respiratory rate of 120 breaths per min, and apply positive-end expiratory pressure (peep;~6 cm h 2 o) and a tidal volume of 0.5 ml. deliver isoflurane continuously at 2% to maintain anesthesia. ! caution perform the surgery with care so as not to cut the blood vessels. if bleeding occurs, stop the bleeding with fine bulldog forceps for microsurgery. 13 place the mouse in the right lateral decubitus position and re-fix its anterior limbs with the tape (fig. 4d) . make an incision in the skin at the left axilla using straight scissors, straight iris scissors, and hooked forceps (fig. 4e) . ! caution carefully change the mouse's position in order to avoid cannula drop off. 14 expose the left lung lobe by surgical intercostal incision between ribs 3 and 4, and keep it exposed by using retractors (fig. 4f) . the brightness of each fluorescent protein during in vivo lung imaging was scored as relative fluorescence intensity compared with fluospheres fluorescent microspheres as an internal standard. for relative intensities of 0-0.2, 0.2-0.6, 0.6-0.9, and >0.9, the brightness scores are represented as +, ++, +++, and ++++, respectively. the brightness of each fluorochrome during in vivo lung imaging was scored as relative fluorescence intensity compared with fluospheres fluorescent microspheres as an internal standard. for relative intensities of 0-0.2, 0.2-0.6, 0.6-0.9, and >0.9, the brightness scores are represented as +, ++, +++, and ++++, respectively. af, alexa fluor; nd, not detected. ! caution perform the surgery with care so as not to cut the blood vessels. if bleeding occurs, stop the bleeding with fine bulldog forceps for microsurgery. c critical because lungs infected with viruses often shrink, secure a large field of surgical view so that the suction window can reach it. 15 place the mouse beneath the objective lens and connect a device to monitor the heart rate of the mouse (we use a labox-1 pulse oximeter). starting up the thoracic vacuum window system • timing 2-3 min 16 turn on the aspirator connected to the thoracic suction window. 17 fix the thoracic suction window to the holding block at a 90°angle and put a round cover glass on the tip of the suction device. ? troubleshooting 18 turn on the suction pressure regulator and adjust the suction pressure to 25-30 mmhg. 19 lower the thoracic suction window gently to immobilize the mouse lungs (fig. 4g,h) . the thoracic suction window should cause the lung to stick to the cover glass because of negative pressure. ! caution carefully move the suction window so as not to scratch the objective. 20 position the objective lens above the thoracic window. 21 put water drops on the cover glass by using a pasteur pipette and lower the objective lens to the thoracic suction window (fig. 4i) . unmixing of spectrum data and analyzing the images • timing 1-2 h per sample 24 to unmix the spectrum data, prepare a reference image of each spectrum in advance. to make a reference image, acquire each fluorescent dye or protein separately without any co-staining and analyze the single fluorescent spectrum. we use the linear unmixing module of the zen software for separating spectrum data; however, other commercial or open-source software is available (table 6 ). 25 subject unmixed time-series stacks to image registration to correct for tissue drifts and respiratory artifacts. this step is critical to certain analyses, such as long-term tracking of individual cells or subcellular structures. in some cases, a reference channel is required for determining the shift and distortion of the objects. in our studies, we use time-series stacks of blood vessels or collagens for such use, because their localizations are constant over time without substantial changes in shape or structure during the observation. ! caution some image registration algorithms may cause spatial distortion. choose algorithms that generate corrected data suitable for your subsequent analyses, especially when examination of the shape and structure of cells and tissues is required. 26 analyze the movies as required for your experiment. troubleshooting advice can be found in table 7 . step 1, infection: 10-20 min anticipated results the imaging system described in this protocol enables the observation of the behavior of virusinfected cells and immune cells in infected lungs in real time. typical images of influenza virus-infected lung are shown in fig. 5a and supplementary video 2. when observing while using a multicolor fluorescent label, it is easier to analyze the detected images if the brightness level of each fluorophore is adjusted to make them similar. it is better to choose fluorescent dyes or proteins that possess high fluorescence stability for long-term observations (tables 2, 4 and 5). we have found that use of ma-cerulean-viruses or ma-venus-viruses for infection produces influenza virus-infected cells with sufficient brightness (table 3) . for labeling immune cells and alveolar cells, we have achieved good results by using the fluorochrome phycoerythrin (pe) for antibody staining and rosa-tdtomato 42 or -mtfp1 33 mice that were crossed with cell-specific cre-expressing mice. if using reporter mice expressing a fluorescent protein such as gfp, which is regulated by an endogenous promoter, the expression level of the fluorescent protein should be confirmed. to visualize the lung structure, we use texas-red dextran or qtracker 655 vascular labels for the red to far-infrared channel. mice die during anesthesia the level of anesthesia is too high decrease the concentration of anesthesia as soon as the mouse shows loss of righting reflex 10 mice regain consciousness during anesthesia the level of anesthesia is too low confirm the concentration of anesthesia; administer the reagents again after a brief pause 15 no heart rate is measured the monitoring probe is mispositioned make sure that the monitoring probe is in the appropriate place 17 the cover glass falls off the cover glass does not hold on the suction device put water droplets on the tip of the suction device and then place the cover glass on it influenza virus-infected lungs are infiltrated by numerous immune cells, including neutrophils and monocytes [43] [44] [45] . an immune cell-specific reporter mouse line can be used to visualize cells infiltrating the alveoli and cells in blood vessels, whereas it is preferable to label intravascular cells by intravenous administration of fluorochrome-conjugated antibodies 5, 46, 47 . consistent with previous reports, intravenously injected antibodies will label only the cells in contact with the blood flow and not those in extravascular regions under our experimental conditions 5 . by administering a fluorescently labeled antibody against neutrophils into neutrophil reporter mice, we can observe the behavior of both the neutrophils infiltrating the influenza-infected lungs and the neutrophils in blood vessels separately (fig. 5b) . to observe the interaction between different kinds of infiltrating immune cells, such as neutrophils and monocytes, double-reporter mice expressing fluorescent proteins with different spectra but similar brightness have a major advantage (fig. 5c and supplementary video 3) . co-infection of the host with different strains of influenza virus can lead to the emergence of reassortant viruses. by infecting mice with color-flu viruses that produce different fluorescence spectra, we detected alveolar epithelial cells that simultaneously expressed two fluorescent proteins in vivo (fig. 6) . visualization of co-infected cells might enable us to better understand the reassortment process of influenza viruses in vivo. in summary, the use of this in vivo imaging system for infected animal and multicolor imaging enables us to analyze pathology and immune cell dynamics at the cellular level, which would not be possible by using conventional histopathology methods. this imaging system thus provides a novel and useful approach for investigating viral pathogenicity. further information on research design is available in the nature research reporting summary linked to this article. the data that support this study are available from the corresponding author upon reasonable request. the matlab scripts are available at https://github.com/kawaokalab/ueki_pnas_2018. for all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or methods section. the exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement a statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly the statistical test(s) used and whether they are one-or two-sided only common tests should be described solely by name; describe more complex techniques in the methods section. a description of all covariates tested a description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons a full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) and variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) for null hypothesis testing, the test statistic (e.g. f, t, r) with confidence intervals, effect sizes, degrees of freedom and p value noted give p values as exact values whenever suitable. for hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes estimates of effect sizes (e.g. cohen's d, pearson's r), indicating how they were calculated our web collection on statistics for biologists contains articles on many of the points above. policy information about availability of computer code to efficiently excite multiple fluorescent proteins and fluorescent dyes simultaneously, the wavelength of the infrared laser was set at 910 nm. all fluorescent spectra between 410 and 695 nm wavelengths were detected using a 20x water immersion lens (carl zeiss ag, germany) and the signals were recorded in lambda image stacks. we use the linear unmixing module of zen software for separating spectrum data. unmixed time-series stacks are subjected to image registration to correct for tissue drifts and respiratory artefacts. a reference channel is required for determining the shift and distortion of the objects. in our studies, we employ time-series stacks of blood vessels or collagens for such use, as their localizations are constant over time without significant changes in shapes or structures during the observation. for manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. we strongly encourage code deposition in a community repository (e.g. github). see the nature research guidelines for submitting code & software for further information. policy information about availability of data all manuscripts must include a data availability statement. this statement should provide the following information, where applicable: -accession codes, unique identifiers, or web links for publicly available datasets -a list of figures that have associated raw data -a description of any restrictions on data availability the data that support this study are available from the corresponding author upon reasonable request. october 2018 field-specific reporting please select the one below that is the best fit for your research. if you are not sure, read the appropriate sections before making your selection. for a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf all studies must disclose on these points even when the disclosure is negative. only one sample was shown as a representative example that can be obtained by using the imaging protocol. data exclusions no data was excluded since one representative image was shown. no repeated measurements were performed in this paper since one image has been shown as a representative image by using the imaging protocol. • microsurgery straight scissors (13.5 cm • microsurgery bulldog forceps (brc, cat. no. 70052-30cii/r) • insulin syringes (0.5 ml, 100 u, 30 gauge × 10 mm; nipro, cat. no. 08277) • pasteur pipettes (bd falcon, cat. no. 357575) • customized surgical retractor • thoracic suction window (sakura seiki, custom made) stage for mounting a thoracic suction window (sakura seiki, custom made) • suction regulator (iwaki, cat. no. 1450050) • cover glass (matsunami glass, cat. no. c013001) • hot plate (hipet, cat • confocal microscope system (zeiss, model no. lsm 780 nlo) • infrared laser (coherent, model no. chameleon vision ii) • 20× water immersion lens (zeiss, plan-apochromat model) • beam-pointing stabilizer (tem messtechnik, model no. aligna 4d system high-efficiency particulate air (hepa) filters (vacushield; pall, cat. no. 4402) • artificial ventilator (shinano, cat • airway pressure monitor (shinano) • gas anesthesia vaporizer • mouse anesthesia induction chamber • mouse anesthesia mask • positive pressure mask (versaflo faceshields softwear iii) • surgical gloves rs seal (roxtec, cat labox-1) highly sensitive real-time in vivo imaging of an influenza reporter virus reveals dynamics of replication and spread consecutive ct in vivo lung imaging as quantitative parameter of influenza vaccine efficacy in the ferret model calcium imaging of single stereocilia in hair cells: localization of transduction channels at both ends of tip links in vivo fluorescence microscopy: lessons from observing cell behavior in their native environment in vivo imaging of the pathophysiological changes and neutrophil dynamics in influenza virus-infected mouse lungs marcello malpighi and the discovery of the pulmonary capillaries and alveoli live imaging 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lymphocyte-specific, cre-mediated mutagenesis in mice genetic analysis of basophil function in vivo lethal influenza infection in the absence of the natural killer cell receptor gene ncr1 high-efficiency type ii cell-enhanced green fluorescent protein expression facilitates cellular identification, tracking, and isolation hyperspectral phasor analysis enables multiplexed 5d in vivo imaging the orfeo toolbox remote sensing image processing software optimizing imaging parameters for the separation of multiple labels in a fluorescence image spectral imaging and linear un-mixing enables improved fret efficiency with a novel gfp2-yfp fret pair blind source separation techniques for the decomposition of multiply labeled fluorescence images image matching as a diffusion process: an analogy with maxwell's demons diffeomorphic demons: efficient non-parametric image registration automated motion artifact removal for intravital microscopy, without a priori information nonrigid registration using free-form deformations: application to breast mr images imart software for correction of motion artifacts in images collected in intravital microscopy automated filtering of intrinsic movement artifacts during twophoton intravital microscopy removing physiological motion from intravital and clinical functional imaging data software tools for single-cell tracking and quantification of cellular and molecular properties cellprofiler: image analysis software for identifying and quantifying cell phenotypes icy: an open bioimage informatics platform for extended reproducible research additional information supplementary information is available for this peer review information nature protocols thanks megan macleod and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. 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 we thank s. watson for editing the manuscript. we thank k. iwatsuki-horimoto, l. wu, s. fukuyama, y. matsuzawa, and k. miyake randomization no randomization is included in this paper since one image has been shown as a representative image by using the imaging protocol. blinding was not relevant to this study which is describing a imaging protocol and anticipated results. we require information from authors about some types of materials, experimental systems and methods used in many studies. here, indicate whether each material, system or method listed is relevant to your study. if you are not sure if a list item applies to your research, read the appropriate section before selecting a response. all antibodies used are commercialized and the fluorescence has been tested in this study. the information is included in table 4 . animals and other organisms policy information about studies involving animals; arrive guidelines recommended for reporting animal research laboratory animals six-ten-week-old c57bl/6 mice (japan slc, inc.) and transgenic mouse lines were used in this study. all animal care and experiments conformed to the guidelines for animal experiments of the university of tokyo, and were approved by the animal research committee of the university of tokyo (pa17-31 and pa17-17). all in vivo imaging studies were performed in the biosafety level 3 facility at the university of tokyo (tokyo, japan), which is approved for such use by the ministry of agriculture, forestry, and fisheries of japan. field-collected samples not applicable. all experiments with mice were performed in accordance with the university of tokyo's regulations for animal care and use and were approved by the animal experiment committee of the institute of medical science, the university of tokyo.note that full information on the approval of the study protocol must also be provided in the manuscript. key: cord-356207-tpn5cg4n authors: beniac, daniel r; andonov, anton; grudeski, elsie; booth, tim f title: architecture of the sars coronavirus prefusion spike date: 2006-07-16 journal: nat struct mol biol doi: 10.1038/nsmb1123 sha: doc_id: 356207 cord_uid: tpn5cg4n the emergence in 2003 of a new coronavirus (cov) responsible for the atypical pneumonia termed severe acute respiratory syndrome (sars) was a stark reminder that hitherto unknown viruses have the potential to cross species barriers to become new human pathogens. here we describe the sars-cov 'spike' structure determined by single-particle cryo-em, along with the docked atomic structures of the receptor-binding domain and prefusion core. supplementary information: the online version of this article (doi:10.1038/nsmb1123) contains supplementary material, which is available to authorized users. and dialysed against pbs. sars-cov-enriched fractions were checked by sds-page and western blotting, and rendered non-infectious by irradiation in a gamma cell on dry ice with a 2 mrad exposure for 90 minutes. the dose was chosen as sufficient for viral inactivation 1 , while retaining antibody 1,2 or enzyme functional activities 3 . previous studies have indicated that viral rna is highly sensitive to radiation and that viruses were by 2 mrad 1 , even when cooled to minimize heat damage to proteins, whereas antigenicity of viral proteins and antibody binding titres were maintained after doses of 3.6 mrad 1 or 5 mrad 3 . irradiated specimens were tested for infectivity by inoculation onto vero e6 cells, and examined for cytopathogenic effects for 10 days, followed by blind passage of the cells and testing for the growth of sars-cov by pcr 4 . cryo-electron microscopy. virus samples (4µl) were applied to glow-discharged holey carbon films supported on 400-mesh copper grids. after blotting immediately for 2-5 seconds with filter paper, grids were plunged into liquid ethane cooled by liquid nitrogen, using a custom built gravity-operated freezing device. specimens were transferred to a tecnai 20 g2 transmission electron microscope (fei) operated at 200kv, equipped with a gatan 626.dh low-temperature specimen holder. observations were made at temperatures of ~ -185°c and images recorded at 29,000x magnification on kodak so -163 electron image film at a dose of 10-20 electrons/å 2 with an exposure of 1-2 seconds. film was developed in kodak d19 for 12 minutes at room temperature. immuno-gold stained samples were imaged at room temperature in the tecnai 20, and digital images were collected using either a gatan msc or amt advantage xr-12 digital cameras. image processing. the exact magnification in the microscope was determined to be 29,968x using a calibration grid ( formvar-carbon coated 400-mesh nickel grids were floated on drops of purified sars-cov (40μl) for 1 minute. all incubations were carried out at 20 °c. grids were then washed in pbs, followed by a 10-minute block in pbs-g-bsa (pbs ph7.2, 0.2% glycine, 2% bsa). after washing in pbs-g (pbs ph7.2, 0.2% glycine), grids were then incubated in primary antibody diluted in pbs-g-bsa or convalescent patient serum diluted in pbs, followed by washes in pbs-g and then incubated in secondary antibody (conjugated to 5 or 10 nm gold), followed again by washing in pbs-g. grids were fixed (1% paraformaldehyde, 1% glutaraldehyde in pbs), washed in deionised water and negatively stained in either 2% uranyl acetate or 2% methylamine tungstate (nanoprobes, yaphank, ny.). in the case of nucleocapsid labelling, specimens were permeabilised by pre-treatment with 0.05% np-40 for in pbs on ice. in all experiments negative controls were run which included omission of the primary antibody to test for non-specific binding. inactivation of lassa, marburg, and ebola viruses by gamma irradiation functional integrity of intravenous immunoglobulin following irradiation with a virucidal dose of gamma radiation controlled gamma-irradiation mediated pathogen inactivation of human urokinase preparations with significant recovery of enzymatic activity detection of airborne severe acute respiratory syndrome (sars) coronavirus and environmental contamination in sars outbreak units eman: semiautomated software for highresolution single-particle reconstructions spider and web: processing and visualization of images in 3d electron microscopy and related fields eman: semiautomated software for highresolution single-particle reconstructions the ribosome at improved resolution: new techniques for merging and orientation refinement in 3d cryo-electron microscopy of biological particles multi-resolution contour-based fitting of macromolecular structures structure of sars coronavirus spike receptor-binding domain complexed with receptor solution structure of the severe acute respiratory syndrome-coronavirus heptad repeat 2 domain in the prefusion state ucsf chimera--a visualization system for exploratory research and analysis eman: semiautomated software for highresolution single-particle reconstructions structure of the hemagglutinin precursor cleavage site, a determinant of influenza pathogenicity and the origin of the labile conformation situs: a package for docking crystal structures into low-resolution maps from electron microscopy key: cord-260834-v254de8k authors: lim, chia chiu; woo, patrick c. y.; lim, theam soon title: development of a phage display panning strategy utilizing crude antigens: isolation of mers-cov nucleoprotein human antibodies date: 2019-04-15 journal: sci rep doi: 10.1038/s41598-019-42628-6 sha: doc_id: 260834 cord_uid: v254de8k antibody phage display has been pivotal in the quest to generate human monoclonal antibodies for biomedical and research applications. target antigen preparation is a main bottleneck associated with the panning process. this includes production complexity, downstream purification, quality and yield. in many instances, purified antigens are preferred for panning but this may not be possible for certain difficult target antigens. here, we describe an improved procedure of affinity selection against crude or non-purified antigen by saturation of non-binders with blocking agents to promote positive binder enrichment termed as yin-yang panning. a naïve human scfv library with kappa light chain repertoire with a library size of 10(9) was developed. the improved yin-yang biopanning process was able to enrich monoclonal antibodies specific to the mers-cov nucleoprotein. three unique monoclonal antibodies were isolated in the process. the yin-yang biopanning method highlights the possibility of utilizing crude antigens for the isolation of monoclonal antibodies by phage display. the blocking effect of ptm buffer and e. coli lysate towards αubi and m13ko7 phages against rubi. initially, a proof of concept was conducted to investigate the binding efficiency of the anti-ubiquitin (αubi) scfv phage towards rubi in the complex microenvironment during biopanning. a total of four different conditions (complex microenvironment) were tested stepwise. first, both αubi and m13ko7 phages were assimilated into different blocking agents: pbs, 2% ptm buffer, e. coli lysate and combinations of 2% ptm buffer with e. coli lysate. phage titres were determined after binding between αubi to rubi and m13ko7 phage to rubi. the immunotube blocking with e. coli lysate and skimmed milk overnight, wash before use; 2. adding the desired amount of antibody phage library into the immunotube; 3. phage library preincubation takes place for 1 h in the immunotube; 4. bring over the preincubated phage library into antigen-coated well for binding; 5. stringent washing and elution of phage with trypsin enzymatic action. www.nature.com/scientificreports www.nature.com/scientificreports/ unbound phages were collected as 'blocked phages' and bound phages as 'eluted phages' (fig. 3) . based on the phage titres, αubi phage showed good binding capacity towards rubi in the presence of 2% ptm and e. coli lysate. some amount of m13ko7 phage was also rescued despite incubation in different sets of blocking agents. nevertheless, it should be highlighted that the combination of ptm buffer and lysate managed to block a large amount of αubi and m13ko7 phage in separate sample sets. the extent of blocking was further determined by calculating the ratios of the difference between blocked αubi in a mixture of ptm buffer and lysate against other sets of blocking agents. there was approximately a 10-fold difference between αubi blocked with a combination of ptm buffer and lysate against the ptm buffer only and lysate only. a 0.83-fold difference was recorded between the ptm buffer and lysate combination against pbs only, where the cut off was 1-fold. the similar ratios of difference were determined for blocked m13ko7 sample sets at a cut off of 1-fold. the amount of blocked m13ko7 in the ptm buffer and lysate combination was higher by 7-fold with respect to m13ko7 in pbs only, 6.73-fold for m13ko7 in lysate only and 0.67-fold for m13ko7 in ptm only. it showed that the combination of ptm buffer and lysate was able to capture more unbound phages than other blocking agents. next, a single round biopanning simulation was conducted in a similar fashion to further investigate the binding capacity of αubi phage towards rubi. αubi and m13ko7 phage were mixed at a ratio of 1:10 and was used for biopanning simulation. the phage titres across 4 samples were determined as 10 8 -10 9 cfu/ml, 10 6 -10 7 www.nature.com/scientificreports www.nature.com/scientificreports/ cfu/ml, 10 8 -10 9 cfu/ml and 10 4 -10 5 cfu/ml for 'blocked αubi' , 'eluted αubi' , 'blocked m13ko7' and 'eluted m13ko7' , respectively ( supplementary fig. s1a ). the binding between αubi phage towards rubi was relatively good despite a higher amount of m13ko7 was present in the sample. the ratios of difference between unbound and eluted fractions for αubi phage across 4 samples were 57-fold, 325-fold, 169-fold and 172-fold (fig. 4a) . the fold change of unbound and eluted m13ko7 across 4 samples were large (10,000-fold, 43,333-fold, 12,916-fold and 40,000-fold). the highest ratio of difference was presented by m13ko7 blocked in ptm buffer, followed by a combination of ptm buffer and lysate and lysate only. this implied that the blocking agents were able to capture the background phages and reduce non-specific binding towards the antigen. lastly, crude rubi was used as the target antigen in a similar biopanning simulation as mentioned above. collectively, the phage titres for unbound αubi phage (10 9 cfu/ml) and unbound m13ko7 (10 9 -10 10 cfu/ml) were slightly higher (about 1-3-fold for unbound αubi phage and 5-13-fold for unbound m13ko7) than those compared to previous simulations using the purified rubi fraction as shown in supplementary fig. s1b . on the other hand, the differences between the eluted αubi phage with eluted m13ko7 were lower compared to the previous simulation. it was 55-180-fold in differences between the eluted αubi phage with eluted m13ko7 for previous simulation whereas the differences dropped to only 2-5-fold, with the exception for sample 3 (300-fold). this was due to the abrupt drop of eluted m13ko7. the binding capacity of αubi phage towards rubi in crude fraction was consistent with previous simulations, as the similar amount of eluted αubi phages was determined to be in the range of 10 6 -10 7 cfu/ml. the highest ratio of difference between unbound and eluted αubi phage was recorded from sample 2 (ptm buffer only) with a 1,000-fold difference followed by a 700-fold difference recorded from sample 4 (ptm buffer and lysate). as for the ratios of difference between unbound and bound m13ko7, the highest fold change was 210,000-fold recorded from sample 3 (lysate only) while the lowest fold change was determined from sample 4 (ptm buffer and lysate) with only 11,000-fold difference (fig. 4b ). there were different trends exhibited from the fold change between unbound and bound αubi phage with m13ko7 but the blocking effects of ptm buffer and lysate were able to be identified. the phage elisa was conducted to measure the performances and effects of different blocking agents on the binding of αubi phages against crude rubi. out of the four samples, the combination of ptm buffer and lysate showed the highest signal (1.226) detected with anti-c-myc-hrp as shown in fig. 4c . this combination of blocking agents was able to control the interference of background phages without compromising the binding capacity of αubi phage. therefore, this combination of blocking agents was used for the next stage of biopanning optimization. lysate preblocking and phage preincubation effects towards αubi and m13ko7 phages against rubi. to further optimize the biopanning conditions against crude antigens, the 'yin-yang' (capture and eliminate) approach was designed to reduce non-specific binding by background phages. the approach utilized e. coli lysate and ptm to block the microwells, followed by incubation of phages with ptm buffer in the prepared microwells. this procedure imposes a capture and elimination effect to the phage mixture, enabling non-specific phages to be preoccupied from interfering in the binding activity. initially, αubi and m13ko7 phages were subjected to binding with purified rubi. the unbound phages were collected as 'blocked phages' and bound phages as 'eluted phages' (fig. 5) . the overall phage titres for unbound m13ko7 were higher compared to www.nature.com/scientificreports www.nature.com/scientificreports/ the previous optimization without lysate preblocking and phage preincubation by approximately 25 to 100-fold. also, the differences of unbound and bound m13ko7 were larger compared to the last optimization by more than 10-fold. therefore, lysate preblocking and phage preincubation was found to reduce the non-specific binding of background phage more effectively. next, a mixture of αubi and m13ko7 (1:10 ratio) was subjected to incubation with ptm buffer in lysate/ ptm-preblocked microwell ('yin' step) prior to binding with purified rubi ('yang' step). a higher number of unbound m13ko7 phage at 10 10 cfu/ml was rescued or determined ( supplementary fig. s2a ). this was a 10-fold increase in comparison to the biopanning process devoid of preblocking and preincubation, indicating that the yin-yang process was more efficient in removing non-specific phage particles. however, higher numbers of eluted m13ko7 phage particles were also obtained indicating more background phages were recovered during the simulation. nevertheless, the bound αubi phage was slightly higher than the bound m13ko7 by 4-32-fold. the www.nature.com/scientificreports www.nature.com/scientificreports/ ratio of difference between unbound and bound αubi phage was determined (fig. 6a ). the highest difference was recorded from sample 1 (pbs only) at 6,800-fold whereas the lowest difference was sample 4 (ptm buffer and lysate) at only 450-fold. the highest fold change between the unbound and bound m13ko7 was determined from sample 2 (ptm buffer) at 1380000-fold, followed by sample 3 (lysate) and sample 4 (ptm buffer and lysate), both were 125,000-fold. this showed that ptm buffer and lysate were suitable for preblocking and phage preincubation. lastly, biopanning was conducted using crude rubi as the target antigen. based on the phage titres in supplementary fig. s2b , the amount of unbound m13ko7 (10 10 cfu/ml) were similar to that of the biopanning simulation using purified rubi. this indicates the efficiency of e. coli lysate and ptm buffer blocking to remove non-specific interaction from crude fractions. as for bound phages, the number of bound m13ko7 (10 4 -10 6 cfu/ml) however was almost the same as the bound αubi phages (10 4 -10 5 cfu/ml) with a difference of only 0.5-4-fold. this pattern was expected, as the difference of bound phages in previous simulation (with pure rubi) was also minimal. notably, the amount of bound m13ko7 for sample 4 (ptm buffer and lysate) was 10-fold higher than αubi phage. this showed a dominant population of empty phages in the eluted phages rather than antibody presenting phages. the highest fold change between unbound and bound αubi phage was recorded from sample 2 (ptm only) at 6,800-fold, followed by sample 1 (pbs only) (5,500-fold) and sample 4 (ptm and lysate) (4,300-fold) (fig. 6b) . on the other hand, the highest ratio of difference between unbound and bound m13ko7 was determined from sample 1 (pbs only) at 716,000-fold, followed by the ptm buffer and lysate combination (254,000-fold). this implied that the 'yin' step was sufficient to deplete the non-specific binding of m13ko7. moreover, the binding capacity of αubi phage towards crude rubi was not affected by the 'yin' effect, but in contrast helped to control the interference of background phages. the eluted phages were amplified and packaged for the subsequent phage elisa to assess the 'yin-yang' effect utilising different blocking agents. out of the four samples, the combination of ptm buffer and lysate showed the highest signal (0.895) detected with anti-c-myc hrp as shown in fig. 6c . despite a drop in the binding of αubi towards the crude rubi that was evident with a lower od 405nm readout, it could be concluded that the 'yin' effect is essential and the use of the ptm buffer and lysate combination was suitable to reduce the non-specific interaction as designed by the 'yin' effect. human naive kappa light chain scfv phagemid library construction. a naïve scfv library was constructed using healthy human samples focusing on the igm v h and v k repertoire. the diversity of the constructed naïve antibody phagemid library was estimated to be 10 9 . colony pcr was performed and resolved by agarose electrophoresis as shown in supplementary fig. s3 . a total of 7 colonies from 10 randomly selected colonies presented a single band of the expected amplicon size at approximately 1,100 bp. this indicated that full-length scfv constructs were incorporated into the phagemid vector at a reasonable success rate of 70%. 'yin-yang' biopanning and polyclonal elisa. the in-house human naïve kappa light chain scfv library was subjected to 'yin-yang' biopanning against crude rmers-np. a higher percentage of blocking agent and an increase in washing stringency were introduced during the biopanning rounds based on the conditions from the earlier optimization steps. in just two rounds of affinity selection, there was an enrichment of specific anti-rmers-np antibodies. supplementary fig. s4 shows the polyclonal elisa result of crude rmers-np biopanning at absorbance 405 nm. a 405nm were slightly low at 0.05 and 0.17 for the first and second round of biopanning respectively. this could be attributed to the low amount of rmers-np being presented in the total crude lysate. the phage titre determination indicates approximately 2.5 folds of enrichment was observed. the phage enrichment was tabulated in supplementary table s1 that complements the polyclonal elisa result. monoclonal elisa. a total of 92 individual clones were selected from the second polyclonal pool for monoclonal elisa assay against crude rmers-np. the positive clones were identified with a cut-off od 405nm value above 0.3 after normalizing with their background values. the readouts of positive controls in the monoclonal elisa were 3.5 and 3.9 respectively (h5 and h11, labelled as p), while the negative controls were below 0.1 (h6 and h12, labelled as n) (fig. 7) . overall, the monoclonal elisa assay resulted in 40 positive binders. the generated human naïve library was able to provide satisfactory enrichment for anti-rmers-np. a preliminary screening by colony pcr was subjected to these 40 positive clones to identify clones with full-length inserts before identification by sequencing. sequence analysis. sequencing results of the selected binders were analysed by imgt/v-quest to determine the identity of the scfv monoclonal antibody clones. only 3 full length antibody clones were obtained that showed unique combinations of heavy chain and kappa light chain families. in addition, 2 clones were found to have deletions at their gene segments but not at the complementarity-determining regions (cdrs) and were categorized as partial clones. then, 3 clones presented with umber and ochre stop codons while 2 truncated clones were also obtained from the anti-rmers-np pool. a compilation of the gene segment usage for the full clones is presented in table 1 . preparation of antibody fragments. the clones a4, c8 and g12 were successfully expressed as inclusion bodies and subjected to solubilization using 8 m urea buffer (ph 8). western blotting using streptavidin-hrp was performed to detect the biotinylated antibody fragments as shown in fig. 8a . the purified recombinant antibody fragments showed a distinct band at the expected size of ~32 kda for ra4scfv and rg12scfv. despite the expected size of rc8scfv is ~32 kda, it ran slightly higher at ~35 kda. www.nature.com/scientificreports www.nature.com/scientificreports/ antigen binding elisa. the preparation of purified rmers-np(his 6 ) and rgfp(his 6 ) for elisa were detailed in the supplementary method 10. the expression profiles of the antigens were included in supplementary result 7. the binding profiles of the recombinant antibody fragments were determined against a range of rmers-np(his 6 ) from 1 μg to 150 μg. each sample set was incubated with 10 μg of purified antibody. in order to assess the cross-reactivity of the antibody towards other his 6 -tagged proteins, rgfp(his 6 ) was used. a total of 10 μg of purified rgfp(his 6 ) was coated and allowed to bind with 10 μg of each antibody clone respectively. www.nature.com/scientificreports www.nature.com/scientificreports/ after normalization. the cross-reactivity profiles of the antibody fragments towards rgfp(his 6 ) at absorbance 405nm after normalizing with their background values are shown in fig. 8f . the data also takes into consideration cross reaction against streptavidin-hrp. overall, the antibody fragments had very little binding towards the control antigen as the readouts were lower than 0.1. therefore, the antibody fragments could be identified as specific towards the rmers-np. to date, antibody phage display still persist as the method commonly used for the generation of recombinant antibodies against various targets for biomedical applications and research 6, 22 . the robustness of bacteriophage to display antibodies has aided to expand the portfolio of target antigens which includes toxins, transmembrane proteins, peptides, haptens and even epitopes 23 . unfortunately, the problems of expression and purification of e. coli produced recombinant proteins are atypical. some recombinant antigens have solubility and purity problems 18 , stability issues, difficult to be immobilized 24 and exist in multiple morphologies 25 . due to these reasons, there is a need to develop an alternative procedure for selection towards relevant antigens presented in a complex mixture especially in situations when rapid antibody development is required or if the purified target is unavailable. therefore, we demonstrated a capture and elimination step prior to affinity selection in order to provide a 'subtractive' effect to the phage population by blocking the undesired binders via binding to other components in the blocking mixture while freeing the positive binders to favour binding towards the relevant antigen. the'yin-yang' panning method is somewhat akin to the negative/positive selection panning strategies whereby negative selection aims to remove unwanted binders and allowing positive selection of targeted binders 24, 26, 27 . the main difference between this study and the previous methods is the approach and target of negative selection. the target for negative selection is the unspecific antibody phage binders that binds to proteins other than the target antigen. in light of this, the blocking mixture is mainly used to deplete the negative binders. in the previous approaches, polyclonal and mabs are used to target the unrelated epitopes presented on phages in order to select and remove them, allowing the remaining phage clones to select for antibodies that have affinity towards specific epitopes 24, 26, 27 . moreover, the 'yin-yang' method offers two rounds of negative selection during a single round of biopanning, one before positive selection and one simultaneously carried out with the positive selection. this creates a competitive situation where a small number of unbound negative phage will compete with a large number of unbound positive phage for the target antigen in the presence of target-unrelated proteins. we demonstrated an inexpensive immobilisation strategy of target-unrelated proteins onto a solid phase for negative selection, whereby unrelated proteins preoccupy non-specific binders, either bound to surface or in phage mixture. the limitation of this method is the inability to remove the non-specific binders prior to binding. however, imposing www.nature.com/scientificreports www.nature.com/scientificreports/ a more stringent washing process during phage elution can compensate for this. therefore, the phages binding to unspecific proteins can be washed away under stringent conditions, subsequently rescuing bound phages that is immobilised on the plate. additionally, this method can also be adapted to membrane-bound proteins by using the western blotting system reported by ding, et al. 28 and liu, et al. 29 . a comparison of other published enrichment strategies and the 'yin-yang' method is provided in table 2 . preliminary optimization was performed in a stepwise manner to investigate the strength of blocking agents towards phage binding capabilities for both antibody phage and phage without antibody using αubi and m13ko7. the use of αubi as a model phage for optimization represents the specific rubi positive binders while m13ko7 represents the population of non-specific binders. the mixture of ptm and e. coli lysate exerts the highest blocking effect for both αubi and m13ko7. nonetheless, the bulk m13ko7 population (about 10 11 cfu/ ml) is expected to saturate the sample thus allowing it to adsorb and bind non-specifically to rubi which can be recovered later. m13ko7 binds at low affinity towards other antigens as reported by lamboy et al. 30 and modifications have been performed to reduce unspecific binding by m13ko7. to simulate the similar conditions during affinity selection, αubi and m13ko7 were mixed to represent a cocktail of library phage for panning against rubi in purified and crude forms 31 . competition is expected to occur between positive binders and non-specific binders against the target antigen. thus, a competitive selection could be imposed by saturating the non-specific binders, m13ko7 with the competitive proteins (lysate and ptm) thereby promoting binding of αubi towards purified rubi 32 . despite the presence of other unknown e. coli proteins in the crude fraction of rubi with the actual concentration of rubi being expected to be lower than the purified rubi, the binding capability of αubi is still unaffected as shown in phage elisa assay upon affinity selection against crude rubi. the blocking effect of ptm and e. coli lysate was the highest among the samples and was able to block m13ko7 from competing with αubi. there was no bias in the titres of packaged phages for elisa assay as they were normalized to 10 11 before assayed. during the 'yin-yang' (capture and elimination) step, preincubation of phage was performed in wells coated with e. coli lysate and subsequently with ptm. proper blocking of the surface with blocking agent such as skimmed milk helps to inhibit unspecific binding [33] [34] [35] and phage preincubation can enhance the reduction of unspecific binders even to the treated surface 36 . the idea of using e. coli lysate proteins as one of the competitive proteins in biopanning originates from the whole cell biopanning approach, where target antigen and un-relevant proteins are presented in the cell lysate mixture 33 . theoretically, it could be expected that non-binders were depleted during the 'yin-yang' step, subsequently allowing free positive binders to initiate binding against the target during affinity selection. the 'yin-yang' step complements the blocking effect by the lysate and ptm, www.nature.com/scientificreports www.nature.com/scientificreports/ thereby allowing more non-binders to be trapped. this was evident as a higher number of unbound m13ko7 was recorded. however, throughout the biopanning simulation against rubi in purified and crude forms, there were increasing numbers of non-binders being rescued, close to the amount of positive binders rescued. this could be attributed to insufficient washing that resulted in the rescue of non-binders together with positive binders during elution 37 . despite this, the rescued phages from the crude rubi were amplified and subjected to phage elisa assay to determine the best performing sample. the best performing sample was the lysate and ptm combination that gave the highest readout after background subtraction. hence, we demonstrated that the 'yin-yang' step is crucial during biopanning against crude antigen while the combination of ptm and lysate can help to control and reduce the disturbance of non-binders during antigen binding. nevertheless, due to the rise of background phage elution, washing stringency should be revised accordingly. the rationale of coating the phage pre-incubation well with lysate and then with ptm is to inhibit the plastic binders from interfering in the optimization steps 38 and for further negative selection. in this work, a naïve human antibody phage library was generated for panning against the mers-cov nucleoprotein. the naïve library was constructed from 52 healthy donors comprising of three ethnic groups. this is aimed to expand the diversity of antibody repertoire for a highly diverse antibody library 39 . the 'single pot' antibody library repertoire has a universal characteristic, which makes it suitable to be employed for antibody selection against a wide range of target antigens 39, 40 . to generate a library of highest possible diversity, size and quality, combinatorial mixing of heavy and light chains was performed to create all possible gene combinations. the v-gene repertoire was amplified using a gene specific primer set with a high coverage of antibody genes 5 . in addition to that, the genetic diversity from donor samples of three ethnic groups may offer additional sequence diversity. cloning efficiency was improved by performing an intermediary cloning to ensure highest incorporation of full antibody gene sequences into the destination phagemid vector. the library generated has a modest diversity of 10 9 that is sufficient for antibody selection. despite naïve libraries have been reported to yield low affinity binders, it is still an important source for antibodies. a correlation between library diversity with antibody affinity was proposed to be directly proportional 41 . even so, the low affinity binders can undergo in vitro affinity maturation to improve their binding affinity and specificity 5, 39, 42 . kappa light chain isotype was solely used with the heavy chain igm isotype repertoire for this naïve library as more kappa antibodies are reported in the human peripheral blood than lambda antibodies. this is because rearrangement of kappa isotypes occurs earlier but when both kappa alleles failed to rearrange productively, this is followed by lambda rearrangement 43 . however, this ratio changes upon encountering antigens, suggesting that light chain rearrangement occasionally takes place in b cell after the onset of somatic hypermutation 44 . this is in line with the structural and physiochemical studies of kappa and lambda antibodies, with both exhibiting differential characteristics during adaptive immune response 43, 45 . however, in this context, we sought to generate a naïve library for kappa antibodies while further studies on differential properties of lambda and kappa antibodies will be carried out later. in this study, the naïve library was subjected to the revised affinity selection to isolate antibodies against rmers-np from a crude preparation. 'yin-yang' biopanning was able to enrich antibodies against crude rmers-np after 2 rounds of selections by 2.5-fold. the low enrichment ratio was expected due to the loss of binders to interfering proteins from the crude fraction. the method is able to impose both the competitive and subtractive effect onto non-specific binders in order to increase the possibility of positive binders to bind the target in extremely low amounts. reproducibility of this method is fairly good as good enrichment was achieved in several attempts using other in-house antibody libraries against different targets (data not shown). mab selection was done and 3 full antibody clones were obtained for this work. based on the sequence analysis, the v-gene combination of clone rmers-np-a4 is ighv4-igkv2, ighv1-igkv2 for rmers-np-c8 and ighv3-igkv1 for rmers-np-g12. the antibody fragments were expressed, purified and their binding specificity determined towards rmers-np. rmers-np-a4 binds specifically to rmers-np with very minimal background signal while rmers-np-c8 and rmers-np-g12 presented some degree of background signal indicating the clones as low performing clones. we noticed a slight difference in the antibody band migration which may likely be due to the introduction of chemical modifications 46 , which in this case is the biotinylation that contributed to the retardation of gel migration. in addition, previous reports examined the efficiency of biotinylation by comparing the gel migration of biotinylated products against non-biotinylated products in the presence of streptavidin and reveal a slower migrating of biotinylated products [47] [48] [49] [50] [51] . despite all three antibody fragments underwent biotinylation, the gel shift effect was more drastic for rc8scfv at approximately 3-4 kda difference in comparison to ra4scfv and rg12scfv. previously reported neutralizing mabs against mers-cov were specific to the receptor-binding domain (rbd) of the spike (s) glycoprotein of mers-cov 52 . the rbd of mers-cov binds to human dipeptidyl peptidase 4 (hdpp4) (cellular receptor) and gains entry into the target cell for viral infection. most of the reported mabs has been focused on the neutralization of rbd to block the interaction of rbd to hdpp4 and extreme potency has been exhibited by mabs targeting rbd. despite hdpp4 is another potential target for neutralization, it is also a particularly important molecule in the immune regulation of t cell activation and chemokine function. therefore, clinical use of it is unadvisable as adverse side effects may arise 53, 54 . a recent report combined mabs specific to rbd and non-rbd (other domains on s glycoprotein) to initiate neutralizing enhancement as different antigenic sites on s proteins are targeted 55 . nevertheless, immune escape is always endeavoured by coronaviruses by expression of mutant s proteins while nucleocapsid proteins have less mutations. the abundant np of mers-cov makes it another potential candidate target other than the s proteins. nps are responsible for viral genomic rna packaging, transcription and assembly 56 . in a previous report, three mabs against mers-cov rbd with exceptional potency was found to originate from the ighv1-69 heavy chain family 57 . interestingly, in this study, we found that rmers-np-c8 clone targeting the np also belongs to ighv1-69 heavy chain family. of note is that the ighv1-69 gene was also found to be utilized by other antiviral antibodies targeting hiv-1, influenza virus and hepatitis c virus 54 . www.nature.com/scientificreports www.nature.com/scientificreports/ this 'yin-yang' method together with the naïve scfv library was able to select antibodies against a complex antigen by providing competitive and subtractive effects to favour binding of positive binders. this permits antigens in different states (complex mixture, low purity, protein cocktails) to gain excess for affinity binding without the limitations of protein purity and yield. it is likely that optimization of this approach is required for different antigens produced in different host cells due to the differences in cell contents. the generated kappa light chain scfv library was able to generate mabs against mers-cov np using the proposed approach with crude rmers-np. therefore, this kappa exclusive naïve library is potentially useful for isolation of antibodies against other interesting antigens in the future. in conclusion, the 'yin-yang' panning together with the kappa naïve scfv library can be used as an alternative selection process for mab generation against challenging target antigens. to prepare rubi and its complementary e. coli lysate, the recombinant plasmid prset-bh6 bearing the ubi gene and the empty plasmid with helper plasmid prare3 were introduced into e. coli bl21 (de3) strain. the transformed cells were grown in 2yt media supplemented with 0.2% (v/v) glucose, 100 µg/ ml ampicillin, 17 µg/ml chloramphenicol and 1 mm isopropyl β-d-1-thiogalactopyranoside (iptg) at 25 °c for 16 h. the bacterial cell pellets were collected and subjected to cytoplasmic extraction with 20 mg/ml lysozyme followed by 4 min sonication on ice. imac purification was performed for rubi using a 1 ml ni-nta agarose fast flow column (ge healthcare). the recombinant plasmid prset-bh6 bearing mers-np gene was expressed in a similar fashion using e. coli c41 (de3) strain and followed by cytoplasmic extraction. the crude proteins and lysates were subjected to sds-page analysis. 'yin-yang' biopanning optimization: blocking effects of ptm buffer and e. coli lysate towards anti-ubiquitin and m13ko7 and the effect of phage preincubation. blocking effects of ptm buffer and e. coli lysate. optimizations were done step-wise to investigate the binding capacity of the antibody towards the antigen in various blocking agents and target-unrelated proteins. initially, a one-step affinity selection was performed separately with αubi and m13ko7 against 10 µg of purified rubi in the presence of various blocking agents. eight microwells were coated with 100 μl of purified rubi in pbs buffer overnight at 4 °c. the wells were washed three times with 0.1% (v/v) tween-20 (pbs-t) and blocked with 300 μl of ptm blocking buffer (2% (w/v) skimmed milk in pbs-t) for 1 h with 600 rpm agitation at room temperature (rt). simultaneously, the αubi phage (10 10 cfu/ml) and m13ko7 (10 11 cfu/ml) were blocked separately with 4 sets of blocking agents in a total volume of 100 μl: i. pbs buffer, ii. 2% ptm, iii. e. coli lysate and iv. 2% ptm and e. coli lysate. the incubation took place for 1 h at rt, with gentle agitation at 600 rpm. then, the antigen-coated wells were washed three times with pbs-t and the blocked phages were transferred into the wells accordingly. the binding took place at rt for 2 h at 600 rpm. the phage mixtures were collected at the end of incubation as 'unbound phages' and the wells were washed three times with pbs-t. the bound phages were eluted by enzymatic digestion with 100 μl of trypsin (10 μg/ml in pbs buffer) for 30 min at 37 °c, static. serial dilutions were performed for both unbound and eluted phages. the serially diluted phages were allowed to infect 200 μl of exponentially growing tg1 culture (od 600nm = 0.5) at 37 °c for 30 min, static. the phage titres were determined by 10 μl spots on ampicillin-glucose-2yt agar and kanamycin-2yt agar plates. the titre data was collected as 'blocked phage' and 'eluted phage' . next, biopanning simulations were carried out using a mixture of αubi (10 10 cfu/ml) and m13ko7 (10 11 cfu/ml) against purified and crude rubi in similar conditions. preparations for antigen-coated wells and ptm-preblocked wells were similar as above. the mixture of αubi and m13ko7 phages was blocked in 4 sets of blocking agents in a total volume of 100 μl: i. pbs buffer, ii. 2% ptm, iii. e. coli lysate and iv. 2% ptm and e. coli lysate. the blocking of phages took place for 1 h with 600 rpm agitation at rt and subsequently transferred into antigen-coated wells for binding. phage titre data for unbound phage and eluted phage was determined. for biopanning simulation against crude rubi, 10 μg of crude rubi was coated onto microwells in pbs buffer. the total protein concentration of crude rubi was measured with nanodrop 2000 (thermo fischer scientific, ma, usa). the procedures were similar as above. www.nature.com/scientificreports www.nature.com/scientificreports/ effect of phage preincubation. the second stage of optimization was performed with preincubation of phage prior to affinity selection against target antigen. then, 100 μl of purified rubi (10 μg) in pbs buffer was coated onto 8 microwells overnight at 4 °c for both αubi and m13ko7 sample sets. concurrently, the same number of wells were blocked with 300 μl e. coli lysate at 4 °c overnight. the antigen-coated wells and lysate preblocked wells were subjected to three times pbs-t washing. the wells were then blocked with 300 μl ptm blocking buffer for 1 h at rt, 600 rpm. the lysate/ptm-coated wells were washed three times with pbs-t and used for phage blocking. αubi and m13ko7 were incubated with various blocking agents in lysate/ptm-coated wells, separately. then, the phages were transferred into antigen-coated wells for binding to occur. the phage titres for both 'blocked phage' and "eluted phage' were determined. for biopanning simulations, a mixture of αubi and m13ko7 was preincubated prior to affinity selection against purified and crude rubi. the rescued phages from biopanning simulations against crude rubi for both optimizations were packaged and amplified. the phages were subjected to phage elisa assay to assess the binding performance in the presence of various blocking agents. the detailed workflow is included in the supplementary methods 1-7. construction of human naïve kappa light chain scfv phagemid library. a total of 52 cdna of healthy human donors from three local ethnic groups (malay, chinese and indian) was used as the starting material for library generation. collection of human blood samples, total lymphocytes isolation, preparation of cdna and library cloning were performed according to lim, et al. 61 with slight modifications. sample collection was performed in accordance with the human ethical approval from the human ethics committee of universiti sains malaysia. all donors were informed about the project and gave their informed consent. all donors are healthy individuals with no known infections and have been physically healthy 3 months before collection. detailed protocol for the library generation is described in supplementary method 8. 'yin-yang' biopanning of human antibody scfv phage library against crude rmers-np. the affinity selection process was separated into 2 steps. the first step was carried out to capture the non-specific phages that are present in the phage library preparation. the second step was biopanning with 'filtered' phages from the previous steps against the target antigen. a total of 0.2 mg of crude rmers-np in 100 µl of pbs buffer was coated to the surface of the microplate wells at 4 °c for 15 h. simultaneously, an immunotube with high binding surface was blocked with equal volumes of e. coli lysate (generated from e. coli c41 (de3) strain with empty prset-bh6) and 5% ptm at 4 °c, rotating for 15 h. all interval washes were performed 3 times with pbs-t. the antigen-coated well was blocked with 300 µl of 5% ptm buffer for 1 h, 600 rpm at rt. 'ying' affinity selection was done in the immunotube with preincubation of library phage (100 µl of library phage at 10 11 cfu/ml, 50 µl of 5% ptm and 50 µl e. coli lysate) for 1 h, rotating at rt. then, the content in the immunotube was transferred into the well and incubated for 2 h, 600 rpm at 37 °c for 'yang' affinity selection. at the end of binding, the wells were washed with 1% pbs-t (pbs buffer with 1% (v/v) tween 20) for 10 times and 25 times for the subsequent rounds of biopanning. the bound phages were eluted with 100 µl of trypsin for 30 min at 37 °c. e. coli tg1 culture was infected with trypsin-eluted phage particles after first round of biopanning. the phage-infected tg1 cells were propagated and the phage particles were packaged and used for the second round of biopanning. polyclonal antibody phage elisa. the trypsin-eluted phages from two biopanning rounds were amplified and packaged. two wells were coated with 0.2 mg of crude rmers-np while concurrently, another two wells were blocked with 5% ptm as background control. the enriched phages were blocked with equal volumes of 5% ptm and lysate in lysate/ptm-blocked immunotubes as described. then, the phage mixtures were incubated in antigen-coated and background wells for 2 h at similar biopanning conditions. anti-m13-horseradish peroxidase (hrp) was used to detect the bound phages for 1 h at rt, 600 rpm and 2,2′-azino-bis(3-ethylbenzothiozoline-6-sulfonic acid) diammonium salt (abts) was used to develop a colorimetric signal. the enrichment pattern was determined at absorbance 405 nm. elisa was carried out using the protocol according to lim, et al. 61 with slight modifications. briefly, phage particles with the highest enrichment was subjected to tg1 infection and 92 single colonies were picked and cultured. the performance of the monoclones was assayed against 0.2 mg of crude rmers-np. 5% ptm was used as the sole blocking agent and three times washing with pbs-t was used at all the interval washes. the monoclonal phage particles were transferred into antigen-coated elisa microplate and incubated for 2 h at 37 °c, 600 rpm. then, anti-m13 hrp was introduced to detect phage binding followed by abts developing solution. the absorbance was measured at od 405nm within 30 min. the detailed protocol is outlined in supplementary method 9. dna sequencing. the positive clones from the monoclonal elisa were cultured at 37 °c and 200 rpm for 15 h. the cell pellets were collected and plasmid extraction was performed using qiaprep spin miniprep kit (qiagen, germany). the purified plasmids were sent for sequencing (first base laboratories sdn bhd, malaysia). the sequencing results were then analysed by imgt/v-quest bioinformatics tool available at imgt ® , the international immunogenetics information system ® 62,63 . a4scfv, c8scfv and g12scfv dna were amplified from their respective plabel vectors and cloned into e. coli expression vector prset-bh6 at restriction sites ncoi and noti (neb, ma, usa). the resulting vectors were named prset-bh6 a4scfv, prset-bh6 c8scfv and prset-bh6 g12scfv. expression of a4, c8 and g12 was performed with shuffle ® t7 with prare3 for in vivo biotinylation. the expression condition for biotinylated a4, www.nature.com/scientificreports www.nature.com/scientificreports/ c8 and g12 was optimized to 24 °c, 140 rpm for 20 h with iptg induction at od 600nm 0.8 at a final concentration of 0.8 mm and 50 μm d-biotin. the inclusion bodies of biotinylated a4, c8 and g12 were solubilized using 8 m urea buffer, ph 8 (8 m urea, 0.1 m nah 2 po 4 , 0.01 m tris). the urea crude fractions were subjected to protein purification using ni-nta agarose gravity column (qiagen, germany) at denaturing conditions. initially, the column was equilibrated with 8 m urea buffer at ph 8 followed by sample loading. then, the column was subjected to washing with 10 ml of 8 m urea buffer (ph 8). step elutions were performed by 8 m urea buffer at ph 6.3, 5.9 and lastly 4.5, 2 ml elution fractions were collected stepwise. the protein fractions were analysed on sds-page. the last 5 fractions from a4, c8 and g12 were combined and subjected to buffer exchange using amicon ® ultra 0.5 ml centrifugal filters (merck, darmstadt, germany). the antibody fragments were dissolved in 1x pbs buffer (ph 7.4). western blotting was then performed using streptavidin-hrp (1:5000) (thermo scientific, ma, usa) to detect and confirm the respective antibody fragments. antigen binding elisa. the purified rmers-np (fused with his 6 -tag only) fractions, purified rgfp (fused with his 6 -tag only) fractions and purified antibody fragments were quantified (detailed in supplementary method 10) . a serial dilution of purified rmers-np(his 6 ) (μg: 1, 5, 10, 25, 50, 150) was coated onto microwells in pbs buffer with a total volume of 100 μl at 4 °c overnight. all interval washes were performed 3 times with pbs-t. the antigen-coated wells were blocked with 2% ptm blocking buffer for 1 h at rt, 600 rpm. the respective antibody fragments (a4, c8 and g12) were resuspended in pbs buffer, with each 100 μl aliquots representing 10 μg. post-blocking, the wells were washed and each well was added with 100 μl of antibody fragments (10 μg). additionally, 3 empty wells were also added 100 μl of antibody fragments as negative control wells. to check the cross-reactivity of antibodies towards his 6 -tagged proteins, 3 wells were coated 10 μg of rgfp(his 6 ) and subsequently allowed to incubate with 100 μl of antibody fragments (10 μg). the antigen-antibody binding took place at 600 rpm, 37 °c for 2 h. then, 100 μl of streptavidin-hrp (diluted at 1:5000 in ptm blocking buffer) was added into each well and incubated for 1 h at rt, 600 rpm to detect antigen-antibody binding. lastly, 100 μl of abts developing solution was added after washing. the absorbance was measured at od 405nm within 1 h. filamentous fusion phage: novel expression vectors that display cloned antigens on 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selection of anti-tumor recombinant antibodies from phage libraries on intact cells targeting membrane proteins for antibody discovery using phage display t.s.l. contributed to the conception of the study, analyzed the data and supervised the study. c.c.l. planned and performed the experiments, collected data and performed data analysis. p.c.y.w. provided important dna material for the study and assisted in data analysis. all authors provided critical feedback to this study and contributed to manuscript writing. supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-42628-6. the authors declare no competing interests.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-013526-6fip93l2 authors: labadie, thomas; roy, polly title: a non-enveloped arbovirus released in lysosome-derived extracellular vesicles induces super-infection exclusion date: 2020-10-19 journal: plos pathog doi: 10.1371/journal.ppat.1009015 sha: doc_id: 13526 cord_uid: 6fip93l2 recent developments on extracellular vesicles (evs) containing multiple virus particles challenge the rigid definition of non-enveloped viruses. however, how non-enveloped viruses hijack cell machinery to promote non-lytic release in evs, and their functional roles, remain to be clarified. here we used bluetongue virus (btv) as a model of a non-enveloped arthropod-borne virus and discovered that the majority of viruses are released in evs. based on the cellular proteins detected in these evs, and use of inhibitors targeting the cellular degradation process, we demonstrated that these extracellular vesicles are derived from secretory lysosomes, in which the acidic ph is neutralized upon the infection. moreover, we report that secreted evs are more efficient than free-viruses for initiating infections, but that they trigger super-infection exclusion that only free-viruses can overcome. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 lipid envelopes surrounding viruses are likely the results of a convergent evolution, potentially acquired during the adaptation of non-enveloped virus to animals [1] . the envelope plays essential roles, such as the fusion with cellular membranes to enter in cells, or the assembly of new virus particles during budding at the plasma membrane. in contrast, non-enveloped viruses developed alternative mechanisms, such as cell entry by membrane penetration [2] and exit by cell lysis. in both cases, free virus particles remain the canonical unit of virus infection, with a single virus particle entering a cell to replicate and amplify its genome. recent studies in the field of non-enveloped viruses have drawn attention on previously unknown cell to cell spread mechanisms, in which released virus particles are not naked. a seminal work on hepatitis a virus revealed that virus particles are released both in enveloped and non-enveloped states, with the envelope protecting virus particles from neutralising antibodies [3] . more recently, it was reported that other non-enveloped lytic viruses, such as coxsackievirus b [4] , hepatitis e virus [5] , poliovirus [6, 7] , polyomavirus [8, 9] , rotavirus and norovirus [10] , can also exit the cells as cloaked in extracellular vesicles (ev). further, it was observed that evs could contain multiple virus particles, allowing infection of a unique cell with many virus particles at a time [7, 10] . this new transmission mechanism, reported as en block transmission, allows viral genome complementation during the infection and could be a regulator of fitness evolution for a virus population [11, 12] . however, the mechanisms by which viruses hijack the host cellular secretion machinery, or the benefit of vesicular transmission are still unclear [11] . understandings these might alter the rigid definition of non-enveloped viruses. to this end, here we investigated ev associated virus release using a model non-enveloped multi-layered capsid virus, bluetongue virus (btv), of the orbivirus genus, with a segmented double stranded rna genome. btv is an insect-borne virus that periodically causes epidemics among ruminants worldwide, with a particularly high mortality rate in sheep. although classified as a non-enveloped virus, btv is able to exit cells by both lytic and non-lytic mechanisms [13] [14] [15] . here, we report that evs containing multiple virus particles are the main infectious unit of btv. our results revealed a mechanism of en block transmission in which both mvbs and the late stages of autophagy are essential to cloak virus particles in secreted lysosomes. our results explain how both forms of the virus (free and vesicular) contribute to its life cycle and demonstrate their importance. further, we observed that ev associated virus induces a more efficient infection and synthesis of viral protein than does infection with free virus particles, while free virus particles are essential to overcome super-infection exclusion. btv egresses infected cells not only by cell lysis but also by a non-lytic mechanism [13, 15] . since btv particles were shown to be associated with mvbs components during trafficking [16] and the purified virus particles are associated with ns3 [15] , a membrane protein, it is possible that multiple btv particles are released within evs. to determine this, we infected sheep cells (pt cell line) and culicoides insect cells (kc cell line) at a multiplicity of infection (moi) of 10, and cell culture supernatants were centrifuged to isolate free virus particles from large evs (~1 μm) which sediment as pellets under the conditions used. viral supernatants were harvested at 24 hours post-infection (hpi) as the released virus content is high at this time point yet there is almost no detectable cell death (s1 fig). virus titrations ( fig 1a) and viral genome quantification by quantitative pcr (fig 1b) both revealed that the majority of infectious virus particles and viral genomes were sedimented after a 10,000xg centrifugation. further, western blot analysis of the structural protein content, both in the pellet and the supernatant revealed that vp2, vp5 and vp7 were at higher levels in the pellet (fig 1c) , consistent with the virus titres. moreover, specific infectivity, defined as the ratio of the number of genomes per pfu [17] , was significantly lower in the 10,000xg pellets when compared to the supernatant (fig 1d) , suggesting that the form of virus present in the pelleted fraction was more infectious than btv present in the supernatant. to localise the btv proteins within purified evs, we used a super resolution microscopy approach that could visualise both structural protein vp7 and non-structural protein ns3 with the phosphocholine bodipy, indicating that btv is associated with lipid membranes in evs ( fig 1e) . moreover, direct observations of both purified evs secreted from infected cells (fig 1f) , and ultrathin sections of evs embedded in agar ( fig 1g) by negative stain electron microscopy revealed the presence of large evs (ranging from 0.5 to 1.5μm) containing multiple virus particles, corroborating our fluorescence imaging observations. altogether, these data established that the majority of btv virus particles are released in large evs secreted from mammalian and insect infected cells in vitro. to further analyse the btv particles found in the 10,000xg supernatant, this supernatant was centrifuged again at 100,000xg, and the infectivity was measured in both the 100,000xg pellet and its supernatant ( fig 1h) . most of the infectivity was then associated with the 100,000xg pellet, that was analysed by electron microscopy for the form of virus present ( fig 1i) . we observed that the majority of virus was in the form of aggregates, made of particles embedded in a lipid membrane, or in the form of naked virus particles. the lipid membrane in the case of the 100,000xg pellet may be a transient lipid membrane acquired from single particles budding at the plasma membrane, as already described [14] , so we refer to the 10,000xg supernatant as the free virus fraction in this report. to determine the origin of evs containing btv virus particles, we tested the presence of specific cellular markers. among the different markers of evs assessed, we determined the presence of annexin a2, the tumour susceptibility gene 101 (tsg101), lc3-i and lc3-ii, and the lysosomal associated protein lamp1 by western blot (fig 2b and s2a fig). note that tsg101 is a mvbs marker and lc3b an autophagy related protein. to identify the cellular mechanisms involved in virus packaging in evs, we first investigated the potential role of autophagy, using the 3-methyladenin (3-ma) that inhibits class iii pi3k activity. we observed that 3-ma failed to significantly decrease the secretion of infectious evs from sheep cells infected at a moi of 10 ( fig 2b) , suggesting that the induction of autophagy is not essential for the secretion of and viral genome quantification (b) in the pellet or in the supernatant after isolation of evs secreted by infected mammalian sheep cells and insect cells. data are presented as mean ± sd. each point indicates the value of independent replicates (n = 3; unpaired t-test, � p<0.05, �� p<0.01, ��� p<0.001, ���� p<0.0001). (c) outer capsid proteins vp2 and vp5, and inner capsid protein vp7, detected in the pellet and supernatant after isolation of evs secreted by infected mammalian cells. (d) specific infectivity, which is the number of genome copies per plaque forming unit, measured for virus particles in the pellet and the supernatant after a 10,000xg centrifugation. data are presented as mean ± sd. each point indicates the value of independent replicates (n = 3; unpaired t-test, � p<0.05). (e) immunofluorescence microscopy images of purified evs secreted from infected (two top rows) or mock (bottom row) mammalian cells labelled with anti vp7 (orange) and ns3 (red) antibodies, and a fluorescent lipid membrane marker (green). scale bar: 2 μm. (f and g) electron microscopy images of whole evs (f) and sectioned evs (g) secreted from infected mammalian cells. yellow arrows indicate the virus particles positions in the sections. scale bar: 100nm. (h) virus particles titration in the pellets and the final supernatant after centrifugation of 10,000xg and 100,000xg. data are presented as mean ± sd. each point indicates the value of independent replicates (n = 3; unpaired t-test, ���� p<0.0001). (i) representative electron microscopy images of btv particles observed in the 100,000xg pellet. scale bar: 100nm. https://doi.org/10.1371/journal.ppat.1009015.g001 virus released in secretory lysosomes infectious evs. however, inhibition of autophagosome-lysosome fusion with chloroquine (cq), led to a significant reduction of infectious evs released as compared to the control ( fig 2b) , indicating that the late steps of autophagy are necessary for infectious evs. in addition, inhibition of mvbs regulator protein hsp90 using geldanamycin in btv-infected cells (moi = 10) also led to a significant reduction of infectivity measured in the evs fraction, as compared to the control, indicating a possible role for mvbs in the release of infectious evs. in contrast, gw4869, a drug that inhibits the release of exosomes (small vesicles~200nm) derived from mvbs, did not affect the secretion levels of evs containing btv ( fig 2b) . as expected, except for gw4869, these drugs also had an inhibitory effect on the virus replication (s2b fig) , affecting virus released in both ev and free viruses, albeit to various levels. these cumulative inhibitory strategies of autophagy and mvbs provide evidences that mvbs and the late stages of autophagy are both involved in the release of evs containing virus particles. however, these evs cannot be exosomes, based on the results obtained with the gw4869 inhibitor. following recent description that autophagy and mvbs share common molecular machinery and organelles such as amphisomes [18] , we asked if evs could be derived from secreted lysosomes after the fusion with amphisomes. therefore, we purified the evs using an isopycnic ultracentrifugation and found that most of the infectivity was associated with the low-density fractions (~1.05 to 1.08 g/ml), along with the cathepsin enzymes, a lysosomal marker ( fig 2c) . interestingly, the ratio of cell surface lamp1 to cytosolic lamp1 was also significantly higher in infected cells (fig 2d) , indicating an increase of lamp1 located at the plasma membrane in infected cells. the presence of viral proteins was then investigated in the lysosomes of btv infected cells. using super resolution microscopy, we observed that in infected cells only, outer capsid protein vp5 as well as lamp1 co-localised with the mvbs marker hsp90 (fig 2e) , indicating a fusion between mvbs and the autophagy compartments. supporting these results, we also observed the co-localisation of lamp1 and vp5 with tsg101, another mvbs marker (s2d fig) , and the co-localisation of vp5 with the lysosomal markers lamp1 and cathepsins, indicating the presence of viral proteins within lysosomes ( fig 2f) . altogether, increased lamp1 localisation at the plasma membrane, a hallmark of increased lysosomal exocytosis [19, 20] , and the presence of btv proteins within lysosomes indicate that btv infection could reprogram lysosomal degradation pathways to facilitate virus secretion. ns3 is the only viral membrane protein and it is likely to be involved in the mechanism of ev secretion. therefore, we tested the presence of virus in evs ( fig 3a) following infection with available ns3 mutants [14, 21, 22] . two of these mutants impair the synthesis of the two ns3 isoforms, respectively ns3 (mutant ns3 m1 ) and ns3a (mutant ns3 m14 ), while another with a modified late domain motif (ns3 gaap ) cannot bind tsg101 and one, unable to bind the outer capsid protein vp2 (ns3 stop211 ). of the four mutant viruses, only btv ns3 m14 significantly reduced virus particle release in evs (fig 3a) . to understand this phenotype, we virus released in secretory lysosomes analysed the localisation of the mutant virus particles in infected cells and observed that the outer capsid protein vp5, and ns3, were still co-localised with the lysosomal markers lamp1 and cathepsin ( fig 3b, s3 fig) , suggesting that the absence of btv ns3 m14 in evs was not due to a defect in virus trafficking. we then examined lysosomes in cells infected with wild type btv (btv wt ) and non-infected cells expressing a lamp1-gfp fusion protein in the presence of a fluorescent lysotracker specific to acidic organelles ( fig 3c) . confocal microscopy revealed a significant decrease of lysotracker fluorescence intensity in the lysosomes of btv wt infected cells, when compared to non-infected cells, suggesting that btv wt neutralises the acidic ph of lysosomes ( fig 3d) . the number of lysosomes detected in infected cells was also significantly higher than in non-infected cells (fig 3e and 3g ). in contrast, cells infected with btv ns3 m14 showed comparable levels of lysotracker fluorescence to non-infected cells, suggesting that btv ns3 m14 was unable to counter lysosomes acidification. to provide further confirmation, cells infected with btv ns3m 14 and non-infected cells were examined in the presence of the lysosomal v-atpase inhibitory drug bafilomycin a1 (fig 3e and 3g ). interestingly, bafilomycin a1 significantly increased the levels of btv ns3 m14 in evs (fig 3h) , while not affecting the intracellular replication of btv ns3 m14 , whereas the drug had a strong inhibitory effect on the replication of btv wt (s2b fig) . consistent with this model, we observed that the intracellular calcium levels of cells infected with btv wt were significantly higher than the calcium levels in btv ns3 m14 and non-infected cells (fig 3i and 3j ). together, our data strongly support that newly synthesised btv particles are released via evs derived from secretory lysosomes after ph neutralisation. along with their cellular origin, the role of the secreted form of non-enveloped viruses remains unclear. to better understand the role of evs during btv infection, we compared the infectivity of evs and free virus particles, by analysing the kinetic of total virus production in cells infected either by evs or free-virus particles. infection of mammalian cells with infectious evs or free virus particles led to a similar amount of virus released from cells at 33 hpi. however, at 9 hpi, we found that cells infected with infectious evs released more virus (fig 4a) , and showed a higher level of viral genome in their cytoplasm ( fig 4b) than cells infected with free virus released in secretory lysosomes virus particles. this suggests that infectious evs are a more efficient form of infecting agents than are free virus particles. to test this possibility, we investigated the kinetic of viral protein synthesis by following the formation of viral inclusion bodies (vibs, virus assembly factories) in the cytosol of cells infected with either evs or free virus particles (moi = 10). we examined the size and number of vibs in infected cells between 6 to 21 hpi, by fluorescence microscopy (fig 4c) . a frequency analysis of the vibs surface area distribution (fig 4d and 4e ) revealed that at 6 hpi, vibs median surface areas were 3.34±1.80μm 2 in free-virus infected cells against 2.75±0.81μm 2 in evs infected cells. at 12 hpi, mean vib size surface was clearly smaller in free-virus infected cells with 3.10±1.52μm 2 as compared to evs infected cells (4.00±1.86μm 2 ). this result suggests that virus-directed protein synthesis is considerably more efficient in evs infected cells. an analysis of the frequency distribution of vibs formed per cell at 6 hpi (fig virus released in secretory lysosomes 4f) indicated that evs infected cells had a geometric mean of 2.7 vibs/cell whereas in free virus infected cells, the geometric mean was 1.1 vibs/cell, supporting the idea that infectious evs allow the formation of multiple viral factories soon after the infection. together, these data indicate that ev infected cells produce more vibs, that expand faster, than vibs produced by free-virus infected cells. thus, in addition to containing the majority of infectious virus particles released from infected cells, evs are also more efficient than free-virus particles to initiate an infection. we next considered if differences between evs and free virus particles are due to cell defence mechanisms, such as super-infection exclusion. although not always understood, this cell defence mechanism in which infected cells become resistant to a second infection has been observed for several viruses. we first evaluated the presence of a super-infection exclusion mechanism in sheep cells infected with btv, using two different viruses, a non-modified btv (btv wt ) and a fluorescent btv (btv unag ) encoding a fluorescent fusion protein vp6-unag. after controlling that btv unag virus particles are also mainly released in evs (fig 5a) , we first infected sheep cells with btv wt, followed by a second infection with btv unag immediately (0 hpi) or at 1, 2, 3, 4 or 5 hpi (fig 5b) . 12 hours after the initial infection with btv wt , cells were then labelled with an anti vp6 antibody. unag fluorescence correspond to the detection of the btv unag virus only, whereas indirect fluorescence with vp6 antibody detects both viruses (fig 5c) , although less efficiently in the case of vp6-unag (fig 5d) . using flow cytometry, we quantified unag fluorescence in cell population gated for vp6 positivity (from the antibody-based detection). these cells were either infected with btv wt (unag negative), btv unag (unag positive) or both viruses (unag positive). we used a constant moi across all experiments, so that the number of btv unag single infected cells should remain constant. therefore, variations of the unag fluorescence level reflect variations of co-infection with btv wt and btv unag and a superinfection exclusion of btv unag . a strong decrease of the unag fluorescence was observed in cells with longer incubation times between the two infections ( fig 5e) . this result provides evidence that btv infection induces super-infection exclusion in sheep cells. we repeated this experiment by infecting sheep cells with btv wt (total virus particles), and then with free-virus particles or evs of btv unag . sheep cells were first infected with btv wt , and then btv unag free virus particles or infectious evs. a super infection exclusion mechanism was observed in cells co-infected with btv wt and evs containing btv unag (fig 5f) , with a significant decrease of unag fluorescence intensity dependent on the incubation times between the two infection. conversely, no significant differences of unag fluorescence were detected in cells with secondary infection of btv unag free virus particles over time ( fig 5g) . altogether, these results indicate that while btv infection led to a super-infection exclusion mechanism in sheep cells, btv free virus particles were able to overcome this mechanism. while recent discoveries regarding evs containing viruses have changed our perception of non-enveloped viruses, such observations were lacking for a non-enveloped arthropod-borne virus. moreover, it has been hypothesised that lipid envelopes are the hallmark of virus adaptation to animals [1] . among all the arthropod-borne viruses infecting vertebrates, arthropodborne reoviruses are the only non-enveloped viruses. based on these results and the recent literature on picornavirus, polyomavirus, calicivirus and rotavirus [3, 7, 10, 23] , it is tempting to suggest that the vesicular transmission could be another example of convergent evolution for a non-enveloped virus adapting to animals. in this study, we demonstrated that the majority of btv virus particles are clustered in evs, secreted by both mammalian and insect cells. despite virus released in secretory lysosomes the absence of envelope, our results highlight the presence of a lipid bilayer protecting virus particles and providing a more efficient infection initiation compared with free virus particles infection. it has been reported that hepatitis a virus and hepatitis e virus exit cells inside exosomes derived from the mvbs [3, 5, 24] , that are characterised by the presence of endosomal compartments and vesicular transport markers, such as flotillin 2, cd63 or members of the escrt protein family. these exosomes are around 50-200nm in size [25] . in contrast, it has been shown that coxsackievirus b and poliovirus are released in vesicles of approximately 500nm in diameter, through an autophagy-mediated mechanism [4, 6, 7] . moreover, a recent study reported that rotavirus egress cells in evs derived from the plasma membrane, independently of mvbs and autophagy, and that norovirus are release in exosomes [10] . a clear picture of different mechanisms used by non-enveloped virus for secretion in evs is lacking. our study contributes to understand better the diversity of release mechanisms. we showed that the lysosomal markers lamp1 and cathepsin are present in infectious evs, in addition to the mvb markers tsg101 and hsp90. based on our results, it is clear that mvbs are involved in the release of btv in evs, because of the presence of the tsg101 in these evs, the co-localisation of tsg101 with viral proteins in infected cells, and the deleterious effect of geldanamycin on the secretion of evs containing virus. however, based on the size of these evs, the failure of inhibition by gw4869, the exosome inhibitor, and the high level of infectious evs released by the btv ns3 gaap mutant (unable to interact with tsg101 [14, 22] ), we conclude that the evs release by our ovine cells cannot be exosomes. however, the presence of lysosomal markers in the evs suggests that they could originate from the fusion between mvbs and autophagosomes that lead to the recently discovered secretory amphisome [18] , virus particles could be subsequently exported in lysosomes after the amphisomes-lysosomes fusion. supporting this mechanism, we observed the presence of the lysosomal cathepsin proteins in the same low-density fractions as the viral particles after isopycnic density centrifugation, as well as co-localisation of the lysosomal markers lamp1 and cathepsin with the viral proteins. interestingly, the btv ns3 m14 virus, which has a mutated first start codon and is only able to synthesis an ns3a isoform of ns3 (lacking the n-terminal 13 amino-acids) [21] , released significantly less virus particles in evs than btv wt . conversely, the btv ns3 m1 mutant (with a mutation in the second start codon preventing the synthesis of the ns3a isoform) showed comparable levels of infectious evs to the btv wt . we did not observe a change in the co-localisation of vp5 or ns3 with the lysosomal markers in cells infected with btv ns3 m14 compared with btv wt . in contrast, we found that lysosome acidity was higher in non-infected cells and cells infected with btv ns3 m14 , compared with btv wt . this suggests that ns3 m14 is unable to counter lysosomal acidification as shown for wt ns3. we were able to revert this phenotype using bafilomycin a1, an inhibitor of the vacuolar type h+-atpase, and showed that lysosomal ph disruption in btv ns3 m14 infected cells restored the secretion of btv particles in evs. this phenomenon suggests that btv infection induces lysosomal disfunctions similar to what is observed in lysosomal storage diseases, commonly associated with an increase of lysosome biogenesis, an increased lamp1 expression at the cell surface, as well as lysosome enlargement [26] [27] [28] . similarly, the levels of free calcium in infected cells were lower in non-infected cells and in cells infected with btv ns3 m14 , compared with btv wt. interestingly, it has been proposed that the endoplasmic reticulum, in close spatial association with lysosomes, regulates the calcium storage in these organelle [29, 30] . we previously showed that ns3 transits though the endoplasmic reticulum (er), and that disrupting ns3 trafficking through the er causes release of immature particles. it would be thus interesting to explore the interplay between ns3, the er and lysosomes in order to determine how ns3 could regulate the secretion of btv particles in evs. altogether, our data support an original model of a virus particles release mechanism involving mvbs and secretory lysosomes. interestingly, this phenomenon is not restricted to viruses, as it has also been described for intracellular uropathogenic e. coli [20] . related with our results, a recent preprint describes how beta-coronaviruses, which are enveloped viruses, could exploit the lysosomes for virus egress by disrupting lysosomal acidification [31] . these results, and ours, reveal a potentially unanticipated wide diversity of egress mechanism using lysosomes by different viral families. additionally, we also showed that evs containing virus particles offer an efficient platform for entry and infection of naive cells. however, we observed that btv infection triggers a superinfection exclusion phenomenon for evs containing virus particles, whereas free virus particles can overcome it, underlying the importance of both viral forms during btv cycle. previously, we showed btv vp2, the outer capsid spike binds sialic acid, a likely receptor for the free virus particles [32] . however, reports from others showed that infection with virus particles cloaked in evs did not necessarily rely on their known virus receptor [7, 8, 23, 33] . here, we discovered evidences of a superinfection exclusion mechanism during btv infectious, supporting the existence of a specific ev receptor. we observed that only free virus particles could enter in cells already infected, suggesting that evs and free virus particles use different entry routes. in such a scenario, the receptor is not required to interact with viral outer capsid proteins, as ev secretion is already an efficient cell-to-cell communication system [34] . however, the difference in virus tropism would certainly have consequences on the virus pathogenicity. it is thus necessary to study the impact of infectious evs on btv induced pathogenicity, as infected ruminants variably respond to btv infection, with cattle being mainly asymptomatic and sheep exhibiting more severe clinical signs, such as haemorrhage and ulcer in the gastro-intestinal tract or muscle necrosis [35] . in natural hosts, btv transmission requires blood feeding midges (culicoides species) to take a blood meal from an infected animal. our results demonstrated that culicoides cells, for which btv is not cytopathic, also secrets evs containing virus particles. it would be thus interesting to investigate whether the uptake of evs secreted by infected insects and evs secreted by infected mammals can infect in mammals or insects respectively. in our electron microscopy study of the viruses present in the 100k pellet we observed the free virus fraction to be heterogeneous by electron microscopy. the observed particles were either naked or aggregated and enveloped in a lipid membrane. it is likely that this membrane is transient and acquired by the budding of individual virions at the plasma membrane, as described previously [13, 14] . however, further investigation will be necessary to understand the importance of this heterogeneity in the virus life cycle. overall, the findings described here highlight an original aspect of virus release leading to the secretion of a non-enveloped virus in host-derived secretory lysosomes, of which the acidic ph is neutralized upon the infection. such mechanism is distinct to the proposed mechanism for the release of rotavirus in evs [10] , suggesting a divergent evolution of insect-borne double-stranded rna viruses. investigation of host-pathogen interactions for insect-borne viruses can significantly enhance our knowledge on the diversity of virus-containing extracellular vesicles origins. this may highlight different cellular tropisms and immunity evasion strategies between the free and vesicular forms of non-enveloped viruses and can have important implications for the development of innovative approaches for virus control. virus released in secretory lysosomes supplemented with 10% fcs and antibiotics (100 units/ml penicillin, 100 mg/ml streptomycin, gibco, life technologies). mammalian cells were incubated at 35˚c in a humidified 5% co2 incubator and insect cells at 28˚c. btv-1 and btvunag (with a segment 9 encoding a vp6-unag fusion protein) stocks were obtained by infecting sheep cells at a multiplicity of 10 plaque forming units per cell (pfu/cell) for 30 min under gentle agitation at room temperature (rt). the inoculum was then removed, and cells were maintained for 48 h in dmem with 2% fcs for 48 h at 35˚c. the harvested supernatants were clarified (10 min at 500xg) twice, and stored at 4˚c. all the ns3 mutants were described in previous studies [14, 21, 22] . stocks for these mutants were amplified as described above for btv wt , and a sanger sequencing of ns3 confirmed the presence of all studied mutations. viral infectivity was estimated using tissue culture infectious dose of 50 per ml (tcid 50 /ml) titration, for which viral suspensions were diluted from 10−1 to 10−12 in d-mem medium and inoculated to bsr cells in 96-well plates. titres were calculated according to the spearman-karber method. for analysis of replication, sheep cells were inoculated with purified evs or free virus particles in 6-well plate (moi = 1) for 1 h at rt under gentle rotation. the inoculum was then removed and replaced by d-mem (1% fcs). cell culture media was harvested at 9, 24, 33 and 48 hpi, clarified by centrifugation (500xg-10 min), and infectivity was quantified by a tcid 50 method. pt infected cells were lysed by three cycle of freeze thawing. lysed cells were resuspended in pbs and cell debris was spun down at 500 g for 5 min. viral rna extraction was then performed on the harvested supernatant as indicated below. extraction of the viral rna was performed with a qiamp viral rna mini kit (qiagen), followed by a reverse transcription using a universal degenerated oligo specific to all 5 0 non-coding sequences of btv-1 segments (btv/uni1; 5 0 gttaaawhdb 3 0 ) and the goscript reverse transcription (rt) system (promega, madison, wi, usa). the cdnas obtained for the segment 7 were then quantified with a real time quantitative pcr (qpcr), using the 2x sybr green qpcr master mix (bimake, houston, tx, usa) and segment 7 specific primers (btv1 s7/322f, 5 0 gacgccagagataccttttac 3 0 ; btv1 s7/478r, 5 0 cttgaatca tatccggaccac 3 0 ) or segment 10 specific primers (btv1 s10/249f, 5' cattcgcatcg-tacgcagaa 3'; btv1 s10/464r, 5' gcttaaacgccacgctcata 3'). for absolute quantification of the cdna copy numbers, a standard curve was produced using a 10-fold serial dilution of a pt7-s7 plasmid of known concentration as a template for amplification. evs were isolated from infected cells cultured in dmem 1% fcs (mammalian cells) or schneider 0 s insect medium (10% fcs), except for the analysis of protein markers in evs by western blot and immunofluorescence, for which cells were cultured in 0% fcs medium in order to avoid detection of evs present in the fcs. to identify the optimal time point for cell culture supernatant harvesting, corresponding to the highest possible viral titre with the lowest cell mortality, we used a cell viability imaging kit based on the detection of total cells number (using dapi staining) versus cells with a compromised plasma membranes (r37609, thermofisher scientific). following harvesting of the, evs were purified from free virus particles using a differential centrifugation protocol adapted from one used on encephalomyocarditis virus [38] . cell debris was first removed by two steps of low speed centrifugation at 500xg for 10 min. large evs virus released in secretory lysosomes were pelleted by centrifuging initial volumes of 1.5 ml at 10,000xg for 30 min. free virus particles remained in the supernatant, and pelleted evs were resuspended in either 50 μl or 1500 μl volumes of d-mem without fcs, depending on the concentration needed for the subsequent analysis. for all quantitative comparisons with free viruses, a correction factors were applied if the ev resuspension volumes were not equivalent to the volume prior centrifugation. when necessary, the 100,000xg centrifugation was performed for 1h at 4˚c using a sw41 ti swinging bucket rotor (beckman coulter). the pellet was then resuspended in 50 μl or 1500 μl of d-mem for subsequent analysis. evs purification by isopycnic gradient centrifugation was performed at 150,000xg for 4 hours at 4˚c, using a sw41 ti swinging bucket rotor (beckman coulter). evs resuspended in 900 μl d-mem and diluted with 100 μl iodixanol (45%) to a final 5% concentration. evs were layered on top of a 10-45% iodixanol discontinue gradient and harvested in 9 fractions, from the bottom of the tube using a syringe. fraction density was calculated by measuring the absorbance at 244nm after a 1:10,000 dilution and using a standard curve obtained by serial dilution of iodixanol. specific infectivity of btv in evs and free virus fractions correspond to the number of particles required to infect a cell. we approximated the particles/ml as the genome copy number/ ml measured by qpcr using s10 specific primers after extraction and reverse transcription of the viral genome. the number of pfu/ml was obtained by virus titration using a plaque assay. proteins were detected from either resuspended evs or cell lysates. cell monolayers were washed twice with a pbs solution and lysed for 30 min at 4˚c with a ripa lysis buffer, 1x protease inhibitor cocktail, and 5 mm edta (78438, thermofisher scientific). cell lysates, or evs, were diluted in 4x nupage lds sample buffer (thermofisher scientific) to a final 1x concentration and incubated at 95˚c for 5 min. proteins were then separated on a 10% sds gel and blotted on a pvdf membrane. when indicated, the membranes were cut horizontally for incubation with different antibodies. the anti vp2, vp5 and vp7 antibodies (made in the pr. roy's laboratory [39] ), anti lc3b (nb600-138455, novus bio), anti annexin a2 (610069, bd bioscience), anti tsg101 (t5701, sigma aldrich), anti lamp1 (ab24170, abcam) and anti pan-cathepsin (sc-376803, santa cruz biotechnology), were incubated overnight at 4˚c, followed by a 2 hours incubation with respective horseradish peroxidase-coupled secondary antibody (anti-mouse igg ab97023, anti-guinea pig igg ab97155 and anti-rabbit igg ab97051, abcam). luminescence was then detected using supersignal west pico plus chemiluminescent substrate (thermo fisher scientific). the imagej software was used to perform linear contrast enhancement. to image evs without sectioning, purified evs were resuspended in d-mem, adsorbed onto a carbon-coated copper grid, and stained with a 1% solution of uranyl acetate. for imaging of evs sections, purified evs were resuspended in 30 μl d-mem (0% fcs) and mixed with a low volume of glutaraldehyde 25% (final concentration = 2.5%) (16220, electron microscopy sciences). after 10 min of fixation at rt, evs were briefly warmed to 37˚c, and 16 μl of evs virus released in secretory lysosomes were dropped on 4 μl of pre-melted low-melting agar (5% in water) and kept at 42˚c before the addition of evs. samples were quickly mixed by pipetting and stored at 4˚c before sectioning. after post-fixation in 1% osmium tetroxide-0.1% sodium cacodylate, evs were dehydrated in increasing concentrations of ethanol and embedded in epoxy resin (taab laboratories equipment ltd., aldermaston, united kingdom). ultrathin sections were stained with reynolds lead citrate and mounted onto nickel grids. images were visualised with a transmission electron microscope (jem-1400, jeol akishima, tokyo, japan). sheep cells in 6 well-plates were infected (moi = 10) with btv-1, 24 hours post seeding. viral or mock inoculum were incubated 30 min at rt under gentle rotation. the inoculum was then removed and replaced by d-mem (1% fcs), and cells were incubated at 35˚c in a humidified incubator (5% co 2 ). at 1 hpi, cell culture medium was replaced with d-mem containing 10 mm 3-methyladenine (m9281, sigma aldrich), 300nm geldanamycin (invivo-gen), 20μm chloroquine (c6628, sigma aldrich), 5μm gw4869 (d1692, sigma aldrich), 1μm bafilomycin a1 (sml1661, sigma aldrich), so that drugs did not interfere with viral entry. these concentrations were selected because they were already validated by previous studies on btv from us and others [3, 20, [40] [41] [42] . at 24 hpi, cell supernatants were harvested for isolation of evs and analysis of infectivity. for intracellular virus particle quantification in presence or absence of the different drugs, the media culture of pt infected cells was removed at 24 hpi, and cell monolayers were rinsed twice with pbs. cells were then lysed by 3 successive cycles of freeze/thaw at -80˚c/35˚c. cell lysates were then resuspended in 500 μl dmem and virus particles were quantified using a tcid 50 method. cell surface protein expression of lamp1 was measured by immunofluorescence using flow cytometry. pt cells were seeded in 6-well plates for 24 h before infection with btv-1 (moi = 10). after 24 hpi, cell monolayers were washed twice with pbs, re-suspended using trypsin-edta (thermo fisher scientific). cells used for analysing the lamp1 cytosolic content were fixed with paraformaldehyde 2% (w/v) for 10 min and permeabilised with a pbs-triton x100 (0.05% v/v) solution for 5 min. the cells were then blocked using bovine serum albumin (pbs-bsa 1%) for 30 min and incubated with rabbit anti lamp1 (ab208943, abcam) targeting the luminal domain, or an isotope control antibody (ab172730, abcam) for 2 hours at room temperature. cells used for analysing the cell surface lamp1 content were labelled with the anti lamp1 antibody for 2 hours at 4˚c prior fixation. then all labelled cells were incubated for 2 h at rt in the presence of a species-specific a488 conjugated secondary antibodies (a21071, thermo fisher scientific). after centrifugation and washing, cells were re-suspended in pbs and analysed with a bd lsr ii flow cytometer (bd biosciences, san jose, ca, usa). for each replicate, 30,000 cells were analysed using the same parameters. flow cytometry data were analysed with flowjo (flowjo llc, ashland, or, usa) software. for the analysis, a gating strategy discriminating doublet cells by plotting fsc-a vs. fsc-h was used, and the threshold of background fluorescence was determined using cells labelled with the isotope control antibody (s2c fig). for imaging, evs were resuspended in d-mem (0% fcs) + fluorescent bodipy phosphocholine analogue at 2 μg/ml (d3793, thermofisher scientific) and incubated overnight at 4˚c. evs were then dropped on poly-l-lysine (p4707, sigma aldrich) pre-coated chambered #1.0 borosilicate coverslips (nunc lab-tek 155411, thermofisher scientific), and adsorbed for 1 hour at 35˚c. the residual medium was removed, and attached evs were fixed with pbs-pfa (2%) for 10 min at rt, rinsed with pbs and permeabilised with pbs-triton x100 (0.05%) for 5 min at rt. evs were then rinsed twice with pbs, incubated for 1 hour at rt with a solution of pbs-bsa (1%), and incubated overnight at 4˚c with primary antibodies anti vp7 and ns3 (made in pr. roy's laboratory), followed by fluorophore a633 and a546-conjugated antibodies (a21071 and a11074, thermofisher scientific) for 2 hours at rt and mounting with aqueous mounting medium (f4680, sigma aldrich). for sheep cells imaging, cells were cultured in chambered tissue culture treated polymer coverslips (μ-slide angiogenesis 81506 or μ-slide 4 well 80427, ibidi), infected with btv-1 (moi = 10) 24 h post-seeding, and at 1 hpi, inoculated with a baculovirus encoding lamp1-gfp at concentration of 10 particles / cell (celllight lysosomes-rfp, bacmam 2.0, thermofisher scientific). at 18 hpi (unless specified), cell monolayers were rinsed twice in pbs, fixed in pbs-pfa (2%) for 10 min, permeabilised in pbs-triton x100 (0.05%) for 5 min, incubated in pbs-bsa (1%) for 30 min. cells were labelled with primary antibodies anti hsp90 (11405-1-ap, proteintech), anti pan-cathepsin (sc-376803, santa cruz biotechnology), anti vp5 and anti ns3 overnight at 4˚c, and labelled with alexa fluor a488, a546 and a633-conjugated antibodies (a11001, a11074 and a21071, thermofisher scientific) for 2 hours at rt. nuclei were labelled with a pbs-hoescht solution at 2 μg/ml for 5 min at rt and samples were finally mounted in mounting medium (f4680, sigma aldrich). labelled cells and evs were analysed with an inverted lsm 880 confocal microscope with airyscan (carl zeiss ltd.), or with an inverted widefield nikon ti eclipse microscope (nikon) for the vibs surface analysis. for the co-localisation analysis, three independent replicates (with more than 90 cells imaged for each replicate) were analysed with a statistical object distance analysis (soda, icy software) on the whole cells [43] . after infection with btv-1 (moi = 10), cells were incubated at rt under gentle rotation for 30 min, before the inoculum was replaced with d-mem containing baculovirus expressing lamp1-gfp at concentration of 10 particles / cell (celllight lysosomes-rfp, bacmam 2.0, thermofisher scientific). at 18 hpi, cells were incubated with hoescht at 2 μg/ml for 5 min at rt, followed by an acidic compartment marker 100nm (lysotracker dnd199, thermofisher scientific) for 1 min at rt. cells were then washed twice with a pbs solution and analysed with a lsm880 confocal microscope. images were analysed with icy (https://icy.bioimageanalysis. org). cells were counted by detection of stained nuclei and lysosomes by detection of the fusion lamp1-gfp fluorescent protein. each lysosome detected was considered as a region of interest, and lysotracker fluorescence intensity detected in these regions were then quantified. after infection with btv (moi = 10), cells were incubated at rt under gentle rotation for 30 min, before the inoculum was replaced with d-mem. at 18 hpi, cells were incubated with hoescht at 2 μg/ml for 5 min at rt, followed by incubation with the fluorescent labelling reagent fluo-4 am (at 1μm in d-mem) for the detection of intracellular calcium (gr3332801, abcam) for 1 hour at 35˚c. fluo-4 am/d-mem solution was then replaced by d-mem only to remove any non-specific binding, and left a further 30 min at 35˚c, followed by two rinsing with a pbs solution and imaged with a lsm880 confocal microscope (fluo-4 am excitation at 488nm). for image analysis, all cells were detected using the hk mean plugvirus released in secretory lysosomes in in icy (https://icy.bioimageanalysis.org) applied on the fluo-4 channel, and detected cells were considered as regions of interest in which the fluo-4am intensity was then measured. cells were infected and fixed at different times post infection as above, and labelled with an anti ns2 (made in pr. roy laboratory) antibody, a secondary alexa fluor a546-conjugated antibody (a11074, thermofisher scientific), and a488-conjugated wheat germ agglutinin as a membrane label (w11261, thermofisher scientific) for 10 min at rt prior cells permeabilisation. microscopy images were analysed with icy, and vibs were detected using the spot detector plugin, which provided ns2 spot surfaces. cell counting was performed based on the number of nuclei detected in images. for data analysis, a cut-off was applied to keep all detected ns2 spots with a surface above 22μm 2 . the frequency distribution of vibs surface values were performed with a bin width of 15μm 2 and the lognormal regression of the frequency distribution of the number of vibs per cells were performed after confirming that data were following a lognormal distribution. the total number of vibs measured at each time points is indicated in the s1 table. for superinfection exclusion analysis, sheep cells in 6 well-plates were infected with btv-1 (btv wt , moi = 5) as described above. simultaneously (0 hpi), or at 1, 2, 3, 4 or 5 hpi, cells were inoculated with purified evs or free virus particles of btv unag (moi = 5) and placed at 35˚c in a humidified incubator (5% co 2 ) for 1 hour. btv unag inoculum was then removed and replaced with d-mem (1% fcs). at 12 hpi (7 hpi of btv unag infection), cell monolayers were washed twice with pbs, re-suspended using trypsin-edta (thermo fisher scientific), fixed with paraformaldehyde 2% (w/v) for 10 min, permeabilised with a solution of pbs-triton x100 (0.05% v/v) for 5 min, and blocked with a pbs-bsa (1%) solution for 30 min. the cells were then incubated with an anti vp6 antibody (made in pr. roy's laboratory) overnight at 4˚c, followed by an incubation of 2 h at rt in the presence of a species-specific alexa fluor a633 conjugated secondary antibodies (a21071, thermo fisher scientific). after centrifugation and washings, cells were resuspended in pbs and analysed with a bd lsr ii flow cytometer (bd biosciences, san jose, ca, usa). for each replicate, 30,000 cells were analysed using the same parameters. the detection of alexa fluor 633 fluorescence corresponded to the total vp6 detected in infected cells, and the unag detected fluorescence to vp6 expressed by btv unag infection only. numerical data were analysed with python (v 3.7.1), the scipy library (v 1.4.1) and prism software version 8.1.2 (graph pad software, san diego, ca, usa). for all sets of data, two-tailed t-tests or anova tests were performed for statistical comparison. when necessary, outlier values removal was performed using the ellipticenvelope from the scikit-learn library (0.23.1). figures were prepared using jupyter notebooks (project jupyter, https://jupyter.org) and inkscape (https://gitlab.com/inkscape/inkscape) cell walls and the convergent evolution of the viral envelope how non-enveloped viruses hijack host machineries to cause infection a pathogenic picornavirus acquires an envelope by hijacking cellular membranes coxsackievirus b exits the host cell in shed microvesicles displaying autophagosomal markers hepatitis e virus egress depends on the exosomal pathway, with secretory exosomes derived from multivesicular bodies nonlytic viral spread enhanced by autophagy components phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses bk polyomavirus hijacks extracellular vesicles for en bloc transmission vesicle-cloaked virus clusters are optimal units for inter-organismal viral transmission extracellular vesicles: vehicles of en bloc viral transmission cooperation between different variants: a unique potential for virus evolution localization of the non-structural protein ns3 in bluetongue virus-infected cells a viral nonstructural protein regulates bluetongue virus trafficking and release bluetongue virus nonstructural protein 3 orchestrates virus maturation and drives non-lytic egress via two polybasic motifs influence of cellular trafficking pathway on bluetongue virus infection in ovine cells viral genome segmentation can result from a trade-off between genetic content and particle stability the interplay between exosomes and autophagy-partners in crime regulated lysosomal exocytosis mediates cancer progression a trp channel senses lysosome neutralization by pathogens to trigger their expulsion interaction of calpactin light chain (s100a10/p11) and a viral ns protein is essential for intracellular trafficking of nonenveloped bluetongue virus nonstructural protein 3 of bluetongue virus assists virus release by recruiting escrt-i protein tsg101 jc virus infected choroid plexus epithelial cells produce extracellular vesicles that infect glial cells independently of the virus attachment receptor hepatitis a virus structural protein px interacts with alix and promotes the secretion of virions and foreign proteins through exosome-like vesicles extracellular vesicle heterogeneity: subpopulations, isolation techniques, and diverse functions in cancer progression lysosome enlargement during inhibition of the lipid kinase pikfyve proceeds through lysosome coalescence a gene network regulating lysosomal biogenesis and function transcriptional activation of lysosomal exocytosis promotes cellular clearance the endoplasmic reticulum, not the ph gradient, drives calcium refilling of lysosomes lysosomes shape ins(1,4,5)p3-evoked ca2+ signals by selectively sequestering ca2+ released from the endoplasmic reticulum β-coronaviruses use lysosomal organelles for cellular egress bluetongue virus coat protein vp2 contains sialic acid-binding domains, and vp5 resembles enveloped virus fusion proteins extracellular vesicles mediate receptor-independent transmission of novel tick-borne bunyavirus extracellular vesicles: a new communication paradigm? the pathology and pathogenesis of bluetongue persistent infection of bhk21/wi-2 cells with rubella virus and characterization of rubella variants the viral envelope is not sufficient to transfer the unique broad cell tropism of bungowannah virus to a related pestivirus picornavirus infection induces temporal release of multiple extracellular vesicle subsets that differ in molecular composition and infectious potential in vitro reconstitution of bluetongue virus infectious cores hsp90 chaperones bluetongue virus proteins and prevents proteasomal degradation autophagy activated by bluetongue virus infection plays a positive role in its replication neutral sphingomyelinases control extracellular vesicles budding from the plasma membrane mapping molecular assemblies with fluorescence microscopy and object-based spatial statistics the authors thank eiko matsuo (kobe university, kobe city, japan) for providing the btv u-nag fluorescent virus. we acknowledge microscopy support from mark turmain (the biosciences em core facility, ucl, london uk). the authors have declared that no competing interests exist. key: cord-003915-kje8lvgl authors: pigeyre, laetitia; schatz, malvina; ravallec, marc; gasmi, leila; nègre, nicolas; clouet, cécile; seveno, martial; el koulali, khadija; decourcelle, mathilde; guerardel, yann; cot, didier; dupressoir, thierry; gosselin-grenet, anne-sophie; ogliastro, mylène title: interaction of a densovirus with glycans of the peritrophic matrix mediates oral infection of the lepidopteran pest spodoptera frugiperda date: 2019-09-17 journal: viruses doi: 10.3390/v11090870 sha: doc_id: 3915 cord_uid: kje8lvgl the success of oral infection by viruses depends on their capacity to overcome the gut epithelial barrier of their host to crossing over apical, mucous extracellular matrices. as orally transmitted viruses, densoviruses, are also challenged by the complexity of the insect gut barriers, more specifically by the chitinous peritrophic matrix, that lines and protects the midgut epithelium; how capsids stick to and cross these barriers to reach their final cell destination where replication goes has been poorly studied in insects. here, we analyzed the early interaction of the junonia coenia densovirus (jcdv) with the midgut barriers of caterpillars from the pest spodoptera frugiperda. using combination of imaging, biochemical, proteomic and transcriptomic analyses, we examined in vitro, ex vivo and in vivo the early interaction of the capsids with the peritrophic matrix and the consequence of early oral infection on the overall gut function. we show that the jcdv particle rapidly adheres to the peritrophic matrix through interaction with different glycans including chitin and glycoproteins, and that these interactions are necessary for oral infection. proteomic analyses of jcdv binding proteins of the peritrophic matrix revealed mucins and non-mucins proteins including enzymes already known to act as receptors for several insect pathogens. in addition, we show that jcdv early infection results in an arrest of n-acetylglucosamine secretion and a disruption in the integrity of the peritrophic matrix, which may help viral particles to pass through. finally, jcdv early infection induces changes in midgut genes expression favoring an increased metabolism including an increased translational activity. these dysregulations probably participate to the overall dysfunction of the gut barrier in the early steps of viral pathogenesis. a better understanding of early steps of densovirus infection process is crucial to build biocontrol strategies against major insect pests. the transmission of parvoviruses predominantly occurs by horizontal routes through inhalation or oral exposure, making interaction with mucosal epithelia a crucial part of their pathogenesis (for review [1] ). the oral route represents a major challenge for viruses as they need to overcome a diversity of barriers to invade their host. indeed, most animal epithelia are covered in their apical surface by a carbohydrate-rich meshwork of various complexity and thickness, the glycocalyx, which can be coated by an additional layer of secreted mucus [2] . these structures constitute successive protective surfaces where viruses aggregate and either access to attachment factors and receptors at the surface of the epithelial cells or are eliminated by luminal or cilia movements [3] . this dual fate depends on the virus affinity for glycans, which must allow to escape the trap of the mucus. the diversity of glycans present on the epithelial surfaces vary between and within species and therefore constitute an important component of the innate immunity and of the species barrier [4] . members of the parvoviridae family are non-enveloped viruses that have a simple capsid with t = 1 icosaedral symmetry protecting a 4-6 kb linear, single stranded (ss) dna genome [5] . they can cause diseases of various severity in a wide range of animals. parvovirus interest in human and animal health or in biomedicine as vectors for gene transfer, has long focused research to understand their cell tropism and entry mechanisms. a number of cellular attachment factors and receptors have been characterized, mostly for vertebrates' parvoviruses. they highlight the importance of glycans in capsid recognition and binding specificity [6] [7] [8] . however, how capsids interact with epithelia remains poorly known, mainly due to difficulties to reconstitute in cellular models the complexity of animal epithelial systems [9, 10] . insect parvoviruses, named densoviruses, can be highly pathogenic, a feature that can both represent threats for insect mass rearing or opportunities for biocontrol against harmful insects as alternative to chemicals. developing methods against infections or tools for biocontrol requires a deep understanding of the mechanisms driving host range and pathogenesis. like their vertebrate counterparts, densoviruses are mainly transmitted orally, gut recognition and binding constitute the primary step of their pathogenesis. the mechanisms determining densovirus specificity are poorly known. depending on species, densovirus replication can be either restricted to or exclude the gut, viral particles being then transported across the epithelium by transcytosis to reach internal organs where replication goes [11] [12] [13] [14] . only one cellular receptor has been so far characterized for a "gut restricted" densovirus. it is a mucin of the gut of the silkworm bombyx mori, whose inactivation makes silkworms resistant to infection with the bombyx mori densovirus type 1 (bmdv) [15] . we have previously reported that gut transcytosis of the junonia coenia densovirus (jcdv) involves a gut specific receptor-dependent mechanism in caterpillars [12] . however, the mechanisms used by viral particles to overcome the successive intestinal barriers of different structures and composition remain elusive. in insects, the intestinal tract is covered by a chitinous acellular layer, which has specific features due to the dual embryonic origin of the gut. indeed, anterior and posterior extremities of the gut are ectodermal and the acellular layer is covered by an impermeable cuticle. the midgut section is endodermal and has no cuticle but is covered in most insects by a semi-permeable membrane, named the peritrophic matrix (pm) (for review [16] ). the pm forms a highly organized lattice of chitin fibrils associated with glycoproteins, mainly peritrophins that have a chitin-binding domain [17, 18] . the midgut is thus the portal of entry for most pathogens and their interaction with the pm a critical step of their pathogenesis. the pm forms pores whose size varies between insect species (e.g., 7-36 nm in lepidoptera and up to 150 nm in coleoptera) and developmental stages [19, 20] . large entomopathogenic viruses have developed specific mechanisms to pass through extracellular matrices including virus-encoded enzymes and specific proteins that are associated with the viral particles of baculo-and entomopoxviruses [21] [22] [23] [24] . how densoviruses cope with the physical barriers that constitute the gut and in particular the pm is so far unknow. due to their small size, it was initially thought they could diffuse passively across the pores of the matrix, but measures of the size of pores, the complexity of the pm and the nature of the interactions between components make this hypothesis unlikely [16, 20] . we previously reported that following oral infection, viral particles of jcdv, a type species ofthe ambidensovirus genus, aggregate on the pm of spodoptera frugiperda caterpillars as a first step of the infection process [12] . such rapid virus concentration on a carbohydrate-rich surface suggested a lectin-like activities of the capsids. although there is no sequence similarity of the unique protein making the surface of the jcdv with lectin domains, its structure displays similarities with cellular carbohydrate binding proteins including lectins, which suggests that capsids could indeed recognize and bind carbohydrates [25] . herein, we used a combination of approaches, including microscopy, biochemistry, proteomics and transcriptomics, to decipher the interaction of jcdv with pm major components (i.e., chitin, glycans and proteins). we found that capsids affinity for the pm might result from multiple interactions with different glycans including chitin and glycosylated proteins. in addition, we showed that jcdv early infection results in (i) an arrest of n-acetylglucosamine (glcnac) secretion by epithelial cells associated with a disorganization of the pm structure mimicking the effect of chitin-binding plant lectin; (ii) substantial changes in the expression of gut genes, which may also contribute to an early gut dysfunction and participate to viral pathogenesis. caterpillars were reared under controlled conditions (25 ± 1 • c, 65 to 70% relative humidity [rh], 16-h light, 8-h dark photoperiod) on a wheat germ-based artificial diet. jcdv was amplified by oral infection. at death, larvae were crushed and virus extraction was processed by clarification and filtration on 0.22 µm to constitute a semi-purified virus stock (jcdv). to obtain a purified viral stock (p_jcdv), the semi-purified virus stock was loaded on optiprep tm (sigma-aldrich, lyon, france) density gradient and dialyzed against phosphate-buffered saline (pbs) 1x as previously described in [13] . viral concentrations were estimated by quantitative pcr (qpcr) as described in [12] and expressed as viral equivalent genomes ([veg]). virus titers were determined by the tissue culture assay method (50% tissue culture infective dose (tcid50) in the permissive ld652 cells as previously described [26] . calcofluor white m2r (calcofluor; f3543), n-acetyl-d-glucosamine (glcnac; a4106), n-acetyl-d-galactosamine (galnac; a2795), d-(+)-fucose (fucose; f8150), d-(+)-mannose (mannose; m6020), mucin from porcine stomach (porcine mucin; m2378) and wga-fitc conjugate (l4895) were all purchased from sigma-aldrich (lyon, france). for in vivo bioassays, third-instar (l3) s. frugiperda caterpillars were individually infected by feeding with jcdv (10 9 veg/caterpillar) or by intrahemocelic injection (10 9 veg/caterpillar). each treatment was applied to a cohort of 24 caterpillars and three independent experiments were performed. calcofluor (0.5% to 3%) was concomitantly administrated with the virus to l3 or l6 caterpillars (as described in the figures). for competition assays, jcdv was incubated with glycans (glcnac, galnac, fucose or mannose at 5 µm and/or 5 mm; for one hour before feeding or injection. in all experiments, control larvae were fed or injected with pbs. caterpillars mortality was recorded each day during 10 days and results were presented as survival rates per day. the time to death was assessed by comparing the survival curves using the kaplan meier method (graphpad prism software, version 7). the significance between groups were analyzed using log-rank (mantel-cox) tests and gehan-breslow-wilcoxon tests. rabbit erythrocytes cells were diluted at 3% (v/v) with 150 mm nacl and treated with 0.5 mg/ml of trypsin (sigma) for 30 min at 37 • c. after 3 washes with 150 mm nacl, 25 µl serially two-fold dilutions of viral inocula (jcdv or p_jcdv; started from 10 11 veg/µl) were mixed with 25 µl of erythrocytes in 96 well microtiter plates for 30 min at 37 • c. positive controls of hemagglutination was performed using 25 µl of wga (1 mg/ml). fifty µl of erythrocytes were subsequently added to each well for 30 min at 37 • c. hemagglutination was read under a light microscope. for calcofluor experiments, pms were isolated from sixth-instar (l6) s. frugiperda caterpillars, previously anesthetized on ice, then opened and washed with phosphate buffered saline (pbs) to remove the food bolus. pms isolated from caterpillars treated with calcofluor were fixed 1 h with 4% paraformaldehyde (pfa), then dehydrated with increasing concentrations of ethanol (50% to 100%) and in 1:1 (100% ethanol and hexamethyldisilazane [hmds]) for 10 min and finally in 100% hmds for 20 min. after overnight evaporation, samples were ultimately coated with platinium and observed with a scanning electron microscope (sem) hitachi s-4800, with an acceleration voltage of 2 kv and a working distance of 5 mm. for ex vivo infection experiments, pms were isolated as described above and incubated for 30 min at room temperature (rt) with jcdv (10 9 veg/µl; 500µl), or with jcdv pre-incubated for 1 h with glycans (glcnac, galnac, fucose or mannose; 5 µm to 5 mm) before infection. the pms were then washed with pbs and fixed with 4% pfa as above. immunolabelling of jcdv particles was performed using a specific rabbit anti-capsid antibody (1:1,000; eurogentec) incubated for 2 h at rt and an anti-rabbit secondary antibody (alexa fluor ® 568; 1:500; invitrogen, carlsbad, ca, usa) incubated for 45 min at rt. labeled pms were then mounted in dako (sigma) on coverslips for observation. for epifluorescence measurements, we worked on a zeiss axioimager z2, equipped with suitable filters for the dyes, using a 40×/1.3 plan-apochromat oil ph3 and a 63×/1.4 plan-apochromat oil ph3 objectives. for structured illumination images, the zeiss apotome-slider was introduced into the field-stop plane of the microscope to improve resolution of images. we used the zen software to operate the microscope and took at least 10 images (10 fields per pm) for each treatment. all images were taken with a cmos orca flash 4.0 b&w camera. images were processed with fiji software. the intensity of fluorescence (arbitrary unit) were measured on epifluorescence images. statistical analyses were performed using the non-parametric kruskal-wallis test (graphpad prism software, version 7, graphpad software, san diego, ca, usa). each experiment was repeated at least three times, and each independent experiment gave similar results. l3 s. frugiperda caterpillars were individually infected by feeding as in section 2.3. twenty four hours later, caterpillars were anesthetized on ice and sacrificed. in parallel, caterpillars were kept until death to insure their infection status. sacrificed caterpillars were fixed in 4% pfa + 2.5% glutaraldehyde for 1h at 4 • c and dehydrated by incubations in increasing concentrations of ethanol (50% to 100%) before progressive embedding in 100% unicryl resin (bb international) as described in [12] . semi-thin sections (1 µm) were cut in the central part of the caterpillars corresponding to the midgut. after 45 min of incubation with wga-fitc (1:300), to label glcnac and the pm, or with phalloidin-fitc (1:300; sigma), to label actin cytoskeleton, and 10 min with dapi (1 µg/ml; invitrogen) to label nuclei, semi-thin sections were mounted and observed with a zeiss axioimager z2 microscope using the structured illumination module zeiss apotome-slider as in 4.5. images were taken with a cmos orca flash 4.0 b&w camera using fixed parameters for all treatments and processed with fiji software. ten µl of chitin beads (new england biolab) were washed three times with pbs and added in 200 µl of virus suspension (10 9 veg/µl in pbs). after 1 h of incubation with gentle rotation, beads were pulled-down by centrifugation (500× g for 5 min à 4 • c), washed five times with pbs, then resuspended in laemmli buffer 2 × (4% sds, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue and 0.125 m tris hcl, ph 7) and heated at 95 • c for 5 min before western blot analysis. two µl (5% of the pull down and 1% of the initial input) was loaded on 4-15% polyacrylamide tris-hcl gel (mini-protean ® tgx™ precast gels, biorad, hercules, ca, usa) and separated by sds-page for 1 h at 150 v. next, samples were transferred to pvdf membrane (immobilon-p, merck) for 2 h at 200 ma. subsequently, the membrane was saturated with 5% of milk in pbs/tween 0.1% (pbst), then incubated 2 h at rt with the primary anti-capsid antibody (1:1,000; see above). after washes in pbst, the membrane was incubated 1 h at rt with an anti-rabbit secondary antibody hrp-conjugated (1:3000; biorad, hercules, ca, usa). proteins were revealed by enhanced chemiluminescence (millipore, burlington, ma, usa) using a chemidoc imager (biorad). peritrophins were extracted from pools of five pms from sixth instar s. frugiperda in 200 µl of laemmli buffer 2x, then boiled and heated at 95 • c for 5 min [27] . after centrifugation at 8000× g for 10 min 4 • c, the supernatant was collected and 1/15 was loaded on 4-15% polyacrylamide tris-hcl gel and separated by sds-page as above. thirty µg of porcine mucins were also loaded on the same gel. gel was stained with page blue (thermo fisher scientific, waltham, ma, usa) to analyze total proteins or with periodic acid-schiff (pas) as described in [28] to analyze glycosylated proteins. proteins were also transferred on nitrocellulose membranes (biorad) for 3 h at 70 v for viral protein overlay binding assay [29] . briefly, the membrane was saturated with 3% bsa in pbst for 2 h at rt, then incubated ovn at 4 • c with jcdv (10 9 veg diluted in pbst containing 1% bsa). after washes in pbst, the membrane was incubated with the rabbit anti-capsid antibody (1:1000; see above), then the anti-rabbit secondary antibody hrp-conjugated, and proteins were revealed by enhanced chemiluminescence as above. protein bands revealed by vopba were cut in sds-page gel stained with page blue (3 replicates, 10 bands) and destained with three washes in 50% acetonitrile and 50 mm triethylammonium bicarbonate (teabc). after protein reduction (with 10 mm dithiothreitol in 50 mm teabc at 60 • c for 30 min in the dark) and alkylation (55 mm iodoacetamide teabc at room temperature for 30 min) proteins were digested in-gel using trypsin (0.5 µg/band, gold, promega, madison, wi, usa) as previously described [30] . digested products were dehydrated in a vacuum centrifuge and resuspended with 0.05% trifluoroacetic acid (tfa) and 2% acn. the generated peptides were analyzed online by nano-flow hplc-nanoelectrospray ionization using a q-exactive plus mass spectrometer (thermo fisher scientific, waltham, ma, usa) coupled to an ultimate 3000 rslc (thermo fisher scientific). desalting and pre-concentration of samples were performed on-line on a pepmap ® pre-column (0.3 mm × 10 mm, dionex, sunnyvale, ca, usa). the capillary reverse-phase column (0.075 mm × 150 mm, acclaim pepmap 100 ® c18, thermo fisher scientific) fitted with an uncoated silica picotip emitter (new objective, woburn, ma, usa) was first equilibrated in solvent a (0.1% formic acid) and a multistep linear gradient of acetonitrile consisting of 0-25% of solvent b (0.1% formic acid in 80% acetonitrile) for 30 min, 25-40% for 5 min and 90% for 8 min, at 300 nl/min was used to elute peptides from. spectra were acquired with the instrument operating in the information-dependent acquisition mode throughout the hplc gradient. survey scans were acquired in the orbitrap system with resolution set at a value of 70,000. up to twelve of the most intense ions per cycle were fragmented and analyzed using a resolution of 17,500. peptide fragmentation was performed using nitrogen gas on the most abundant and at least doubly charged ions detected in the initial ms scan and an active exclusion time of 45 s. for all full scan measurements with the orbitrap detector a lock-mass ion from ambient air (m/z 445.120024) was used as an internal calibrant as described [31] . analysis was performed using the maxquant software (version 1.5.5.1) [32] . all ms/ms spectra were searched using andromeda against a decoy database consisting of a combination of s. frugiperda databases [33] and 236 classical contaminants, containing forward and reverse entities. the following settings were applied: spectra were searched with a mass tolerance of 7 ppm (ms) and 0.5 th (ms/ms). enzyme specificity was set to trypsin. up to two missed cleavages were allowed and only peptides with at least seven amino acids in length were considered. carbamidomethylation was set as fixed cystein modification and oxidation was set as variable methionine modification for searches. fdr was set at 0.01 for peptides and proteins. sequences which found homology were annotated according to the gene ontology (go) terms and classified using blast2go software (https://www.blast2go.com/; [34] ). the enrichment in go terms compared to the s. frugiperda reference (predicted proteins from the ogs2.2 genome; gouin et al., 2017) was analyzed with the same software (fdr set at 0.01). fourth instar s. frugiperda caterpillars were orally infected or not with jcdv (10 12 veg per caterpillar; twenty caterpillars per condition). at 1-day p.i. or 3 days p.i., caterpillars were anesthetized on ice and dissected. the midguts were washed in pbs to eliminate the food bolus and the pms. trachea, malpighi tubes and visceral muscles were removed and the epithelia were incubated with 2.5% trypsin for 30 min to dissociate the tissues. after washes, gut cells were lysed in 800 µl of trizol ® reagent (invitrogen) for total rna extraction according to the manufacturer's instructions. total rna amount and purity were checked by using spectrophotometrer nanodrop nd-1000 (thermo scientific) and the integrity of total rna was analyzed by capillary electrophoresis (2100 bioanalyzer instrument, agilent, santa clara, ca, usa). we used digital gene expression (dge) method that generates short sequences (tags) specific for mrna [35] [36] [37] . four dge libraries were constructed from midgut total rna extracted from s. frugiperda caterpillars infected (or not) for 1 and 3 days. sequence tag preparation was done with illumina's digital gene expression tag profiling kit according to the manufacturer's protocol (version 2.1b) as described in [36] . for fourth libraries, 10 µg of total rna were incubated with oligo-dt beads. first-and second-strand cdna syntheses were performed using superscript ii reverse transcription kit according to the manufacturer's instructions (invitrogen). the cdnas were cleaved using the nlaiii anchoring enzyme. subsequently, digested cdnas were ligated with the gex adapter 1 containing a restriction site for mmei. the second digestion with mmei was performed, which cuts 17 bp downstream of the catg site. at this point, the fragments detach from the beads. the gex adapter 2 was ligated to the 3 end of the tag. a pcr amplification with 15 cycles using phusion polymerase (finnzymes) was performed with primers complementary to the adapter sequences to enrich the samples for the desired fragments. the resulting fragments of 85 bp were purified by excision from a 6% polyacrylamide tbe gel. the dna was eluted from the gel using spin-x cellulose acetate filter (0.45 µm), precipitated, resuspended in 10 mm tris-hcl (ph 8.5) and quantified using nanodrop 1000 spectrophotometer. cluster generation was performed after applying 4 pm of each sample to the individual lanes of the illumina 1g flowcell. after hybridization of the sequencing primer to the single-stranded products, 18 cycles of base incorporation were carried out on the 1g analyzer according to the manufacturer's instructions. image analysis and base calling were performed using the illumina pipeline, where sequence tags were obtained after purity filtering. we could assign 63% of the tags (supplementary materials tables s2-s4) out of which 54% correspond to multiple matches and were discarded from functional analysis with go. functional annotation was performed using biotag software (skuld-tech, grabels, france). the statistical value of dge data comparisons, as a function of tag counts, was calculated by assuming that each tag has an equal chance of being detected. differential expression of the tag counts of the infected vs. mock conditions was performed to obtain a list of up-and down-regulated tags for each condition. tags for which differential expression was ≥ 5 fold change were assigned using the reference databases for s. frugiperda [33, 38] . sequences with homology were annotated according to the go terms and classified using blast2go software (https://www.blast2go.com/; [34] ) and represented at level 2 of biological process and molecular function. the enrichment in go terms compared to the s. frugiperda reference (predicted transcripts from the ogs2.2 genome) was analyzed with the same software (fdr set at 0.01). the junonia coenia densovirus (former jcdnv) corresponds to the complete genome of the oxford isolate (genbank accession number kc883978.1). early studies have estimated that the average pore size of the pm is around 8 nm in s. frugiperda caterpillars, which might exclude 25 nm densovirus particles [39] . to assess the pm barrier function against densovirus infection, we disrupted its structure by feeding third instar (l3) larvae with sub lethal doses (0.5%) of the chitin-binding agent calcofluor white prior jcdv infection [40] [41] [42] [43] [44] . we then measured larval mortality rates daily. results showed that jcdv infected larvae pre-treated with calcofluor displayed a significant shorter median time to death (lt50) compared to untreated infected larvae (7 vs. 9 days p.i. for control larvae; p < 0.01) ( figure 1a and supplementary materials figure s1 ), supporting that the pm can limit jcdv infection. noteworthy, an increased larval mortality was also observed at late time post-treatment with calcofluor in mock-infected caterpillars compared to untreated controls (18% at 10 days p.i.), confirming a detrimental inhibition of chitin assembly on larval development [41] . larvae (n = 24) were infected individually by feeding with jcdv (10 9 veg/caterpillar) concomitantly with 0.5% (5 µg) of calcofluor or pbs as a control. caterpillar mortality was recorded once a day during 10 days and results were presented as survival rates per day. three independent experiments were performed, each independent experiment gave similar results, one is represented here. the log-rank (mantel-cox) and the gehan-breslow-wilcoxon tests were used to determine statistical significance. p values of less than 0.05 were considered significant (**, p < 0.01). pbs refers to control (pbs-treated and non-infected) caterpillars; calcofluor refers to calcofluor-treated and non-infected caterpillars; jcdv refers to jcdv-infected caterpillars and jcdv+calcofluor to calcofluor-treated and jcdv-infected caterpillars. (b) calcofluor disrupts the pm integrity. sem images of pm ultrastructure isolated from l6 caterpillars fed with pbs (0%), 0.5% (5 µg) or 3% (30 µg) of calcofluor. endoperitrophic face is shown (bars, 300 nm). we analyzed the effect of calcofluor on the pm integrity by scanning electron microscopy (sem). because the pm of l3 larvae has a gel-like structure that cannot be manipulated, we took l6 larvae for this experiment as the pm is thick and solid at this stage and can be easily dissected. l6 caterpillars were fed with up to 3% calcofluor (not lethal at 24 h post treatment) and pms were isolated 24 h post-treatment and prepared for sem analysis. as shown by figure 1b , pms from control larvae displayed a highly organized structure, similar to pms from caterpillars treated with 0.5% calcofluor. by contrast, pms from caterpillar fed with 3% calcofluor had a clear disrupted structure with enlarged pores, confirming that calcofluor binding to chitin fibrils compromised the integrity of the matrix. the rapid recognition of the pm by jcdv capsids suggests that their affinity for glycans is important for the oral infection process. to test this hypothesis, we first assayed the capsid ability to agglutinate erythrocytes, a feature displayed by vertebrate parvoviruses [45, 46] . we performed a typical hemagglutination assay, adding serial dilutions of the virus inoculum to rabbit erythrocytes ( figure 2 ). the first dilutions (1:2 and 1:4) of jcdv triggered a strong hemolysis of erythrocytes suggesting a toxic effect of the viral inoculum, ie capsids or some host-derived component associated with the inoculum. it is worthy to note that we use semi-purified inoculum as it mimics naturally occurring infections. jcdv was therefore further purified on a density gradient (p_jcdv) and similarly assayed for hemagglutination. a clear hemagglutination was obtained with p_jcdv, supporting that toxicity is likely due to a host-derived component that can be eliminated during the purification process. hemagglutination with p_jcdv was obtained up to the third dilution (hemagglutination titer of 1:8), which indicates a rather weak interaction of the capsids with glycans at the surface of (mammalian) erythrocytes. to better understand capsid affinity for glycans, we performed a competition bioassay using monomeric glycans as jcdv-binding competitors. jcdv binding was revealed with an immunofluorescence staining on the pms using a specific anti-capsid antibody. we quantified this fluorescence as a proxy of binding and competition. we first performed competition ex vivo on isolated pms incubated with jcdv in the presence of four monosaccharides commonly found in insects [47] , ie n-acetyl-d-glucosamine (glcnac), which is the monomeric unit of chitin, n-acetyl-d-galactosamine (galnac), d-fucose and d-mannose (figure 3 ). we first verified with a dot blot assay that capsids interaction with monosaccharides were not interfering with antibody recognition, which validated the competition bioassay (supplementary materials figure s2 ). as shown in figure 3 , jcdv binding resulted in an intense fluorescence signal on the pms (left panel), which was similarly competed away by the four monosacharides and within a similar concentration range (0.5 mm to 5 mm). we noted that fluorescence quantification did not result in a strictly linear dose-dependent effect. to further test the role of glycans in jcdv pathogenesis, we carried out these competition bioassays in vivo (figure 4 and supplementary materials figure s3 ). we mixed jcdv with each monosaccharide prior infection, fed caterpillars with these inocula and calculated mortality rates daily as in figure 1 . results showed that oral infection with 5 mm (but not 5 µm) of each monosaccharide significantly delayed the median time to death (lt50) of caterpillars (7 vs. 6 days; p < 0.05 for 5 mm) ( figure 4a and supplementary materials figure s3a ,b), further supporting that pm recognition is the first step of jcdv oral infection. to confirm these results and reveal jcdv binding and competition for binding the pm in vivo, we carried out midgut semi-thin sections and immunofluorescence as above. caterpillars were infected with jcdv mixed or not with 5 mm glcnac and sacrificed at 2 h p.i. for midgut isolation and preparation. as shown in figure 4b , we observed a red fluorescence signal in untreated infected caterpillars that typically lines the pm. in addition, labelling was also observed in the lumen, likely revealing jcdv interaction with food bolus and/or microbial components. both signals were strongly and specifically decreased following competition with glcnac ( figure 4b) , showing that different glcnac-containing glycans in the gut lumen can recognize the capsids. control caterpillars were fed with pbs. three independent experiments were performed, each independent experiment gave similar results, one is represented here. the log-rank (mantel-cox) and the gehan-breslow-wilcoxon tests were used to determine statistical significance. p values of less than 0.05 were considered significant (ns, non-significant; * p < 0.05; ** p < 0.01; *** p < 0.001). (b) immunolabeling of midgut semithin transversal sections 2 h after ingestion of jcdv alone or jcdv (10 9 veg/caterpillar) incubated for 1 h with 5 mm of glcnac before oral infection. control caterpillars were fed with pbs. the pm is shown by an arrowhead. phalloidin-fitc is in green, jcdv is in red, and nuclei are labeled with dapi (blue). bars, 30 µm. lum, midgut lumen; hemol, hemolymphatic compartment. (c) survival curves of caterpillars (n = 24) infected by injection of jcdv alone (10 9 veg/caterpillar) or of jcdv incubated for 1 h with 5 mm of each glycan (glcnac, galnac, fucose or mannose) before infection. control caterpillars were injected with pbs. three independent experiments were performed, each independent experiment gave similar results, one is represented here. the log-rank (mantel-cox) and the gehan-breslow-wilcoxon tests were used to determine statistical significance, p > 0.05 were considered non-significant (ns). pbs refers to control (pbs-treated and non-infected) caterpillars; 'jcdv' to jcdv-infected caterpillars; 'jcdv + glcnac', 'jcdv + galnac', 'jc + fucose' and 'jc + mannose' refer to caterpillars infected with jcdv incubated with glcnac, galnac, fucose or mannose, respectively, before infection. last, we studied whether such "stickiness" was specifically required by the densovirus to cross the gut, i.e., for oral infection. jcdv infection of target cells (eg epidermis, trachea, hemocytes) proceeds by a receptor-dependent mechanism different from intestinal cells [12] . these cells express glycan structures of various complexity that are attached to the cell surface or secreted and forming extracellular matrices, whose glycans might be similarly targeted by jcdv for attachment. we performed competition bioassays in vivo, bypassing the midgut by injecting caterpillars with jcdv mixed or not with 5 mm of each monosaccharide. interestingly, the median time to death was similar for all conditions ( figure 4c and supplementary materials figure s3c ; p > 0.05), showing that none of the monosaccharides competed with jcdv infection proceeding by the systemic route. altogether these results show that jcdv capsid is a carbohydrate-binding protein and this feature is required for oral infection to target the pm of caterpillars. we next wanted to determine which component of the pm, i.e., chitin and/or glycosylated proteins were involved in capsid interactions, using biochemical assays. we first tested capsid physical interaction with chitin using a pull-down assay with chitin beads. jcdv from purified or semi-purified inocula were incubated with chitin beads, pulled-down by centrifugation and subsequently revealed by western blot using a specific jcdv anti-capsid antibody. figure 5a shows jcdv pull-down by chitin beads and we did not observed difference between inocula (purified vs. semi-purified), which confirmed that capsids can interact directly with chitin. second, we tested virus interaction with pm proteins using a viral overlay binding assay (vobpa). total proteins were extracted from isolated pms, separated with sds-page and either stained with page blue and periodic acid schiff (pas) in order to visualize total and glycosylated proteins respectively, or blotted onto nitrocellulose membranes for vopba. we included porcine mucins as a control of highly (o-)glycosylated proteins. at the first glance, vopba revealed that jcdv binds to most if not all the pm proteins labelled by page blue and pas combined, although with different intensities ( figure 5b ). interestingly, no binding was observed with the porcine mucins which might support some specificity for insect glycans. more specifically, a set of jcdv-interacting proteins was identified at high molecular weights (>250 kda) including proteins with a pattern similar to porcine mucins. these proteins were labelled with page blue and pas or only with pas suggesting that they are mostly and probably highly glycosylated ( figure 5b ). interestingly, proteins at 180 kda displayed a higher intensity as they are in a relative lower amount (according to page blue), which suggests higher affinity for jcdv. proteins interacting with jcdv were detected at 150-200 kda and 25-60 kda, and corresponding to proteins with low or no glycosylation (according to pas staining). thirty µg of porcine mucins were also loaded in the gel as a control of highly o-glycosylated proteins. proteins were then stained with page blue or periodic acid schiff (pas, pink) to visualize total or glycosylated proteins, respectively, and transferred to nitrocellulose membranes for probing with jcdv and anti-jcdv capsid antibody. proteins interacting with jcdv capsids were finally revealed by enhanced chemiluminescence (black arrowheads on the vopba jcdv membrane); the corresponding positions of these bands were reported on the page blue and pas gels and indicated as well by black arrowheads on the right of these gels. in total, 10 bands representing jcdv interacting proteins are reproducibly obtained with vopba. noteworthy, each band probably include several proteins and/or isoform/glycoform of the same proteins. proteins corresponding to these 10 bands were next analyzed by lc-ms/ms mass spectrometry. we only considered 155 proteins that were shared between 3 replicates ( figure 6a) , out of which 138 were annotated in the reference genome of s. frugiperda [33] . these proteins are pm structural proteins (i.e., peritrophins including intestinal mucins) and pm-associated proteins (enzymes, i.e., serine proteases and aminopeptidases n (apn) (supplementary materials table s1 ). gene ontology (go) annotation confirmed the enrichment in proteolytic activities (particularly serine-type endopeptidases) and chitin synthesis, which are consistent with the pm composition and the gut function ( figure 6b ). interestingly among the set of proteins >150 kda, we identified intestinal mucins, an atp binding cassette a type 5 (abca5) transporter and aminopeptidases n (supplementary materials table s1); the latter being known receptors for a number of viruses and for the cry toxins from bacillus thuringiensis [49] [50] [51] [52] . [33] . (b) go terms enrichment for the 138 common annotated pm proteins interacting with jcdv (in green), compared to the reference in grey (predicted proteins from ogs2.2 s. frugiperda genome) (fdr set at 0.01). specific enrichment in jcdv interacting proteins is considered when the green bars exceed the greys (controls). the 138 common pm proteins were assignated to the go terms using blast2go software. these results show that jcdv capsids can recognize and bind to the different components of the pm including chitin and several highly glycosylated proteins, both structural components of the pm (mucin, peritrophins) or associated proteins (enzymes). jcdv recognition and binding to glycans of the pm concentrates viral particles close to the epithelial surface, which raised questions about the mechanism involved to cross over and reach the midgut receptor(s). we hypothesized that capsids aggregation on the matrix can result in its disorganization, in a way similar to chitin-binding wheat germ agglutinin (wga) lectin or calcofluor [53] . to test this hypothesis, we used fluorescent wga-labelling (wga-fitc) to label chitin and thus examine chitin fibrils formation and pm organization. third-instar caterpillars were fed with jcdv and then sacrificed at 1 day p.i. to dissect and prepare midguts for semi-thin sections and wga labelling. as shown in figure 7 , the labelling of the pm (green) lined the apical surface of the epithelium in non-infected larvae (pbs condition, figure 7 , upper panel). in addition, we observed a specific labelling at the apex of columnar cells, probably corresponding to microvillar secretion of glcnac from these cells. by contrast, the labelling lining the epithelium appeared discontinuous following infection (figure 7, lower panel) , displaying a disorganized pattern reminiscent of the pm structure observed for caterpillars fed with the wga lectin [53] . moreover, intracellular labelling was no longer observed in the sections from infected caterpillars suggesting an arrest of glcnac secretion from the cells following early infection, i.e., before we can detect virus replication in subepithelial tissues [13] . these results thus support the hypothesis that jcdv binding on the pm and transcytosis is associated with a loss in its integrity, which might reveal gut dysfunction. to determine the midgut response following jcdv break in, we analyzed the transcriptomic response. we used digital gene expression (dge) based on the serial analysis of gene expression approach [35] . this method involves the sequencing and quantification of end tagged short cdna fragments (i.e., tags), which enables quantitative differential gene-expression analysis. we built four cdna libraries from midguts of mock-and infected larvae (supplementary materials tables s2 and s3) . tag sequences were mapped to the genome and transcriptome of s. frugiperda ( [33, 38] ) and to the jcdv genome. none of the tags were assigned to viral transcripts, which is consistent with jcdv pathogenesis excluding replication in midgut cells [13] . pie charts represented go assignment corresponding to unique transcripts at 1 and 3 days p.i. that displayed a differential expression at least 5-fold up-or down-regulated (supplementary materials figure s4a,b) . interestingly, the distribution of go terms was roughly similar at 1 and 3 days p.i., suggesting that the overall intestinal response to jcdv oral infection was poorly affected by virus replication going on in subepithelial tissues (supplementary materials figure s4a ,b). we observed only go terms enrichment for the over-represented transcripts at 1-day p.i., more specifically in functions involved in metabolic processes including translation (i.e., regulation of biological processes, response to stimuli and signaling) at 1 day p.i., that might indicate that jcdv intrusion induces a rapid metabolic response in the gut (figure 8 ). interestingly, these changes did not change significantly at 3 days p.i. suggesting that the gut response is rapidly initiated by jcdv transcytosis and was not affected by the viral replication that takes place in underlying tissues. we did not observe any significant activation of genes involved in "inflammation" nor in the canonical gut immune response. however, we observed an increased expression in cytochrome p450 and catalase genes that might indicate a response of the cells to the ongoing infection. table s4 ), we only observed few changes including a trehalose transporter and an intestinal mucin, both down-regulated from day 1 p.i., and a chitinase up-regulated at day 3 p.i.. except for an aminopeptidase n, no gene corresponding to the jcdv interacting proteins identified by vopba displayed a transcriptional change. we did not observe any significant activation of genes involved in "inflammation" nor in the canonical gut immune response. however, we observed an increased expression in cytochrome p450 and catalase genes that might indicate a response of the cells to the ongoing infection. altogether these results show that jcdv infection induces rapid changes in the gut, particularly in translation and metabolism, within 24 h p.i.. both increased molecular activities might favor viral invasion by supporting the increased energetic demand associated with virus replication in target tissues. interestingly, the canonical midgut immune system did not detect jcdv break in and transport across the epithelium. how densoviruses cope with the forest of glycans that constitute extracellular matrices and decorates insect cell surfaces has been so far a neglected step of their early pathogenesis. results presented here show that jcdv capsids display carbohydrate-binding properties that insure recognition of the peritrophic matrix and determines caterpillars oral infection. we found that capsids can bind to the different components of the pm and their agglutination on the pm surface is associated with the disruption of its organization. furthermore, we showed that this primary step of infection of caterpillars results in a series of physiological changes in the midgut including an arrest of chitin synthesis by epithelial cells. the pm is an obligatory binding platform for capsids to avoid elimination and get closer to the epithelial cell surface where receptor recognition can occur. however, strong attachment to glycans composing the pm would trap capsids there and thus impair their physical connection with the receptor(s). therefore, a first hypothesis is that the "stickiness" of the capsids is balanced to bind and unbind glycans. we used competitions assays with monosaccharides to test this "bind and release" hypothesis and our results showed that indeed, capsids have an affinity for glycans, although the concentration range of the monosaccharides (mm) we tested likely indicates their poor affinity for the capsids. as these monosaccharides could compete capsids away from the pm further suggests that glycan-capsids interaction are probably of low affinity. the issue for bound viral particles is then to move across the pm. our experiments show that capsid binding results in a structural disorganization of the pm similar to effects induced by chitin-binding lectin wga and calcofluor. such capsid-induced disruption of the pm thus favors a second hypothesis, involving a "saturate and pass through" mechanism, where bound capsids are not released but open a way for viral particles to cross over. such "cooperative" mechanism of the capsids to overcome the pm is supported by the fact that pm disruption is enhanced by virus concentration and decreased as caterpillars age. such developmental resistance of s. frugiperda has been also reported following baculovirus infections and a high synergism with calcofluor was obtained at late instars (e.g., > 60-fold at 4th instar vs. 3 to 6-fold at 2nd and 3rd instars) [54] .the structure and the composition of the pm can vary as caterpillars grow and feed, or between populations, which might impact virus-pm interactions and consequently insect susceptibility [16, 17, 55] . whether pm disruption results from mechanical stresses on chitin fibers similar to calcofluor and probably wga lectins or from an enzymatic activity of the capsids (i.e., of vp4) remains to be analyzed more thoroughly. understanding the early interaction of jcdv with the glycans of the pm within species, i.e., along the larval development is of importance to develop biocontrol strategies against insect pests. whether or not the pm could contribute to the species barrier against densovirus infection is unknown. a better understanding of the structure and the glycan composition of the pm in s. frugiperda together with comparative studies in different lepidopteran species are essential to go further on the role of the pm in densovirus infection. structure-fonction studies of the capsid of parvoviruses infecting vertebrates, in particular for species in the genera protoparvovirus and dependovirus, have highlighted the importance of glycans recognition on tissue tropism, pathogenicity, and host range adaption [6, 45, [56] [57] [58] [59] . regarding densoviruses, information and capsid structure-function studies is poor [60] . we performed preliminary assays with a glycan array from the consortium for functional glycomics (http: //www.functionalglycomics.org/fg/, as gosselin-grenet, unpublished data). although this array represents mammalian glycans, the specific recognition of jcdv capsids by paucimannose, which are particularly abundant in insects, suggests some specificity in the interaction of the capsid with glycans. an "insect array" would be of interest to explore glycan ligands affinity and specificity for densovirus capsids. morevover, insects are particularly "handly" animal models for structure-function assays in vivo. we showed that jcdv early infection triggers an arrest of glcnac secretion, which might considerably weaken the pm and explain the disorganized pattern observed with chitin-labelling at 1 day p.i. interestingly, these changes are observed before we could detect jcdv transcription/replication in primary targeted cells [13] , suggesting that these effects are induced as a consequence of the transport of the viral particles across the epithelium. it has been reported that specific drugs that disorganize microtubules induce an arrest of chitin synthesis [41, 61] . we speculate that jcdv transcytosis might induce some stress on the cytoskeletal network, similar to microtubules disorganizing drugs. it is worthy to note that chitin synthesis arrest was not associated with a drop in the expression of chitin synthase genes. however, we cannot exclude to have missed enzymes due to some lack in the annotation of the genome of s. frugiperda [33] . last, our results showed that jcdv capsids can also interact with components of the luminal compartment including food and bacteria. the competition of the capsid with monosaccharides for binding the pm, suggests that food components could interfere with infection. indeed, plants contain compounds that can interfere with pm synthesis, which has been shown to consequently affect baculovirus infection [55] (chen et al., 2018) . regarding bacteria, it has been shown recently that the pm controls commensal bacteria, and conversely that its synthesis and integrity can be microbiota-dependent, i.e., the gut microbiota inducing the expression of components of the peritrophic matrix [62, 63] . so, it is plausible that food and microbiota can modify the outcome of the densovirus infection, either by directly competing for binding the pm, or indirectly by modulating the composition of the pm. the binding of densovirus capsids to a wide array of glycans questioned about the role of this "stickiness" in the whole infection cycle including transmission. groundbreaking articles have shown the role of microbiota polysaccharides including glcnac, on the infectivity and thermostability of picornaviruses [64] [65] [66] , whose capsid share structural similarity with parvoviruses [25] . it is tempting to speculate that densovirus "stickiness" can similarly impact their transmission, which might participate to their success if only among arthropods that occupy extremely diversified ecosystems [67] . such consideration could also apply to parvoviruses going through faecal-oral route and environmental contamination [58, 68] . more generally, stickiness is a major issue for most viruses to and mathematical models have been applied to influenza. they predict that a maximum stickiness favors a maximum fitness [69] . however, trade-off probably exists in biological systems with an optimal "stickiness" that must be found to infect and leave a host for transmission. densoviruses can be highly pathogenic for insect pests and vectors, which have long stimulated their interest as biocontrol agents or genes vectors [70] . they are considered today with a renewed interest as solutions to control harmful insects are lacking, which encourages efforts to understand their pathogenesis and their specificity. altogether our results suggest that pm glycans are crucial interacting components of the early jcdv pathogenesis. exploring their diversity and their complexity in insects can also provide important cues on the extend of the mechanisms that determine densovirus specificity. supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/11/9/870/s1, figure s1 . calcofluor increases s. frugiperda caterpillars susceptibility to densovirus oral infection. figure s2 . virus interaction with monosaccharides did not interfere with anti-capsid antibody recognition. figure s3 . replicates of survival curves of caterpillars infected by jcdv. figure s4 . jcdv oral infection induces midgut gene expression modulation. table s1 . annotation of pm proteins interacting with jcdv in vopba. table s2 . characteristics of dge libraries 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peritrophic matrix regulates midgut homeostasis and prevents systemic infection of malaria vector mosquitoes pgrp-ld mediates a. stephensi vector competency by regulating homeostasis of microbiota-induced peritrophic matrix synthesis intestinal microbiota promote enteric virus replication and systemic pathogenesis interactions between enteric bacteria and eukaryotic viruses impact the outcome of infection bacteria and bacterial envelope components enhance mammalian reovirus thermostability discovery of parvovirus-related sequences in an unexpected broad range of animals transmission ecology of canine parvovirus in a multi-host, multi-pathogen system how sticky should a virus be? the impact of virus binding and release on transmission fitness using influenza as an example viral delivery of dsrna for control of insect agricultural pests and vectors of human disease: prospects and challenges we are grateful to c. gibard, r. bousquet and g. clabot for their great help with insect rearing and the piq quarantine plateform from vectopole sud. we especially acknowledge e. jublanc and c. cazevieille for their skillful technical assistance and the imaging facility mri, member of the national france-bioimaging infrastructure supported by the french national research agency (anr-10-inbs-04, pia «investments for the future»). we warmly thank m. agbandje-mckenna from the cfg consortium (university of florida) for the glycan array and p. clair from the qphd platform (montpellier genomix). m. younes, and d. piquemal are also acknowledged for their support in the sage analysis. special thanks to p.a. lafon for his help for statistical analyses. l. pigeyre is a doctoral fellow of the french ministry for higher education and research/ephe. key: cord-026866-0hlre9i6 authors: perruzza, lisa; jaconi, stefano; lombardo, gloria; pinna, debora; strati, francesco; morone, diego; seehusen, frauke; hu, yue; bajoria, sakshi; xiong, jian; kumru, ozan selahattin; joshi, sangeeta bagai; volkin, david bernard; piantanida, renato; benigni, fabio; grassi, fabio; corti, davide; pizzuto, matteo samuele title: prophylactic activity of orally administered flid-reactive monoclonal siga against campylobacter infection date: 2020-06-09 journal: front immunol doi: 10.3389/fimmu.2020.01011 sha: doc_id: 26866 cord_uid: 0hlre9i6 campylobacter infection is one of the most common causes of bacterial gastroenteritis worldwide and a major global health threat due to the rapid development of antibiotic resistance. currently, there are no vaccines approved to prevent campylobacteriosis, and rehydration is the main form of therapy. secretory immunoglobulin a (siga) is the main antibody class found in mucous secretions, including human milk, and serves as the first line of defense for the gastrointestinal epithelium against enteric pathogens. in this study, we describe the prophylactic activity of orally delivered recombinant siga generated from two human monoclonal antibodies (caa1 and ccg4) isolated for their reactivity against the flagellar-capping protein flid, which is essential for bacteria motility and highly conserved across campylobacter species associated with severe enteritis. in an immunocompetent weaned mouse model, a single oral administration of flid-reactive siga caa1 or ccg4 at 2 h before infection significantly enhances campylobacter clearance at early stages post-infection, reducing the levels of inflammation markers associated with epithelial damage and polymorphonuclear (pmn) cells infiltration in the cecum lamina propria. our data indicate that the prophylactic activity of caa1 and ccg4 is not only dependent on the specificity to flid but also on the use of the siga format, as the immunoglobulin g (igg) versions of the same antibodies did not confer a comparable protective effect. our work emphasizes the potential of flid as a target for the development of vaccines and supports the concept that orally administered flid-reactive siga can be developed to prevent or mitigate the severity of campylobacter infections as well as the development of post-infection syndromes. campylobacter infection is one of the most common causes of bacterial gastroenteritis worldwide and a major global health threat due to the rapid development of antibiotic resistance. currently, there are no vaccines approved to prevent campylobacteriosis, and rehydration is the main form of therapy. secretory immunoglobulin a (siga) is the main antibody class found in mucous secretions, including human milk, and serves as the first line of defense for the gastrointestinal epithelium against enteric pathogens. in this study, we describe the prophylactic activity of orally delivered recombinant siga generated from two human monoclonal antibodies (caa1 and ccg4) isolated for their reactivity against the flagellar-capping protein flid, which is essential for bacteria motility and highly conserved across campylobacter species associated with severe enteritis. in an immunocompetent weaned mouse model, a single oral administration of flid-reactive siga caa1 or ccg4 at 2 h before infection significantly enhances campylobacter clearance at early stages post-infection, reducing the levels of inflammation markers associated with epithelial damage and polymorphonuclear (pmn) cells infiltration in the cecum lamina propria. our data indicate that the prophylactic activity of caa1 and ccg4 is not only dependent on the specificity to flid but also on the use of the siga format, as the immunoglobulin g (igg) versions of the same antibodies did not confer a comparable protective effect. our work emphasizes the potential of flid as a target for the development of vaccines and supports the concept that orally administered flid-reactive siga can be developed to prevent or mitigate the severity of campylobacter infections as well as the development of post-infection syndromes. keywords: secretory iga, monoclonal antibodies, prophylaxis, campylobacter, flagellar-capping protein, flid campylobacter is an established cause of diarrhea worldwide and has recently been added to the who list of bacteria whose antibiotic resistance might pose a global threat to human health (1) . campylobacter's epidemiology differs between high-income countries, where infections are sporadic, and low-and middle-income countries, in which the infection is common in early childhood and a major cause of lifethreatening acute watery diarrhea in infants (2) . considered as a leading zoonosis, campylobacter infection is mainly associated with the consumption of contaminated undercooked animal meat (poultry being the primary bacteria reservoir), water, or unpasteurized milk (3). campylobacter jejuni and campylobacter coli are the major causes of campylobacter enteritis in humans, and despite the clinical relevance of other emerging species (4), they will unlikely be eclipsed in terms of prevalence and magnitude of the disease. campylobacteriosis typically results in an acute, gastrointestinal illness characterized by watery or bloody diarrhea, fever, weight loss, and cramps that last on average 6 days (3, 5). although considered a self-limiting disease, the severe dehydration associated with campylobacter enteritis represents a significant cause of death among newborns and children particularly in developing countries (6) . furthermore, c. jejuni infections has been consistently linked with the onset of autoimmune conditions such as guillain-barré syndrome (gbs) (7, 8) and inflammatory bowel disease (ibd) (9) . despite a yet incomplete understanding of campylobacter pathogenesis, flagellum-mediated motility is widely presumed pivotal for virulence and pathogenicity, as demonstrated in both experimental animal models and in human healthy volunteer studies (10) . nevertheless, flagellin (flaa), the major constituent of the flagellum, is not highly conserved even within the same c. jejuni species, and its heavy glycosylation pattern varies greatly depending on the strain and growth phase (11, 12) . in addition, a recombinant non-glycosylated form of c. jejuni flagellin was shown to be poorly immunogenic in clinical trials (2) . moreover, the possibility to use c. jejuni as vaccines has been limited by the risk of gbs development associated with ganglioside mimicry of bacterial lipo-oligosaccharide (los) (2) . due to these shortcomings, there are currently no vaccines approved to prevent campylobacter infection. rehydration is the main form of therapy, and albeit antibiotics have been shown to be beneficial in severe infections, they are often not recommended due to the rapid development of antibiotic resistance (1) . despite recovering from infection, continuous exposure of infants in low-income countries to intestinal pathogens, including campylobacter, has been linked to environmental enteropathy (ee)/environmental enteric dysfunction (eed), a subclinical chronic inflammation of the small intestine. this inflammation is associated with malabsorption of nutrients, growth faltering, impaired cognitive development, changes in microbiota, and reduced responsiveness to oral vaccination (13) . secretory immunoglobulin a (siga) represents the most abundant class of antibodies produced in humans and serves as the first line of defense at the level of intestinal mucosa against enteric toxins and pathogens (14) . typically, a siga consists of two monomeric iga linked tail to tail by disulfide bonds via a 15-kda protein called joining (j) chain and further complexed with a heavily glycosylated secretory component (sc) (15) . while iga monomers and the j chain are synthesized, assembled, and secreted by plasma cells in the lamina propria, the sc consists of the ectodomain of the polymeric immunoglobulin receptor (pigr) that remains bound to polymeric iga (piga) following transcytosis across the enterocyte (16) . the sc protects the antibody from proteolytic degradation, confers innate recognition functions, and contributes to appropriate tissue localization by anchoring the immunoglobulin to the mucus lining the epithelial surface (17, 18) . siga acts primarily by preventing adhesion of pathogens to epithelial cells through receptor blockade, steric hindrance, and/or immune exclusion, eventually facilitating their clearance from the intestinal lumen via peristaltic and mucociliary activities (14, 19) . furthermore, antigen-bound siga can be transported back into peyer's patches (pps) subepithelial dome (sed) by microfold (m) cells in a process known as reverse transcytosis, which promotes migration and activation of follicular dcs, thus conditioning the inflammatory response associated to infection by enteric pathogens (14, 20) . siga is also the main antibody class found in mucous secretions, including human milk, in which the ig portion of the molecule is produced by mammary gland plasma cells that originate in the small intestine (entero-mammary pathway) (21) . the provision of siga by breastfeeding has been reported to be critical for the protection against enteric pathogens especially in early infancy, when the contribution of endogenous production is limited (22) (23) (24) . in this study, an immunocompetent mouse model of campylobacter infection was used to evaluated the prophylactic activity of orally delivered recombinant siga generated from human monoclonal antibodies isolated and selected for their reactivity against the flagellar-capping protein flid, which is pivotal for bacteria motility (25), and highly conserved across campylobacter species frequently associated with severe neonatal enteritis (26) . we demonstrated that, regardless of the iga isotype, prophylactically administered flid-reactive siga can enhance campylobacter clearance at early stages post-infection, dramatically reducing the levels of inflammation markers associated with epithelial damage and polymorphonuclear (pmn) cells infiltration. our data indicate that the prophylactic activity of these antibodies is not only dependent on the specificity to flid but also on features strictly associated with the siga format as the igg versions of the same antibodies did not produce a comparable beneficial effect. flid amino acid sequences from c. jejuni (n = 38) and c. coli (n = 17) isolates were retrieved from genbank and aligned using mega software to obtain a consensus sequence. condon optimization and synthesis of the corresponding nucleotide sequence was outsourced (genscript), and the construct was subcloned in pet21a in frame with the c-term his tag. the plasmid was transformed to escherichia coli rosetta strain. gene expression was induced by the addition of 1 mm isopropyl-beta-d-thiogalactopyranoside (iptg) to log-phase grown bacteria. after 5 h of induction at 37 • c, bacteria were collected by centrifugation and stored at −20 • c for 16 h before being resuspended in sonication buffer (50 mm nah 2 po 4 , 300 mm nacl, 10 mm imidazole, and ph 7.4) and being sonicated 4 × 30 s in ice. lysed bacteria were spun (14,000 × g, 1 h, and 4 • c), and the supernatants were filtered and then loaded on a nickel column (hitrap chelating hp, ge healthcare). column was washed with washing buffer (50 mm nah 2 po 4 , 300 mm nacl, 10 mm imidazole, and ph 7.4) followed by washing buffer + 0.1% triton-x114 and washing buffer again. bound protein was eluted from the column with elution buffer (50 mm nah 2 po 4 , 300 mm nacl, 500 mm imidazole, and ph 7.4) and dialyzed for 16 h at 4 • c in dulbecco's phosphate-buffered saline (dpbs). protein size was measure on size exclusion column (hiload 16/600 superdex 75 pg; ge healthcare), and the concentration was measured before storage at −20 • c. flid-reactive monoclonal antibodies caa1 and ccg4 were isolated, as previously described (27, 28) , from iga + and igg + memory b cells derived from tonsillar donors who had given written informed consent, following approval by the cantonal ethical committee of cantone ticino, switzerland (ce3536 2019-02061). the sequences for the variable regions of the mabs were obtained from memory b cell clone messenger rnas (mrnas) following reverse transcription pcr (rt-pcr) and sequencing. the vh region of each mab was cloned into a plasmid encoding iga1, iga2 allotype m(2) (29), or igg1 heavy chains. the constructs were transiently transfected in expi293 cells together with those for the corresponding light chains and, in the case of siga, also with those encoding for the joining chain (j) and the ectodomain of pigr (sc) (29) . after 48 h, cells were supplemented with supplement medium and further incubated for 5 days under shaking. next, cells were spun down, and the supernatant was filtered before being purified using captureselect tm iga affinity matrix (thermo fisher). siga assembly was appraised by ultrahigh performance liquid (uhpl) chromatography using acquity beh sec guard column (waters) and by denaturing nonreducing gel using siga purified from human milk (mp biomedicals) as positive control. the purified immunoglobulins were quantified with pierce tm rapid gold bca protein assay kit (life technologies) and then aliquoted and store at −80 • c. n-glycan oligosaccharide analysis was performed as previously described (30) . briefly, a glycoworks rapifluor-ms n-glycan kit (waters corporation, milford, ma) was used to identify and quantify n-linked glycans following the manufacturer's instructions. fluor-ms n-glycan analysis was performed using an agilent 1,260 infinity ii hplc system equipped with a 1,260 fld detector (agilent, santa clara, ca) and an agilent 6,230 electrospray ionization time-of-flight mass spectrometer (agilent, santa clara, ca). a hilic advancebio glycan mapping column (120 å, 2.1 × 150 mm, 2.7 µm), operated at 45 • c, was used to separate various n-glycans. fifty microliters of prepared samples was injected into liquid chromatographymass spectrometry (lc-ms) system, with a flowrate of 0.6 mg/ml and a gradient run time of 55 min. fluorescence was obtained using excitation and emission wavelengths of 265 and 425 nm, respectively. ms was acquired simultaneously from 400 to 2,000 m/z at a constant scan rate of one spectrum per second. n-glycans were assigned based on m/z values using the n-glycan glycobase database, and nglycan quantification was calculated on integration of the fluorescence chromatogram. total carbohydrate analysis was performed as previously described (30) . briefly, a glycoprotein carbohydrate estimation kit (thermo-fisher #23260) was used to determine the total carbohydrate content (both n-and o-linked oligosaccharides) in mab samples as a percentage of total protein mass. lysozyme, bovine serum albumin (bsa), ovalbumin, apo-transferrin, fetuin, and α1-acid glycoprotein were used as glycoprotein standards to construct a standard curve. size exclusion chromatography (sec) was performed as previously described (30) . briefly, a shimadzu prominence ultrafast liquid chromatography hplc system equipped with a diode array detector (with absorbance detection at 214 nm) was equilibrated at 0.5 ml/min flowrate in 0.2 m sodium phosphate buffer, ph 6.8 for at least 2 h. ten microliters of each mab (10 µg total protein) was injected and separated by a tosoh tskgel g4000swxl column (8 µm particle size, 7.8 mm id × 30 cm) with the corresponding guard column operated at ambient temperature (tosoh biosciences) using a 30-min run time. gel filtration molecular weight standards (bio-rad, hercules, ca) were injected before and after the mab samples to ensure integrity of the column and hplc system. potential presence of larger aggregates was determined by running mab samples with and without the sec column (i.e., protein percentage recovery). data were analyzed using lc-solution software (shimadzu, kyoto, japan). sedimentation velocity analytical ultracentrifugation (sv-auc) was performed as described previously (30) using a proteome lab xl-i (beckman coulter) analytical ultracentrifuge equipped with a scanning ultraviolet-visible optical system. all experiments were performed at 20 • c after at least 1 h of equilibration after the rotor reached 20 • c. sv-auc was performed at a rotor speed of 40,000 rpm and with detection at 280 nm. the data were analyzed using sedfit software (dr. peter schuck, nih). intrinsic tryptophan fluorescence spectroscopy measurements as a function of temperature and polyethylene glycol (peg) relative solubility measurements were both performed as described previously (30) . for fluorescence spectroscopy measurements, 20 µl of each mab sample was diluted to 0.2 mg/ml in pbs, ph 7.2 and loaded into a 384-well plate (hard-shell 384-well pcr plates) and overlaid with 2 µl of silicon oil (thermo fisher scientific, waltham, ma). samples were excited at 295 nm, and the emission spectra were recorded from 300 to 450 nm with an integration time of 100 ms using a fluorescence plate reader equipped with a charge-coupled device (ccd) detector (fluorescence innovations, minneapolis, mn). temperature ramps were programmed from 10 to 100 • c with an increment of 2.5 • c per step. the mean center of spectra mass peak algorithm was used to analyze the data to determine the shift in fluorescence peak position as a function of temperature. with respect to relative solubility measurements, various concentrations of peg 10,000 solutions ranging from 0 to 25% w/v were prepared with a final mab concentration of 0.2 mg/ml. samples were incubated overnight at room temperature in the dark in 384-well plates with 30 µl in each well. the next day, the plates were centrifuged for 15 min at ∼1,200×g, and the supernatant was transferred to a clean 384-well plate. relative protein concentration in each well was then measured at 214 nm using a spectramax m5 plate reader (molecular devices). thermal melting temperature values (t m ) and % peg midpoint calculations were performed using origin v 9.1 software (originlab, northampton, ma). quantification of recombinant siga and igg by elisa was performed by coating 96-well plates with goat anti-human iga or igg (southernbiotech). two-fold serial dilutions of the mabs in duplicates were incubated for 1 h at room temperature. in the case of siga, detection was performed using human pigr biotinylated antibody biotinylated anti-human sc antibody (r&d system) followed by incubation with streptavidin-ap, while goat anti-human igg-ap (southernbiotech) was used for igg. siga purified from human milk (mp biomedicals) and rituximab (mabthera) were used as quantification standards for siga and igg, respectively. binding of caa1, ccg4 siga, and igg counterparts to the recombinant antigen was measured by elisa using flidcoated 96-well-plates following the same detection protocol described above. epitope binning was tested by biolayer interferometry (bli) using octet red96 (fortebio) immobilizing recombinant flid antigen (5 µg/ml) onto aps biosensor (pall fortebio) for 7 min. after incubation with blocking buffer (bb), the sensor was incubated with caa1 siga2 (7.5 µg/ml) for 7 min before being further incubated with ccg4 siga2, caa1 siga2, or bb for additional 7 min. binding of siga2 to flid antigen in denaturing and reducing conditions was tested by western blot. briefly, 5 µg of recombinant flid was loaded on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) nupage 12% bis-tris protein gel (thermo fisher scientific) in the presence of a reducing agent. blotting was performed using iblot system (invitrogen), and the membrane was blocked with 5% milk (biorad) in tris-buffered saline with tween 20 (tbst) for 1 h at room temperature (rt). the mabs were biotinylated by incubation with 20 mm biotin using the ez-link nhs-peo solid phase biotinylation kit followed by desalting with zeba spin desalting columns (thermo scientific). the membrane was incubated with a 1% blocking grade blocker (biorad) in tbst containing 20 µg/ml of biotinylated mabs followed by washes in tbst and by incubation with streptavidin-horseradish peroxidase (hrp). detection was performed using ecl detection kit (ge healthcare) on fusion fx7 (witec ag). sensitivity to igase was evaluate by incubating caa1 siga1 and siga2 with recombinant iga protease from neisseria gonorrhoeae (igase pro-pro-y-pro-mo bi tec). briefly, 100 µg of caa1 siga1 and siga2 were incubated at 37 • c for 18 h with 1.5 µg of igase or with reaction buffer alone. the post-incubation products were visualized in a denaturing non-reducing gel with himark prestained protein standard (thermo fisher). c. jejuni 81-176 and c. coli nctc 11437 strains were purchased from the american type culture collection (atcc) and public health england bacteria collection, respectively. the campylobacter species were grown from glycerol stocks in mueller-hinton agar (sigma aldrich) (15 g/l) supplemented with vancomycin and trimethoprim (10 µg/ml) (sigma-aldrich) for 96 h at 40 • c in microaerophilic jar with oxoid tm campygen tm 2.5 l sachet (fisher scientific). in vitro binding of the different flid-reactive siga to a pure culture of c. jejuni was measured by flow cytometry. fifty microliters of each mabs (20 µg/ml) was incubated with 50 µl of c. jejuni 81-176 (10 7 cfu/ml) for 1 h at 4 • c. after washing with pbs 2% fetal bovine serum (fbs), bacteria were incubated for 30 min with biotin-conjugated anti-human iga (southern biotech, cat. no. 2050-08). immunoglobulin-bound bacteria were then stained with streptavidin-pb at 4 • c for 30 min. samples were washed with pbs 2% fbs, resuspended in 2% paraformaldehyde and acquired on lsr fortessa flow cytometer (bd biosciences, franklin lakes nj, usa) using forward scatter (fcs) and side scatter (ssc) parameters in logarithmic mode. data were analyzed using the flowjo software (treestar, ashland, or, usa) or facs diva software (bd biosciences, franklin lakes nj, usa). binding of caa1 and ccg4 siga on the poles of c. jejuni was also confirmed by microscopy. briefly, 30 µg of caa1, ccg4, and hgn194 siga2 were incubated with 10 6 cfu of c. jejuni 81-176 for 1 h at 37 • c in microaerophilic conditions. next, bacteria-mabs complexes were washed and further incubated with antihuman iga-af647 conjugated (1:200, jackson immunoresearch, cat. no. 109-606-011) and sytobc (1:1,000, thermo fisher scientific, cat. no. s34855) for additional 30 min before being poured in microscope slides. images were acquired with a leica sp5 laser scanning confocal microscope with a 488-and 633-nm excitation. the resulting fluorescence emission was collected using 490-627-nm (for sytox green) and 636-770 nm (for alexa 647) windows. samples were imaged with a 63× objective (n.a. 1.4) in xy optical sections of 61.51 µm (1,024 × 1,024 pixels) with a pixel size of 60.1 nm. to improve lateral resolution, the pinhole was set to 0.5 au. animal experiments were performed in accordance with the swiss federal veterinary office guidelines and authorized by the cantonal veterinary office (ti-09-2019). twentyone-day-old c57bl/6 female mice were purchased from charles river laboratories. mice were housed, five per cage, under standardized conditions (20 ± 2 • c, 55 ± 8% relative humidity, 12 h light/dark cycle), at the institute for research in biomedicine. food and water were available ad-libitum, and mice were monitored daily. in order to measure c. jejuni specific antibody binding to the mouse microbiota, stools were collected and homogenized in sterile pbs (0.01 g of stools in 100 µl pbs). samples were centrifuged for 5 min at 400 g to pellet large debris, and then, the supernatant were centrifuged at 8,000 g for 5 min to pellet bacteria. the supernatant was removed, and the bacterial pellet was resuspended in 150 µl (50 µg/ml) of the different anti-campylo mabs and incubated at 4 • c for 30 min. after washing, bacteria were incubated at 4 • c for 30 min with biotin-conjugated anti-human iga (southern biotech, cat. no. 2050-08) and syto bc (1:1,000, thermo fisher scientific, cat. no. s34855). bacteria were washed once and then incubated with streptavidin-pb and, at the end, resuspended in 2% paraformaldehyde in pbs for flowcytometry analysis. all the samples were acquired on an lsr fortessa flow cytometer (bd biosciences, franklin lakes nj, usa) using fcs and ssc parameters in logarithmic mode. data were analyzed using the flowjo software (treestar, ashland, or, usa) or facs diva software (bd biosciences, franklin lakes nj, usa). differences in localization and persistence of siga vs. igg in the gastrointestinal (gi) tract of 21-day-old c57bl/6 mice were evaluated by testing the pk of the two species in the three gi subcompartments: small intestine, cecum, and colon. weaned mice were administered with a pbs solution containing 150 µg of campylobacter-irrelevant antibody hgn194, which targets hiv gp120 and presents no cross-reactivity with the murine microbiota, in the form of siga2m(2) or igg. a control group was administered only with pbs. at 2, 4, and 8 h postoral administration, five animals per group were euthanized, and the content of the three different intestinal tracts was collected and resuspended in 1.5 ml pbs. the samples were then homogenized, and after centrifugation, the supernatants were collected and stored at −20 • c. the detection of human iga or igg in the different parts of the gi tract was performed by elisa. briefly, 96-well plates were coated with goat antihuman iga or igg antibodies (southern biotechnology: 2050-01 and 2040-01) and blocked with 1% bsa blocking buffer. plates were then incubated with 2-fold serial dilutions (1:2 to 1:256) of each intestinal sample for 1 h at room temperature. after four washes with pbs + 0.05% tween 20, the presence of the antibodies was detected by incubation with goat anti-human lambda antibody ap conjugated (southern biotechnology: 2070-04) for 1 h at room temperature followed by 15 min incubation with p-nitrophenylphosphate (pnpp) and detection at 405 nm. taking into account the different numbers of lambda light chains possessed by the polymeric immunoglobulin, to quantify the amount of siga or igg in the samples, two distinct standard curves were built using hgn194 siga and hgn194 igg as standards, respectively. no signals were detected testing the samples from the control animals administered only with pbs. the inflammation status of mice was evaluated by measuring the levels of lipocalin-2 (lcn-2) in fecal supernatants via elisa assay (r&d systems, duoset elisa mouse lipocalin-2/ngal) 24, 48, and 72 h post-infection. briefly, 0.01 g feces was resuspended in 100 µl in pbs, centrifuged for 10 min at maximum speed, and diluted before performing the elisa assay, according to manufacturer's instructions. ceca from all animals were examined at necropsy, fixed in 10% neutral buffered formalin for at least 48 h prior to embedding in paraffin, and stained with hematoxylin and eosin (h&e). pathological scores were determined in a blinded manner using a previously described scoring scheme (31) . briefly, each tissue section was assessed for (1) submucosal edema (0, no change; 1, mild; 2, moderate; 3, severe); (2) crypt hyperplasia (0, no change; 1, 1-50%; 2, 51-100%; 3, >100%); (3) goblet cell depletion (0, no change; 1, mild depletion; 2, severe depletion; 3, absence of goblet cells); (4) epithelial integrity [0, no pathological changes detectable; 1, epithelial desquamation (few cells sloughed, surface rippled); 2, erosion of epithelial surface (epithelial surface rippled, damaged); 3, epithelial surface severely disrupted/damaged, large amounts of cell sloughing; 4, ulceration [with an additional score of 1 added for each 25% fraction of tissue in the cross-section affected up to a maximum score of 8 (4 + 4) for a tissue section that had entirely lost its crypt structure due to epithelial cell loss and immune cell infiltration]; (5) mucosal mononuclear cell infiltration (per 400× magnification field) (0, no change; 1, <20; 2, 20-50; 3, >50 cells/field); (6) submucosal pmn and mononuclear cell infiltration (per 400× magnification field) (1, <5; 2, 21-60; 3, 61-100; 4, >100 cells/field). a maximum score under this scale is 24. statistical analyses were performed using graphpad prism v7.04 (graphpad software, la jolla, ca, usa). mann-whitney u test was used to determine the statistical significance of the results. a p < 0.05 was considered significant in all cases. different campylobacter's proteins were evaluated as potential targets to prevent infection on the basis of (i) the presence of surface exposed epitopes, (ii) role in the bacteria pathogenesis, (iii) ability to stimulate an immune response in the context of natural infection, and (iv) level of amino acid sequence conservation within but not limited to the c. jejuni species. the flagellar-capping protein flid, also known as hookassociated protein 2 (hap2), is a 70-kda protein with high sequence conservation across the c. jejuni species (26) (supplemental figures 1a,b ). based on structures and selfoligomerization mechanisms characterized for other flagellated pathogens (25, 32), campylobacter flid oligomers form the cap protein complex located at the tip of the flagellum, which controls the distal growth of the filament by regulating the assembly of the flagellin molecules. due to its functional role in filament elongation, flid-deficient mutants exhibit defects in bacterial motility (25). the potential of flid as a promising target is strengthened by the proposed involvement in cell adherence (33) and the immunogenicity in chickens during natural infection. accordingly, it has been suggested as a vaccine candidate for the prevention c. jejuni in broilers (26, 34) . flid was therefore selected as a candidate target antigen for monoclonal antibody (mab) development. the frequencies of igg + and iga + flid-reactive memory b cells in 50 tonsillar samples of swiss origin were evaluated using the antigen-specific memory b cell repertoire analysis (ambra) (35) (supplemental figure 2) . igg + and iga + memory b cells from selected tonsils were then immortalized under clonal conditions (28) , and culture supernatants were screened using a 384-well plate-based high-throughput platform to identify b-cell clones expressing flid-reactive antibodies. using this approach, memory b cell clones producing two human monoclonal antibodies, namely caa1 and ccg4, were isolated and selected based on their specificity and affinity for recombinant flid. caa1 was originally isolated as an iga1 encoded by v h 3-48/d2-15/j h 3 with a 21-amino acid hcdr3 and v k 1-39/j k 5, whereas ccg4 was isolated as an igg3 encoded by v h 3-9/d1-7/j h 1 bearing a shorter 11-amino-acid hcdr3 and v l 3-27/j l 3. humans present two different iga subclasses that differ mainly for the length and glycosylation of the hinge region. iga1 possesses a 13-amino-acid longer hinge than iga2 containing up to five o-linked glycans at serine and threonine residues. the longer hinge not only confers to iga1 antibodies higher flexibility and a longer fab reach but is also linked to the sensitivity to a specific class of bacterial proteases known as iga1 proteases. bearing a shorter hinge region lacking proline-serine and/or proline-threonine peptide bonds, iga2 is instead resistant to these proteases (36) . in addition, when compared to igg, igm, and iga1, the iga2 isotype has the unique characteristic to undergo reverse transcytosis by contacting dectin-i receptor on the surface of pps m cells (20) . both the cα1 region and the glycosylation pattern appear pivotal for the interaction at the basis of this mechanism, which has been suggested as a strategy to boost adaptive immunity against pathogens (37) . the recombinant siga1, siga2, and diga2 versions of the flid-reactive mabs were generated by transient transfection of expi293 cells and were then purified by affinity chromatography. various physicochemical measurements were performed to monitor key structural attributes of recombinant flid-reactive polymeric igas as summarized in supplemental figure 3a . the conformational stability of the mabs was determined by monitoring their overall tertiary structure as a function of temperature (intrinsic tryptophan fluorescence spectroscopy), and a 6-7 • c lower thermal melting temperature (tm) value was observed for siga1 when compared to siga2 and diga2 (supplemental figure 3a) . the difference in conformational stability could be due to structural differences in the hinge region between the two subtypes. the relative solubility of the mabs was evaluated by a peg precipitation assay, and diga2 was found to be relatively more soluble compared to siga1 and siga2, a result of which suggests that the presence of the secretory component protein may affect mab solubility at neutral ph. molecular size analysis by sedimentation velocity analytical ultracentrifugation (sv-auc) revealed a heterogeneous distribution of species for each mab (supplemental figure 3a) . we observed that siga2 had a considerably higher relative percent composition of larger molecular weight species than the other two mabs (as compared to the percent main peak). as expected, diga2 had a later elution time when compared to siga1 and siga2 in size exclusion chromatography (supplemental figure 3b) . each of the caa1 iga molecules were heavily glycosylated with carbohydrates ranging from ∼31-39% of the total mass of each mab. as expected by sharing the same iga backbone, the nlinked oligosaccharide structural types were overall very similar in siga2 and diga2 antibodies (supplemental figure 3c ). differently to these antibodies, caa1 siga1 lacked g0f + gn, g1f -gn, gif + gn and g1f + nana, while it contained g2f2 and man4g12 + nana (supplemental figure 3c) . finally, incubation of caa1 siga1 and siga2 with recombinant igaase from n. gonorrhoeae confirmed the differences in sensitivity to bacterial iga proteases of the two isotypes, which have been associated to the length and sequence of the hinge regions (supplemental figure 3d) . based on its resistance to bacterial iga proteases and the ability to undergo reverse transcytosis, the iga2 scaffold was initially selected over the iga1 for our studies, and both caa1 and ccg4 siga2 were further characterized in vitro. to include a control antibody that was not reactive with the antigen in our experiments of campylobacter infection, we also generated the siga2 version of the hgn194 mab, which targets an epitope on the v3 loop of hiv-1 env glycoprotein (38) . caa1 and ccg4 siga2 molecules maintained the original flid binding activity, displaying similar ec 50 (caa1 = 0.08; ccg4 = 0.12 µg/ml) for the flagellar protein ( figure 1a) . epitope binning studies by bli indicated that the binding of one antibody to the antigen did not prevent the binding of the other mab (figure 1b) , pointing to the recognition of two distinct epitopes. in addition, caa1, but not ccg4, recognized flid by western blot analysis under denaturing and reducing conditions, confirming the binding to structurally different antigenic determinants (figure 1c) . accessibility of the two mabs to these epitopes in the flagellum was assessed by flow cytometry using two flid-divergent historical isolates c. jejuni 81-176 and c. coli nctc 11437. caa1 and ccg4 stained both pathogens, thus confirming epitope accessibility and breadth ( figure 1d) . consistent with flid localization, confocal imaging on c. jejuni 81-176 confirmed that the binding of the two mabs occurred at the cell poles ( figure 1e ). the murine intestine is highly resistant to campylobacter due to intense competition exerted by the rich gut microbiota and to a certain level of tolerance, which limits inflammation (39) (40) (41) . to overcome the challenge represented by the resident microbiota, pre-treatment via oral gavage with vancomycin, for which campylobacter species are inherently resistant (42) , is largely adopted. although the pretreatment allows robust bacterial colonization in the cecum, it does not appear to enhance the pathology in adult wild-type mice since minimal signs of inflammation were previously observed (31) . higher susceptibility to c. jejuni infection of infant wild-type mice in comparison to adult animals has been previously reported and linked to significant differences in the microbiota composition (43) . to set up a model that would allow us to evaluate the prophylactic activity of our mabs under immunocompetent conditions, we evaluated the sensitivity to c. jejuni 81-176 infection of pups (12 days old), weaned (21 days old), and adult (56 days old) c57bl/6 mice. animals were pre-treated with vancomycin via oral gavage to deplete the murine microbiota before infection with c. jejuni 81-176 at 10 8 cfu/mouse. campylobacter isolation from stools at 6 days post-infection revealed a ∼2 log higher increase in shedding from 21-day-old animals than from 12-and 56-day-old mice (figure 2a) . in line with these results, weaned mice displayed significantly higher intestinal inflammation as measured by the levels of lipocalin-2 in the stools and histological score values in the cecum in comparison to pups and adult mice (figures 2b,c) . since antibiotic pretreatment should provide comparable ecological niches for infection in the different animals, other factors could be accountable for the different sensitivity to c. jejuni infection displayed by the three groups of mice. analysis of murine iga in the stools of 12-, 21-, and 56-day-old animals revealed different concentrations among the groups (figure 2d ). a phylogenetic tree built using neighbor-joining method with jukes-cantor distance measurement is shown to provide the amino acid distance of flid between the two historical isolates tested (in red). one representative experiment out of three is represented. (e) binding of caa1 siga to c. jejuni as observed in confocal microscopy. bacteria were stained using syto bc (green), whereas caa1 was detected using anti-human iga af647 conjugated (red). interestingly, weaned mice presented negligible levels of iga in the stools in comparison to both pups and adult mice. this observation is consistent with the transition between the exogenous supply of maternal antibodies provided along with the milk (12-day-old mice) and the beginning of the endogenous production that is established in adult animals (i.e., 56-day-old mice). therefore, a likely important factor in the susceptibility of weaned mice to c. jejuni infection could be the lower concentration of secretory iga due to the relative immaturity of intestinal immune system and the depletion of maternal antibodies in these animals. based on these observations, weaned mice were selected as an immune competent animal model to study the prophylactic activity of flid-reactive mabs. since off-target binding of caa1 and ccg4 to the murine microbiota would result in reduced bioavailability and thus activity against pathogens in a prophylactic setting, we evaluated potential cross-reactivity of the recombinant siga with the microbiota of weaned mice. stools from animals orally infected with c. jejuni, c. coli, or pbs (mock infected) were collected 24 h post-infection and incubated with the two flid-reactive mabs and the control hgn194. ccg4 displayed limited levels of cross-reactivity with the murine microbiota (6.62%), which were comparable with those observed for the control antibody (1.11%) (supplemental figure 4a) . conversely, caa1 was binding the microbiota to a higher extent (22%), suggesting a possible fabmediated recognition of antigens in the commensal bacteria that present structures similar to flid (supplemental figure 4a) . nevertheless, for both flid-reactive mabs, the percentage of igacoated bacteria dramatically increased in the stools of infected animals confirming the prompt recognition of the c. jejuni and c. coli species (caa1, 79.2 and 73.9%; ccg4, 70.7 and 55.4%). interestingly, we also observed an increase in hgn194 binding to the bacteria in the stools of these animals (26.5 and 26.4%) that could be consistent with the innate binding activity of iga and secretory component against various pathogens (44, 45) . finally, to examine the conditions for testing the prophylactic efficacy of the mabs, we evaluated the pharmacokinetics of orally administered siga in different gastrointestinal tracts of weaned mice. the campylobacter-irrelevant hgn194 siga2, which displayed negligible cross-reactivity with the murine microbiota (supplemental figure 4a) , was administered as a single oral gavage of 150 µg in pbs (≈15 mg/kg), and its concentration in the different intestinal subcompartments was measured at 2, 4, and 8 h post-administration (supplemental figure 4b) . hgn194 siga concentration in the cecum was maintained almost constant within the first 4 h post-administration and then tended to rapidly decrease by 8 h. the human antibody was not detectable at 12 or 24 h post-administration (data not shown). we next evaluated the prophylactic activity of orally administered flid-reactive recombinant siga2 in immunocompetent-weaned mice infected with c. jejuni 81-176. as such, vancomycin pretreated animals were administered with a single oral gavage of 200 µg/mouse of caa1, ccg4, hgn194 siga2, or pbs 2 h before oral infection with 10 8 cfu/mouse of c. jejuni 81-176. treated animals and the corresponding control groups were monitored for 72 h, during which the severity of infection and degree of inflammation were recorded. of note, analysis of the stools from treated mice revealed a trend characterized by higher campylobacter shedding at 24 h post-infection followed by a significant decrease over time. conversely, untreated and hgn194-treated groups presented lower shedding at early time points followed by a consistent cfu increase at 48 h post-infection ( figure 3a) . these results suggest that that caa1 and ccg4 could prevent or reduce the ability of the pathogen to adhere to the surface of the mucosal epithelium, thus facilitating the clearance of bacteria via peristalsis or mucociliary activity at early stages postinfection. this hypothesis is further supported by the significant lower levels of lipocalin-2, a marker of intestinal inflammation linked to epithelial damage and neutrophil infiltration, recorded at 72 h post-infection in the stools of caa1-and ccg4-treated animals in comparison to the control groups (infected/nontreated and infected/hgn194-treated groups) ( figure 3b ). in addition, animals treated with a single administration of flidreactive mabs presented levels of pmn cells infiltration and histological score values in the cecum significantly lower than the hgn194-treated group (figures 3c,d) , which indicates that the activity exerted by caa1 and ccg4 is largely fab mediated. similar findings were observed in animals administered 2 h before infection with higher or lower campylobacter infection doses (10 7 or 10 9 cfu/mouse). indeed, in both cases caa1-administered animals presented significantly higher bacterial clearance than the untreated group (supplemental figures 5a,b) and levels of intestinal inflammation similar to those of noninfected mice (supplemental figures 5c,d) . furthermore, no significant differences were observed in the reduction in bacterial shedding, pmn infiltration in the cecum lamina propria, and lipocalin-2 levels in the stools when caa1 siga2 was provided 2 h before infection or when it was premixed with c. jejuni 81-176 before oral administration to the animals (supplemental figures 5e-g) . taken together, these results indicate that prophylactic administration of flid-specific caa1 and ccg4 siga2 increase protection from campylobacter infection by accelerating bacterial clearance at early stages after infection, thus significantly reducing inflammation. since longer fab reach, higher flexibility, and different glycosylation may affect the cross-linking ability and/or persistence of the polymeric ig in the intestine, we next investigated whether the two iga isotypes may exert different prophylactic activities in our mouse model of campylobacter infection. caa1 siga1 and siga2 displayed comparable binding to flid (supplemental figure 6a) , and no significant differences in reactivity to campylobacter species in vitro or with murine microbiota ex vivo were observed between the two formats (supplemental figures 6b,c) . the prophylactic activity of the two isotypes was then tested in the murine model of campylobacter infection. in line with our previous findings, animals administered with the flid-reactive mabs displayed higher campylobacter shedding at early time postinfection followed by decrease over time, while infected non-treated animals produced an opposite trend ( figure 4a) . although caa1 siga2 accelerated shedding faster than siga1 as confirmed also by the percentage of human iga-coated bacteria in the stools (supplemental figure 6d) , both subclasses were equally capable to limit inflammation in infected animals as shown by the levels of lipocalin-2 in the stools, pmn infiltration, and the corresponding histological score in the cecum at 72 h postinfection (figures 4b-d) . overall, our results indicate that differences in flexibility and glycosylation associated to the two human iga subclasses do not have an impact in the prophylactic activity exerted by caa1 in the in vivo model. although siga are the most abundant antibodies expressed in association with the intestinal mucosa and the first line of defense against enteric pathogens, they are characterized by a complex structure, and their development as drugs might result challenging in comparison to igg-based monoclonal antibodies. because the activity of the campylobacter-reactive mabs was previously shown to be dependent on the specificity for flid, we generated caa1, ccg4, and the campylobacterirrelevant antibody hgn194 as human igg1 and evaluated their prophylactic activity in comparison to their corresponding siga2 counterparts. since glycosylation might affect the ability of mabs to interact with mucin on the mucosal surface, the localization and persistence of hgn194 igg1 in the murine intestinal tract were appraised by administering the antibody with a single oral gavage to weaned mice and then by measuring the its concentration in the different intestinal subcompartments after 2, 4, and 8 h (supplemental figure 4c) . as in the case of hgn194 siga2, at every time point, the highest concentration of the igg1 was detected in the cecum; however, in this intestinal subcompartment, the igg1 concentration tended to decrease faster than for siga2, with a significant reduction already at 4 h post-administration (supplemental figures 4b,c) . next, we evaluated the prophylactic activity of the flidreactive mabs caa1 and ccg4 as igg1 or siga2 both administered to weaned mice 2 h before infection with c. jejuni 81-176. interestingly, while animals treated with siga2 antibodies displayed the previously observed pattern characterized by higher shedding at 24 h postinfection followed by a significant decrease at 48 and 72 h, the groups treated with the igg version of the same antibodies revealed trends similar to the non-treated groups ( figure 5a and supplemental figure 7a ). the importance of the siga format for the in vivo efficacy was further confirmed by the lower ability of caa1 and ccg4 igg to limit inflammation in comparison to their polymeric counterparts, as shown by pmn cells infiltration in the cecum and lipocalin-2 levels in the stools of the infected animals at 72 h post-infection (figures 5b,c and supplemental figures 7b,c) . overall, no significant differences in the histological scores were observed between the mice treated with the flid-reactive igg antibodies and the non-treated animals ( figure 5d and supplemental figure 7d) . conversely, the siga2 versions of the antibodies were able to replicate the beneficial effect previously observed, maintaining the histological score in the cecum to values significantly lower than both non-treated and igg-treated mice (figure 5d and supplemental figure 7d) . taken together, these data indicate that the caa1 and ccg4 igg have limited prophylactic activity when orally administered prior to campylobacter infection. the reduced activity of caa1 and ccg4 igg might be dependent on both the lower stability and persistence in the gastrointestinal tract and/or on the decreased "bonus effect of multivalency" at the basis of the cross-linking activity associated with the polymeric format. the aim of this study was to evaluate the ability of orally administered recombinant siga, derived from selected flidreactive monoclonal antibodies, to prevent infections by campylobacter species frequently associated with severe neonatal gastroenteritis. despite its pivotal role in bacteria motility, epithelial cells adherence (25, 33) , and the high level of conservation among c. jejuni (supplemental figure 1) , flid has never been assessed to date as a potential target for therapeutic mabs. consistent with the immunogenicity previously reported for this antigen (26, 34) , our results indicate that flid-reactive mabs can be isolated from the human memory b cell repertoire. further development of these antibodies, namely, caa1 and ccg4, resulted in recombinant siga that preserved the ability to bind flid on the campylobacter's flagellum of both c. jejuni and c. coli species (figure 1d) . genetically manipulated animals characterized by an exacerbated inflammatory response to bacteria, such as sigirr or il10 − / − , have been proposed as models to study campylobacter pathogenesis (31, 46) . these mutations, however, dramatically alter the murine immune system to an extent that even the presence of commensal microbes can potentially result in spontaneous enterocolitis (47) . the sensitivity of wild-type mice to campylobacter infection has consistently been linked to age-dependent variations in the composition of the resident microbiota (43) . of note, our findings indicate that different levels of iga in the murine intestine also play a role in campylobacter pathogenesis in mice. in particular, we observed that weaned mice represent the most permissive wild-type age group and that this is likely due to the transition between the exogenous iga supply provided by the maternal milk and iga endogenous production ( figure 2d) . moreover, giallourou et al. described a weaned mouse model of c. jejuni that can recapitulate enteropathy and diarrhea (48) . in the current study, we established a weaned mouse model of campylobacter infection to evaluate the prophylactic activity of the mabs based on bacterial shedding and inflammatory biomarkers (figures 2a-c) . in this animal model, we documented a consistent effect following a single prophylactic administration of flid-reactive siga 2 h before c. jejuni infection, which is characterized by significantly higher campylobacter shedding at the early stage of infection followed by a rapid decline at later time points (figure 3a) . our hypothesis is that by binding flid, the siga can limit bacteria motility and ability to adhere to the surface of the mucosal epithelium, thus accelerating their clearance via peristalsis. this idea is further strengthened by the fact that animals administered with flid-reactive mabs display considerably lower levels of inflammatory biomarkers in the cecum and stools than the control groups, which present an opposite shedding trend. flid-reactive mabs efficacy relies on the specific recognition of the bacterial surface antigen, as an irrelevant antibody in the same format does not produce similar beneficial effects (figures 3a-d) . further studies will elucidate whether these antibodies may act primarily by limiting bacteria motility (cross-linkage), by limiting attachment to cell receptors (steric hindrance), by limiting secretion via the t3ss of proteins that contribute to host cell invasion (e.g., ciab) or via a combination of all these activities. interestingly, similar prophylactic activity was observed when caa1 was premixed with c. jejuni or administered 2 h before infection, thus confirming siga stability during the passage through the small intestine and their persistence in the cecum (supplemental figures 5e-g) . despite the fact that siga2 showed a slightly higher ability to accelerate bacterial shedding at early stage of infection in comparison to the siga1 counterpart (figure 4a) , differences in length and glycosylation of the hinge region between the two human iga isotypes do not appear to significantly undermine the ability of the polymeric immunoglobulins to prevent inflammation (figures 4b-d) . however, iga2 might represent the most promising backbone for development of orally deliverable mabs due to resistance to iga proteases expressed by different pathogens (supplemental figure 3d ) and to the unique ability to undergo reverse transcytosis (20) , which in turn could contribute to boost a potential vaccinal effect (37) . the prophylactic activity of flid-reactive caa1 and ccg4 was significantly decreased when these mabs were administered as igg1. indeed, in the igg1 format, the mabs were neither able to boost campylobacter shedding at early time postinfection nor able to limit inflammation to an extent comparable with their corresponding siga (figure 5 and supplemental figure 7) . the superior efficacy of caa1 and ccg4 as siga might be related to higher resistance to proteases in the small intestine (30, 49) , higher localization and better persistence in the cecum (17, 18) , and/or to the mechanisms of action (i.e., cross-linking) associated with the polymeric immunoglobulin structure (14, 19) . in summary, our results indicate that the flagellar-capping protein flid represents a promising target for the development of campylobacter-reactive mabs capable to exert a prophylactic activity dependent on both their affinity to the antigen and on features strictly related to the siga format. although improvement in water sanitation and healthcare conditions are pivotal to reduce campylobacter's impact in infants in middle-and low-income countries, these findings suggest that the development of orally deliverable recombinant siga targeting flid might represent a novel strategy to prevent or mitigate both disease severity and the development of postinfection syndromes. the datasets generated for this study are available on request to the corresponding author. the animal study was reviewed and approved by swiss federal veterinary office guidelines and the cantonal veterinary office (ti-09-2019). who publishes list of bacteria for which new antibiotics are urgently needed status of vaccine research and development for campylobacter jejuni the 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roles in immunity and mucosal homeostasis in the gut the structure and dynamics of secretory component and its interactions with polymeric immunoglobulins receptor-mediated transcellular transport of immunoglobulin: synthesis of secretory component as multiple and larger transmembrane forms the polymeric immunoglobulin receptor: bridging innate and adaptive immune responses at mucosal surfaces high-avidity iga protects the intestine by enchaining growing bacteria dectin-1 is essential for reverse transcytosis of glycosylated sigaantigen complexes by intestinal m cells breastfeeding: maintaining an irreplaceable immunological resource protection of breast-fed infants against campylobacter diarrhea by antibodies in human milk protection against campylobacter diarrhea: role of milk iga antibodies against bacterial surface antigens self-oligomerizing structure of the flagellar cap protein flid and its implication in filament assembly evaluation of flagellum-related proteins flid and fspa as subunit vaccines against campylobacter jejuni colonisation in chickens rapid development of broadly influenza neutralizing antibodies through redundant mutations an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus mucosal immune defense: immunoglobulin a preformulation characterization and stability assessments of secretory iga monoclonal antibodies as potential candidates for passive immunization by oral administration a novel mouse model of campylobacter jejuni gastroenteritis reveals key pro-inflammatory and tissue protective roles for toll-like receptor signaling during infection bacterial flagellar capping proteins adopt diverse oligomeric states host cell binding of the flagellar tip protein of campylobacter jejuni characterization and antigenicity of recombinant campylobacter jejuni flagellar capping protein flid clonal dissection of the human memory b-cell repertoire following infection and vaccination the iga1 proteases of pathogenic bacteria secretory iga as a vaccine carrier for delivery of hiv antigen to m cells analysis of memory b cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from hiv-1-infected individuals novel murine infection models provide deep insights into the "menage a trois" of campylobacter jejuni, microbiota and host innate immunity modification of intestinal microbiota and its consequences for innate immune response in the pathogenesis of campylobacteriosis campylobacter jejuni colonization of mice with limited enteric flora mechanisms of antibiotic resistance in campylobacter species campylobacter jejuni infection of infant mice: acute enterocolitis is followed by asymptomatic intestinal and extra-intestinal immune responses glycosylation of human iga directly inhibits influenza a and other sialic-acidbinding viruses binding of lactoferrin and free secretory component to enterotoxigenic escherichia coli the role of serine protease htra in acute ulcerative enterocolitis and extra-intestinal immune responses during campylobacter jejuni infection of gnotobiotic il-10 deficient mice c57bl/6 and congenic interleukin-10-deficient mice can serve as models of campylobacter jejuni colonization and enteritis a novel mouse model of campylobacter jejuni enteropathy and diarrhea increased risistance of immunoglobulin a dimers to proteolytic degradation after binding of secretory component genomic characterization of the guillain-barre syndrome-associated campylobacter jejuni icdccj07001 isolate campylobacter jejuni strain cg8421: a refined model for the study of campylobacteriosis and evaluation of campylobacter vaccines in human subjects campylobacter jejuni lipopolysaccharides in guillain-barre syndrome: molecular mimicry and host susceptibility the authors would like to thank dr. antonio pellanda, dr. elena scotti, dr. stefano ermanni (eoc-ospedale regionale di lugano, switzerland), and dr. nicola melik (eoc-ospedale regionale di locarno, switzerland) for providing tonsillar samples from patients undergoing tonsillectomy. we would also like to thank omar vandal and jeremy blum for helpful advice throughout the execution of the study. lp, fg, dc, and mp conceived the study. lp and mp initiated the study design. sj, gl, dp, fst, rp, and fb helped with implementation. dm and fse provided expertise in microscopy and immunohistochemistry, respectively. yh, sb, jx, ok, sbj, and dv performed the physiochemical characterization of the recombinant mabs. lp, fg, dc, and mp outlined and wrote the manuscript, whereas ok, sbj, and dv contributed to its finalization. all authors approved the final manuscript. the remaining 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 perruzza, jaconi, lombardo, pinna, strati, morone, seehusen, hu, bajoria, xiong, kumru, joshi, volkin, piantanida, benigni, grassi, corti and pizzuto. 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-295416-y3lvcjqd authors: eichinger, katherine m.; kosanovich, jessica l.; gidwani, sonal v.; zomback, aaron; lipp, madeline a.; perkins, timothy n.; oury, tim d.; petrovsky, nikolai; marshall, christopher p.; yondola, mark a.; empey, kerry m. title: prefusion rsv f immunization elicits th2-mediated lung pathology in mice when formulated with a th2 (but not a th1/th2-balanced) adjuvant despite complete viral protection date: 2020-07-29 journal: front immunol doi: 10.3389/fimmu.2020.01673 sha: doc_id: 295416 cord_uid: y3lvcjqd respiratory syncytial virus (rsv) remains the most common cause of lower respiratory tract infections in children worldwide. development of a vaccine has been hindered by the risk of developing enhanced respiratory disease (erd) upon natural exposure to the virus. generation of higher quality neutralizing antibodies with stabilized pre-fusion f protein antigens has been proposed as a strategy to prevent erd. we sought to test whether there was evidence of erd in naïve balb/c mice immunized with an unadjuvanted, stabilized pre-fusion f protein, and challenged with rsv line 19. we further sought to determine the extent to which formulation with a th2-biased (alum) or a more th1/th2-balanced (advax-sm) adjuvant influenced cellular responses and lung pathology. when exposed to rsv, mice immunized with pre-fusion f protein alone (pref) exhibited increased airway eosinophilia and mucus accumulation. this was further exacerbated by formulation of pref with alum (aluminum hydroxide). conversely, formulation of pref with a th1/th2-balanced adjuvant, advax-sm, not only suppressed rsv viral replication, but also inhibited airway eosinophilia and mucus accumulation. this was associated with lower numbers of lung innate lymphocyte cells (ilc2s) and cd4+ t cells producing il-5+ or il-13+ and increased ifnγ+ cd4+ and cd8+ t cells, in addition to rsv f-specific cd8+ t cells. these data suggest that in the absence of preimmunity, stabilized pref antigens may still be associated with aberrant th2 responses that induce lung pathology in response to rsv infection, and can be prevented by formulation with more th1/th2-balanced adjuvants that enhance cd4+ and cd8+ ifnγ+ t cell responses. this may support the use of stabilized pref antigens with th1/th2-balanced adjuvants like, advax-sm, as safer alternatives to alum in rsv vaccine candidates. respiratory syncytial virus (rsv) is the most common cause of lower respiratory tract infections (lrti) in children worldwide with nearly every child infected by 2 years of age (1) (2) (3) . in children >5 years of age, rsv causes an estimated 33 million acute lrti annually, with over 3 million episodes requiring hospitalization (4) . in 2017, the global cost estimate for inpatient and outpatient rsv lrti management in young children (< 5 years of age) was ∼4.8 billion euros (equivalent to ∼$5.2 billion usd) (5) . in addition to young children, rsv is a common cause of severe respiratory disease in the elderly and those who are immunocompromised (6) (7) (8) . given that severe rsv disease affects ages spanning infancy to geriatrics, it is clear that natural rsv infection does not induce long-lasting immunity and individuals are re-infected throughout their lives (9, 10) . thus, rsv immunization has the potential to boost rsv immunity and alleviate the morbidity associated with repeated rsv infections across all age groups. however, despite the immense economic and healthcare burden posed by rsv infection, there is currently no licensed rsv vaccine. the discovery and stabilization of the prefusion conformation of the rsv f protein (pref) re-ignited hopes for an rsv vaccine due to its ability to elicit potent neutralizing antibodies (11) . a number of studies have demonstrated the protective potential of high levels of rsv neutralizing antibody and as such, boosting serum neutralizing antibody levels has been an important objective of rsv vaccine research (12, 13) . the incorporation of adjuvants into vaccine formulations can enhance the vaccine's effect as well as reduce antigen concentrations and the number of immunizations required for a protective effect (14) . differential stimulation of various pattern recognition receptors can further shift the immune response toward vaccine antigens to promote th1-or th2-type immune responses. based on the known th2-bias associated with early rsv infections, it is imperative to understand the extent to which preventative rsv vaccine adjuvants shift the th1/th2 balance. in use since the 1930's, aluminum salts have long been recognized for their ability to enhance immunogenicity and boost antibody production (15) . alum adjuvant was used in the formalin-inactivated rsv (fi-rsv) trials of the 1960's that produced enhanced respiratory disease (erd) requiring hospitalization upon natural rsv exposure in 80% of vaccinees (16) . subsequent investigations into the cause of fi-rsv-induced erd have suggested that alum exacerbated the t helper type 2 (th2) pathology associated with erd (17) . other studies have disputed alum's role and have instead suggested that formalin inactivation of rsv resulted in poor neutralizing antibody development and immune complex deposition (18, 19) . reports have also demonstrated that formalin-inactivation of rsv resulted in post-fusion f (postf) protein being the predominant protein presented on the surface of the virion (20) . the implication of this data is that pref is more representative of live rsv and therefore, may be less likely than postf subunit vaccines to induce pathology. moreover, in a naïve cotton rat model, both pref and postf immunization elicited protective rsv immunity without inducing alveolitis when paired with the th1-skewing toll-like receptor 4 agonist (tlr4), glucopyranosyl lipid a (gla) (21) . these results suggest that more th1-biased adjuvants may provide a safe alternative to alum in models of pref immunization. importantly, th1skewing adjuvants, including tlr9 agonists, have recently been fda-approved for use in other vaccine systems, like hepatitis b (22) . advax-sm is an adjuvant comprised of delta inulin polysaccharide formulated with the tlr9 agonist, cpg oligodeoxynucleotides (cpg odn). the advax-sm adjuvant system has demonstrated greater th1-skewing properties when formulated with live rsv immunization (23) and ameliorated th2-related airway eosinophilia in a model of immunization against severe acute respiratory syndrome (sars)-associated coronavirus (24) . in the immunization studies presented here, we conducted a detailed evaluation of the protective capacity of rsv pref antigen alone or combined with the th1/th2-balanced adjuvant, advax-sm (pref/advax-sm), or the th2-skewing adjuvant, alum (pref/alum/aluminum hydroxide) in naïve balb/c mice. these studies evaluated immunogenicity, efficacy, innate and adaptive immune responses, and safety at acute and convalescent time points following rsv challenge. despite pref/alum immunization generating higher neutralizing antibody titers, both pref/advax-sm and pref/alum had undetectable viral replication, while pref alone lacked the immunogenicity to fully protect from rsv infection in naïve animals. pref/advax-sm induced more balanced th1/th2 immunity characterized by the generation of neutralizing antibody, a mean pref-specific igg2a/igg1 ratio >2, rsv f-specific and cytotoxic cd8+ t cells, and th1 cd4+ t cells. importantly, pref/advax-sm immunization protected from increased inflammation and mucus production, even when compared to pbs controls. in contrast, pref alone and pref/alum generated robust th2 immunity evidenced by pref-specific igg2a/igg1 ratios <1 and increased il-5+ and il-13+ cd4+ t cells. interestingly, despite undetectable viral replication at 4dpi, pref/alum immunization induced large populations of type 2 innate lymphoid cells (ilc2) in the lung producing the th2-type cytokines, il-5 and il-13. combined, the th2 immunity of pref/alum was consistent with enhanced inflammation featuring airway eosinophils and increased mucus production. overall, our observations have significant implications for the rsv vaccine field demonstrating that higher neutralizing antibody titers, while protective, are not implicitly tied to rsv pref vaccine safety and more th1/th2balanced adjuvants may generate protective responses while eliciting a desirable safety profile in naïve mice. animal studies were carried out in accordance with the university of pittsburgh's iacuc guidelines for the use and care of laboratory animals. seven to eight week old balb/cj female mice were purchased from the jackson laboratory (bar harbor, me). female mice were immunized via intramuscular (i.m.) injection with 50 mcl of vehicle (naïve and pbs), stabilized rsv prefusion protein (pref; 10 mcg; calder biosciences) alone, or formulated with advax-sm tm (pref/advax-sm; 1 mg/mouse; vaxine pty ltd, bedford park, australia) or alum (pref/alum; 2 mg/ml). specifically, alhydrogel adjuvant 2%, an aluminum hydroxide wet gel suspension from invivogen, was used in these studies. advax-sm is composed of microparticles of polyfructofuranosyl-d -glucose (delta inulin) combined with cpg55.2-odn (5 ′ atcgactctcgagcgttctc-3 ′ ), which was synthesized by genedesign (osaka, japan). the immunized mice were boosted 3 weeks later with their respective vaccine formulations. at 9 weeks post-prime, mice were challenged intranasally (i.n) with rsv l19 (5 × 10 5 pfu/gm) and culled at 4 or 8 days post-infection (dpi) using 100% isoflurane and cervical dislocation. rsv l19 was propagated and viral titers quantified as previously described (25) . all animal studies were approved by the university of pittsburgh institutional animal care and use committee; protocol #20047209. bronchoalveolar lavage (bal) was collected through intratracheal instillation of hbss + edta. bal samples were centrifuged and the soluble fraction stored at −80 • c for cytokine analysis and the cellular fraction analyzed via flow cytometry. cytokine concentrations were determined using the bio-plex pro tm mouse cytokine 23-plex assay (biorad, ca), per manufacturer's protocol. the right lung was harvested and enzyme-digested into a single cell suspension for flow cytometry, as described previously (26, 27) . where indicated, bal cells and lung homogenate were stimulated ex-vivo for intracellular cytokine detection. briefly, live cells from bal and lung homogenate were enumerated using a hemacytometer and trypan blue. for intracellular cytokine staining of lung homogenate or bal, 1 million cells in duplicate (singular for bal) were plated in a cd3-coated (5 mcg/ml, biolegend) 96-well flat-bottomed tissue culture plate in 200 mcl of 10% rpmi supplemented with cd28 (2 mcg/ml) and incubated at 37 • c overnight. after overnight stimulation with cd3/cd28, lung homogenate underwent a secondary stimulation with pma (1:1,000), ionomycin (1:1,000), and brefeldin a (1:1,000) for 2 h prior to t cell surface and intracellular flow staining. to obtain intracellular ilc2 cytokine staining, lung homogenate (3 million cells) was plated in 24-well tissue culture plates and stimulated with pma (30 ng/ml), ionomycin (500 ng/ml), and brefeldin a (1:1,000) in 10% rpmi at 37 • c for 3 h prior to surface and intracellular flow staining. bal cells were surface stained with combinations of the following (clone): molecular probes live/dead fixable blue, cd16/32 (2.4g2), siglec-f (e50-2440), f4/80 (t45-2342), cd11b (m1/70), ly6g (1a8), cd4 (gk1.5), cd8α (53-6.7), cd44 (im7) (bd biosciences, ca), cd11c (n418), cd19 (6d5), and tcr β (h57-597) (biolegend, ca). lung homogenate was surface stained with combinations of the following antibodies: cd16/32 (2.4g2), lineage cocktail, cd45 (30-f11), st2 (dih9), il-7rα (a7r32), cd19 (6d5), tcr β (h57-597) (biolegend), cd4 (gk1.5), and cd8α (53-6.7) (bd bioscience). following surface staining, cells were fixed and permeabilized for intracellular staining with bd cytofix/cytoperm tm solution kit (bd biosciences) according to the manufacturer's protocol. intracellular cytokines were stained with a combination of the following: cd206 (c068c2), il-5 (trfk5), ifnγ (xmg1.2), granzyme b (qa16a02) (biolegend), and il-13 (ebio13a) (thermofisher scientific, ma). where indicated and prior to surface staining, bal samples were incubated with rsv a strain f-protein 85−93 mhc i pentamer (h-2kd kyknavtel; proimmune, fl) to identify rsv f 85−93 -specific cd8 + t cells. samples were run on a bd lsrfortessa managed by the united flow core of the university of pittsburgh. data was analyzed using flowjo v10 software (flowjo, llc, or). cell populations were defined as follows: eosinophils (siglec f+/ f4/80+/ cd206lo/-/ cd11b+); neutrophils (siglec f-/ cd11bhi/ ly6g+/ cd11c-/lo); monocytes (siglec f-/ f4/80+/ cd11c+/ cd11b+); ilc2s (lin-/ cd45+/ st2+/ il-7rα+); t cells (± cd19-/ tcr β +/ cd4+ or cd8+). a lps-treated negative control was used to set the gate for rsv a strain f-protein 85−93 mhc i pentamer+ cd8+ t cells. pre-challenge serum was collected via submandibular bleed 2-3 days prior to rsv challenge and separated using gel-z serum separator tubes (sarstedt, germany). serum was stored at −80 • c until heat inactivation (56 • c for 30 min) and neutralizing antibody titers were performed. serial dilutions of heat inactivated serum (50 mcl in phenol-free mem supplemented with 5% fbs and pen/strep, invitrogen) were incubated for 2 h in a 37 • c co 2 incubator in a 96well plate format with 100 pfu/well line 19 rsv-renilla luciferase virus (provided by martin moore) in 50 mcl phenol free mem medium as above. after 2 h, hep-2 cells were trypsinized and a total of 2.5 × 10 4 cells were added per well in 25 mcl of phenol free mem with fbs and antibiotics as above. cells were incubated for a total of 64-66 h at 37 • c, 5% co 2 and luciferase readout was then obtained using the renilla-glo luciferase kit (promega) according to the manufacturer's instructions. luciferase activity (luminescence) was measured using a novostar plate reader after a 15 min incubation at 25 • c. all plates were run in duplicate and averaged. co-star 96-well, high binding elisa plates were coated with rsv pref at a concentration of 5 mcg/ml overnight at 4 • c. each plate included standards of either mouse igg1 or igg2a (invitrogen) at 10 and 2 mcg/ml in a 2-fold dilution series for intra-plate quantification of signal on uncoated wells. plates were then washed with pbs, and blocked for 1 h at 37 • c with 1% bsa in pbs. heat-inactivated serum samples were diluted 1:500 in 1% bsa in pbs for the first well, and then 3-fold serially diluted a total of 3 times. serum was incubated on the plates for 1 h at 25 • c, followed by three washes with pbs 0.05% tween-20, and secondary antibody incubation with anti-igg1 or anti-igg2a (isotype specific, bd pharmingen), respectively at a 1:10,000 dilution for 30 min at 25 • c in 1% bsa. 1-step tmb (thermo scientific) was used to develop the plates and the reaction was quenched by the addition of 4 n h 2 so 4 . plates were read at 450 nm in a novostar plate reader. data analysis was performed in excel and data points were interpolated from the linear region of the standards on each individual plate. samples were run in duplicate and the data presented represents the average values from both runs. left lungs were gravity filled with 10% formalin at 4 and 8 days post rsv challenge, as previously described (28) . the mcgowan institute for regenerative medicine (university of pittsburgh, pa) stained and processed the preserved lungs. periodic acid-schiff (pas) stained lungs were assessed by two pathologists blinded to treatment groups to quantify airway mucus production, according to previously published methods (26) . briefly, a score of 0-4 was given to all airways (average 50) with the following scale: 0 = no pas+ cells; 1 = 1-25% pas+ cells; 2 = 26-50% pas+ cells; 3 = 51-75% pas+ cells; 4 = 76-100% pas+ cells. scores were averaged and the total percentage of pas+ airways were graphed along with a more detailed breakdown of the proportion of each severity score (0-4) (# of airways of individual severity scores/total airways scored). standard hematoxylin and eosin (h&e) staining was performed on lung sections and scored by two pathologists (oury and perkins) blinded to treatment groups, according to previously described methods (29) . in short, each field (average 28 fields) in the lung was observed with a light microscope (x200 magnification) and scoring was based on the percentage of lung tissue affected according to the following scale: 0 = no inflammation, 1 = up to 25%, 2 = 25-50%, 3 = 50-75%, and 4 = 75-100%. scores were averaged and reported as a proportion of the sum of scores divided by the total number of fields counted. statistics were performed with graphpad prism 8 software (graphpad software, la jolla, ca). results in the figures are displayed as the mean ± sem. neutralizing antibody data was analyzed by nonlinear regression to obtain ic 50 values, which were compared between immunization groups using anova with a tukey's post-test, with pbs serving as the control. statistical significance was determined between each immunization cohort using anova with tukey's multiple comparison test between all groups (note: naive animals were not included in analysis but are graphed for reference). comparisons of the inflammatory and % pas+ scores within immunization groups over time were made using t-tests corrected for multiple comparisons with the holm-sidak method (α = 0.05). p-values <0.05 were considered significant. data is representative of 2 separate experiments. the th2-biased humoral immunity of pref/alum and the greater th1-skewed humoral immunity of pref/advax-sm protect against rsv replication the objective of this study was to determine the capacity of rsv pre-fusion (pref) vaccines formulated with different adjuvants to generate neutralizing antibody, prevent virus replication, and protect from pulmonary pathology following rsv challenge. to that end, 8 week old balb/cj mice were immunized twice with pbs (vehicle control), pref alone, or pref formulated with the th1/th2-balanced adjuvant, advax-sm (pref/advax-sm), or the t helper type 2 (th2)-skewing adjuvant, aluminum hydroxide (pref/alum). six weeks after their final immunization, sera were collected from mice in each group prior to viral challenge with rsv line 19; a control group of pbs-immunized mice received vehicle only (naïve) ( figure 1a) . a comparison of pref-specific igg2a and igg1 in prechallenge sera provided initial evidence supporting the roles of advax-sm as a more th1-biased adjuvant and alum as a th2polarizing adjuvant; production of igg2a and igg1 subclasses are reflective of their respective th1 and th2 biased immune responses in mice (30) . pref/advax-sm immunization produced higher titers of igg2a than all immunization groups tested ( figure 1b ) and greater igg1 compared to pbs ( figure 1c ). the mean igg2a/igg1 ratio of pref/advax-sm mice was > 2 and significantly higher than pref or pref/alum animals, indicating a more th1-skewed humoral response ( figure 1d ). in contrast, pref/alum immunization produced negligible titers of igg2a ( figure 1b) and instead elicited increased titers of igg1 ( figure 1c) , with a resulting igg2a/igg1 ratio < 1 ( figure 1d ). similar to pref/alum, pref alone elicited an igg2a/igg1 ratio < 1, indicating th2-dominant antibody responses in both groups. to assess differences in protective antibody responses between immunization groups, neutralizing antibody titers were measured in pre-challenge sera and rsv lung titers were quantified at 4 days post-infection (dpi). mice immunized with pref/alum generated greater neutralizing antibody titers than all other immunization groups ( figure 1e) . despite lower neutralizing antibody titers in mice immunized with pref/advax-sm, as compared to pref/alum, both immunization groups had undetectable rsv in their lungs at 4 dpi ( figure 1f ). in contrast, pref alone generated measurable neutralizing antibody titers in 50% (n = 4/8) of the animals and only 38% (n = 3/8) had undetectable rsv lung titers ( figure 1f ). all immunized mice had lower viral lung titers when compared to pbs controls following rsv challenge, however, only mice figure 1 | the th2-biased humoral immunity of pref/alum and the th1 skewed-humoral immunity of pref/advax-sm protect from rsv challenge. at 7-8 weeks of age, female adult balb/cj mice were immunized with phosphate buffered saline vehicle control (naïve & pbs), prefusion rsv f protein alone (pref), or pref formulated with advax-sm (pref/advax-sm) or alum (pref/alum) as depicted in (a). six weeks post-boost, pre-challenge serum was collected and mice were subsequently intranasally challenged with vehicle (naïve) or 5 × 10 5 pfu/gram of rsv line 19. animals were culled for sample collection at 4 or 8 days post infection (dpi) (a). pre-challenge serum was analyzed for pref-specific igg2a (b), igg1 (c), igg2a to igg1 ratios (d) and neutralizing antibody titers (e). at 4 dpi, left lungs were harvested and virus quantified using standard h&e plaque assays (f). data are represented as mean ± sem (n = 6-8 mice per group);*p < 0.05, **p < 0.01, and ****p < 0.0001. due to a lack of pre-existing pref-specific antibody, pbs was not included in the analysis in (d). immunized with pref/alum or pref/advax-sm achieved full viral protection at 4 dpi. to evaluate potential immunopathology in mice that received th2-skewing immunizations (pref alone and pref/alum) as compared to the greater th1-biased regimen (pref/advax-sm), left lungs were harvested from immunized mice at 4 dpi and stained with h&e to evaluate inflammation. representative images (10x) taken from animals in each immunization group demonstrated increased inflammation in all immunization groups relative to pbs controls, with pronounced perivascular inflammation seen in pref-and pref/alum-immunized animals (figures 2a-e) . inflammatory scores increased at 4 dpi in all groups that received pref-formulated immunizations but only pref alone and pref/alum elicited significantly greater inflammation compared to pbs (figure 2f) . to determine the contribution of innate cellular responses to the enhanced figure 2 | pref and pref/alum immunization elicited enhanced pulmonary inflammation characterized by robust airway eosinophil recruitment. naive mice were immunized and challenged with rsv as described in figure 1 . at 4 dpi, left lungs were formalin filled, paraffin embedded and sectioned for staining with h&e. each panel represents an individual mouse from the indicated group (scale bar 100 µm) (a-e). two blinded, independent pathologists scored all slides as described in the methods. scores between the two investigators were averaged and data is represented as mean ± sem (n = 3 mice) (f). at 4 dpi, bal was collected and eosinophils (g), neutrophils (h), and monocytes (i) were identified via flow cytometry. data are represented as mean ± sem (n = 6-8 mice per group);*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. yellow arrows highlight perivascular inflammation and blue arrows highlight peribronchial inflammation. inflammation observed in pref-and pref/alum-immunized groups, bronchoalveolar lavage (bal) was collected at 4 dpi and analyzed via flow cytometry (gating strategy for discriminating innate immune cells is shown in supplementary figure 1) . immunization with pref alone and pref/alum elicited a dramatic recruitment of eosinophils to the airways following rsv challenge, whereas eosinophil populations remained at baseline in pref/advax-sm-immunized mice ( figure 2g ). neutrophils were increased in the bal of pref-and pref/advax-smimmunized mice compared to pbs controls ( figure 2h ) and total monocytes were greater in pref-immunized animals as compared to all other groups tested (figure 2i) . taken together these results show that increased airway eosinophils paralleled increases in inflammation scores in mice immunized with pref alone and pref/alum, whereas neutrophils and monocytes were more pronounced in the unadjuvanted pref group. human rsv disease is characterized by both inflammation and extensive mucus production. thus, to determine if the enhanced inflammation seen in pref-and pref/alum-immunized groups was also associated with enhanced mucus production, left lungs were harvested at 4 dpi and periodic acid-schiff (pas) stained to analyze mucus metaplasia. representative images (10x) were taken from a sample within each immunization group to visualize the extent of mucus production (figures 3a-e) . a majority of airways from naïve ( figure 3a) , pbs (figure 3b) , and pref/advax-sm immunization groups ( figure 3d ) had little to no pas+ staining, while pref alone ( figure 3c ) and pref/alum ( figure 3e ) groups had extensive pas+ staining. consistent with the representative lung sections, pref-and pref/alumimmunization groups had higher percentages of pas+ airways compared to all other groups tested ( figure 3f) . to discriminate the degree of airway mucus production, the level of severity was reported for each group, whereby no pas+ staining in the airway yielded a score of "0, " the frequency of airways with 1-25% pas+ staining received a score of "1, " 26-50% pas+ yielded a score of "2, " 51-75% pas+ airways received a score of "3, " and 76-100% pas+ staining received a score of "4." four days after diluent (figure 3g ) or rsv challenge, pbs ( figure 3h ) and pref/advax-sm ( figure 3j ) immunization groups had the largest percentage of unaffected airways and a small proportion of airways with mild pas+ staining (1 score). in contrast, pref-( figure 3i ) and pref/alum-immunized mice ( figure 3k ) had smaller percentages of unaffected airways and greater frequency of airways with higher severity scores (2-4 scores). pref/advax-sm immunization produced th1-dominant immunity with increased rsv f-specific cd8+ t cells to delineate the relationship between distinct th cell subsets and associated pulmonary inflammation and mucus production, figure 3 | increased mucus production parallels enhanced inflammation in mice immunized with pref alone or pref/alum. mice were immunized and challenged with rsv as described in figure 1 . at 4 dpi, lungs were formalin filled, paraffin embedded and sectioned for staining with pas. each panel represents an individual mouse from the indicated group (scale bar 100 µm) (a-e). to quantify the extent of pas staining, lungs were scored as previously described in the methods. scores were averaged and the total percentage of pas+ airways were graphed (n = 3) (f); **p < 0.01 and ***p < 0.001. a more detailed breakdown of scores for each cohort is provided, calculated as a proportion i.e., number of airways of each severity score (0-4) divided by the total number of airways, according to the methods (g-k). th1-and th2-type cytokines were quantified from bal. ifnγ, the canonical th1 cytokine, was highest in pref/advax-sm mice at 4 dpi (figure 4a) , whereas levels of the th2-associated cytokines, il-5 ( figure 4b ) and il-13 ( figure 4c) , were similar between pref-advax-sm-immunized mice and control groups (naïve and pbs). conversely, pref-and pref/alum-immunized mice had increased concentrations of il-5 and il-13 with no appreciable increase in ifnγ levels in these groups. to identify cellular sources contributing to the th1associated cytokine bias of pref/advax-sm and the th2associated cytokine bias of pref and pref/alum immunization, ilc2 and cd4+ t cell populations were analyzed from lung homogenate at 4 dpi via flow cytometry (gating strategy to discriminate t cell populations and ilc2s are shown in supplementary figures 2, 3 , respectively). consistent with their th2 cytokine profiles, pref/alum immunization elicited greater ilc2 populations ( figure 4d ) following rsv challenge compared to all other groups. though ilc2s trended higher in pref alone immunization than control and pref/advax-sm groups, the difference was not significant. moreover, pref/alum-immunized mice had increased il5+ ( figure 4e ) and il-13+ ( figure 4f ) ilc2 populations as compared to pref/advax-sm-immunized mice and pbs controls; once again, increased trends in the pref alone group did not achieve significance. in conjunction with increased ilc2s, pref/alumimmunized mice had marked increases in il-5-producing cd4+ t cells in lung homogenate ( figure 4h ). pref-immunized mice had similar trends in increased il-5+ cd4+ t cells but were not significantly different compared to pbs controls. in contrast, pref/advax-sm-immunized mice had the largest population of ifnγ+ cd4+ t cells (figure 4g ) with distinctly increased ifnγ+:il-5+ cd4+ t cell ratios (figure 4i ) as compared to pref-and pref/alum-immunized mice. a similar increase in the ratio of ifnγ+:il-5+ cd4+ t cells was observed in the pbs group. lastly, due to the importance of cd8+ t cells in clearing rsv, general cd8+ t cell populations and rsv f 85−93 -specific cd8+ t cells were compared in mice from each immunization group. mice immunized with pref/advax-sm had increased total cd8+ t cells as compared to pref-immunized mice and pbs controls ( figure 4j) . moreover, only pref/advax-smimmunized mice had increased rsv f 85−93 -specific cd8+ t cells, which were greater than all other groups tested ( figure 4k) . collectively, these results identified ilc2s and cd4+ t cells as figure 4 | pref/advax-sm immunization produced th1-dominant immunity with increased rsv f-specific cd8+ t cells. at 4 dpi, first wash samples were harvested from the airways for quantification of ifnγ (a), il-5 (b), and il-13 (c) by luminex. right lungs were harvested and homogenized for quantification of ilc2s (d) and ilc2 intracellular cytokine staining of il-5 (e) and il-13 (f) by flow cytometry. intracellular production of ifnγ (g) and il-5 (h) by cd4+ t cells from lung homogenate were quantified by flow cytometry and the ratio of ifnγ/il-5-producing cd4+ t cells was calculated (i). total cd8+ t cells (j) and rsv f 85−93 -specific cd8+ t cells (k) were measured from bal. data are represented as mean ± sem (n = 6-8 mice per group);*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. frontiers in immunology | www.frontiersin.org cellular sources of th2-type cytokines associated with pref-and pref/alum-immunized mice following rsv challenge. in stark contrast, pref/advax-sm immunization elicited an increase in ifnγ-producing cd4+ t cells and rsv f 85−93 -specific cd8+ t cells in response to rsv exposure that likely contributed to viral protection. to determine if immunization with pref, pref/alum, or pref/advax-sm expedites disease resolution, innate inflammatory cells and lung inflammation were examined later at 8 days post-rsv challenge. representative images (10x) were taken from h&e stained lung sections from each immunization group (figures 5a-e) . each rsv-challenged group displayed enhanced inflammation that was predominately perivascular in nature with non-significant reductions in inflammation in the immunization groups compared to the pbs group ( figure 5f ). similar to the 4-day time point, pref/alum mice maintained greater airway eosinophil populations ( figure 5g ) as compared to pref/advax-sm-immunized mice and pbs controls. neutrophils were elevated in pbs controls and the pref/alum group compared to pref/advax-sm-immunized mice, although no significant difference was observed in the pbs group (figure 5h) . pref-immunized and pbs control groups had the greatest number of monocytes in the bal when compared to pref/advax-sm-immunized mice ( figure 5i) . overall, populations of innate inflammatory cells had largely resolved in the bal of pref/advax-sm-immunized mice by 8 dpi. in pbs controls, increases in neutrophils and monocytes likely contributed to the dramatic increase in inflammation between 4 and 8 dpi, whereas inflammatory scores for the groups immunized with pref remained largely unchanged (figure 5j) . these results suggest that inflammation and recruitment of inflammatory cells dramatically increased in pbs controls, innate inflammatory cells largely resolved in the pref/advax-sm group, and pref/alum immunization failed to resolve airway eosinophilia by 8 days post-rsv challenge. to determine if mucus production resolved or worsened in immunized mice over the course of infection, left lungs were collected and pas-stained at 8 dpi. representative images (10x) were taken from samples in each group (figure 6a-e) . readily discernable mucus was observed in pbs ( figure 6b ) and pref/alum ( figure 6e ) lung sections, with a lower frequency of pas+ staining seen in mice immunized with pref-alone ( figure 6c ). quantitative analysis of the percentage of pas+ figure 5 | inflammation worsens over time in unimmunized compared to immunized groups. at 8 dpi, left lungs were formalin fixed and stained with h&e to assess inflammation. each panel represents an individual mouse from the indicated group (scale bar 100 µm) (a-e). two blinded, independent pathologists scored all slides and scores were averaged. data is represented as mean ± sem (n = 3-4 mice) (f). bal was collected at 8 dpi for quantification of eosinophils (g), neutrophils (h), and monocytes (i) via flow cytometry. data are represented as mean ± sem (n = 6-8 mice). inflammatory scores from 4 and 8 dpi were compared for each immunization groups over time (j). data are represented as mean ± sem (n = 3-4 mice); *p < 0.05 and **p < 0.01. airways revealed that pbs controls and pref/alum-immunized groups had the highest proportion of pas+ airways (figure 6k) . pref/advax-sm immunized mice maintained a low percentage of pas+ airways through 8 dpi. a more detailed analysis revealed that >50% of airways were pas+ in pbs-( figure 6g ) and pref/alum-immunized mice ( figure 6j ) and these groups had the largest proportions of airways that received higher severity scores (scores of 2-4) relative to all other groups (figure 6f-j) . at 8 dpi, the majority of airways in the prefimmunization group were unaffected and when pas+ was observed, it was generally mild (score of 1; figure 6i ). mice immunized with pref/advax-sm (figure 6j ) had the largest percentage of pas-free airways, with a smaller proportion of airways receiving higher severity scoring (scores of [2] [3] [4] . similar to what was seen with inflammation over time, pbs controls demonstrated a distinct enhancement of mucus production between 4 and 8 dpi (figure 6l) , while pref/alum immunization elicited a high degree of pas+ staining throughout the rsv time course. in contrast, by 8 dpi, pref-immunized mice resolved some of the high proportion of pas+ airways observed at 4 dpi, while mice immunized with pref/advax-sm maintained low levels of mucus production. together, these data suggest that the high degree of mucus produced by 8 dpi in pbs controls is mitigated more effectively in mice immunized with pref/advax-sm as opposed to pref/ alum immunization. based on the changes in lung pathology observed between immunization groups at 8 dpi, we asked whether there was an associated change in th phenotypes among immunization figure 6 | mucus production persisted in the lungs of pref/alum-immunized mice at 8 dpi and remained low in mice immunized with pref/advax-sm. at 8 dpi, left lungs were formalin fixed and stained with pas to assess mucus production. each panel represents an individual mouse from the indicated group (scale bar 100 µm) (a-e). a more detailed breakdown of scores for each cohort is provided, calculated as a proportion i.e., number of airways of each severity score (0-4) divided by the total number of airways, according to the methods (f-j). to quantify the extent of pas staining, lungs were scored as previously described in the methods. scores were averaged and the total percentage of pas+ airways were graphed. total percentage pas+ airways from 8 dpi are shown in (k) (n = 3-4) and are further compared over time between 4 and 8 dpi in each group in (l); **p < 0.01. (figure 7a) , while mice immunized with pref alone and pref/alum had larger populations of il-5+ cd4+ t cells ( figure 7b) . additionally, il-13+ cd4+ t cell populations in pref/alum immunized mice were significantly higher than pref/advax-sm animals ( figure 7c ). examination of cd8+ t cell populations revealed greater granzyme b+ ( figure 7d) and ifnγ+ cd8+ t cells + (figure 7e ) in the airways of pref/advax-sm immunized mice and pbs controls, with pbs mice having significantly more ifnγ+ cd8+ t cells than the other immunization groups tested. in addition to cellular analysis in the bal, t cells and ilc2s were measured in lung homogenate to address potential differences in cell localization. in accordance with their role as early immune responders, ilc2 populations ( figure 7f ) had contracted by 8 dpi and no differences were detected between immunization groups. moreover, populations of il-5+-( figure 7g ) and il-13+-producing ilc2s ( figure 7h) were similar between all groups. finally, th phenotypes were examined at 8 dpi in lung homogenate. pref/advax-sm immunized mice had the highest number of ifnγ+ cd4+ t cells (figure 7i ) compared to other groups, while il-5+ cd4+ t cells ( figure 7j) were greatest in pref-vaccinated mice. consistent with the data at 4 dpi, the cd4+ ifnγ+:il-5+ ratio ( figure 7k ) was higher in pref/advax-sm immunized mice as compared to all other groups tested. this demonstrates the persistent th1-dominant immune response elicited by pref/advax-sm immunization. here, we explored the ability of a rsv pref subunit proteinbased immunization to protect naïve balb/c mice from rsv infection and pulmonary pathology when formulated with either a th2-or more th1/th2-balanced adjuvant. alum has been in use in human vaccines since the early 20th century and is well recognized for its th2-skewing and antibody boosting properties. as expected, pref/alum immunization produced th2-biased immunity with high titers of pref-specific igg1 and neutralizing antibody, which were associated with undetectable viral replication following rsv challenge. while pref alone lacked the immunogenicity to offer complete rsv protection, likely due to low neutralizing antibody production, prefimmunized mice generated th2-type immune responses similar to mice immunized with pref/alum. on the other hand, advax-sm is a delta-inulin polysaccharide adjuvant formulated with cpg55.2 odn, a tlr9 agonist. the combination of delta inulin and tlr9 agonism has been reported to generate a th1/th2balanced adjuvant response in models of sars-associated coronavirus, japanese encephalitis virus, and west nile virus (24, 31, 32) . in models of rsv immunization, tlr9 agonists formulated with formalin-inactivated rsv have increased immunogenicity while ameliorating pulmonary pathology and reducing airway hyperresponsiveness normally exacerbated by fi-rsv immunization (33, 34) . in one report, an intramuscular immunization of live rsv formulated with advax-sm protected from rsv infection with increased neutralizing antibody titers and greater rsv-specific igg2a/igg1, but had similar lung inflammation as unadjuvanted control mice, suggesting the th2 polarization of the live rsv vaccine may have overcome the greater th1 bias of the cpg oligonucleotide (23) . these studies support the idea that tlr9 agonists may be combined with stabilized pref proteins to induce a more th1-biased response with an acceptable safety profile and highlight the importance of evaluating adjuvant safety as well as efficacy when developing vaccine strategies. in our study, mice immunized with pref/advax-sm elicited a greater th1-type response, while still generating high levels of pref-specific igg2a and igg1 antibody. despite producing lower neutralizing antibody titers than pref/alum immunized, pref/advax-sm mice were resistant to rsv infection. moreover, the discreet th profiles produced by pref/alum and pref/advax-sm generated distinct differences in pulmonary pathology. the th2 profile of pref/alum-immunized mice was associated with enhanced pulmonary inflammation and mucus production at 4 dpi and continued to have the greatest proportion of pas+ airways as late as 8 dpi. the immunopathology associated with pref/alum immunization occurred in spite of high neutralizing antibody titers and undetectable viral replication. previous studies of fi-rsv-associated erd have demonstrated that poor avidity and affinity maturation resulted in non-protective antibody development and th2-associated immunopathology (19) . furthermore, it has been suggested that alum did not alter the erd profile induced by fi-rsv immunization (18) . however, in our study, alum adjuvant exacerbated the pathology induced by immunization with pref alone despite protection from rsv infection. in contrast, the th1-type responses generated by immunization with pref/advax-sm elicited less overall inflammation with low levels of mucus production. importantly, pref/advax-sm immunization protected mice from the worsening inflammation and mucus production that occurred between 4 and 8 dpi in pbs controls. taken together, these data indicate that rsv pref immunization formulated with th1-skewing adjuvants, like advax-sm, provide protection against rsv infection in naïve balb/c mice as compared to alum by inhibiting viral replication without eliciting enhanced pulmonary pathology. though ige was not measured in these studies, alum, as opposed to cpg-odn, adjuvanted antigens have been associated with an increased production of ige (35) . future studies will be needed to address the role of ige following immunization with rsv pref adjuvanted with alum. neutralizing antibodies have been shown to reduce the severity of rsv disease (12, 13) . moreover, the risk of reinfection is inversely correlated to the level of serum neutralizing antibodies (36) . as such, boosting serum neutralizing antibody titers has been an important objective of rsv vaccination, especially since the discovery and subsequent stabilization of the pre-fusion conformation of rsv f protein (11) . rsvneutralizing activity in human sera is primarily derived from pref-specific antibodies (37, 38) . thus, the use of stabilized pref as a vaccine antigen has sparked new hope that high neutralizing antibody titers can be produced to provide long-term protection. pre-clinical studies in young and aged mice have demonstrated the neutralizing antibody boosting-potential of rsv pref when formulated with both th2-and th1/th2-skewing adjuvants (39) . in agreement with our data, pref immunization alone has been shown to lack sufficient immunogenicity to increase neutralizing antibody titers above a protective threshold (39) . our study expanded these findings by directly challenging pref-immunized mice and demonstrated incomplete protection and enhanced lung pathology following rsv infection in association with little to no measurable neutralizing antibody production (i.e., only 50% of pref-immunized mice produced any measurable neutralizing antibody). earlier work found that higher titers of neutralizing antibody were associated with rsv pref immunization formulated with th1/th2-balanced adjuvants (39) . in contrast, our results show that the th2-dominant immune response of pref/alum immunization produced higher neutralizing antibody titers as compared to the th1-biased immune response elicited by pref/advax-sm immunization. importantly, however, both pref/alum and pref/advax-sm immunization fully protected mice from rsv infection with a complete absence of detectable viral replication in the lungs at 4 dpi. additional studies will be required to determine if lower neutralizing antibody production, as seen in pref/advax-sm, affects the durability of protection against rsv challenge. despite evidence of the correlation between neutralizing antibody in the serum and protection from severe rsv disease (12, 13, 36, 37) , other data suggests that high serum levels of neutralizing antibody provide insufficient protection from rsvdisease or reinfection in some individuals (40) (41) (42) . therefore, rsv vaccines that rely on neutralizing antibody as the sole correlate of protection against rsv disease may not provide universal or long-lasting immunity. numerous studies have demonstrated the importance of t cells in clearing rsv in primary rsv infection (43) (44) (45) . in mouse models of primary infection, cd8+ t cells are critical for viral control and both the number of cd8+ t cells and their production of ifnγ are crucial for effective clearance (46) (47) (48) . despite conferring protection against rsv replication, immunopathology has been linked to ifnγ-producing cd8+t cells during primary infection (47) and in memory responses resulting from immunization (49) . in contrast, our data shows that immunization with pref/advax-sm protects against rsv infection and elicits rsv f-specific and ifnγ-producing cd8+ t cells without inducing additional pulmonary pathology. alternatively, by 8 dpi, a robust ifnγ+ cd8+t cell response was induced in pbs-immunized control mice that was further associated with increased inflammation. these divergent outcomes in lung pathology associated with ifnγ+ cd8+t cells may be explained by the presence of pre-existing neutralizing antibody in pref/advax-sm as compared to the lack of pre-existing neutralizing antibody in pbs controls. another model of rsv vaccination has demonstrated that pre-existing neutralizing antibody can protect mice from severe immunopathology caused by potent memory ifnγ-producing cd8+t cells (50) . therefore, rsv vaccination approaches that combine pref with th1/th2-balanced adjuvants may prove to be efficacious and safe through the combination of increased neutralizing and rsv-specific antibody production and memory ifnγ+ cd8+t cell responses. like cd8+ t cells, research has demonstrated the importance of cd4+ t cells in the control of rsv infection and their role in inducing pathology (43) (44) (45) . the 1960's vaccine trials of formalin-inactivated rsv formulated with alum (fi-rsv) demonstrated the sobering potential for vaccine-associated erd. upon natural rsv exposure, 80% of vaccinees developed erd, requiring hospitalization and two children died (16) . investigations into the causes of erd using multiple animal models have revealed th2 cd4+ t cells to be closely linked to the increased inflammation, mucus, and airway hyperresponsiveness observed in erd (51) (52) (53) (54) (55) . in our study, both pref alone and pref/alum immunization elicited th2 cd4+ t cell profiles associated with enhanced inflammation, airway eosinophils, and extensive mucus production. interestingly, despite their similar th2 phenotypes, pref-immunized mice resolved airway neutrophils, eosinophils, and mucus more rapidly than pref/alum immunized mice. this resolution occurred in pref-immunized mice despite their poor neutralizing antibody production and detectable rsv replication, which was likely the cause of increased inflammatory innate cell recruitment as compared to pref/alum immunization. whereas, mice immunized with pref/alum had high titers of neutralizing antibody and undetectable rsv in the lung, yet persistent airway mucus and inflammation. in contrast to data showing the ability of pre-existing antibody to temper the immunopathology induced by ifnγ+ cd8+t cells (50) , our data suggests that pre-existing neutralizing antibody alone may not modulate the immunopathology associated with th2 cd4+ t cells in a similar manner. in addition to increases in th2 cd4+ t cells, pref-but especially pref/alum-immunization was associated with large populations of ilc2s producing il-5 and il-13 in the lung. ilc2s are the dominant innate lymphoid cell in the lung and are well recognized for their ability to produce large amounts of type 2 cytokines upon stimulation (56) . in primary rsv, ilc2 expansion and activation result from damage to airway epithelial cells via rsv infection and subsequent release of il-33 (57) and/or thymic stromal lymphopoietin (tslp) (58). however, in pref/alum immunized animals, ilc2s increased and became activated in spite of effective rsv neutralization and undetectable virus in the lung at 4 dpi. our data suggests that an alternative mechanism may contribute to the induction of ilc2 responses in pref/alum immunized mice. a murine model of nippostrongylus brasiliensis demonstrated that crosstalk between ilc2s and antigen-specific th2 cd4+ t cells occurs, promoting ilc2 proliferation and il-13 production (59). this ilc2-cd4+ t cell crosstalk was shown to be mutually beneficial to both cell types' maintenance, expansion, and cytokine production. future studies are necessary to understand how pref/alum immunization contributes to ilc2 expansion and activation. however, this is the first study to suggest a relationship between ilc2 activation and th2-associated immunopathology following pref/alum immunization in naïve mice. in conclusion, our studies demonstrated that pref/advax-sm and pref/alum immunizations provided equivalent protection from rsv infection but elicited dramatically different outcomes regarding lung pathology. the th2 immunity induced by pref/alum immunization was associated with increased inflammation and abundant mucus production that persisted through 8 dpi. in addition to il-5-and il-13-producing cd4+ t cells, pref/alum immunization was also associated with increased il-5-and il-13-producing ilc2 populations. this previously unrecognized relationship between pref/aluminduced th2 immunity and ilc2 activation in the absence of rsv replication may have important implications for rsv vaccine development. it suggests that despite eliciting high titers of neutralizing antibody, the extensive th2 immune bias generated by pref/alum may still lead to overt pathology in naïve individuals. it has been suggested that rsv exposure prior to immunization may mitigate the overwhelming th2 immunity and lung pathology observed here with pref/alum, however, considering the important safety concern, further studies are needed to confirm this hypothesis. in contrast to the th2-skewed immunity of pref/alum, the combination of neutralizing antibody production, th1 immunity, and cytotoxic ifnγproducing cd8+t cells induced by pref/advax-sm provided vigorous protection from rsv replication without inducing pathology. taken together, these data suggest that rsv pref protein subunit vaccinations formulated with th1/th2-balanced adjuvants may provide complete rsv protection with a desirable safety profile. future studies will be needed to elucidate safety and protection of preimmune mice when immunized with pref alone or when formulated with a th1-vs th2-biased adjuvant. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the animal study was reviewed and approved by the university of pittsburgh institutional animal care and use committee. kei, jk, and kem contributed to the overall study design, execution, and interpretation of data and writing of the paper. sg and az contribute through development of novel assays for generation of data. ml contributed to data interpretation, figure development, and writing. my contributed to data generation, study design, and analysis. tp and to contributed to the analysis and interpretation of data. cm and np contributed to study design. all authors contributed to the article and approved the submitted version. viral and host factors in human respiratory syncytial virus pathogenesis respiratory syncytial virus-associated hospitalizations among children less than 24 months of age global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial 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model of enhanced pulmonary pathology induced with a formalin-inactivated rsv vaccine but not a recombinant fg subunit vaccine rsv vaccine-enhanced disease is orchestrated by the combined actions of distinct cd4 t cell subsets vaccine-enhanced respiratory syncytial virus disease in cotton rats following immunization with lot 100 or a newly prepared reference vaccine respiratory synctial virus infection in balb/c mice previously immunized with formalin-inactivated virus induces enhanced pulmonary inflammatory response with a predominant th2-like cytokine pattern group 2 innate lymphoid cells in pulmonary immunity and tissue homeostasis for clinical pharmaceutical sciences, university of pittsburgh school of pharmacy. this work benefitted from special bd lsrfortessatm funded by nih 1s10od011925-01 (pi: borghesi). np (vaxine pty ltd., bedford park, australia) generously provided advax-sm but did not otherwise provide financial assistance for this project. development of advax-sm adjuvant was supported by national institutes of health contracts hhsn272201400053c, hhsn272200800039c and ai061142. this publication's contents are solely the responsibility of the authors and do not necessarily represent the official views of the national institutes of health, national institute of allergy and infectious diseases. we would like to pay special thanks to dr. kevin legge for his thoughtful and constructive feedback during the writing of this manuscript. conflict of interest: np is affiliated with vaxine pty ltd., which hold commercial interests in advax adjuvants cm and my are affiliated with calder biosciences, which holds commercial interests in the stabilized pref protein. the remaining 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 eichinger, kosanovich, gidwani, zomback, lipp, perkins, oury, petrovsky, marshall, yondola and empey. 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-000354-05lnj3w0 authors: de vries, erik; tscherne, donna m.; wienholts, marleen j.; cobos-jiménez, viviana; scholte, florine; garcía-sastre, adolfo; rottier, peter j. m.; de haan, cornelis a. m. title: dissection of the influenza a virus endocytic routes reveals macropinocytosis as an alternative entry pathway date: 2011-03-31 journal: plos pathog doi: 10.1371/journal.ppat.1001329 sha: doc_id: 354 cord_uid: 05lnj3w0 influenza a virus (iav) enters host cells upon binding of its hemagglutinin glycoprotein to sialylated host cell receptors. whereas dynamin-dependent, clathrin-mediated endocytosis (cme) is generally considered as the iav infection pathway, some observations suggest the occurrence of an as yet uncharacterized alternative entry route. by manipulating entry parameters we established experimental conditions that allow the separate analysis of dynamin-dependent and -independent entry of iav. whereas entry of iav in phosphate-buffered saline could be completely inhibited by dynasore, a specific inhibitor of dynamin, a dynasore-insensitive entry pathway became functional in the presence of fetal calf serum. this finding was confirmed with the use of small interfering rnas targeting dynamin-2. in the presence of serum, both iav entry pathways were operational. under these conditions entry could be fully blocked by combined treatment with dynasore and the amiloride derivative eipa, the hallmark inhibitor of macropinocytosis, whereas either drug alone had no effect. the sensitivity of the dynamin-independent entry pathway to inhibitors or dominant-negative mutants affecting actomyosin dynamics as well as to a number of specific inhibitors of growth factor receptor tyrosine kinases and downstream effectors thereof all point to the involvement of macropinocytosis in iav entry. consistently, iav particles and soluble fitc-dextran were shown to co-localize in cells in the same vesicles. thus, in addition to the classical dynamin-dependent, clathrin-mediated endocytosis pathway, iav enters host cells by a dynamin-independent route that has all the characteristics of macropinocytosis. influenza a virus (iav) is an enveloped, segmented negativestrand rna virus infecting a wide variety of birds and mammals. as its first step in infection iav attaches to host cells by the binding of its major surface protein, the hemagglutinin (ha), to sialic acids, which are omnipresent on the glycolipids and glycoproteins exposed on the surfaces of cells. where the structural requirements for this interaction have been studied in great detail, much less is known about whether and how the attachment to specific sialylated receptors (e.g. to n-linked glycoproteins, olinked glycoproteins or gangliosides or even to specific receptors within these groups) affects the subsequent endocytic steps. obviously, knowledge about the repertoire of endocytic pathways that can successfully be used by iav will increase our insights into cell and species tropism of iav. in turn, this will contribute to our understanding of the requirements for the generation of novel viruses with pandemic potential that can arise by exchange of rna segments between currently circulating human serotypes and an animal virus during occasional co-infection in a human or an animal host. clathrin mediated endocytosis (cme) has for long been identified and studied as the major route of iav cell entry [1, 2] and is, by far, the best characterized endocytic pathway. evidence obtained from live cell imaging has revealed the de novo formation of clathrin-coated pits at the site of virus attachment [3] and the requirement for the adapter protein epsin 1, but not eps15, in this process [4] . still, specific transmembrane receptors linking viral entry to epsin 1 or to other adapters have not been identified although a recent study performed in cho cells indicated the specific requirement for n-linked glycoproteins in iav entry [5] . some recent papers provided indications for the utilization of alternative entry pathways by iav. studies in which cme was obstructed by pharmacological or genetic intervention indicated the ability of iav to enter host cells via alternative endocytic routes [4, 6, 7] . also live cell imaging revealed the simultaneous availability of entry routes involving non-coated as well as clathrin-coated pits [4] . however, this alternative iav entry route has not been characterized in any detail and requirements for any specificity in receptor usage apart from the need for the proper sialic acid moiety have not been established. during the past decades quite a variety of endocytic pathways have been identified in eukaryotic cells [8, 9, 10] . their occurrence, abundance and mechanistic details appear to vary between cell types, tissues and species and their utilization by viruses as a route of entry makes them an important factor in host and cell-type permissiveness for infection [11, 12] . besides by cme, different viruses have been shown to enter cells via caveolae, macropinocytosis or other, less well described, routes [11, 12] . most often, the selection of a specific endocytic route is linked to the utilization of a specific receptor that facilitates traveling via that particular route. nevertheless, many receptors allow flexibility by their capacity to enter through multiple pathways. for iav, an additional level of complexity to the dissection of potential entry routes is added by the apparent lack of an iav-specific protein receptor. a full experimental characterization of the iav entry pathways will benefit from separation of the iav entry pathways into routes that can be studied independently. whereas co-localization with clathrin is an established marker for endocytosis via this route, the complete lack of unique markers for macropinosomes or most other endocytic compartments [13, 14] complicates such studies. furthermore, crucial to any study concerning endocytic pathways is the abundantly documented fact that such pathways are highly dependent on experimental cell culture conditions [15] [16] [17] [18] [19] . pathways that are constitutive in one cell type may be absent or inducible by specific experimental conditions in other cell types. moreover, the manipulation of specific endocytic pathways may result in up or down regulation of other specific pathways. here we have established entry assay conditions that allow dissecting cell entry of iav into a dynamin-dependent (dyna-dep) and a dynamin-independent (dyna-ind) component. dynamin is a large gtpase forming multimeric assemblies around the neck of newly formed endocytic vesicles. gtp hydrolysis is required for pinching off of the vesicles [20] . whereas cme is completely dependent on dynamin, several other endocytic routes do not require dynamin [21] . we performed an extensive characterization of the dynamin-independent iav entry route using pharmacological inhibitors as well as by expressing dominant-negative mutants and applying sirna induced gene silencing as tools. taken together the results identify a pathway that closely resembles macropinocytosis as a novel entry pathway for iav. to identify and characterize potential non-cme entry routes taken by iav, we adapted a luciferase reporter assay [22] to enable the quantitative determination of infection or entry by measuring the activity of secreted gaussia luciferase. twentyfour hours prior to infection hela cells were transfected with a plasmid (phh-gluc) allowing constitutive synthesis (driven by the human poli promoter) of a negative strand viral rna (vrna) encoding a gaussia luciferase under control of the untranslated regions (utrs) of the np segment of influenza a/wsn/33 (h1n1) (hereafter called iav-wsn) np segment. upon iav infection, the combined expression of the viral polymerase subunits and np will drive transcription of luciferase mrna from the negative strand vrna and subsequent synthesis of gaussia luciferase. a dose-response curve demonstrating the applicability of the assay to inhibitor screening (fig. 1a) was obtained for bafilomycin a1 (bafa1), a known inhibitor of iav entry [23] . bafa1 acts upon the vacuolar-type h(+)-atpase, thus preventing endosomal acidification and thereby trapping iav in peri-nuclear immature endosomes with a lumenal ph that does not permit viral membrane fusion. remarkably, dynasore, a small molecule inhibitor of the gtpase dynamin 2 that is crucial for endocytic vesicle formation in clathrin-and caveolin-mediated endocytosis [8] as well as in a poorly described clathrin-and caveolin-independent endocytic pathway [8, 19] , did not give significant inhibition (fig. 1b) . bafa1 specifically inhibits iav during the entry phase as demonstrated in fig. 1c . the continuous presence of 10 nm bafa1 (added to the cells 1 hr prior to infection) for 16 hrs completely prevents infection. in contrast the addition of bafa1 at 1 hr or 2 hrs post infection resulted in high levels of luciferase activity (again measured at 16 hrs p.i.) that were 63% or 90% respectively of the control to which no bafa1 was added, indicating that entry was essentially completed within 2 hrs. the last bar of fig. 1c shows that the inhibition by bafa1 is reversible as withdrawal of the inhibitor after 2 hrs resulted in high levels of infection. the specific effect of bafa1 on iav entry was confirmed by confocal microscopy demonstrating that bafa1, as expected, traps iav particles in a peri-nuclear location, presumably in nonacidified endosomes (fig. 1d) . bafa1 was subsequently exploited to establish a specific iav entry assay (hereafter further referred to as the gluc-entry assay). hela cells transfected with phh-gluc were inoculated with iav at a range of mois and incubated for 2 hrs after which the entry medium was replaced by complete growth medium containing 10% fcs and 10 nm bafa1 to prevent any further entry of virus. entry was indirectly quantified by determination of luciferase activity after further incubation for 14 hrs demonstrating a quantitative correlation between infection dose and luciferase activity across a wide range of mois (fig. 1e) . the indirect gluc-entry assay was next tested for its capacity to examine the effects of inhibitors on iav entry. dynasore or bafa1 (fig. 1f) were included in the medium (dmem containing 10% attachment to and entry into a host cell are the first crucial steps in establishing a successful virus infection and critical factors in determining host cell and species tropism. influenza a virus (iav) attaches to host cells by binding of its major surface protein, hemagglutinin, to sialic acids that are omnipresent on the glycolipids and glycoproteins exposed on the surfaces of cells. iav subsequently enters cells of birds and a wide variety of mammals via receptormediated endocytosis using clathrin as well as via (an) alternative uncharacterized route(s). the elucidation of the endocytic pathways taken by iav has been hampered by their apparent redundancy in establishing a productive infection. by manipulating the entry conditions we have established experimental settings that allow the separate analysis of dynamin-dependent (including clathrin-mediated endocytosis) and independent entry of iav. collectively, our results indicate macropinocytosis, the main route for the non-selective uptake of extracellular fluid by cells, as an alternative iav entry route. as the dynamindependent and -independent iav entry routes are redundant and independent, their separate manipulation was crucial for the identification and characterization of the alternative iav entry route. a similar strategy might be applicable to the study of endocytic pathways taken by other viruses. fcs) during entry (the first 2 h of infection) and were removed when the inoculum was replaced by growth medium containing bafa1. concentrations up to 80 mm dynasore did not inhibit entry which is in agreement with the result shown in fig. 1b . in contrast, 1.25 nm bafa1 already inhibited entry for more than 60% (fig. 1f) . as a control, dynasore was also added at 2 hrs post infection to analyze whether the drug affected iav replication during the post entry phase. as expected, 80 mm dynasore did not significantly inhibit iav replication when present from 2 to 16 hrs p.i. (fig. 1f ). thus, with the gluc-entry assay we can study the effect of specific inhibitors on iav entry in a quantitative manner, at least as long as the inhibitors do not irreversibly affect iav replication during the post entry phase. furthermore, the lack of inhibition of iav entry by dynasore demonstrates that under these experimental conditions iav is able to enter cells via a pathway that is fully redundant to any dynamindependent (dyna-dep) entry route, including the classical cme pathway. also when iav travels via this novel dynamin-independent (dyna-ind) route, iav apparently enters via low ph compartments as entry is fully sensitive to bafa1. as factors present in serum are known for their potential to induce specific endocytic pathways, we further explored the conditions required for the novel dyna-ind iav entry pathway (using the gluc-entry assay) by inoculating cells in pbs in the presence of increasing concentrations of fetal calf serum (fcs). whereas dynasore completely inhibited entry in pbs, inclusion of 5% and 10% fcs resulted in increasing levels of dynasore resistant entry ( fig. 2a) , suggesting the existence of a serum-inducible dyna-ind iav entry pathway. this effect was not caused by inactivation of dynasore during the experiment as vesicular stomatitis virus (vsv), which enters cells by cme [24, 25] , was still sensitive to 80 mm dynasore in the presence of 10% fcs (fig. 2b) . in agreement herewith, the uptake of transferin, known to occur via cme, was inhibited by dynasore regardless of the hela cells were grown on glass cover slips and infected with iav (strain wsn; moi of 10) and fixated after 30 min, 3 hrs or 6 hrs (column 1, 2 or 3 respectively). infection was performed in 0.2% dmso (upper row panels) or in the presence of 10 nm bafa1 (lower row panels). the nucleus was visualized by dna staining with topro-3 (red). iav infection was visualized by staining with monoclonal antiserum directed against np (green). in the absence of inhibitor, iav localized to the nucleus after 3 hrs, while new virus particles spread to the cytoplasm after 6 hrs. bafa1 (lower row panels) caused accumulation of incoming virus particles at a peri-nuclear location. (e) quantitative determination of iav entry by a single-cycle gluc-entry assay. hela cells (10,000 cells/well in dmem supplemented with 10% fcs) were transfected with phh-gluc 24 hrs prior to infection with a serial dilution of infectious iav particles (plotted on the x-axis). two hours after infection 10 nm bafa1 was added to block any further entry. cells were incubated for a further 14 hrs to allow expression of luciferase activity (y-axis; relative light units, rlu). (f) effect of dynasore and bafa1 on iav entry in the gluc-entry assay. dynasore (dy, dark grey bars; 20, 40 or 80 mm) or bafa1 (light grey bars; 1.25, 2.5 or 5 nm) were present from 1 hr prior to infection (strain wsn; moi 0.5) to 2 hrs p.i. after which the inhibitor-containing medium was replaced with medium containing 10 nm bafa1 to block any further entry. cells were incubated for a further 14 hrs to allow the quantitative expression of luciferase activity (y-axes; rlu relative to the control infection without inhibitor). whereas bafa1 displayed dose-dependent inhibition of iav entry, dynasore did not significantly inhibit iav entry. presence of fcs (fig. s2, panel a) . as expected, both dyna-dep entry in pbs and dyna-ind entry in the presence of 10% fcs and 80 mm dynasore required sialic acid receptors for efficient entry as pre-treatment of hela cells with neuraminidases almost completely abolished entry via either pathway (fig. 2c ). the kinetics of the dyna-dep and dyna-ind entry pathways were compared by performing a time-course experiment in which iav entry was terminated by the addition of 10 nm bafa1 at different time points (fig. 2d) . in comparison to entry via the dyna-dep pathway (the only pathway available in pbs) entry in the presence of fcs (when presumably both the dyna-dep and dyna-ind entry pathways are available) showed similar kinetics. in contrast, entry via the dyna-ind pathway (which is the only pathway that is active in the presence of 10% fcs and 80 mm dynasore) was slower. the difference was most prominent after 15 min, while after 4 hrs similar levels of entry were reached. to validate and extend these results we visualized the reduction of the number of infected cells by immunoperoxidase staining using an antibody against np (fig. 3) . a number of different cells of mammalian and avian origin were infected for 2 hours at an moi of 1 in pbs with or without serum. after 2 hours the inoculum was replaced by growth medium containing 10% fcs and 10 nm bafa1 and the expression of np was examined after 14 hours later. after incubation in pbs, staining was completely prohibited by the presence of 80 mm dynasore whereas in the presence of serum dynasore had no effect. a serum-inducible, dyna-ind route of entry was thus functional in all five cell lines, including the human epithelial airway carcinoma cell line a549. to confirm our results and to obtain further proof for the utilization of dyna-dep and dyna-ind entry routes by iav, we additionally used an iav virus-like particle (vlp) direct entry assay [26] . these vlps contain iav ha and na in their envelope and harbor a beta-lactamase reporter protein fused to the influenza matrix protein-1 (blam1), which allows the rapid and direct detection of entry, independent of virus replication. upon fusion of viral and endosomal membrane, blam1 gains access to the cytoplasmically retained fluorigenic substrate ccf-2 that, after cleavage by blam1, shifts to a shorter fluorescent emission wavelength that can be detected by flow cytometry. entry into hela cells was performed in the absence or presence of 10% fcs using vlps containing ha and na either from iav-wsn (having a strict alpha 2-3 linked sialic acid binding specificity) or from the pandemic 1918 iav (ha from a/ newyork/1/18, binding to alpha 2-3 and alpha 2-6 linked sialic acids; na from a/brevigmission/1/18). entry of vlps of both iav strains was severely inhibited by dynasore when no serum was added to the inoculum (fig. 4a, 4d) , whereas the presence of 10% fcs rendered entry completely dynasore resistant. (fig. 4b, 4e ). quantification of vlp entry is shown in fig. 4c and f. importantly, to confirm the existence of the serum-inducible entry pathway by a method that is independent of dynasore, we used sirna induced silencing of dynamin 2. fig. 4g shows that two different sirnas had a significant inhibitory effect (48 hrs after sirna transfection) on entry of the renilla luciferaseencoding pseudovirus wsn-ren [27] in hela cells in the absence of fcs, whereas the presence of 10% serum no reduction in entry levels was observed, confirming the results obtained with dynasore. knockdown of dynamin 2 protein levels (48 hrs after sirna transfection) was analyzed by western we conclude that a dyna-ind entry pathway can be induced by serum in different cell types from several species. the evidence was obtained using both replication-dependent (gluc-entry assay and immunodetection of infected cells) and replication-independent assays (entry of vlps), the latter allowing immediate detection of the fusion-mediated delivery of viral m1 protein into the cytoplasm. cascade blue). in the histograms entry is displayed by a shift to higher fluorescence (the grey area represents background fluorescence of noninfected cells). (c and f) quantification of facs results. background fluorescence was subtracted from each measurement (geometric mean) and data were normalized to vlp entry in optimem without dynasore (dy) (red curve of panel a and and d). vlp entry was not inhibited by dynasore in presence of 10% fcs whereas the access of blam1 to its ccf2 substrate in the cytoplasm was blocked by dynasore in pbs. vlp entry was more efficient in the presence of serum. (g) effect of downregulation of dynamin 2 by sirna silencing. serum-inducible dyna-ind entry was analyzed in hela cells that were transfected 48 hrs prior to infection with two different sirnas targeting dynamin-2 (dyna). sirna treated cells were infected with the pseudovirus wsn-ren in pbs (grey bars) or in pbs containing 10% fcs (black bars) and luciferase activity was determined after 16 hrs post infection (y-axis; rlu relative to infection of cells transfected with a scrambled sirna). entry of pseudovirus wsn-ren (moi 0.5) was reduced by 50% to 70% when entry was performed in pbs (grey bars) whereas entry was not significantly affected in the presence of 10% serum (black bars). (h) western blot showing the knockdown of dynamin 2 (in comparison to tubulin) at 48 hrs after transfection with sirnas. (i) quantification of the residual levels of dynamin 2 (dyna) mrna (determined by quantitative rt-pcr) and protein (determined by densitometric scanning of the western blot) 48 hrs after sirna transfection. data were normalized to 18s rna (rt-pcr) or tubulin protein levels and calculated relative to the levels obtained after transfection with a scrambled sirna that served as a control. doi:10.1371/journal.ppat.1001329.g004 inhibitors of growth factor receptor tyrosine kinases and actomyosin network dynamics reduce dyna-ind entry of iav the dyna-ind entry pathway was further characterized by inhibitor profiling using an 80-compound kinase inhibitor library. serum-induced dyna-ind entry was examined in 10% fcs using the gluc-entry assay. 80 mm dynasore was added in order to block cme and any other potential dyna-dep entry pathways. this allowed the independent inhibitor profiling of the novel pathway by avoiding the potentially masking effect of the presence of redundant entry pathways. cells were preincubated with the kinase inhibitors (10 mm) for 1 h at 37uc and then inoculated with virus (moi 0.5) in the presence of 10% fcs and 80 mm dynasore for 2 h at 37uc (dyna-ind entry). in parallel, inoculations were also done in pbs to compare the effects of the inhibitors on dyna-dep entry. after 2 hr the medium and inhibitor were replaced by full growth medium containing 10% fcs and 10 nm bafa1 to allow the subsequent expression of gluc activity under identical conditions for the dyna-ind and -dependent entry assay. six kinase inhibitors appeared to act non-discriminatively, inhibiting both dyna-dep and dyna-ind entry (fig. 5a ): the protein kinase c (pkc) inhibitors ro 31-8220, rottlerin (both displaying moderate cytotoxicity, result not shown) and hypericin, which have all three been previously identified as iav inhibitors [28, 29] ; the highly cytotoxic pan-specific serine/threonine protease inhibitor staurosporine; the irreversible pi-3 kinase inhibitor wortmannin and the receptor tyrosine kinase inhibitor tyr9. in order to investigate whether some of these inhibitors affect iav replication during the post-entry phase, we performed the same experiments but now adding the kinase inhibitors after viral entry. four of the inhibitors thus appeared to induce significant inhibition of post-entry processes (fig. 5a ). although unlikely, we cannot formally exclude that post-entry processes specific for only one of the two entry pathways are affected. interestingly, whereas no specific dyna-dep entry inhibitors were identified, 15 inhibitors (none displaying cytotoxic effects, data not shown) caused significant (p,0.05) inhibition (.5-fold) of dyna-ind entry (fig. 5b ). this included inhibitors of the calmodulin dependent kinases myosin light chain kinase (mlck) and camkii and seven inhibitors of different growth factor receptor tyrosine kinases. in contrast to the three non-specific pkc inhibitors mentioned above, the pkc inhibitors bim-1 and hbdde appeared to have a specific inhibitory effect on dyna-ind entry. the specific effect of these drugs on dyna-ind entry is not only shown by the lack of inhibition of dyna-dep entry in pbs, but also by the observation that none of the fifteen compounds induced .2-fold inhibition when added post-entry (at t = 2 hr post infection). the kinase library screen was repeated on a549 human epithelial lung carcinoma cells in order to confirm the results in a potentially more natural host cell line. the inhibition profiles obtained were very similar to those found for hela cells with the exception of the strong effect of ag879 (99% inhibition) and moderate effects of ag825 (39% inhibition) and tyr51 (68% inhibition) on dyna-dep entry. (fig. 5c) . mlck inhibitors ml-7 and ml-9 have been reported to be highly specific for their target kinase [30] . phosphorylation by mlck activates non-muscle myosin ii light chain, indicating that a functional actomyosin network might be essential for dyna-ind entry of iav. this was further examined by testing the effect of blebbistatin, an inhibitor of myosin ii heavy chain activity, and of several inhibitors that affect actin dynamics by disrupting actin microfilaments (cytochalasin b and d), by enhancing actin polymerization (jasplakinolide) or by inhibiting actin polymeriza-tion (latrunculin a). actin inhibitors were used at the minimal concentration required to induce clearly visible changes in the actin cytoskeleton as pre-determined by staining with fitcphalloidin (results not shown). whereas the inhibitors did not affect dyna-dep entry (fig. 6a) using gluc-entry assay, all inhibitors as well as ml-7 and ml-9 significantly inhibited dyna-ind entry (fig. 6b) . next, hela cells were transfected with plasmids encoding dominant negative or wildtype rab5 fused to green fluorescent protein (rab5 dn and rab5 wt in fig. 6 ) 24 h prior to infection with iav. rab5 is a small gtpase found in association with several endosomal compartments and crucial for the function and maturation of early endosomes. it is required for the trafficking of a wide range of endocytic cargo following different routes, including dyna-dep as well as dyna-ind routes [31] . entry of iav has been shown to require rab5 [32] . consistently, we found that hela cells expressing rab5 dn (as identified by gfp fluorescence, fig. 6c ) were much less susceptible to productive iav infection (as judged by indirect immunofluorescence using alexa-488 labeled np antibodies) than cells transfected with several dynamin-independent endocytic pathways have been described [8, 19] . of these, macropinocytosis has been demonstrated to be stimulated by growth factors present in serum and to depend on actin dynamics [12] [13] [14] . yet, studies on macropinocytosis are hampered by a lack of specific inhibitors, cargo, membrane markers and characteristic morphology. amiloride and the more potent derivative eipa are inhibitors of epithelial sodium channels (enac) as well as of several other na+/h+ antiporters. eipa has often been used as a hallmark inhibitor that specifically inhibits endocytosis via the macropinocytic pathway [14] . whereas dyna-dep entry of iav was not inhibited by eipa (fig. 7a) , dyna-ind entry was fully blocked eipa (fig. 7b) . the existence of redundant entry pathways in the presence of 10% fcs is clearly demonstrated by the marginal inhibition by either eipa or dynasore whereas the combination of eipa and dynasore resulted in strong inhibition both in the gluc-entry assay (fig. 7c ) and in the direct vlp entry assay ( fig. 7d and e) . supplementary fig. s1 shows that other cell lines, including the human lung epithelial cell line a549, display similar iav inhibition patterns for eipa and dynasore. consistently, virus production displayed a similar inhibitor sensitivity profile (fig.7 f and g) as virus entry indicating that the entry pathways we characterized lead to a productive infection. clearly, vlps and viral particles follow similar redundant entry pathways, distinguishable in a dyna-dep and a dyna-ind pathway, the latter being sensitive to eipa and dependent on actomyosin function. one characteristic of macropinocytosis is the nonselective uptake of large amounts of extracellular solutes [33] . therefore, the uptake of soluble fitc labeled dextran (fdx) into relatively large vesicles (0.3 to 5 mm) has often been applied as a morphological marker for macropinosomes. using this marker we found that the addition of 10% fcs to the culture medium slightly increased the uptake of fdx into hela cells (fig. 8a) . notably, the distribution of fdx changes in response to serum from a random distribution into a more granular pattern. at high magnification and at color settings adjusted to higher intensity it could be seen that these fdx granules were free of actin staining (by phalloidin) indicating that they were in the lumen of vesicles (result not shown). interestingly, in the presence of iav (moi of 10) the uptake of fdx into vesicles was clearly enhanced. at a higher magnification viral particles could be found to co-localize in fdx loaded vesicles as well as outside these vesicles (fig. 8b) . phalloidin staining of actin was used to demonstrate that many virus particles localized to actin-rich protrusions at the periphery of the cell. the uptake of fdx was studied in a quantitative manner by flow cytometry (fig. 8 c) . a moderate, but reproducible shift to higher fdx fluorescence was observed at 37uc when virus was added in presence of 10% fcs whereas such a shift was absent when no serum or virus was added. this result confirms the observations by confocal microscopy (fig. 8a) which showed that the combined presence of fcs and iav increases the uptake of fdx as compared to fcs alone. in a control experiment the uptake of fdx in 10% fcs in presence of iav was shown to be specifically inhibited by eipa, but not by dynasore (fig. s2, panel b) . in contrast, transferrin uptake, which serves as a specific marker for cme, was affected by dynasore, but not by eipa (fig. s2, panel a) . in conclusion, serum induces the uptake of fdx into large vesicles, which can be further enhanced by the addition of iav particles that, after entry, co-localize in part with these vesicles. these results indicate the utilization of a macropinocytic pathway for entry of iav, which is consistent with the observed sensitivity of the seruminducible dyna-ind entry of iav and vlps to eipa. macropinocytosis has been implicated in the entry of several viruses [12, 14] . however, differences in susceptibility to inhibitors suggest that distinct forms of macropinocytosis might be used by different viruses [34, 35] . by screening specific inhibitors in the gluc-entry assay using dyna-ind entry conditions we evaluated the possible involvement of a few signaling cascades that have been implicated in the induction of macropinocytosis. serum-inducible macropinocytosis has been shown to be activated via a myriad of signaling cascades initiated by growth factors binding to transmembrane tyrosine kinase receptors [14, 17, 36, 37] , consistent with the results shown in fig. 5 . a prominent downstream effect of these signaling cascades is the activation of p21 associated kinase 1 (pak1) which in turn can activate a number of different pathways leading to actin network rearrangements that can ultimately lead to the induction of macropinocytosis [38] . fig. 9a -b shows that 20 mm ipa3, an inhibitor of pak1 [39] , specifically inhibits background fluorescence from fdx binding to the outside of cells was determined by performing the same experiment at 4uc (at which no endocytosis takes place) and was subtracted from the mean fluorescence intensity obtained at 37uc to determine the amount of fluorescent fitc-dextran that was internalized at 37uc. data were plotted relative to fitcdextran uptake in pbs in absence of iav. doi:10.1371/journal.ppat.1001329.g008 dyna-ind entry of iav. activation of pak1 in response to growth factor stimulation often involves upstream signal transduction by members of the rho sub-family of small gtpases like cdc42 and/or rac1 [34, 40, 41] . alternatively, activated cdc42 and rac1 can induce actin rearrangements independently of pak1 [34, [40] [41] [42] [43] by direct interaction with wasp or wave family proteins, respectively [44, 45] . however, inhibitors of cdc42 (pirl1 [46] ), rac1 (nsc23766 [47] ) or n-wasp (wiskostatin [48] ) did not display inhibitory effects on dyna-ind or dyna-dep entry of iav (fig. 9c-d) . instead, pirl1 and wiskostatin induced a significant, concentration dependent increase of entry. this stimulatory effect was not observed for the control vaccinia virus strain wr, which enters cells via a rac1dependent, macropinocytotic pathway [43] (fig. 9e) , indicating that this effect is specific for iav. the results suggest a requirement for pak1 in dyna-ind entry of iav that does not require activation by either cdc42 or rac1. growth factor inducible activation of the tyrosine kinase src has also been linked to the induction of macropinocytosis [49] [50] [51] ; consistent with this observation the src inhibitor pp2 [52] specifically inhibited dyna-ind entry of iav (fig. 9a-b) . remarkably, 17-aageldanamycin, a specific inhibitor of the chaperone protein hsp90 [53] , also caused specific inhibition of dyna-ind entry (fig. 9a-b) . hsp90 affects the folding and activity of many proteins but the recent demonstration of direct activation of the catalytic activity of src by hsp90 [54] provides another indication of the involvement of src in dyna-ind endocytosis of iav. in conclusion, like for other viruses utilizing a macropinocytic entry pathway, pak1 seems to play a crucial role in dyna-ind entry by iav. however, this pathway is independent of rac1 or cdc42 but may require src, either upstream and/or downstream of pak1. the data presented in this study demonstrate for the first time that iav can enter cells via dyna-ind macropinocytosis in addition to the previously described dyna-dep classical cme pathway [1, 2] . several lines of evidence indicate that the dyna-ind entry route of iav that we identified corresponds with macropinocytosis. first of all, the entry pathway is dependent on the presence of serum, a well-known inducer of macropinocytosis. second, iav colocalized in vesicles with soluble fitc-dextran, a marker for macropinocytosis. third, dyna-ind iav entry was sensitive to the amiloride-derivative eipa, the hallmark inhibitor of macropinocytosis [14, [55] [56] [57] [58] . fourth, this iav entry pathway is sensitive to inhibitors or dominant-negative mutants affecting actomyosin dynamics. fifth, the specific inhibition of dyna-ind iav entry by a number of inhibitors of growth factor receptor tyrosine kinases as well as downstream effectors thereof also points at the involvement of macropinocytosis. finally, macropinocytosis is independent of dynamin [12, 14, 19] . despite this extensive list of arguments, viral entry by macropinocytosis needs to be considered with caution. the characteristics of the dyna-ind route of cell entry by iav are similar, but not identical to the macropinocytic entry routes taken by other viruses, like two different strains of vaccinia virus and by coxsackie virus b [34, 35] . as is shown in table 1 and discussed in more detail below, the macropinocytic pathways used by each of these viruses have a few unique characteristics. this may very well reflect the growing notion that macropinocytosis represents a number of differentially induced and regulated processes, rather than being a single endocytic pathway [13, 14] . macropinocytosis has collectively been described as an inducible form of endocytosis by which fluid-phase cargo travels via non-coated, relatively large and heterogeneous organelles that have emanated from extensive protrusions (e.g lamellar ruffles, circular ruffles or retracting blebs) of the plasma membrane [13] . in the case of dyna-ind iav entry more extensive studies using electron microscopy will be required to study the morphology of membrane protrusions with which iav may associate. in addition, live cell imaging microscopy will be required to characterize the exact itinerary that is taken by iav virions traveling via a macropinocytic process. this is especially important as different routes of iav entry are likely to converge at some point in the endocytic pathway. although unlikely, co-localization of iav particles with fluid-phase dextran as shown in fig. 8b may thus represent a situation occurring after convergence of several different routes. the use of microscopy to study macropinocytosis is however complicated by the lack of specific membrane-associated markers for any early step of this endocytic process. a model (fig. 10 ) based on our results explains the key steps involved in the macropinocytic entry pathway of iav, which are described in more detail below. by manipulating the inoculation conditions we were able to experimentally dissect iav entry into a dyna-dep and dyna-ind route. the dyna-ind route required the presence of 10% fcs in the entry assay medium. previously, a strict dependency on a dyna-dep entry route for iav was concluded from experiments with a cell line expressing an inducible dominantnegative mutant of dynamin 2 [59] . in that study, as well as in other entry studies of iav, entry was performed in dmem containing 2% serum or bsa. also in our hands 2.5% serum ( fig. 2a) or 0.2% bsa (result not shown) was not sufficient to allow dyna-ind entry. we are currently investigating which serum component is responsible for the observed effects on iav entry. dialysis of fcs (mw cut off .10 kda) did not affect its capacity to induce dyna-ind endocytosis (result not shown), indicating that low molecular weight solutes are not responsible for the observed effect. our evidence for a dyna-dep and a serum inducible dyna-ind entry route is based on the use of pharmacological (dynasore, a highly specific inhibitor of dynamin) as well as genetic (sirna directed against dynamin 2) tools, ruling out the possibility that the inhibitory effect of dynasore was due for instance to absorption of the inhibitor by serum components. whereas dynasore resulted in near 100% inhibition of dyna-dep entry, only 65% inhibition was observed upon sirna induced silencing of dynamin 2 indicating that the residual levels of dynamin 2 that remain after 48 hrs of silencing still support a low level of dyna-dep entry (fig. 4h) . reversible inhibitors like dynasore [60] offer a major advantage for characterization of iav entry pathways. they can be applied for a limited period thus preventing the secondary adaptive effects of cells that may occur in response to long-term down regulation of a gene product by genetic methods like sirna interference. both entry routes were consistently identified by a viral entry assay quantified by virus induced expression of a luciferase reporter as well as by a vlp entry assay allowing direct analysis of the membrane fusion mediated entry step. the consistent performance of an ha with a strict preference for binding to a2-3 linked sialic acids (from iav-wsn; our unpublished data) and an ha also binding to a2-6 linked sialic acids (from 1918 iav [61] ) in the vlp entry assay indicates that both pathways can be utilized by has of different specificity and may therefore be relevant to avian as well as human iav infections. consistently, serum-inducible dyna-ind entry was observed both in avian df1 cells and in a human lung epithelial carcinoma cell line a549 (fig. 3) . the dyna-dep and dyna-ind iav entry pathways were found by our quantitative assays to be fully redundant. in the presence of serum, the combination of dynasore (inhibiting dyna-dep entry) and eipa (inhibiting dyna-ind entry) completely abolished entry whereas either drug alone had no effect. eipa, an inhibitor of plasma membrane na+/h+ exchangers, has been shown to invariably inhibit macropinocytosis [14, [55] [56] [57] [58] . as other routes of endocytosis are generally not affected, eipa is considered as a hallmark inhibitor of macropinocytosis [14] , although results obtained with eipa should be considered with care as long as a mechanistic explanation for its effect on macropinocytosis is not yet fully clear [62] . occasionally, a moderate two-to three-fold inhibition by dynasore alone was observed (result not shown) indicating that the capacity of the serum-inducible entry pathway is somewhat variable, possibly depending on slight variations in serum quality and factors like cell distribution in the wells that have been reported to influence viral infection [63] . a redundancy in the utilization of cme as well as a clathrin-independent route for entry of iav has been visualized previously by quantitative live cell imaging [4] . both routes were operative simultaneously in the same sample and the specific down-regulation of cme did not affect the total number of entry events. in response to specific extra-cellular signals (e.g. serum induction), changes in the actomyosin network occur that give rise to membrane protrusions required for macropinosome formation [13] . compounds inhibiting actin polymerization (cytochalasin b and d), depolymerization (jasplakinolide) or sequestering soluble actin (latrunculin a) all specifically inhibited dyna-ind iav entry. in addition, the requirement for myosinii activity was established by a specific inhibitor (blebbistatin) of myosin ii atpase activity and by the expression of a dominant negative mutant of myosiniia heavy chain. also, the regulation of myosinii activity by phosphorylation of myosin light chain through the action of mlck is suggested by the inhibitory effect of mlck inhibitors ml-7 and ml-9 as well as by the similar effect of an expressed mlck dominant negative mutant. recently, a function for the actin cytoskeleton in iav entry was reported to be required for the entry into polarized epithelial cells but not for entry into non-polarized cells [64] . when using the low-serum conditions used in that paper (2% fcs), we only observed dyna-dep entry that was not affected by actin dynamics inhibitors. perhaps, the polarized cells permit dyna-ind entry at lower serum concentrations. the changes in actin network dynamics that can lead to the formation of macropinosomes can be triggered by a number of signaling cascades. actin dynamics are induced by the activation of growth factor receptor tyrosine kinases by their respective figure 10 . a model for iav entry by macropinocytosis. the model summarizes the inhibitory (red boxes) or stimulatory (blue boxes) effects of compounds on dynamin-independent iav entry. the effect of over-expression of dominant-negative mutants is indicated by red-lined boxes. the pathway requires the presence of serum factors in the entry medium and results in the enhanced uptake of dextran and its co-localization with iav in large vesicles (green boxes). we hypothesize that the interaction of serum factors and/or iav with receptor tyrosine kinases (rtks) is the primary signal for the induction of macropinocytosis. a number of rtks have been shown to be involved in this process in different cell lines. remarkably, a recently published genome-wide sirna screen of iav infection identified the fgf receptor as a host factor required for influenza virus replication [22] . activation of rho family gtpases cdc42 and/or rac1 has been shown to be essential for signal transduction leading to macropinocytosis in many cases [13, 14] but inhibitors are without effect or are stimulatory in the case of iav entry. downstream effectors of rho family gtpases include scaffold proteins like n-wasp and wave and protein kinases like pak1. macropinocytic entry of iav however seems to require a rho family gtpaseindependent pak1 activation mechanism. in addition, src family kinases, which can be directly activated by rtks, play a role. pak1 and src have previously been linked to the activation of macropinocytosis via their effect on changes in actomyosin dynamics, a process which is crucial to any form of macropinosome formation [13, 14] . apart from n-wasp-or wave-containing macromolecular assemblies other actin binding proteins can induce such changes (e.g. cortactin, which can be activated by src [81] ) and thereby induce the formation of one of the different plasma membrane protrusions that can result in the formation of macropinosomes. in addition to an effect on the formation of plasma membrane protrusions and subsequent macropinosome formation, inhibitors can also affect downstream trafficking and maturation of macropinosomes which might be actindependent, but this is not depicted in the scheme. doi:10.1371/journal.ppat.1001329.g010 growth factor ligands that are normally present in serum [12] [13] [14] 17, 36, 37] the signal transduction cascades that link activation of growth factor receptor tyrosine kinases to actin remodeling and macropinocytosis are only beginning to be revealed. the specific inhibition of dyna-ind entry of iav by ipa3, an inhibitor of pak1, provides proof for the involvement of these cascades. pak1 is a key serine/threonine kinase regulating actin network dynamics but its crucial function in several pathways of endocytosis as well as numerous other cellular processes does not make it a very specific marker [65] . even so, macropinocytosis has consistently been demonstrated to require pak1 activation, both in the induction of the process and/or in further downstream trafficking events of macropinosomes [13, 14] . growth factor dependent activation of pak1 has most often been demonstrated to depend on upstream activation of small gtpases rac1 or cdc42 [34, 40, 41] . different strains of vaccinia virus were recently shown to induce their uptake by macropinocytosis via activation of either rac1 or cdc42 [34] . activation of rac1 has been linked to the induction of macropinocytosis via actin network-mediated formation of lamellipodia and/or circular ruffles whereas cdc42 has most often been implied in the formation of filopodia [44] . an inhibitory effect of the rac1 inhibitor nsc23766 or the cdc42 inhibitor pirl1 on iav entry, however, could not be demonstrated. remarkably, cdc42 inhibitor pirl1 enhanced iav entry and a similar effect was observed by wiskostatin, an inhibitor of n-wasp which functions directly downstream of cdc42 as a scaffolding complex required for the activation of actin polymerization leading to filopodia formation. similarly, the macropinocytosis-like entry pathway taken by coxsackie b virus was also shown to require pak1 activity that was independent of rac1 activation [35] . direct examination of the magnitude and timing of the activation of pak1 will be required to obtain more insight in the involvement of this complex pathway. the induction of macropinocytosis by a pak1dependent mechanism has been associated with ruffling at the cell membrane [12, 14, 15, 37] . the identification of sub-membranous regions with increased actin staining by phalloidin has been interpreted as evidence for ruffling. this was not unambiguously identified by confocal microscopy in the experiments presented in fig. 8 and fig. s2 and needs to be investigated in depth by life cell imaging techniques. in agreement with our observation that the dyna-ind entry of iav was inhibited by pp2, an inhibitor of src family kinases, the non-receptor tyrosine kinase c-src has been shown to function as a key signaling intermediate in the induction of macropinocytosis via a mechanism independent of rac1 or cdc42 [49] [50] [51] . downstream effects of c-src on actin networks proceed, amongst others, via phosphorylation of cortactin by c-src resulting in accelerated macropinosome formation [50] . c-src has been shown to associate with macropinosomes [49, 51] , both during their formation and their trafficking, while c-src kinase activity is required for macropinocytosis following egf stimulation of hela cells [49] . interaction of hsp90 with c-src was recently shown to induce c-src kinase activity [54] . also hsp90 has been demonstrated to associate with macropinosomes, while its specific inhibitor geldanamycin reduced the membrane ruffling that preceded macropinocytosis [66] . thus, the inhibition of iav entry via macropinocytosis by aa-geldanamcyin may very well involve the effects of hsp90 on c-src. as detailed above, the dyna-ind entry pathway of iav shares many characteristics with the endocytic pathway macropinocytosis. this is corroborated by the observation that iav particles and dextran colocalize in large vesicles in the presence of fcs. several viruses have recently been reported to enter cells via macropinocytosis [12, 14] . apart from common factors like the requirement for pak1 activation, actin dynamics and independence of dynamin, virus specific details have been described [34, 35] (table 1 ). in part these might be contributed to differences in experimental conditions (e.g. cell types tested) but diversity in the molecular mechanisms by which macropinocytosis can be induced and executed is likely to exist and to be exploited by viruses. whereas vaccinia virus is able to trigger its own macropinocytic uptake [34, 43] , we have described a macropinocytosis pathway that is operational under conditions that are activated by components in serum. still, this does not exclude signaling induced by virus-host cell interactions, which are for instance suggested by the significant increase of fitc-dextran uptake in the presence of iav. the possible requirement for costimulatory signals from serum components and virus imposes an additional layer of complexity on the analysis of iav entry via dyna-ind pathways. influenza viruses cause respiratory infections by targeting the epithelial cells lining the respiratory tract. these surfaces are covered by a mucous layer composed of a variety of small solutes and glycoproteins derived among others from goblet cells [67] . this semi-fluid layer in turn conditions the underlying cells and determines their physiological state, including the activities of their uptake and secretion pathways. it will be important to determine to what extent the dyna-dep and dyna-ind iav entry pathways are operational under the conditions prevailing along the respiratory tract. current knowledge on the protein composition of the fluids covering the respiratory epithelium is rapidly expanding by the application of proteomic methods to determine the protein composition of bronchial alveolar lavage fluids (balf). these studies have extended the previous notion that balf is highly similar in composition to serum. for example, just as for the serum proteome more than 85% of the total protein mass of the balf proteome is accounted for by albumin, immunoglobulins, transferring, a1-antitrypsin and haptoglobin. in addition, many other proteins have been identified both in serum and in balf including growth factors that can bind to growth factor receptor tyrosine kinases [68] [69] [70] . thus, balf is likely to harbor, just as serum, the protein factors that can activate signaling pathways that are crucial for the induction of dyna-ind entry of iav. in agreement herewith, macropinocytosis has been described as a functional entry pathway of haemophilus influenzae into primary human bronchial epithelial cells [71] although the factors involved in signaling the process have not been identified yet. in addition to infecting the respiratory tract, iav has been shown to be able to cause systemic infections involving multiple organs. this has mainly been studied in avian infections [72, 73] or by infection of mice with human-derived h1n1 or h3n2 iavs [74] but is poorly documented for human infections and may have been underestimated thus far. obviously, during potential systemic spreading of iav, the serum-rich conditions that we have demonstrated here to enable the use of alternative entry pathways will be encountered and may contribute to such spreading. mdck, a549, df-1 and hela cells were maintained in complete dulbecco's modified eagle's medium (dmem) (lonza, biowittaker) containing 10% (v/v) fetal calf serum (fcs; bodinco b.v.), 100 u/ml penicillin, and 100 mg/ml streptomycin. chinese hamster-e36 cells were maintained at 37uc in a-minimal essential medium (gibco) supplemented with 10% (v/v) fcs, 100 u/ml penicillin, and 100 mg/ml streptomycin. cells were passaged twice weekly. influenza a/wsn/33 (h1n1) (iav-wsn) was grown in mdck cells. briefly, ,70% confluent mdck cells were infected with iav-wsn at a moi of 0.02. supernatant was harvested after 48 hr of incubation at 37uc and cell debris was removed by centrifigutation (10 min at 2000 rpm). virus was stored at 280uc and virus titers were determined by measuring the tcid 50 on hela cells. the iav-wsn luciferase pseudovirus (wsn-ren) system has previously been described [27] . briefly, wsn-ren pseudovirus harbors a ha segment in which the ha coding region is replaced by renilla luciferase. the pseudovirus is produced in a mdck cell line that stably expresses the ha of iav-wsn. wr-luc, a firefly luciferase encoding vaccinia virus (strain wr) was previously described [75] . vsv-fl, a firefly luciferase encoding vsv virus was also previously described [76] . stocks of bafilomycin a1 (bafa1), dynasore, cytochalasin d, cytochalasin b, blebbistatin, 17-aa-geldanamycin, ml-7, ml-9, pp-2, 5-(n-ethyl-n-isopropyl)amiloride (eipa), ipa-3 (all obtained from sigma-aldrich), latrunculin a (enzo), jasplakinolide, wiskostatin, nsc23766 (all obtained from calbiochem) and pirl1 (chembridge) were prepared in dimethylsulfoxide (dmso). all stocks were stored at 220uc. a kinase inhibitor library composed of 80 kinase inhibitors was obtained from biomol (2832a[v2.2]). hela cells (10,000 cells/well in 96-well plates) were treated with 2 munits of vibrio cholerae neuraminidase (roche) in 50 ml phosphate-buffered saline (pbs) for 2 hr. after washing with pbs cells were infected with iav as described. virus-like particles (vlps) were produced as described [26] . briefly, 293t cells were transfected using lipofectamine 2000 (invitrogen) with pcaggs-blam1 (encoding a beta-lactamase reporter protein fused to the influenza matrix protein-1), pcaggs-ha (encoding ha derived from either a/newyork/ 1/1918 or iav-wsn) and pcaggs-na (encoding iav neuraminidase [na] derived from either a/brevigmission/1/18 or iav-wsn) and maintained in optimem. supernatants were harvested 72 h after transfection and centrifuged to remove debris. vlps were used for inoculation of cells without further concentration. vlps were incubated for 30 min at 37uc with trypsin/tpck for activation of ha. mdck or hela cells grown to near confluency in 24-well plates were inoculated with 250 ul of vlps after pre-treatment of the cells with inhibitors as indicated. infection was synchronized by centrifugation at 1500 rpm for 90 min at 4uc and was performed by further incubation at 37uc for 2 h in the absence or presence of 10% fcs and inhibitors as indicated. detection of beta-lactamase activity was performed as described [25] by loading cells with ccf2-am substrate (invitrogen) and subsequent analysis by flow cytometry on a lsrii flow cytometer (becton dickinson). typically 10,000 events were collected and analyzed using flowjo 8.5.2 software. the reporter construct phh-gluc was derived from plasmid phh-fluc [22] by replacing the firefly luciferase coding region with the gaussia luciferase coding region of pgluc-basic (new england biolabs). unique spei and xbai restriction sites were introduced into phh-fluc using the quikchange xl site-directed mutagenesis kit (stratagene) and oligonucleotides spe4262 (5-9gcctttctttatgtttttggcactagtcattttaccg-atgtcactcag), spe4263 (59-ctgagtgacatcggtaa-aatgactagtgccaaaaacataaagaaaggc), xba4260 (59-gtatttttctttacaatctagactttccgcccttc-ttgg) and xba4261 (ccaagaagggcggaaagtctag-attgtaaagaaaaatac). a spei site was introduced by sitedirected mutagenesis in pgluc-basic directly following the start codon of the gaussia luciferase coding sequence. the unique spei -xbai fragment of pgluc-basic was subsequently cloned into the spei-xbai site of phh-fluc resulting in plasmid phh-gluc. cells were seeded in 96-well plates at a density of 10,000 cells/ well and transfected the next day with 10 ng phh-gluc using lipofectamine 2000 (invitrogen) according to the manufacturer's protocol. after 24 hrs the transfected cells were treated with inhibitors and infected as indicated. at 16 hr p.i. samples from the supernatant were assayed for luciferase activity using the renilla luciferase assay system (promega) according to the manufacturer's instructions, and the relative light units (rlu) were determined with a berthold centro lb 960 plate luminometer. wr-luc and vsv-fl were used to inoculate hela cells (10,000 cells/well) at an moi of 2, in complete dulbecco's modified eagle's medium (dmem) (lonza, biowittaker). after 7 hr the luciferase activity was detected using the steadyglo assay kit (promega). the addition of 10% (v/v) fcs did not change infection levels for both viruses. cells were fixed with 3.7% paraformaldehyde (pfa) in pbs and subsequently permeabilized with 0.1% triton-x-100 in pbs. after blocking with normal goat serum iav-infected cells were incubated for 1 h with a monoclonal antibody directed against the nucleoprotein (np) (hb-65; kindly provided by dr. ben peeters). after washing, the cells were incubated with a 1:400 dilution of alexa fluor 488-or 568-labeled goat anti-mouse igg (molecular probes) secondary antibody for 1 h. nuclei were subsequently stained with topro-3 and after three washing steps, the coverslips were mounted in fluorsave (calbiochem). actin was stained using phalloidin labeled with alexa fluor 633. the immunofluorescence staining was analyzed using a confocal laser-scanning microscope (leica tcs sp2). fitc, gfp or alexa fluor 488 were excited at 488 nm, alexa fluor 568 at 568 nm, and topro-3 at 633 nm. hela cells were grown in 24-well plates on glass coverslips (50,000 cells/well). prior to fitc-dextran uptake cells were serum-starved for 2 hr in pbs. fitc-dextran (mw70,000, sigma-aldrich) was incubated with hela cells (final concentration of 0.5 mg/ml) in 500 ml pbs or in pbs containing 10% fcs in the absence or presence of iav (strain wsn; moi 10; concentrated and purified by centrifugation through a 15 to 30% sucrose gradient with a 50% sucrose cushion at the bottom) at 37uc. after 15 min cells were washed 4 times with pbs at 4uc, fixed with 3.7% pfa in pbs and subsequently permeabilized with 0.1% triton-x-100 in pbs. slides were stained for examination by confocal microscopy as described above. for quantification of fitc-dextran uptake 1.5610 5 hela cells were infected with iav-wsn (moi 10) in suspension in a volume of 1 ml in the presence of fitc-dextran (1 mg/ml). infections were performed for 15 min in pbs (containing 2% bsa to reduce unspecific binding of fitc-dextran) or in pbs containing 10% fcs at 37uc or at 4uc (control for binding of fitc-dextran to cells in the absence of endocytosis). mock-infected samples were analysed in parallel. infection was terminated by addition of 3 ml ice-cold pbs followed by three washes with cold pbs and fixation with 3.7% pfa. 20,000 cells were analyzed by facs and results were represented as the mean fluorescence which was plotted relative to the uptake in the mock-infection in pbs (after subtraction of background fluorescence obtained at 4uc). the effect of dynasore and eipa on dextran and transferrin uptake hela cells (grown on glass cover slips) were incubated at 4uc for 1 hr with 50 mg/ml alexa633-labeled transferin (invitrogen) in pbs. after 1 hr the medium was replaced by pbs or pbs supplemented with 10% fcs containing iav (strain wsn; moi 10) and 0.5 mg/ml fitc-dextran (sigma; 70 kda) and cells were transferred to 37uc for 15 min. after 15 min cells were fixed and stained as described above and examined by confocal microscopy. cells were fixed with 3.7% pfa in pbs and subsequently permeabilized with 0.1% triton-x-100 in pbs. peroxidase was visualized using an aec substrate kit from vector laboratories. iav-positive cells were detected using bright-field light microscopy. two sirna duplexes targeting different sites within the coding sequences of dynamin 2 were obtained from ambion inc (15581 (dynamin 2 sirna 1) and 146559 (dynamin 2 sirna2)). a scrambled sirna (ambion inc.) was taken along as a control for non-specific effects of the transfection procedure and was used for normalization. one day after seeding in 96-well plates (6,000 cells/well), the hela cells were transfected with a final concentration of 10 nm sirna using oligofectamine (invitrogen). 48 h after transfection, the cells were inoculated with the wsn-ren pseudovirus (moi 0.5) in pbs or in pbs containing 10% fcs. after 2 h of infection the entry medium was replaced by complete growth medium containing 10 nm bafa1 to prevent further entry. at 16 h post infection intracellular renilla luciferase expression was determined as described above. each sirna experiment was performed in triplicate. cell viability was not affected as determined by performing a wst-1 cell-viability assay (roche). functional knockdown of dynamin 2 mrna levels was performed by quantitative rt-pcr. using a taqman gene expression assay for dnm2 (hs00191900_m1, ambion) and using 18s rna (hs03928985_g1, ambion) as a control for normalization. the comparative ct-method was used for quantification of the results [77] . reduction of dynamin 2 protein levels was determined by western blotting using polyclonal goat-anti-dynamin 2 c18 (santa-cruz sc-6400). a monoclonal against alpha-tubulin (dm1a, sigma t9026) was used to detect tubulin for normalization. results were quantified by densitometric scanning of the dynamin 2 and tubulin signals displayed in fig. 4h . hela cells were grown in 24-well plates on glass coverslips (50,000 cells/well) for 24 hrs. cells were then transfected (1 mg of dna with lipofectamine 2000 as described above) with plasmids encoding wild-type or dominant-negative (dn) human mlck fused to gfp [78] , wild-type or dn rab5 fused to gfp [79] , or myoii-tail or myoii-head domain fused to gfp [80] . 24 hr after transfection cells were inoculated with iav-wsn (moi 1) in pbs or in pbs containing 10% fcs and 80 mm dynasore. 4 hr after infection cells were fixed and stained for examination by confocal microscopy as described above. an unpaired student's t-test was used for detemination of statistically significant differences. the use of the term significant in text refers to a comparison of values for which p,0.05. studies on the mechanism of influenza-virus entry into cells infectious entry pathway of influenza virus in a canine kidney cell line assembly of endocytotic machinery around individual influenza viruses during viral entry epsin 1 is a cargo-specific adaptor for the clathrinmediated endocytosis of influenza virus influenza virus entry and infection require host cell n-linked glycoprotein influenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis endocytosis of influenza viruses mechanisms of endocytosis molecular mechanisms of clathrin-independent endocytosis endocytosis unplugged: multiple ways to enter the cell virus entry: open sesame virus entry by endocytosis defining macropinocytosis virus entry by macropinocytosis ruffles induced by salmonella and other stimuli direct macropinocytosis of bacteria phosphatidylserine (ps) induces ps receptor-mediated macropinocytosis and promotes clearance of apoptotic cells origin, originality, functions, subversions and molecular signaling of macropinocytosis clathrin-independent endocytosis: a unique platform for cell signaling and pm remodeling pathways of clathrin-independent endocytosis dynamin, endocytosis and intracellular signaling ctbp3/ bars drives membrane fission in dynamin-independent transport pathways virus-inducible reporter genes as a tool for detecting and quantifying iav replication involvement of the vacuolar h(+)-atpase in animal virus entry role of clathrin-mediated endocytosis during vesicular stomatitis virus into different host cells vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization an enzymatic virus-like particle assay for sensitive detection of virus entry human host factors required for influenza virus replication virucidal activity of hypericin against enveloped and non-enveloped dna and rna viruses modulation of influenza virus replication by alteration of sodium ion transport and protein kinase c activity the specificities of protein kinase inhibitors: an update rab proteins as membrane organizers differential requirements of rab5 and rab7 for endocytosis of influenza and other enveloped viruses vaccinia virus strains use distinct forms of macropinocytosis for host-cell entry virus-induced abl and fyn kinase signals permit coxsackievirus entry through the epithelial tight junctions phosphoinositide metabolism during membrane ruffling and macropinosome formation in egf-stimulated a431 cells membrane ruffling and signal transduction regulation of macropinocytosis by p21-activated kinase-1 an isoform-selective, small-molecule inhibitor targets the autoregulatory mechanism of p21-activated kinase the small gtp-binding protein rac regulates growth factor-induced membrane ruffling activation of rho gtpases by cytotoxic necrotizing factor 1 induces macropinocytosis and scavenging activity in epithelial cells ras-related gtpases and the cytoskeleton vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells rho gtpases and actin dynamics in membrane protrusions and vesicle trafficking regulation of actin dynamics by wasp family proteins the cdc42 inhibitor secramine b prevents camp-induced k+ conductance in intestinal epithelial cells rational design and characterization of a rac gtpase-specific small molecule inhibitor chemical inhibition of n-wasp by stabilization of a native autoinhibited conformation role of src-family kinases in formation and trafficking of macropinosomes src triggers circular ruffling and macropinocytosis at the apical surface of polarized mdck cells ) c-src trafficking and co-localization with the egf receptor promotes egf ligand-independent egf receptor activation and signaling modulation of the fcepsilon receptor i signaling by tyrosine kinase inhibitors: search for therapeutic targets of inflammatory and allergy diseases ansamycin inhibitors of hsp90: nature's prototype for anti-chaperone therapy geldanamycin inhibits tyrosine phosphorylation-dependent nf-kappab activation distinct endocytotic pathways in epidermal growth factor-stimulated human carcinoma a431 cells adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake a clathrin independent macropinocytosis-like entry mechanism used by bluetongue virus-1 during infection of bhk cells kaposi's sarcoma-associated herpesvirus utilizes an actin polymerizationdependent macropinocytic pathway to enter human dermal microvascular endothelial and human umbilical vein endothelial cells early stages of influenza virus entry in mv-1 lung cells: involvement of dynamin dynasore, a cell-permeable inhibitor of dynamin a single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity amiloride inhibits macropinocytosis by lowering submembranous ph and preventing rac1 and cdc42 signaling population context determines cell-to-cell variability in endocytosis and virus infection role of the actin cytoskeleton during influenza virus internalization into polarized epithelial cells an emerging role for p21-activated kinases (paks) in viral infections histone deacetylase 6 regulates growth factor-induced actin remodeling and endocytosis mucus and mucins mapping the lung proteome in cystic fibrosis proteomics and the lung: analysis of bronchoalveolar lavage fluid proteome analysis of bronchoalveolar lavage in lung diseases infection of primary human bronchial epithelial cells by haemophilus influenza: macropinocytosis as a mechanism of airway epithelial cell entry a mouse model for the evaluation of pathogenesis and immunity to influenza a (h5n1) viruses isolated from humans biological heterogeneity, including systemic replication in mice, of h5n1 influenza a virus isolates from humans in hong kong multiorgan distribution of human influenza a virus strains observed in a mouse model expression of the firefly luciferase gene in vaccinia virus: a highly sensitive gene marker to follow virus dissemination in tissues of infected animals carrier cell-based delivery of an oncolytic virus circumvents antiviral immunity mouse hepatitis coronavirus replication induces host translational shutoff and mrna decay, with concomitant formation of stress granules and processing bodies myosin ii light chain phosphorylation regulates membrane localization and apoptotic signaling of tumor necrosis factor receptor-1 dynamin and rab5 regulate grk2-dependent internalization of dopamine d2 receptors myosin iia is involved in the endocytosis of cxcr4 induced by sdf-1a cortactin signalling and dynamic actin networks the following persons are gratefully acknowledged for providing us with plasmids. dr. a. pekosz for plasmid phh-fluc; dr. m yanez-mo for miia-egfp ''head'' and ''tail'' constructs; dr. p. gallagher for dn-mlck-egfp; dr. p. van der sluijs for dn-s43n-rab5a-egfp. we thank dr. m. esteban for providing us with vaccinia wr-luc virus and dr. j. bell for providing vsv-fl virus. dr. b peeters is acknowledged for a gift of monoclonal antibody against iav np (hb-65). confocal images were acquired at the center for cellular imaging (cci) in the faculty of veterinary medicine, utrecht university and we thank dr. r. wubbolts for help and technical advice. key: cord-335121-ro3x3qa3 authors: ingram, george a.; al-yaman, fadwa title: a comparative assessment of four serological methods used in the detection and measurement of anti-parasite antibodies in the serum of the amphibian, bufo viridis date: 1988-04-30 journal: international journal for parasitology doi: 10.1016/0020-7519(88)90147-6 sha: doc_id: 335121 cord_uid: ro3x3qa3 abstract antibodies against crithidia fasciculata choanomastigotes were detected in green toad (bufo viridis) sera by direct agglutination, indirect haemagglutination (iha), complement-fixation test (cft) and enzyme-linked immunosorbent assay (elisa), correlation coefficients (r) were calculated for comparisons between each of the techniques and regression formulae derived in order to convert antibody levels as determined by one immunological method to that of another. the highest mean titre obtained by elisa was approximately 1.5–3.5 times greater than those obtained by the other techniques whilst cft gave the lowest values. iha and elisa titres were affected by different preparations of the crithidial antigen extracts. highly significant r values were determined for control sera when iha was compared to elisa (r > 0.79), and to both cft and elisa with immune animals (r > 0.96). elisa would seem most applicable for screening other lower vertebrates for anti-parasite antibodies especially in areas of human disease prevalence. amphibians are frequently parasitized by various protozoans present in body fluids and tissues and in some cases the parasites cause serious and debilitating diseases (abrams, 1969; roudabush & coatney, 1937) . trypanosomatid flagellates, in particular trypanosomes, have been reported in and isolated from the blood of frogs, toads and newts (bardsley & harmsen, 1973; woo & bogart 1986) . throughout the investigations into trypanosomatid infection in humans and mammals, sera have been examined for both anti-parasite antibodies and parasite antigens using various immunodiagnostic techniques (strickland & hunter, 1982; tiru & hennessen, 1985) . however there is a dearth of information pertaining to the use of serodiagnostic methods to detect kinetoplastid infections and resultant antibody production in poikilothermic vertebrates with only reptiles having been studied (dollahon, hager & hua, 1983; ingram & molyneux, 1983a , b. 1984a . in this paper we present the results of acomparative assessment of four serological tests (direct agglutination, indirect haemagglutination, complement-fixation test and elisa) used to detect and to determine the levels of antibodies in the sera of green toads (b. viridis), used as experimental models. these amphibians had been injected with the choanomastigotes of c. fusciculutu, a trypanosomatid flagellate. the parasites were chosen because of their ease of culture under laboratory conditions and more importantly because of their presence in the blood and alimentary canal of ranid anurans under natural conditions (smyth & smyth, 1980) . to the authors' knowledge, this is the first report of the use of an immunoenzyme method to detect antibodies in amphibians. furthermore, there are no previous data concerning anti-parasite antibody detection in amphibian serum. materials and methods injecrion und serrr. before injection of the parasites. a small sample of blood was taken by cardiac puncture from each of the toads and inspected for any current infection (with naturally-occurring trypanosomatid flagellates) by smear, wet mount preparation and slope culture as described elsewhere (ingram & molyneux, 1983a. b ). in addition. blood smears were also examined by an immunoenzyme method reported previously by ingram and molyneux (1 y84b, c) but using a rabbit anti-c., firscic~tkctcr serum/swine anti-rabbit immunoglobulins antiserum/ peroxidase-rabbit antiperoxidase/amino-ethylcarbazole substrate system. furthermore, the gut contents and pbs extracts of randomly selected insects and worms respectively were also examined microscopically and by culture for the presence of trypanosomatids. the choanomastigotes in culture overlay were centrifuged at 250 x g for 5 min. the overlay was removed and the parasites were washed three times in phosphate buffered saline-pbs. ph 7.3 (i 45 mm-naci; 7.53 mm-na?hpo, and i 30 mm-nah,po,. 2h,o in de-ionized water). the number of parasites was counted and the suspension adjusted with pbs to give the required dose for injection purposes.toads were given a single intraperitoneal (ip) injection of 16 x io" choanomastigotes in pbs. control animals consisted of those given pbs ip and normal, uninjected toads. the toads were anaesthetized. bled, killed and their weights and lengths noted. the parasite-and pbs-injected animals were bled at 7-day intervals. the uninjected controls were sampled at random intervals throughout the duration of the experiment. in order to detect parasite infection. blood was examined in a similar manner to that obtained from prrinjected animals and the uninjected controls. the sera were isolated and stored at -20°c. specificifv. the promastigotes and procyclics of lershmania herfigi her@ and trpanosoma brucei brucei respectively, both kinetoplastid flagellate species related to c'. ~uscic~ulrtu, were used in the antibody assays to examine for possible non-specific reactions in the toad sera. antisera preparation. rabbit anti-toad serum was produced by an immunization schedule as described previously (ingram & alexander. 1976 ) and the immunoglobulin tttres of the antisera obtained were estimated by either countercurrent electrophoresis or elisa (ingram & molyneux, 1983a). antigen extract. parasite antigen extracts were produced in two ways for use in the immunological techniques. the choanomastigotes were centrifuged at 1700 x g for io min in cold pbs. ph 7.3. the pellet formed was resuspended in pbs and then washed and centrifuged a further three times. prior to use, the cells were subjected to either freezing and thawing (f&t) or sonication (son) treatments. in the former case the pellet was resuspended in chilled pbs, broken up by mixing and the parasites f&t at 3-min periods for 20 min. the material wsas then centrifuged to remove cell debris and the supernatant protein concentration measured by the lowry method. alternatively, after addition of cold buffer, the pellet was disrupted by ultrasonication for three 4-min intervals whilst the mixture was kept chilled. the sonicated material was then left overnight at 5 "c to further remove any protein. it was then centrifuged at 2900 x g for i5 min and the amount of soluble protein in the supernatant determined. agglutination assay two-fold serial dilutions of toad sera. inactivated by heating at 3x"c for 20 min to destroy naturally-occurring complement activity, were preparcd with pbs. ph 7.3 (containing 24x mr+naci). to each dilution wsas added an equal volume of choanomastigotes (i 5 x io cells ml-') and the mixtures incubated at 30°c for 45 min. the direct agglutination (da) end point titre was regarded as that dilution in which visible agglutination was observed when compared to the pbs/parasite controls. normal toad sera were examined for the presence of natural haemagglutinins against sheep crythrocytes (she) before commencing the iha test as described by weir (iy7x). she were tanned with 3.3 x it)-' mg ml-' tannic acid and coated with either f&t or son ant&n extract containing 0.y mg ml-' protein. doubling dilutionr of inactivated sera were made and to each was added the same volume of 2% tanned and coated she.the test samples were incubated at 37°c for 45 min followed by{ overnight at 4c. the samples were then examined and the degree of haemapglutination assessed. untanned, tanned and antigencoated she were used as controls. antigen. toad antisera and either bvs or gpc were mixed together and fixation allowed to occur overnight at 5 "c. sensitized she were then added and the mixtures incubated at 37°c for 60 min after which the end-points were scored. following further incubation for 12 h at sc, the samples under test were re-examined. the complement-fixing antibody (cfa) titre was taken as that dilution which gave 50% hacmolysis. ellsa. a modification of the method of chandler. cox. premier & hurrell (i yx2) was used. the concentrations of antigen. rabbit anti-toad antiserum and enzyme-conjugate used for the elisa were determined by chequerboard titration. the f&t and son antigen extracts (containing i mg ml-' protein) were prepared in 0.053 m-carbonate/bicarbonate coating buffer ph y.6: c'. fi~scicrtlartr was adjusted to 2.5 x 10" cells ml-' in coating buffer and the three antigen preparations were separately dried onto the plates by overnight incubation at 30°c. after washing with pbs (containing o.os% tween 20) ph 7.3, a range of two-fold serial dilutions of control and immune sera were added to the appropriate wells and the plates were reincubated at 37°c for 30 min. they were washed again. rabbit anti-toad antiserum (elisa titre i : 1024) at a dilution of 1 :7s was added and the plates were incubated as before. after re-washing, sheep anti-rabbit i@ immunoglobulin comugated to urease. diluted i in 120. was dispensed into the wells and the plates were similarly incubated. after a final three washes with pbs/tween followed by four washes with distilled water, urease substrate (sera-i.ab, crawley. u.k.) was added to the wells. the plate\ were subjected to a final incubation at 37°c for 30~40 min and the reaction halted by the addition of i % thiomersal solution (v/v). the end point antibody titre was considered to be that dilution which was visually different from the appropriate reference controls included with each experimental run. the mean toad serum antibody titres, s.e.m. and ranges in the control and c. fasciculuta -injected groups, together with the number of sera-containing detectable antibodies, are given in table 1 . the mean value, range in titres and number of animals in which antibodies were detected were lowest for the cft and highest by elisa. of the 29 control and 38 parasiteinjected toads, 93 and 92%, respectively contained antibodies detectable by elisa. when all four immunological assays were applied to each individual serum, in all cases antibodies were detected by at least two or more of the methods used. however the background 'positive antibody' titres in the control animals ranged from 0 to 2-j depending on the technique employed. therefore values higher than 2-' were considered to be positive for the parasiteinjected toads. furthermore comparisons between control and immune sera for each method revealed significant differences in antibody titres (p< 0.01; student's t-test). moreover, 'antibody' levels were not significantly different between the two control groups (p> 0.05). the titres against l. hertigi and t. brucei varied from 0 to 2-j and from 0 to 2-1 in the control and immune sera, respectively. the results for each technique were compared in turn with those for the other methods and the half matrix of the pearson product-moment correlation coefficients (r) calculated for all experimental animals ( table 2 ). the significance of each r value was tested by the t-test. in the case of the control sera, r values ranging from 49 to sy% (p< 0.01) were calculated whilst overall higher correlations were determined for sera from immune animals which varied from 82 to 99% (all i'< 0.00 1). in order that the results of the present study can be used by other investigators for comparative purposes with different experimental models, regression formulae to convert the antibody titres as determined for each technique to those of another are given in table 3 . the regression equations, based on the rectilinear relationship y = mx + c, were only calculated for the highest mean titre found for each of the four immunological methods. the mean antibody titres against c. fasciculata and the number of control and immune sera containing detectable antibodies were the lowest for da and cft, intermediate for iha and highest for the elisa method (table 1) . these findings may reflect the different sensitivities of the immunological techniques used (voller & de savigny, 1 y8 1). the classical agglutination test has often been used for antibody titration in amphibian immunobiological studies (cooper, 1976 ). significant correlations (p < 0.0 1) were obtained for both control (range 4y-76%) and immune sera (83-y2%) when the da titres were compared to the values found for the other methods. although da is simple to perform, preconditions of the test include antigen-type specificity, the non-immobilization and non-autoagglutination of parasites and usually the use of living cells. in the current study, loss of cell motility was observed in some instances and the possibility of inclusion of nonviable cells in others cannot be ruled out. nevertheless caution must be taken in the interpretation of natural 'antibody' levels in view of the detection of 'antibodies' in normal bvs against l. hertigi and t. brucei with titres within the range found for c. fuscicdata. positive results for normal bvs would suggest the presence of low amounts of specific immunoglobulins induced by a current infection with or previous exposure to c. fasciculata parasites. however, the low levels of naturally-occurring 'antibodies' in normal bvs may also have been stimulated by the environmental presence of micro-organisms or other trypanosomatid flagellates which non-specifically cross react with shared cell wall carbohydrate antigenic determinants (andrews, reilly, ferris & hanson, 1965; schnaidman, yoshida, gorin & travassos, 1986; sharabi & gilboa-garber, 1980 ). in the present study, the low levels of 'antibody' in sera from the control groups are not likely to be caused by a current infection because no increase in titres were found throughout the 1 o-week duration of the experiment. the lack of detection of c. fasciculata in blood microscopically or by culture coupled with the finding of parasite antigen(s) in blood up to 2 weeks postinjection by the immunoenzyme method suggests that 8. grids possesses an efficient immune system responsible for the rapid elimination of antigen. therefore it was not possible in the work reported here to correlate the level of parasitaemia and antibody titres. however the exposure of b. grids to c. fasciculata evoked a specific immune response and resulted in significantly increased levels of serum antibody. under normal environmental situations, it is feasible that naturally-occurring immunoglobulins in bvs restrict parasite numbers to below a potential infectious threshold. the finding of serum antibody titres above those of normal background levels in amphibians or other animal hosts in similar habitats or areas endemic for certain diseases would indicate current infection with parasites, other infectious agents or pathogens within a population. no haemolytic anti-complementary activities were detected in normal bvs against the antigen extracts unlike previous reports for amphibians (gigli & austen, 197 1) . whereas the cft is frequently nonspecific and inconvenient for handling many samples, it can utilize crude, soluble parasite antigen extracts. as with da cross reactivity can often lead to false positive results. when the results obtained by cft were compared with each other and with iha and elisa, the lowest range of correlations, although significant and similar to those determined for the da comparisons, were found for control bvs (50-77%; igm which is an efficient complement fixer and also a good agglutinin (atwell & marchalonis, 1976; yamaguchi. kurashige & mitsuhashi, 1973) . however, the 'antibodies' in normal bvs would be present in limited amounts, fix complement less effectively and hence result in low background cfa values. this could also account for the lowest correlations found when the cfa titres were compared to those of the other methods for the controls. the source of complement seems to be important for the efficient fixation of toad antibodies. the use of toad serum as homologous complement source gave a higher mean value and usually slightly higher individual endpoint titres in both control and immunized animals compared to the antibody levels obtained with heterologous gpc. homologous serum has proved a reliable source of complement for the fixation of immunoglobulins in other anurans (alexander & steiner, 1980; sekizawa, fujii & katagiri, 1984) . however the use of commercially available gpc is also known to initiate good fixation in amphibian species (lallone, chambers & horton, 1984 : romano, geczy & steiner, 1973 ). in the current study, 50% correlation (p < 0.0 1) and 92"/,, (p< 0.00 1) were found for control and immune sera. respectively when the different complement sources were compared. low (so-77%, controls) and high (86-94x, immunized) but significant (i'< 0.01) r values were determined in comparisons between iha and cft. iha is prone to lack specificity in some cases and, in contrast to the cft, usually requires highly purified antigen preparations but is easier to perform. elisa gave the highest percentage positive titres in all the samples examined and appears to be the most sensitive of the techniques used to detect anticrithidial antibodies in toad sera. elisa was easy to peform, specific, an important factor which affects the values of the antibody titrcs is the preparation of the antigen extract (crouch & raybould, 1983; pappas. hajkowski, cannon & hockmeyer. 1984) . son antigen preparations gave higher titres than f&t antigen extracts. however, the coating of elisa plates with whole cells produced the highest values. in the work reported here, similar batches of antigen were used thus reducing potential variations due to different preparations. it is of interest to note that a comparison between iha and elisa control titres revealed 79-89% (p < 0.00 i ) significant correlations and similar numbers of positive animals. this implies that the above two techniques have similar sensitivities in the screening of normal bvs for anti-parasite antibodies. nevertheless the method of antigen preparation ma> not be a salient criterion for antibody estimation in immune sera because correlation values of over 89% (f'< 0.001) were found when iha titres were compared to those of elisa. elisa seems appropriate for use in serodiagnostic surveillance programmes. applied to different amphibian species or other aquatic and semi-aquatic lower vertebrates, to detect antibodies stimulated against diverse parasite environmental pathogens. furthermore this technique would be of value in screening lower vertebrates for potential carriers or reservoirs of infective kinetoplastid tlagellates or indeed other pathogenic micro-organisms. such information may reflect the health status of animals within a population and aid in the determination of specific epidemiological and aetiological features of zoonoses and epizootics prevalence. ac~k~f~~*,lef~~.lg~l?~e~~t,s-'i'his work was undertaken whilst one of us (gai) was on an eec funded project contracted to the british council. in part subcontracted to the university of salford, for the development of the faculty of science at yarmouk university. jordan. ga1 would like to thank dr s. k. abdul-hafez for the use of his laboratory facilities for part of the work, dr n. ishmail for assistance with the bleeding of the animals and dr ellen lee of the university of salford computing centre for advice on the use of the computer for statistical analysis of the results. diseax\ in an amphihian colony the first component of complement from the bullfrog runa cutrsheianu: functional properties of cl and isolation of subcompon-entclq leptospiral agglutinins in sera from southern illinois herpetofauna immune response in xenopua iuevis and immunochemical properties of the serum antibodies. immunology 24: 109-l 1x. key: cord-335591-r0x8yaqj authors: ohnishi, kazuo title: establishment and characterization of monoclonal antibodies against sars coronavirus date: 2007-11-28 journal: sarsand other coronaviruses doi: 10.1007/978-1-59745-181-9_15 sha: doc_id: 335591 cord_uid: r0x8yaqj immunological detection of viruses and their components by monoclonal antibodies is a powerful method for studying the structure and function of viral molecules. here we describe detailed methods for establishing monoclonal antibodies against severe acute respiratory syndrome coronavirus (sars-cov). b cell hybridomas are generated from mice that are hyperimmunized with inactivated sars-cov virions. the hybridomas produce monoclonal antibodies that recognize viral component molecules, including the spike protein (s) and the nucleocapsid protein (n), enabling the immunological detection of sars-cov by immunofluorescence staining, immunoblot, or an antigen-capture elisa system. in addition, several s protein-specific antibodies are shown to have in vitro neutralization activity. thus the monoclonal antibody approach provides useful tools for rapid and specific diagnosis of sars, as well as for possible antibody-based treatment of the disease. the outbreak of severe acute respiratory syndrome (sars) in 2003 ultimately led to 8000 people becoming infected, 916 of whom died. the causative agent was identified as sars coronavirus (sarscov) (1,2) . even though the epidemic ended, the threat of reemergence persists, compounded by the absence of an established vaccine. one of the critical issues in controlling a pandemic is a system for early diagnosis that distinguishes sars from other ohnishi types of pulmonary infections. based on clinical experience, several options have been considered in the quest to develop the capacity to accurately diagnose sars-cov infection, including molecular biology techniques and serological tests such as antigen-capture elisa assay and immunofluorescence assay to detect virus-infected cells in respiratory swabs (3-7) . the preparation of monoclonal antibodies (mabs) is considered to be especially valuable for serological testing. here we describe a method for the successful establishment and characterization of mabs against sars-cov structural components. these mabs enable the general immunological detection of sars-cov by methods such as immunofluorescent staining, immunoblotting, and immunohistology, in addition to the construction of a highly sensitive antigen-capture sandwich elisa (6). 1. sars-cov, strain hku-39849, is expanded, purified, and inactivated by the method described in chapter 11. 2. balb/c mice, 8-to 10-week-old females, (japan slc, shizuoka, japan). 3. freund's complete adjuvant. 4. freund's incomplete adjuvant. the viruses generally elicit strong humoral immune response in mice and hence are good antigens for obtaining the mabs. the sars-cov also raises a high titer of serum antibody in mice. however, the immunization protocol should be optimized to obtain the desired specificity and quality of mabs of interest. the choice of parameters for immunization, such as the type of adjuvant, pretreatment of viral antigens (inactivation procedures), antigen dose, route of immunization, and the strain of mice, profoundly affect the properties of resulting mabs. for example, we found that inactivation of sars-cov with uv or formaldehyde gave rise to different immunoglobulin isotypes in mice (8). in addition, the choice of host animals for the immunization affects the mode of epitope recognition. with the common fusion-partner cell lines such as ns-1, p3u1, or sp2/o, a variety of mouse strains, even different species such as rat and hamsters, are usable for the donor of antigen-specific b cells. however, balb/c mice are the most commonly used immunization host because the most efficient fusion-partner myelomas are derived from balb/c strain. the protocol described here is a general procedure to obtain mabs against viral antigens, though the choice of parameters, i.e., the immunization protocol, hybridoma production and so on, should be optimized for each purpose (see note 1). 1. balb/c mice are immunized subcutaneously with 20 g of uv-inactivated purified sars-cov using freund's complete adjuvant (fca). 2. after 2 weeks, the mice are boosted with subcutaneous injection of 5 g of uvinactivated sars-cov using freund's incomplete adjuvant (fia). 3. on day 3 after the boost, sera from the mice are tested by elisa (see section 3.3) for the antibody titer against sars-cov. 4. the two mice showing the highest antibody titer are further boosted intravenously with 5 g of the inactivated virus 14 days after the previous boost. 5. if the antibody titer is lower than expected, the booster injection can be repeated several times before the final boost. three days after the final boost, spleen cells from immunized mice are fused with sp2/o-ag14 myeloma by the polyethylene glycol method of kosbor et al. (9) (see section 3.2, step 5. this protocol gave rise to more than 40 candidate hybridomas, some of which react with s and n protein of sars-cov ( table 1) . 1 . for 1 to 2 h before cell-fusion, 2 g of peg in a sterile glass tube is melted in a microwave oven and dissolved in 2.4 ml of rpmi1640 (abbreviated as rpmi hereafter), prewarmed to 37 • c, by repeated pipetting. the peg solution is kept at 37 • c for at least 1 h. 2. one or two spleens from mice are excised and the spleen cell suspension is prepared by passing through a sterile stainless mesh or by smashing with two slide glasses. the cells are washed by centrifugation (270 × g for 5 min) twice with 5% fcs/rpmi and once with rpmi. 3. the log-phase growing sp2/o-ag14 myeloma cells are washed twice with 5% fcs/rpmi and once with rpmi. 4. count the cells and mix them to a ratio of splenocyte:sp2/o-ag14 = 3:1. spin the cells down and remove the supernatant thoroughly. 5. add 0.5 ml peg/rpmi solution to the cell pellet slowly (taking about 60 sec), loosening of cell pellet using the tip of a pipette. stir the cell clumps gently with a pipette for additional 90 sec. the cells should be seen as small aggregates in this step. 6. add 10 ml of prewarmed (37 • c) rpmi slowly, taking 3-4 min for the first 1 ml drop by drop, and then taking about 6 min for the remaining 9 ml. 7. incubate the tube at 37 • c for at least 20 min. 8. wash the cells twice with prewarmed rpmi. 9. suspend the cells with hat-medium to a concentration of 3-5 × 10 4 sp2/o-ag14 cells/ml and plate to 0.2 ml/well of the 96-well plates. 10. feed the cells with hat-medium by changing two-thirds of the volume of the wells on the days 3, 7, and 12. 11. the hybridoma colonies should be seen by microscope on days 3-7 and some fast-growing colonies should be recognizable by eye from day 7. generally, if the immunization and cell fusion step was successful, more than 70% of the wells contain hybridoma colonies. when the sizes of the colonies becomes one-tenth to one-fifth of the 96-well bottom area, the first screening of positive clones should be taken by elisa (see section 3.3). 12. the hybridoma cells in the elisa-positive wells are recovered and cloned by a limiting dilution method as follows. cells are counted, diluted with hatmedium, and plated into 96-well plates so that one well contains three or ten cells; the left-half of the plate contains three cells/well and the right-half of the plate contains ten cells/well. 13. the hybridoma colonies will be discernible after 7-10 days. then the elisa test should be performed again and the elisa-positive wells containing single colonies are selected. 14. the cells from the selected wells are expanded in a 24-well plate and adapted to ht-medium for at least 1 week. 15. the cells are further adapted to the hybridoma medium for 1 week. if necessary, the cells are further adapted to a serum-free hybridoma medium. 16. the hybridomas are now ready for collecting the mabs. the aliquots of the cells are resuspended in freezing buffer and stored at -135 • c or in liquid nitrogen. the first screening is conducted by elisa using sars-cov-infected vero e6 cell lysate as the antigen. in this first screening, the uninfected vero e6 cell lysate is used as the antigen for a negative control. in the case of the assay for the test bleed of immunized mice, the serial dilution of the sera with pbs-tween (start from 1/20 dilution) is used in place of hybridoma culture supernatants. 1. hybridomas are grown in about 2 liters of serum-free hybridoma medium to the late-log phase/early stationary phase. 2. the culture supernatants are harvested by centrifugation at 1700 × g for 20 min, and 1/100 volume of 1 m tris-hcl (ph 7.4) and 1/500 volume of 10% nan 3 are added. the following steps are carried out at 4 • c (in a cold room) or on ice. 3. the protein g-sepharose 6b column (2-5 ml bed volume) is preequilibrated with pbs and the supernatant is loaded at a flow rate of about 1 drop/5 sec. this step takes 2-3 days. 4. the column is washed with pbs and the bound antibody is eluted with glycine/hcl solution. in this step, 1-ml fractions are collected into an eppendorf tube that contains 0.1 ml of 1 m tris-hcl (ph 8.0). 5. after measuring the od 280 of the fractions, the protein-containing fractions are pooled. an equal volume of saturated (nh 4 ) 2 so 4 is gradually added to the pooled fraction. the solution is kept on ice overnight. 6. precipitated proteins are collected by centrifugation at 2300 × g for 30 min and the pellet is dissolved with a small volume (1-2 ml) of pbs, dialyzed against pbs (three changes of 500 ml, more than 3 h each), 7. the dialyzed sample is centrifuged at 15,000 × g for 20 min, aliquoted, and stored at -20 • c. this procedure generally gives 2-10 mg of purified mab from a 2-liter culture of hybridomas. 1. the protein concentration of the purified antibody is determined by od 280 using a molecular extinction coefficient equal to 1.4 (for immunoglobulins). 2. the protein concentration is adjusted to 2 mg/ml with pbs, and 1 ml is dialyzed against 50 mm sodium bicarbonate buffer (ph 8.5) at 4 • c. 3. dissolve 1 mg of sulfo-nhs-lc-biotin in 1 ml of distilled water, and quickly add 74 l of this solution to the antibody solution. mix well and stand in ice for 2 h. 4. dialyze the solution against pbs at 4 • c overnight with three changes of 500 ml pbs dialysis solution, each for more than 3 h. 5. the sample is transferred to an eppendorf tube and centrifuged at 15,000 × g, 4 • c for 20 min. 6. the supernatant is recovered and the protein concentration is determined by od 280 as above. the aliquots are stored at -20 • to -80 • c. avoid repeated freeze-thawing. the working solution can be stored at 4 • c for months to years. preservatives such as sodium azide can be added to 0.05% if necessary. 1. uv-inactivated purified sars-cov is electrophoresed and blotted to pvdf membrane by the standard procedure (see note 5). 2. the blotted pvdf membrane is blocked with starting block tm solution for 1 h at room temperature. 3. the membrane is reacted with the first mabs diluted to 1 g/ml (see note 4) with 10% starting block tm /pbs-tween for 1 h at room temperature. this incubation is done by placing the pvdf membrane in the hybridization bag with 5 ml of antibody solution. 4. the membrane is washed with excess volume (about 20-30 ml) of pbs-tween three times for 5 min each time in an appropriate container. 5. the membrane is reacted with peroxidase-conjugated f(ab ) 2 fragment anti-mouse igg diluted to 1:20,000 by 10% starting block tm /pbs-tween (see note 4). the incubation is done in the hybridization bag for 1 h at room temperature. 6. the membrane is washed thoroughly with pbs-tween four times for 5 min each time. 7. after washing, the membrane is placed on 3 mm filter paper and the liquid is removed but not dried. the membrane is then soaked with west femto a:b = 1:1 mixture (in case of minigel size, i.e., 7 × 9 cm, 0.8-1.0 ml of a:b mixture is required). the membrane should be completely soaked with excess volume of a:b mixture. 8. the membrane is picked up by a flat-tip forceps and placed in between two transparent polyester sheets (see note 6). 9. the membrane/transparent sheet sandwich is placed in an x-ray film cassette with film and exposed for an appropriate time to obtain the best signals. an example of the result is shown in fig. 2 , in which the purified sars-cov proteins (0.5 g/lane) are electrophoresed with sds-page, blotted to pvdf membrane, and detected by anti-s and anti-n mabs. for the diagnosis of sars-cov infection, serological tests such as immunofluorescence assay (ifa, described above) and antigen-capture elisa assay are two good options for detecting virus in, e.g., respiratory swabs or in virus-infected cells. by utilizing established mabs against sars-cov, it is possible to construct a highly sensitive antigen-capture sandwich elisa test system for detection of sars-cov. the sandwich elisa consists of two mabs, an antigen-capturing antibody and a detecting antibody, which recognize different epitopes of the target antigen. the antigen-capturing antibody is immobilized on the elisa plate and captures the viral antigens in the test sample. the detecting antibody is normally labeled with a signal-producing enzyme such as a peroxidase or a phosphatase. the following method is the basic procedure for constructing a sandwich elisa (see note 7). chamber slides: lab-tek 8-well glass chamber slides ifa-staining buffer: 1% bsa (sigma) in pbs paraformaldehyde solution: 4% paraformaldehyde is freshly dissolved in pbs by heating to 70 • c pbs/tritonx-100 solution: 0.1% triton x-100 in pbs dapi solution: for the stock solution, 4,6-diamidino-2-phenylindole hydrochloride (dapi, invitrogen) is dissolved with dw at 5 mg/ml and stored at -20 • c mounting solution: fluoromount g (southernbiotech immunoblot with monoclonal antibodies 1. blot membrane: pvdf membrane blocking reagent: starting block tm detecting (second) antibody: peroxidase-conjugated f(ab ) 2 fragment anti-mouse igg (h + l) (use with 1:20,000 dilution chemiluminescent reagent: supersignal west femto antigen-capture sandwich elisa 1. high-binding immunoassay microplate: immulon-2 (dynatech labs, va, usa) or the equivalent blocking solution: 1% ova in pbs-tween detecting (second) reagent: ␤-d-galactosidase-labeled streptavidine (zymed galactosidase substrate: 4-methy-lumbelliferyl-␤-d-galactoside (sigma) reaction stop solution: 0.1 m glycine-naoh purified mab for the antigen-capture is immobilized on an immulon-2 microplate by incubating 4 g/ml antibody in the elisa-coating buffer at 4 • c overnight the microplate is blocked with 1% ova for 1 h at room temperature or overnight at 4 • c the plate is washed three times with pbs-tween the uv-inactivated purified sars-cov samples (see note 1), which are serially diluted with 1% ova/pbs-tween, are added to the wells and incubated for 1 h at room temperature after washing three times with pbs-tween, wells are reacted with biotinylated detecting (second) mab (0.1 g/ml) for 1 h at room temperature after washing three times with pbs-tween, wells are reacted with ␤-dgalactosidase-labeled streptavidine for 1 h at room temperature after washing four times with pbs-tween, fluorescent substrate 4-methylumbelliferyl-␤-d-galactoside is added and incubated for 2 h at 37 • c the fluorescence of the reaction product, 4-methyl-umbelliferone (4-mu), is measured using fluoroscan ii (flow laboratories inc., inglewood, ca) at excitation and emission wavelengths of 355 and 460 nm identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study detection of sars-associated coronavirus in throat wash and saliva in early diagnosis immunological detection of severe acute respiratory syndrome coronavirus by monoclonal antibodies sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with severe acute respiratory syndrome formalin-treated uv-inactivated sars coronavirus vaccine retains its immunogenicity and promotes th2-type immune responses in vitro stimulated lymphocytes as a source of human hybridomas using antibodies: a laboratory manual monoclonal antibodies: a practical approach cleavage of structural proteins during the assembly of the head of bacteriophage t4 the author would like to thank drs. fumihiro taguchi and shigeru morikawa for their advice and discussion, dr. koji ishii for providing recombinant sars-cov proteins, and ms. sayuri yamaguchi for her technical assistance. this work was supported by grant from the ministry of public health and labor of japan. figure 3 shows an example of an antigen-capture elisa system for sars-cov, in which two anti-n mabs, skot-8 (coating mab) and biotinylated skot-9 (detecting mab) are used. this sandwich elisa detects sars-cov protein at a concentration as low as 40 pg/ml (6). 1. for further understanding and detailed explanations of the hybridoma methodology, good textbooks are available (10,11). 2. the validation for the complete inactivation of the virus is crucial. for a detailed account see chapter 11 in this volume. 3. in this step the culture supernatants are diluted to one-half in order to reduce the background of the elisa. if the background is still high, dilution of the culture supernatants can be one-fifth to one-tenth or more with pbs-tween. 4. the titration of the mabs and the second reagent is very important for obtaining the best results. it often varies from 1/100 to 1/100,000, and it should be determined for each mab. 5. the sds-polyacrylamide gel electrophoresis (page) is carried out by the method of laemmli (12). the detailed conditions, such as the concentration of the gel or reducing/nonreducing and so on, should be changed case by case. generally, loading 0.5-10 g/lane of purified virus fraction would be enough for the detection of the viral antigen by the chemiluminescence method. 6. we conveniently utilize transparent sheets such as those used for an overhead projector (ohp-sheet). 7. in order to obtain good sensitivity and specificity of the test system, the optimization of the combination of two antibodies, i.e., coating mab and detecting mab, is required. this can be done by the elisa with a matrix of candidate coating-mabs and detecting-mabs (6). key: cord-334165-7gfk554m authors: stadlbauer, daniel; amanat, fatima; chromikova, veronika; jiang, kaijun; strohmeier, shirin; arunkumar, guha asthagiri; tan, jessica; bhavsar, disha; capuano, christina; kirkpatrick, ericka; meade, philip; brito, ruhi nichalle; teo, catherine; mcmahon, meagan; simon, viviana; krammer, florian title: sars‐cov‐2 seroconversion in humans: a detailed protocol for a serological assay, antigen production, and test setup date: 2020-04-17 journal: curr protoc microbiol doi: 10.1002/cpmc.100 sha: doc_id: 334165 cord_uid: 7gfk554m in late 2019, cases of atypical pneumonia were detected in china. the etiological agent was quickly identified as a betacoronavirus (named sars‐cov‐2), which has since caused a pandemic. several methods allowing for the specific detection of viral nucleic acids have been established, but these only allow detection of the virus during a short period of time, generally during acute infection. serological assays are urgently needed to conduct serosurveys, to understand the antibody responses mounted in response to the virus, and to identify individuals who are potentially immune to re‐infection. here we describe a detailed protocol for expression of antigens derived from the spike protein of sars‐cov‐2 that can serve as a substrate for immunological assays, as well as a two‐stage serological enzyme‐linked immunosorbent assay (elisa). these assays can be used for research studies and for testing in clinical laboratories. © 2020 the authors. current protocols in microbiology published by wiley periodicals llc. basic protocol 1: mammalian cell transfection and protein purification basic protocol 2: a two‐stage elisa for high‐throughput screening of human serum samples for antibodies binding to the spike protein of sars‐cov‐2 severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the virus that causes coronavirus disease 2019 (covid19; often written , emerged in late 2019 in wuhan, china (wu et al., 2020; zhu et al., 2020) . rapid, global spread of the virus is presently causing a pandemic. currently, no drugs or antivirals are available and countermeasures are limited to non-pharmaceutical interventions (npis). nucleic acid−based tests for detection of the virus during acute disease are in use worldwide corman et al., 2020) . however, the development of serological assays is lagging due to lack of suitable reagents. serological assays are needed to perform serosurveys aimed at determining the real infection rate and infection fatality rate in a given population. furthermore, they are useful to characterize the immune response to the virus in a detailed qualitative and quantitative manner. serological assays are also of immediate practical use. they can be used to identify individuals who were infected (including severe, mild, and asymptomatic cases) and who are now potentially immune. a recent study in non-human primates showed that re-infection, at least in the small number of animals used in the study, does not occur (bao et al., 2020) once antibody responses have been mounted. infection with coronaviruses circulating in human populations, such as hku, nl63, etc., also leads to immunity that protects from re-infection for months to years (callow, parry, sergeant, & tyrrell, 1990) . therefore, individuals who have mounted an immune response to sars-cov-2 are likely immune, which means that they are unlikely to transmit the virus to others. as an example, healthcare workers who are immune could potentially care for covid19 patients with minimal risk to themselves, their colleagues, and other patients. in addition, the use of convalescent plasma may serve as a valuable treatment option for patients with severe covid19, especially in the absence of other options. a serological assay is critical for identifying potential plasma donors. the surface glycoprotein of the virus, termed the spike (s) protein, mediates attachment of the virus to human cells via its receptor-binding domain (rbd; wrapp et al., 2020) and mediates fusion of viral and cellular membranes. antibodies binding to the spike protein, and especially to the rbd domain, can neutralize sars-cov-2. therefore, we used different recombinant spike protein preparations as the antigens for our elisa. we reported in our earlier work that individuals not exposed to sars-cov-2 are completely naïve to the spike protein, and their serum samples show little or no reactivity in an elisa (amanat et al., 2020) . it is, therefore, easy to distinguish between exposed/immune and naïve individuals. community in the future. not every aspect of these protocols has been optimized in detail, and we provide notes and comments whenever further optimizations and testing are recommended. mammalian expression plasmids for the generation of the recombinant proteins are available from the corresponding author and from bei resources. this protocol can be used for both expression vectors: the one expressing secreted rbd as well as the one expressing a soluble, trimeric version of the sars-cov-2 spike protein. expression levels of the rbd are very high in our hands (>20 mg/l culture), while expression levels for the full-length spike are lower (approximately 4 to 5 mg/l). therefore, we use the recombinant rbd for initial screening elisas and the full-length spike for confirmatory elisas (as described in basic protocol 2). the expression vector constructs were described previously (amanat et al., 2020) . in brief, the sequences used for both proteins are based on the genomic sequence of the first isolate, wuhan-hu-1, which was released on january 10, 2020 (genbank: mn908947.3). sequences were codon-optimized for mammalian cell expression. the full-length spike protein sequence was modified to remove the polybasic cleavage site, which is recognized by furin, and to add a pair of stabilizing mutations (figure 2 ). these two modifications were included to enhance the stability of the protein based on published literature (amanat et al., 2020) . the plasmids are grown in e. coli at 37°c (or 30°c) at 225 rpm in luria-bertani (lb) broth with ampicillin (lb-amp) in shaker flasks overnight. high-quality plasmid dna can be obtained using commercially available maxiprep kits (ideally with an endotoxin-removal step). importantly, other cell lines (293t, cho, etc.), other media, transfection reagents, and more sophisticated protein purification methods might be used as alternatives if available. rbd = receptor-binding domain of sars-cov-2 (nr-52306) pbs = phosphate-buffered saline rt = room temperature (18°to 25°c) mem = minimum essential medium 2015) and seeded at a density of 600,000 cells/ml in expi293 expression medium. the viability of the cells must be greater than 90% at all times. 2. cells are passaged every 3 to 4 days and incubated in an orbital shaking incubator at 37°c with shaking at 125 rpm and 8% co 2 . see current protocols article phelan and may (2015) for basic cell culture techniques. a maximum cell density of 4−5 × 10 6 cells/ml is recommended, at which point cells should be immediately passaged. 3. 600 × 10 6 cells are suspended in 200 ml (3 × 10 6 cell/ml) of expi293 expression medium in a 1-l erlenmeyer flask. transfections are performed according to manufacturer's instructions. 4. 12 ml of opti-mem is added to two 50-ml sterile polypropylene conical tubes: one tube receives 200 μg (1 μg/μl final dilution in the total volume of culture) of respective plasmid dna (for rbd or full-length spike), while the other tube receives 640 μl of expifectamine transfection reagent from the kit. 5. the contents of both 50-ml tubes are mixed together and incubated at rt for 10 min to prepare the transfection mixture, after which the transfection mixture is added dropwise to the cells slowly, over a course of a few seconds using a micropipette while swirling the cells in a circular pattern. 6. cells are then returned to the shaking incubator. 7. at 16 hr post-transfection, 1.2 ml of expifectamine 293 transfection enhancer 1 and 12.0 ml of expifectamine 293 transfection enhancer 2 from the kit are added to the culture, and subsequently the culture is returned to the shaking incubator. 8. at 3 days post-transfection, the cells are harvested and centrifuged 20 min at 4000 × g, 4°c. 9. the supernatant is filtered using a 0.22-μm stericup filter; the cell pellet can be discarded. alternatively, cells can be spun down at 200 × g for 10 min, supernatant can be collected, and the same cells can be resuspended in 200 ml of fresh expi293 expression medium and returned to the shaking incubator for another 3 days. this makes it possible to collect more protein in the fresh supernatant (the cells continue to express the protein) and can be used to increase protein yield. the protein integrity needs to be verified in the same way as for the initial protein harvest. this alternate strategy works well with the rbd, but is less suitable for the full-length spike (we have detected protein degradation in that case). 10. continue to process the supernatant and purify protein immediately. alternatively, if the supernatant is stored, it must be kept at 4°c (and for no longer than overnight/16 hr) in order to prevent denaturation of the protein at room temperature. these steps can be replaced by more advanced purification methodologies, for example, if an äkta purifier is available. the methods described below work even in labs not geared toward protein purification. see current protocols article petty (1996) for additional detail on metal-chelate affinity chromatography. 11. prior to use, ni-nta resin (6 ml per 200 ml culture) is washed once with fresh pbs (transfer resin into a 50-ml tube and fill up with pbs), then spun 10 min at 2000 × g, 4°c. 12. once the centrifugation is complete, the pbs is discarded and resin is resuspended with the cell culture supernatant and inverted two or three times. 13. the resin is then incubated with the supernatant for 2 hr on a shaker (65 rpm) at rt. 14. two clean 5-ml polypropylene columns are loaded with the supernatant-resin mixture and then washed with one column volume of wash buffer four times. 15. columns are then eluted using the elution buffer. 16. four fractions are collected from each column by incubating the resin in the column with 3 ml of elution buffer for each fraction. incubate resin with elution buffer for 5 min after each addition of elution buffer. 17. eluate is collected directly in a 50-ml polypropylene conical tube placed on ice. the total volume of eluate should be 24 ml from the two columns. more columns can be used to speed up the purification time, depending on the volume of the culture. 18. eluate is spun through 10-kda amicon ultra centrifugal filter units (for rbd) or 50-kda amicon ultra centrifugal filter units (for full-length spike) at 4000 × g for 30 min (or longer if eluate takes longer to pass through the membrane) at 4°c or until only 200 to 300 μl remain in the unit. amicon filter units should be equilibrated with pbs before use. 19. pbs is added twice to the amicon ultra centrifugal filter unit from step 18 and the unit is spun at 4000 × g for 30 min at 4°c or until only 200 to 300 μl remain in the unit. stadlbauer et al. current protocols in microbiology 20. finally, the protein is collected from the amicon ultra centrifugal filter unit, its concentration is measured (e.g., using the bradford protein assay or similar methods; see current protocols article; lovrien & matulis, 2005) , and a denaturing sds-page (4% to 20% gradient; see current protocols article: manns, 2011 ) is run to check the integrity of the purified protein. 21. after the elution step, protein should always be kept on ice or stored at 4°c. for storage longer than 24 hr, protein should be frozen at −80°c to avoid degradation. a concentration of 2 mg/ml and an aliquot size of 50 to 200 μl is recommended. the purpose of this protocol is to describe the procedure for measuring human antibody responses to the recombinant receptor-binding domain (rbd) of the spike protein or fulllength spike protein of sars-cov-2 and to ensure the reproducibility and consistency of the obtained results. we developed this as a two-stage elisa in which the first stage ('a' steps below) includes relatively high-throughput screening of samples in a single serum dilution against the rbd (which expresses very well and therefore can be produced in greater quantities). this is followed by a second stage ('b' steps below) in which positive samples from the first stage undergo a confirmatory elisa against the full-length spike protein (which is harder to express; therefore there is usually less available). for the second stage, a dilution curve is performed. typically, if only one operator is available, screening elisas can be run in the morning (760 samples/10 plates per run) and confirmatory elisas can be run in the afternoon (140 samples/10 plates per run). of note, we describe the assay here as it is set up in our laboratory. we use a plate washer and a plate reader, but no automated system. the protocol can be adapted to use with an automated liquid handler. in addition, one of the difficulties in setting up the assay is the availability of appropriate negative and positive controls. negative controls are easier to come by, and can be serum pools taken before 2020. positive controls can be convalescent samples from covid19 patients or monoclonal antibodies (mabs) like cr3022 (ter meulen et al., 2006; tian et al., 2020) . if no human sera or mabs are available, mouse mabs, mouse sera against sars-cov-2, other animal sera against sars-cov-2, or anti−his tag antibodies (the proteins are histagged) can be used. however, in this case, a different secondary antibody for the species from which the primary antibody is derived is needed for the positive control. also, we recommend generating large batches of positive controls, which can be used for many runs. the positive control should be selected to result in a strong signal (recommend od 490 of about 2.0), and should be clearly distinguishable from the negative controls. elisas can be run with either serum or plasma. caution: before starting to work with covid19 samples, please consult with your local biosafety officer regarding which precautions, personal protective equipment and protective measures are required. definitions elisa = enzyme-linked immunosorbent assay pbs = phosphate-buffered saline current protocols in microbiology rbd screening elisa 1a. coating elisa plates (day 1). i. thaw the required number of vials of antigen (sars-cov-2 rbd protein) to coat 96-well microtiter elisa plates at a concentration of 2 μg/ml. once thawed, mix by gently vortexing vials before diluting in 1× pbs. prepare approximately 5 ml for each plate to be coated. ii. coat plates with 50 μl of diluted protein per well using a multichannel pipettor and a reservoir. lightly tap plates against surface to ensure protein is evenly coating the bottom of every well. iii. incubate at 4°c overnight. always keep a plate cover on top of coated plates during all steps of the protocol! plates can likely be stored at 4°c for up to 1 week, but this needs to be validated locally to ascertain that it does not change the results. 2a. heat inactivation of samples (day 1, this is a general safety precaution for work with human serum). caution: we have not tested if this procedure inactivates sars-cov-2; please consult with your local biosafety officer to discuss proper safety precautions. i. set the water bath to 56°c. once temperature is reached, place the serum/plasma samples in the water bath and immediately start the timer for 1 hr. ii. remove samples when the timer goes off. do not leave samples at 56°c for longer than 1 hr. ii. using an automated plate washer, wash coated elisa plates three times with pbs-t. iii. add 200 μl blocking solution to all wells of the plates, starting the timer for 1 hr (do not exceed 4 hr) after completing the first plate. place plates in a 20°c (rt) incubator until step 6a. 4a. pre-diluting samples (day 2). i. in a biological safety cabinet, set up sterile 1.5-ml microcentrifuge tubes to predilute serum samples at a 1:5 ratio. ii. add 40 μl of sterile 1× pbs to all tubes. iii. gently vortex each serum sample to mix and add 10 μl to the microcentrifuge tubes, vortexing once more. do this for all remaining samples including the positive and negative controls. the volume of serum not needed in these 'a'steps will be stored and used for the 'b'steps, below. 5a. setting up dilution plates (day 2). i. calculate and prepare at least 30 ml of pbs-t + 1% (w/v) milk powder. ii. prepare one dilution plate (separate flat-bottomed cell culture plate) per antigencoated plate prepared. iii. add 180 μl of pbs-t containing 1% milk to all wells of the dilution plate (including blank wells). iv. leaving columns 1 and 12 as blanks, add 20 μl of pre-diluted sample (or control) to the designated wells. this results in a final serum dilution of 1:50. v. continue until all samples and controls have been added to the designated wells. see reference plate layout in figure 3 . 6a. transferring serum dilution (day 2). i. after the blocking incubation in step 3a, substep iii, remove elisa plates from the rt incubator and throw off the blocking solution. tap the plates dry on a kimwipe or other absorbent material. ii. using a multichannel pipettor, pipette up and down four to six times in the wells of the first row of the dilution plate to mix. iii. transfer 100 μl from each well of the first row of the dilution plate to the corresponding wells in the elisa plate. change tips and continue to transfer the second row of the dilution plate to the elisa plate in the same manner. iv. start the timer for 2 hr as soon as the contents of all of the rows have been transferred to the first elisa plate. v. place plates in a 20°c (rt) incubator. do not exceed 4 hr of incubation at rt before proceeding to step 7a. 7a. incubating with secondary antibody (day 2). i. after 2 hr incubation at rt, wash the elisa plates from step 6a three times with pbs-t using an automated plate washer. ii. dilute anti-human igg (fab-specific) hrp-labeled secondary antibody 1:3000 in pbs-t containing 1% milk. prepare at least 5 ml per plate. iii. add 50 μl of the secondary antibody solution to all wells of the plate using a multichannel pipettor. be sure to avoid touching the walls of the wells with the pipette tips, to avoid carry-over and high background signals. stadlbauer et al. current protocols in microbiology iv. start the timer for 1 hr (stay in a range of 50 to 65 min) as soon as the secondary antibody has been added to the first plate. v. place plates in a 20°c (rt) incubator. 8a. developing and reading plates (day 2). i. after 1 hr, wash plates three times with pbs-t using an automated plate washer. ii. prepare sigmafast opd solution and calculate amount needed. one set of tablets (1 gold + 1 silver tablet) dissolved in 20 ml water for injection (wfi) can be used for two 96-well plates. iii. fully dissolve one gold tablet in 20 ml wfi. do not add silver tablet to solution until ready to start adding to the plates. the opd solution needs to be prepared immediately before use. iv. add 100 μl opd solution to all wells of the plate. begin the timer for 10 min as soon as opd has been added to the first row of the first plate. do not exceed 10 min of developing before stopping the reaction. v. to stop the reaction after exactly 10 min, add 50 μl of 3 m hcl to all wells. ideally, read plates immediately after adding hcl. vi. read elisa plates in a plate reader at an absorbance of 490 nm (immediately after adding hcl) and record data. samples that exceed a certain od 490 cutoff value (proposed cutoff: od 490 = 0.15 to 0.2, or mean of negative controls plus 3 times the standard deviation of the negative controls) are assigned as presumptive positives and will be tested in the confirmatory elisa using full-length spike protein ('b' steps, below) . the od 490 cutoff has to be experimentally determined and depends on assay background and noise. spike confirmatory elisa 1b. coating elisa plates (day 1). i. thaw the required number of vials of antigen (sars-cov-2 spike protein) to coat 96-well microtiter elisa plates at a concentration of 2 μg/ml. once thawed, mix by gently vortexing vials before diluting in 1× pbs. prepare approximately 5 ml for each plate to be coated. ii. coat plates with 50 μl of diluted protein per well using a multichannel pipettor and a reservoir. lightly tap plates against surface to ensure protein is evenly coating the bottom of every well. iii. incubate at 4°c overnight. always keep a plate cover on top of coated plates during all steps of the protocol! plates can likely be stored in 4°c for up to 1 week but this needs to be validated locally to ascertain that it does not change the results. 2b. blocking elisa plates (day 2). i. calculate to prepare at least 30 ml of blocking solution per plate. ii. using an automated plate washer, wash coated elisa plates three times with pbs-t. iii. add 200 μl blocking solution to all wells of the plates, starting the timer for 1 hr (do not exceed 4 hr) after completing the first plate. iv. place plates in a 20°c (rt) incubator until step 4b. 3b. pre-diluting samples (day 2). retrieve 1:5 pre-diluted samples from step 4a, above, to be tested and confirmed (samples that are above certain threshold in rbd screening elisa based on a set od 490 value; see annotations after step 8a, substep vi). confirmatory spike elisa reference plate layout. the sample layout on the elisa plate is shown, including the serial dilution steps that need to be performed. wells designated for positive (+) and negative (−) controls are indicated. 4b. performing serial dilutions (day 2). i. calculate and prepare at least 20 ml of pbs-t + 1% (w/v) milk powder per plate. ii. after the blocking incubation in step 2b, substep iii, remove plates from the rt incubator and throw off the blocking solution. tap the plates dry on a kimwipe or other absorbent material. iii. using a multichannel pipettor, add 120 μl of pbs-t containing 1% milk to all wells of each plate. iv. leaving columns 1 and 12 as blanks, add an extra 51 μl of pbs-t containing 1% milk to wells only in columns 2 and 7. wells of column 2 and 7 will be the sample wells. v. add 9 μl of 1:5 pre-diluted sample (final dilution 1:100 on the plate) to the first well in column 2 and continue to add samples to all 8 wells. vi. in column 7, add samples to wells a through f. vii. transfer positive and negative controls into wells g and h respectively. see reference plate layout in figure 4 . viii. with the multichannel pipettor, pipette up and down four to six times in column 2 to mix. discard these tips. with new tips, transfer 60 μl (3-fold dilution) from column 2 to column 3, and pipette up and down four to six times to mix. repeat this until column 6; discard 60 μl before column 7. ix. taking fresh tips, mix column 7 by pipetting up and down four to six times. repeat the same process of transferring, mixing, and discarding tips from columns 7 to 11. once column 11 is reached, discard 60 μl. x. start the timer for 2 hr once the first elisa plate has been serially diluted. do not exceed 4 hr of incubation at rt before proceeding to step 5b. xi. place plates in a 20°c (rt) incubator. current protocols in microbiology 5b. incubating with secondary antibody (day 2). i. after 2 hr of incubation at rt, wash the plates from step 4b with pbs-t using the automated plate washer. ii. dilute anti−human igg (fab-specific) hrp-labeled secondary antibody 1:3000 in pbs-t containing 1% milk. prepare at least 5 ml per plate. iii. add 50 μl of the secondary antibody solution to all wells of the plate using a multichannel pipettor. be sure to avoid touching the tips of the pipette to the walls of the well. iv. start the timer for 1 hr (stay in a range of 50 to 65 min) as soon as the secondary antibody has been added to the first plate. v. place plates in a 20°c (rt) incubator. 6b. developing and reading plates (day 2). i. after 1 hr, wash plates three times with pbs-t using an automated plate washer. ii. prepare sigmafast opd solution and calculate amount needed. one set of tablets (1 gold + 1 silver tablet) dissolved in 20 ml water for injection (wfi) can be used for two plates. iii. fully dissolve one gold tablet in 20 ml wfi. do not add silver tablet to solution until ready to start adding to the plates. the opd solution needs to be prepared immediately before use. iv. add 100 μl to all wells of the plate. begin timer for 10 min as soon as opd has been added to the first row of the first plate. do not exceed 10 min of developing before stopping the reaction. v. to stop the reaction after exactly 10 min, add 50 μl of 3 m hcl to all wells. vi. read elisa plates in plate reader at an absorbance of 490 nm and record data. true positive samples will have a signal higher than the negative control plus 3 standard deviations of the negative controls in at least two consecutive dilutions. 31.74 g nah 2 po 4.· h 2 o 70.16 g nacl 64.0 g imidazole (sigma-aldrich # i5513 or equivalent; final concentration is 235 mm) 4 l distilled water store at room temperature up to 4 months use distilled water filtered using a 0.22-μm stericup vacuum filtration system. 45 l distilled water 5 l 10× pbs (corning tm #46013cm or equivalent)) 50 ml tween 20 (fisher bioreagents #bp337-500 or equivalent) store at room temperature for to 4 months wash buffer (4 l) 31.74 g nah 2 po 4· h 2 o 70.16 g nacl 5.44 g imidazole (sigma-aldrich # i5513 or equivalent; final concentration is 20 mm) store at room temperature up to 4 months use distilled water filtered using a 0.22-μm stericup vacuum filtration system. the protein expression and purification methods (basic protocol 1) described in this article are based on well-established techniques. the expression plasmids and protein sequences have been optimized to increase protein stability and yield (amanat et al., 2020) . plasmids can be requested from the krammer laboratory or can be found on bei resources. the elisa protocol (basic protocol 2) has been designed to allow for highthroughput screening of many samples per day, followed by a confirmatory step to verify presumptive positive results. the elisa assay itself is based on well-established protocols and has been optimized for the use of sars-cov-2 antigens. the most common problem for the transfection (basic protocol 1) is low cell viability before performing the transfection. the cells need to be 90% to 95% viable. the absence of antibiotics/antifungals requires good sterile technique to prevent contamination. sterile plasmid preparations are also recommended, and it is important to add the enhancer to the shaking flasks 16 hr post-transfection. for the protein purification, we recommend always using fresh ni-nta resin to prevent cross-contamination with other proteins. harvested supernatant should be ideally processed immediately to ensure protein integrity. to make filtering of the supernatant easier, an additional centrifugation step (after pelleting the cells) is recommended to pellet residual cells and other particles. when performing buffer exchange using amicon ultra centrifugal filter units, make sure to use the right-size cutoff (use smaller cut-off for rbd). it is recommended that purified protein be diluted to a concentration of about 2 mg/ml. storage at higher concentrations may result in aggregation of protein. for the elisa (basic protocol 2), performing all of the washing steps and adhering to the incubation times are important to achieve low background reactivity. most critical are the incubation times for the secondary antibody and the substrate (opd and hcl for stopping the reaction). in addition, touching wells with tips when transferring secondary antibody and substrate can result in higher background and possibly false positive wells, and needs to be avoided. in preparing the opd, it is also important to dissolve the gold tablet fully and only add the silver tablet right before the substrate is added to the elisa plate. we expect expression levels of the rbd to be above 20 mg per l of culture cells and expression of the full-length spike protein to be approximately 4 mg per l of 293fs, using a gravity-flow protein-purification strategy. when running the sds-page to confirm protein integrity, clear single bands are expected for the rbd and full-length spike at around 25 to 30 kda and ∼190 kda, respectively. additionally, elisas with positive and negative controls (e.g., monoclonal antibodies) are performed to confirm correct protein folding. we expect a good binding profile for the positive control and low-to-no background reactivity for the negative control. basic protocols 1 and 2 can be completed in about 6 days. basic protocol 1 takes about 4 days. growing up a cryostock of 293f cells, bringing them to passage 4 (recommended before transfection), and obtaining a sufficient cell number would take another few days; this is not taken into account in the protocol. basic protocol 2 takes at least 2 days (antigen coating on day 1 and running the elisa on day 2). the screening elisa could be performed in the morning and the confirmatory elisa in the afternoon, or the assays can be done on consecutive days. a serological assay to detect sars-cov-2 seroconversion in humans. medrxiv reinfection could not occur in sars-cov-2 infected rhesus macaques. biorxiv the time course of the immune response to experimental coronavirus infection of man molecular diagnosis of a novel coronavirus (2019-ncov) causing an outbreak of pneumonia detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr assays for total protein sds-polyacrylamide gel electrophoresis (sds-page) of proteins. current protocols in microbiology basic techniques in mammalian cell culture human monoclonal antibody combination against sars coronavirus: synergy and coverage of escape mutants potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody cryo-em structure of the 2019-ncov spike in the prefusion conformation a new coronavirus associated with human respiratory disease in china a novel coronavirus from patients with pneumonia in china we thank dr. raffael nachbagauer (icahn school for medicine at mount sinai) and dr. aubree gordon (university of michigan) for critical reading and constructive comments. development of this protocol was partially supported by the niaid centers of excellence for influenza research and surveillance (ceirs) contract hhsn272201400008c.philanthropic donations in support of our work are much appreciated, since the reagents stadlbauer et al. current protocols in microbiology are shared free of charge with the scientific community. please contact vanesa saric (vanesa.saric@mountsinai.org) for further information. key: cord-347661-q9lgliph authors: zevenhoven-dobbe, jessika c.; wassenaar, alfred l. m.; van der meer, yvonne; snijder, eric j. title: production of monospecific rabbit antisera recognizing nidovirus proteins date: 2007-11-28 journal: sarsand other coronaviruses doi: 10.1007/978-1-59745-181-9_16 sha: doc_id: 347661 cord_uid: q9lgliph the importance of monospecific antisera for the experimental analysis of viral proteins is undisputed. they make it possible to identify and analyze the target protein against a background of a large number of other proteins, either in whole fixed cells or in cell lysates. this chapter describes our experience with the production of such rabbit antisera directed against proteins of coronaviruses and other nidoviruses. the use as antigens of either synthetic peptides (coupled to a carrier protein) or proteins expressed in escherichia coli is described, and detailed protocols for immunization and preparation of test bleeds are provided. for screening of the immune response following immunization, detailed protocols for three commonly used techniques are described, all of which are based on the use of infected cells or cells expressing the protein of interest, side by side with appropriate controls. the in situ immunodetection of the target in fixed cells by immunofluorescence microscopy is described, as are protocols for techniques that can be applied to cell lysates containing the target protein (western blotting and immunoprecipitation). the latter techniques are performed in combination with polyacrylamide gel electrophoresis, thus allowing confirmation of the molecular weight of the target that is recognized by the antiserum. coronaviruses, and other nidoviruses such as arteriviruses, produce one of the largest sets of viral polypeptide species among the rna viruses. this feature is related to the polycistronic nature of their genome, which contains up to 12 open reading frames and, in particular, to the polyprotein strategy used to produce the large viral replicase/transcriptase, the complex set of nonstructural proteins that is responsible for replication and transcription of the nidovirus genome [see (1) and the references therein]. in coronaviruses (fig. 1) , the larger of the two replicase polyproteins (pp1ab) can be more than 7000 amino acids long and is processed into 15 or 16 cleavage products, now commonly referred to as nonstructural protein (nsp) 1 to 16 (2,3) . in arteriviruses, despite their much smaller replicase size (pp1ab is "only" ∼3100 to 3900 amino acids), the complexity is not very different and replicase processing yields 13 or 14 cleavage products. the regulated autoproteolysis of nidovirus replicase pp1a and pp1ab is driven by two to four proteinases that reside in the orf1a-encoded part of the replicase. cleavage sites for coronavirus and arterivirus proteinases have been identified for several prototypic nidoviruses through a combination of theoretical and experimental methods and can now be confidently predicted for other members of these virus groups. since an additional 4 to 12 proteins are expressed from subgenomic mrnas produced from the 3 -end of the viral genome ( fig. 1) , including the structural polypeptides responsible for virion formation, the proteome of the average nidovirus consists of between 20 and 30 protein species. it should be noted that intermediates of replicase polyprotein processing, which have been described for various nidoviruses, are not taken into consideration in this count and are likely to even further increase the repertoire of viral polypeptides produced in infected cells. replicase precursors and processing intermediates in fact complicate certain types of analyses, such as those that rely on microscopy, since antibodies will usually not discriminate among precursors, intermediates, and processing end products. in the infected cell, many nidovirus proteins are targeted to specific locations (fig. 2) , some of them even to multiple specific locations. over a period of about 15 years, our laboratory has been involved in the production and characterization of polyclonal rabbit antisera directed against specific structural and, in particular, nonstructural proteins of nidoviruses (4-8). our experience in this area is summarized in this chapter. the usefulness and importance of monospecific antibodies for the experimental analysis of almost any protein in biology is undisputed. in particular, when depicted is the cytoplasmic replication cycle, which starts with virus entry and release of the genome into the cytoplasm. subsequently, the genome is translated to produce replicase polyproteins pp1a and pp1ab, which are cleaved to yield 16 nonstructural proteins (2). a complex for viral rna synthesis is assembled on cytoplasmic double-membrane vesicles (dmvs). this complex is involved in genome replication and the synthesis of a nested set of subgenomic mrnas, which are required to express the genes in the 3 -proximal third of the genome. translation of the smallest subgenomic mrna yields the viral nucleocapsid protein (n) that assembles into new nucleocapsid (nc) structures together with newly generated genome rna. other subgenomic mrnas encode viral envelope proteins that are (largely co-translationally) inserted into membranes of the host cell ' the target protein has to be studied against a background of a large number of other proteins, either in whole fixed cells or in cell lysates, specific antibodies can provide a rapid and reliable method for discriminating signal from noise. three commonly used antibody-based detection techniques, which are also important for screening of the immune response during antiserum production, are discussed below: • in situ immunodetection of the target in fixed cells, either by immunofluorescence (if) microscopy or immunoelectron microscopy (iem). immunofluorescence microscopy images of vero-e6 cells infected with sars-coronavirus illustrating the expression of four important viral genes and the different subcellular localization of their products. nonstructural protein 3 (nsp3) is one of the replicase subunits expressed from the genomic rna, revealing the localization of the membrane-bound viral replication complex. spike protein s localizes to different compartments of the exocytic pathway. membrane protein m accumulates in the golgi complex, in particular early in infection. nucleocapsid protein n is a largely cytoplasmic protein. • detection of the target by western blotting (wb), i.e., electrophoresis of a cell lysate containing the target and transfer of the (denatured) proteins to a solid carrier such as nitrocellulose or polyvinylidene fluoride (pvdf) membranes. • immunoprecipitation (ip) of the target from a cell lysate, often used in combination with radioactive labeling of protein synthesis in the cells for a specific time prior to cell lysis. although in several techniques experimental data obtained with monoclonal antibodies can be superior, in particular because of reduced background signal, the generation and screening of hybridoma cell lines requires a considerable investment, in terms of both labor and capital. furthermore, in the context of specific research questions, polyclonal antisera may sometimes even be preferred over monoclonal antibodies since they contain a mixture of immunoglobulin molecules, derived from different b-cell lines in the immunized animal, often recognizing multiple epitopes of the target protein. moreover, if desired, the chances of cross-reactivity of the antiserum with related proteins, e.g., from different strains of the same virus or from closely related other virus species, are considerably better for polyclonal antisera, particularly when they have been raised using a larger and/or conserved part of the target protein. obviously, the primary goal during polyclonal antiserum production in laboratory animals is to obtain a reasonable volume of a high-affinity antiserum. for rabbits, if necessary, bleeds of ∼15-20 ml can be obtained at 4-week intervals, and the final bleed can yield up to 50 ml of serum from about 80 ml of whole blood. since monoclonal antibodies are routinely produced in mice, the use of other species such as rabbits also creates the possibility of convenient in situ double-labeling experiments (see below). in such experiments, using speciesspecific secondary antibodies, a polyclonal rabbit antiserum against the target of choice and, for instance, a mouse monoclonal antibody recognizing a cellular protein can be used in combination to detect both proteins in the same specimen simultaneously. 7 the (obvious) standard prerequisites for the production of a polyclonal rabbit antiserum are: (i) an antigen, (ii) a (pathogen-free) rabbit to be immunized, (iii) a test method to detect the response, and (iv) a permit for animal experiments. the last relates to institutional and/or governmental regulations controlling animal use, which differ from country to country and are not discussed here in detail. important considerations are the choice of adjuvant, method and site of administration of the antigen, sedation of animals, maximum volume of test bleeds, and various safety precautions regarding animals and personnel. two types of antigens have been used in our studies: synthetic peptides and proteins expressed in and purified from escherichia coli. the production of antigens in e. coli are not covered in any detail in this chapter and the reader is referred to the extensive literature on this topic published elsewhere (and in chapter 13 in this volume). e. coli allows for the cheap production of large amounts of antigen, but clearly such antigens have to be purified prior to use for immunization. this can be facilitated, e.g., by the use of a variety of tags (such as fusion to glutathione-s-transferase, maltose-binding protein, or the commonly used hexahistidine tag) for which convenient affinity resins are available. reasonably pure antigens (>80% pure) are required and in the case of larger tags one should consider removing the tag proteolytically to ensure that the immune response will be directed against the target rather than against its tag. if the e. coli expression product turns out to be insoluble, which will usually interfere with its straightforward affinity purification, it may still be possible to purify a reasonably pure protein sample from inclusion bodies by repeated extraction of this material with increasing concentrations of urea (or guanidium isothiocyanate). as long as small amounts of this material can be used (i.e., the protein concentration is sufficiently high), after washing of the protein pellets with pbs, such urea-containing samples can be used for mixing with the adjuvant and immunization without the need for dialysis. synthetic peptides offer the advantage of being pure, and with certainty they contain exclusively the amino acid sequence of the selected target. since they normally cover only 10 to 25 residues of this target, their sequence has to be selected with care (see below). it is not straightforward to confidently predict antigenic peptides from an amino acid sequence. several programs to support this activity can be found on the internet and algorithms for this purpose are included in most dna/protein analysis software (9-11). if the structure of the target is known, peptides located on its surface are to be preferred. entirely polar and helical peptides should be avoided. in our experience, peptides located at (or close to) the n-or cterminus of the target also have a high probability of being immunogenic. this was true in particular for peptides derived from the nidovirus replicase polyproteins, which were usually selected on the basis of known or predicted cleavage sites (although based on relatively small numbers; success rate with terminal peptides was around 75% and with internal peptides only around 25%). the peptides to be synthesized usually are 10-25 amino acids long. peptides that are very hydrophobic may be more difficult to handle (e.g., poor solubility prior to coupling). to protect peptides against host proteases and thus increase their stability, the c-terminal carboxyl group can be replaced with an amide group during synthesis. (this may not be advisable when using peptides that normally form the c-terminus of a protein.) to facilitate coupling to bsa used as the carrier protein (see below), one or two lysine residues can be added to one side of the peptide (to the n-terminus when targeting the c-terminus of a protein, and vice versa). synthetic peptides are available from a variety of commercial or in-house sources. in our institute, peptides are synthesized by solid-phase strategies on an automated multiple peptide synthesizer (syroii, multisyntech, witten, germany). the purity of the peptides is determined by analytical reversed-phase hplc and should be at least 80%. the identity and homogeneity of the peptides is confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and analytical reversed-phase chromatography. freeze-dried peptides, dissolved peptides, or coupled peptides in solution are best stored at -85 • c. synthetic peptides have to be coupled to a carrier protein to enhance their immunogenicity. bovine serum albumin (bsa) and keyhole limpet hemocyanin (klh) are the two most commonly used carrier proteins. we have always relied on bsa (a very soluble and stable plasma protein) and a coupling reaction using glutaraldehyde, which links the amino groups of carrier and synthetic peptide (in lysine residues and at the amino terminus of the peptide). for example, crosslinking at lysine residues can be represented as: note that the above calculations are based on a number of assumptions and should be considered as a rule of thumb. clearly, since the sequence is known, the mr of the peptide can be calculated more precisely. however, since we usually add extra lysines to the peptide there will be different ways in which it can be coupled to the carrier and a variety of complexes will be formed (bsa "trees" with a varying number of peptide "branches" scattered all over the backbone), presenting the epitope in many different ways. 3 . to the peptide-bsa mixture, add pbs to give a final volume of 670 l; then (carefully, slowly, and while mixing) add 330 l of a freshly prepared 0.3% glutaraldehyde solution (equaling approximately 10 mol). 4. incubate the coupling reaction on a roller device at room temperature for 1 h to allow cross-linking of peptide and carrier protein. 5. add 200 l of 1-m glycine in order to quench the remaining free glutaraldehyde and continue rolling for 1 h. 6. dialyze the coupling reaction overnight against pbs to remove small molecules such as uncoupled peptide, glycine, and glycine-glutaraldehyde. dialyze two or three times against at least 500 volumes of pbs at 4 • c. the final step can be overnight. usually, after dialysis, the concentration of peptide-bsa complex is around 4 mg/ml and the solution is ready for direct use as an antigen in immunization. when using synthetic peptides or proteins purified from e. coli, adjuvants containing immunostimulatory molecules are applied to enhance the immune response to the antigen. freund's adjuvants have been used in our studies; freund's complete adjuvant (fca) for the initial immunization and freund's incomplete adjuvant (fica) for subsequent booster immunizations. fca (but not fica) contains heat-inactivated mycobacterium tuberculosis or mycobacterium butyricum (or extracts thereof), which stimulate both cellular and humoral immunity. the water-in-oil emulsion that is the basis for fca guarantees the slow release of the antigen from the site of immunization, but it should be noted that the mineral oil component is quite toxic and induces granulomatous reactions. mix fca/fica with the antigen-containing solution to form a "toothpaste-like" stable emulsion. see below for details. in our standard protocol, ∼200 g of antigen (peptide-bsa complex) is used for the primary immunization, and the same amount for subsequent booster reactions. 1 . before use, mix the fca, e.g., gently on a roller device to get the bacteria into suspension. for the primary immunization, the antigen (200-250 g) is diluted with pbs to give a final volume of 0.8 ml pbs, which is mixed with 0.4 ml of fca. transfer this mixture to a 3-ml syringe (luer-lock). use a p1000 micropipette; put the tip in the opening of the syringe, and slowly pull the plunger back. in the same manner, fill a second syringe with 0.4 ml of fca and 0.4 ml of air (to promote the formation of the emulsion). 2. connect the two syringes with a three-way stopcock (fig. 3) . mix the contents of the two syringes until a thick, white emulsion is obtained. this means that the suspension is converted into an oily mass that includes the water. this emulsion, when properly prepared, is stable and will not disperse when a droplet is put into pbs. the emulsion will ensure the slow release of the antigen into the animal. 3. disconnect the syringes and use the antigen suspension the same day. optionally, the syringes can be stored for later use, overnight at 4 • c or for longer periods at -80 • c. 4. for booster immunizations fica is used instead of fca. material for two boosts can be prepared in one action by doubling the volumes above. filled syringes can be stored at -80 • c for prolonged periods of time. young adult new zealand white rabbits (preferably females, weighing between 2 and 3 kg) have been used routinely in our studies (fig. 3) . the young age of the animals is thought to be particularly important to ensure a strong primary response to the antigen. although it is often advised to use two animals per antigen, in our experience-with just a few exceptions-both animals generally give the same (positive or negative) result, although titers of the antiserum and background signal(s) may vary between such duplicates. still, if the number of animals available or housing capacity is limited, and also for financial and ethical reasons, it may be wiser to use a single animal per antigen and-in the event of failure-return to such an antigen in a subsequent round of immunizations. the rabbits are injected subcutaneously at three or four places on their back (fig. 3) . given the strong inflammatory response induced by fca, the animals are monitored closely following the primary immunization. the interval between the primary immunization and the first booster, and between subsequent boosters, is about 4 weeks. it is essential to obtain a preimmunization bleed on (or before) the day of the primary immunization, which will be used to assess the response against the antigen (see below). the standard schedule for immunization and bleeding would look like this: bleeds should be limited to 15% of the animal's total blood volume and a 4-week recovery period should be allowed. as a "rule of thumb," the blood volume of rabbits is about 56 ml/kg of body weight-assuming the animal is mature, healthy, and adequately nursed. in our experience, a (good) response is unlikely (but not impossible. . .) if there is no positive signal after two or three boosts. although there have been some exceptions to this rule, it is advisable-for financial reasons as well as ethical considerations-to terminate the experiment after ∼70 days and, if necessary, try again with an additional animal or an alternative antigen later on. 1. collect 15-20 ml of blood (usually from an ear vein of the rabbit; fig. 3 ) in a 50-ml centrifuge tube and allow complete clotting of the blood in a water bath at 37 • c for 1 h. 2. use a pasteur pipette with a melted tip to gently liberate the clot from the wall of the tube. be sure to cause minimal lysis of red blood cells, to ensure a clear serum sample later on. subsequently, leave the clot to shrink overnight at 4 • c (fig. 3) . 3. centrifuge the tube at 1300 × g for 10 min to further reduce the volume of the clot. 4. carefully, with a pipette, remove as much serum as possible from the tube without touching/damaging the clot; in case of doubt, use a second collection tube for the last few ml (also an additional spin might help), which may be less clear and might be saved as a backup sample only. 5. prepare some smaller and some larger aliquots of the serum and store at -20 • c or -80 • c. sera can be kept for many years, but repeated freezing and thawing should be avoided. also, it is advisable to use tubes with a screw cap lid containing a rubber seal, which will minimize evaporation during prolonged storage. store a small sample (0.5 to 1 ml) at 4 • c for testing. 6. working stocks of antisera (in screw cap tubes) can be kept at 4 • c for many months; if desirable, 0.05% (final concentration) of sodium azide (highly toxic!) can be added as a preservative, but in our experience this is not necessary if sera are kept cool and handled with care. it is advisable to spin antisera (in particular the less clear ones) for 1 min at full speed in a microcentrifuge prior to use. the native proteins that are the targets for antiserum production are usually highly structured molecules and obviously the reactivity of the antiserum will be influenced by the conformation of the antigen used for immunization. in particular, when synthetic peptides are used the structural resemblance between antigen and target may be limited. this probably explains why-in our experiencepeptides derived from the termini of the target protein, which are often less structured than the protein core, generally work better than internal peptides. although an elisa approach, based on the antigen used for immunization, can be employed for an initial screening of the immune response in the animal, this result may be relatively meaningless when it comes to the question of whether the antiserum will ultimately react with the viral protein target. one will in fact be measuring the response against the combination of peptide and carrier protein or, in the case of immunization with proteins expressed in and purified from e. coli, against the target and contaminants present in the antigen. all the immunizations we carried out with bsa-coupled peptides produced sera that reacted positively in an elisa using plates coated with the uncoupled peptide, but less than half of those turned out to be useful in if, wb, or ip. therefore, it is advisable to screen immediately using samples containing the native, full-length viral protein target. moreover, during this screening process, the conformation of the target is an issue. when screening formaldehyde-fixed whole cells by microscopy techniques the target is fixed but structured. during wb analysis proteins are subjected to denaturing conditions and fixed to the membrane used for blotting, whereas ips are done in solution and can be performed under both native and denaturing conditions. in fact, the level of denaturation in ip experiments can be easily influenced by varying the percentage of sds in the ip buffer. often, ips with antisera raised using synthetic peptides work better in the presence of relatively high (0.25-0.5%) concentrations of sds, probably because the epitope in the denatured target resembles the antigen used for immunization more closely under these conditions. an additional significant advantage is the fact that the higher sds concentrations will strongly reduce the background ip signal and result in a much cleaner analysis (see below). in our experience, antisera that work well in if assays usually also work well in wb and ip. conversely, sera that are negative in if may still be highly reactive in wb and/or denaturing ip. consequently, it is important to test new antisera in at least two of these three assays. in our daily practice, we have usually relied on if for preliminary screening and wb for confirmation. there is an additional reason to confirm the if results by subsequent wb or ip analysis: in if assays antisera can sometimes show a strong reaction with cellular proteins (fig. 4) , either-at low dilutions-because of the lack of a specific response against the target or-at higher dilutions-because of an unexpected cross-reactivity cov nsp3 (4) , which was very specific in vero-e6 cells (see fig. 5 ), resulting in a strong, punctate nuclear labeling in bhk-21 cells that expressed the ace-2 receptor for sars-cov. (d) sars-cov-infected cells labeled several weeks after fixation and embedded using prolong mounting fluid. an aspecific nucleolar background labeling was observed, especially in the green range of the fluorescence spectrum. the specific, much brighter signal is derived from an anti-nsp3 labeling using a rabbit antiserum that was directly coupled to alexa fluor-488 (4). with cellular proteins. wb or ip analysis provides important information about the molecular mass of the target that is recognized, and may thus prevent premature conclusions about the specificity of the antiserum. the correct assessment of specificity is also aided by the inclusion of two important controls: the preimmunization serum (which should obviously not show the same signal) and mock-infected control cells (which might reveal that it is in fact a cellular target that is being recognized). when performing initial screening with if assays, it is practical to use cell cultures that have been infected at an moi of 0.5-1 and therefore contain a mixture of virus-infected and mock-infected cells in the same specimen. semiconfluent cells seeded on glass cover slips (10-mm-diameter) are the preferred specimens for initial testing. the cells can either be infected with the target virus or transfected with a vector expressing the target protein. obviously, cells should be fixed at a time point when the target protein is convincingly expressed. the most reactive rabbit antisera that we have produced can be used in if assays at dilutions of between 1:1000 and 1:5000. for initial testing, however, dilutions on the order of 1:100 to 1:500 are advised. 1 . wash the cells once with pbs and fix the cells at room temperature with 3% paraformaldehyde in pbs for at least 30 min (or overnight). 2. wash the cells once with pbs containing 10 mm glycine (coverslips in sealed dishes can be stored at 4 • c in pbs for many weeks). 3. using sharp tweezers, carefully transfer coverslips to the wells of a prelabeled 24well cluster containing pbs-glycine. be sure to remember, every time you handle the coverslips, on which side there are cells. while in the cluster, cells should always be facing upward. 4. permeabilize the cells at room temperature for 10 min in pbs containing 0.1% triton x-100. 5. in 5 min, wash the cells three times with pbs-glycine and leave them in the last wash step. 6. dilute the primary antiserum (initial dilutions, e.g., 1:100 and 1:500) in pbs containing 5% fcs; 50 l per coverslip (10-mm-diameter) will be needed. 7. cut a large piece of parafilm, place it in a larger dish, label the position of the various samples, and place 50-l drops of the antiserum dilutions on the parafilm. one by one, take the coverslips from the 24-well cluster, remove excess pbs by touching a tissue to the side of the coverslips, and place the coverslips on the drops with the cells facing the antiserum. 8. incubate for 30-60 min at room temperature (or 37 • c); make sure the samples do not dry out during this incubation (e.g., by placing a wet tissue in the dish). 9. return the coverslips to the 24-well cluster; in 20 min wash three times with pbsglycine and leave them in the last wash step. 10. prepare a dilution of the fluorescently labeled secondary antibody. optimal dilutions of conjugates should be determined separately using a well-defined primary antiserum. again 50 l per coverslip will be needed. optionally, 1 g/ml of hoechst 33258 can be added to this dilution for staining of nuclear dna. 11. incubate and wash as described under steps 7 to 9. 12. take the coverslips from the 24-well cluster. embed the specimens onto glass microscopy slides in a mounting fluid [e.g., mowiol or prolong (molecular probes)]. avoid air bubbles in the mounting fluid by slowly and carefully sliding the coverslip on a small drop (∼5 l) of mounting fluid. 13. store the specimens in the dark at 4 • c. the mounting fluid should harden at least overnight before high magnification lenses and immersion oil are used. 14. analyze the specimens in a fluorescence microscope using the filter sets required for the label attached to the secondary antibody. double-labeling experiments can be done to compare the localization of two (or more) proteins of interest in the same cell by combining a rabbit antiserum recognizing one protein and, e.g., a mouse monoclonal antibody recognizing a second target. obviously, the two primary antisera have to be detected with suitable conjugates carrying different fluorescent labels, preferably with wellseparated emission spectra. following initial optimization (testing of different dilutions, balancing of the two signals, and controls for specificity of primary and secondary antibodies), one can usually carry out the labeling using a twostep approach. first, specimens are simultaneously incubated in a combined dilution of the two primary antibodies and, subsequently, after extensive washing, in a combined dilution of the two conjugates. in case of background and/or specificity problems, however, it may be necessary to perform the labeling in four consecutive steps. in if assays, double labeling with two antisera from the same species is only possible when one of the two is directly coupled to a fluorescent group. we have recently been successful (fig. 5) (4) in purifying the ig fraction from small volumes (2-3 ml) of rabbit antisera using a commercially available protein a column and have subsequently conjugated these antibodies to alexa fluor-488. the if assay then consisted of three incubation steps: (i) incubation with the uncoupled rabbit antiserum, (ii) incubation with the anti-rabbit conjugate recognizing the first antibody, and (iii) incubation with the alexa fluor-488-coupled second rabbit antiserum. the order of these steps is very important to avoid binding of the anti-rabbit conjugate to the directly labeled second rabbit antiserum. during western blotting (wb) analysis samples containing the protein of interest are separated according to size in acrylamide gels and transferred to a membrane, which is subsequently incubated with the antiserum. protein bands reacting with the antiserum are detected using a secondary antibody and accompanying assay (a variety of enzyme-linked or fluorescent conjugates is available; here we use a peroxidase-coupled secondary antibody). as in the case of if assays, lysates to be tested can be derived from cells that are either infected or transfected with an appropriate expression vector. for infection lysates, we use a high multiplicity of infection to avoid a mixture of infected and uninfected cells in the sample. cells are lysed in protein lysis buffer, which leave the nuclei intact and allow their removal by centrifugation. for initial testing, an amount of lysate equaling ∼5 × 10 5 cells per 5 cm gel width (or 10-15 slots) can be used. alternatively, purified protein (e.g., expressed in e. coli) can be used (100-500 ng is fig. 6 . example of a standard test of a rabbit antiserum in western blot and immunoprecipitation. the antiserum used here was raised against a domain in the c-terminal region of pp1a of the arterivirus eav (8) and recognizes a large set of processing intermediates and end products, which are indicated by arrowheads: (a) western blot analysis: eav-and mock-infected cell lysates were prepared at 8 h postinfection, run on an sds-polyacrylamide gel, blotted to pvdf membrane, and incubated with the postimmune serum at a 1:1000 dilution. detection was with an anti-rabbit igg hrpo conjugate and a chemiluminescence assay. [reproduced from (13).] (b) immunoprecipitation: eav-infected cells were labeled with 35 s-methionine/cysteine for 3 h, from 5-8 h postinfection and lysates were used for ip using 5 l of antiserum per sample. following the ip, samples were run on an sds-polyacrylamide gel and signal was detected using autoradiography. as specificity controls, the preimmune serum was tested on eav-infected cells and the postimmune serum was tested on mock-infected cells. furthermore, for the left panel the binding of the antiserum to the antigen was done in the presence of 0.1% sds, whereas 0.5% sds was used for the right panel. the comparison illustrates how higher sds percentages can reduce the background signal and improve the overall picture. usually sufficient). most antisera can be used at a 1000-fold dilution or higher, but we advise starting the testing with a 1:500 dilution (fig. 6a) . protein-antibody complexes are then purified by having them bind to beads carrying the ig-binding proteins a or g on their surface, which are subsequently spun down and washed repeatedly to remove unbound proteins. the simplest form of protein a carrying beads are formalin-fixed staphylococcus aureus cells (e.g., pansorbin; calbiochem), but alternatively protein a or g coupled to sepharose can be used. usually, metabolically labeled protein lysates (e.g., 35 s-methionine-labeled) are used for immunoprecipitation studies and samples are run on sds-page gels that can then be used for autoradiography. in contrast to wb analysis, immunoprecipitation relies on recognition of the target in solution. depending on the conditions used, proteins can either be close to their native confirmation, partially denatured, or completely denatured (e.g., in the presence of high concentrations of sds). thus, the conformation of the antigen used for immunization may influence the results. in our experience, linear antigens such as synthetic peptides may yield antisera that work considerably better in immunoprecipitation assays carried out under stringent denaturing conditions (e.g., 0.5-1% sds), which will often also reduce the background in the assay (fig. 6b) . nevertheless, the optimal concentration of sds should be determined for every antigen-antiserum pair since the optimum may be as low as 0.1% sds, e.g., when using antibodies recognizing mainly (or exclusively) conformational epitopes. furthermore, the amount of antiserum in the immunoprecipitation assay is important. for initial screening of rabbit antisera we usually test 1 and 5 l of the antiserum in a 300 l total ip volume. coverslips with infected cells or cells expressing the protein of interest. 2. fixative: 3% paraformaldehyde in pbs pbs: see section 2.1, step2 pbs with 5% fetal calf serum (fcs) pbs with 10 mm glycine protein lysis buffer (20 mm tris-hcl amersham biosciences, hybond p, #rpn303f) 10x western blot transfer buffer (wtb): 250 mm tris, 1.92 m glycine pbs: see section 2.1, step 2 pbs-tm with 1% bsa) anti-rabbit igg horseradish peroxidase conjugate, e.g., swine-anti-rabbit igg hrpo amersham biosciences, ecl plus western blotting detection kit ip buffer (20 mm tris-hcl weak wash buffer a (20 mm tris-hcl ph 7.6; 150 mm nacl laemmli sample buffer (lsb): 50 mm tris-hcl, ph 6.8; 100 mm dtt; 10% glycerol formalin-fixed staphylococcus aureus cells; calbiochem #507858) or protein a/g sepharose beads wash the cells with cold pbs and-to a 10-cm 2 dish with 1-2 × 10 6 cells-add 300 l of cold protein lysis buffer transfer the lysate to a labeled microfuge tube and spin down the nuclei for 2 min at full speed in a microcentrifuge transfer the supernatant to the new tube, leave the pellet (nuclei, often barely visible) behind and add 1/100 the lysate can now be used for wb or stored in the -20 • c/-80 • c freezer prepare an sds-page gel (or minigel) of a suitable acrylamide percentage (depending on the size of the protein of interest) and run: (i) the lysate containing the protein of interest, (ii) a control lysate (mock-infected or untransfected cells), and (iii) a molecular weight marker. several sets of these samples can be run on one gel to try different antiserum dilutions, or wider slots can be used and strips cut with the right samples after blotting with a pencil mark one side of the membrane; this is the side of the membrane that will face the gel during blotting and, subsequently, the solutions during incubation. prewet the pvdf membrane in methanol for 5 min; never touch the pvdf membrane with bare fingers; always use gloves! dilute 10x wtb to give 1x wtb and incubate the membrane in 1x wtb for 10 min take the gel from between the glass plates and briefly wash it in 1x wtb build the electroblot stack-from cathode to anode-with the following layers: three sheets of whatman paper presoaked in wtb (and optionally one sheet of whatman paper soaked in wtb with 0.1% sds), the equilibrated gel, the equilibrated and marked membrane :500) and the preimmune serum as a control (1:500) in pbs-tmb and incubate the blots for 1 h at room temperature while swirling dilute the peroxidase-conjugated secondary antibody (swine-anti-rabbit igg hrpo) in pbs-tmb and incubate the blots for 1 h at room temperature while swirling prepare the solutions for the chemiluminescence assay according to the manufacturer's instructions gently "semidry" the blot with some whatman paper (let the fluid run off; do not really dry it!) put the chemiluminescence solution on the plastic foil and incubate the blot with the "protein side down" for 5 min remove the excess of fluid with some whatman paper and wrap the blot in plastic foil expose an x-ray film to the blot for 1-2 min, develop the film, and check if a longer exposure is required. to avoid overexposure of the film, it may be wise to wait about 30 min before making the first exposure take 50 l of 35 s-labeled cell lysate in protein lysis buffer (12) (equaling about 2-4 × 10 5 cells; obviously the expression level of the target protein is to be considered as well); add 250 l of ip buffer and the antiserum (1 or 5 l). incubate these ∼300-l samples overnight wash the pansorbin cells (25 l for each sample) by mixing with an equal volume of ip buffer, spin down for 20 sec at full speed in a microcentrifuge, and resuspend the pansorbin cell pellet in the original volume (again add 25 l of washed pansorbin cells to each immunoprecipitation and incubate at 4 • c for more than 1 h while swirling spin down for 1 min at full speed in a microcentrifuge, remove the supernatant, and add 500 l of weak wash buffer a; vortex until the pellet is completely resuspended. (optionally, this step can be repeated once to get a cleaner result spin down for 1 min at full speed in a microcentrifuge again and repeat the same washing procedure with weak wash buffer b resuspend the pellet in 30 l of lsb by pipetting up and down before loading the samples on an sds-page gel, denature at 96 • c for 3 min. (for some proteins, e.g., very hydrophobic ones, it may be better not to heat the samples and just leave them in lsb for 15 min or longer spin the pansorbin cells down for 2 min at full speed in a microcentrifuge; do not resuspend the pellet! transfer the supernatant to a new tube and load part of this sample onto an sds-page gel following sds-page, process the gel for autoradiography or phosphorimager analysis according to standard protocols and expose for 1-7 days not all bleeds of the same rabbit have the same concentration of antibody and thus the dilutions to be used can vary mounting solutions can sometimes have strange side effects, especially when the cells are not freshly fixed. for example, the use of prolong gold mounting fluid can generate a green nonspecific nucleolar signal when microscopy slides used for embedding are not clean, wipe them first with a tissue with 70% ethanol, next with water, and then with a dry paper towel/tissue. when ethanol is not removed, a (sometimes bright) orange background may result occasionally, antisera judged to be negative in if using paraformaldehyde-fixed cells can become positive using methanol-fixed cells (or the other way round) however, methanol fixation is less gentle and generally the morphology of the cell is less well preserved. fixation is performed for 10 min at -20 • c (using ice-cold 100% methanol or 95% methanol/5% acetic acid) and cells are subsequently transferred to and kept in pbs-glycine. methanol immediately dissolves all cellular membranes topley and wilson's microbiology and microbial infections: virology volume virus-encoded proteinases and proteolytic processing in the nidovirales unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex nuclear localization of nonstructural protein 1 and nucleocapsid protein of equine arteritis virus the putative helicase of the coronavirus mouse hepatitis virus is processed from the replicase gene polyprotein and localizes in complexes that are active in viral rna synthesis processing of the equine arteritis virus replicase orf1b protein: identification of cleavage products containing the putative viral polymerase and helicase domains proteolytic processing of the replicase orf1a protein of equine arteritis virus prediction of protein antigenic determinants from amino-acid-sequences the antigenic index-a novel algorithm for predicting antigenic determinants semiempirical method for prediction of antigenic determinants on protein antigens structural proteins of equine arteritis virus proteolytic maturation of replicase polyprotein pp1a by the nsp4 main proteinase is essential for equine arteritis virus replication and includes internal cleavage of nsp7 the authors would like to thank jan wouter drijfhout and willemien benckhuijsen (lumc department of immunohaematology) for advice and assistance on peptide design and synthesis. we are grateful to the staff of the lumc animal facility for almost 15 years of pleasant collaboration and reliable housing and handling of the rabbits used for antiserum production. this work was supported (in part) by the european commission int the context of the activities of the euro-asian sars-dtv network (sp22-ct-2004-511064). key: cord-332221-6ea6gz9s authors: li, guiping; zhou, lijuan; zhang, can; shi, yun; dong, derong; bai, miao; wang, rong; zhang, chuanfu title: insulin-like growth factor 1 regulates acute inflammatory lung injury mediated by influenza virus infection date: 2019-11-26 journal: front microbiol doi: 10.3389/fmicb.2019.02541 sha: doc_id: 332221 cord_uid: 6ea6gz9s the acute inflammatory lung injury is an important cause of death due to influenza a virus (iav) infection. insulin-like growth factor 1 (igf1) played an important role in the regulation of inflammation in the immune system. to investigate the role of igf1 in iav-mediated acute inflammatory lung injury, the expression of igf1 and inflammatory cytokines was tested after iav a/puerto rico/8/1934 (h1n1; abbreviated as pr8) infection in a549 cells. then, a balb/c mouse model of pr8 infection was established. on days 3, 5, 7, and 9 post-infection, the mice lung tissue was collected to detect the expression changes in igf1 mrna and protein. the mice were divided into four groups: (1) pbs (abbreviation of phosphate buffered saline); (2) pr8 + pbs; (3) pr8 + igf1; and (4) pr8 + ppp (abbreviation of picropodophyllin, the igf1 receptor inhibitor). the body weight and survival rate of the mice were monitored daily, and the clinical symptoms of the mice were recorded. on day 5 post-infection, the mice were sacrificed to obtain the serum and lung tissues. the expression of inflammatory cytokines in the serum was detected by enzyme linked immunosorbent assay; lung injury was observed by hematoxylin-eosin staining; the viral proliferation in the lung was detected by real-time quantitative pcr; and the protein expression of the main molecules in the phosphatidylinositol-3-kinases/protein kinase b (pi3k/akt) and mitogen-activated protein kinase (mapk) signaling pathways was detected by western blot. it was found that igf1 expression is upregulated in a549 cells and balb/c mice infected with pr8, whereas igf1 regulated the expression of inflammatory cytokines induced by pr8 infection. overexpression of igf1 aggravated the iav-mediated inflammatory response, whereas the inhibition of igf1 receptor reduced such inflammatory response. the phosphorylation of igf1 receptor triggered the pi3k/akt and mapk signaling pathways to induce an inflammatory response after iav infection. therefore, igf1 plays an important immune function in iav-mediated acute inflammatory lung injury. igf1 may provide a therapeutic target for humans in response to an influenza outbreak, and inhibition of igf1 or igf1 receptor may represent a novel approach to influenza treatment. the acute inflammatory lung injury is an important cause of death due to influenza a virus (iav) infection. insulin-like growth factor 1 (igf1) played an important role in the regulation of inflammation in the immune system. to investigate the role of igf1 in iav-mediated acute inflammatory lung injury, the expression of igf1 and inflammatory cytokines was tested after iav a/puerto rico/8/1934 (h1n1; abbreviated as pr8) infection in a549 cells. then, a balb/c mouse model of pr8 infection was established. on days 3, 5, 7, and 9 post-infection, the mice lung tissue was collected to detect the expression changes in igf1 mrna and protein. the mice were divided into four groups: (1) pbs (abbreviation of phosphate buffered saline); (2) pr8 + pbs; (3) pr8 + igf1; and (4) pr8 + ppp (abbreviation of picropodophyllin, the igf1 receptor inhibitor). the body weight and survival rate of the mice were monitored daily, and the clinical symptoms of the mice were recorded. on day 5 post-infection, the mice were sacrificed to obtain the serum and lung tissues. the expression of inflammatory cytokines in the serum was detected by enzyme linked immunosorbent assay; lung injury was observed by hematoxylin-eosin staining; the viral proliferation in the lung was detected by real-time quantitative pcr; and the protein expression of the main molecules in the phosphatidylinositol-3-kinases/protein kinase b (pi3k/akt) and mitogen-activated protein kinase (mapk) signaling pathways was detected by western blot. it was found that igf1 expression is upregulated in a549 cells and balb/c mice infected with pr8, whereas igf1 regulated the expression of inflammatory cytokines induced by pr8 infection. overexpression of igf1 aggravated the iav-mediated inflammatory response, whereas the inhibition of igf1 receptor reduced such inflammatory response. the phosphorylation of igf1 receptor triggered the pi3k/akt and mapk signaling pathways to induce an inflammatory response after iav infection. therefore, igf1 plays an important immune function in iav-mediated acute inflammatory lung injury. igf1 may provide a therapeutic target for humans in response to an influenza outbreak, and inhibition of igf1 or igf1 receptor may represent a novel approach to influenza treatment. keywords: influenza virus, igf1, acute inflammatory lung injury, pi3k/akt, mapk introduction influenza is an acute infectious disease caused by influenza virus infection, which is primarily characterized by respiratory damage. moreover, it is associated with serious features (e.g., acute onset, wide spread, strong contagiousness, and great harm), which seriously threatens human health. studies have shown that a series of symptoms and consequences caused by the influenza virus are not caused by the direct action of the influenza virus itself but are rather due to the inflammatory injury caused by the excessive activation of the immune response induced by an influenza virus infection (oda et al., 1989; taubenberger and morens, 2006; basler and aguilar, 2008; li and cao, 2017) . during the process of an iav infection, there are different cytokine waves from the initial early responses of monocyte chemotactic protein-1 (mcp-1), interleukin-8 (il-8), and interferon-alpha/beta/kappa (ifn-α/β/κ) to the later responses of il-1α/β, il-6, il-18, tumor necrosis factor-alpha (tnf-α), ifn type i, macrophage inflammatory protein-1 alpha/beta (mip-1α/β), mip-3α, mcp-3, and interferon-inducible protein-10 (ip-10), which are mainly secreted by infected macrophages in the lower respiratory tract (julkunen et al., 2001; xagorari and chlichlia, 2008; jewell et al., 2010; gu et al., 2019) . the modest cytokine response contributes to the induction of antiviral and th1-type immune responses in the body; however, excessive inflammatory responses can be harmful. for example, the main reason for the high pathogenicity of the h5n1 avian influenza virus is the exaggerated generation and secretion of excessively high levels of pro-inflammatory cytokines, known as a "cytokine storm" (kobasa et al., 2007; allen et al., 2009) . in 1918, the "spanish flu" also caused fatal inflammatory damage to the lung tissue by triggering an overreaction of the human immune response (ocana-macchi et al., 2009 ). this inflammatory injury to the lung tissue is both an important cause of death due to influenza virus infection and a major cause of lung infections caused by severe acute respiratory syndrome (sars), sepsis, and aspiration pneumonia (imai et al., 2005; nicholls and peiris, 2005) . currently, immunosuppressive agents (e.g., glucocorticoids) are often used in clinical practice to inhibit inflammatory cytokine responses, thereby blocking disease progression and improving the clinical therapeutic effect. the literature reports that 51-69% of patients with severe h1n1 influenza are treated with glucocorticoids, which have a specific therapeutic effect (aikawa et al., 2012) ; however, glucocorticoids systemically inhibit the immune function of the infected individual. moreover, the long-term treatment with such medication can induce or aggravate the infection, causing a variety of complications and serious side effects (e.g., femoral head necrosis). therefore, it is imperative to develop a deep understanding of the pathogenesis of influenza virus and to identify new strategies of treating influenza more safely and effectively. insulin-like growth factor 1 (igf1) belongs to the insulinlike growth factor family, which also includes growth hormone (gh), insulin-like growth factor ii (igf2), insulin-like growth factor 1 receptor (igf1r), insulin-like growth factor ii receptor (igf2r), and insulin-like growth factor binding protein 1-6 (igfbp1-6; jogie-brahim et al., 2009) . this family plays an extremely important role in the process of cell growth, differentiation, and apoptosis (stewart and rotwein, 1996; granata et al., 2004) . igf1 mainly functions by binding to igf1r, a transmembrane protein composed of two α domains and two β domains (siddle et al., 2001) . the α domain binds to igf1 to activate the β domain (siddle et al., 2001) . since the β domain has tyrosine kinase activity, it can promote the phosphorylation of the substrate hepatocyte growth factor (hgf), docking protein insulin receptor substrate (irs), vascular endothelial growth factor (vegf), and growth factor receptor binding protein 2 (grb2; wilden et al., 1992; hubbard, 1997) . some phosphorylated substrates activate the downstream phosphatidylinositol-3-kinases/protein kinase b (pi3k/akt) and mitogen-activated protein kinase (mapk) signaling pathways to regulate a range of biological responses (myers et al., 1992; egan et al., 1993) . recent studies have found that igf1 plays an important role in the regulation of inflammation in the immune system. igf1 mrna expression in the bronchial cells of asthmatic patients was significantly higher than that of normal people and was significantly associated with fibrosis in epithelial cells (hoshino et al., 1998) . the associated mechanism is that igf1 binds to the receptor and activates the pi3k/akt signaling pathway and induces akt activation, which further activates the downstream il-17-mediated inflammatory pathway (lee et al., 2014) . in addition, the levels of serum igf1 protein in patients with type 2 diabetes are significantly higher than those in healthy people. obesity is closely related to the pathogenesis of type 2 diabetes, as long-term obesity will lead to il-6 and il-17-mediated chronic inflammation (cohen and leroith, 2012; ge et al., 2013) . in non-autoimmune inflammatory contact dermatitis, igf1 relieves the inflammatory response by recruiting regulatory t cells to release the anti-inflammatory cytokine, il-10 (johannesson et al., 2014) . in the nervous system, igf1 recruits anti-inflammatory proteins to protect nerve cells from degeneration (arroba et al., 2016) . other studies have shown that igf1 plays an important role in regulating the function of lactating buffalo oocytes and preventing inflammation induced by post-partum genital tract infections. a concentration of 50 ng/ml igf1 acts on lipopolysaccharides (lps; 1 μg/ml)-infected buffalo granulosa cells, reducing the expression of the inflammatory cytokines, il-6, tnf-α, and il-1β, as well as decreasing akt phosphorylation in pi3k/akt signaling pathway and extracellular-regulated kinase 1/2 (erk1/2) phosphorylation in the mapk signaling pathway (onnureddy et al., 2015) . however, whether igf1 plays a significant role in mediating inflammation and pathology during influenza infection and its associated mechanism remains unknown. in this study, we found that igf1 mrna and protein increased after influenza virus infection. overexpression of igf1 aggravated cytokine expression during infection by influenza, while blocking of igf1 production in mice treated with igf1r inhibitor, decreased immunopathology. the phosphorylation level of igf1r was elevated after influenza virus infection, triggering the pi3k/ akt and mapk signaling pathways to induce an inflammatory response. thus, igf1 could regulate influenza virus-mediated acute inflammatory lung injury, which may provide a therapeutic target for humans in response to an influenza outbreak. the human alveolar epithelial cell line a549 is particularly sensitive to influenza virus infection and has been widely used as a good in vitro model to study influenza virus for nearly 20 years. the cell line a549 was purchased from the american type culture collection (atcc, usa) and propagated in dulbecco's modified eagle's medium (dmem; life technologies, usa) supplemented with 10% fetal bovine serum (fbs; hyclone, usa) at 37°c in a 5% co 2 incubator. the mouse adapted influenza a virus (iav) a/puerto rico/8/1934 (h1n1; abbreviated as pr8) was kindly provided by prof. shihui sun (beijing institute of microbiology and epidemiology) and propagated in 9-to 11-day-old spf chicken embryos. the allantoic fluid was collected and titrated to determine the 50% tissue culture infection dose (tcid 50 ) in a549 cells and the median lethal dose (ld 50 ) in mice following the reed-muench method (reed and muench, 1938) . specific pathogen free (spf) grade female balb/c mice aged 6-8 weeks (body weight: 18-20 g) were purchased from the experimental animal center of the military medical research institute. amplification of human igf1 open reading frame (orf; guangzhou genecopoeia biotechnology co., ltd.) using primers containing xba i and xho i restriction sites (forward: 5′-tgctctagaatgggaaaaatcagcagtct-3′; reverse: 5′-ccgctcgagctacatcctgtagttcttgt-3′) ligated into a pcdna3.1 expression vector, constructing pcdna3.1-igf1. the pcdna3.1-igf1 vector was transfected into a549 cells with lipo2000. the cell line overexpressing igf1 was screened with g418 (500 μg/ml). the human igf1 shrna lentiviral particles (sc-37193-v) were purchased from santa cruz company. the total cellular rna was extracted using trizol (invitrogen, cat: 15596-026). the cdna was synthesized by reverse transcription using a tianscript rt kit (tiangen, cat: kr104), followed by quantitative pcr (qpcr) using sybr premix ex taq ii (takara, cat: rr820a). the primer sequences that were used are presented in table 1 . when detecting the viral proliferation in the lungs of mice, a realtime fluorescent quantitative pcr probe method was used, and the probe sequence was fam-tgcagtcctcgctcac tgggcacg-bhq1. the primer sequence of matrix protein 1 (m1) was forward: 5′-gaccratcctgtcacctctgac-3′; reverse: 5′-gggcattytggacaaakcgtctacg-3′. gapdh was selected as the internal reference, and the results were analyzed using the 2 −△△ct method. the reaction conditions were set as follows: step 1: 95°c for 30 s; step 2: 95°c for 5 s, 60°c for 30 s, 40 cycles; and step 3: dissolution curve analysis. for the cultured cells, the total cellular protein was extracted using the whole cell lysate (beijing comwin, cat: cw0074) containing a protease inhibitor cocktail (roche, germany). the protein was quantified using a bicinchoninic acid (bca) protein assay kit (beijing comwin, cat: cw0014), and a 30 μg protein sample was obtained for polyacrylamide gel electrophoresis (page). the lung tissues from six mice of each group were mixed and ground in liquid nitrogen, and then the total cellular protein was extracted using the whole cell lysate (beijing comwin, cat: cw0074) containing a protease inhibitor cocktail (roche, germany). protein was quantified using a bca protein assay kit (beijing comwin, cat: cw0014). a 50 μg protein sample was obtained for page and subsequently transferred to a polyvinylidene fluoride (pvdf) membrane. the primary antibodies of rabbit anti-glyceraldehyde phosphate dehydrogenase (gapdh) and goat anti-igf1 (abcam) were diluted to 1:2,000. the secondary antibodies of horseradish peroxidase (hrp)-goat anti-rabbit immunoglobulin g (igg) and hrp-rabbit anti-goat igg (zsgb-bio) were diluted to 1:5,000. the signals were detected using a western hpr substrate peroxide solution (millipore). the quantification of western blot analysis was performed by using image j software, and the protein expression levels were normalized to gapdh levels. the level of cytokine expression was detected using mouse igf1 elisa kit, il-6 elisa kit, tnf-α elisa kit, ifn-γ elisa kit, and il-1β elisa kit (all purchased from r&d systems). for viral infection, a549 cells were washed with phosphate buffered saline (pbs) and subsequently infected with pr8 at the multiplicity of infection (moi) of 0.5 or 1.0 in infection medium, which was dmem supplemented 0.5 mg/ml n-p-tosyl-phenylalanine primer 5′-3′ chloromethyl ketone (tpck)-treated trypsin (sigma, usa) and 0.3% bovine serum albumin (bsa; sangon, china). after 1 h, cells were incubated with fresh infection medium at 37°c for the indicated times. balb/c mice were intraperitoneally injected with sodium pentobarbital (50 mg/kg), and the mice were induced into a deep anesthetic state and administered a nasal inhalation of phosphate buffered saline (pbs) or pr8 (diluted 10-fold, 30 μl/mouse, 50ld 50 ). in the pr8 + igf1 group, igf1 (20 μg/ kg, peprotech) was intraperitoneally injected 6 h before infection, and after infection, igf1 was intraperitoneally injected every 24 h. the pr8 + ppp group was intraperitoneally injected with the igf1 receptor inhibitor, and picropodophyllin (ppp, 20 mg/kg, selleck) 6 h before infection and after infection ppp was intraperitoneally injected every 12 h. after the continuous administration for 14 days, changes in the clinical symptoms of mice were observed daily, and changes in the body weight and survival were recorded. on days 3, 5, 7, and 9 after pr8 infection, six mice of each group were sacrificed every time. the blood was taken by excising the eyeballs, and the lung tissues were collected. the blood was kept at room temperature for 1 h and centrifuged at 11,000 g for 15 min. the serum was collected and aliquoted and stored at −80°c for later use. all animal experimental procedures were approved by the animal care and use committee of the academy of military medical sciences (amms; id: syxk2012-05) and were carried out in strict accordance with the guidelines. all experiments involving the live virus were performed in an approved biosafety level 2 facility. after removing the whole lung tissue of the mice, damage to the lung tissue was observed. the degree of lung injury visible to the naked eye was dark red due to edema. the area ratio of lung injury to the total lung tissue was estimated. each sample was estimated by at least three different individuals, and the average was obtained. finally, the lung injury area of six mice in each group was counted. the wet weight of the lung tissue was weighed. lung index = lung wet weight/ body mass. all experiments were repeated at least three times. data were expressed as means ± standard deviation (sd). the graphical representation of the data was performed using graphpad prism 5 software. the grayscale analysis of the western blot images was performed using image j software by comparing the integrated density of the igf1 band with the control gapdh. the statistical analyses were performed using spss 20.0 statistical software with two-tailed student's t test for two comparisons, *p < 0.05; **p < 0.01; and ***p < 0.001. a p below 0.05 was considered statistically significant. to detect the regulation of igf1 by iav, a549 cells were infected with pr8 (10 4.58 tcid 50 /ml) at different multiplicity of infection (moi) of 0.1, 0.5, or 1.0, and the level of igf1 mrna and protein expression was detected at 12, 24, 48, and 72 h post-infection. as shown in figure 1a , compared with the mock-infected controls, the level of igf1 mrna expression in a549 cells increased gradually at 12, 24, and 48 h after pr8 infection at a moi of 0.5. the level of igf1 mrna expression peaked at 48 h post-infection, which was 6.35 ± 0.30 times higher than that of the mock controls. the level of igf1 mrna expression decreased slightly at 72 h post-infection but still reached 4.23 ± 0.18 times that of the mock group. as shown in figure 1b , in a549 cells infected with different mois of pr8, the level of igf1 mrna expression increased following the increasing of moi at 48 h post-infection. at a moi of 1.0, the level of igf1 mrna increased to 7.49 ± 0.38 times that of the mock group. the level of igf1 protein expression was also upregulated at 12, 24, 48, and 72 h post-infection of pr8 ( figure 1c ) and following the increasing of moi at 48 h post-infection in a549 cells ( figure 1d) . to compare the trend in igf1 variability more intuitively, the grayscale analysis of figures 1c,d was performed and plotted according to the ratio of the integrated density of igf1/gapdh. figures 1e,f show that the level of igf1 protein expression in the a549 cells following pr8 infection was consistent with the level of igf1 mrna; however, such upregulation times were lower than the level of mrna. compared with the mock group, the level of igf1 protein expression peaked 1.82 times at 48 h after pr8 infection at a moi of 0.5 ( figure 1e) . the level of igf1 protein expression was also increased following the increasing of moi. at a moi of 1.0, the level of igf1 protein expression reached 2.26 times that of the mock group at 48 h post-infection ( figure 1f) . the above results indicate that igf1expression is upregulated in pr8-infected a549 cells. to investigate the role of igf1 in the inflammatory response to influenza virus infection, we established two stable a549 cell lines, in which igf1 is overexpressed or inhibited. using pcdna3.1-igf1 to overexpress igf1, figure 2a showed that the level of igf1 mrna was increased 15.28 ± 1.84 times that of the control group. the expression of igf1 in the cells was inhibited by lentiviral packaged shrna (lenti + shigf1), after which the level of igf1 mrna was reduced to 0.21 ± 0.087 times that of the control group (figure 2a) . as shown in figure 2b , the level of igf1 protein was also increased in stable a549 cell line with pcdna3.1-igf1 and inhibited in stable a549 cell line with lenti + shigf1. the stably transfected a549 cell lines were infected with pr8 at a moi of 0.5. after 24 h, real-time quantitative pcr (qpcr) was used to detect the changes in cytokine expressions [il-6, il-8, il-10, monocyte chemotactic protein-1 (mcp-1, or ccl2), tnf-α, and il-1β], which were related to the inflammatory response. the results in figure 2c showed that compared with the mock controls, the cytokine levels were significantly increased following pr8 infection in control cells (pr8 + pcdna3.1-con group and pr8 + lenti + con group). igf1 overexpression further increased the level of cytokine mrna expression, whereas the levels of cytokine expression in the pr8-infected cells with igf1 knocked down (pr8 + lenti + shigf1) were significantly decreased far lower than the pr8-infected control cell (pr8 + lenti + con group), as well as the uninfected mock group. especially the lowest ccl2 and tnf-α mrna of pr8 + lenti + shigf1 group were downregulated to less than half that of the mock group. these results indicate that the overexpression of igf1 aggravated the inflammatory response induced by influenza virus infection, whereas the inhibition of igf1 expression can alleviate this inflammatory response. to detect whether influenza virus can also regulate igf1expression in vivo, iav pr8 (diluted 10-fold, 30 μl/mouse, 50ld 50 ) was intranasally inoculated into balb/c mice. mice were sacrificed on days 3, 5, 7, and 9 post-infection to obtain mice lung tissue and serum, and the samples of six mice in every group were mixed. the level of igf1 mrna expression in the lung tissue of mice was detected by qpcr. as shown in figure 3a , the level of igf1 mrna expression in the lung tissue gradually increased at days 3, 5, and 7 after pr8 infection. on day 7 post-infection, igf1 mrna increased to 5.81 ± 0.623 times that of the control group and decreased slightly on day 9 post-infection. a western blot was used to detect changes in igf1 protein expression in the lung tissue following pr8 infection ( figure 3b ). the grayscale analysis by comparing the integrated density of the igf1 band with the control gapdh showed that the trend in the level of igf1 protein expression was figure 1c , and plotted as the ratio of the integrated density of igf1/ gapdh; (f) grayscale analysis of figure 1d , and plotted as the ratio of the integrated density of igf1/gapdh. data were the means ± sd from three independent experiments. **p < 0. consistent with that of the mrna data ( figure 3c) . the content of igf1 in the serum of mice was detected by enzyme linked immunosorbent assay (elisa). as shown in figure 3d , compared with the pbs control group, the content of igf1 protein in serum increased significantly on day 5 post-infection of pr8 and decreased on day 9. these results were also consistent with the qpcr and western blot findings. to detect whether igf1 regulates acute inflammatory lung injury in pr8 infection, balb/c mice were tested as follow. balb/c mice were randomly divided into four groups: (1) pbs; (2) pr8 + igf1; (3) pr8+ ppp; and (4) pr8 + pbs. the pbs group was intranasally inoculated with 30 μl pbs, whereas the other three groups were intranasally inoculated with 30 μl influenza virus pr8 (50ld 50 ). from 6 h before the inoculation to day 14 post-infection, the mice in the pr8 + igf1 group were intraperitoneally injected with igf1 protein (20 μg/kg/24 h); pr8 + ppp mice were injected intraperitoneally with an igf1 receptor inhibitor, picropodophyllin (ppp, 20 mg/kg/12 h); and the pr8 + pbs group was injected with pbs (30 μl/24 h). the clinical symptoms of the mice were observed daily for 14 days, and changes in body weight and survival were recorded. six mice of each group were sacrificed at day 5 post infection of pr8, and the serum and lung tissues of each group were collected and mixed. the extent of lung injury and inflammatory cell infiltration of the mice in each group was observed by histologically. changes in the level of serum cytokines were detected by elisa. a western blot was used to detect the changes in the expression of major proteins in the igf1/igf1r-related signaling pathways. there was no change in the general appearance of the pbs control group mice. in the pr8 + pbs group, flu-like symptoms began to appear on day 3 after pr8 inoculation, such as reduced activity, ruffled fur, and slight weight loss. on day 5 postinfection, these conditions worsened with 20% weight loss, canthus secretion, hunched back. the symptoms of the pr8 + igf1 group were more severe than those of the pr8 + pbs group, whereas the pr8 + ppp group displayed milder clinical symptoms than that of the pr8 + pbs group (figure 4a) . figure 4b showed that the body weight of the mice decreased significantly on day 3 post-infection of pr8, and the body weight of the mice on day 9 decreased to the lowest. although the changes in body weight were similar between the pr8 + ppp group and pr8 + pbs group, the survival rate of the pr8 + ppp group was dramatically increased up to 75% compared with the survival rate of the pr8 + pbs group was only 25%, with the first death not occurring until the day 8 post-infection ( figure 4c ). the first death in the pr8 + igf1 group was accelerated to day 5 post-infection, and all mice have succumbed to the infection by day 8. these findings indicated that the intraperitoneal injection of igf1 promoted the death of pr8-infected mice, whereas the inhibition of igf1 increased the survival rate of pr8-infected mice. figure 5a showed that in iav pr8-infected mice, the lungs exhibited differential degrees of damage, and the color of the damaged parts changed from pink to dark red, with the presence of edema. the extent of lung injury in the pr8 + igf1 group was significantly more severe than that of the pr8 + pbs group, as the lung color was darker, and the lesion area was larger. the degree of lung injury in the pr8 + ppp group was significantly less severe than that of the pr8 + pbs group. figures 5b,c showed that the lung index of uninfected mice was approximately 1%. compared with the pr8+ pbs group, the lung index of the pr8 + igf1 group increased significantly from 1.953 ± 0.074 to 2.515 ± 0.121%, and the area of lung injury also increased from 0.38 ± 0.042 to 0.78 ± 0.069, approximately twice that of the control group. in the pr8 + ppp group, the lung index decreased to 1.393 ± 0.032%, and the lung injury area also decreased to 0.25 ± 0.062, which were both lower than that of the pr8 + pbs group. as shown in figure 5d , the lung structure of the mice in the pbs group was clear, with intact pulmonary alveoli, clear alveolar septum, and without hemorrhaging and inflammatory cell infiltration. the pulmonary alveoli of the pr8 + pbs, pr8 + igf1, and pr8 + ppp groups exhibited varying degrees of damage. the level of inflammatory cell infiltration in the lung tissue of the pr8 + igf1 group was greater than that of the pr8 + pbs group, and there was lower inflammatory infiltration in the pr8 + ppp group than the pr8 + pbs group. whether igf1 regulates the viral replication of iav, qpcr was used to detect changes of the viral matrix protein 1 (m1) expression in the lungs of pr8-infected mice, which could indirectly reflect the viral load. the viral load in the lungs of the pr8 + igf1 group was significantly increased to nearly two-fold of the pr8 + pbs group ( figure 5e) . however, the viral load of the pr8 + ppp group and the pr8 + pbs group was similar, indicating that igf1 responded to influenza infection via the host immune response rather than targeting viral replication. the cytokine content in the serum of pr8-infected mice was detected. figures 6a-d showed that there was a significant increase in the serum inflammatory following pr8 infection. figure 5 | lung injury following pr8 infection in balb/c mice. groups of balb/c mice (n = 6, females, 6-8 weeks) were intranasally infected with pr8 (50ld 50 ), treated with igf1 or ppp, and at day 5 post-infection, the mice lungs were examined for changes in (a) morphology, (b) lung injury, (c) injury areas, (d) histopathology, and (e) viral load. data were the means ± sd from three independent experiments. **p < 0.01 vs. pr8 + pbs group by t test. moreover, the inflammatory cytokine levels in the intraperitoneal injected igf1 protein group were further increased, in which il-1β was highly variable, and there was no statistical difference with the pr8 + pbs group. the levels of inflammatory factors ifn-γ, tnf-α, il-6, and il-1β in the serum of the pr8 + ppp group were significantly decreased, and il-1β was lower than that of the uninfected mice. the results further confirm that igf1 induced an inflammatory response following influenza virus infection, which can be alleviated by inhibiting igf1. to explore the mechanism by which igf1 regulates acute inflammatory lung injury induced by iav infection, a western blot was used to detect the expression of molecules in key signaling pathways in the lung tissue of pr8-infected mice (50ld 50 pr8). each sample was from six mice in a group and was tested for three times. figures were representative of three independent experiments. figure 7a showed that the phosphorylation level of igf1r increased gradually following pr8 infection, peaked at day 7 post-infection, and then decreased slightly on day 9, which was consistent with the changes in the trend of igf1 protein expression ( figure 3b ). figures 7b-d showed that the pi3k/akt and mapk signaling pathways were activated following pr8 infection; p-akt expression was upregulated in the pi3k/akt signaling pathway; and p-p38 and p-jnk expression were upregulated in the mapk signaling pathway. the levels of p-akt and p-p38 expression were further increased in the pr8 + igf1 group. the expression level in the pr8 + ppp group was between that of the pbs and pr8 + pbs groups. there was no significant difference in the level of p-jnk expression between the groups infected with influenza virus. the results indicated that the administration of igf1 protein following influenza virus infection promoted the activation of the pi3k/akt and mapk signaling pathway. intervention with the igf1r inhibitor ppp inhibited influenza virus mediated pi3k/akt and mapk signaling pathways. these results suggest that igf1 affected the expression of key proteins associated with mapk and the pi3k/akt signaling pathway in response to iav-mediated inflammation. in this study, we investigated that igf1 played a significant role in mediating inflammation and pathology during iav infection. igf1 expression was upregulated in a549 cells and balb/c mice infected with pr8, which modulated inflammatory cytokine expression. igf1 overexpression aggravated influenzamediated inflammatory responses, whereas the inhibition of igf1 expression reduced such inflammatory responses. the phosphorylation of igf1r triggered the pi3k/akt and mapk signaling pathways to induce inflammation. thus, inhibiting igf1 or igf1r expression to block downstream signaling may be a novel treatment strategy for influenza infection. following an influenza virus infection, a series of immunological responses are initiated. first, the host's innate immune system, consisting of interferon, cytokines, macrophages, neutrophils, nk cells, and dendritic cells (dcs), all rapidly respond to the viral infection and activate adaptive immunity (gu et al., 2019) . although the innate immune response is important, the host typically eliminates the viral infection with the aid of the adaptive response (bonilla and oettgen, 2010) . in humoral immunity, neutralizing antibodies can prevent the adsorption of the virus, via opsonization, and primarily interact with extracellular free viruses (murasko et al., 2002) . activated th1 cells release various cytokines (i.e., ifn-γ and tnf), activate macrophages and nk cells to induce further inflammatory reactions, and promote the proliferation and differentiation of cytotoxic t lymphocytes (ctls), which play an important role in antiviral infection (julkunen et al., 2001; farsakoglu et al., 2019) . however, studies have shown that while an inflammatory response helps to clear pathogens, excessive inflammatory reactions do not only protect the host organism but also cause the additional damage. many studies have shown that the cause of death from an influenza virus infection is the pulmonary respiratory syndrome induced by inflammation caused by an excessive reaction of the body's immune system (blach-olszewska and leszek, 2007) . in the present study, changes in the level of igf1 mrna and protein expression in a549 cells were detected. the results showed that igf1 mrna and protein expression were significantly altered following iav infection. to further confirm the important role of igf1 in iav-mediated inflammation and the associated signaling mechanism, igf1 protein was administered as an intervention to pr8-infected mice. compared with the untreated infected mice, the infected mice administered igf1 displayed more severe clinical symptoms; in particular, the serum levels of ifn-γ, tnf-α, and il-1β were significantly increased, and lung damage was more serious. igf1 is known to function by binding to the receptor, igf1r. in this study, the igf1r inhibitor ppp was used to inhibit igf1r function, thereby disrupting its downstream signaling capacity. by monitoring the clinical symptoms and inflammation indicators in mice, it was found that during the same period of viral infection, when ppp was used to inhibit igf1, the clinical symptoms of mice were alleviated; the lung injury area of the mice was reduced; the degree of lung injury was reduced; the lung index was significantly decreased; the serum levels of inflammatory cytokines ifn-γ, tnf-α, il-1β, and il-6 were significantly decreased; and the survival rate increased from 25 to 75% compared with the untreated group. the viral proliferation in the lungs of mice treated with igf1 was significantly increased; however, the viral proliferation of the mice treated with ppp was similar to that of the pr8 + pbs group. thus, the inhibition of igf1 only affects the immune response but has no significant effect on iav replication. thus, the inhibition of igf1, rather than antiviral pathways that affect viral replication, may provide a new therapeutic avenue for influenza that limits detrimental inflammatory responses to infection. this is consistent with the previously reported effective treatments for influenza-mediated inflammation, which prolong the survival of older influenza-infected individuals, compared to those treated with antivirals (pillai et al., 2016) . the inflammation-associated pi3k/akt and mapk signaling pathways are known to be activated in response to influenza virus infection (dong et al., 2014; hirata et al., 2014) . although igf1 primarily regulates cell growth and apoptosis through the pi3k/akt and mapk signaling pathways, whether igf1 plays a role in these pathways in the context of influenza virus-mediated inflammation remains unknown. in this study, a western blot was used to detect the expression of key proteins in the pi3k/akt and mapk signaling pathways. the results showed that p-igf1r was upregulated following pr8 infection; p-akt expression was upregulated in the pi3k/akt signaling pathway; and p-p38 and p-jnk expression were upregulated in the mapk signaling pathway compared with the pbs group. the expression of p-akt and p-p38 in the pr8 + igf1 group was higher than that of the pr8 + pbs group, and the expression in the pr8 + ppp group was between that of the pbs and pr8 + pbs group. there was no significant difference in the level of p-jnk expression between the groups infected with influenza virus. this suggested that igf1 affected the expression of key proteins associated with p38 in the mapk and the pi3k/akt signaling pathways in the context of influenza virusmediated inflammation, which confirmed our hypothesis. therefore, we conclude that igf1 expression is upregulated following influenza virus infection, and igf1 receptor phosphorylation is elevated, triggering two signaling pathways downstream of pi3k/akt and mapk to induce inflammation. thus, the inhibition of igf1 or igf1r expression to block such downstream signaling pathways may represent a novel approach for the treatment of influenza virus infection. the data used to support the findings of this study are available from the corresponding author upon request. the animal study was reviewed and approved by academy of military medical sciences (amms; id: syxk2012-05). chz and gl contributed to conceptualization. chz, gl, and lz contributed to methodology. lz, caz, and ys contributed to investigation. rw and chz contributed to writing the original draft. rw and chz contributed to writing, reviewing, and editing. chz contributed to funding acquisition. lz, dd, and mb contributed to resources. rw, gl, and chz helped in supervision. glucocorticoid: major factor for reduced immunogenicity of 2009 influenza a (h1n1) vaccine in patients with juvenile autoimmune rheumatic disease the nlrp3 inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna autophagy resolves early retinal inflammation in igf1-deficient mice progress in identifying virulence determinants of the 1918 h1n1 and the southeast asian h5n1 influenza a viruses mechanisms of over-activated innate immune system regulation in autoimmune and neurodegenerative disorders adaptive immunity obesity, type 2 diabetes, and cancer: the insulin and igf connection a dual character of flavonoids in influenza a 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responses the authors thank prof. shihui sun (beijing institute of microbiology and epidemiology) for kindly providing the iav a/puerto rico/8/1934 (h1n1). key: cord-339694-sp212tai authors: jiang, xinpeng; hou, xingyu; tang, lijie; jiang, yanping; ma, guangpeng; li, yijing title: a phase trial of the oral lactobacillus casei vaccine polarizes th2 cell immunity against transmissible gastroenteritis coronavirus infection date: 2016-03-28 journal: appl microbiol biotechnol doi: 10.1007/s00253-016-7424-9 sha: doc_id: 339694 cord_uid: sp212tai transmissible gastroenteritis coronavirus (tgev) is a member of the genus coronavirus, family coronaviridae, order nidovirales. tgev is an enteropathogenic coronavirus that causes highly fatal acute diarrhoea in newborn pigs. an oral lactobacillus casei (l. casei) vaccine against anti-transmissible gastroenteritis virus developed in our laboratory was used to study mucosal immune responses. in this l. casei vaccine, repetitive peptides expressed by l. casei (specifically the mdp and tuftsin fusion protein (mt)) were repeated 20 times and the d antigenic site of the tgev spike (s) protein was repeated 6 times. immunization with recombinant lactobacillus is crucial for investigations of the effect of immunization, such as the first immunization time and dose. the first immunization is more important than the last immunization in the series. the recombinant lactobacillus elicited specific systemic and mucosal immune responses. recombinant l. casei had a strong potentiating effect on the cellular immunity induced by the oral l. casei vaccine. however, during tgev infection, the systemic and local immune responses switched from th1 to th2-based immune responses. the systemic humoral immune response was stronger than the cellular immune response after tgev infection. we found that the recombinant lactobacillus stimulated il-17 expression in both the systemic and mucosal immune responses against tgev infection. furthermore, the lactobacillus vaccine stimulated an anti-tgev infection th17 pathway. the histopathological examination showed tremendous potential for recombinant lactobacillus to enable rapid and effective treatment for tgev with an intestinal tropism in piglets. the tgev immune protection was primarily dependent on mucosal immunity. lactic acid bacteria (lab) are a group of gram-positive bacteria that includes species of lactobacillus, leuconostoc, pediococcus and streptococcus. consumed for centuries, lab has enjoyed a long and safe association with humans and animals for healthy food. over the past decade, there has been increasing interest in the use of lab as mucosal delivery vehicles. the application of lab stems from research into effective strategies to deliver vaccine antigens, which may come into contact with the mucosal tissues for the first time, such as the intranasal, oral and genital mucosal surfaces (lavelle and o'hagan 2006; malik et al. 2007 ). mucosal delivery of therapeutics or vaccines for chronic diseases and infections of mucosal origin could increase their potency and specificity. because the mucosal immune system builds an effective iga barrier in no more than 3 days, contact with the mucosal tissues will neutralize the pathogenic microorganism outside of the host. there are abundant studies in the field of lab vaccines. one major advantage of the use of lab as a delivery vehicle for vaccines is that the bacteria can elicit both antigen-specific secretory immunoglobulin (ig) a and an effective systemic immune response. the specific igas have the same function as the neutralizing iggs. some candidate lab vaccines elicited antigen-specific iga responses in faeces, saliva or bronchoalveolar and intestinal lavage fluids (wells and mercenier 2008) . additionally, lactobacilli are probiotics that may confer health benefits to the host (bengmark and gil 2006; corthesy et al. 2007; isolauri et al. 2002; saarela et al. 2000) , and there is accumulating evidence that lactobacilli are effective at preventing intestinal disease in both humans and animals due to their ability to inhibit pathogen adhesion to the intestinal wall and prevent inflammatory processes (blum and schiffrin 2003; de vrese and marteau 2007; ouwehand 2007; sartor 2005; sheil et al. 2007 ). because the porcine digestive tract is similar to the digestive tract of human infants with respect to anatomical and histological features and digestive physiology (kararli 1995; oswald et al. 2003; tadros et al. 2003) , the pig has been used as an animal model to study gastrointestinal diseases of human infants (gunzer et al. 2002) and vaccination studies against these diseases. coronaviruses (covs) comprise a large family of enveloped, positive-stranded rna viruses that infect a broad range of animal hosts as well as humans. well-known representatives are porcine transmissible gastroenteritis virus, porcine respiratory cov, porcine epidemic diarrhoea virus, canine cov, feline cov, bovine cov, human covs, severe acute respiratory syndrome-associated cov, murine hepatitis virus (mhv), avian cov infectious bronchitis virus (ibv) and turkey cov (tcov). the most famous and critical coronavirus is the middle east respiratory syndrome virus found in africa and asia (siddell et al. 1983 ). this study investigates whether mucosal immunization is effective in stimulating a protective immune response against cov infection. transmissible gastroenteritis coronavirus (tgev), which is a member of the genus coronavirus, family coronaviridae, and order nidovirales, is an enteropathogenic coronavirus that causes highly fatal acute diarrhoea in newborn pigs (cavanagh 1997) . the viral rna consists of a single strand comprised of three major structural proteins: a phosphorylated nucleoprotein (n protein) and two glycoproteins (the membrane (m) and the spike (s) proteins) (schwegmann-wessels and herrler 2006). the s protein has four sites (a, b, c and d). both the a and d sites were demonstrated to induce tgev-neutralizing antibodies (di-qiu et al. 2011) ; however, the a site is highly glycosylated and thus is not suitable for expression in the lab prokaryotic expression vector. additionally, the different tgev sites induce different immune responses. following infection with virulent transmissible gastroenteritis coronavirus, isolated mesenteric lymph node cd4+ t cells mounted a specific proliferative response against infectious or inactivated purified virus upon secondary in vitro stimulation (anton et al. 1996) . the peptide n321 defines a functional t helper epitope that elicits t cells capable of collaborating with b cells specific for different tgev proteins (anton et al. 1995) . the most important finding is that oral immunization with a recombinant lactobacillus vaccine and infection with tgev elicit various immune responses, such as humoral immunity and cellular immunity. here, an oral lactobacillus casei vaccine against anti-transmissible gastroenteritis virus developed in our laboratory was used to study the mucosal immune response (jiang et al. 2014) . in this l. casei vaccine, repetitive peptides expressed by l. casei (specifically the mdp and tuftsin fusion protein (mt)) are repeated 20 times and the d antigenic site of the tgev spike (s) protein is repeated six times. the pig model was developed to study intestinal mucosal immune responses (ruan and zhang 2013) . probiotic feed supplementation may benefit the animal host directly by preventing infection and combating the causative agent of the intestinal disorder by balancing the disrupted equilibrium of the enteric flora and augmenting the host's immune responses. however, lab vaccine has not received national law or certification for human or animal use against coronaviruses. the first clinical trial to use recombinant lab demonstrated that the containment strategy for l. lactis expressing recombinant il-10 was effective against crohn's disease (braat et al. 2006) . our laboratory has researched the lab vaccine for more than one decade, and we have developed many lab vaccines in the field of piglet diarrhoea (di-qiu et al. 2011; liu et al. 2011; qiao et al. 2009; yigang and yijing 2008) . this study is the last step to obtain new drug certification in china. this lactobacillus vaccine has been demonstrated to increase the treg population in the mouse model (jiang et al. 2014) . tregs effectively depressed t and b cell proliferation, and some studies demonstrated that this regulation also depressed proliferation in inflammatory bowel disease. our study investigated whether the recombinant lactobacillus vaccine gradually increased the breg and treg populations during the immunization process at the first step of immunization (unpublished data). the intestinal immune system of the pig maintains its ability to mount an active immune response against pathogens and exhibits tolerance to at least food antigens and probably commensal flora through an extremely complex network of cellular and humoral immune interactions. consequently, it is important to elucidate the immunological inductive sites of the protective mucosal immune response following oral immunization in pigs. this vaccine could induce tgev antibody immune responses in both the humoral and mucosal immune systems. mdp and tuftsin possess substantial immunopotentiating properties and can induce cellular-mediated immune responses upon oral administration in mice. however, their use in oral vaccines against tgev challenge in the pig host may have different results. furthermore, the only host (and target of the vaccine) of the transmissible gastroenteritis coronavirus is the weaning piglet. there have been no clear reports concerning whether tgev infection stimulates th1 or th2type immune response. the relationship between humoral and cellular immune responses is not clear; moreover, whether the systemic or mucosal lymphoid response will be the primary immune response following oral immunization is unknown. at present, we are not certain which pathway the lactobacillus vaccine will provoke against tgev infection. our study is the first to analyse immunity in response to oral immunization and tgev infection. virus, bacterium and cell line the l. casei atcc 39392 strain used in this study was deposited in atcc and is a plasmid-free strain grown in man-rogosa-sharpe (mrs) medium (sigma) at 37°c without shaking. the recombinant l. casei (pg:612-20mt6d) was generated in previous study (jiang et al. 2014) . chloramphenicol (cm) and kanamycin (sigma) were each used at a final concentration of 10 μg/ml. tgev was previously isolated and purified in our laboratory. swine testicle (st) cells were cultured in dulbecco's modified eagle's medium (dmem, gibco) supplemented with 10 % foetal bovine serum (fbs, gibco) at 37°c with 5 % co2. the virus stocks were clarified by centrifugation at 800×g for 10 min to remove cell debris, titrated using the cytopathic effect assay and then stored in aliquots at −80°c until needed. tgev-seronegative crossbreed (large white) piglets were obtained from a local breeding farm after birth. the piglets were housed separately in specialized cages that were maintained in sterile stainless steel isolators (five piglets/isolator) and fed commercial sterile milk and water. four groups (n = 5 each) of piglets were orally dosed with 10 10 cfu of pg:612-20mt6d in 1 ml of pbs or pbs alone (jiang et al. 2014) ; this formulation was used to immunize piglets via an intragastric route in a different immunization protocol. the first group was immunized with pg:612-20mt6d in 1 ml for priming. the second group was immunized with pg:612-20mt6d in 2 ml for priming. the third group was immunized with pg:612-20mt6d in 2 ml for 48 consecutive hours. the forth group was immunized with pg:612-20mt6d in 1 ml for 48 consecutive hours. the control group was immunized with pbs. the piglets were handled and maintained under strict ethical conditions according to international recommendations for animal welfare. seven days after immunization, serum samples were prepared from collected blood samples. the intestinal mucus was collected by rectal swab and subsequently homogenized for 30 min in 400 μl of sterile pbs (ph 7.4) containing 0.01 mol/ l edta-na2 and then incubated for 12 h at 4°c. clear extracts of all samples were collected by centrifugation at 3000×g for 10 min and stored at −80°c with protease inhibitors for subsequent analysis. enzyme-linked immunosorbent assay (elisa) plates were coated for 18 and 12 h at 4°c with full tgev and the vp4 protein, respectively, which were previously isolated and purified in our laboratory. cultured st cells were used as a negative antigen control. after the wells were blocked for 12 h at 4°c with pbs containing 5 % skim milk, the plates were washed three times with pbs + tween 20 (0.1 %) (pbst). mucus and serum (diluted 1:50) samples were added to the wells in triplicate and incubated for 1 h at 37°c. afterwards, the plates were washed three times with pbst, and a horseradish peroxidase-conjugated goat anti-pig igg or iga antibody (invitrogen) was added to each well (1:5000) and incubated for an additional 1 h at 37°c. after another round of washing, colour development was accomplished using ophenylenediamine dihydrochloride as a substrate, and the absorbance was measured at 490 nm. naive purified spleen t cells (5 × 10 5 cells/ml) were cultured in 24-well tissue culture plates and stimulated with 1.0 μg ml −1 of plate-bound anti-cd4(pe) antibody (abcam) in complete rpmi 1640 medium. single-cell suspensions were stimulated in culture with ionomycin (2 μg ml −1 ) in the presence of monensin (2 μm) for 4-6 h of culture. the cells were surface labelled and then fixed, permeabilized and intracellularly labelled with ifn-γ and il-4 antibodies as previously described (moore-connors et al. 2013; zhou et al. 2009 ). for th1 and th2 differentiation, the cells were stimulated in the presence of 10 ng ml −1 anti-ifn (fitc) and 10 ng ml −1 anti-il-4 (fitc) (abcam) antibodies. the cells were labelled with carboxyfluorescein succinimidylester (cfse) according to a previous protocol (jiang et al. 2014) . the data were acquired by gating on the cd4 + cell population with a facscalibur cytometer. the sequential loss of cfse fluorescence was used to measure cell division and proliferation. groups were housed in the same facility but separated by room and ventilation system. pigs in each room were confined by pens on a solid floor that was rinsed daily, fed a balanced diet ad libitum based on weight and provided free access to water. tgev-challenged pigs received a 5 ml dose of 1 × 10 5 plaque-forming units (pfu)/ml via oral-gastric gavage on 0 days post-inoculation (dpi). pigs in the control group were administered volume-matched virus-free cell culture media. the control, immunized and no challenge group pigs were randomly selected for necropsy on the fourth day. to assess histological changes in the intestinal tissues, both the intestine and other major organs were examined at necropsy. after 48 h of fixation in 10 % neutral buffered formalin, tissue sections were trimmed, processed, and embedded in paraffin, sectioned, stained with haematoxylin and eosin (h&e) and then examined for pathological changes by light microscopy using a model microscope (olympus, tokyo, japan). real-time rt-pcr (qrt-pcr) was employed to determine the amount of tgev and cytokine gene products (isgs) in rectal swab samples and the intestinal tissues using a cfx96tm real-time pcr detection system (bio-rad). total rna was extracted from faecal samples and splenic and intestinal tissues using viral rna extraction and total rna extraction kits (intron) according to the manufacturer's instructions. the extracted rna was subjected to real-time qrt-pcr using a one-step sybr® qrt-pcr reagent kit (takara, shiga, japan). following reversetranscription of the viral rna at 45°c for 20 min, the resulting cdnas were used for real-time pcr amplification. a standard curve was generated by plotting threshold values against serially diluted plasmid dna encoding the fragment of the tgev spike protein (lee et al. 2011 ). all determinations were performed using data from wells evaluated in duplicate to ensure reproducibility. the copy number of the experimental samples was determined by interpolating the threshold cycle values using the standard curve. real-time quantitative pcr was utilized to quantify the products of interest (trl-2, −4, −9, il-4, il-17, ifn-γ and tgf-β) relative to the quantity of messenger rna (mrna) in the total rna isolated from the splenic and intestinal tissues (dirisala et al. 2013; kiros et al. 2011) ; the specific primers are listed in table 1 . the livak method (δδct method) was used to calculate the fold change compared to the β-actin gene control. gene expression data were expressed relative to unimmunized and uninfected piglets. to phenotype immune cells in the spleen and mesenteric lymph nodes, single-cell suspensions were isolated and labelled with fluorochrome-conjugated antibodies. to determine the cell type and the frequency of ifn-γ and il-4-producing th1 and th2 cells, single-cell suspensions were stimulated in culture with ionomycin (2 μg ml −1 ) in the presence of monensin (2 μm) for 4-6 h. the cells were surface labelled to detect cd4 and then fixed, permeabilized and intracellularly labelled with ifn-γ and il-4 antibodies as previously described (moore-connors et al. 2013; zhou et al. 2009 ). comparison of the piglets' iga and igg titres was conducted by a paired t test. the th cell, cytokine expression and faecal pedv rna shedding titres among litters were compared using one way analysis of variance (anova) followed by duncan's multiple range test. the mucosal immune response of the piglets was evaluated by measuring the iga response in diluted intestinal lavage fluid post-intragastric immunization. as shown in fig. 1a , the newborn piglets that received 2 ml of recombinant lactobacillus pg:612-20mt6d had the highest mucosal iga levels after immunization. the iga levels at 48 h in the piglets that received 4 ml were slightly lower than the newborn piglets that received 2 ml throughout the process. the vaccine doses also provoked systemic immunity based on the serum analysis and elicited specific igg responses from the immunized piglets (fig. 1b) . newborn piglets that received 2 ml of the vaccine also had the highest specific igg level. the specific igg titre reached as high as 1:1600. finally, the antibody kinetics in the serum and intestinal lavage samples from the animals showed that the specific antibody igg and iga levels were increased on the seventh and eighth days and the titre was decreased during the last week without immunization. to analyse the effect of recombinant lactobacillus pg: 612-20mt6d on t helper cells, we evaluated t helper cell polarization. throughout the process, we utilized the model of immunization described here. as shown in fig. 2 , the immune response balance mediated by th1 and th2 was broken in favour of th1-mediated immunity. the pg:612-20mt6d/l. casei group exhibited 80 % protection within 4 days of challenge with tgev (pg:612-20mt6d/l. casei) (fig. 3) . in contrast, the control group piglets immunized with pbs all died after tgev challenge. all piglets that died/were killed were emaciated and had yellow faeces coating the skin and hair. in some piglets, the intestinal lumens were filled with large amounts (approximately 50-70 ml) of yellowish foamy fluid. in other piglets, the walls of the small intestine were transparent and thin and the intestinal lumens were empty. no significant gross lesions were observed in other major organs (lung, kidney, liver and heart). formalin-fixed intestinal tissue sections from piglets treated with different treatment modalities were blindly analysed for histopathological changes associated with tgev infection. according to the histopathological analysis, the small intestine samples from the three groups (positive, negative and immunized groups) showed obvious differences. as indicated in fig. 4 , different degrees of pathological changes were detected after infection, especially in the positive group where serious damage was observed. the representative pathological changes included intestinal villi hyperaemia, atrophy and destruction. in the pbs infection group, the jejunum villi were severe atrophied and destroyed, and the ilea exhibited severe lymphocyte proliferation in the lamina propria. the recombinant lactobacillus group showed jejuna with intact villi but low-grade hyperaemia and lymphocyte proliferation, and the ilea exhibited lymphocyte proliferation in the lamina propria. both the pbs and vaccine groups had severe inflammatory responses. the negative control piglets that were not infected showed normal histology. the sequences of the two primers were checked using the ncbi blast software, and no significant alignment with any other animal virus gene was found piglets inoculated with virulent tgev shed the virus for 12 h, followed by profuse diarrhoea that led the piglets to the verge of death 1-4 days after inoculation. the tgev load shed in the diarrhoea was 3.2 × 10 5 copies at the 12th hour, peaked at 1.72 × 10 6 copies at the 48th hour and then decreased until death in the pbs group (fig. 5) . the recombinant lactobacillus vaccine group (pg:612-20mt6d) exhibited the same trend. the tgev copy number was 10 5 at the 12th hour, and the copies reached a peak at the 48th hour. the copy numbers were similar and followed the same trend after reaching the peak. however, the copy numbers in the pg:612-20mt6d group were significantly lower than in the pbs group. next, we investigated the generation of lactobacillus vaccineinduced regulatory cells after infection in piglets. as shown in immunized with pg:612-20mt6d and pbs piglets were orally challenged with tgev. tgev-challenged pigs received a 5-ml dose of 1 × 10 5 plaqueforming unit (pfu)/ml via oralgastric gavage on days postinoculation. pigs (control group) were administered volumematched virus-free st cell culture media. all piglets were euthanized at 5 days for necropsy examination fig. 6 , there was a marked increase in the production of il-4 in cd4+ t cells. the th1 immune response induced by the vaccine was seriously broken in favour of th2-mediated systemic and mucosal immunity post-infection. the systemic th2 immune response was higher than the mucosal th2mediated immune response. after tgev infection, the body activated more th2 to protect itself in response to the infection. as shown in fig. 7 , toll-like receptor (tlr) expression was detected in the splenic lymphocytes (sl) and mesenteric lymph node cells (ml) in the piglets in the pbs and vaccine groups. tlr-2 was higher in the vaccine group than in the pbs group. in contrast, there were no significant differences between tlr-4 and tlr-9. however, the three tlrs exhibited the same trends in the mesenteric lymph node cells compared with the pbs and lactobacillus vaccine. tgev infection induced tlr expression and especially enhanced tlr-2 and tlr-4 expression, but the expression levels in the lactobacillus vaccine group were significantly lower than the levels in the pbs group. cytokine expression in the splenic lymphocytes and mesenteric lymph node cells was also analysed and compared in our study. the ifn-γ, il-4 and il-17 levels in the splenic lymphocytes from the lactobacillus vaccine group showed marked changes, whereas no significant difference was observed in the level of tgf-β. tgev infection stimulated cytokine expression in the pbs group, including ifn-γ and il-4. the vaccine group did not express a notably higher level of ifn-γ and il-4 compared with the pbs group. the tgf-β expression level in the mesenteric lymph node cells was lower in the vaccine group than in the pbs group; the same trend was observed with ifn-γ and il-4. the vaccine group provoked higher il-17 expression than the pbs group following tgev infection. the il-17 expression levels in both the splenic lymphocytes probiotics are well known to have additive effects on human health in terms of improving the gut microflora and modulating immune responses (villena et al. 2006) . studies have also reported that probiotic feeding results in an increased spleen mass, followed by higher levels of total serum proteins, increased globulin levels and enhanced production of secretory iga (dock et al. 2004) . in humans, lactobacilli colonize the distal small bowel and the large intestine. different probiotic bacteria possess various mechanisms, including adhesins and/ or coaggregation factors, which aid in adhesion and colonization (friedrich 2013) . during this period, immunization with recombinant lactobacillus is crucially important on the effects of immunization, such as the timing of the first immunization in the protocol and the dose. from these results, we show that the immune response in response to the first priming immunization dose is better than the response to the second immunization because the priming dose is better at initiating adhesion and colonization in the piglet. interestingly, the mucins are large glycoproteins that are the major organic components of mucus. the mucin protein content of the mucus is 20 %, whereas the carbohydrates comprise 70 to 80 % by weight. intestinal mucin has been shown to inhibit the replication of rotavirus in vitro (chen et al. 1993; superti et al. 1993 ). additionally, a high dose of lactobacillus adversely affects the immunization. there is some evidence of diarrhoea after a double dose of the lactobacillus vaccine, but from this result, we find that the two-dose immunization is still the best immunization plan. the reason for the diarrhoea after immunization is the overdose of lactobacillus, which is a type of microbe that causes a disturbance in the intestinal microbial flora for short time. many enteric pathogens must first adhere to the intestinal epithelial cells to initiate intestinal disease. limiting access of the pathogens to intestinal epithelial cells is one strategy to prevent disease that has been investigated previously. for example, the competitive inhibition of bacterial adherence by mimicry of receptors on the apical surface of enterocytes using oral administration of sialylated glycoproteins has been shown to protect newborn calves from the enterotoxigenic e. coli strain k99 (mouricout et al. 1990 ). lactobacillus must colonize the gut soon after birth; therefore, the vaccine could play a role in non-specific immunity. probiotic strains with a high adherence capacity have been demonstrated to enhance the immunoglobulin a response to rotavirus (kaila et al. 1992) . in this study, we observed a significant increase in the anti-tgev iga titre in the intestinal tract of piglets administered recombinant l. casei. furthermore, we showed that the diarrhoea state of piglets administered l. casei was significantly lower than that of piglets administered saline. in the murine model of ifv infection, the virus moves from the upper respiratory tract to the lower respiratory tract (hori et al. 2001) . hrv-vaccinated and lactobacillus acidophilus-fed pigs had a significantly higher magnitude of hrv-specific iga and igg antibody-secreting cell responses in the ileum and serum igg antibody and virus neutralizing antibody titres compared to hrv-vaccinated pigs without l. acidophilus colonization (zhang et al. 2008) . our immunization stimulated the same systemic specific igg titres. the specific antibody response neutralized the challenged tgev, and the load of tgev in the diarrhoea was significantly decreased. the mucosal immune response formed the first barrier function to neutralize tgev. in large scale swine farm surveillance, lower piglet birth weight and higher within-litter variability in birth weight were factors associated with higher losses from birth to weaning (yuan et al. 2015) . during tgev infection, it is likely that the stronger piglets obtained more iga than their non-immunized counterparts and thus were more likely to survive until the intestinal villi regenerated and immunity developed. during the histopathological analysis, some damage was observed in the small intestine. for instance, the villus wall of the control groups was thin and almost transparent, probably due to atrophy. lymphocyte proliferation in the intestinal lamina propria was also found in some piglets administered an oral dose of recombinant lactobacillus, indicating that recombinant lactobacillus induced a mucosal immune responses in the piglets. taken together, our data show the tremendous potential for recombinant lactobacillus to enable rapid and effective treatment for tgev infection with intestinal tropism in piglets. we evaluated the specific t cell immune responses induced by the recombinant l. casei vaccine compared with the pbs control group in the piglets. we demonstrated that l. casei significantly enhanced the immunogenicity of the tgev vaccine as indicated by the significantly higher magnitude of specific ifn-γ-producing cd4+ t cell responses. there has reported that mice fed recombinant l. casei with the adjuvant mdp and tuftsin have significantly higher th1 and th17 production than control group mice (jiang et al. 2014) . these results were the same and indicated that l. casei had a strong potentiating effect on both the cellular and humoral immunity induced by the oral l. casei vaccine. similarly, a previous study showed that oral intake of l. fermentum cect5716 fig. 7 cytokines and tlr expression. the rna of splenic lymphocyte (sl) and mesenteric lymphocyte (ml) in immunized piglets, pg:612-20mt6d and pbs groups, were used to analysed the cytokines and tlr expression. ifn-γ, il-4, il-17, tgf-β, tlrs expressing were detected in splenic lymphocyte (sl) and mesenteric lymph cells (ml) piglets, such as pbs and vaccine groups. *significant difference by student t test (p < 0.05). data shown were compared using one-way analysis of variance (anova) followed by duncan's multiple range test. representative for three independent experiments significantly enhanced serum th1 type cytokine production and influenza-specific iga antibody responses to an intramuscular influenza vaccine in adults (olivares et al. 2007) . the mesenteric lymph nodes were primarily used to analyse tgev infection and the immunoprotection provided by the lactobacillus vaccine in terms of mucosal immunization and infection. ifn-γ induction by tgev results from interactions between an outer membrane domain of el and the pbmc membrane (charley and laude 1988) ; however, these authors did not study the expression of il-4 by pbmcs. the expression of ifn-γ was higher than il-4 in the immunized group, and the th1/th2 balance was broken in our study. after immunization with recombinant lactobacillus, ifn-γ played a major role in the mucosal immune response. however, after tgev infection, the systemic and local immune responses shifted from th1 to th2. the systemic humoral immune response was stronger than the cellular immune response after tgev infection. this is the first study to demonstrate that tgev infection polarized the immune response to th2 immunity and that recombinant lactobacillus could weaken tgev infection in the form of th2 immunity. from these results, we found that the immunization did not polarize th2 immunity more seriously compared to the pbs control group. the proteinbased sars coronavirus vaccine boost induced similar levels of th1 and neutralizing antibody responses that protected vaccinated mice from subsequent sars-cov challenges but induced stronger th2 and ctl responses (zheng et al. 2008) . the uv-inactivated sars coronavirus vaccine retained its immunogenicity and promoted th2type immune responses (tsunetsugu-yokota et al. 2007 ). the activation of th2 responses such as il-10 stimulate b cell proliferation, which can produce specific and nonspecific anti-infection antibodies (grodeland et al. 2015) ; similarly, both t and b cells have functions following lactobacillus vaccination. the production of il-9 by th2 cells results in the proliferation of mast cell growth, and il-13 stimulates epithelial cell growth (tukler henriksson et al. 2015) . the proliferation of epithelial cells is crucial for tgev infection. the th2 response could also stimulate the production of mucus by epithelial cells (zhang et al. 2015) . il-17 cells play an essential role in mhv-induced immunopathology, and ifn-γ is important for maintaining the immune balance between th1 and th17 responses during acute viral infection (yang et al. 2011 ). however, the th1/th2 balance in the negative control group was different than the balance in the immunized group. tgev affects both systemic and local cellular immunity without immunization. il17 has been reported to have both pro-and anti-inflammatory effects (lafdil et al. 2009; nagata et al. 2008) . our results showed that the immunized piglets provoked il-17 expression form both the systematic and mucosal immune responses after tgev infection. compared with the mucosal immune response, il-17 expression in the mesenteric lymph nodes was markedly lower than the expression in the spleen cells. however, il-17 expression in the pbs group was lower than the expression level in the immunized groups, indicating that the lactobacillus vaccine group activated il-17 expression during tgev infection. swine-origin influenza a virus-infected patients exhibited rapid lymphopenia, t cell activation and a preferential loss of the th17 subset during the early stage of acute infection (jiang et al. 2010) , which was consistent with the first reports that swine-origin virus inhibited th17 proliferation after infection. our study also found the same phenomenon after coronavirus infection in swine compared with the immunized group. the most important finding was that the oral recombinant lactobacillus vaccine stimulated th17 cell proliferation. the proliferation was involved in cytokine and chemokine production, neutrophil recruitment, promotion of t cell priming and antibody production (dirisala et al. 2013) . th17 responses are protective against lethal influenza virus infection in il-10deficient mice (mckinstry et al. 2009 ). in contrast, a deleterious role of il-17 has been proposed to contribute to the acute lung injury associated with il-17-mediated neutrophil recruitment during influenza virus infection (crowe et al. 2009 ). there were significant changes in the il-4 and ifn-γ expression levels in the mesenteric lymph node cells compared with the pbs group. moreover, il-4 expression was higher than ifn-γ expression in the spleen cells. the expression of il-4 and ifn-γ indicated that the recombinant lactobacillus effectively inhibited inflammation after tgev infection. in this study, we also evaluated tlr-2, tlr-4 and tlr-9 expression in piglets immunized with recombinant l. casei and then challenged with tgev. previously, tlr expression in pigs has been studied only at the mrna level in lymphocytes using real-time pcr because antibodies against porcine tlrs are not currently available. tgev infection did not induce tlr-2, tlr-4 and tlr-9 expression in the spleen cells. however, tlr expression was significantly different in the mesenteric lymph node cells compared with the pbs and lactobacillus vaccine groups. tlr expression was extremely high in the pbs group compared with the other groups. the cytokine and tlr expression levels in the splenic lymphocytes are indicators of systemic immunity, whereas the expression in the mesenteric lymph node cells was associated with the local and mucosal immune responses. the expression levels of all tlrs in mesenteric lymph node cells were higher in the pbs group than the vaccine group, suggesting that the lactobacillus vaccine effectively inhibited tlr expression. the recombinant lactobacillus groups exhibited jejuna and ilea with lymphocyte proliferation in the lamina propria. transmissible gastroenteritis (tge) coronavirus infection resulted in antibody production from primed mesenteric lymph node cells following an in vitro boost with viral antigen (berthon et al. 1990 ); as an intestinal infectious disease, tgev would attack the intestinal tissue and local immune system. exposure of pigs to tgev or prcv results in distinct disease patterns related to differences in tissue tropism (saif 1996) . however, the increased frequencies of tlr-2 and tlr-9 expression in pigs may simply be due to the significantly higher counts of l. casei or mdp and tuftsin, which may translate to an increased magnitude of tlr agonists available to stimulate the host mucosal immune system. it is likely that the higher lab count in the lab plus hrv group played a more pertinent role in the significant increases in tlr2 and tlr9 expression (wen et al. 2009 ). taken together, tgev immune protection was primarily dependent on the mucosal immune response. systemic immunity did not play a key role after tgev infection. interestingly, il-17 expression in the vaccine group was significant higher than il-17 expression in the pbs group challenged with tgev, and th17 played an anti-inflammatory role in mucosal immunity. il-17 also stimulated intestinal epithelial cell differentiation and growth. in conclusion, our study suggests that the recombinant lactobacillus vaccine provokes specific mucosal and systemic immune responses to protect piglets from infection. iga played a dominant role in the mucosal immune response after tgev challenge. therefore, the histopathology and rna copy numbers directly demonstrated that the lactobacillus vaccine was effective for tgev infection. the protective efficacy was significantly higher, which would have great value in 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cytokine responses induced by lactobacilli colonization and human rotavirus infection in gnotobiotic pigs interferon-gamma negatively regulates th17-mediated immunopathology during mouse hepatitis virus infection construction of recombinant lactobacillus casei efficiently surface displayed and secreted porcine parvovirus vp2 protein and comparison of the immune responses induced by oral immunization within-litter variation in birth weight: impact of nutritional status in the sow probiotic lactobacillus acidophilus enhances the immunogenicity of an oral rotavirus vaccine in gnotobiotic pigs tmem16a-mediated mucin secretion in il-13-induced nasal epithelial cells from chronic rhinosinusitis patients studies of sars virus vaccines critical role of the interleukin-17/interleukin-17 receptor axis in regulating host susceptibility to respiratory infection with chlamydia species acknowledgments this work was supported by the national natural science foundation of china (31472226). conflict of interest the authors declare that they have no competing interests.ethical statement the piglets were handled and maintained under strict ethical conditions according to international recommendations for animal welfare. this article does not contain any studies with human participants performed by any of the authors. key: cord-295033-5fd9bu60 authors: parma, y.r.; chacana, p.a.; rogé, a.; kahl, a.; cangelosi, a.; geoghegan, p.; lucchesi, p.m.a.; fernández-miyakawa, m.e. title: antibodies anti-shiga toxin 2 b subunit from chicken egg yolk: isolation, purification and neutralization efficacy date: 2011-09-15 journal: toxicon doi: 10.1016/j.toxicon.2011.07.009 sha: doc_id: 295033 cord_uid: 5fd9bu60 shiga toxins (stx1 and stx2) are the main virulence factors of enterohemorrhagic escherichia coli (ehec), a foodborne pathogen associated with diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. the aim of this study was to evaluate the antibodies against stx2 obtained from egg yolks of laying hens immunized with a recombinant stx2b subunit. a high specific response in serum was observed 25 days after the first immunization and igy antibodies were extracted from day 47th and purified from egg yolk. a concentration of 0.84 mg of total igy/ml of egg yolk was obtained, of which 8% were antigen specific. the ability of anti-stx2b igy to recognize stx2b and stx2 either in solid-phase or in solution were evaluated and compared with anti-stx2b rabbit antibodies by western blotting and elisa. the protective efficacy of igy against stx2 was determined by in vitro and in vivo experiments. the results show that igy was able to recognize stx2b and stx2 in denatured conditions, attached to a solid-phase and free in solution. the anti-stx2b igy could effectively block the biological activity of stx2 on vero cells and protect mice from stx2 challenge. the data suggest that immunization of hens with stx2b could be a strategy to obtain at low cost a relatively high concentration of anti-stx2 egg yolk igy, able to neutralize stx2 lethal activity. igy technology could be an useful tool for research, diagnosis and therapy of ehec infection. shiga toxins (stx1 and stx2) are the main virulence factors of enterohemorrhagic escherichia coli (ehec), a foodborne pathogen associated with diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. the aim of this study was to evaluate the antibodies against stx2 obtained from egg yolks of laying hens immunized with a recombinant stx2b subunit. a high specific response in serum was observed 25 days after the first immunization and igy antibodies were extracted from day 47th and purified from egg yolk. a concentration of 0.84 mg of total igy/ml of egg yolk was obtained, of which 8% were antigen specific. the ability of anti-stx2b igy to recognize stx2b and stx2 either in solid-phase or in solution were evaluated and compared with anti-stx2b rabbit antibodies by western blotting and elisa. the protective efficacy of igy against stx2 was determined by in vitro and in vivo experiments. the results show that igy was able to recognize stx2b and stx2 in denatured conditions, attached to a solid-phase and free in solution. the anti-stx2b igy could effectively block the biological activity of stx2 on vero cells and protect mice from stx2 challenge. the data suggest that immunization of hens with stx2b could be a strategy to obtain at low cost a relatively high concentration of anti-stx2 egg yolk igy, able to neutralize stx2 lethal activity. igy technology could be an useful tool for research, diagnosis and therapy of ehec infection. ó 2011 elsevier ltd. all rights reserved. enterohemorrhagic escherichia coli (ehec), a subset of shiga toxin producing e. coli (stec), are important human foodborne pathogens (kaper et al., 2004) . transmission is most frequently associated with the consumption of contaminated food or unpasteurized dairy products and infections can also be acquired through person-to person contact (griffin, 1995) . the clinical manifestations of ehec infections range from watery diarrhea, or hemorrhagic colitis (hc), to the most severe outcome, the life threatening hemolytic uremic syndrome (hus) (nataro and kaper, 1998) . hus occurs in about 5-10% of individuals, primarily very young and elderly subjects (rivas et al., 2010) . although ehec infections are a serious problem in developed countries, argentina has the highest incidence of hus in the world, with approximately 500 new cases observed each year in children under 5 years of age (rivas et al., 2010) . stec also referred to as verotoxin producing e. coli (vtec), produce cytotoxins structurally related to those produced by shigella dysenteriae type 1. shiga toxins (stx1 and stx2) are the main virulence factors involved in the pathogenesis of hus and belong to the class known as ab5 toxins composed of one a subunit and five identical b subunits. the a subunit (32 kda), possesses enzymatic rna n-glycosidase activity that hydrolyzes the n-glycoside bond of adenosine of the 28 s rrna of 60 s ribosomes, and hence inhibits protein synthesis; b subunits (7 kda) are mainly involved in receptor binding (jeong et al., 2010) . stec strains produce stx1, stx2 (or their variants), or both of these toxins. although the mechanisms of action of stxs are thought to be similar, cytotoxicity of stx2 may be stronger than that of stx1; the 50% lethal dose in mice of purified stx2 is 1 ng, whereas stx1 has a 50% lethal dose of 400 ng (tesh et al., 1993) . additionally, epidemiological data indicate that stx2 producing strains are more frequently associated to severe illnesses such as hus than stx1 producing strains (karmali et al., 1985) . due to their extreme toxicity, shiga toxins are classified in category b of bioterrorism agents by the center for disease control and prevention (cdc, 2009) . immunoglobulins are widely used for a variety of purposes, such as in diagnostic tests, purification columns, and passive immunotherapy (kim et al., 2010) . therefore, research and diagnostic community constantly demand new alternatives and procedures to produce cost-effective antibodies. the use of laying hens to produce polyclonal antibodies is an alternative to the use of mammals, such as rabbits and, since more than two decades, egg yolk antibodies (igy) are a low cost and ethical alternative (schade et al., 1996; schade and chacana, 2007) . compared with the stressful bleeding of mammals to obtain serum, igy can be easily obtained non-invasively from the egg yolk. from the economical point of view, the amount of antibodies produced by a single hen is similar to that of a large mammal such as a sheep or goats, whereas maintenance costs are much lower (schade et al., 2005; zhang, 2003) . igy from serum is actively transferred into the yolk by a receptor -mediated process (schade et al., 2005) and the amount of the immunoglobulin varies between 100 and 250 mg per egg (schade et al., 1996) . thus, a substantial amount of antibody can be produced from just one hen (up to 40 g of total igy per chicken per year), of which 1-10% is expected to be specific to the antigen of interest (mine and kovacs-nolan, 2002) . in contrast to mammalian igg, igy antibodies do not activate mammalian complement, do not cross-react with fc receptors, mammalian rheumatoid factor, or human anti-mouse antibodies, thus eliminating false-positive results in serological assays (schade et al., 2005) . also, chickens are able to develop a better response against mammalian antigens, due to the phylogenetic distance between mammals and birds (schade et al., 2005) . at present, there are no established specific prophylaxis or therapy strategies for ehec borne disease or the prevention of its complications (orth et al., 2008; tzipori et al., 2004) , nor early and reliable predictors of the severity of the disease. but, the rapid diagnosis of stec infection and early intervention before the onset of systemic diseases are desirable to prevent or ameliorate toxin-related complications, including hus, as a proper supportive treatment can be determined. besides, rapid laboratory diagnosis and subtyping of stec isolates leads to prompt detection of outbreaks and implementation of control measures (cdc, 2009) . in this work, polyclonal antibodies were raised in chicken against the stx2b subunit. the antibodies were extracted from egg yolk, purified and analyzed for their binding and neutralizing capabilities against stx2 holotoxin. the comparative performance of anti-stx2b igy was evaluated by immunoblotting, elisa and in vitro and in vivo neutralization against polyclonal antibodies obtained from immunized rabbits. the stx2 holotoxin, corresponding to stx2-edl 933 variant (krüger et al., 2011) , was obtained from stec 026:h11 strain t 59-2 isolated from cattle by parma et al. (2000) . briefly, the bacteria were grown in luria bertani (lb) broth at 37 c with shaking at 150 rpm until od 600 nm reached 0,3. the culture was treated with mitomicin c (0.5 mg/ml) and incubated overnight at 37 c with shaking. after centrifugation at 10,000 â g, 10 min at 4 c, the supernatant was filter-sterilized and aliquots were stored at à20 c. a negative control for the sandwich elisa assay was prepared performing the same protocol with e. coli dh5a strain. stx2b subunit was produced by a cloning method based in site-specific recombination reactions that mediate the integration and excision of phage lambda into and from the e. coli chromosome (gateway cloning technology, invitrogen). a dna fragment encoding stx2b was obtained by pcr amplification using dna extracted from the reference strain e. coli edl 933 and the following pairs of synthetic primers that include recognition sites (attb) for the recombination: 5 0 ggggacaagtttgtacaaaaaagcaggcttctagaa gaaggagatatacatatgaagaagatgtttatggcggtt 3 0 (attb1-forward, sequence corresponding to attb1 is underlined). 5 0 ggggaccactttgtacaagaaagctgggtggatcctta tcagtgatggtgatggtgatgccgaccttcgatgtcattattaa actgcactt 3 0 (attb2-reverse, sequence corresponding to attb2 is underlined and his-tag codons are in italics). the accuracy of the final dna construction was confirmed by dna sequencing of different clones. the plasmid obtained was called pdeststx2b. for expression of recombinant stx2b subunit, bl21-ai ô e. coli cells were transformed by pdeststx2b. isolated colonies were then grown with shaking at 200 rpm at 37 c to mid exponential phase (od 600 nm: 0,6) in 200 ml of luria bertani (lb) broth supplemented with 100 mg/ml ampicillin (sigma aldrich). the cultures were induced with l (þ) arabinose (sigma aldrich) at a final concentration of 0.2% and incubated with shaking at 37 c overnight. the stx2b was purified by affinity chromatography under native conditions using a ni-nta column (qiagen). the eluted fractions were exchanged with pbs (ph 7.4) by repeated dilution and concentration by using centrifugal ultrafiltration with vivaspin 20 (3000 mwco, sartorius). protein yield was determined by the bradford assay using bsa as the standard protein, purity and presence of the recombinant protein in the elution fractions were determined by sds-page and western blot, respectively. one gel was stained with coomassie blue and the other was electrotransferred onto a nitrocellulose membrane (hybond ecl, amersham pharmacia biotech). membranes were blocked overnight with 5% skimmed milk in pbs-tween 20 (pbs-t) 0.1% at 37 c and then washed three times with pbs-t. this was followed by an incubation with 1:3000 dilution of anti-his-tag antibody (invitrogen) in pbs-t and then with a 1:1000 dilution of the anti-mouse ecl secondary antibody (sigma aldrich). after a washing step, the membrane was developed with the ecl western blotting detection kit (amersham pharmacia biotech), according to the manufacturer's instructions. the blot was exposed for 1 min to an x-ray film (super rx; fuji photo). serum samples were collected from the wing vein 7 days after each immunization. eggs were collected daily starting one week after the third immunization and stored at 4 c until further processing. egg yolks were pooled and frozen for purification of igy antibodies. hens were housed in individual cages before the beginning of laying, maintained on 12/12 h light/dark cycle and received food and water ad libitum. two new zealand white male rabbits (3 kg) were immunized subcutaneously with 150 mg of purified recombinant stx2b emulsified with fca at multiple sites in the dorsal region. subsequent booster immunizations were done with an equivalent dose of stx2b emulsified with fia at days 20, 40, 60 and 80. blood was collected from the marginal ear vein after the second booster. final bleeding of the anaesthetized animals was done by cardiocenthesis. animals were housed individually with ad libitum water access and commercial rabbit food was supplemented with hay and carrots once a week. prior to the first immunization, serum samples from hens and rabbits and egg yolks samples were taken and used as pre-immune negative controls. presence of specific antibodies in chicken and rabbit sera was tested in a dot blot assay, using recombinant stx2b subunit as antigen. egg yolk antibodies were purified by the water dilution method (wd) (akita and nakai, 1992) . briefly, egg yolk content was mixed with water in a 1:5 dilution and kept at à20 c for at least 72 h and thereafter thawed at 4 c. the disrupted emulsion was centrifuged at 8000 â g, 15 min at 4 c. the liquid phase containing the igy was filtered through a gauze tissue and ammonium sulfate was added (0.24 g/ml supernatant). after centrifugation at 10,000 â g, 15 min at 4 c, the pellet was resuspended with ammonium sulfate 2 m and centrifuged again. finally, the pellet was resuspended and dialyzed against pbs (ph 7.4) at 4 c. for rabbit sera antibodies, blood samples were kept overnight at room temperature, sera were separated by centrifugation and thereafter stored at à20 c until use. igg was precipitated with ammonium sulfate (45% saturation) and stirred overnight at 4 c (spira et al., 1987) . after centrifugation at 10,000 â g for 3 min at 4 c, supernatants were discarded and pellets were dissolved and dialyzed against pbs (ph 7.4). pre-immune serum antibodies from non-immunized chickens and rabbits were also processed. purity of igy and igg was checked by sds-page and western blot and protein concentration was determined by the bradford assay, using bsa as the standard protein. specific antibodies were purified using hitrap nhsactivated hp 1 ml columns (ge). a coupling procedure was done using recombinant stx2b (1.24 mg in coupling buffer) according to the manufacturer's protocol. about 4 ml of either igy or igg antibodies isolated as described in section 2.3, were applied onto the column. unbound material was eluted with 10 column volumes of binding buffer (75 mm tris-hcl ph 8.0). bound antibodies were eluted with 10 column volumes of elution buffer (100 mm glycine, 0.5 m nacl ph 2.7) in 0.5 ml fractions into collection tubes containing 100 ml 1 m tris-hcl ph 9.0. protein concentration in the eluted fractions was determined by the bradford assay. recombinant stx2b and stx2 holotoxin were separated by 12.5% sds-page (under reducing conditions) and transferred onto a nitrocellulose membrane (hybond ecl, amersham pharmacia biotech). the membrane was blocked overnight at 4 c with 5% skimmed milk in pbs-t 0.1%, and incubated with a 1:5000 dilution of either igy or rabbit serum in pbs-t for 1 h at 37 c. after washing, the membrane was incubated with horseradish peroxidase-conjugated goat anti-chicken igy (1:6000) or goat anti-rabbit igg (1:5000) for 1 h at 37 c. finally, membranes were revealed using dab/h2o2 system (pierce). microplates (nunc maxisorp) were coated overnight at 4 c with 100 ng/well of recombinant stx2b or stx2 holotoxin in carbonate/bicarbonate buffer ph 9.6 and blocked at 37 c for 1 h with 5% skimmed milk in pbs-t 0.05%. after a washing step, igg and igy (or pre-immune samples) were 2-fold serially diluted in pbs-t and incubated at 37 c for 1 h. previously, both antibodies were properly diluted to give the same starting protein concentration determined by the bradford assay (initial concentration: 1.4 mg/ml). plates were washed and incubated with horseradish peroxidase-conjugated goat antichicken igy (1:6000) or goat anti-rabbit igg (1:5000) for 1 h at 37 c. the plates were washed with pbs-t and developed with abts/h2o2 system (pierce) and absorbance was read at 405 nm. antibody titers were defined as the reciprocal of the dilution of igy and igg antibodies corresponding to a od 405 nm of 0,5. for chicken sera, 4-fold serial dilutions were made and antibody titers were defined as the reciprocal of the highest dilution of anti-stx2b generating a signal about 2-fold higher than the pre-immune serum. microplates (nunc, maxisorp) were coated overnight at 4 c with 100 ml of igy or igg at 500 ng/well in carbonate/bicarbonate buffer ph 9.6. after washing with pbs-t 0.05%, plates were blocked with 5% skimmed milk in pbs-t for 1 h at 37 c. wild type stx2 holotoxin 3-fold serially diluted in pbs-t was added and incubated at 37 c for 1 h. as a negative control, dh5a supernatant was used in the same dilutions as stx2. plates were washed and incubated with a 1:1000 dilution of horseradish peroxidaseconjugated anti-stx2b igg antibodies, coupled in the lab by using ez-link plus activated peroxidase (thermo scientific). plates were incubated 1 h at 37 c and then developed with opd (sigma) and absorbance was read at 490 nm. minimun concentration of stx2 detected by igy and igg was defined as the reciprocal of the highest dilution generating a signal about 2-fold higher than that of dh5a supernatant. african green monkey kidney (vero) cells were plated at 10 4 /well on 96-well plates in dmem medium containing 10% fetal bovine serum and incubated overnight at 37 c under 5% co 2 . cytotoxic dose 50% (cd 50 ) was calculated from dose-response curves geometrically as the reciprocal of the toxin dilution causing 50% reduction in cellular viability. igy and igg antibodies (initial concentration: 1.4 mg/ml) (or pre-immune samples) were serially diluted 2-fold in dulbecco's modified eagle medium (dmem) and pre-incubated for 1 h at 37 c with an equal volume of stx2 holotoxin (8 cd 50 ) (final stx2 concentration: 4 cd 50 ). for the toxin control, serially 10-fold dilutions of stx2 in dmem without antibody were prepared. the stx2-igy and stx2-igg mixtures and stx2 alone were added to the vero cell monolayer (100 ml/well) and incubated for 48 h at 37 c under 5% co 2 . the viability of the vero cells was determined by crystal violet staining (gentry and dalrymple, 1980) and absorbance was read at 490 nm.percentage of neutralization was calculated by using the following formula: od (toxin þ antibody) -od (toxin only)/od (no toxin) -od (toxin only) â 100. results were expressed as percent of neutralization compared with total igg or igy concentration in mg/ml. all data represent the average of triplicate assays. nih mice (anlis-malbrán) of 10-14 g, were allocated randomly over the groups. during the test period mice were housed in wire-topped plastic cages with a layer of sawdust as bedding. cages were located in a room with controlled lighting (14 h/day), constant temperature (18-20 c) and constant relative humidity (55-65%). mice toxicity of holotoxin preparation was determined. serially doubling dilutions were made in saline solution and the 5 successive dilutions, expected to cover the 0-to-100% mortality range, were tested in separate groups of 4 mice each. median lethal dose (ld 50 ) was calculated by the method of reed and muench (1938) . two-fold dilutions of igy and igg diluted in pbs (initial concentration: 1.4 mg/ml) were incubated with 5 ld 50 of wild type stx2 holotoxin for 1 h at 37 c. groups of four mice were injected intravenously (iv) with 0.5 ml of each immunoglobulin dilution-stx2 mixture. the mice were monitored over the next 4 days and symptoms of disease and death were recorded. control mice injected with only stx2 holotoxin were included in each assay and the results were used to confirm the l þ /5 test dose of the toxin and to correct the antitoxin values obtained. control mice (non-injected or injected with saline solution) were also included. antibody titers were calculated by the spearman-karber method (markus et al., 1995) . all procedures involving animals were reviewed by the animal care and use committee at the national institute of agriculture technology. the average yield of purified recombinant stx2b protein from 6 independent 200 ml culture preparations was 1.3 mg (6.5 mg/l). sds-page analysis of the purified stx2b is shown in fig. 1a . one band between 7 and 14 kda was observed in the eluted fractions. this band likely corresponds to the recombinant stx2b monomeric conformation of the protein (theoretical mw: 11 kda) (fig. 1b) . densitometry analysis of the protein bands on the sds-page gels revealed >98% purity in the purified protein preparations. hens had a different profile of antibody production, although in both birds the highest specific titer was found after the second immunization (day 25th, titer: 64,000). in hen n 1, specific titer diminished after the third (titer: 16,000) and fourth booster (titer: 4000) but it was increased with the last booster to approximately the same level of specific antibody than after the second immunization. in hen n 2, specific antibodies remained constant after 2nd and 3rd immunization (titer: 64,000) but diminished significantly after the final boosters (titer: 4000). highly purified total antibodies preparations were obtained as most of the other proteins present in sera and egg yolk could be removed ( fig. 2a and b) . the final affinity purification of specific antibodies revealed that approximately 8% of total igy and 6% of total igg were antigen specific. igy and igg antibodies isolated as described in section 2.3 were used at the same protein concentration for subsequent elisa and in vitro and in vivo neutralization assays. western immunoblots (fig. 3, lanes 1 and 3) and indirect elisa (fig. 4a) indicate that igy and igg were able to recognize both denatured and native forms of recombinant stx2b protein, respectively. elisa titers were higher for igy (30,189) than for igg (5219). specific anti-stx2b polyclonal antibodies obtained from chicken egg yolk and rabbit sera recognized not only the denatured and native form of b subunit, used for immunization but the antibodies were also able to recognize the denatured and native wild type stx2 holotoxin in western blot (fig. 3) , indirect elisa (fig. 4) and sandwich elisa (fig. 5) . unspecific signal was observed with pre-immune igy and igg antibodies, probably due to the recognition of e. coli proteins from the supernatant of stx2. specific titer of chicken igy and rabbit igg against the holotoxin were of 1573 and 2149, respectively. in the sandwich elisa, soluble native stx2 holotoxin was detected by igy and igg. the holotoxin could be detected in a 1/25 dilution when igy was used as capturing antibody, whereas using igg coupled to the elisa plate it was possible to detect the holotoxin in a 1/75 dilution. the cytotoxicity of stx2 holotoxin in vero cells was 2000 cd 50 /ml and the lethality in mice was 3165 ld 50 /ml. the cytotoxicity assay based in vero cells was used to test if anti-stx2b antibodies from both species were able to prevent the toxin activity in vitro. both igy and igg antibodies neutralized the cytotoxic effects on vero cells (fig. 6) . the data obtained show that igg antibodies were 4 times more efficient than igy antibodies since a concentration of 1.09 mg/ml was able to neutralize 100% of the toxin cytotoxicity compared with igy antibody that needed 4.38 mg/ml. vero cells that were treated with the mixture stx2-pre-immune antibodies or stx2 alone subsequently died (data not shown). in addition, neutralizing activity was evaluated in vivo during 4 days with native stx2 holotoxin that had been previously incubated with igy and igg antibodies. all animals that received only native stx2 (5 ld 50 ) died at the third day of the experiment, whereas mice that received 8.75 mg/ml of igy antibody survived (fig. 7b) . in concordance to the results observed in the in vitro assay, neutralizing capability of igg was higher than igy antibodies. this can be easily visualized with 0.55 mg/ml of igg ( fig. 7a ) that prevented 3 of 4 mice from dying at the end of the experiment compared with mice that received igy at the same concentration that all could not survive to the third day of the experiment (fig. 7b) . neutralizing activity was calculated by the spearman-karber method, as the antibody dilution that prevented mortality in 50% of the animals, resulting in 1:640 and 1:4305 dilution for igy and igg antibodies, respectively. in the present report, specific egg yolk igy antibodies with binding and neutralizing capabilities against the wild type stx2 toxin were obtained after immunization of laying hens. the antigen that was injected consisted in the recombinant b subunit stx2b, obtained with a yield of 6.5 mg/l culture, a relatively elevated production compared with the 0.1 mg/l and 0.7 mg/l reached in the procedure described by acheson et al. (1995) and marcato et al. (2001) , respectively. in this work, significant levels of specific antibodies were measured by elisa after 25 days of initiating immunization, showing that stx2b (w11 kda) is immunogenic for chickens. however, in some other mammal species as human (ludwig et al., 2001) and mice (imai et al., 2004) , stx2b was found to be a poor immunogen. marcato et al. (2001) reported that high specific antibody titers against stx2b in rabbits could only be achieved including endotoxin in the antigen preparation. so it can be hypothesized that a selective pressure to minimize a long-term immunity against the b subunit was favored in host species (johannes, 2002) . although a high antibody response was achieved at the beginning of immunization, elisa results from chicken sera showed that levels of specific antibodies were not constantly maintained in the immunized birds, in contrast to the results of pauly et al. (2009) . it is possible that conjugation of stx2b with a carrier may be necessary (marcato et al., 2005) although wang et al. (2010) obtained a high titer of specific antibodies against b subunit from shiga toxin type 1 without conjugation to a carrier and the response remained constant over one year. this suggests a possible negative effect of b subunit from shiga toxin type 2 in laying hens. to the best of our knowledge, there is no information available regarding to this fact, though it has been reported that the stx holotoxin and the b subunit have harmful effects, including lethality, in different mammal species (huang et al., 2010) . early studies have established that chickens tend to be more resistant against toxins than other species (bengtson, 1924) . the polyclonal chicken antibodies were tested in classical immunological assays, recognizing not only the denatured toxin, but also the native holotoxin and the b monomer either in solid-phase or in solution. the stxb subunit binding capability of egg yolk igy was comparatively higher than rabbit igg. however, recognition of the b subunit in the context of the whole assembled toxin was similar for igy and igg. in general, ideal antigenic b-cell epitopes are hydrophilic, surface orientated and flexible because in most natural environments, hydrophilic regions tend to reside on the surface of proteins, while hydrophobic regions are found hidden in the interior of the protein. then, a possible justification for the difference in binding is that igy antibodies recognized epitopes in the isolated b subunit that became inaccessible in the holotoxin. in addition to the ability for binding to the native toxin, the chicken antibodies were able to neutralize crude stx preparation, both in vitro (cell culture cytotoxicity assay) and in vivo (mouse bioassay). wang et al. (2010) also evaluated the neutralizing capability of anti-stx igy antibodies by in vitro and in vivo experiments, but they tested the protective efficacy against stx1. in this work, we demonstrate that hens, and also rabbits, can produce neutralizing igy antibodies against stx2, which is associated to more severe course of illness (karmali et al., 1985) and when administered systemically it is about 400 times more lethal to mice than stx1 (tesh et al., 1993) . also in our work, we completely neutralize the activity of shiga toxin in vitro and in vivo with an igy concentration of about 700 times lower than that used in wang et al. (2010) . others authors have similarly generated neutralizing chicken antibodies against ricin toxin (pauly et al., 2009) , botulinum toxins (gomez et al., 2006; pauly et al., 2009) , clostridium difficile toxins, rabies and viper venom (motoi et al., 2005) . in this work, rabbit anti-stx2b igg antibodies were about 4 times more effective in stx2 neutralization than egg yolk antibodies (at a similar mass) in vitro and in vivo. however, to obtain 4 mg of specific anti-stx2b antibody from one hen the collection of only 5 eggs was enough, while to reach the same amount of specific igg, the exsanguination of one rabbit was necessary. total igy yield per egg was 8.4 mg although gassmann et al. (1990) described a yield of 64 mg igy per egg. however, there was less difference in the amount of specific antibodies per egg: 0.67 mg from this work and 2 mg from gassmann et al. (1990) . another group (bizhanov and vyshniauskis, 2000) reported a recovery rate of 2-10 mg of anti-sendai virus igy per ml of egg yolk. antibiotic therapy is not recommended for food poisoning caused by enterohemorrhagic e. coli infection, because increases the risk of serious complications, such as hemolytic uremic syndrome, due to the release of shiga toxin from killed bacteria and by inducing expression of stx through replication of phages that carry stx genes (kozlov et al., 1988) . therefore, alternative therapeutic approaches, such as inhibiting shiga toxin activity or absorption from the intestine, are required. oral administration of igy has proved successful for treatment of a variety of gastrointestinal infections, such as bovine and human rotaviruses, bovine coronavirus, yersinia ruckeri, c. difficile, salmonella spp., edwardsiella tarda, staphylococcus and pseudomonas, (mine and kovacs-nolan, 2002; schade et al., 2005) . chicken egg yolk has previously been used as an inexpensive and effective source of igy antibodies for the passive immunization or treatment of piglets suffering enterotoxigenic f4(k88)þ (marquardt et al., 1999) and f18abþ (imberechts et al., 1997) e. coli infections, dental caries due to streptococcus mutans in humans (smith et al., 2001) and porcine epidemic diarrhea virus (pedv) infection in piglets (kweon et al., 2000) . therefore, anti-stx igy could be an economic alternative for prophylactic and therapeutic treatment of ehec infections or stx exposure, either alone or combined with other products. furthermore, therapeutic use of igy in primates was effectively studied by leclaire et al. (2002) using neutralizing igy antibodies against the highly toxic staphylococcal enterotoxin b (seb). neutralization of stx may also be useful to decrease cattle colonization by stec (hoffman et al., 2006) , which is considered the main reservoir of the bacteria. since stx plays a role in both colonization and systemic disease, passive administration of anti-stx antibodies to reduce or prevent ehec infection in people has also been proposed (krystle et al., 2010) . this was demonstrated in mouse models with anti-stx2 antibodies administered before infection (donohue-rolfe et al., 1999) . in subsequent evaluations of the efficacy of such passive therapy, anti-stx antibodies were shown to provide protection against stec-mediated illness and death even when administered up to 4 days after bacterial challenge (donohue-rolfe et al., 1999; yamagami et al., 2001) . besides, igy is widely used in many applications ranging from immunofluorescence, immunohistochemistry, immuno-enzyme techniques (elisa), western blotting and immunoelectrophoresis (lee et al., 2002; tini et al., 2002) . these antibodies are capable to detect multiple bacterial and parasitic organisms such as acanthamoeba spp., helicobacter pylori (shin et al., 2002) , microsporidia (young et al., 2007) , e. coli o157:h7 (sunwoo et al., 2006) . in conclusion, although further studies should evaluate different adjuvants and immunization plans in chickens in order to maximize the anti-stx2b igy production, this work shows that igy technology is a promising alternative to be applied in the detection of stec and the prophylaxis or treatment of hemolytic uremic syndrome. there are no conflicts of interest related to this study. expression and purification of shiga-like toxin ii b subunits immunoglobulins from egg yolk: isolation and purification studies on organisms concerned as causative factors in botulism a comparison of three methods for extracting igy from the egg yolk of hens immunized with sendai virus recommendations for diagnosis of shiga toxin-producing escherichia coli infections by clinical laboratories antibody based protection of gnotobiotic piglets infected with escherichia coli o157:h7 against systemic complications associated with shiga toxin 2 efficient production of chicken egg yolk antibodies against a conserved mammalian protein quantitative microtiter cytotoxicity assay for shigella toxin production of chicken antibodies against botulinic toxin a escherichia coli o157:h7 and other enterohaemorrhagic escherichia coli bovine immune response to shiga-toxigenic escherichia coli o157:h7. clin shiga toxin b subunits induce vwf secretion by human endothelial cells and thrombotic microangiopathy in adamts13-deficient mice production of secretory immunoglobulin a against shiga toxin-binding subunits in mice by mucosal immunization chicken egg yolk antibodies against f18 ab fimbriae of escherichia coli inhibit shedding of f18 positive e. coli by experimentally infected pigs in vitro and in vivo protective efficacies of antibodies that neutralize the rna n-glycosidase activity of shiga toxin 2 the epithelial cell cytoskeleton and intracellular trafficking. i. shiga toxin b-subunit system: retrograde transport, intracellular vectorization, and more pathogenic escherichia coli the association between idiopathic hemolytic uremic syndrome and infection by verotoxin producing escherichia coli performance of an enzyme-linked immunosorbent assay for detection of clonorchis sinensis infestation in high-and low-risk groups the primary structure of the operons coding for shigella dysenteriae toxin and temperature phage.h30 shiga-like toxin verotoxins in bovine and meat verotoxin-producing escherichia coli isolates: type, number of variants, and relationship to cytotoxicity neutralizing antibodies to shiga toxin type 2 (stx2) reduce colonization of mice by stx2-expressing escherichia coli o157:h7 immunoprophylactic effect of chicken egg yolk immunoglobulin (igy) against porcine epidemic diarrhea virus (pedv) in piglets protection against bacterial staphylococcal enterotoxin b by passive vaccination vitro studies of chicken egg yolk antibody (igy) against salmonella enteritidis and salmonella typhimurium antibody response to shiga toxins stx2 and stx1 in children with enteropathic hemolytic-uremic syndrome immunoprophylactic potencial of cloned shiga toxin 2b subunit recombinant shiga toxin b-subunit-keyhole limpet hemocyanin conjugate vaccine protects mice from shigatoxemia an alternative approach to the optimal design of an ld 50 bioassay passive protective effect of egg-yolk antibodies against enterotoxigenic escherichia coli k88þ infection in neonatal and earlyweaned piglets chicken egg yolk antibodies as therapeutics in enteric infectious disease: a review production of rabies neutralizing antibody in hen's eggs using a part of the g protein expressed in escherichia coli diarrheagenic escherichia coli prevention and treatment of enterohemorrhagic escherichia coli infections in humans virulence genotypes and serotypes of verotoxigenic escherichia coli isolated from cattle and foods in argentina. importance in public health monitoring of laying capacity, immunoglobulin y concentration, and antibody titer development in chickens immunized with ricin and botulinum toxins over a two-year period a simple method of estimating fifty per cent endpoints chapter 10: diarrheagenic escherichia coli in argentina specificity of chicken (igy) versus rabbit (igg) antibodies raised against cholecystokinin octapeptide (cck-8) chicken egg yolk antibodies (igytechnology): a review of progress in production and use in research and human and veterinary medicine egg yolk compounds -livetin fractions use of egg yolk-derived immunoglobulin as an alternative to antibiotic treatment for control of helicobacter pylori infection passive transfer of immunoglobulin y antibody to streptococcus mutans glucan binding protein b can confer protection against experimental dental caries tsh binding proteins in rat and human serum detection of escherichia coli 0157:h7 using chicken immunoglobulin y comparison of the relative toxicities of shiga-like toxins type i and type ii for mice generation and application of chicken egg-yolk antibodies antibody therapy in the management of shiga toxin-induced hemolytic uremic syndrome passive protection of purified yolk immunoglobulin administered against shiga toxin 1 in mouse models efficacy of postinfection treatment with anti-shiga toxin (stx) 2 humanized monoclonal antibody tma-15 in mice lethally challenged with stx-producing escherichia coli chicken-derived igy recognizes developing and mature stages of loma salmonae (microsporidia) in pacific salmon, oncorhynchus spp the use of gene specific-igy antibodies for drug target discovery the study received financial support from anpcyt pict 0861/08 and pict 33774. key: cord-327568-5vo4nmei authors: tosini, fabio; ludovisi, alessandra; tonanzi, daniele; amati, marco; cherchi, simona; pozio, edoardo; gómez-morales, maria angeles title: delivery of sa35 and sa40 peptides in mice enhances humoral and cellular immune responses and confers protection against cryptosporidium parvum infection date: 2019-05-15 journal: parasit vectors doi: 10.1186/s13071-019-3486-8 sha: doc_id: 327568 cord_uid: 5vo4nmei background: cryptosporidium parvum is a major cause of diarrhea in children and ruminants at the earliest stages of life. maternal antibodies represent the main shield of neonate mammals for most of the infections. two recombinant antigens (sa35 and sa40), portions of two c. parvum proteins, were tested for their ability to induce immune responses in adult mice and for protection on neonate balb/c mice born from females immunised by mucosal delivery of both peptides. methods: adult balb/c mice were intraperitoneally immunised with sa35 and sa40, separately or mixed, and their immune response was characterised. furthermore, balb/c pregnant mice were immunised by mucosal delivery with an sa35/40 mix, before and during pregnancy. soon after birth, their offspring were infected with two doses (1 × 10(5) and 5 × 10(3)) of c. parvum oocysts and the parasitic burden was determined at 5 and 9 days post-infection. results: intraperitoneal immunisation with sa35 and sa40 induced specific igg and igg1 in serum, specific iga in the intestinal mucosa, increase of cd3+/cd4+ and cd30+ cells in splenocytes, which produced ifn-γ. neonates born from immunised mice and infected with 1 × 10(5) oocysts showed a significant reduction of oocysts and intestinal forms (23 and 42%, respectively). a reduction of all parasitic forms (96%; p < 0.05) was observed when neonates were infected with 5 × 10(3) oocysts. conclusions: sa35 and sa40 peptides induce specific humoral and cell-mediated immune responses to c. parvum in adult mice. moreover, mucosal administration of the sa35/40 mix in pregnant mice reduces c. parvum burden in their litters. the cryptosporidium genus includes 38 species that infect a wide range of vertebrates, including many mammals. humans are also susceptible to these parasites, and domesticated and wild ruminants, particularly livestock, represent important sources of zoonotic transmission [1] . in mammals, cryptosporidium spp. mainly affect the gut, causing gastrointestinal symptoms and diarrhoea. approximately 90% of human infections are caused by cryptosporidium parvum and cryptosporidium hominis, although over 20 species can infect humans [2] . in immunocompetent patients, cryptosporidiosis is a self-limiting infection of the small intestine that causes watery diarrhoea and can last up to ten days, whereas the infection can become a chronic and life-threatening disease in immunocompromised individuals such as aids subjects [3] . parasites & vectors *correspondence: fabio.tosini@iss.it; mariaangeles.gomezmorales@iss.it european union reference laboratory for parasites, istituto superiore di sanità, rome, italy transmission occurs via the faecal-oral route through the ingestion of oocysts contaminating food or water. oocysts are highly resistant to environmental conditions, chlorination and other sterilisation treatments, and contamination of water plants can therefore cause massive outbreaks [4] . it follows that the food and agriculture organisation and the world health organisation consider protozoa of the genus cryptosporidium to be one of the most significant food-borne parasites [5] . in livestock husbandry, c. parvum is a major cause of severe diarrhoea among neonate calves and lambs, resulting in substantial costs for farmers. a recent study on diarrhoea conducted on calves younger than one month reported that c. parvum is responsible, as the sole agent, for 37% of diarrhoeal infections and for 20% of coinfections with other intestinal pathogens [6] . in recent years, infections by c. parvum and c. hominis have emerged as significant causes of infantile diarrhoea. an extended case-control study, referred to as the global enteric multicenter study (gems) [7] , has shown a high prevalence of cryptosporidium spp. among children in developing countries, ranking these protozoa among the four pathogens responsible for the majority of diarrhoea cases in children younger than five years [8] . the prevalence of these parasites is higher in infants aged less than 11 months and, in this age range, cryptosporidium spp. is the second most common cause of death associated with diarrhoea, after rotavirus [9] . the susceptibility of neonates and children to cryptosporidium spp. is not completely understood. in the nursing period, the innate immune response of enterocytes, which is usually triggered by the stimulation of toll-like receptor 4 (tlr4), is almost completely inhibited so as to favour the establishment of commensal flora in the gut [10] . given that the immune response to cryptosporidium spp. begins with tlr4 stimulation and the consequent activation of the nf-kappa b pathway [11] , the temporary inhibition of the innate enterocyte response could promote the proliferation and dissemination of cryptosporidium spp. in the neonatal intestine. at present, nitazoxanide is the only drug approved for cryptosporidiosis by the us food and drug administration (us-fda), but this drug cannot be used in children younger than one year and is ineffective in immunodeficient patients [12] . a vaccine for cryptosporidiosis is not yet available, and immune protection of neonates is difficult to achieve because of their early-age immune status. however, various cryptosporidium spp. proteins, particularly those considered to be virulence factors, have been proposed as possible vaccine candidates [13, 14] . in this context, the passive immunisation of neonates might be an alternative to a conventional vaccine. indeed, passive immunity provided through hyperimmune bovine colostrum has been shown to establish an appreciable level of protection in human patients with aidsassociated cryptosporidiosis [15, 16] . more recently, the protective role of maternal anti-cryptosporidium antibodies has been demonstrated in two natural contexts. a survey of a child cohort in bangladesh reported that the presence of anti-cryptosporidium iga in breast milk protects neonates from cryptosporidiosis [17] . a second study, conducted in an endemic area of tanzania on a cohort of breastfeeding mothers and their infants (0 to 6 months), has shown that the infection rate increases with a reduction in maternal milk ingestion [18] . this study investigates the feasibility of an immunological preventive treatment to impede or reduce cryptosporidiosis in newborn mammals. as a preliminary step, two recombinant antigens, sa35 and sa40 [19] , were inoculated in adult mice to achieve the onset of a specific immune response. a group of female mice were then immunised by mucosal delivery of the same antigens prior to and during their pregnancies in parallel with a control group treated only with the adjuvant. the offspring of these mice were then infected with two different doses of c. parvum oocysts (1 × 10 5 and 5 × 10 3 ) and the parasite burden was measured by flow cytometry, microscopic count and a quantitative pcr assay. our results indicated that neonates born from immunised mothers had a reduced parasitic load compared to the neonates born from the control mice. cryptosporidium parvum oocysts (isolate code issc6) were collected from faeces of experimentally infected calves and purified by centrifuging with sucrose and percoll ® (sigma-aldrich, saint louis, mo, usa) densitygradients [20] . cryptosporidium parvum-crude extract (cce) was obtained as previously described [21] . heatlabile enterotoxin (lt) was obtained from chiron spa (siena, italy), and ketavet and xylazine solutions were provided by the animal care unit of the istituto superiore di sanità in rome, italy. a rabbit anti-c. parvum igg polyclonal antibody, which recognises all stages of c. parvum [22] , was used for ifa assays. the recombinant peptides sa35 (mw, 35 kda) and sa40 (mw, 40 kda) represent antigenic portions of two microneme proteins of c. parvum named cpa135 (cgd7_1730) and gp900 (cgd7_4020), respectively [23, 24] . protein id codes refer to sequences in the cryptodb database, release 37 [25] . the two peptides were purified as previously described [26] , dialysed in phosphate buffered saline (pbs) plus 50% glycerol and stored in aliquots at −20 °c. both peptides were used as a sole immunising agent or as a 1:1 mixture of the two antigens (referred to hereafter as the sa35/40 mix). eight-to ten-week-old balb/c female mice (charles river laboratories, milan, italy) were housed in the animal care unit of the istituto superiore di sanità in accordance with european directive 63/2010. the in vivo protocol was approved by the italian ministry of health (approval no. 8/2014-b, 15 january 2014). three groups of five female mice each were immunised intraperitoneally: one group with 5 μg of sa35, a second group with 5 μg of sa40, and a third group with the sa35/40 mix (5 μg plus 5 μg of sa35 and sa40, respectively). a control group (n = 5) was injected with an equal volume of pbs instead of antigens, according to the same immunisation schedule, which was as follows: day 0, peptides or pbs were administered in freund's complete adjuvant (sigma-aldrich); day 14, the same amount of peptides or pbs were inoculated using freund's incomplete adjuvant (sigma-aldrich); day 28, the last booster was provided using the same quantity of peptides or pbs without adjuvant. the mice were bled on days 0, 7, 14, 21, 28 and 35 following initial immunisation. individual sera were stored at −20 °c. faecal samples for an iga assay were collected weekly from each mouse separately, weighed and dissolved in 1.0 ml of pbs with 0.1% sodium azide per 100 mg of faecal material, and a cocktail of protease inhibitors (sigma-aldrich) was added at a ratio of 1:20 to each sample. the samples were then vortexed for 5-10 min and centrifuged to remove debris; supernatants were collected and stored at −70 °c. levels of igg, igg1 and iga in serum samples and iga in faecal samples specific to c. parvum were determined by elisa. levels of igg and iga specific to sa35 and sa40 peptides were also determined by elisa. microtiter plates (nunc-immuno plate polysorp ™ , sigma-aldrich, roskilde, denmark) were blocked using 200 μl per well of 1% w/v bsa in pbs, at room temperature (rt) for 90 min and coated with 1 µg/ml of recombinant peptides (sa35 or sa40) or 5 µg/ml of cce in a 50 mm carbonate/bicarbonate buffer with a ph of 9.6, and incubated at 37 °c for 90 min. serum samples were diluted in pbs with 0.5% w/v bsa and 0.05% v/v tween-20. supernatants of faecal samples were diluted as described above. plates were washed three times with 0.05%-tween-20 in pbs after incubation with sera, and washes were repeated after incubation using conjugated antibodies. dilutions of 1:50 for sera and 1:10 for faecal supernatants were established to be the optimal working conditions. these dilutions were then added in duplicate to wells and incubated overnight at 4 °c. as negative controls, the corresponding pre-immune faecal and serum samples from each mouse were used at the same dilution in elisa. bound antibodies were detected by incubation with 1:500 dilutions of biotin-conjugated rat monoclonal anti-mouse igg, igg1, and iga antibodies (bd pharmingen tm , san diego, ca, usa) at rt for 5 h followed by 2.5 mg/ml avidin-peroxidase (sigma-aldrich) for 30 min. the peroxidase substrate tmb (3,3′,5,5′-tetramethylbenzidine, kierkegaard and perry laboratories, gaithersburg, md, usa) was added to each well and the optical density was measured by a multiskan spectrum elisa reader (thermo scientific, vantaa, finland) at 450 nm. elisa was performed for each isotype using the same protocol. at the end of the immunisation schedule, the mice were sacrificed and their spleens were harvested under sterile conditions and disrupted using a syringe. the erythrocytes were lysed, and the splenocytes were then suspended in complete medium [rpmi-1640 containing 10% foetal bovine serum, 25 mm hepes, 2 mm l-glutamine, 100 u/ml penicillin, 100 µg/ml streptomycin, 1 mm sodium pyruvate, 5.5 × 10 −5 m 2-mercaptoethanol and 0.1 mm non-essential amino acids, all from hyclone laboratories (logan, ut, usa)]. the final cell concentration for proliferation analysis was 1 × 10 6 cells/ml and cells were settled in flat-bottom 96-well plates (costar corporation, cambridge, ma, usa). for cytokine and phenotypic analysis, cells were suspended at 2 × 10 6 cell/ ml in 5 ml tubes (becton dickinson, franklin lakes, nj, usa). cell cultures were stimulated with 5 μg/ml of cce, 1 μg/ml of sa35 or 2.5 μg/ml of sa40 and incubated in 5% co 2 at 37 °c for five days. cell cultures for negative controls were grown without any stimulants, whereas positive control cells were stimulated with 1.0 μg/ml of concanavalin a. lymphocyte proliferation was measured after 12 h of culture by 3 h-thymidine incorporation in the presence of 0.5 µci/well [ 3 h]-thymidine (amersham life science, buckinghamshire, uk) sampled in triplicate. proliferation was expressed as a stimulation index (si) (i.e. counts per minute of cce-stimulated cells divided by counts per minute of unstimulated cells). an si above 3.0 was considered positive. for the cytokine analysis, culture supernatants were harvested after five days of culture and stored at −70 °c until assayed. cytokine levels (ifn-γ, il-4, il12) were measured in culture supernatants by elisa. essentially, 96-well plates (nunc-immuno tm plate maxisorp tm surface, sigma-aldrich, roskilde, denmark) were coated with a solution of 1 μg/ml (in 0.1 m na 2 hpo 4 , ph 9.0) of monoclonal rat anti-mouse cytokine antibody (bd pharmingen). after overnight incubation at 4 °c, the plates were washed with pbs-t and blocked with 1% bsa (sigma-aldrich) in pbs for 2 h at 37 °c. after washing, serial dilutions of culture supernatants and standards, i.e. recombinant mouse cytokines, were added to the wells and incubated overnight at 4 °c. the plates were then washed and the appropriate biotin-conjugated rat antimouse cytokine antibody (bd pharmingen) was added to the well and incubated for 2 h at 37 °c. after washing and the addition of hrp-conjugated streptavidin (bd pharmingen) for 2 h at 37 °c, the tmb (kirkegaard & perry laboratories) substrate was added to the wells and absorbance was measured at 450 nm after 10-20 min. standard curves were generated for each cytokine using the corresponding murine recombinant standard. the minimum detection level for each cytokine by elisa was < 15 pg/ml for ifn-γ, < 4 pg/ml for il-4, and < 15 pg/ ml for il-12. to analyse the lymphocyte subsets after incubation with cce, the splenocyte suspension from each mouse was washed with pbs containing 2% bsa, and further re-suspended in 100 µl of the same buffer. the cells were then incubated with 5 µl of the following fluorescein isothiocyanate (fitc) or phycoerythrin (pe) conjugated monoclonal antibodies to murine leukocyte differentiation molecules: anti-cd3, anti-cd4, anti-cd8, anti-cd25, and cd30 (bd pharmingen). the cells were analysed for single and dual fluorescence assay in a fluorescence-activated cell-sorter (facscalibur; becton dickinson, franklin lakes, nj, usa) and data were acquired by cell-quest software (becton dickinson). the instrument was set to measure the fluorescence intensities of forward-angle light scatter (fsc), side-angle light scatter (ssc-h), fitc (fl1), pe (fl2) and fitc (fl1). cells incubated with fitc-and pe-conjugated mouse igg1/igg2a served as isotype control. the proportion (%) of cells expressing a given molecule was determined as the average of three replicas. two groups (experimental and control groups) of five mice each were anaesthetised with ketavet (100 mg/ml) and xylazine (20 mg/ml) at 50 mg/kg and 3 mg/kg, respectively. the mice from the experimental group were then immunised intranasally with 15-20 μl of pbs containing 1 μg of lt with 5 μg of sa35/40 mix, whereas the mice from the control group were inoculated intranasally with 15-20 μl of pbs containing 1 μg of lt. after seven days, the same immunisation protocol was repeated. then, bedding from cages housing male mice was transferred to the cages housing females for 48 h to induce oestrus. subsequently, one male and two females were kept in the same cage for 72 h and the females were examined daily for the presence of copulatory plugs. the day of plug detection was assumed to be day 0 of pregnancy [27] . immunisations were repeated at days 7, 14, and 21 of pregnancy ( fig. 1 ). blood samples were taken by tail bleeding on days 0, 7, 14, 21, 28, 35 and 230 after initial immunisation. individual sera were stored at −20 °c until analysis. faecal samples were collected at the same times and treated and stored as described above. specific igg and iga to c. parvum were measured in serum and faecal samples by elisa. proliferation assays and phenotypic analysis of spleen cells were performed as described above. to study the protection induced by mucosal immunisation, six litters of three-day-old balb/c mice (39 mice) were used. two litters, which were born from mothers immunised intranasally with sa35/40 with lt, were infected with 10 5 oocysts and then sacrificed five [28] and nine days post-infection (p.i.), respectively [29] . another litter born from an intranasally immunised mother was infected with 5 × 10 3 oocysts and sacrificed at nine days p.i. the remaining three litters were the respective control groups. all suckling mice were experimentally infected by the oral route using oocysts suspended in 20 µl of pbs with a 24-gauge gavage needle and kept with their mothers. suckling mice were anaesthetised before euthanasia and the whole intestine was collected. lymphocytic phenotypic analysis was performed on spleens from mice born from intranasally immunised mothers, infected with 10 5 oocysts and sacrificed five days later. the entire intestinal content was collected after washing of the intestinal tract with pbs. samples were vortexed for 30 s with a 1-min pause. the supernatant was centrifuged at 2500×g for 10 min at 4 °c. the pellet containing oocysts and free forms of the parasite was then suspended in 1 ml of pbs and vortexed for 30 s. each sample was divided in two aliquots: one aliquot was tested by an immunofluorescence assay, i.e. flow cytometry or indirect immunofluorescence assay (ifa), whereas the second aliquot was used to extract dna for qpcr (see below). using flow cytometry, the number of oocysts in the faecal sample was evaluated using an acquisition gate based on the forward-angle light scatter (fsc-h) characteristic and the fluorescence intensities of fitc (on fl1 detector) of a positive control [30, 31] . the positive control consisted of an oocyst suspension in pbs incubated with the anti-c. parvum igg. flow cytometry analysis was performed three times for each mouse and the number of oocysts was expressed as the mean ± sd for each mouse group. intracellular parasites were counted in the ileum homogenate from each mouse [28, 32] . homogenisation was carried out individually in 1 ml of pbs for 1 min at 20×g using a potter homogeniser and further diluted 1:5 in pbs. the suspension was then filtered using a 30-µm filcon syringe (beckton dickinson) and incubated with the anti-c. parvum igg and an anti-rabbit fitc antibody for 30 min at rt. intracellular parasites were counted using flow cytometry as above. the number of intracellular stages was expressed as the mean ± sd for each mouse group. when significant differences between the experimental and control groups were not found, the number of oocysts was also evaluated using ifa (see below). for ifa, the oocyst suspension was mixed with 20 µl of a solution of 0.5 µg/ml anti-c. parvum igg in pbs. the mixture was kept in the dark at 4 °c for 30 min and then centrifuged for ten min at 2500×g. the pellet was resuspended in 1 ml of pbs, and 20 µl of an anti-rabbit fitc antibody was added. the samples were kept in the dark at 4 °c for 30 min and then centrifuged at 2500×g for 10 min. the pellet was suspended in 1 ml of pbs and transferred to a polystyrene tube and kept at 4 °c. all samples were analysed on the day of oocyst collection. the number of oocysts was evaluated by fluorescence microscopy (zeiss axioplan 2; carl zeiss, jena, germany) at 400× magnification [31] . after washing (see above), the intestines from mice infected with 5 × 10 3 oocysts were fixed in 4% formaldehyde in pbs for 4 h for histological examination, which was performed using the following procedure. the ileum portion was cut and washed in tap water overnight, dehydrated in increasing concentrations of ethanol from 50 to 100%, placed in 100% xylol and finally included in paraffin. the paraffinated tissue was cut using a leica rm2125 rts microtome (leica biosystems, nussloch, germany) into microscopic slices of 4-6 μm, distributed on microscopic slides and dried overnight at 37 °c. the slices were then deparaffinated by immersion in 100% xylol for 5 min and re-hydrated with decreasing concentrations of ethanol from 100 to 50% and a final wash in distilled water for 2 min. antigen retrieval from fixed tissue was performed using the following "pressure cooking protocol": the microscopic slides were placed in a stainless steel slide rack in the boiling citrate buffer (10 mm sodium citrate, 0.05% tween 20, ph 6.0) in a pressure cooker heated by a hot plate; the lid was then closed for steaming at high pressure for 90 s, and the pan was then rapidly cooled using running tap water on the lid. immediately after the slides were prepared for indirect immunofluorescence assay (ifa), blocked using 2% foetal calf serum (fcs) in pbs for 1 h at room temperature, incubated for 1 h with anti-c. parvum rabbit serum [26] diluted 1:500 in 2% fcs in pbs, and washed three times (5-min wash) with pbs to remove unbound antibodies. secondary incubation was conducted for 1 h at room temperature using goat anti-rabbit antibody conjugate with fluorescein (fitc) (biorad, hercules, ca, usa) diluted 1:1000 in 2% fcs in pbs, and the slides were then washed as above and sealed using prolong antifade (thermo fisher scientific, waltham, ma, usa). microscopic inspection of the tissue slices was conducted using a fluorescence microscope (zeiss axioplan 2) at 1000× magnification and digital images were obtained using axiovision software (carl zeiss). for qpcr, aliquots of whole intestinal content from mice infected with 5 × 10 3 oocysts (see above) were centrifuged, suspended in 1 ml of 50% ethanol in pbs and stored at −20 °c until dna extraction. each aliquot was centrifuged, washed once with pbs and resuspended in 100 µl of lysis buffer (10 mm dtt, 1.8 mg/ml proteinase k in pbs), transferred to a 96-well plate and incubated for 1 h at 55 °c. for automated extraction, the plates were transferred to a biosprint 96 apparatus (qiagen, hilden, germany) using the protocol for blood samples and one-for-all vet kit reagents (qiagen), and dna was eluted in a final volume of 50 µl. dna was also extracted in the same way from referenced dilutions of purified oocysts in triplicate to obtain samples with 10 5 , 10 4 , 10 3 , 100, 10, 1 and a theoretical 0.1 oocysts, and used for a standard curve based on the median crossing threshold (ct) in qpcr experiments. dna samples from infected mice and referenced dilutions were analysed using real-time pcr assay (qpcr) based on the cowp gene [33] . this assay was tested for sensitivity using reference samples, obtaining positive curves with a single oocyst (ct = 36 in three out of three replicas) and up to a theoretical 0.1 oocysts (ct = 39 in one out of three replicas), and samples producing less than 40 ct were considered positive. cowp primers were synthesised by primm (milan, italy), and taqman probe and cowp probe labelled with 5′-hexachlorofluorescein (hex; λ em = 553 nm) were from tib molbiol (berlin, germany). each pcr reaction was completed in a final volume of 25 μl, comprising 12.5 μl of lightcycler 480 probes master mix containing fast-start taq dna polymerase, a proprietary buffer including deoxynucleotide triphosphates (dntps), mgcl 2 at a final concentration of 3.2 mm, 300 nm of each primer, 500 nm of taqman probe, and 5 µl (1/20 of whole intestinal content) as dna sample. all reactions were performed on a 96-well plate in a lightcycler 480 pcr system (roche diagnostics, mannheim, germany) in triplicate and three negative controls were included in each plate. the pcr conditions were: 10 min of incubation at 95 °c followed by 40 cycles of 95 °c for 15 s and 60 °c for 1 min. fluorescence data (three data real-time points) were collected at the end of each cycle. to compare each experimental group with the relevant control group, the mann-whitney u-test was used. a p-value < 0.05 was considered significant. the ip immunisation of adult balb/c mice to a single antigen (sa35 or sa40) or to a mixture of the two antigens (sa35/40 mix) induced specific anti-cryptosporidium igg in serum after day 14 following initial administration. later, the level of specific igg progressively increased over time, reaching the highest response 35 days after initial immunisation (sa35, u ( fig. 2c ). as expected, sera from mice immunised with sa35, sa40 or sa35/40 mix reacted with their respective homologous antigens by elisa (data not shown). specific anti-cryptosporidium iga were detected in faeces from day 7 after initial immunisation until the last sampling (day 35) with all the three antigen formulations (u (0) = 2, z = −2.50672, p = 0.0079 for sa35, sa40 and sa35/40 mix; fig. 2d ). no specific iga response was detected in faeces at day 0 in the control mice. cce induced proliferation in splenocytes from sa35, sa40 and sa35/40 mix in ip immunised mice. the highest responses to cce were obtained in splenocytes from mice immunised with sa35 alone or the sa35/40 mix. splenocytes from immunised and control mice responded to concanavalin a. single peptides also induced proliferation in splenocytes from mice immunised with homologous peptides (fig. 3) . cce induced ifn-γ production in the splenocytes from all groups of immunised mice. splenocytes from mice immunised with sa35 or sa40 produced higher levels of ifn-γ after stimulation with homologous peptides than mice immunised with the sa35/40 mix and stimulated with the same mix. il-12 production was observed in all mouse groups; the highest level of il-12 production was observed in splenocytes from mice immunised with sa35. minimal basal production of il-4 was detected in spleen cells from some groups of immunised mice (table 1) . induced cell populations in splenocytes were analysed at day 35 after initial immunisation. the percentages of cd3+ and cd4+ t cells were similar in mice immunised with sa35, sa40 and the sa35/40 mix and were significantly higher than in non-immunised control mice. however, the percentage of cd8+ t cells was similar among all groups, including the controls. moreover, the percentage of activated cd4+ lymphocytes co-expressing the high affinity interleukin-2 receptor (cd25) was decreased in all immunised groups, particularly in the sa35 immunised group (u (0) = 5, z =, 2.80224, p = 0.0022; fig. 4 ). the mucosal delivery of sa35/40 mix in female balb/c mice induced specific anti-cryptosporidium igg (mainly igg1) in serum 21 days after initial immunisation. the igg level increased over time from day 28 until day 35. high igg levels were detected until the last observation on day 230 (versus control group u (0) = 2, z = −2.50672, p = 0.01208 for igg and igg1; fig. 5a , b). a low specific iga level was detectable in serum until the end of the experiment (230 days after initial immunisation; fig. 5c ). in faeces, specific anti-cryptosporidium igg and iga began to increase on the day 28 following the first delivery and then over time (versus control group u (0) = 3, z = 2.64733, p = 0.0022 and u (0) = 5, z = −2.80224, p = 0.00512 for igg and iga, respectively). no specific igg or iga responses were detected in faeces at day 0 or in the control mice (fig. 5d, e) . cce and cona induced proliferation in splenocytes from mice immunised intranasally with the sa35/40 mix. splenocytes from control mice responded only to concanavalin a (fig. 6) . the cytometric analysis was performed in cce-stimulated splenocytes from both adult mice immunised intranasally and their litters. spleens were harvested from adult mice at day 236 following initial immunisation. the percentages of cd3+/cd4+ and cd30+ t cells were increased in adult mice treated with the sa35/40 mix. the same results were obtained with the cce-stimulated splenocytes from passively immunised litters infected with 10 5 oocysts (table 2) . when infected with 10 5 oocysts, passively immunised suckling mice showed similar levels of reduction at day 5 and day 9 following infection. in particular, a significant reduction of 23% of excreted oocysts (u (20) = 38, z = −2.76956, p = 0.0028) and 42% of intracellular forms (u (14) = 23, z = −2.43588, p = 0.00734) in the ileum was observed using flow cytometry (fig. 7a) . infection of neonates was also undertaken with a lower dose of oocysts in order to get closer to a natural infective dose using 5 × 10 3 oocysts. the histological examination of the ileums of infected mice did not show any evidence of the infection (i.e. parasitophorous vacuoles) in the analysed sections from neonates born from immunised females (fig. 8c, d) . by contrast, newborns from non-immunised females had numerous parasitophorous vacuoles disseminated along the epithelial lining of the intestinal lumen (fig. 8c, d) . indeed, a quantitative analysis of the entire intestinal content at day 9 p.i. showed a striking 96% reduction in parasites in newborns from immunised females (u (4) = 12, z = 2.65165, p = 0.00402), as evaluated by microscopic counting after ifa (fig. 7b) . to confirm the overall reduction in parasitic forms in passively immunised newborn mice infected with the lower infective dose (i.e. 5 × 10 3 oocysts), the intestinal contents of these newborns were evaluated using a realtime qpcr assay. as shown in fig. 9 , ct values in faecal samples from suckling mice born from immunised mothers ranged between 34 and 40, i.e. equivalent to 5-0.1 oocysts. four out of 6 samples showed a ct value higher than 37, thus with less than one oocyst equivalent in their content. ct values for the control mice ranged between 27 and 33, i.e. equivalent to 10-100 oocysts. therefore, qpcr analysis confirmed that the parasitic load in the intestinal contents of newborns from immunised mothers was extremely low when newborns were infected with the lower dose of 4.5 × 10 3 oocysts. control of neonatal cryptosporidiosis is a challenging task because of the lack of appropriate drugs and the difficulties in providing effective immunisation in the early phase of life. here, we explored the protective effect of anti-c. parvum maternal antibodies transferred to newborns through the placenta during gestation and through the colostrum in the first days of life. to this end, two recombinant peptides (sa35 and sa40) were first immunologically characterised by ip injection in adult mice to evaluate their potential as immunogens. both peptides, separately and in a mixed formulation, induced specific igg and iga responses. sa35 and sa40 peptides have been previously described as immunodominant antigens involved in the maintenance of t-cell response in healthy c. parvum-sensitised individuals [20] . it is important to note that the amino acid sequences of sa35 and sa40 peptides do not show similarities with mammalian proteins, but, by contrast, are also highly conserved in other cryptosporidium species and particularly in other human pathogens (data not shown). therefore, it is most likely that antibodies for sa35 and sa40 also react with the strictly related proteins of c. hominis and c. ubiquitum. in this study, it has been demonstrated that ip immunisation with these recombinant peptides is able to support an effective cell-mediated immunity in response to specific stimuli: cce-, sa35-and sa40-induced specific proliferation in splenocytes from all immunised adult mice. however, maximum proliferative responses to cce were obtained in splenocytes from mice following ip immunisation with sa35 or the sa35/40 mix (fig. 3) . the induced cell populations showed a higher percentage of cells expressing cd3+ and cd4+. moreover, splenocytes from mice immunised with sa35 showed a significant reduction in the percentage of regulatory cells (cd4+/cd25+) compared to the control group, whereas the others did not (fig. 4) . overall, the three antigen formulations (sa35, sa40 or sa35/40 mix) induced ifnγ, il-12 and, to a lesser extent, il-4 in immunised mice (table 1) . what is noteworthy is that stimulation with the homologous antigen resulted in the highest level of production of these cytokines even when the splenocytes also responded to stimulation with cce (table 1) . experimental studies and clinical cases have demonstrated that activation of cd4+ lymphocytes and production of ifn-γ are crucial for parasite clearance [34] [35] [36] [37] . indeed, ifn-γ production is one of the essential functions of cd4+ cells in response to cryptosporidium antigens [38, 39] . with regard to humoral response, all three antigenic formulations (sa35, sa40 or sa35/40 mix) induced specific igg, igg1 and secretory iga (fig. 2) . the production of these transferable antibodies is a fundamental requirement in conferring protective immunity for neonates. therefore, the antigenic sa35/40 mix was used for mucosal immunisation of female mice in order to evaluate the protective effect on their offspring. the notes: adult female balb/c mice were mucosally immunised with sa35/40 mix. cells were stimulated with c. parvum crude extract and were gated for 90-95% cd45 + . analysis of splenocytes from adult mice was carried out on the last day of the experiments. the number of mice is shown in fig. 1 . newborn mice born from intranasally immunised mothers were infected with 10 5 oocysts and sacrificed five days later the comparison between c. parvum-infected littermates born from immunised mothers and their respective controls, i.e. c. parvum-infected littermates born from mothers immunised only using a mucosal adjuvant, showed that maternal protection is highly effective with the lower infective dose (5 × 10 3 ), since there was a 96% reduction in parasitic burden (fig. 7) . in adult females, mucosal immunisation with the sa35/40 mix leads to a long-lasting (33 weeks) humoral response at systemic and intestinal level (fig. 5) . moreover, splenocytes from balb/c mothers specifically proliferated in response to cce and the resulting population showed an increase in the percentages of cd3+/cd4+ and cd30+ cells ( table 2) . the experimental infection of neonate mice born from immunised mothers showed that when neonates were infected with a massive dose of oocysts (1 × 10 5 ), the protective effect on the progeny is measurable but ineffective (−23% of excreted oocysts). conversely, a drastic reduction (96%) in oocyst emission was observed when neonates were infected with a lower dose (5 × 10 3 oocysts), which still represents a huge amount given the small weight (about 1 g) of newborn mice (fig. 7) ; in fact, this dose is comparable to a dose of 30 × 10 7 oocysts in a 60 kg human. these oocyst numbers were tested in the first instance to allow a quantitative analysis of excreted oocysts and of all parasitic forms using flow cytometry, although the 5 × 10 3 dose yielded parasite amounts below the sensitivity threshold for the instrument. to overcome this technical limitation, ifa/microscopic counting and qpcr were used to evaluate the minimal quantity of parasites in infected mice. moreover, dna samples for qpcr were prepared using an ad-hoc automated method in order to reduce handling errors. cell-mediated immunity showed unexpected signs of activity in splenocytes harvested from litters from immunised mothers, as there was an increase in the percentage of cd3+-cd4+ cells as well as cd30+ cells (table 2) . similarly, t-cell responses were identified after maternal vaccination in newborn mice of a plasmodium yoelii model, although there was no parallel increase in antibody response [40] . recent studies indicate that during pregnancy, maternal molecules such as inflammatory cytokines, as well as microbial products, are transferred in utero to the foetus and this influences the foetal immune system [41] . thus, neonatal hosts may compensate for an insufficiency in adaptive immune responses by having a heightened capacity to mount certain types of innate immune responses [42] . attempts at passive immunisation through administration of hyperimmune colostrum have been performed in humans and in other mammals. indeed, hyperimmune bovine colostrum was given to aids patients with prolonged cryptosporidiosis, resulting in a reduction in diarrhoea and an improvement in symptoms [15, 16] . in livestock, passive immunisation with recombinant antigens has shown a certain efficacy in protecting passively immunised calves and kids [43, 44] . more recently, heterologous protection was also successfully tested through administration of hyperimmune ovine colostrum in neonate mice [45] . unlike previous studies, this study combined the use of a mixture of two defined antigens extensively characterised as immunogens. a further peculiarity of this study is the mucosal immunisation of females to confer protection for their progeny. maternal immunisation protocols for other pathogens have been applied successfully to prevent neonatal infectious diseases in various contexts. with regard to livestock, a vaccine for rotavirus, coronavirus and escherichia coli (rotavec-corona ® , msd) is commonly used to immunise heifers and preparturient cows. this treatment induces a higher titre of specific antibodies for these pathogens in colostrum and milk [46] and has significant efficacy in reducing diarrhoea in neonate calves [47] . in humans, maternal immunisation with tetanus toxoid has fig. 9 quantification of cowp gene dna copies by qpcr in the intestinal content of neonate mice infected with 5 × 10 3 cryptosporidium parvum oocysts. values are expressed as the mean of cycles for crossing thresholds (ct), and each sample was tested in triplicate. standard curve (black dots) was obtained from dna extracted from the following oocysts dilutions: 1 × 10 5 (1), 1 × 10 4 (2), 1 × 10 3 (3), 100 (4), 10 (5), 1 (6) and 0.1 (7) . blue dots represent ct values from control mice. red dots represent ct values from immunised mice. values from control mice are between 33 and 29 cycles, equivalent to dna amounts from 6 to 100 oocysts, whereas values from immunised mice are between 39 to 35 cycles, equivalent to dna amounts from 0.1 to 5 oocysts been the first example of effective prevention for neonates [48] and it has been extensively applied as part of the who maternal and neonatal tetanus elimination (mnte) program. successful immunisations have been also achieved with influenza virus [49] and with the acellular pertussis antigen vaccine that protects infants from clinical pertussis [50] . therefore, maternal immunisation can be an effective strategy to limit neonatal cryptosporidiosis before the development of a mature immune system. the sa35 and sa40 peptides enhance humoral and cellmediated immune responses to c. parvum in adult mice. mucosal immunisation of pregnant mice with a mixture of sa35 and sa40 antigens (sa35/40 mix) resulted in a remarkable reduction in parasite load in the gut of their infected offspring. overall, this result represents the proof of concept that maternal immunisation can effectively prevent neonatal cryptosporidiosis. genetic diversity and population structure of cryptosporidium molecular epidemiologic tools for waterborne pathogens cryptosporidium spp. and giardia duodenalis a review of the global burden, novel diagnostics, therapeutics, and vaccine targets for cryptosporidium a massive outbreak in milwaukee of cryptosporidium infection transmitted through the public water supply world health organization. who estimates of the global burden of foodborne diseases bovine cryptosporidiosis: impact, host-parasite interaction and control strategies the global enteric multicenter study (gems): impetus, rationale, and genesis burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the global enteric multicenter study, gems): a prospective, case-control study use of quantitative molecular diagnostic 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prospective cohort study identification and characterisation of three antigenic proteins from cryptosporidium parvum sporozoites using a dna library expressing poly-histidine tagged peptides convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over 100m website views per year • at bmc experimental cryptosporidiosis in hamsters cryptosporidium parvum-specific cd4 th1 cells from sensitized donors responding to both fractionated and recombinant antigenic proteins characterization and immunolocalization of a cryptosporidium protein containing repeated amino acid motifs a new modular protein of cryptosporidium parvum, with ricin b and lccl domains, expressed in the sporozoite invasive stage a novel multi-domain mucin-like glycoprotein of 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infants publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we are very grateful to dr silvia vendetti for her helpful suggestions on the mucosal delivery mouse model. we would like to thank simone m. cacciò for the critical reading of the manuscript. all data presented in this manuscript are available at the istituto superiore di sanità. not applicable. the authors declare that they have no competing interests.received: 28 november 2018 accepted: 6 may 2019 key: cord-340125-il35gs97 authors: jayapal, manikandan; tay, hwee kee; reghunathan, renji; zhi, liang; chow, kah kiong; rauff, mary; melendez, alirio j title: genome-wide gene expression profiling of human mast cells stimulated by ige or fcεri-aggregation reveals a complex network of genes involved in inflammatory responses date: 2006-08-16 journal: bmc genomics doi: 10.1186/1471-2164-7-210 sha: doc_id: 340125 cord_uid: il35gs97 background: mast cells are well established effectors of ige-triggered allergic reactions and immune responses to parasitic infections. recent studies indicate that mast cells may play roles in adaptive and innate immunity, suggesting an innovative view of the regulation of immune responses. here, we profiled the transcriptome of human mast cells sensitized with ige alone, or stimulated by fcεri aggregation. results: our data show that among 8,793 genes examined, 559 genes are differentially regulated in stimulated mast cells when compared with resting/unstimulated mast cells. the major functional categories of upregulated genes include cytokines, chemokines, and other genes involved in innate and adaptive immune-responses. we observed the increased expression of over 63 gene-transcripts following ige-sensitization alone. our data was validated using real-time-pcr; elisa and western blot. we confirmed that ige alone does not trigger mast cell-immediate responses, such as calcium signals, degranulation or protein-phosphorylation. conclusion: this report represents a substantial advance in our understanding of the genome wide effects triggered by "passive sensitization" or active stimulation of human mast cells, supporting mast cells' potential involvement in a wide range of inflammatory responses. mast cells are best known for their role in immunoglobulin e (ige)-dependent allergic responses as one of the most powerful reactions of the immune system [1] . recent studies suggest that mast cells may also be involved in innate and adaptive immunity by producing high levels of chemokines and cytokines [1] [2] [3] . mast cells are derived from haematopoietic progenitor cells that enter nearly all vascularized tissues, where they complete their maturation and, in some cases, can migrate into epithelia [1] [2] [3] [4] . after appropriate activation, such as the aggregation of the high affinity ige receptor (fcεri), mast cells can produce a range of pro-inflammatory mediators, including cytokines and chemokines [5, 6] . crosslinking of fcεri-bound ige with multivalent antigen initiates the activation of mast cells by promoting the aggregation of fcεri [7, 8] . this fcεri-dependent cell activation process has three major outcomes: degranulation, secretion of preformed mediators stored in cytoplasmic granules -these granules contain vasoactive amines, neutral proteases, proteoglycans and some cytokines and growth factors; the de novo synthesis of pro-inflammatory lipid mediators (such as eicosanoids); and the synthesis and secretion of cytokines and chemokines [4] [5] [6] [7] [8] . mast cells are regarded as key effector cells in ige-associated immediate hypersensitivity reactions and allergic conditions, as well as in certain immune responses to parasites [9] . because the ige dependent release of proinflammatory mediators can begin within minutes of antigen challenge, the crucial role of mast cells in acute allergic reactions, such as anaphylaxis and acute attacks of atopic asthma, is now widely accepted [4, 5, 7, 8] . on the other hand, the role of mast cells in chronic inflammation and other long-term tissue changes that are observed in some ige associated diseases, including asthma, still debated [10] . however, in recent studies it is suggested that mast cells can markedly enhance antigen-dependent airway hyper reactivity, airway eosinophil infiltration, and the increased number of proliferating cells in the airway epithelium [11] [12] [13] [14] . these reports and other lines of evidence, suggest that a key role of mast cells in igeassociated immune responses is to amplify both acute and long-term local tissue responses to relatively weak biological signals [5, 9, 11, 12] . moreover, through their ability to release immunoregulatory cytokines, and perhaps through other mechanisms, mast cells might also influence the development, strength and persistence of thelper 2 cell-associated immunity. however, such immunoregulatory functions of mast cells have not been fully characterized. mast cells have also been proposed to play a role in mediating bacterial clearance by releasing cytokines, and by ingesting and killing opsonized bacteria, suggesting that mast cells have a physiological role in modulating host defenses against infectious agents [13] . mast cell products, such as tryptase and histamine, can influence the immune-response by recruiting neutrophils or by activating other immune-effector cells [14] . these previous findings show that mast cells, apart from being principal players in allergies, appear have effects in the initiation and regulation of innate immunity. there is increasing evidence demonstrating that the innate and adaptive immune systems cooperate through shared signaling mechanisms, and very recently it was shown that mast cells may regulate the recruitment of t-cells to lymph nodes [15] . several groups have carried out limited gene expression profiles of mast cells, without any stimuli [16] [17] [18] ; stimulated by cytokines [19] ; triggered by antigens and in response to the inhibition of calcium-atpase [20] ; or stimulated by tlr4 [21] ; or focused on the cytokine/ chemokine profile triggered by fcεri aggregation [21] [22] [23] . a recent study has shown that ige alone can induce the release of the interlukin-8 (il-8) and of the monocyte chemoattractant protein 1 (mcp-1) [24] . however, many aspects of the genetic responses triggered by ige-antigen on human mast cells are still unclear, such as the overall genetic responses to ige sensitization alone, or widegenome expression profile triggered by fcεri aggregation. thus, we decided to analyze the differential gene expression profile of 8,793 fully-annotated genes, after ige sensitization, and following fcεri aggregation. the data we present here show that, over 559 genes were differentially regulated in the stimulated cells, when compared to unstimulated cells (basal). a substantial number of genes were regulated by ige sensitization alone; and following fcεri aggregation, a wide range of genes were triggered, including genes for cytokines, chemokines, transcription factors, anti-apoptosis, and several genes involved in innate and acquired immunity. some of the most prominent findings are the upregulation of proinflammatory cytokines and chemokines, involved in innate and adaptive immunity. we also observed the upregulation of several receptors involved in innate immune reactions. we confirmed that ige alone does not trigger mast cell-immediate responses, such as calcium signals, degranulation or protein-tyrosine phosphorylation, whereas fcεri aggregation did indeed trigger these immediate responses. thus, these results represent a substantial advance in our understanding of the genomewide effects triggered by the physiological "passive sensitization" or "active stimulation" of human mast cells, and suggest that mast cell activation may not only play a pivotal role in allergic responses, but may influence the regulation of other inflammatory immune responses. perhaps the most important factor for mast cell development, survival and proliferation is stem-cell factor (scf or c-kit ligand) [4] , although several lines of evidence also indicate il-3 as a crucial factor for mast cell development, survival and proliferation [4] . human mast cells are very difficult to isolate in numbers that would allow for wide-genome expression profiling studies: for that reason we isolated cd34+ progenitor hematopoietic cells from umbilical cord blood and differentiated them to mast cells by culturing with the cytokines scf and il-3. after culturing cd34 + progenitor cells with cytokines for 5-6 weeks, cells were characterized as mast cells by flow cytometry as positive for the specific mast cell marker intracellular chymase; positive c-kit/cd117 + and fcεri + , and by toluidine blue staining ( figure 1 ). purity was estimated at >97%. our aim is to study the global gene expression pattern induced by ige sensitization and fcεri aggregation on human mast cells. gene expression analysis using cdna or oligo-dna microarrays has proven to be a sensitive method to develop and refine the molecular determinants of several human disorders, including cancer and autoimmune diseases. we analyzed the expression pattern of 8,793 transcripts from the stimulated mast cells, and compared the expression patterns with control/unstimulated samples. the complete gene expression data of our experiments, representing 8,793 probeid is available at the ncbis gene expression omnibus [25] , and is accessible through geo series accession number gse1933 (25) . the microarray analysis revealed that 760 genes (~8.6%) were differentially expressed between resting and stimulated mast cells with statistical significance (p ≤ 0.05), which were hierarchically clustered ( figure 2 ). because of the relatively large number of genes that were differentially regulated, we focused on genes that were upregulated by at least a 2-fold in any time point of mast cell stimulation. of the 760 genes, 58 genes were initially upregulated (at least 2-fold) by ige-sensitization alone (table. 1), and a total of 115 genes were overexpressed (by 2-fold or more in at least one time point), during the time course of mast cell activation by ige-alone or after crosslinking fcεri (table. 1 ). in order to examine the global characteristics of these genes, we used the gene ontology consortium database for biological processes [26] . using this database we analyzed the genes that were upregulated by at least 2fold, thus, allowing us to separate the 115 genes, into the following functional families: (a) cytokines and cytokine receptors; (b) chemokines and chemokine receptors; (c) other immunoregulatory genes; (d) cell proliferation and anti-apoptosis; (e) adhesion and cytoskeleton remodeling; (f) transcription factors and regulators of transcription; (g) signal transduction; (h) genes involved in other cellular functions "others" (table 1 & figure 3 ) our study revealed that substantial changes in gene expression in response to monomeric-ige sensitization alone. in order to ensure that the human ige (ige, cat: 30-ai05, lot number a01071004, fitzgerald, concord, ma), used in this study was indeed monomeric ige, prior to each experiment; we run a sample of the ige through nondenaturing polyachrylamide-gel-electrophosys (nonreducing-page). no aggregates were observed at any time (data not shown). we focused the analysis of our data on genes that were upregulated by at least 2-fold, over basal levels, and identified 58 genes that were increased by ige sensitization alone (table. 1 ). we then separated them into different categories, based on their biological function (determined by public databases). among the most prominent findings was the upregulation of genes coding for the cytokines il-1β (3.3 fold), il-6 (2.7 fold), and csf1 (1.6 fold); genes coding for the chemokines il-8 (cxcl8) (2.1 fold), mip1β (ccl4) (3.5 fold), mcp3 (ccl7) (2.1 fold), groα (cxcl1) (2.3 fold) and groγ (cxcl3) (1.6 fold), were also upregulated. other than these, several genes coding for other receptors involved in immune-responses; immunoregulatory genes; adhesion and/or cytoskeleton remodeling; regulators of apoptosis; signal transduction; transcription factors; were also upregulated by monomeric ige (table. 1 ). thus, these results suggest that "passive" sensitization of mast cells, with monomeric ige, may not only prime mast cells to be ready for the challenge to come, but that mast cells may also have the potential to purity of mast cells an interesting finding in our study was upregulation of genes coding for the pyrogenic and proinflammatory cytokines il-1β, il-6 ( table. 1a). one of the key roles for il-1β is to trigger the upregulation of key proinflammatory proteins [27] . we also observed the upregulation of ptx3 (pentaxin-related gene) ( table. 1c): ptx3 is a protein involved in inflammation that is rapidly upregulated by il-1β. other cytokine genes that were upregulated are csf1 (a growth promoting cytokine), and the cytokine receptors il1r1, il27ra, tnfrsf9, tnfrsf12 and il1rn (table. 1a). we also found that the level of expression of genes coding for the chemokines il-8 (cxcl8), ccl7 (mcp3), ccl4 (mip1β) and cxcl1 (groα) was increased (table 1b) . il-8 plays a major role in inflammatory responses mainly due to its ability to recruit and activate neutrophils. ccl7, ccl4 and cxcl1 are known to recruit monocytes, nk, basophils, dendritic cells and th2 cells. moreover, our data also show that several other chemokine genes were upregulated during mast cell stimulation: these include ccl5 (rantes), cxcl3 (groγ), and the chemokine receptor ccrl2 (table. 1b). these data suggest a key role for fcεri in triggering an a wide range of inflammatory responses, as the over-expression of cytokines and chemokines is a prerequisite to triggering inflammation, including vascular permeability, and leukocyte and lymphocyte recruitment, differentiation and activation. several genes involved in innate and adaptive immuneresponse were upregulated at least by 2-fold during mast cell stimulation: these include the toll like receptor 2 (tlr2), as well as several genes of the tnfα signaling pathways, including tnfaip6 (table. 1c). these findings support previous reports of the role of mast cells in innate immunity and antibacterial activity [21] . transcripts of genes lif, cd69, and cd83 were also upregulated, as were the major histocompatibility complex genes (hla-dqb1, and hke2) and inhibitory receptor of igg, fcgr2b (table. 1c). the generation of transcripts for such genes suggests that mast cells can acquire characteristics typical of cells involved in innate and adaptive immune responses. several genes involved in cell proliferation and survival, such as pdgfa (platelet-derived growth factor alpha polypeptide), pdgfb (platelet-derived growth factor beta polypeptide), pbef1 (pre-b-cell colony-enhancing factor), tieg (tgfb inducible early growth response), and insig1 (insulin induced gene 1) were upregulated, as well as several anti-apoptosis genes including tnfaip3 changes in gene expression in human mast cells following ige sensitization and fcεri aggregation figure 2 changes in gene expression in human mast cells following ige sensitization and fcεri aggregation. changes in expression over control of human cord blood derived mast cells that were activated by ige sensitization and fcεri crosslinking for different time points (2 hr, 6 hr and 12 hr). labelled crna from cell of each time point were hybridized to human genome focus array and signals were scanned after fluidics. the data was analyzed as described in material and methods and analysis revealed differential expression of 760 genes between resting and stimulated mast cells with statistical significance (p ≤ 0.05). agglomerative average-linkage hierarchical clustering of the five different experimental conditions was obtained for selected groups of genes using genespring 7.0. each colored box represents the normalized expression level of a given gene in a particular experimental condition and is colored according to the color bar. the data represent average of four independent experiments. sensitised 2h 6h 12h fold change color bar table 1 (table. 1d ). this supports the fact that, in the initial stages of mast cell activation, several mediators produced are mainly for cell proliferation and survival [28] . thus, fcεri aggregation may enhance mast cell proliferation and survival, perhaps owing to the autocrine effects of the cytokines, growth factors, and antiapoptotic proteins, triggered by fcεri aggregation. nm_002984.1 ccl4 chemokine (c-c motif) ligand 4 3.5 4.7 1.6 -3.0 nm_001511.1 cxcl1 chemokine (c-x-c motif) ligand 1 2.3 2.5 1.1 -2.9 nm_006273.2 ccl7 chemokine (c-c motif) ligand 7 2.1 3.6 5.3 2.2 nm_002090.1 cxcl3 chemokine (c-x-c motif) ligand 3 1.6 2.5 1.6 -1.8 nm_002985.1 ccl5 chemokine (c-c motif) ligand 5 1.6 1.8 2.1 1.8 af015524 ccrl2 chemokine (c-c motif) receptoranother functional characteristic of immune-cell activation is the coordinated expression of genes involved in cell adhesion and cytoskeleton remodeling (table. 1e). of particular importance are the genes coding for proteins involved in cell motility, cytokinesis, endocytosis and exocytosis. we found at least 2-fold upregulation of various genes coding for proteins involved in cell adhesion, such as flrt2 (fibronectin leucine rich transmembrane protein 2), kal1 (kallmann syndrome 1 sequence), cd151 (cd151 antigen), and alcam (activated leukocyte cell adhesion molecule); as well as for several gene-transcripts involved in cytoskeleton remodeling, including rasal1 (ras protein activator like 1), arhe (ras homolog gene family, member e), arf6 (adp-ribosylation factor 6), and flnb (filamin b, β-actin binding protein 278) (table. 1e). the expression of genes involved in cell adhesion and cytoskeleton remodeling is an essential step in immunecell activation. resting immune cells have cytoskeletal structures that sequester antigen, chemokine, and adhesion receptors in accessible regions of the plasma membrane. upon activation, reorganization of the actin cytoskeleton leads to the formation of supramolecular activation clusters, bringing receptors and costimulatory molecules together, as well as important adaptor proteins that promote the sustained activation of the cell. stimulation of immune-effector cells through their antigen receptors initiates cell cycle entry and changes the gene expression pattern, a response generally referred to as "activation". we found that the genes for several transcription factors were upregulated during ige-sensitization and fcεri aggregation, including the transcription factors most active during an immune response, such as nfκb and nfat (table. 1f). we observed an increase in the transcripts for the nuclear factor of kappa light polypeptide genes 1, alpha, and epsilon (nfκb 1, 1a, and 1e), and pie chart showing the percentage distribution of the upregulated genes figure 3 pie chart showing the percentage distribution of the upregulated genes. a. percentage distribution of the total amount of genes upregulated. all the genes, observed to be upregulated at least 2-fold at any given time point, were distributed, according to their biological function described in b the nuclear factor of activated t cells, nfatc1 (table. 1f ). other transcription factors upregulated were the oncogenes myc (v-myc myelo-cytomatosis viral oncogene homolog), and maff (v-maf musculo-aponeurotic fibrosarcoma oncogene homolog f) ( table. 1f). interestingly, the activities of nfκb and nfat together are responsible for the transcription of many proinflammatory genes, including several genes coding for cytokines and chemokines [29, 30] . during mast cell activation, many signaling molecules are engaged in diverse responses, ranging from calcium release from internal stores, degranulation, the generation of lipid-derived proinflammatory mediators and the production of cytokines and chemokines. in our study we observed that a substantial number of genes coding for intracellular signaling proteins were upregulated, by at least 2-fold ( (table. 1g ). moreover, we show here the upregulation of genes that code for oxidized low density lipoprotein receptor (olr1), and for the low density lipoprotein receptor (ldlr) ( table. 1g), indicating a potential role for mast cells in cholesterol homeostasis. of particular interest is the upregulation of the gene coding for the lipid kinase, sphingosine kinase 1 (sphk1) ( table. 1g). we and others have previously reported that sphk1 plays a critical role in the intracellular signaling pathways triggered by fcεri in mast cells [28, 31] , and coordinates several physiological responses triggered by activated mast cells. we confirmed our microarrays findings by real time pcr on selected genes such as il-1β il6, il-8, mcp3, rantes and sphk1, utilizing an aliquot of the same rna sample that was used for the microarray experiments ( figure 4) . the results showed that the messenger rna for the selected genes follows a similar pattern of expression to that observed with the oligo-dna microarray experiment, thus confirming the results and the quality of the data obtained with the high-density microarrays. mast cell activation also results in the sustained de novo production of pro-inflammatory cytokines and chemokines both of which may contribute to the inflammation and pathology underlying allergic disease as well as in innate and acquired immunity. the amounts of these cytokines were measured by elisa and depicted in figure 5a . fcεri-triggered generation of il-1β, il-6, il-8, ccl7 (mcp3) and ccl5 (rantes), whereas ige sensitization alone triggered smaller amounts of il-1β and mcp3, high amount of il-8, and very less amounts of il-6 and rantes ( figure 5a ). we verified the differential expression of sphk1 by western blot analysis ( figure 5b ). its levels of expression were found to be consistent with that of microarray as well as real time pcr results. thus, these data together with real time pcr data validate microarray results. mast cell activation via fcεri triggers exocytosis of granules containing pre-formed inflammatory mediators in a tyrosine kinase and calcium dependent manner. here we studied, whether monomeric-ige alone, may activate fcεri intracellular signaling pathways, leading to physiological responses of mast cells, by analyzing the overall tyrosine phosphorylation; fluctuations in cytosolic ca 2+ concentration; and degranulation by measuring β-hexosaminidase release (fig. 6a,b &6c) . we show here that in our experimental setting monomeric-ige alone is not able to trigger any changes on the overall protein-tyrosine phosphorylation patterns compared with resting cells; nor was it able to trigger calcium release from internal stores; neither degranulation. on the other hand fcεri-aggregation did indeed trigger all these responses (fig. 6a,b &6c ). binding of ige to fcεri enhances the cell surface expression of fcεri, as a result, its ability to promote the stabilization/accumulation of fcεri on the mast cell surface in the presence of continued basal levels of protein synthesis [32, 33] . it is possible that most of the enhanced ige dependent functions that are observed after antigen or anti-ige-induced fcεri aggregation, in cells that have been sensitized with ige, are a consequence of the higher level of fcεri expression. however, a controversial question remains as to whether monomeric ige can also have more direct effects on mast cell functions. many studies over the years have shown no evidence that the binding of monomeric ige can induce detectable signaling or production of mediators by mast cells. however, some groups have reported that monomeric ige can enhance mast cell survival and trigger cytokine production [24, 28, 33] . in concon sen 2h 6h 12h trast, a recent study by matsuda et al [24] , fail to find any ability of ige to enhance mast cell survival on withdrawal of scf. interestingly, the study by matsuda et al, also showed that ige sensitization alone can induce the upregulation of cytokines and chemokines at the protein level, namely il-8 and mcp1 [24] . in agreement to this, we show that il-8 is induced by ige alone at the mrna as well as at the protein level (table 1, and figures 4 and 5) , in contrast we could not detect any significant increment on mcp-1 levels, we can speculate that this difference could be due to the different amounts ige used (1 µg/ml vs 2.5 µg/ml). however, we also show the upregulation of various chemokines, including the mcp-1-related protein mcp-3, which was also upregulated by ige alone (table 1, and figures 4 and 5) . in our present study, we found that several genes related to proliferation were upregulated by ige alone (table 1) ; however, whether these genes, if fully transcribed, may be able to trigger mast cell proliferation in the absence of scf is not known. the observation that ige alone can induce the upregulation of a substantial number of genes encoding for cytokines and chemokines, has profound implications in our understanding of the role of mast cell in inflammation. cytokines and chemokines share many activities, including the ability to induce fever and shock syndrome in animal models [27] . cytokine and chemokine production are universal components of a wide range of disease states, including immune-complex-mediated conditions such as nephritis [34] , arthritis [35] , and acute graft rejec[36] . these data suggest a potential role for mast cells in triggering, or at least contributing to, strong inflammatory responses. in is also interesting to mention that, several genes encoding for transcription factors were upregulated by monomeric-ige and fcεri aggregation. perhaps the most prominent of these transcription factors, is nfκb. nfκb represents a family of related proteins which dimerize to form transactivating complexes [35] . nfκb dimmers are sequestered in the cytoplasm by interaction with inhibitory proteins (the iκbs). various stimuli activate kinase signaling cascades that result in the phosphorylation and degradation of iκb, thereby releasing nfκb to translocate to the nucleus, where it activates transcription of target genes. many studies have emphasized the role of this transcription factor in regulating genes at critical points in immune-cell development and activation [37] . many nfκb targets are antiapoptotic [38] , which may explain the importance of the nfκb pathway in oncogenesis and resistance to chemotherapy [39, 40] . during an immuneresponse several genes are triggered by the nfκb, these include genes coding for the various proinflammatory molecules, such as mips, il-1β, il-6, il-8, tnfα, groα and other cytokines, chemokines and cell adhesion molecules icam, vcam and selectins [41] . as immune cells progress through development and respond to antigenic challenge, they trigger signal transduction pathways that alter their cellular functions and the activity of transcription factors, changing their effector functions and their gene expression profiles. during mast cell activation, many signaling molecules are engaged in diverse responses, ranging from calcium release from internal stores, degranulation, the generation of lipidderived proinflammatory mediators and the production of cytokines and chemokines. in our study we observed that a substantial number of genes coding for intracellular signaling proteins were upregulated, by at least 2-fold, during mast cell stimulation (table. 1g). interestingly, we observed a substantial upregulation of the mrna for sphingosine kinase 1 (sphk1), even by ige alone, this upregulation was also confirmed at the protein level. sphingosine kinases are novel enzymes that phosphorylate sphingosine (a membrane lipid), to generate the bioactive molecule sphingosine-1-phosphate (spp), which is implicated in several inflammatory responses. we and others have previously reported that sphk1 plays a critical role in the intracellular signaling pathways triggered by fcεri in mast cells [28, 31] , and coordinates several physiological responses triggered by activated mast cells. we showed that sphk1 is involved in the calcium signals triggered by fcεri aggregation in human mast cells, as well as playing a critical role for mast cell degranulation [31] . previously, we reported a pivotal role for mast cell activation: tyrosine phosphorylation, calcium sig-nals, degranulation figure 6 mast cell activation: tyrosine phosphorylation, calcium signals, degranulation. a. analysis of overall protein phosphorylation on tyrosine residues. upper panel; overall tyrosine-phosphorylation pattern was analysed in cell extracts from: control unstimulated cells (basal); cells treated with ige alone for (ige) for 5 min; and after fcεri crosllinking for 5 min (xlfcεri). lower panel; the blots were probed for α-tubulin (control for equal loading). results shown are representative of four separate experiments. b. levels of intracellular free calcium. intracellular calcium measurements of mast cells following addition of ige alone (ige); and following the addition of the anti-human ige, to igesensitized cells (xlfcεri), the intracellular calcium levels were analyzed in a continuous reading for the timesstated in the graph. results shown are the mean plus the standard deviation of triplicate measurements and are representative of four separate experiments. c. degranulation. b-hexosaminidase release was determined from control-unstimulated mast cells (basal); following monomeric-igesensitization for 30 minutes (ige); and following fcεri aggregation by addition of the anti-human ige to sensitized cells for 30 minutes (xlfcεri). results shown are the mean plus the standard deviation of triplicate measurements and are representative of four separate experiments. [42, 43] , showing that sphk is key in triggering calcium release from internal stores, and the activation of the phagocyte nadph oxidase. moreover, very recently we demonstrated the role of sphk1 in inflammatory responses triggered by the anaphylatoxin c5a in human neutrophil and macrophages. these responses include: calcium signals, degranulation, cytokine production and chemotaxis triggered by c5a [44, 45] . the observation that the gene encoding for sphk1 is activated during ige-sensitization of mast cells, coupled to the findings above may indicate a key role for sphk1 in mast cell triggered responses. the significance of this research supports the notion that, activation of mast cells appear to be linked to a wide range of pathologies, not only in allergies (as is widely recognized), but potentially in other inflammatory conditions. the method of global gene expression analysis using cdna or oligo-dna microarrays has proven to be a sensitive method to identify and define/redefine the molecular determinants of several human disorders, including cancer and autoimmune diseases, and has provided us with signatures of the immune response [41] . using this technology, complemented with powerful analytical methods, we compared the gene expression profiles of human mast cells stimulated by ige sensitization, and from a series of time points of fcεri aggregation, with unstimulated/control human mast cells. whether changes in gene expression, under these conditions, are representative of a pathological state is not currently known. it is also not known whether ige/antigen and fcεri aggregation will trigger the same set of genes in an organism, where a number of events may be activating mast cells at the same time. however, taken together, our data brings us better insights into the molecular basis of mast cell activation, and provides meaningful information, regarding the mechanisms by which mast cell activation may contribute to the overall activation of the immune response, having clinical implication for improving not only allergic conditions but potentially other inflammatory diseases, where mast cells may play a role. this study is an attempt to elucidate the molecular mechanisms which mast cells undergo during "priming" ige sensitization and full activation by fcεri aggregation in a global perspective. in conclusion, our present study provides information that mast cells, by generating a broad range of cytokines and chemokines, may be a potent contributor of the immune response by recruiting and/or activating other immune-effector cells including the activation of lymphocytes that may, in turn, continue the spreading of the inflammatory response. moreover, changes in the gene expression pattern of transcription factors, intracellular signaling molecules, and cytoskeletal remodeling and anti-apoptosis pathways occur, which would also contribute to the amplification of the inflammatory response. mast cells are well established innate immune-effector cells, and there is mounting evidence to, at least, suggest that mast cells may contribute to the development of acquired immunity [46, 47] , whether in host defense or in allergic or autoimmune diseases. it will be pivotal to define in more detail whether and under which circumstances mast cells may influence the development and/or magnitude of acquired immune responses. unless specifically stated all materials and reagents were purchased from sigma-aldrich (singapore). human umbilical cord blood (cb) samples were collected from normal full-term deliveries of informed individuals with formal consents, meeting the universality institutional review board guidelines, for research using human samples. cd34 + haematopoietic progenitor-cells were harvested using macs cell isolation kit (miltenyi biotec), following the manufacturer's instructions. the isolated cd34 + haematopoietic progenitor-cells were cultured for 5-6-weeks in the presence of 100 ng/ml of stem cell factor (scf cat: 300-07, peprotec, rocky hill, nj), and for the first week this was supplemented with 10 ng/ml of interleukin-3 (il-3 cat: 200-03, peprotec, rocky hill, nj). cells were shown to be differentiated by staining them for specific mast cell markers as follows: for mast cells chymase, with an anti-human chymase mab (igg1-mab1254, chemicon, temecula, ca), and fitc-conjugated secondary antibody (anti-mouse igg-fitc, sigma-aldrich, singapore); for c-kit with anti-human c-kit mouse monoclonal pe-conjugated (cat. no. 555714; clone yb5.b8, bd biosciences -pharmigen, singapore), isotype control anti-mouse ige-pe (cat. no. 555749; clone mopc-21, bd biosciences -pharmigen, singapore); and for fcεri cell-surface expression with antihuman fcεri polyclonal (rabbit-igg-ab31494; abcam, cambridge, uk) and fitc-conjugated secondary antibody (anti-rabbit igg-fitc, sigma-aldrich, singapore) also used as isotype control, and analyzed immediately using a coulter epics-elite esp flow cytometer (beckman, germany). purity was estimated at >97%. the differentiated mast cells were plated in 6 well plates and allowed to rest for 24 hr. after differentiation, mast cells were plated in 6 well plates and allowed to rest for 24 hr. cells in all wells, except the control well, were sensitized with human monomeric ige (1 µg/ml, ige, cat: 30-ai05, lot number a01071004, fitzgerald, concord, ma) overnight. fcεri aggregation was carried out by incubating the cells with monoclonal mouse-anti-human ige (1 µg/ml, anti-human-ige, cat: mca2115, clone 4c3, serotec, oxford, uk) at 37°c, for 2 hr, 6 hr and 12 hr. rna was extracted from all the samples using the qiagen rneasy mini kit (qiagen, valencia, ca). integrity of rna was checked by formamide gel electrophoresis; quantification of rna was carried out by measuring the a 260 nm . labeling and hybridization was carried out as previously described [46] . briefly, 8 µg of total rna from each sample was used to synthesize double stranded cdna using t7-(dt24) oligonucleotide primer and superscript reverse transcriptase (invitrogen). the resultant cdna was purified and 1 µg of purified cdna was labeled with biotin by transcription in vitro. the labeled crnas were, fragmented in the presence of metal ions and then hybridized to hg-focus array (affymetrix), following hybridization the gene chips were washed and stained after which the chips were scanned by gene array scanner (agilent technologies). data collection and analysis was carried out using micro-array suite 5.0 (mas) (affymetrix). the absolute data (signal intensity, detection call and detection p-value) were exported into genespring v7.0 (silicon genetics, redwood city, ca, usa) software for analysis by parametric test based on cross gene error model (pcgem). the anova approach has been used to find differentially expressed genes (p < 0.05). the benjamini and hochberg false discovery rate multiple testing correction was applied. agglomerative average-linkage hierarchical clustering of the five different experimental were obtained for selected groups of genes with gene spring 7.0 software (silicon genetics, redwood city, ca, usa) using standard correlation as similarity matrix. real-pcr was performed, as previously described [46] , using 1 µg of total rna from the same samples used for microarray experiments. pcr was performed for transcripts of il-1β (primers: for amplicon detection, the light cycler rna master sybr green kit (roche) was used as described by the manufacturer. pcrs were performed in a lightcycler ® instrument (roche) as follows: reverse transcription at 61°c for 20 min, initial denaturation at 95°c for 2 min; amplification for 45-65 cycles of denaturation (95°c, 5s, ramp rate 20°c/s), annealing (optimal temperature, 5s, ramp rate 20°c/s) and extension (72°c, product length [bp]/25 s, ramp rate 2°c/s). a single online fluorescence reading for each sample was taken at the end of extension step. quantitative results were expressed by identification of the second derivative maximum points, which marked the cycles where the second derivatives of the fluorescence signal curves are at maximum. these points were expressed as fractional cycle numbers. then, these cycle numbers were plotted against the logarithm of the concentrations of serially 2-fold diluted standard samples to obtain a standard curve. the concentrations of unknown samples were calculated by extrapolation from this standard curve. positive sample specificity was confirmed by determining the melting curve (95°c, 5s, ramp rate 20°c/ s; 68°c, 15s, ramp rate 20°c/s; 95°c, 0s, ramp rate 0.1°c/s, continuous measurement). supernatants from control cells, cells sensitized, and cells following fcεri aggregation, were collected and stored at -20c until use. il-1β, il-6, il-8, mcp-3 and rantes levels in the supernatants were evaluated using elisa (r&d systems inc., mn, usa) following the manufacturer's instructions. western blots were carried out as previously done [31] . briefly, 40 µg of lysate for each sample was resolved on 10% polyacrylamide gels (sds-page) under denaturing conditions and then transferred to 0.45 µm nitrocellulose membranes. for overall tyrosine phosphorytaion, the blots were probed using a specific monoclonal anti-phosphotyrosine primary antibody (p-tyr, sc-7020, santa cruz, ca, usa), and an anti-mouse hrp-conjugated secondary antibody (anti-mouse igg-hrp, a-4416, sigma). bands were visualized using the ecl western blotting detection system (amersham, singapore). for sphk1 expression, the blots were probed using a rabbit polyclonal anti-sphk1 primary antibody (anti-sphk1, x1627p, exalpha, ma, usa), and hrp-conjugated secondary antibody (anti-rabbit igg-hrp, sc-2004, santa cruz, ca, usa). for loading control the blots were probed with a monoclonal anti α-tubulin (anti-α-tubulin, sc-5286, santa cruz, ca, usa), and an anti-mouse hrpconjugated secondary antibody (anti-mouse igg-hrp, a-4416, sigma). bands were visualized using the ecl western blotting detection system (amersham, singapore), and quantified by densitometry analysis. cytosolic calcium was measured as described previously [31] . briefly, cells were loaded with 1 µg/ml fura2-am (molecular probes, leiden, the netherlands) in pbs, 1.5 mm ca 2+ and 1 % bsa. after removal of excess reagents by dilution and centrifugation (in pbs), the cells were resuspended in pbs containing 1.5 mm ca 2+ and 1 % bsa, for 30 min; or in pbs containing 1.5 mm ca 2+ , 1 % bsa, and human-monomeric ige (1 µg/ml) for sensitization, for 30 min. after removal of excess ige by dilution and centrifugation (in pbs), the cells were resuspended in 1.5 mm ca 2+ supplemented pbs and warmed to 37°c in the cuvette; unsensitazed cells were placed in the cuvette and cytosolic calcium was measured before and after the addition of monomeric ige. ige-sensitized cells were placed in the cuvette and fcεri was crosslinked by addition of mouse-anti-human ige (1 µg/ml). fluorescence was measured at 340 and 380 nm. degranulation was measured using as previously described [31] . briefly, an aliquot of cells was resuspended in pbs containing 1.5 mm ca 2+ and 1 % bsa, and incubated with monomeric ige for 30 min at 37°c. another aliquot of cells was resuspended in pbs containing human-monomeric ige (1 µg/ml) for sensitization, 1.5 mm ca 2+ and 1 % bsa, for 30 min. after removal of excess ige by dilution and centrifugation (in pbs), the cells were resuspended in 1.5 mm ca 2+ supplemented pbs, and fcεri was crosslinked by addition of mouseanti-human ige (1 µg/ml) to cells for 30 min at 37°c. following the incubation, 50 µl of supernatant, was incubated with 200 µl of 1 mm p-nitrophenyl n-acetyl-β-dglucosaminide for 1 hr at 37°c. the total β-hexosaminidase concentration was determined by a 1:1 extraction of the remaining buffer and cells with 1% triton x-100; a 50 µl aliquot was removed and analyzed as described. reactions were quenched by addition of 500 µl of 0.1 m sodium carbonate buffer. the enzyme concentration was determined by measuring the od at 400 nm. β-hexosaminidase release was represented as a percent of total enzyme. to analyze the expression of the intracellular mast cell chymase, 1 × 10 6 cells were washed with ice cold pbs, fixed and permeabilised using the fix and perm reagents from caltag (caltag laboratories, burlingame, ca) as follows: after washing, samples were resuspended in 100 ul of reagent a (fixation medium) and incubated for 15 min at rt. the cells were then washed twice with ice-cold pbs, and resuspended in 100 ul of reagent b (permeabilization medium) and incubated for 15 min at rt. cells were washed twice and resuspended in 100 µl of pbs/1% fbs and 5 µl of the anti-human chymase mab (igg1-mab1254, chemicon, temecula, ca) was added and samples were incubated for 20 min at rt. samples were washed twice in ice-cold pbs, then resuspended in pbs/ 1% fbs and 5 µl of fitc-conjugated secondary antibody (anti-mouse igg-fitc, sigma-aldrich, singapore) was added, and incubated in dark for an 30 min at rt. samples were washed twice with ice-cold pbs and resuspended in 100 µl of pbs/1% fbs for immediate analysis. to analyze the cell surface expression of ckit and fceri, the samples were initially processed as above except that the permeabilisation step was omitted. for c-kit the primary antibody was an anti-human c-kit mouse monoclonal (mca1841, clone 104d2, serotec, oxford, uk), and the secondary antibody was a fitc-conjugated (antimouse igg-fitc, sigma-aldrich, singapore). for fcεri cell-surface expression, the cells were labeled with the primary anti-human fcεri polyclonal (rabbit-igg-ab31494. abcam, cambridge, uk), and the secondary antibody was a fitc-conjugated (anti-rabbit igg-fitc, sigma-aldrich, singapore). all the samples were analyzed by flow cytometry was using a facscalibur machine (bd biosciences), and the data analysed using the cell quest™ pro software. mast cells in innate immunity mast cells to the defense mast cells in autoimmune disease mast cells the diverse potential effector and immunoregulatory roles of mast cells in allergic disease transcriptional response of human mast cells stimulated via the fcεri and identification of mast cells as a source of il-11 the receptor with high affinity for ige signalling through the high-affinity ige receptor fcεri roles of mast cells and basophils in innate and acquired immunity complexity and redundancy in the pathogenesis of asthma: reassessing the roles of mast cells and t cells mast cells can amplify airway reactivity and features of chronic inflammation in an asthma model in mice an essential role of mast cells in the development of airway hyperresponsiveness in a murine asthma model mast cells phagocytosis of fimh expressing enterobacteria histamine-induced activation of human lung macrophages mast cell-derived tumor necrosis factor induces hypertrophy of draining lymph nodes during infection human mast cell transcriptome project identification of novel mast cell genes by serial analysis of gene expression in cord blood-derived mast cells gene expression screening of human mast cells and eosinophils using high-density oligonucleotide probe arrays: abundant expression of major basic protein in mast cells comparative cytokine profile of human skin mast cells from two compartments strong resemblance with monocytes at baseline but induction of il-5 by il-4 priming gene expression profiling of ca 2+ -atapase inhibitor dtbhq and antigen-stimulated rbl-2h3 mast cells identification of specific gene expression profiles in human mast cells mediated by toll-like receptor 4 and fcεri gene expression profiles for fcεri, cytokines and chemokines upon fcεri activation in human cultured mast cells derived from peripheral blood marked increase in cc chemokine gene expression in both human and mouse mast cell transcriptomes following fcε-receptor i cross-linking: an interspecies comparison monomeric ige enhances human mast cell chemokine production: il-4 augments and dexamethasone suppresses the response interleukin-1 induces a shock-like state in rabbits: synergism with tumor necrosis factor and the effects of cyclooxygenase inhibition monomeric ige stimulates signaling pathways in mast cells that lead to cytokine production and cell survival nf-kappa b and rel proteins: evolutionary conserved mediators of immune responses generic signals and specific outcomes: signaling through ca 2+ , calcineurin, and nf-at dichotomy of ca 2+ signals triggered by different phospholipid pathways in antigen stimulation of human mast cells minimal requirements for ige mediated regulation of surface fcεri karasuyama h: drastic up-regulation of fcεri on mast cells is induced by ige binding through stabilization and accumulation of fcεri on the cell surface increased il-12 release by monocytes in nephritic patients the physiological and pathophysiological role of chemokines during inflammatory and immunological responses differential roles of il-1 and tnf-α on graft versushost disease and graft versus leukemia rel/nf-κb transcription factors: key mediators of b-cell activation constitutive nfκb maintains high expression of a characteristic gene network, including cd40, cd86, and a set of antiapoptotic genes in hodgkin/reed-stemberg cells control of oncogenesis and cancer therapy resistance by the transcription factor nf-κb calcium mobilization via sphingosine kinase in signalling by the fcεri antigen receptor signatures of the immune response fcγri coupling to phospholipase d initiates sphingosine kinasemediated calcium mobilization and vesicular trafficking a molecular switch changes the signalling pathway used by the fcγri antibody receptor to mobilize calcium anaphylatoxin signaling in human neutrophils: a key role for sphingosine kinase antisense knockdown of sphingosine kinase 1 in human macrophages inhibits c5a receptordependent signal transduction, ca 2+ signals, enzyme release, cytokine production and chemotaxis mast cells in the development of adaptive immune responses mast cells in allergy and autoimmunity: implications for adaptive immunity melendez aj: expression profile of immune response genes in patients with severe acute respiratory syndrome this work was supported by a bmrc-young investigator award (r-185-000-044-305). we thank a-k fraser-andrews for proofreading the manuscript. mj carried out data analysis and prepared the microarraydata table and figures. hkt isolated the progenitor cells, differentiated the mast cells and carried out the receptor crosslinking. rr carried out the rna extraction, labeling and microarray hybridization. lz carried out rt-pcr. kkc and mr provided the cord blood. ajm designed the study and drafted the manuscript. all authors read and approved the final manuscript. key: cord-333639-usgpe1cz authors: zuwala, kaja; riber, camilla f.; løvschall, kaja borup; andersen, anna h.f.; sørensen, lise; gajda, paulina; tolstrup, martin; zelikin, alexander n. title: macromolecular prodrugs of ribavirin: polymer backbone defines blood safety, drug release, and efficacy of anti-inflammatory effects date: 2018-04-10 journal: j control release doi: 10.1016/j.jconrel.2018.02.012 sha: doc_id: 333639 cord_uid: usgpe1cz macromolecular (pro)drugs hold much promise as broad-spectrum antiviral agents as either microbicides or carriers for intracellular delivery of antiviral drugs. intriguing opportunity exists in combining the two modes of antiviral activity in the same polymer structure such that the same polymer acts as a microbicide and also serves to deliver the conjugated drug (ribavirin) into the cells. we explore this opportunity in detail and focus on the polymer backbone as a decisive constituent of such formulations. fourteen polyanions (polycarboxylates, polyphosphates and polyphosphonates, and polysulfonates) were analyzed for blood pro/anti coagulation effects, albumin binding and albumin aggregation, inhibitory activity on polymerases, cytotoxicity, and anti-inflammatory activity in stimulated macrophages. ribavirin containing monomers were designed to accommodate the synthesis of macromolecular prodrugs with disulfide-exchange triggered drug release. kinetics of drug release was fast in all cases however enhanced hydrophobicity of the polymer significantly slowed release of ribavirin. results of this study present a comprehensive view on polyanions as backbone for macromolecular prodrugs of ribavirin as broad-spectrum antiviral agents. viruses are fascinating products of evolution and life-threatening pathogens at the same time. indeed, these non-cellular assemblies of macromolecules and lipids are simpler than even minimalistic replicating cells but can infect living organisms in both plant and animal kingdoms. throughout history, viral infections repeatedly had a devastating effect on human society and even in modern times, viral outbreaks create an enormous socio-economic burden [1, 2] . recent examples include the outbreaks of coronavirus-associated southeast asian and middle eastern respiratory syndromes (sars, mers) as well as the ebola and zika virus outbreaks. to counter this, interdisciplinary efforts have been made over the past decades in the design and development of antiviral drugs. in large part, this was spurred by the spread of the human immunodeficiency virus (hiv) [3] . currently, there are nearly one hundred antiviral drugs approved by the us fda [4] . nevertheless, significant challenges remain. it is now understood that a significant limitation of current strategies is that countermeasures to viral pathogens are most typically developed individually and specifically to each emerging pathogen. an aspect that came into the focus of research attention is the development of "broad-spectrum antiviral agents" [5] . such agents can constitute prophylactic, [6] preventative, [7, 8] or curative [9, 10] measures. prophylaxis (vaccination) holds a great appeal but universal antiviral vaccines are yet to be developed [6] . in fact, the overall majority of pathogenic viruses have no corresponding vaccination product. in turn, curative measures are highly attractive in their own right. however, while replicational cycles of viruses are similar, there is a great variability between viral enzymes and other proteins that could serve as drug targetsmaking the design of universal antiviral agents challenging [4] . interferon is an endogenous protein that serves as a potent stimulatory signal to human immunity and acts as a non-specific, broadly acting antiviral agent. however, viruses developed multiple interferon inhibitors which compromise induction or activity of interferon [11] . moreover, due to its side effects, its clinical use is limited [12] . several small molecule drugs also serve as broadly acting antivirals. of these, ribavirin (rbv) has a long history of medicinal use and appears on the world health organization list of essential medicines for both adults and children [13, 14] . however, this drug also has a dose limiting toxicity. several other experimental drug leads are in different stages of (pre)clinical development [15] [16] [17] [18] . finally, preventative antivirals that act through neutralization of viruses and/or blocking virus cell entry appear to be highly promising as broad-spectrum antiviral agents [19] [20] [21] . a notable class of such agents is polymers. in the early 1940s, it was first observed that viruses interact with polymers [22] and inhibit virus infectivity [23] . over subsequent decades, this effect was shown to be affecting viral pathogens across the viral genus and families, and observed for polymers diverse in their structure, natural and synthetic, positively or negatively charged [24] [25] [26] [27] [28] [29] . de clercq et al. proposed that activity of the negatively charged polymers, at least in vivo, is indirect and proceeds via stimulation of interferon production [30] . however, antiviral activity proceeds potently and efficaciously in vitro, in absence of interferon, and the direct contact between polymers and the virus with ensuing neutralizing activity is now well-documented. inherent skepticism towards polyanions and other polymers as antivirals [31] originates in the failures of hallmark clinical trials for these agents both when administered systemically or as topical microbicides [7] . specifically for systemic administration, it has been concluded that preventative antiviral effect would have to rely on a sustained, high concentration of these agents in blood not achievable without toxic effects [31] . however, other polymers are successfully commercialized as antivirals, specifically as lubricants in condoms acting as microbicides with activity against hiv and herpes simplex viruses [32] . nucleic acid based polyanions progress through clinical trials as agents against hepatitis b virus with potential of further development as broad-spectrum antiviral activity [20] . it is important to note that failed clinical trials are indeed discouraging yet important, game-changing developments have occurred in polymer chemistry in recent decades. specifically, novel polymerization techniques are now available to afford polymers with well-controlled molar mass and architecture and a significantly broader available polymer functionality (side chains) [33, 34] . another notable advancement includes the development of polymeric virucidal agents that compromise stability of the viral envelope and exert permanent damage to the virion [35, 36] . together, the broad spectrum of activity of polymers against viral pathogens and the chemical versatility of these agents renders this class of antiviral agents unique and attractive for deeper investigation. in our past studies, we developed broad-spectrum antiviral agents based on macromolecular prodrugs (mp) of ribavirin. when conjugated to polymers, ribavirin did not accumulate in the red blood cells which in vivo is the main side effect of the drug [37] [38] [39] . mp of ribavirin broadened the therapeutic window of the drug (in vitro) [37] and were effective against diverse viruses such as hcv (in a replicon cell culture model), measles, and influenza, in the latter case revealing antiviral effects in chicken embryo model [40, 41] . we also experimentally confirmed the link between ribavirin and the synthesis of nitric oxide in macrophages, with relevance to the treatment of hepatitis [42] . key to successful delivery of ribavirin using mp was the development of a disulfide-containing linker that releases the drug from the prodrug upon cell entry [43] . from a different perspective, we also focused on preventative measures and conducted a systematic study of polymers with anionic charge as inhibitors of virus cell entry [21] . our broad study considered 14 polymers, infectious zika virus, hiv, and hsv, as well as pseudotyped viral particles for ebola, lassa, lyssa, sars, and other viruses. the main finding of our work was that anionic charge alone (be it carboxylate, phosphate/phosphonate, or sulfonate) did not endow the polymer with broad-spectrum antiviral activity. decisive factor for such activity was the enhanced (but balanced) hydrophobicity of the macromolecule. identified lead candidate polymers exhibited antiviral activity against all the enveloped viral pathogens, including the zika virus. for the latter, we presented evidence of direct contact between the polymer and the viral particle and concurrent inhibition of viral infectivity [21] . one intriguing possibility lies in the prospect of combining the curative and the preventative antiviral activity within the same macromolecular antiviral agent [40, 44] . such agents represent a combination therapy wherein the carrier exerts extracellular preventative effect [21] whereas the conjugated drug, once released inside the cell, is an intracellularly active therapeutic [37] . it is also possible that polyanions have an intracellular antiviral effect of their own, that is the inhibition of viral polymerases, [40, 45, 46] presumably through competitive electrostatic interaction with the protein. in this work, we explore the design of such combination therapy in detail. we focus on the choice of the macromolecular backbone as a carrier for the conjugated drug and analyze blood coagulation, binding to albumin, albumin aggregation, inhibitory activity on polymerases, and cytotoxicity for polymers differed by their anionic charge (carboxylates, phosphates and phosphonates, sulfonates). further, we synthesize ribavirin-containing counterparts to the polyanions, investigate kinetics of drug release for the resulting macromolecular prodrugs, and investigate their use for intracellular drug delivery of ribavirin in an anti-inflammatory model in macrophages. as a result, we identify polymers and macromolecular prodrugs that are devoid of blood anti-coagulation activity but are strong as inhibitors of polymerases and efficacious as delivery vehicles for ribavirinthus being attractive for the development of broad-spectrum antiviral agents. we also identified polymers that are benign and devoid of any activity in these tests thus being attractive as "stealth" materials for diverse biomedical applications [47] . results of this study would be important for the advancement of biomedical engineering, specifically with regards to development of antiviral therapies and drug delivery techniques. homopolymers of 14 different anionic monomers with variations in the anionic functionality (carboxylic acid, sulfonic acid, phosphonic acid and phosphate, fig. 1 ) were obtained through the reversible addition-fragmentation chain transfer (raft) polymerization [21] . synthetic approach to the synthesis of the corresponding macromolecular prodrugs was chosen to proceed via copolymerization of the drug containing monomer and that corresponding to the carrier polymer. towards this end, rbv containing monomers were designed to be compatible with polymerization of diverse co-monomers, acrylic and methacrylic, hydrophilic and hydrophobic (fig. 2 ). of these, the synthesis of the hydrophobic methacrylate monomer (monomer 1 in fig. 2 ) was reported in our previous publications [43] . acrylic counterparts were obtained via similar protocols with minor variations. design consideration also concerned polarity of co-monomers, an aspect that exerts limitations to the choice of the solvent for polymerization. polarity of the applied anionic monomers varied from those with a hydrophilic character (phosphates/phosphonates) to those with well pronounced hydrophobic character (ethylacrylic acid, propylacrylic acid). rbv monomers were therefore designed to mimic the polarity of the anionic comonomers through the use of silyl protecting groups to render the monomer more hydrophobic or using deprotected, hydrophilic 2′,3′-hydroxyl containing monomers. in total four different rbv prodrug monomers were applied (fig. 2) , allowing for the copolymerization to be performed under hydrophobic and hydrophilic conditions with either acrylate/acrylamide or methacrylate/methacrylamide containing monomers. in each case, the monomer structure also comprised a disulfide bond for intracellular degradation, coupled to a self-immolative linker for drug release [48] . due to the variations in monomer type and monomer polarity, a unique set of reaction conditions was necessary for each copolymerization including the choice of raft agent, solvent (or solvent mixture), reaction time, initiator, and purification procedure (table 1, for full details see experimental section). synthesized macromolecular (pro)drugs were analyzed by 1 h nmr during synthesis to assess the conversion of the two monomers and in the purified form to confirm the absence of unreacted monomers. polymers were also characterized through size exclusion chromatography (using an 8-angle static light scattering detector) to determine the molar mass and dispersity of the polymer samples (table 1) . we acknowledge that (co)polymers exhibited variability in their degree of polymerization (molar mass). for this reason, analyses presented below do not take into account the likely, non-negligible influence of molar mass on the properties of the polymers. in doing so, we assume that the polymer structure is the factor determining the polymer properties whereas the molar mass is of secondary importance. indeed, our recent publication on the antiviral activity of the polymers revealed clear structure-activity correlations with regards to the polymer functionality whereas no clear correlation with the polymer molar mass was observed [21] . blood pro/anticoagulation is the most known side effect of polyanions upon their systemic administration. indeed, depending on structure, polyanions may exhibit anticoagulation effect, [49] as is wellknown for heparin, [50] a polysaccharide approved by the us fda specifically for the purpose of blood anticoagulation. other polyanions such as polyphosphates (nucleic acids) are reported to be pro-coagulants [51] . no interference with blood coagulation is a highly desired safety feature of injectable formulations and identification of coagulation-inert polymers was one of the aims of this study. we investigated activated partial thromboplastin time (aptt) in presence of the synthesized polyanions. the platelet-depleted plasma from healthy donors was incubated with polyanions at concentrations of 10 or 100 mg/l and the aptt was measured (fig. 3 ). polysulfonates and pvbza at 100 mg/l enhanced blood coagulation time to a value over 150 s, similarly to heparin, and this indicates that they are strong blood anti-coagulants. this was also observed with pvpa, which doubled the aptt value. in contrast, other polyphosphates and polyphosphonates papa, pmpa, paep and pmep had minor if any influence on blood coagulation time. of the carboxylates, paa and pmaa exhibited a noticeable anti-coagulation effect. in contrast, peaa and ppaa, two polymers with an enhanced hydrophobicity but decreased anionic character compared to paa and pmaa (higher pka) had no effect on blood coagulation time at 10 mg/l. taken together, results in fig. 3 reveal that six out of the tested 14 polymers (polyphosphates, polyphosphonates, peaa and ppaa) appear to have no inhibitory activity with regards to the blood coagulation times. albumin is the most abundant protein in human plasma. it has numerous physiological functions that include the maintenance of table 1 polymerization conditions and macromolecular characteristics of polyanions and macromolecular prodrugs used in this work. for chemical structures and details of synthesis of the rbv containing monomers 1 through 4, see fig. 1 and experimental section, respectively. chain transfer agents were cyanomethyl dodecyl trithiocarbonate (cdtc), (4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid) (cdpa), cyanomethyl methyl(phenyl)carbamodithioate (cmpd), 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (dcma) or 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (cppa); polymerization initiators were azobisisobutyronitrile (aibn), 2,2′-azobis(4-methoxy-2.4-dimethyl valeronitrile) (v-70) or 4,4′-azobis(4-cyanovaleric acid) (acva). m n : number-average molar mass, sec-mals: size exclusion chromatography with multi-angle light scattering detection, dp: degree of polymerization. values of dn/dc were calculated from sec/mals measurements assuming full mass recovery; dp values for ppaa, ppaa-rbv, and pvbza (marked with a * symbol) are calculated from m n (calc). experimental conditions for anionic homopolymers are taken from ref. 21 osmotic pressure and the transport of hydrophobic solutes through the blood. this protein is safe-guarded by the body and physiological mechanisms exist to ensure that albumin is not degraded but retained in the body. as a result, albumin blood residence time reaches a phenomenal value of 3 weeks [52] . albumin binding is among the most successful methodologies in biomedicine to extend the circulation times of solutes and this has been achieved to a range of drug molecules, from small molecule anticancer drugs [53] to peptide hormones and proteins [54] . recently, we observed that synthetic polymers (peaa) can bind albumin and this afforded hepatic deposition of the polymer [55] . albumin binding for peaa is not altogether surprising in that this protein is well known to bind hydrophobic solutes and anionic solutes, both features being emphasized in the structure of peaa. herein, we aimed to investigate albumin binding for the library of anionic polymers, with and without conjugation of ribavirin. albumin has several binding sites, most notable of which are the so-called sudlow i and sudlow ii sites [56, 57] . binding of solutes to these sites can be interrogated using fluorescent probes that exhibit site-specific biding to albumin, specifically dansyl asparagine (sudlow i) and dansyl sarcosine (sudlow ii) [56] . fluorescence of these probes is significantly decreased upon displacement from the protein globule, and this presents itself as a facile methodology to quantitate binding of albumin with the polymers. polymers were mixed with albumin at 5 μm (0.33 g/l) concentration of the protein and 10 molar equivalents of the polymer to albumin. the immediate observation from the results of these experiments ( fig. 4) is that polymers differed significantly in their capacity to interact with albumin via the two nominated sites. polyphosphonate polymer papa brought about no decrease in the fluorescence of probes indicating no detectable polymer interaction with albumin. papa also exhibited no anticoagulation activity (fig. 3) , and together these data suggest that papa is a blood-safe carrier for diverse drug delivery applications. albumin binding for the structurally similar pmpa as well as the phosphate analogues paep and pmep was also not pronounced and at 10-fold mole excess of the polymer to albumin, probe fluorescence decreased by no more than 50%. polysulfonates also exhibited minor association with albumin and afforded no change in fluorescence for the sudlow i binding probe and a change in fluorescence for the sudlow ii probe within 50%. exception to this was the most hydrophobic of these polymers, styrenic psvbs which showed a 75% probe displacement. in contrast, polycarboxylates exhibited a strong tendency for binding with albumin and exhibited a highly efficacious probe displacement. binding was not site specific and probes were displaced from both sudlow i and ii sites. in the case of paa, displacement of probes for both sudlow i and ii sites was nearcomplete indicating a strong binding. for this polymer, incorporation of rbv abrogated polymer binding to albumin. this illustrates that paa binding to albumin is structure-programmed and change to this macromolecule such as addition of rbv interferes with albumin binding. pmaa, peaa and ppaa also exhibited strong probe displacement capacity albeit not as pronounced as paa. interestingly, unlike paa-rbv, peaa-rbv and ppaa-rbv were strong albumin binders. albumin binding was also analyzed by sec-mals. this experiment confirmed the difference between the polymers in their capacity to binding albumin and highlighted further changes in the result of the polymer-protein association, fig. 5 . elution of albumin was hardly altered upon its incubation with pamps (polysulfonate) taken at an equimolar concentration to albumin and there was minor protein aggregation in the presence of this polymer. this observation was similar for the protein incubation with papa and all polyphosphonates and polyphosphates as well as for pvsa and pspa. in contrast, for the carboxylates paa, pmaa, ppaa, pvbza and the hydrophobic polysulfonate psvbs, polymer-protein interaction afforded strong aggregation and formation of high molar mass products (mda). finally, upon incubation with peaa, a strong sudlow i/ii probe displacing polymer, elution of albumin was shifted to shorter times indicating increased molar mass of the protein, as would be expected for a polymer-protein adduct, with only minor protein aggregation. peaa is a unique polymer and albumin binding without noticeable aggregation was not observed fig. 4 . polyanions and macromolecular prodrugs exhibit structure-dependent binding to albumin via sudlow i/ii sites. interaction with albumin was quantified using fluorescent probe displacement assay using dansyl asparagine and dansyl sarcosine as probes for sudlow i and sudlow ii sites, respectively. fluorescence intensities of the probe in the presence of polyanions were normalized to that in control experiments. 100% probe fluorescence indicates no probe displacement and 0% probe fluorescence implies quantitative displacement and indicates strong interaction between the polymer and albumin. results are presented as average of three independent experiments ± sd. statistical significance compared to the control samples was evaluated via one-way anova with dunnett's multicomparison post-hoc analysis. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. for other polymers studied in this work. next, we examined macromolecular prodrugs with regards to the kinetics of drug release. rbv conjugation was designed such as to be reversed upon the scission of the disulfide linkage, specifically upon cell entry. intracellular environment is characterized by a relatively high concentration of a thiol containing tripeptide, glutathione (gsh), [58] and this effectively triggers the degradation of disulfide linkages inside the cells [59] [60] [61] . accessibility of the disulfide within the structure of macromolecular prodrugs can vary due to the difference in the size of the side groups functionalities. polymers may thus exhibit significant differences in the drug release kinetics. we have previously investigated the release of rbv and other drugs conjugated to polymers via the same linkage as used in this study (disulfide trigger paired with a self-immolative linker) via hplc [43, 46, 62] . herein, we established an 1 h nmr-based method to quantify the release of rbv as illustrated in fig. 6 on an example of papa-rbv. macromolecular prodrug was incubated in the presence of gsh. disulfide reshuffling initiates the spontaneous cyclization of the self-immolative linker with ensuing release of the pristine drug, ribavirin (fig. 6, a) . chemical shift of the ribavirin protons exhibits a well-defined migration in the 1 h nmr spectrum and with time, the signal corresponding to the polymer-bound drug decreases whereas that for the free rbv increases (fig. 6, b) . integration of the corresponding peaks provides a facile means to quantify rbv bound to the polymer and released from the prodrug. release of rbv from the macromolecular prodrug was fast and within 1 h, over 75% of the drug was released from the macromolecular carrier (fig. 7) . this kinetics profile is highly advantageous, significantly faster than that reported for the peptide-based linkers between the drug and the carrier, [63] and allows for rapid drug release upon prodrug cell entry. we note that data in fig. 7 are rather similar to the drug release data collected in our prior studies via hplc [43, 46, 62] and this serves to validate the established herein nmr-based approach to monitor drug release. 1 h nmr based method to quantify drug release was applied to the macromolecular prodrugs based on other polyanions as well as a nonionic carrier, phpma, fig. 8 . within 2 h of observations, all mp exhibited a pronounced release of their payload. a significant finding from this experiment was that hydrophobicity of the polymer chain has a pronounced effect on the kinetics of drug release. in contrast, no such correlation appears to exist with regards to the charge of the polymer (anionic vs charge neutral) or the nature of the negative charge on the polymer. indeed, structurally similar phpma and pmaa as well as papa and paep released ribavirin quantitatively within 2 h revealing no difference in drug release kinetics. however, relatively small structural change from pmaa to peaa or from peaa to ppaa afforded a significant decrease in the drug release. similarly, a single methyl group variation from papa to pmpa afforded a pronounced drop in the amount of rbv released within 2 h. a likely explanation to this phenomenon is the more compact, less hydrated and thus less accessible polymer coil for increasingly hydrophobic polymers which hinder gsh from initiating drug release. , as well as a significantly slower release from the methacrylamide based phosphonate relative to the acrylamide based phosphonate. all data are displayed as mean ± standard deviation from three independent experiments. for comparison a oneway anova (analysis of variance) was performed, followed by a tukey's post hoc test. ***: p ≤ .001. therapeutic and/or side effects elicited by the polyanionic (pro) drugs investigated herein would likely manifest themselves due to the interaction of the polymers with other macromolecules such as proteins. the spectrum of polyanionic endogenous compounds is rather large, and competition [64] for electrostatic interactions with these polymeric partners is highly likely. in the context of antiviral performance of the polymers, we [65] [46] and others [45] have shown that polyanions can act as efficient inhibitors of polymerases. charge neutral polymers were devoid of such activity [65] pointing to the anionic charge of the polymer (that is, electrostatic interaction with the target) as the origin of the observed phenomenon. polymers synthesized in this work were analyzed as inhibitors of two types of polymerase enzymes, namely a dna-dependent dna polymerase (replicase) and an rnadependent dna polymerase (reverse transcriptase), fig. 9 . despite being similar in carrying multiple anionic charges, polymers differed markedly in their ability to interfere with the performance of polymerases. polyphosphates and polyphosphonates were nearly devoid of any inhibitory activity in these assays and regardless of the polymer concentration, polymerases remained uninhibited. together with the data presented above, e.g. papa appears to have unique "stealth-like" properties with no-interference in coagulation assays, albumin binding, and competition for binding with polymerases. in contrast, polycarboxylates and polysulfonates exhibited a dose-dependent polymerase inhibition. a rather surprising finding is that high anionic character alone does not make the polymer a strong inhibitor and in the homologous row of carboxylates, it was peaa that exhibited the strongest inhibitory activity (complete inhibition of reverse transcriptase activity at 1 mg/l polymer concentration). this observation echoes our recent findings on the apparent unique pairing of negative character and hydrophobicity of the polymer backbone that renders peaa an efficacious inhibitor of e.g. hepatitis c virus intracellular replication [55] and a lead polymer with broad-spectrum antiviral activity [21] . similar observation can be made for the negatively charged pvbza, also characterized by pronounced hydrophobicity of the chain due to the aromatic ring in each repeat unit of the polymer. finally, as a test for activity of the polymers in cell culture, we evaluated polyanions and their counterparts conjugated to rbv as inhibitors of inflammation in stimulated macrophages. our previous findings revealed that paa-rbv inhibited the synthesis of an inflammatory marker nitric oxide (no) in the lipopolysaccharide (lps)stimulated macrophages whereby both the polymer and the released drug elicited their individual anti-inflammatory activity [65] . we tested the library of polyanions presented above and quantified inflammatory responses in cells incubated with polymers in a range of concentrations from 0.32 to 200 mg/l. independently, metabolic activity of the cells (indicative of cell viability) was measured by the presto blue assay. these experiments therefore provide information on both, toxicity of the polymers to macrophages in cell culture and anti-inflammatory activity of the polymers. none of the tested polymers, with or without conjugated ribavirin, exhibited noticeable toxicity at concentrations up to 40 mg/l, fig. 10 . the overall majority of polymers were also devoid of activity with regards to inflammation. however, several polymers comprised notable exceptions and were efficacious inhibitors of inflammation without associated toxicity to the cells. specifically, among the polyanions, pmaa, peaa, pvbza and psvbs afforded significant decrease in the levels of no produced by macrophages. an apparent unifying structural feature of these four polymers is that these are the most hydrophobic of the polyanions used in this work. decreased inflammatory response by pvbza and psvbs was observed already at 8 mg/l making these polymers most potent of the tested polyanions. it is also striking that fig. 9 . activity of the polymerase enzyme in the presence of polyanions (1 or 10 mg/l in the reaction mixture) expressed in % of de novo synthesized nucleic acid relative to the un-inhibited polymerase reaction. statistical significance compared to control was evaluated via a one-way anova with dunnett's multicomparison posthoc analysis. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. these two polymers, as well as peaa, afforded a highly efficacious decrease in the synthesis of no with a near-complete suppression of inflammation without associated toxicity to the cells. this comes in stark contrast with the rbv based treatment which could only afford ca. 50% reduction in the levels of no at sub-toxic concentrations of the drug [37] . it is worthy of note that polyanions are typically deemed to have restricted cell entry [66] . however, confocal laser scanning microscopy evaluation of the polymer uptake by macrophages revealed pronounced polymer-associated fluorescence inside the cells (fig. 11) . this was true for all polymers: polycarboxylates, polyphosphates and polyphosphonates, and polysulfonate. contrary to expectations, cell entry does not appear to be decisive in the observed activity of the polymers. macromolecular prodrugs of rbv were synthesized for the (meth) acrylate/(meth)acrylamide type of anionic monomers thus excluding pvbza, pvpa, psvbs, and pvsa. both pmaa and peaa displayed an inherent, structure-driven effect in the anti-inflammatory assay and in these cases it is therefore not possible to discern the effects elicited by the polymer or the intracellularly released rbv. it is likely that both the polymer and the released drug contribute to the overall anti-inflammatory response in macrophages for pmaa-rbv and peaa-rbv. surprising other leads in this assay were macromolecular prodrugs based on pmpa and pamps. in both cases, statistical significance of anti-inflammatory effects was observed at concentrations at and above 40 mg/l. this effect was efficacious and had minor if any associated toxicity. to further probe the mechanism of activity of the polymers in this assay, we investigated potential antagonism of the polyanions with lps. indeed, the latter has anionic component to it and competion with polyanions for interaction with the receptor is not inconceivable. furthermore, prior reports suggested that sulfated polyaromatic suramin can antagonize lps [67] . to investigate this on the example of peaa, lps stimulation of macrophages was performed using increasing concentration of lps in the presence or absence of a fixed concentration of the polymer (fig. 12) . in this assay, antagonism at the receptor in absence of intracellular effects would manifest itself as a shift in the potency of lps without a change in efficacy. in turn, intracellular effects in absence of receptor antagonism would mean a decreased efficacy of the inflammatory response with no change in the potency of lps. experimental data suggest that both effects are likely observed (fig. 12) and peaa exerts its activity through both, receptor antagonism and intracellular effects. indeed, the polymer affords statistically significant decrease in the inflammation and also a change, albeit a very minor one, in the ic 50 value for lps with regards to stimulation of macrophages (0.42 vs 0.24 mg/l in the presence of peaa). results of this study provide a comprehensive view on polyanions as the backbone for macromolecular broad-spectrum antiviral therapeutics. together with the results of the screen of these polyanions for inherent antiviral activity, [21] presented data provide a suggestive view on the utility of polyanionic macromolecular prodrugs of ribavirin as antiviral agents (summarized in table 2 ). first, we wish to evaluate the validity of our assumption that (table 2) can mask or skew the observed effects and lead to false negatives or misinterpretation of data. furthermore, terminal groups arising from the different raft agents used in this study to produce the polymers may also considerably change polymer properties [68] . with regards to the latter point, we note that in our hands, macromolecular prodrugs of rbv prior to and after removal of their cleavable end-groups revealed no difference in the antiinflammatory activity [42] . in this work, to accommodate the synthetic diversity of polymers, we used five different raft agents (table 1) and we see no correlation of polymer activity (table 2 ) and the raft agent used for the polymer synthesis. similarly, experimental results presented in this work appear to show no correlation with the polymer molar mass. blood anti-coagulation effect of the polymers (fig. 3) shows strong dependence on the nature of the polymer charged functionality (sulfonates > carboxylates > phosphates and phosphonates) but not with m n (dp). albumin binding results (probe displacement results, fig. 4 ) illustrate that polycarboxylates are strong albumin binders despite the variation of molar mass while phosphate-containing polyanions are weak binders, even the highest molar mass papa. drug release kinetics for macromolecular prodrugs (fig. 8) shows a statistically significant correlation with the polymer hydrophobicity but not with polymer dp. polymers most active in rbv delivery were not those characterized by the lowest or the highest dp (in fact, the lowest and the highest dp polymers were not effective in this screen) suggesting that polymer structure, not the number of repeat units in the structure, is decisive in the utility of the polymer as a carrier for the conjugated drug. without doubt, examples of strong, pronounced effects of molar mass on the biomedical properties of polymers are numerous and importance of this factor should not be overlooked. in our own prior work, with regards to the delivery of rbv, we found that high molar mass restricted the polymer cell entry and in doing so limited the observed therapeutic effects [39, 65] . not disregarding the importance of these observations, the data of this study strongly suggest chemical structure of the polymer is a dominant factor in its properties (at least in the experiments presented in this study) whereas polymer chain length is of secondary importance. the first important conclusion with regards to the activity of polymers is that the lead polymer structures with inherent broad-spectrum antiviral activity (peaa, pvbza, psvbs) [21] appear to also be strong anti-coagulants and strong albumin binders (figs. 3, 4) . in turn, weak anti-coagulants and weak albumin binders are also weak inhibitors of virus infectivity. this conclusion may not be altogether surprising in that both anti-coagulation and antiviral effects are likely due to the interaction between the polymers and a proteinaceous target (for antiviral effectsglycoproteins of the virus capsid). as such, this result implies that polyanionic macromolecular (pro)drugs are not well suited for systemic administration to exert antiviral activity in circulation. from a different perspective, the three leads with broad-spectrum antiviral activity [21] are also shown in this work to be inhibitory to the activity of polymerases and to possess inherent activity within macrophages, in absence of cytotoxicity. this observation is intriguing in that these three polymers may therefore be inhibitory to viruses both extraand intracellularly, in part fulfilling the desired combination of antiviral effects. in our view, these polymers become lead candidates for optimization and design of microbicides for non-systemic, localized antiviral treatments. regretfully, design of ribavirin mp was only considered in this work for (meth)acrylates and (meth)acrylamides; we are currently investigating the synthesis of mp based on pvbza and psvbs as a next step in the optimization these antiviral (pro)drugs. also worthy of note is the discovery of a family of polyanions that are devoid of any inhibitory activity in the screens we have performed, namely the polyphosphates and polyphosphonates. these polymers appear to have only weak and in most cases minimal interaction with the proteinaceous targets in our assays. due to this, as well as their high solubilizing power (as is well used in the design of low molar mass prodrugs [53] ) and inherent affinity to bone [69] , these polymers are highly attractive as carriers for diverse drug delivery applications and specifically for bone targeting. the results are an average of three independent experiments n = 3 ± sd. lower standard error bars were omitted for clarity. the statistical difference in inflammation between peaa and non-peaa treated cells was compared using unpaired student's t-test. p < 0.01**. summary of experimental results of this study and the screen for inherent broad-spectrum antiviral activity (**, taken from ref. [21] ). results are denoted as "+" if the polymer exhibited the nominated property (being increasingly pronounced on a scale from + to +++), "−" if the polymer is devoid of this property or effect, and "n/a" if the polymer was not analyzed in this test. anti-inflammatory activity is denoted '+' if the effect is observed with minimal cytotoxicity of treatment. all chemicals were purchased from sigma aldrich and used without further purification unless stated otherwise. ribavirin was purchased from apichemistry. 1 h nmr and 13 c nmr were recorded with a bruker biospin gmbh 400 mhz nmr spectrometer. multiplicities are indicated by abbreviations. s = singlet, bs = broad singlet, d = doublet, t = triplet, p = pentet, m = multiplet. hrms was recorded on a bruker maxis impact-tof-ms with electrospray ionization (esi+). size-exclusion chromatography (sec) was performed using a system comprising a lc-20ad shimadzu hplc pump, a shimadzu rid-10a refractive index detector and a wyatt dawn heleos 8 light scattering detector along with a spd-m20a pda detector, equipped with either 1) a hema-bio linear column with 10 μm particles, a length of 300 mm and an internal diameter of 8 mm from mz-analysentechnik in series with a ohpak sb-803 hq shodex column with the dimensions 8.0 × 300 mm a particle size of 6 μm or b) mz-gel sdplus linear column with 5 μm particles length of 300 mm and an internal diameter of 8 mm from mz-analysentechnik providing an effective molecular weight range of 1000-1.000.000 or c) superose 6 increase 10/300 gl from ge healthcare with a pore size of 7 um, a particle size of 8.6 um, an inner diameter of 10 mm and a length of 300 mm providing an effective molecular weight range of 5,000-5,000,000 da with an exclusion limit > 40,000,000 da. the solvent used was either a) 0.01 m pbs filtered through a 0.1 μm filter with 300 ppm sodium azide at 1.0 ml/ min at 40°c or b) dmf with 10 mm libr, at 1.0 ml/min at 40°c or c) 0.01 m pbs filtered through a 0.1 μm filter with 300 ppm sodium azide, at 0.75 ml/min at 30°c. values of dn/dc used for molar mass calculations were established by wyatt mals/astra software assuming full mass recovery. plate reader experiments were performed in black 96well optiplates on an enspire 2300 multilabel reader (perkin elmer®). monomer 1 (800 mg, 1.11 mmol, 1 equiv.) was dissolved in dry thf (20 ml) under a n 2 atmosphere. tea·3hf (0.905 ml, 5.55 mmol, 5 equiv.) was added and the reaction was stirred at room temperature for 24 h. the product was purified by flash column chromatography meoh/dcm 0:1 to 1:4. residual triethylamine was removed by dissolving the crude mixture in dcm, washing with nh 4 cl twice, once with water, and once with brine. the organic phase was dried over sodium sulfate, filtered, and concentrated in vacuo yielding the pure product. 1 tea (8.05 g, 79.55 mmol, 2 equiv.) was added over a cold (0°c) solution of 2-hydroxyethyl disulfide (12.28 g, 79.64 mmol, 2 equiv.) in dcm (150 ml) under n 2 atmosphere. acryloyl chloride (3.60 g, 39.77 mmol, 1 equiv.) was added dropwise maintaining the temperature at 0°c. the reaction mixture was heated slowly to room temperature and stirred for 1 h. the reaction was quenched and washed twice with nh 4 cl and brine. the organic phase was dried over sodium sulfate, filtered, and concentrated in vacuo. the product was purified by flash column chromatography pentane/etoac 70:30 to 60:40 yielding the pure product as a light yellow liquid (2.91 g, 35%). 1 a solution of ((2r,3r,4r,5r)-3,4-bis((tert-butyldimethylsilyl)oxy)-5-(3-carbamoyl-1h-1,2,4-triazol-1-yl)tetrahydrofuran-2-yl)methyl (4nitrophenyl) carbonate (4.11 g, 6.25 mmol, 1 equiv.) in dry dcm (120 ml) was added over a cold solution of 2-((2-hydroxyethyl)disulfaneyl)ethyl acrylate (2.69 g, 12.89 mmol, 2 equiv.), diea (2.50 g, 19.34 mmol, 3 equiv.) and dmap (0.157 g, 1.29 mmol, 0.20 equiv.) in dry dcm (350 ml) under n 2 atmosphere. the reaction mixture was stirred at room temperature for 40 h resulting in a conversion of 85%. the reaction was quenched and washed twice with nh 4 cl and brine. the organic phase was dried over magnesium sulfate, filtered, and concentrated in vacuo. the product was purified by flash column chromatography pentane/etaco 60:40 to 50:50. residual p-nitrophenol was removed by basic alumina column chromatography etoac/meoh 95:5 yielding the pure product as a colorless solid (1.20 g, 31%). 1 monomer 4 was synthesized following the protocol described above with an in situ deprotection performed without purification of the monomer 3. thus, the crude mixture obtained according to the protocol for the synthesis of monomer 3 was dissolved in thf (80 ml) under n 2 atmosphere. tea•3hf (4.45 g, 27.60 mmol) was added and the reaction was stirred for 72 h. the solvent was removed in vacuo and the product was purified by flash column chromatography dcm/meoh 100:0 to 90:10. residual tea was removed by dissolving the product in chloroform and washing twice with nh 4 cl yielding the pure product (0.430 g, 24%). 1 experimental details for the syntheses of ethylacrylic acid, propylacrylic acid, 2-methacrylamidoethyl phosphate, 2-acrylamidoethyl phosphate, 2-methacrylamidoethyl phosphonic acid and 2-acrylamidoethyl phosphonic acid were performed according the protocols published previously [21] . anionic homopolymers were synthesized as previously described in ref. dcma (2.57 mg, 0.00705 mmol, 1 equiv.) and v-70 (5.1 mg, 0.00165 mmol, 0.2 equiv.), were dissolved in methanol (0.2 ml). monomer 3 was dissolved in dmf (0.2 ml), amps dissolved in water (0.4 ml) was added. five rounds of freeze-pump-thaw were performed before the ampule was flame sealed and the reaction performed at 60°c for 64 h. the reaction mixture was diluted with a ph = 10.8 nahco 3 / na 2 co 3 buffer and purified by dialysis followed by lyophilisation to obtain the pure product (60 mg recovered). 1 4.1.14. poly(2-methacrylamidoethyl phosphonic acid-co-rbv) 2-methacrylamidoethyl phosphonic acid (0.115 g, 0.598 mmol, 200 equiv.) was dissolved in acetate buffer (0.3 ml). monomer 2 (0.0339 g, 0.0688 mmol, 23 equiv.) dissolved in meoh (0.15 ml) was added together with cdpa (0.00299 mmol, 1 equiv.) and acva (0.0015 mmol, 0.5 equiv.) stock solutions. methanol (0.2 ml) was added giving a slightly unclear solution. five rounds of freeze-pumpthaw were performed before the ampule was flame sealed and the reaction performed at 60°c for 72 h. the reaction mixture was diluted with a ph = 10.8 nahco 3 /na 2 co 3 buffer and purified by dialysis followed by lyophilisation to obtain the pure product (24 mg). 1 monomer 4 (0.126 mmol, 24 equiv.), cdpa (0.00214 g, 0.0053 mmol, 1 equiv.), and v-70 (0.00265 mmol, 0.5 equiv.) dissolved in methanol (0.25 ml) was added to 2-acrylamidoethylphosphonic acid (0.0949 g, 0.53 mmol, 100 equiv.) dissolved in water (0.1 ml). six rounds of freeze pump thaw were performed before the ampule was flame sealed and the reaction was performed at 30°c for 68 h. the reaction mixture was diluted with a ph = 10.8 nahco 3 /na 2 co 3 buffer and purified by dialysis, obtaining the pure product upon lyophilisation. 1 cdpa (0.961 g, 0.0024 mmol, 1 equiv.), v-70 (0.367 g, 0.00119 mmol, 0.5 equiv.), and monomer 2 (27 mg, 0.0548 mmol, 23 equiv.) were dissolved in methanol (0.3 ml) and added to 2-methacrylamidoethyl phosphate (100 mg, 0.478 mmol, 201 equiv.) dissolved in water (0.1 ml). the mixture was slightly unclear. five rounds of freeze pump thaw were performed before the ampule was flame sealed and the reaction performed at 30°c for 64 h. a small amount of precipitate was observed upon reaction, the mixture did not change notably in viscosity. the reaction mixture was diluted with a ph = 10.8 nahco 3 /na 2 co 3 buffer and purified by dialysis. a second dialysis was performed in 1:1 ethanol/water yielding the pure product which was lyophilized. 1 monomer 4 (0.129 mmol, 24 equiv.), cdpa (0.0022 g, 0.00545 mmol, 1 equiv.), and v-70 (0.00273 mmol, 0.5 equiv.) dissolved in methanol (0.25 ml) was added to 2-acrylamidoethyl phosphate (0.105 g, 0.538 mmol, 99 equiv.) dissolved in water (0.1 ml). six rounds of freeze pump thaw were performed before the ampule was flame sealed and the reaction performed at 30°c for 68 h. the reaction mixture was diluted with a ph = 10.8 nahco 3 /na 2 co 3 buffer and purified by dialysis. a second dialysis was performed in 1:1 ethanol/ water yielding the pure product upon lyophilization. 1 the competition assay was performed in 96-well plates, and fluorescence analyzed (excitation wavelength 360 nm, emission wavelength 465 nm) on a plate reader. polymers were pre-dissolved in minimal amounts of dmso and then diluted in 0.01 m pbs (ph 7.4). stock solutions of human serum albumin (hsa) and fluorescent probes (dansyl asparagine/dansyl sarcosine) were prepared in 0.01 m pbs (ph 7.4). the components were mixed and pbs added to obtain a total volume of 100 μl in each well, with the following concentrations: hsa 5 μm, dansyl sarcosine 5 μm or dansyl asparagine 15 μm, and polymer at 50 μm, corresponding to 10 equivalents compared to albumin. upon mixing the plates were incubated at 37°c for 1 h to allow the system to equilibrate. the data represents three independent experiments, which each included three technical replicates. polymers and albumin (2 g/l) were dissolved in 0.01 m pbs with 300 ppm nan 3 in equimolar amounts. the samples were incubated for 24 h at room temperature and then directly injected into the sec-mals system equipped with a biocolumn and using pbs as an eluent. results were analyzed using the wyatt software astra 6. a sample of albumin with no polymer added was used as reference in order to compare the albumin peak both with regards to the determined molecular weight and the elution times observed. ribavirin containing polymers were pre-dissolved in minimal amounts of ddmso (if necessary) and diluted in 0.01 m pbs (ph 7.4) prepared from d 2 o. glutathione (gsh) was also dissolved in 0.01 m pbs (ph 7.4) prepared from d 2 o. an aliquot of the polymer solution was mixed with an aliquot of the gsh solution to obtain a 0.4 ml sample containing 2.5 g/l of polymer and 12.5 mm gsh. the sample was incubated for the described time upon which ellmann's reagent was added in equimolar amounts compared to gsh to quench the reaction. the quenched samples were analyzed by 1 h nmr spectroscopy as described in the main text. time point measurements were repeated at least three times for each polymer. raw 264.7 macrophage cell line (tested negative for mycoplasma) was used to determine the anti-inflammatory activity of polyanions. the cells were seeded on 96-well flat-bottomed plate at initial density of 2 · 10 4 cells per well in 100 μl of media: high glucose dmem, stable lglutamine, without phenol red (gibco, 21063029) supplemented with 10% fbs and penicillin/streptomycin. three hours after the cells were seeded, the polymers were added at indicated concentrations in 100 μl of the media and incubated with the cells for 24 h. after 24 h, media was removed and the cells were washed with pbs. subsequently, the cells were stimulated with lps (sigma, l2654) for 24 h. the level of nitric oxide was quantified via griess assay. briefly, 50 μl of the cell supernatant was mixed with 50 μl of sulfanilic acid (10 g/l, 5% phosphoric acid). after 5 min of incubation, 50 μl of n-1-napthylethylenediamine dihydrochloride (1 g/l) was added and absorbance (548 nm) was measured using a plate reader (bmg labtech, ortenberg, germany). the level of nitrite was quantified against sodium nitrite standard curve and normalized to the negative control which was lpsstimulated cells only. cell viability was measured using prestoblue viability reagent (invitrogen, a13261). briefly, the cells were incubated with medium containing 10% volume of prestoblue reagent. after 1 h of incubation time the fluorescence (570 nmexperimental wavelength, 600 nmreference wavelength) was measured on the plate reader (bmg labtech, ortenberg, germany). the cell viability was normalized to the viability of the cells incubated with pure media. the blood obtained from healthy donors was collected into sodium citrate 3.2% tubes. platelet poor plasma (ppp) was obtained by centrifuging the blood at 3100g for 25 min at 20°c. the polymers were dissolved in pbs buffer containing 5% dmso. polymers in the solution were mixed with ppp in volume:volume ratio 1:9 resulting in the final polymers concentration of 10 and 100 mg/l. prothrombin time was measured using mrx owren's pt reagent (medirox, ghi 131-10) and activated partial thromboplastin time was measured using dade actin fs (siemens, b4218-100). all measurements were performed on sysmex cs-2100i. pbs and pbs with 5% dmso were used as a negative control. heparin (sigma, h3393) was used as a positive control. to analyze inhibitory effect of polymers on dna-dna polymerase activity, quantity pcr (qpcr) reaction was performed using power sybr green pcr master mix (life technologies) according to the protocol provided by the manufacturer. for one sample 10 μl of green pcr master mix were mixed with forward. 5′-ggtctctctggttagaccagat-3′ and reverse primer 5′-ctgctagagattttccacactg-3′, 0.46 ng of phxb2-env plasmid (nibsc, programme eva centre for aids reagents, reference number: arp206) and a polymer diluted in pbs to a desired concentration. primers were complementary to ltr upstream element of gag. the program used was 95°c for 5 min followed by 45 cycles with 95°c for 12 s, 62°c for 26 s and during the last cycle, a melting curve was made. a level of inhibition of taq polymerase was calculated by comparing to a positive control without polymer as 100% of enzyme activity. influence of polymers on activity of reverse transcriptase was determined using enzchek reverse transcriptase assay kit (life technologies). the reaction mixture was prepared according to the protocol provided by the manufacturer. subsequently polymers diluted in pbs were added at the given concentration, and then 5 u of mulv reverse transcriptase (life technologies, cat. nr. n8080018). the reaction was performed at 37°c for 1 h. nucleic acids were stained with picogreen dye and fluorescence was measured on a plate reader (bmg labtech, ortenberg, germany). cellular uptake of polyanions in macrophages was evaluated by confocal laser scanning microscopy. coverslips (thermofisher scientific) placed in 24 well plates were coated with 0.01% poly-l-lysine in water (sigma-aldrich) at 37°c for 2 h, and washed twice with pbs ph 7.4 (gibco). 10 5 raw 264.7 cells (mycoplasma negative) were seeded on each coverslip in the 24-well plate in 500 μl media (high glucose dmem, stable l-glutamine (gibco) supplemented with 10% fbs and penicillin/streptomycin). 24 h after seeding, media was removed from wells, and 500 μl of 40 mg/l of fluorescently labelled polyanion diluted in media was added to the well. 24 h after addition of polyanions, cells on coverslips were processed for confocal microscopy: coverslips were washed twice with pbs and fixed with 1% paraformaldehyde in pbs for 30 min at rt, followed by washing twice with pbs. coverslips were stained with 1 μg/ml dapi in pbs for 10 min, and dapi was removed by washing twice with pbs. cell membrane was stained with 50 μg/ml concanavalin a conjugated to alexa fluor® 633 (lifetechnologies) for 30 min at rt. following this staining step, coverslips were washed twice with pbs and transferred, cells facing down, to a microscopy slide (vwr), and then the cells were submerged in a prolong™ diamond antifade mountant (thermofisher scientific). slides were visualised using a zeiss lsm700 confocal with 63×/1.4 oil dic objective, using excitation line 405 nm for dapi, 488 nm for fitc and 633 nm for alexa fluor® 633 with no spectral overlap in collected emission. emerging viral diseases: confronting threats with new technologies global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the global burden of disease study the design of drugs for hiv and hcv approved antiviral drugs over the past 50 years combating emerging viral threats advances in antiviral vaccine development topical microbicides to prevent hiv: clinical drug development challenges microbicides and other topical strategies to prevent vaginal transmission of hiv broad-spectrum antiviral agents broad-spectrum agents for flaviviral infections: dengue, zika and beyond the use of interferon-alpha in virus infections is the 25-year hepatitis c marathon coming to an end to declare victory? ribavirin -current status of a broad spectrum antiviral agent antiviral agents active against influenza a viruses broad-spectrum antiviral that interferes with de novo pyrimidine biosynthesis nitazoxanide: a first-in-class broad-spectrum antiviral agent a novel peptide with potent and broad-spectrum antiviral activities against multiple 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structures, focusing on those of green seaweeds guide to anticoagulant therapy: heparin: a statement for healthcare professionals from the extracellular rna constitutes a natural procoagulant cofactor in blood coagulation the neonatal fc receptor, fcrn, as a target for drug delivery and therapy prodrugs in medicinal chemistry and enzyme prodrug therapies materials and methods for delivery of biological drugs synthetic polymer with a structuredriven hepatic deposition and curative pharmacological activity in hepatic cells the interaction of ms-325 with human serum albumin and its effect on proton relaxation rates crystal structure analysis of warfarin binding to human serum albumin -anatomy of drug site i glutathione homeostasis and functions: potential targets for medical interventions disulfide-cleavage-triggered chemosensors and their biological applications drug delivery strategy utilizing conjugation via reversible disulfide linkages: role and site of cellular reducing activities ligand-targeted drug delivery disulfide reshuffling triggers the release of a thiol-free anti-hiv agent to make up fast-acting, potent macromolecular prodrugs prodrug strategies in anticancer chemotherapy competitive reactions in solutions of poly-l-histidine, calf thymus dna, and synthetic polyanions: determining the binding constants of polyelectrolytes macromolecular prodrugs of ribavirin: concerted efforts of the carrier and the drug tools of gene transfer applied to the intracellular delivery of non-nucleic acid polyanionic drugs inhibition of lipopolysaccharide and il-1 but not of tnf-induced activation of human endothelial-cells by suramin phosphorus-containing polymers: a great opportunity for the biomedical field we wish to acknowledge financial support from the danish council for independent research, technology and production sciences, denmark (anz; project dff -4184-00177). key: cord-343409-oao75pzy authors: hayward, joshua a; tachedjian, mary; cui, jie; field, hume; holmes, edward c; wang, lin-fa; tachedjian, gilda title: identification of diverse full-length endogenous betaretroviruses in megabats and microbats date: 2013-03-27 journal: retrovirology doi: 10.1186/1742-4690-10-35 sha: doc_id: 343409 cord_uid: oao75pzy background: betaretroviruses infect a wide range of species including primates, rodents, ruminants, and marsupials. they exist in both endogenous and exogenous forms and are implicated in animal diseases such as lung cancer in sheep, and in human disease, with members of the human endogenous retrovirus-k (herv-k) group of endogenous betaretroviruses (βervs) associated with human cancers and autoimmune diseases. to improve our understanding of betaretroviruses in an evolutionarily distinct host species, we characterized βervs present in the genomes and transcriptomes of megaand microbats, which are an important reservoir of emerging viruses. results: a diverse range of full-length βervs were discovered in megaand microbat genomes and transcriptomes including the first identified intact endogenous retrovirus in a bat. our analysis revealed that the genus betaretrovirus can be divided into eight distinct sub-groups with evidence of cross-species transmission. betaretroviruses are revealed to be a complex retrovirus group, within which one sub-group has evolved from complex to simple genomic organization through the acquisition of an env gene from the genus gammaretrovirus. molecular dating suggests that bats have contended with betaretroviral infections for over 30 million years. conclusions: our study reveals that a diverse range of betaretroviruses have circulated in bats for most of their evolutionary history, and cluster with extant betaretroviruses of divergent mammalian lineages suggesting that their distribution may be largely unrestricted by host species barriers. the presence of βervs with the ability to transcribe active viral elements in a major animal reservoir for viral pathogens has potential implications for public health. retroviruses (family retroviridae) are a diverse and widely distributed family of rna viruses distinguished by their use of a viral rna-dependent dna polymerase (reverse transcriptase; rt) and ability to integrate into the genomes of their cellular hosts [1] . in addition to the existence of infectious viral particles that are horizontally transmitted between hosts (exogenous retroviruses), the capacity of retroviruses to integrate into the host germline also generates vertically transmissible endogenous retroviruses (ervs) [1, 2] . ervs may or may not be capable of producing infectious viral particles, and germline integration over the course of multiple generations typically leads to the accumulation of mutations that render them defective and non-functional [2] . the retroviral family is composed of seven genera: alpharetrovirus, betaretrovirus, gammaretrovirus, deltaretrovirus, epsilonretrovirus, lentivirus, and spumavirus [3] . the genomic organization of retroviruses is classified as either 'simple' or 'complex' , with simple retroviruses encoding the structural polyproteins gag and env, and the functional polyproteins pro and pol [4] . complex retroviruses encode additional accessory and regulatory proteins with diverse functions that typically establish and maintain virus replication and pathogenesis [5] . the core elements of all retroviruses are flanked by a pair of typically untranslated nucleotide regions at their 5′ and 3′ ends. in the provirus, formed by integration of the viral cdna into the host cell chromosome, these regions are referred to as 'long terminal repeats' (ltr) [4] . exogenous retroviruses of zoonotic origin have been associated with disease in humans, the most notable being human immunodeficiency virus (hiv) [6] . other retroviruses such as human foamy virus (hfv) and human t-cell leukemia virus (htlv) are known to be capable of infecting humans [7, 8] . the retroviruses most recently associated with human disease are betaretroviruses. the up-regulation of gene products derived from the human endogenous retrovirus-k (herv-k) group of betaretroviruses has been linked to a diverse range of cancers such as those of the breast, ovaries, and prostate alongside other significant human maladies [9, 10] . the genus betaretrovirus consists of the type b and type d groups of exogenous and endogenous retroviruses and the herv-k group of endogenous retroviruses. among the exogenous, infectious members of the genus are the type b mouse mammary tumour virus (mmtv), the type d jaagsiekte sheep retrovirus (jsrv), which causes pulmonary carcinoma in sheep, and the type d mason-pfizer monkey virus (mpmv) which causes wasting and immunosuppression in new-born rhesus monkeys [11] [12] [13] . all betaretroviruses utilize variants of the lysine trna primer binding site (pbs) and encode a deoxyuridine triphosphatase (dutpase), within their pro gene which functions as a nucleocapsid-dutpase fusion protein [14] [15] [16] . type b and type d betaretroviruses differ in several respects including their complement of accessory factors, virion morphology, strategies for rna nuclear export, and the length of their ltr regions. type b betaretroviruses contain spherical viral cores and have ltrs of~1,200 nucleotides while type d contain cylindrical viral cores and have ltrs of~300 nucleotides. the prototypical type b betaretrovirus, mmtv, encodes the accessory proteins regulator of export of mmtv mrna (rem) and negative acting factor (naf ), which have roles in viral mrna export, protein synthesis and gene expression [17] [18] [19] , in addition to the virulence factor, superantigen (sag) [20] . the type d retrovirus jsrv has been shown to encode the trans-acting factor rej which has a role in protein synthesis and may assist rna nuclear export [21] . while no distinct oncogenes or sag-like virulence-associated proteins are known to be encoded by type d betaretroviruses, the env protein of jsrv is associated with oncogenesis [13, 14] . there are two major strategies employed by betaretroviruses to export unspliced or partially spliced viral rna from the nucleus that use distinct export pathways. complex betaretroviruses such as mmtv employ a hiv rev-like accessory protein encoded within the env gene that binds and facilitates export of intron containing retroviral rna by recruitment of the cellular karyopherin export factor, chromosome region maintenance 1/exportin 1 (crm1/xpo1) [17, 19] . simple betaretroviruses such as mpmv contain a constitutive transport element (cte) within the nucleotide sequence at the 3′ end of the retroviral genome that recruits a cellular binding factor, tap (nuclear rna export factor 1; nxf1) which mediates nuclear export [22, 23] . importantly, ervs provide a unique opportunity to study the evolutionary history of this family of viruses as they are essentially genetic 'fossils' of past retroviral infections [2, 24] . as such, their existence serves as an indication of the potential host range of a given retroviral lineage and may be interpreted as evidence for the possible existence of exogenous retroviruses that have yet to be isolated. indeed, previous studies have reported a number of endogenous betaretroviruses (βervs) in species for which no exogenous betaretrovirus has yet been identified. these include mammalian species as diverse as primates, horses, rats, lemurs, and an australian marsupial, the common brushtail possum [25] [26] [27] . there are over 1,100 known species of bats (order chiroptera), accounting for approximately 20% of all mammalian species [28] . bats are relatively divergent from other mammals, having branched off from the perissodactyla (containing horses) approximately 88 million years ago (mya) [29] . they are divided into two major groups: megabats (suborder megachiroptera) which are mainly fruit-eating, and microbats (suborder microchiroptera), small insectivores that navigate by means of echolocation [30] . notably, bats harbour over 100 viral species from a diverse range of virus families including the paramyxoviridae, coronaviridae, herpesviridae, rhabdoviridae, arenaviridae, togaviridae, flaviviridae, orthomyxoviridae, reoviridae, bunyaviridae, filoviridae, and picornaviridae [31] . bats, belonging to the mammalian superorder laurasiatheria, are a major viral reservoir that is evolutionarily distinct from another major viral reservoir, rodents, which together with primates belong to the superorder euarchontoglires [29, 32] . bats have recently gained attention as they have been implicated in numerous newly emerging diseases of humans caused by viruses such as sars-coronavirus, hendra virus, nipah virus, and the ebola virus [33] [34] [35] . this track record of zoonotic transmission of previously unknown viral pathogens from bats to humans has prompted calls for a proactive approach to future emerging diseases originating in bats [30] . to this end a natural history survey of bats has begun, and we have recently reported the discovery of diversified defective endogenous gammaretroviruses in both mega-and microbats [36, 37] . previous studies of βervs have tended to focus on isolated viruses, although a report on the βervs of murid hosts indicated that the genus betaretrovirus might possess a diverse and previously unrecognized range of sub-types extending beyond the classical type b/type d paradigm [25] . using transcriptome and genome analyses of the megabats pteropus alecto (black flying fox) and pteropus vampyrus (large flying fox), and the microbats myotis lucifugus (little brown bat), rhinolophus megaphyllus (eastern horseshoe bat), and rhinolophus ferrumequinum (greater horseshoe bat), we herein examine βervs present in a diverse range of bat species. in conjunction with phylogenetic analyses, we incorporated the diversity of genomic organizations and the use of specific lysine trna pbs to identify eight distinct groups of betaretroviruses. to determine if bats contained and expressed a full suite of integrated endogenous betaretroviral genes we generated and analyzed transcriptome databases of p. alecto, r. megaphyllus, and r. ferrumequinum. gag, pol, and env protein sequences were translated from the genomes of extant betaretroviruses: mmtv, jsrv, mpmv, squirrel monkey retrovirus (smr), and simian retrovirus (srv). local tblastn searches were conducted to determine if the transcriptomes contained nucleotide sequences that, when translated into any of their six reading frames, contained significant protein sequence similarity to the betaretoviral protein query sequences. because the variation in length between different transcripts causes difficulty when interpreting relatedness if similarity is expressed as a percentage identity, the significance of the similarity levels observed was determined on the basis of the e-value (probability of random sequence identity) of the blast hits. each transcriptome was found to contain mrna sequences with notable similarity (e-values < 1×10 -10 ) to the betaretroviral proteins gag, pol, and env, with the exception of the r. ferrumequinum transcriptome in which no betaretroviral gag-like transcripts were identified (table 1) . reciprocal blastx searches of the transcript hits with the lowest e-values (i.e. the top hits presented in table 1 ) against the ncbi non-redundant protein database returned predominantly betaretroviral hits. the majority of the mrna sequences identified within the bat transcriptomes were partial, not being of sufficient length to reveal an entire gag, pol, or env gene sequence. as a point of reference, the nucleotide sequence lengths of mpmv gag, pol, and env are 1,974, 2,583, and 1,758, respectively, while the majority of the transcripts identified in the blast analyses were <1,000. the p. alecto transcriptome was found to contain two retroviral transcripts 5,433 and 5,830 nucleotides in length which overlap each other by 3,152 bases with 100% sequence identity. the extent of overlap and perfect identity indicated that the two sequences likely represented a fulllength retroviral genomic sequence >8,103 bases in length that was later determined through phylogenetic analysis to be a βerv. this full-length p. alecto βerv genomic transcript was named paerv-βa (pteropus alecto endogenous retrovirus -betaretrovirus a) ( figure 1 ). in addition to paerv-βa, different transcripts covering the length of one distinct betaretroviral pol transcript (papol-01) most closely related to jsrv pol and one env transcript (paenv-01) similar to type c gammaretrovirus and mpmv-like type d betaretrovirus env were identified in the p. alecto transcriptome. a single transcript (rfenv-01) covering the length of a gammaretrovirus-like env gene most similar to rd114 env was identified in the r. ferrumequinum transcriptome. these transcripts were incorporated into the subsequent phylogenetic analyses. the paerv-βa sequence was found to begin 25 nucleotides upstream of the gag start methionine and contains all of the expected core retroviral genes along with the betaretroviral dutpase domain ( figure 1 ). all of the genes were found to be defective as they each contained frameshift mutations. in addition, the pol and env genes contained premature stop codon mutations. identification of a 19 nucleotide polypurine tract (ppt) allowed the delineation of the beginning of the unique 3′ (u3) region. conserved retroviral active site motifs were present in the protease (dxg), reverse transcriptase (ddd), and integrase (dde) domains. the major homology region (mhr; nucleotide coordinates 1,456 -1,496) and zinc fingers (nucleotide coordinates 1,752 -1,805 and 1,866 -1,919) conserved in gag were also present. two additional orfs were identified; the first overlaps the 3′ end of the pro gene while the second overlaps the u3 region. however, protein translations of the orfs compared to the publicly accessible protein family (pfam) database revealed no known protein domains. in addition, blastp analysis of the translations against the ncbi non-redundant protein database yielded no hits. later identification of closely related p. vampyrus βervs (pverv-βj and pverv-βk) indicated that the orf overlapping the u3 region was not legitimate. given the successful identification of betaretrovirus-like nucleotide sequences in the transcriptomes, we sought to mine the publicly available genomes of p. vampyrus and m. lucifugus for full-length endogenous betaretroviruses. the aforementioned extant betaretoviral protein sequences together with the retroviral mrna sequences identified in the bat transcriptomes were used to conduct tblastn and tblastx searches on the p. vampyrus and m. lucifugus genomes. these searches revealed a number of hits in the genomes that contained betaretroviral gag, pol, and env genes. full-length ervs were delineated by the identification of retroviral gag, pol, and env sequences positioned next to each other and located between a pair of ltrs. in total, we identified 11 full-length βervs in p. vampyrus and six in m. lucifugus (table 2 ). these bat βervs contain all of the expected core elements and the betaretrovirus-specific dutpase domain. as retroviruses were previously categorized based on the specific trna that anneals to their pbs required for initiation of reverse transcription, we determined the specific trna used by all identified bat βervs through nucleotide alignment with known mammalian lysine trna sequences (additional file 1: figure s1 ). the pbs was intact and could be identified in the majority of the bat βervs, and all but one (mlerv-βe) was found to harbour a pbs complementary to either trna lysine 1,2 (lys 1,2) or trna lysine 3 (lys 3) typical of betaretroviruses. reciprocal blastp searches confirmed that the gag, pol, and env of these full-length ervs were more similar to known betaretroviral proteins than those of other retroviral genera with pol sequence similarities ranging from 64% to 76% (additional file 2: table s1 ). all of the bat βervs possessed ltrs of 300-500 nucleotides in length, as expected for type d betaretroviruses with the exception of pverv-βb with ltr length typical of type b betaretrovirues (1265 bp) ( table 2 ). each bat βerv was found to contain a ppt immediately upstream of their 3′ ltr regions. we analyzed each pro and pol figure 1 a schematic representation of paerv-βa. two transcripts were identified in the p. alecto illumina sequenced transcriptome that overlapped by 3,152 nt with 100% sequence identity which were used to assemble the paerv-βa genomic sequence. indicated are the retroviral genes gag, pro, pol, and env, which have been rendered defective by random mutation since integration. also shown are the key enzymatic active sites of the viral protease (d×g), reverse transcriptase (ddd), and integrase (dde); the betaretroviral dutpase domain in pro; two unique open reading frames (orfs); the polypurine tract (ppt); and the (unique 3') (u3) region. orf* does not appear to be genuine, but rather has arisen as a result of an insertion mutation that has disrupted a stop codon. gene and identified the expected enzymatic active site motifs in the retroviral protease (d×g), reverse transcriptase (ddd), and integrase (dde) domains. the gag gene of each βerv contained the expected mhr and zinc-knuckles. while the m. lucifugus genome sequencing coverage was relatively high (7× coverage), the p. vampyrus genome has only been sequenced to 2.6x coverage. the nature of a low-coverage genome such as this means that within the assembled 'scaffolds' there occasionally exist stretches of nucleotides of ambiguous identity. in this regard, several of the bat βervs reported herein contain short 'non-sequenced regions' (nsr) ( table 2) . as a result, the pbs present in pverv-βa and the mhr of pverv-βb could not be identified as they contained nsrs overlapping those elements. to confirm that each βerv was the product of a retroviral integration event, the four-nucleotide repeats known as genomic target site duplication (tsd) sequences that flank the proviruses were identified (additional file 2: table s2 ). tsds were identified for all proviral βervs with the exception of pverv-βd and f whose 5′ ltrs were masked by nsr, the genome size is given for the proviral version of the βervs. § the genome size of paerv-βa is uncertain as the known sequence begins 25nt upstream of the gag gene and does not include the (unique 5') region. b the core retroviral genes gag, pro, pol, and env that contain frameshift or premature stop mutations are described as 'defective' , those that contain neither of these are described as 'intact' in bold font. c the pro open reading frame (orf) of each βerv was found to encode a betaretroviral dutpase protein domain. d the number of orfs that do not code for the core genes and are 300 nucleotides or greater in length. e the length of the long terminal repeats (ltrs). * for those βervs whose 5′ and 3′ ltr lengths differ, the value of the 5′ ltr is given. f the specific lysine (lys) trna complementary to the primer binding site (pbs) for each βerv is given. † the specific identity of the pbs of mlerv-βe is uncertain. nsr: non-sequenced region. pverv-βk whose 3′ ltr appears to be truncated, and pverv-βb which is the sole βerv to have intact and unambiguous ltrs yet no identifiable tsds. to determine if closely related clusters of βervs were generated as a result of post-integration chromosomal duplication events, we compared their flanking chromosomal dna through a blastn analysis (additional file 2: table s3 ). one pair of bat βervs (pverv-βk and pverv-βj) was found to have homology in the chromosomal regions immediately up-and downstream of the proviruses. pverv-βk and pverv-βj appear to have arisen as a result of a duplication of a single integrated provirus. the truncation of the 3′ ltr of pverv-βk suggests a chromosomal duplication event. next, we examined the phylogenetic relationships of the bat βervs identified in our analysis of the bat genomes and transcriptomes (table 2) . accordingly, the gag, pol, and env of the full-length bat βervs were aligned with those of known exogenous and endogenous betaretroviruses and phylogenetic trees were estimated for each ( figure 2 ). in all three trees a great diversity of bat βervs was observed, with individual βervs clustering with members of the type d (e.g. mpmv and jsrv), type b (e.g. mmtv), and herv-k groups. the close relationship between viral sequences derived from transcriptomes and some endogenous viral sequences mined from bat genomes suggests that at least some of the bat βervs have the ability to transcribe. notably, a number of bat βervs (pverv-βj, k and paerv-βa), together with several exogenous betaretroviruses, were found to possess env sequences that formed a cluster so highly divergent, and more closely related to gammaretroviruses, as to require omission from the initial betaretroviral env tree bootstrap values are denoted as ** >90%; * >70% and < 90%. the trees are midpoint rooted for purposes of clarity only. βerv proteins of p. vampyrus and p. alecto are highlighted in red text. βervs of m. lucifugus are highlighted in blue text. the clades within the gag and pol trees highlighted with a grey background (γ-env) contain betaretroviruses whose env sequence is not sufficiently closely related to the env of other betaretroviruses to be included in the env tree. ( figure 2c ). finally, we also found some evidence for within-genome recombination (e.g. mlerv-βc, d and e) as reflected in the phylogenetic incongruence between the gag and pol and env trees. our reciprocal tblastx searches indicated that the p. alecto erv (paerv-βa) and two of the p. vampyrus ervs (pverv-βj and k) encoded env sequences that were more similar to gammaretroviral env, while still possessing gag and pol sequences that closely resembled those of known betaretroviruses (see above). to confirm this observation we undertook a phylogenetic analysis of the env sequences of known gammaretroviruses and betaretroviruses, together with the newly identified βerv env sequences ( figure 3 ). this analysis confirmed previous observations [12, 38] that the env sequences of some extant type d betaretroviruses, namely mpmv, smr and simian retrovirus serotypes 1 and 4 (srv1 and srv4), cluster with gammaretroviral env, as do those of pverv-βj, k, paerv-βa, paenv-01 (env sequence derived from p. alecto), and rfenv-01 (env sequence derived from r. ferrumequinum). other type d retroviruses such as jsrv and the enzoonotic nasal tumor viruses (entv) of sheep and goats did not fall into this cluster. this indicates that a recombination event has occurred, in which a sub-lineage of type d betaretroviruses acquired a gammaretroviral env gene. our analysis of the full-length bat βervs revealed an unexpected diversity of genomic organizations, as a number were found to contain unique orfs. some of these orfs were in alternative reading frames within the core element domains and others were either upstream of gag, or downstream of env. furthermore, the differential use of trna lys 1,2 and trna lys 3 was not found to be restricted to either type b or type d betaretroviruses. rather, it appears that a switch between the two has occurred multiple times throughout the history of the genus. this diversity of genomic organization was used in conjunction with the phylogenetic analyses of gag, pol, and env (with prime consideration given to the highly conserved pol phylogeny) and the trna usage to identify eight distinct groups within the betaretrovirus genus ( figure 4 ). the eight betaretroviral subgroups that we propose are distinguished from each other by major evolutionary differences such as deep phylogenetic divergence with strong bootstrap support (>90% of trees resolving the clade), significant mutations in key genetic features such as a switch to the use of a different pbs, or the presence of retroviral genes from a different genus. group i (represented by herv-k113) consists of the herv-k group of endogenous betaretroviruses which contain a pbs similar to trna lys 1,2 and have a deep phylogenetic divergence from other betaretroviruses. no known exogenous betaretroviruses or bat βervs currently reside in group i. group ii (represented by mlerv-βa) consists of a phylogenetic cluster of endogenous bat βervs that branched off from group i early in betaretroviral history. three bat βervs are included in this group. the pbs of mlerv-βa and mlerv-βb are complementary to trna lys 1,2 and lys 3, respectively, while the trna usage of pverv-βa is unknown as a 100 nucleotide nsr overlaps its pbs. mlerv-βa contains a large 1,493 nucleotide insertion within its 5′ ltr that contains a 323 codon orf. this insertion presumably arose post-integration and the nature of this genetic element is unknown. a pfam domain search and blastp analysis of the translation of the orf against the ncbi non-redundant protein sequence database did not identify any known protein domains or similarity to any known protein. group iii (represented by mlerv-βc) consists of microbat ervs that possess a phylogenetically divergent pol (bootstrap support >90%) and a pbs complementary to trna lys 3. within this group is mlerv-βc, the first fully intact bat βerv to be identified, and which raises the possibility that exogenous members of group iii may yet exist as undiscovered infectious betaretroviruses. group iv (represented by mlerv-βe) appears to have diverged as a part of the type b betaretroviral lineage. however, the precise phylogenetic position of group iv's sole member, mlerv-βe, is not supported by high bootstrap support in any of the trees. furthermore the precise identity of its pbs is uncertain. the pbs does not appear to be specifically complementary to either lys 1,2 or lys 3 trna, but rather it appears to be complementary to an alternative mammalian lysine trna. there are presently no known extra copies of mlerv-βe within the m. lucifugus genome. mlerv-βe is distinguished by its possession of a unique orf upstream of gag. this orf begins within the 5′ ltr and terminates three nucleotides upstream of the gag start methionine, within the same reading frame. orfs upstream of gag may be relevant to gag expression considering that murine gammaretroviruses encode an alternative n-terminally extended version of gag, glyco-gag, that has a role in the promotion of viral replication [39, 40] . no promoter elements or tata boxes were predicted to exist upstream of the orf, however a tata box is predicted within the orf coupled with a possible start methionine downstream, encoding a potential 84 amino acid protein. group v (represented by pverv-βb) consists of archetypically structured type b betaretroviruses (mmtv-like) that contain long ltrs (~1,200 bases). it is possible that the extension of the 3′ ltr has facilitated the emergence of orfs in this location as in the case of mmtv's sag gene. in this regard, pverv-βb has an orf within its 3′ ltr. this orf is 123 codons in length, much shorter than mmtv's sag protein, which is 320 amino acids long. while it is possible that the orf was longer at integration and has simply been interrupted by stop codon mutations since that time, a tblastn analysis of mmtv's sag protein against the 3′ ltr of pverv-βb did not reveal any significant sequence similarity. also in this group is eqerv, an endogenous horse betaretrovirus, which does not contain a sag gene or sag-like orf within its 3′ ltr [27] . group vi (represented by pverv-βd) consists of jsrv-like type d betaretroviruses that contain short ltrs (~300 bases) and env protein sequences that do not phylogenetically cluster with those of the gammaretrovirus genus. members of this group harbour a pbs complementary to trna lys 1,2 and may or may not contain additional orfs within their core element domains, as is the case for jsrv and entv's orf-x located within pol, and pverv-βd, which has an orf overlapping the 3′ end of the env gene. group vii (represented by pverv-βf) consists wholly of bat βervs. group vii members are phylogenetically type d-like and are primarily distinguished by a pbs complementary to trna lys 3 as opposed to trna lys 1,2 which is the expected pbs complementarity for type d betaretroviruses. also, several bat βervs in this group possess a unique orf upstream of gag that is distinct from that of group iv's mlerv-βe. this orf begins within the 5′ ltr and terminates 26 nucleotides upstream of the gag start codon. promoter elements and tata boxes are predicted to exist upstream of this orf. as there were differences in the start position of this orf in the various group vii bat βervs (pverv-βe -i), likely due to random mutation since integration, a nucleotide alignment of the region was generated (additional file 1: figure s2 ). the alignment demonstrated that the consensus orf contained a possible start methionine that would code for a 101 amino acid protein. one member of this group, pverv-βe, is almost fully intact as it does not appear to contain any frameshift mutations and only a single premature stop codon within the pro gene. group viii (represented by pverv-βj) consists of mpmv-like type d betaretroviruses. the distinguishing feature of this group is the possession of an encoded env polyprotein that phylogenetically clusters with those of gammaretroviruses rather than those of other betaretroviruses. the bat βervs in this group have an additional feature which is an orf beginning 40 bases downstream of the env stop codon and terminating 15 bases into the 3′ ltr. this is exemplified in pverv-βj. a nucleotide sequence alignment of the extreme 3′ region (additional file 1: figure s3 ) of the closely related pverv-βj, k, and paerv-βa generated a consensus sequence that contained this orf and revealed that the equivalent orf sequences in pverv-βk and paerv-βa are respectively interrupted by a frameshifting deletion mutation and stop mutation. this orf contains a possible start methionine that would generate a 90 amino acid protein. this alignment also indicated that the alternative orf* in paerv-βa (figure 1 ) was likely to be an artifact as the u3 region contained an eight nucleotide insertion that disrupts a stop codon which, if the insertion did occur after integration, has generated an artificial orf. the paerv-βa genome was derived from illumina based transcriptome sequencing while the pverv-βj and pverv-βk genomes were derived through wholegenome shotgun/sanger sequencing. accordingly, each method can be used to orthogonally verify the other. a full alignment of the three proviruses (additional file 3: figure s4 ; demonstrating 96.66% nucleotide identity between pverv-βj and pverv-βk and 93.77% between pverv-βk and paerv-βa) supports the veracity of these proviral sequences and provides further evidence that the group viii βervs are likely derived from a single integration event. the unique orfs identified in the bat βervs of all groups were subjected to a blastp analysis against the ncbi non-redundant protein database and pfam domain search. however, no blast hits or known protein domains were identified. to determine if the groupings we had assigned were congruent with known functional differences between retroviruses with respect to betaretroviral rna nuclear export strategies, we analyzed the bat βervs, alongside known exogenous and endogenous betaretroviruses, for evidence of motifs indicative of the major export strategies (additional file 2: table s4 ). to this end we employed a computational analysis to search for the presence of nuclear localization signals (nls) and nuclear export signals (nes) common to the retroviral rev-like proteins used in the archetypal rev/rev-responsive element (rre) equivalent export mechanism. we also searched for the presence of tap-binding elements (tbe) within and downstream of the env gene, which would imply the utilization of the cte export pathway, and for direct nucleotide repeats (dr) and inverted nucleotide repeats (ir) that might suggest the formation of stem-hairpin-loop structures known to be associated with the cte [23] . while a number of βervs were predicted to contain either an nls or an nes, only mlerv-βb and pverv-βb were found to contain both. these βervs broadly cluster with herv-k and mmtv, which respectively encode the rev-like proteins rec and rem, and the presence of both nls and nes points to the possibility that they encode rev-like proteins and make use of the crm1 nuclear rna export pathway. the majority of the βervs in group vii were found to contain tbe, indicating that the original exogenous forms of these retroviruses likely utilized the nuclear export pathway accessed by the cte. we used an analysis of the ltrs to estimate the time since integration of the bat βervs. this analysis evaluated the extent of the difference between the nucleotide sequences of the 5′ and 3′ ltrs of each βerv, which are expected to be identical at the time of integration. the number of nucleotide differences between the 5′ and 3′ ltr is assumed to be proportional to the time since integration, although this may be compromised by such factors as gene conversion [41] . under this assumption, all βervs integrated into the genomes of the ancestors of modern bats within a wide time range of between 3.2 and 36.3 million years ago (mya), and hence long after the divergence of bats from other mammalian lineages (table 3) . this, in turn, suggests that (i) that the original exogenous forms of these βervs targeted ancient bats, and (ii) there has been a continual integration of betaretroviruses into bat genomes during their evolutionary history. we coupled our analysis of the genomic features of the bat βervs with the phylogenetic patterns observed in the gag, pol, and env trees (with primacy given to the phylogeny of the highly conserved polymerase sequences) to generate a hypothetical series of events that may have led to the current state of diversity in the genus betaretrovirus ( figure 5 ). our analysis indicates that while the ancient progenitor betaretrovirus likely made use of a trna lys pbs, its specific identity is uncertain. groups i and ii appear to have branched off together early in betaretroviral history. this has led, in the case of the herv-k betaretroviruses, to the emergence of distinct genetic elements such as the np9 and rec proteins, whose current endogenized forms have possible roles in tumorgenesis [42, 43] . group iii's phylogenetic position places its point of divergence after the split of groups i and ii but prior to the split between the type b and type d lineages. the divergence between type d and type b βervs seems to have occurred as a result of their differential use of trna lys 1,2 and trna lys 3, respectively. within the type b lineage are groups iv and v which, although possibly splitting after the divergence of type b and type d, differ in the length of their ltrs, their trna usage, and their additional genetic elements. within the type d lineage an early event appears to have been a recombination between a betaretrovirus and a gammaretrovirus, which has caused a divergence between jsrv-like and mpmv-like type d betaretroviruses. in this split, group viii appears to have diverged from groups vi and vii through the acquisition of a gammaretroviral env gene. group vii later diverged from group vi by a switch from the use of trna lys 1,2 to trna lys 3 and differentiation of their additional orfs. we searched for the expression of betaretroviral genes in the transcriptomes of the megabat p. alecto and the microbats r. megaphyllus and r. ferrumequinum. through this analysis we determined that betaretroviral genes were being transcribed into mrna within each species and we identified that a full-length genomic transcript of a betaretrovirus (paerv-βa) was being expressed in p. alecto. as each of the genes of paerv-βa were found to contain mutations that likely rendered them non-functional, it seems reasonable to conclude that the transcript was expressed from a defective βerv rather than a functional exogenous betaretrovirus. it is important to note that we cannot exclude the possibility that the reported paerv-βa transcript was derived from multiple similar sequences during transcriptome assembly and due to recombination between similar transcripts during cdna synthesis or pcr as published [44] . our analysis of the genomes of the megabat p. vampyrus and the microbat m. lucifugus revealed that they contain a genetically diverse range of full-length βervs. in the case of m. lucifugus this included an intact βerv (mlerv-βc) that did not contain any mutations that would clearly render the gene products non-functional. however, it should be noted that as revealed by the ltr analysis, nucleotide substitutions have occurred in the mlerv-βc sequence. while the critical enzymatic active site motifs are intact, whether or not the nucleotide substitutions that have occurred in the coding domains would have a detrimental effect on the functionality of the gene products is not known. in analyzing the genetic content of the full-length βervs for the presence of orfs, aside from those coding for the core genes, we set a minimum cut-off of 100 [25] . nd: not dated; these βervs could not be dated using this method. pverv-βd and pverv-βf contained non-sequenced regions within their 5′ ltr, while pverv-βg and pverv-βk contained bulk deletions within their 3′ ltrs. codons to limit the amount of incidental non-coding orfs that would be identified. however, many retroviral accessory and regulatory genes, such as rec and np9 of herv-k and vpr and tat of hiv-1, are shorter than 100 codons and are often encoded over the span of two exons. despite the high minimum cut-off, it is striking that the bat βervs possessed a diverse array of additional orfs. while we cannot confirm that these are indeed protein coding domains, much less speculate on their function, the existence of similar elements is not without precedent among the betaretroviruses. one example is the 'orf x' of jsrv, the function of which is unknown but it has been found to be broadly conserved amongst jsrv isolates [45] . several of the orfs we identified overlap the proviral ltrs, which consist of typically untranslated regions. this is also not unprecedented, with a prime example being the sag gene of the betaretrovirus mmtv, which is situated entirely within the u3 region of the 3′ ltr. the presence of unique orfs in βervs may indicate the evolution of novel retroviral genes whose products have regulatory or accessory functions required for the retroviral life-cycle and/or pathogenesis. in addition to the βervs reported in this study we noted the presence in both mega-and microbats of betaretrovirus-like retroelements that resemble βervs but lack the env gene; these were not investigated further (data not shown). we reported each βerv as a distinct entity. nevertheless it is reasonable that some of their number, particularly the βervs within each of groups vii and viii, represent a common progenitor infectious betaretrovirus that has undergone duplication events via retrotransposition or recombination since an original, single integration event. for example, the integration time of pverv-βj coupled with its similarity to paerv-βa and pverv-βk may mean that these βervs originated from a single integration into the genome of the common ancestor of p. vampyrus and p. alecto and that at least a single duplication event has occurred within p. vampyrus (or the common ancestor). however, it is also arguable that multiple integrations of closely related infectious retroviruses separated from each other by perhaps a small number of infectivity cycles occurred. we attempted to address this question by a comparative analysis of the flanking genomic dna located immediately up-and downstream of the proviruses and by identifying the tsd that border each provirus and arise as a by-product of the integration mechanism [46] . unique tsd indicate distinct integration events. in the case of the group viii βervs a tsd for pverv-βk could not be identified as its 3′ ltr appears to be truncated. this may indicate that it is a copy of pverv-βj that has arisen through a chromosomal duplication event. this appears to be confirmed by the identification of genomic dna bordering pverv-βj that is homologous to genomic dna flanking pverv-βk. as paerv-βa is a genomic transcript it does not contain tsd. in the case of group vii βervs all of the identifiable tsd differ from one another, indicating separate integration events. additionally, no flanking genomic dna homology was identified amongst the members of the group. notably, both the phylogenetic and ltr analyses revealed a great diversity of βervs in bat genomes. our molecular clock dating suggested that the earliest viral incorporation event occurred at approximately 36 mya which is older than the separation of the megabats and microbats studied (around 20 mya) [28] . in addition, it is clear that some of the βervs present in bat genomes were vertically transmitted from their ancestors; e.g. mlerv-βa and pverv-βa are grouped together and are of similar age having been integrated approximately 30 mya. however, it is also the case that many of the bat βervs formed via independent viral invasion and incorporation as they have different phylogenetic positions as well as different estimated ages of integration. in addition to their genomic diversity, we observed that a number of phylogenetic clusters within the genus differed in their more fundamental aspects. specifically, the use of trna lys 1,2 or trna lys 3 was not restricted to the divide between type b and type d betaretroviruses, and a clade that was distinct in both gag and pol trees possessed a gammaretroviral env gene. this prompted us to define eight sub-groups (group i-viii) within the genus that accounted for these fundamental differences in the context of phylogenetic divergences at the amino acid level of the core polyproteins. our ltr analysis also revealed that bats have been infected with betaretroviruses for most of their evolutionary history. this supports the notion that bats are a potential reservoir for infectious betaretroviruses. a previous study reported a short, partial retroviral sequence (cperv-β5, ac138156) in the genome of the microbat carollia perspicillata (seba's short-tailed bat) [25] . however, this sequence contained large deletions, was missing the entire pro and pol genes, and only fragments of the gag and env genes remained. the partial env of cperv-β5 most closely matched the env of the betaretrovirus smr and on that basis it was reported as a betaretroviral sequence. in this study, we report a series of complete βervs in mega-and microbat genomes representing the breadth of the genus betaretrovirus. although cperv-β5 does contain a lysine trna-specific pbs, without a pol gene to phylogenetically differentiate it or the presence of the characteristically betaretroviral dutpase domain within pro, it cannot be known with certainty whether it is a group viii betaretrovirus or a gammaretrovirus. the study by ballie et al. [25] and a recent study by anai et al. [47] both noted the similarity between the env of type c gammaretroviruses and some type d betaretroviruses which was attributed to a likely recombination event. we have shown that the betaretroviruses, which possess a gammaretrovirus-like env, form a single clade in both gag and pol phylogenies. this indicates that a single recombination event produced these group viii betaretroviruses. furthermore, the typical mammalian gammaretroviral use of trna proline and glycine-specific pbs and the absence of dutpase domains from their pro genes [14] can be used to infer that the nature of the recombination event was the insertion of a type c gammaretroviral env gene into a type d betaretrovirus. previous studies also determined a recombinatorial origin for the type d env [12, 38] . however, this conclusion was reached prior to the sequencing of the genome of jsrv [48] , which does not possess a gammaretrovirus-like env, and its subsequent classification as a type d retrovirus. as such, it was hypothesized that it was this recombination event that gave rise to the type d lineage of betaretroviruses [12, 38] . our analysis aimed to provide a clarification of the differences between, within, and outside of the type b and d groups of betaretroviruses. accordingly, we suggest that the fundamental feature giving rise to the division between the type b and d lineages may have been the use of different primer binding sites, not the possession or not of a type c env gene, which appears to be a more recent and more significant lineage divergence within the type d group. ballie et al. [25] described seven groups within the genus betaretrovirus. these groupings were made solely on the basis of pol gene nucleotide sequence similarity. while manually determining amino acid sequences from genes that contain frameshift mutations is difficult, when the manual reconstruction is closely informed by the alignment of each translated frame against known betaretroviral polymerases, amino acid sequence reconstruction is a viable option. as such, our phylogenetic analyses differ from those undertaken previously in that they are based on amino acid sequence alignments, and our groupings are based on differences in the fundamental genomic features in addition to phylogenetic clustering. tristem [49] reported on the identification and classification of the highly diverse endogenous retroviruses present in the human genome (hervs) and suggested that trna pbs specificity, in addition to the polymerase phylogeny of endogenous retroviruses, should inform their classification. this is because even if the ervs of a given species cluster together in phylogenies, the use of different trna pbs may be evidence of separate origins. indeed, that study made the assumption that hervs with alternative pbs homologies were derived from cross-species transmissions. with this in mind, we analyzed the pbs sequences of the identified βervs and used this information to aid and inform the delineation of our grouping scheme. mammalian cells restrict the export of intron containing mrna from the nucleus to the cytoplasm, and betaretroviruses have been found to utilize two different mechanisms to circumvent this restriction and export unspliced genomic rna and singly-spliced env mrna. the type b betaretrovirus mmtv, and the herv-k endogenous retroviruses are known to use rem and rec, respectively, which are hiv rev-like export proteins, that possess equivalent mechanisms of action [17, [50] [51] [52] . the type d betaretroviruses mpmv and srv make use of the cis-acting cte, which in the absence of a retroviral accessory protein, recruits cellular proteins to effect nuclear export of intron containing viral rna [22, 23] . this apparent dichotomy has been complicated by recent lines of investigation that have found that i) mmtv likely possesses a second, rem-independent mechanism for the export of singly-splice env mrna [52] ; and ii) the type d betaretrovirus jsrv contains both a cte and a rev-like protein, rej, which while found to possess a primary function related to gag synthesis, also enhances rna export in some cell types [21, 53] . this indicates that betaretroviruses may make use of multiple export mechanisms, possibly providing some measure of redundancy to promote productive replication in different contexts. we conducted a computational analysis to predict the presence of rna export motifs that would indicate which mechanism was utilized by each βerv. we found that bat βervs, clustering with betaretroviruses known to utilize the crm1 export pathway, typically contained one or both of the nls and nes motifs, suggesting that they too encode a rev-like protein. it was not surprising that some βervs were predicted to contain one motif but not the other, as random mutation since integration is expected to interfere with sequence-based motif prediction. it is also possible that the nes of some betaretroviral rev-like proteins (such as is the case for herv-k rec) are encoded at the exon boundary and/or within a frame different to that used by env, making the prediction of nes from the env protein sequence challenging. a number of βervs in group vii were found to contain retroviral tap-binding motifs, defined as published [23] , implicating their use of the cte:tap export pathway. the presence of putative nls and nes in some group vii βervs suggests that rev-like elements may also be present. as rev-like proteins are encoded within the env gene, the recombination event that replaced the betaretroviral env with a gammaretroviral env and gave rise to group viii would have caused the incidental loss of any encoded rev-like protein. such a lineage would only have remained viable if it either possessed an alternative mechanism for export, or never made use of a rev/rre equivalent export mechanism in the first place. that rev-like proteins are widely distributed amongst the betaretroviruses suggests that it is not unreasonable that the progenitor of group viii did possess a rev-like protein. this possibility is supported by the existence of the rej protein of jsrv, as jsrv clusters alongside group viii in the type d lineage. in addition, several bat βervs in groups vi and vii contain putative nls and nes motifs, suggesting that members of these groups contain rev-like elements. if group viii did lose a rev-like protein upon acquisition of a gammaretroviral env, then two explanations for the lineage's survival are apparent: i) the recombination event was confined to env and the betaretroviral cte possessed by mpmv and srv, which is located immediately downstream of env, already existed as a redundant export mechanism and remained after the event, or ii) the recombination event included the nucleotide sequence downstream of the env gene, and a putative cte-like element was acquired in the process. with regard to the second possibility it is important to note that the mrna nuclear export mechanism of gammaretroviruses has not been elucidated and the proposal of a cte-like element remains hypothetical. however, this notion is supported by the observation that accessory proteins have not been reported for gammaretroviruses, expression of unspliced and singly-spliced viral mrna would require nuclear export, and that a cte-like cisacting nuclear export element would necessarily be located in singly-spliced env mrna. in either event, our analysis leads to the surprising implication that the betaretroviruses are part of a fundamentally complex retroviral genus and that one lineage, group viii, has evolved through gene replacement into a simple retrovirus sub-group that does not possess any distinct accessory proteins or virulence factors. using the phylogenetic analysis of retroviral pol sequences we proposed a pathway through which the genus betaretrovirus may have evolved from its progenitor. this hypothetical evolutionary history paints an interesting picture of a broad and diverse retroviral genus whose distribution may be largely unrestricted by host species barriers. the βerv members of a number of groups are represented in hosts who are distantly related, such as group viii, which contains host species from bats, primates, rodents, and marsupials. this suggests that cross-species transmission of betaretroviruses is a likely and common occurrence, such that betaretroviruses may be particularly adept at evading host defences. this possibility is intriguing, particularly in light of the wide array of additional orfs found within the genus that hint at the existence of as yet undiscovered betaretroviral accessory and virulence factors; these could, for example, act as countermeasures to circumvent the action of host intracellular restriction factors that are known to act as barriers to cross-species transmission [54] . the wide distribution of diverse βervs in bats and rodents suggests that these two largest groups of mammals play a major role as both hosts and cross-species transmitters for betaretroviruses. bats and rodents are globally distributed, appearing on all continents with the exception of antarctica [30, 55] . as such it appears reasonable to postulate that they have both played a large role in the global spread and evolution of betaretroviruses. we have demonstrated the presence of a range of βervs in mega-and microbats that possess a diversity that cannot be confined to the classical type b/type d division. among their number we identified an intact βerv that may be capable of producing infectious virions, and our ltr analysis indicates that betaretroviruses have been circulating in bat populations throughout their evolution and likely still do. our evidence that bats have carried a range of exogenous infectious betaretroviruses and that cross-species transmission has been commonplace has important implications for disease emergence. indeed, the reported association between the betaretrovirus mmtv and human breast cancer and primary biliary cirrhosis may mean that betaretroviral zoonosis is already causing disease in humans [56] [57] [58] . urban expansion into the natural habitats of bats is gradually increasing the amount of overlap between bat and human environments, and with it the amount of contact between bats and humans [59] . in many countries the practice of hunting bats as a source of consumable bushmeat is common [60] . these circumstances provide the opportunity for retroviral transmission between bats and humans. we propose that the transmission of a betaretroviral infection from bats into humans is possible. as such, it is imperative to continue to survey those viruses present in bats. approval for the use of bat tissue was granted by the australian animal health laboratories animal ethics committee (protocol aec1281) and by the animal ethics committee of east china normal university (approval number 20110224). p. alecto transcriptome datasets were generated from the non-stimulated thymus tissue of a healthy male juvenile bat and the pooled total rna obtained from mitogen-stimulated spleen, white blood cells, and lymph node and the unstimulated thymus and bone marrow obtained from one pregnant female and one adult male as described previously [61] . the p. alecto transcriptome is accessible through the ncbi sequence read archive (http://www.ncbi.nlm.nih.gov/traces/sra/) [sra: srp008674]. the r. ferrumequinum transcriptome was generated using whole brain tissue as published [37] . the p. alecto and r. ferrumequinum transcriptomes were sequenced using the illumina next-generation sequencing (ngs) platform as described previously [37, 61] . the p. alecto transcriptome was assembled using velvet, oases, and mira software packages as described previously [61] . the r. ferrumequinum transcriptome was assembled using the brujin graph and soapdenovo software packages as described previously [37] . the generation of the r. megaphyllus transcriptome was conducted as follows: four wild bats, (one female and 3 male) were caught in the booloumba creek caves in queensland, australia in november 2006 and tissues from brain, kidney, large and small intestines, liver, lung, spleen, heart, skin, bone and reproductive organs were pooled and stored in rnalater (ambion). total rna was isolated from the 12 pooled bat tissues using the qiagen rneasy kit. dna was prepared from purified total rna (2.5 μg per cdna reaction) using the evrogen mint cdna synthesis kit (cat # sk001) but with a modified oligodt adapter primer containing the recognition sequence for gsui (5′ agcagtggtatcaacg cagagt ctggag(t) 20 vn). the cdna was normalized with a duplex specific nuclease (dsn) using a modification of the protocol described in the evrogen trimmer cdna normalization kit (cat # nk001). after the second limited pcr amplification (12 cycles) with the m2 primer, pcr buffer, primers and enzyme were removed using the machery nagel nucleospin ii kit. dna was then digested overnight with gsui to remove the 3′ polya tail adapter sequence so as to remove stretches of homopolymer ts and as which can effect the 454 sequencing run due to cross-talk (homopolymer flash). five micrograms of normalized amplified double stranded cdna was purified using the machery nagel nucleopsin kit with the selective removal of the gsui digested 43 base pair (bp) 3′polya adapter sequence using a modification of the binding conditions. library preparation for roche 454 sequencing for the gs flx platform was performed by the australian genome research facility ltd, st lucia, queensland with sequence output of 74 mb, 374,360 single-end reads with an average read length of 239 bp. clc genomics workbench version 4.5.1 (clc bio, aarhus, denmark) was used to trim reads based on quality and to remove the evrogen normalization primer sequence, subsequent 337,805 reads were de novo assembled using clc genomics workbench default settings and blast databases were prepared using either de novo assembled or trimmed unassembled reads. the gag, pol, and env genes of each genome sequence were translated into protein sequences using the clc main workbench 6.6 (clc bio). to identify the transcripts of interest we used the tblastn function of the clc main workbench incorporating the following parameters: blosum62 matrix, word size = 3, e-values < 1×10 -10 , gap costs of existence 11, extension 1, and low complexity filtered. to confirm that the transcripts identified were more similar to betaretroviruses than other retroviral genera we performed a reciprocal blast analysis of each transcript against the ncbi non-redundant protein database (http://blast.ncbi.nlm. nih.gov/blast.cgi) using the blastx function of the clc main workbench with the following parameters: blosum80 matrix, word size = 3, e-values < 1 × 10 -10 , gap costs of existence 10, extension 1, low complexity filtered, and limit by entrez query = viruses. annotated sequences of the full-length betaretroviral sequences included in the phylogenetic analyses (paerv-βa, papol-01, paenv-01, and rfenv-01) are included as additional file 4. we generated the genomic sequence of paerv-βa using two transcripts identified in the p. alecto transcriptome during the initial blast analysis which were aligned using the clc main workbench and trimmed by 245 and 401 nucleotides at the 5′ and 3′ extremities of their overlapping region, respectively. to determine the presence of full-length βervs in mega-and microbats we retrieved the genomes of m p. vampyrus and m. lucifugus from the ensembl database (http://www.ensembl.org/index.html). we searched for genomic sequences with similarity to the aforementioned extant betaretroviral proteins by conducting a tblastn analysis of the genomes using the clc main workbench with the following parameters: blosum62 matrix, word size = 3, e-values < 1×10 -10 , gap costs of existence 11, extension 1, and low complexity filtered. we searched for genomic sequences with similarity to the betaretroviral transcripts identified in the bat transcriptomes by conducting a tblastx analysis of the genomes using the clc main workbench with the following parameters: blosum80 matrix, word size = 3, e-values < 1×10 -10 , low complexity filtered. to sort fulllength from fragmented βervs and various other retroelements within the blast output, a script was created using microsoft office excel 2003 (microsoft corporation, redmond, usa) that compared the blast data for the gag, pol, and env analyses and identified scaffolds that emerged as a hit in each. the long terminal repeats (ltrs) which were used to delineate the full-length βervs were identified by subjecting each identified gene scaffold to a blastn analysis in which the entire sequence was aligned with itself to identify repeated sequences using the following parameters: word size = 11, match score = 1, mismatch score = −3, gap costs of existence 5, extension 2, and low complexity filtered. transcription promoter elements within the 5′ ltrs of the βervs were predicted using the online promoter predictor tool nnpp 2.2 [62] (http://www.fruitfly.org/ seq_tools/promoter.html). tata boxes were predicted using the hamming-clustering method through the online hctata tool [63] (http://zeus2.itb.cnr.it/~webgene/ wwwhc_tata.html). poly(a) signal sites were predicted using the hamming-clustering method through the online hcpolya tool [63] (http://zeus2.itb.cnr.it/~webgene/ wwwhc_polya.html). primer binding sites were identified by an alignment of the genomic nucleotide sequence between the 5′ ltr and the beginning of the gag gene of each βerv against the university of strasbourg's online trna database [64] (http://trna.bioinf.uni-leipzig. de/dataoutput/search) using the associated blast tool (default parameters). open reading frames (orfs) were identified within each βerv using the clc main workbench. the dutpase protein domains and nucleocapsid zinc knuckles were identified by subjecting the translated gag and pro genes to a protein family (pfam) domain search [65] through the clc main workbench using the publicly accessible pfam database (http://pfam. sanger.ac.uk/). the conserved major homology region (mhr) of gag and enzymatic active sites of the retroviral protease (dxg), reverse transcriptase (ddd), and integrase (dde) were identified through a protein sequence alignment, using the create alignment function of the clc main workbench, between the gag, pro, and pol of each bat βerv against those of the aforementioned extant betaretroviruses. prediction of rna export elements nls and nes were predicted by analyzing the env, or if known, the rev-like protein sequence of each betaretrovirus. nls were predicted using the online tool cnls mapper [66] (http://nls-mapper.iab.keio.ac.jp/ cgi-bin/nls_mapper_form.cgi) with a prediction score threshold of 3.0. nes were predicted using the online tool netnes 1.1 [67] (http://www.cbs.dtu.dk/services/ netnes/). the strength of each nes prediction within the env/rev-like protein is defined as strong if the scores for the neural network model and hidden markov model, together with the overall nes score, are above the algorithm-assigned threshold. the strength is weak if one of the scores is below the threshold. no nes is predicted for proteins in which more than one score is below the threshold. tbe, dr, and ir were identified by subjecting the nucleotide sequence within and downstream of env ending at the poly(a) signal site within the 3′ ltr of each betaretrovirus to a blastn analysis in which the sequence was aligned against itself to identify repetitive elements using the following parameters: word size = 11, match score = 1, mismatch score = −3, gap costs of existence 5, extension 2, and low complexity not filtered. all nucleotide and protein alignments were conducted using the create alignment function of the clc main workbench except where stated otherwise. to determine the evolutionary relationships among the different bat betaretroviruses we inferred the phylogenetic relationships among the gag, pol and env amino acid sequences. all of the reference sequences were downloaded from ncbi (additional file 2: table s5 ) and aligned with bat sequences using muscle [68] . we employed the gblocks program [69] to remove regions of high sequence diversity and hence uncertain alignment. phylogenetic relationships were then inferred using the maximum likelihood (ml) method available in phyml 3.0, employing spr (subtree pruning and regrafting) branch-swapping [70] and incorporating 1,000 bootstrap replications to determine the robustness of each node. the prottest 2.4 program [71] was used to select the best-fit model of amino acid substitution, which was found to be lg+i+г for all data sets. a time-scale for βerv evolution was established as described previously [36] and employing the bayesian markov chain monte carlo method (mcmc) available in the beast v1.7 package [72] . we first acquired the genomic substitution rates (r) for mega-and microbats. for this, divergence times of mega-and microbats were taken from the fossil record [28] and used to calibrate date estimates for the rest of the species tree, assuming an uncorrelated lognormal relaxed molecular clock. all phylogenetic trees were inferred using the gtr substitution model and the yule speciation prior, and the beast analyses were run until all relevant parameters converged, with 10% of the mcmc chains discarded as burn-in. the estimated substitution rates were then used to calculate the age of each βerv using the following formula: t=(d/r)/2, where t is the invasion time of each βerv (million years), d is the number of differences per site among the both 5′ and 3′ ltrs, and r is the genomic substitution rate (substitutions per site per year). the genbank accession numbers of the retroviruses used in this study are listed in additional file 2: table s5 . additional file 1: figure s1 . alignment of extant and bat betaretroviral primer binding sites (pbs). the pbs of bat endogenous betaretroviruses and those of known extant and exogenous betaretroviruses are aligned and grouped according to the specific lysine trna complementary to the pbs. *the pbs complementarity of mlerv-βe is uncertain. figure s2 . alignment of the orf present in the group vii endogenous betaretroviruses (βervs) of bats. the region from the beginning of the 5 ′ ltr to the beginning of the gag gene of each group vii bat βerv was aligned and a consensus sequence generated. the annotations belong to the consensus sequence and depict the 5 ′ ltr, predicted promoter element and tata boxes, the pbs complementary to trna lys3 (lys 3 pbs), and an open reading frame (orf). figure s3 . annotated alignment of the group viii endogenous betaretroviruses (βervs) of bats. the region from the end of the env gene to the 3 ′ long terminal repeat (ltr) of each group viii bat βerv was aligned and a consensus sequence generated. the annotations belong to the consensus sequence and depict an open reading frame (orf), the beginning of the 3 ′ ltr, and mutations in paerv-βa and pverv-βk that influence the presence of orfs. additional file 2: table s1 . comparison of βerv polymerase sequences to those of known betaretroviruses. table s2 . identification of the target site duplications (tsd) flanking endogenous betaretroviruses. table s3 . comparison of the 5 ′ and 3 ′ flanking regions of phylogenetically clustered βervs. table s4 . analysis of betaretroviral rna export motifs. table s5 . genbank accession numbers and ensembl database locations of the retroviruses used in this study. historical introduction to the general properties of retroviruses studies of endogenous retroviruses reveal a continuing evolutionary saga retroviral virions and genomes converging strategies in expression of human complex retroviruses origin of hiv-1 in the chimpanzee pan troglodytes troglodytes persistent zoonotic infection of a human with simian foamy virus in the absence of an intact orf-2 accessory gene human t-cell leukemia virus type 1 (htlv-1) and leukemic transformation: viral infectivity, tax, hbz and therapy identification, characterization, and comparative genomic distribution of the herv-k (hml-2) group of human endogenous retroviruses expression of human endogenous retrovirus type k envelope protein is a novel candidate prognostic marker for human breast cancer mouse mammary tumor virus p75 and p110 cux1 transgenic mice develop mammary tumors of various histologic types nucleotide sequence of mason-pfizer monkey virus: an immunosuppressive d-type retrovirus sheep retrovirus structural protein induces lung tumours retroviral taxonomy, protein structures, sequences, and genetic maps barabás o: dutpase and nucleocapsid polypeptides of the mason-pfizer monkey virus form a fusion protein in the virion with homotrimeric organization and low catalytic efficiency flexible segments modulate co-folding of dutpase and nucleocapsid proteins mouse mammary tumor virus encodes a self-regulatory rna export protein and is a complex retrovirus naf, a trans-regulating negativeacting factor encoded within the mouse mammary tumor virus open reading frame region a novel, mouse mammary tumor virus encoded protein with rev-like properties a maternally inherited superantigen encoded by a mammary tumour virus identification and mutational analysis of a rej response element in jaagsiekte sheep retrovirus rna a small element from the mason-pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication rev-independent retroviral constitutive transport element evolved from cellular tap(nxf1)-binding sequences endogenous viruses: insights into viral evolution and impact on host biology multiple groups of endogenous betaretroviruses in mice, rats, and other mammals endogenous type d retrovirus in a marsupial, the common brushtail possum (trichosurus vulpecula) characterization of a full-length endogenous betaretrovirus, eqerv-beta1, in the genome of the horse (equus caballus) a molecular phylogeny for bats illuminates biogeography and the fossil record comparative analysis of bat genomes provides insight into the evolution of flight and immunity bats: important reservoir hosts of emerging viruses antiviral immune responses of bats: a review primitive early eocene bat from wyoming and the evolution of flight and echolocation hendra and nipah viruses: different and dangerous fruit bats as reservoirs of ebola virus bats are natural reservoirs of sars-like coronaviruses identification of diverse groups of endogenous gammaretroviruses in mega and microbats discovery of retroviral homologs in bats: implications for the origin of mammalian gammaretroviruses receptor interference groups of 20 retroviruses plating on human cells gag-related polyproteins of moloney murine leukemia virus: evidence for independent synthesis of glycosylated and unglycosylated forms moloney murine leukemia virus glyco-gag facilitates xenotropic murine leukemia virus-related virus replication through human apobec3-independent mechanisms on the estimation of the insertion time of ltr retrotransposable elements a novel gene from the human endogenous retrovirus k expressed in transformed cells human endogenous retrovirus rec interferes with germ cell development in mice and may cause carcinoma in situ, the predecessor lesion of germ cell tumors analysis of 454 sequencing error rate, error sources, and artifact recombination for detection of low-frequency drug resistance mutations in hiv-1 dna an accessory open reading frame (orf-x) of jaagsiekte sheep retrovirus is conserved between different virus isolates retroviral dna integration: viral and cellular determinants of target-site selection infectious endogenous retroviruses in cats and emergence of recombinant viruses nucleotide sequence of the jaagsiekte retrovirus, an exogenous and endogenous type d and b retrovirus of sheep and goats identification and characterization of novel human endogenous retrovirus families by phylogenetic screening of the human genome mapping project database identification of a rev-related protein by analysis of spliced transcripts of the human endogenous retroviruses htdv/herv-k rec (formerly corf) function requires interaction with a complex, folded rna structure within its responsive element rather than binding to a discrete specific binding site indik s: identification of the remresponsive element of mouse mammary tumor virus jaagsiekte sheep retrovirus encodes a regulatory factor, rej, required for synthesis of gag protein antiretroviral restriction factors rodent evolution: back to the root mouse mammary tumor virus-like sequences in human breast cancer human mammary tumor virus in inflammatory breast cancer cloning the human betaretrovirus proviral genome from patients with primary biliary cirrhosis distribution and activity of bats at local and landscape scales within a rural-urban gradient bats as bushmeat: a global review the immune gene repertoire of an important viral reservoir, the australian black flying fox application of a time-delay neural network to promoter annotation in the drosophila melanogaster genome hamming-clustering method for signals prediction in 5′ and 3′ regions of eukaryotic genes trnadb 2009: compilation of trna sequences and trna genes the pfam protein families database systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs analysis and prediction of leucine-rich nuclear export signals muscle: multiple sequence alignment with high accuracy and high throughput improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments new algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of phyml 3.0 prottest: selection of best-fit models of protein evolution bayesian phylogenetics with beauti and the beast 1.7 submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution additional file 3: figure s4 . unannotated alignment of the full proviral genomes of the group viii endogenous betaretroviruses (βervs) of bats.additional file 4: annotated sequences of paerv-βa, papol-01, paenv-01, and rfenv-01. the authors declare that they have no competing interest.authors' contributions mt, jah, jc, and gt conceived the study. jah, jc and mt performed the analyses, mt generated the r. megaphyllus transcriptome, hf collected bats from which tissue was obtained to generate the transcriptome data. all authors contributed to the writing of the paper. all authors have read and approved the submission of the manuscript. key: cord-298224-flyx85lr authors: hibbitts, alan j.; ramsey, joanne m.; barlow, james; macloughlin, ronan; cryan, sally-ann title: in vitro and in vivo assessment of pegylated pei for anti-il-8/cxcl-1 sirna delivery to the lungs date: 2020-06-27 journal: nanomaterials (basel) doi: 10.3390/nano10071248 sha: doc_id: 298224 cord_uid: flyx85lr inhalation offers a means of rapid, local delivery of sirna to treat a range of autoimmune or inflammatory respiratory conditions. this work investigated the potential of a linear 10 kda poly(ethylene glycol) (peg)-modified 25 kda branched polyethyleneimine (pei) (pei-lpeg) to effectively deliver sirna to airway epithelial cells. following optimization with antiglyceraldehyde 3-phosphate dehydrogenase (gapdh) sirna, pei and pei-lpeg anti-il8 sirna nanoparticles were assessed for efficacy using polarised calu-3 human airway epithelial cells and a twin stage impinger (tsi) in vitro lung model. studies were then advanced to an in vivo lipopolysaccharide (lps)-stimulated rodent model of inflammation. in parallel, the suitability of the sirna-loaded nanoparticles for nebulization using a vibrating mesh nebuliser was assessed. the sirna nanoparticles were nebulised using an aerogen(®) pro vibrating mesh nebuliser and characterised for aerosol output, droplet size and fine particle fraction. only pei anti-il8 sirna nanoparticles were capable of significant levels of il-8 knockdown in vitro in non-nebulised samples. however, on nebulization through a tsi, only pei-peg sirna nanoparticles demonstrated significant decreases in gene and protein expression in polarised calu-3 cells. in vivo, both anti-cxcl-1 (rat il-8 homologue) nanoparticles demonstrated a decreased cxcl-1 gene expression in lung tissue, but this was non-significant. however, pei anti-cxcl-1 sirna-treated rats were found to have significantly less infiltrating macrophages in their bronchoalveolar lavage (bal) fluid. overall, the in vivo gene and protein inhibition findings indicated a result more reminiscent of the in vitro bolus delivery rather than the in vitro nebulization data. this work demonstrates the potential of nebulised pei-peg sirna nanoparticles in modulating pulmonary inflammation and highlights the need to move towards more relevant in vitro and in vivo models for respiratory drug development. oligonucleotide therapeutics offer a unique opportunity for accurate and specific disease treatment at a genetic level. to date, these have been investigated in a variety of endogenous and infectious conditions [1] [2] [3] . of these, sirna has gained renewed prominence following the successful clinical approval of patisiran (onpattro™) by alnylam pharmaceuticals for hereditary ttr-mediated amyloidosis (hattr) [4] . in part, the approval was based on patisiran's ability to rapidly and accurately reach its target cells (hepatocytes) by utilising the most appropriate delivery method (i.v. perfusion) [5] . when the i.v. route is not practical or effective, utilising local delivery routes offer a means of vastly increasing the targeting of oligonucleotides and thereby improving the specificity and efficacy [6] . local delivery is particularly promising as an approach for oligonucleotide therapeutics targeting respiratory diseases. this is especially true for episodes of acute pulmonary inflammation, including acute asthmatic episodes and pathogenic respiratory conditions such as acute respiratory distress syndrome (ards) [7, 8] . the urgent need to mitigate the effects of respiratory viruses has become especially relevant with the emergence of the covid-19 pandemic. covid-19 is clinically manifest in the lungs in~50% of patients and mortality is strongly linked to cytokine storm-induced ards [9] [10] [11] . in these cases, a fast-acting specific treatment of short duration may be more favourable than a continuous, systemic depression of the innate immune system in an otherwise healthy individual. one potential target for cytokine-induced respiratory distress is the chemotactic pro-inflammatory cytokine il-8. il-8 is known to be secreted from pulmonary epithelial cells and is elevated in covid-19 patients [12, 13] . furthermore, anti-il-8 therapy is currently under investigation in phase ii clinical trials using i.v.-delivered anti-il-8 monoclonal antibodies in covid-19 patients [14] . however, the effective delivery of sirna to the lungs has been hampered by poor delivery device performance. this is especially true in the case of the nebulised delivery of sirna. nebulisation offers the benefits of a non-invasive, easy to use device with a large level of flexibility in the dose delivered. it has long been established that air-jet and older ultrasonic nebulisers are unsuited to sirna delivery due the high cost of the therapeutic cargo and their known deficiencies in output efficiency [15] . previous tests have indicated that air-jet and ultrasonic nebuliser devices have a considerable discrepancy in their ability to effectively form droplets of the desired size [16] . to address this, there has been a relatively new "3rd generation" of nebulisers developed known as vibrating mesh nebulisers (vmns) (air-jet and ultrasonic nebulisers being the 1st and 2nd generations, respectively). these nebulisers function in a manner modified from previous ultrasonic nebulisers, whereby a plate containing approximately 1000 tapered holes vibrates at a frequency of roughly 128 khz, thereby causing the ejection of liquid droplets [17] . vibrating mesh nebulisers are now becoming widespread and there is investigation into their use for delivery of sensitive therapeutic cargoes, such as proteins [18] , mirna and anti-mirna target site-blocking oligonucleotides [19, 20] , mrna [21] and sirna [22, 23] . furthermore, the bulk of recent publications indicate that vmns deliver more drug to the patient with significantly less wastage, than the more prevalent air-jet nebulisers [24] [25] [26] . in parallel to effective device-borne delivery to the lungs, the successful development of a locally delivered sirna therapy for pulmonary conditions must contend with the challenges of the lung micro-environment. major barriers to local pulmonary delivery include the mucociliary clearance action of the ciliated epithelial cells and the presence of mucus and alveolar fluid in different parts of the airways [27, 28] . particles that are deposited on the ciliated cells are rapidly removed by muco-ciliary clearance and are eventually coughed up or swallowed. this mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles [29] . efforts to overcome the mucus barrier and improve the efficacy of delivered drugs has led to the development of "mucus penetrating particles". previous approaches to modifying cationic polymers for sirna delivery have drawn inspiration from naturally occurring molecules such as viral proteins [30] . of particular interest is the use of poly(ethylene glycol) (peg)-modified nanoparticles, which has demonstrated much potential in this regard [31] . recent research within our group has focused on developing nanoparticle carriers to better deliver sirna to the pulmonary epithelium [23, 32, 33] . specifically, we have previously developed a block co-polymer of 25 kda branched polyethyleneimine (pei) conjugated to a high degree (80%) with 10 kda linear poly(ethylene glycol) (peg). this pei-lpeg was capable of higher levels of gene knockdown in fully polarised calu-3 epithelial monolayers compared to the pei controls, with limited nanomaterials 2020, 10, 1248 3 of 23 cellular toxicity evident [32] . in this study, this pei-lpeg polymer will be further investigated for its potential to deliver anti-il-8 sirna to the lungs via a vibrating mesh nebuliser. given its prominent role in modulating pulmonary inflammation, the successful nebulisation of anti-il-8 sirna would serve as a promising proof of concept in combatting pulmonary inflammation at the genetic level. to achieve this, pei-lpeg sirna and pei sirna nanoparticles were nebulised using a vibrating mesh nebuliser, and their post-nebulisation stability was assessed in terms of their physicochemical properties and their ability to facilitate gene knockdown. cell studies first focused on validating the sequence specificity of anti-il-8 sirna in non-nebulised transfections. following this, the post-nebulisation efficacy was investigated following passage through a twin-stage impinger (tsi). this was first validated at a genetic level using the glyceraldehyde 3-phosphate dehydrogenase (gapdh) house-keeping gene before assessing the changes in il-8 protein levels. finally, anti-cxcl-1 (rat il-8 homologue [34] ) sirna nanoparticles were assessed in an in vivo pilot study using a rat model of acute pulmonary inflammation involving intra-tracheal intubation, sirna delivery and subsequent lipopolysaccharide (lps) challenge. all the cell culture reagents were obtained from invitrogen corporation/thermofisher scientific (ca, usa) unless otherwise stated. the calu-3 bronchial epithelial cell line was obtained from the american tissue type culture collection (atcc) and used at passages 20-40. all the poly (ethylene glycol) molecules were obtained from iris biotech (marktredwitz, germany). the sirna sequences for human gapdh, β-actin and il-8 were obtained from qiagen uk and were diluted 1:10 in te buffer as directed. the specific sequences remained the proprietary knowledge of qiagen. the sirna sequences for rat cxcl-1 (5 uaacgagauauuuaacgccccc 3 ) were obtained from (riboxx gmbh, radebeul, germany), and the sigenome non-targeting sirna #2 (5 uaaggcuaugaagagauac 3 ) scrambled sequence controls were obtained from dharmacon (now horizon discovery, cambridge, uk). all the other general chemicals and reagents used were of the highest grade possible and were obtained from sigma-aldrich company ltd. (wicklow, ireland) unless otherwise stated. branched 25 kda pei was modified with 10 kda linear peg by the reaction of succinimidyl-activated peg (peg-ssa) with pei under slightly basic aqueous conditions as previously described [32] . briefly, 1 g of 25 kda pei was dissolved in 25 ml of phosphate buffered saline (ph 8). following this, 500 mg of 10 kda linear peg-ssa was dissolved in 5 ml of 99.9% dimethyl sulfoxide (dmso). this was added dropwise with stirring to the pei solution. the reaction proceeded for 4 h at room temperature before being stopped by the addition of 60 ml of deionised water. the reaction mixture was transferred to cellu•sep h1 membranes (25 kda mwco) (orange scientific, braine-l'alleud, belgium) and dialysed overnight in excess deionised water to remove unreacted components before being lyophilised. the final products were then analysed for form and purity using 1 h and 13 c nmr spectroscopy (bruker avance 400) and gel permeation chromatography (gpc) (perkin elmer, dublin, ireland) with electronic light scattering detection (agilent technologies, cork, ireland). pei and pei-peg polymer-sirna complexes were formed using pei nitrogen to rna phosphate (n/p) in ratios of 15 and 7. briefly, the appropriate amount of polymer was added to an sirna solution (20 µm) to yield a final concentration of 2 µm sirna, vortexed for 10 s and incubated for polyplex formation for 30 min. following this, the solutions were diluted using pbs. the sirna nanoparticles were nebulised and the droplet size distributions, described by a volumetric median diameter (vmd), were measured by a malvern spraytec particle size analyser for the droplets produced (malvern instruments ltd., malvern, worcestershire, uk) with the rt sizer software (version 5.60). a 5 l/min vacuum flow was implemented through the system, ensuring a laminar flow and reducing the artificial droplet size growth through collision with other droplets. the vacuum also ensured that the droplets passed through the laser beam only once. the centre of the emitted aerosol plume was directed through the centre of the laser beam to increase the accuracy of data acquisition. data acquisition was performed as previously described [23] , beginning when beam obscuration exceeded 3% and continued until the end of dosing. the data acquisition rate was set to 500 hz, which is 500 individual readings per second taken to characterise the droplet size distribution. the data reported for each individual measurement is an average of the individual readings recorded over the course of the dose. in order to verify the accuracy of the generated data, the spraytec analyser's laser diffraction apparatus was tested with a reference reticle (malvern instruments ltd., malvern, worcestershire, uk). the droplet size is described by volumetric median diameter (dv 50 ) and the fine particle fraction (fpf) (percentage of droplets less than 5 µm in size). the surface tension of the sirna nanoparticle solutions was also examined using a ring/plate tensiometer (lauda scientific gmbh, lauda-königshofen, germany) at room temperature according to the manufacturer's instructions. in all cases, the changes in output rates and fluid surface tension were compared to pbs controls. the pei and pei-peg sirna nanoparticles were formed as previously described using 7.5 µg of sirna and diluting with pbs to a final volume of 5 ml polyplex. the sirna samples were nebulised into a glass impinger at 60 l/min using an aerogen ® pro vibrating mesh nebuliser (aerogen, galway, ireland). before nebulisation, pbs was added to the upper (stage a, 3 ml) and lower (stage b, 7 ml) stages. washes were quantitatively collected using pbs with the device, throat, stage a and stage b made to 5, 5, 10 and 25 ml respectively. a size analysis of the sirna polyplexes was performed using a malvern nano-zs zetasizer. pre-nebulisation samples underwent a 1 in 10 dilution in dh 2 o before addition of pbs. post-nebulisation, rinsed samples were concentrated by centrifuging out excess pbs using centrisart 1 centrifugal uf units (20 kda mwco, sartorius). despite this, due to the high level of dilution that occurred and the strong ionic character of pbs, it was not possible to accurately measure the zeta potentials of the post nebulisation samples. size analysis programmes consisted of five separate scans which contained a minimum of 15 sub-scans. calu-3 cells were seeded in 12 mm transwell chambers at 5 × 10 5 cells/well cultured in complete media of 50:50 dmem:ham's f-12 (10% fbs and 1% pen/strep) in at a liquid-liquid interface for 48 h and for a further 10-12 days at an air/liquid interface. a trans-epithelial electrical resistance (teer) value of >500 ωcm 2 was taken as a sign of tight junction formation. on the day of transfection, 0.6 µg/well (360 nm) of sirna nanoparticles in pbs were formed as previously described. the sirna nanoparticles were administered directly to each well and incubated for 24 h at 37 • c and 5% co 2 . following this, the apical (600 µl) and baso-lateral (1.2 ml) layers of the transwells were aspirated off and collected for il-8 elisa analysis (invitrogen via bio-sciences, dublin, ireland) according to manufacturer's instructions and later normalised to account for differences in dilution. sequence specificity was demonstrated by use of non-complexed pei or pei-lpeg and gapdh-sirna negative controls. in addition to this, the teer value fluctuations over the 24 h of transfection were recorded using an evom voltohmmeter (world precision instruments, stevenage, uk) at various time points for analysis. briefly, a baseline reading was established prior to transfection by equilibrating the apical layer in pbs for 45 min at 37 • c prior to reading. following the addition of the sirna nanoparticle pbs, readings were taken at 30 min and at 1, 2, 4 and 24 h and compared to the pbs-only treated controls. calu-3 cells were seeded in 6.5 mm transwell plates at a density of 2.5 × 10 5 cells/well using the previously described methods for calu-3 culture in transwell plates at liquid-liquid and air-liquid interfaces. cell monolayers were used for the transfection studies once a teer of >500 ωcm 2 was established [35] . a glass tsi was assembled as previously described by grainger et al. [35] , apart from the absence of solution in the lower chamber. an aerogen ® pro vibrating mesh nebuliser was sealed into place with parafilm at the entrance of the tsi ( figure 1a (i)). the adapter piece ( figure 1a (ii)) was removed and parafilm was wrapped around the base of the connecting tube to produce an attachment surface for the transwell insert. the transwell insert was then pushed onto the connecting tube until the tapered internal walls of the insert fastened firmly onto the parafilm ( figure 1b) . thus, air flowing down the connecting tube was diverted through the lateral ports of the insert. this caused particles to exit the air stream through inertial impaction and deposit onto the cell layer. following this, preformed pei/pei-lpeg sirna nanoparticles at a concentration of 360 nm were delivered using either 0.6, 1.2 or 2.4 µg of sirna per well. the vacuum pump was run at 60 l/min and allowed to run for 30 s past complete sample nebulisation. the nebuliser was rinsed by nebulising 3 times with 500 µl of pbs between doses with no transwell attached to the lower section. furthermore, the tsi itself was disassembled and rinsed out with sterile deionised water when switching between the pei and pei-lpeg samples. following the nebulisation of all samples, the cells were incubated for 24 h at 37 • c and 5% co 2 . gapdh sirna was first delivered to validate the system using gene expression prior to the delivery of the anti-il-8 sirna and the protein expression analysis. nanomaterials 2020, 10, 1248 5 of 24 in addition to this, the teer value fluctuations over the 24 h of transfection were recorded using an evom voltohmmeter (world precision instruments, stevenage, uk) at various time points for analysis. briefly, a baseline reading was established prior to transfection by equilibrating the apical layer in pbs for 45 min at 37 °c prior to reading. following the addition of the sirna nanoparticle pbs, readings were taken at 30 min and at 1, 2, 4 and 24 h and compared to the pbs-only treated controls. calu-3 cells were seeded in 6.5 mm transwell plates at a density of 2.5 × 10 5 cells/well using the previously described methods for calu-3 culture in transwell plates at liquid-liquid and air-liquid interfaces. cell monolayers were used for the transfection studies once a teer of >500 ωcm 2 was established [35] . a glass tsi was assembled as previously described by grainger et al. [35] , apart from the absence of solution in the lower chamber. an aerogen ® pro vibrating mesh nebuliser was sealed into place with parafilm at the entrance of the tsi ( figure 1a (i)). the adapter piece ( figure 1a (ii)) was removed and parafilm was wrapped around the base of the connecting tube to produce an attachment surface for the transwell insert. the transwell insert was then pushed onto the connecting tube until the tapered internal walls of the insert fastened firmly onto the parafilm ( figure 1b) . thus, air flowing down the connecting tube was diverted through the lateral ports of the insert. this caused particles to exit the air stream through inertial impaction and deposit onto the cell layer. following this, preformed pei/pei-lpeg sirna nanoparticles at a concentration of 360 nm were delivered using either 0.6, 1.2 or 2.4 μg of sirna per well. the vacuum pump was run at 60 l/min and allowed to run for 30 s past complete sample nebulisation. the nebuliser was rinsed by nebulising 3 times with 500 μl of pbs between doses with no transwell attached to the lower section. furthermore, the tsi itself was disassembled and rinsed out with sterile deionised water when switching between the pei and pei-lpeg samples. following the nebulisation of all samples, the cells were incubated for 24 h at 37 °c and 5% co2. gapdh sirna was first delivered to validate the system using gene expression prior to the delivery of the anti-il-8 sirna and the protein expression analysis. following 24 h of incubation, the basal secretions were collected and the apical layer of cells was washed and collected with an equivalent amount of sterile pbs. the gapdh gene knockdown was then assessed using real-time rt-pcr. rna was extracted using the rneasy micro kit (qiagen, uk) following the manufacturer's instructions. cdna for the real-time pcr was synthesised from rna using the high capacity reverse transcription kit (applied bio-systems via bio-sciences, dublin, ireland) according to the manufacturer's instructions. a quantitative pcr was performed in 200 µl tubes using a rotor-gene 6000 (corbett research, uk, now qiagen, manchester, uk) thermal cycler with the real-time detection of fluorescence. the pcr was conducted in a volume of 25 µl using rotor-gene sybr green rt-pcr kit (qiagen, manchester, uk). the percentage knockdown of gapdh was then quantified using the comparative quantitation software supplied by corbett research for the rotorgene. in all the experiments, human β-actin was used as a housekeeping gene to normalise the expression. in the case of anti-il-8 sirna delivery, the protein levels were assessed as previously described using an il-8 elisa. ) administration of anaesthetic (xylazine 10 mg/kg and ketamine 85 mg/kg) and were returned to their home cage until the anaesthetic took effect. a tail and toe pinch were used to assess the depth of anaesthesia. the anaesthetised rats were positioned supine on a biolite rodent intubation stand (kent scientific, torrington, ct, usa) suspended from the front incisors. the tongue was extended and moved to one side using forceps. a small animal laryngoscope was used to illuminate the vocal cords, epiglottis and opening to the trachea as well as to hold the tongue in place. once the airway of the rat had been visualised, a penn-century ia-1c microsprayer ® (penn-century inc, wyndmoor, pa, usa) with a flexible stainless steel delivery tube of 0.64 mm (23-gauge) attached was used to access the lungs for sirna nanoparticle or lps delivery. prior to endotracheal intubation, pbs, lps or 75 µg of sirna nanoparticles (200 µl) were loaded into the microsprayer. this was achieved by aspirating the entire pre-prepared 200 µl volume into the microsprayer and attaching 4 spacer clips corresponding to 50 µl each attached to the handle of the microsprayer. the dead volume was subsequently expelled by pressing down on the microsprayer handle until only 200 µl remained. once the rat had been successfully intubated, approximately 200 µl of the sample was delivered by pressing down the handle of the microsprayer quickly and steadily to completion and holding for approximately 10 s to ensure full release of the sample. the microsprayer was immediately removed and the rats were administered 15 µl of 5 mg/ml atipamezole hydrochloride (sedastop™) diluted to 150 µl using sterile pbs via i.p. injection to aid recovery and were placed on their sides in a heated incubator until consciousness was regained. the rats were then moved to a room with constant temperature set at 37 • c for 21 h with access to food and water ad libitum. then, 21 h after the initial treatment, the rats underwent the intratracheal administration of either 200 µl of sterile pbs or 1 mg/ml of lps (from e. coli 055:b5) by the same procedure. the animals were not administered sedastop™ on this occasion and were placed in the incubator for 3 h. after 3 h, the rats were sedated again if necessary and then sacrificed via cervical dislocation for tissue harvesting. once the rats were euthanised, the thoracic cavity was opened. the lungs and heart were excised en bloc. one bronchus was clamped to allow bal collection from one side of the lungs only. 1 ml nanomaterials 2020, 10, 1248 7 of 23 of saline was added to one side of the lungs through the trachea using a 20-gauge catheter with the needle removed and a 5 ml syringe. saline was collected and a further 1 ml of fresh saline was added. this procedure was repeated until the lung had been washed with 5 ml of saline. the remaining lobes were collected for rt-pcr in 5 ml of rnalater™ (thermofisher via bio-sciences, dublin, ireland). the collected bal fluid was centrifuged at 1× g for 10 min at 4 • c. supernatants were collected as bal fluid and stored at −80 • c. the cell pellets were resuspended in 1 ml of red cell lysis buffer and centrifuged at 1× g for 10 min at 4 • c. the supernatants were discarded and the cell pellets were resuspended in 100 µl of saline. an amount of 10 µl of cell suspension was mixed with 90 µl of trypan blue and the total cell counts taken using a haemocytometer. the remainder of the cell suspension was spun for 5 min at 500 rpm onto shandon coated microscope slides (fisher scientific, dublin, ireland) using shandon filter cards and shandon cytospin ® 2. the cells were fixed and stained using speedy-diff 203 cell stain kit (clin-tech ltd., guildford, uk). briefly, the slides were immersed for a few seconds in speedy-diff fixative (coloured methanol) five times then immersed for a few seconds in speedy-diff a (buffered eosin y) at least 5 times followed by blotting and washing in pbs and finally immersed in speedy-diff b (buffered azur/methylene blue) at least 5 times, blotted and washed. the slides were dried and the differential cell counts were obtained using a ceti light microscope (medline scientific, chalgrove oxon, uk) at 100× magnification under oil immersion. the macrophages and neutrophils were counted in 5 random fields and the total differential cell counts were calculated. a rat demonstration 7-plex ultra-sensitive kit assay (mesoscale discovery, rockville, md, usa was used to simultaneously detect ifn-γ, il-1β, il-4, il-5, il-13, kc/gro/cxcl-1 and tfn-α cytokines. the assay was set up and run according to the manufacturer's instructions. briefly, 25 µl of provided diluent 6 was added to each well and the plate was sealed and incubated at room temperature with vigorous shaking (300-1000 rpm) for 30 min. an 8-point serial dilution of rat demonstration 7-plex calibrator blend (40000-0 pg/ml) was prepared in 1% bsa in pbs (for bal fluid calibration). the bal fluid samples were mixed with 1% bsa to reduce protein adherence to the micro-tubes. standards and bal fluid (25 µl) were added in duplicate to wells and the plate was sealed and incubated for 2 h at room temperature with vigorous shaking. the plate was washed 3 times with pbs-t followed by the addition of 25 µl of 1× detection antibody solution. the plate was resealed and vigorously shaken at room temperature for 2 h. the washing 3 times in pbs-t was repeated. an amount of 150 µl of 2× read buffer t (4× read buffer t diluted in dh 2 o) was added to each well and the plate was immediately analysed on a sector imager. standard curves were produced and the concentrations of analytes for each sample determined using the msd discovery workbench ® software. the fixed lung tissue was embedded in paraffin and the sections were mounted onto slides. the paraffin was removed by immersing slides in histochoice ® clearing agent for at least 30 min. the tissue sections were hydrated by immersing them for a few seconds in 100% ethanol followed by 90%, 70% and 50% ethanol and finally dh 2 o. after hydration, the sections were stained with haematoxylin for 15 min followed by rinsing in dh 2 o. the sections were differentiated in 1% acid alcohol to remove excess haematoxylin by immersing for a few seconds in 50% ethanol, then 70% ethanol followed by acid alcohol (1% concentrated hydrochloric acid in 70% ethanol (v/v)). the sections were then immersed in 70% ethanol followed by rinsing in dh 2 o. the sections were immersed in alkaline ammonia water (0.2% potassium bicarbonate and 2% magnesium sulphate) and rinsed by running under tap water for 10 min. following rinsing, the sections were stained with eosin (5 min) nanomaterials 2020, 10, 1248 8 of 23 and then immersed for a few seconds in dh 2 o. the sections were then dehydrated by immersing for a few seconds in 50%, then 70%, 90% and finally 100% ethanol followed by a short immersion in histochoice ® clearing agent. coverslips were mounted onto the slides with dpx mounting medium. the lung sections were examined for inflammation by light microscopy. the degree of neutrophil-rich inflammation was scored as − (absent), +/− (very mild), + (mild) or ++ (moderate). scores were then assigned per treatment group. photomicrographs were acquired for each "score" using a nikon eclipse e600 microscope at 40×, 200× and 600×. all the samples were run in triplicate and the experiment was repeated on 3 independent occasions unless otherwise stated. statistical significance was determined for in vitro assays using the graphpad prism 5 software and the one or two-way anova method of statistical analysis using bonferroni multiple comparison tests analysis in all cases. for the in vivo work, significance was calculated using the kruskal-wallis test and dunn's post-hoc test. the data was expressed ± the standard deviation at all times (sd). the results were deemed to be statistically significant (*) where the p values were found to be <0.05, very significant (**) at p < 0.01 and extremely significant (***) at p < 0.001. following purification, the pei-lpeg was analysed via gpc, where it was found that the final product demonstrated a faster elution time than either the pei or lpeg starting materials ( figure s1 ). this indicated a larger molecular weight co-polymer had been effectively synthesised. confirmation of the successful conjugation was also carried out using 13 c nmr, which highlighted the presence of the carboxylic bonding between the pei and lpeg at 164 ppm ( figure s2 ). finally, the % grafting was estimated using 1 h nmr and the integration of the pei present at 2.5 ppm and the peg present at 3.5 ppm ( figure s3 ). this was approximately 80% grafted, as was previously the case when the initial in vitro work was reported [32] . in order to establish an effective inhaled sirna therapy, it is important to examine the effect of nebulisation on the sirna nanoparticle size and to establish that, on reaching their site of action, the sirna nanoparticles were of the desired size for cell endocytosis. the sirna nanoparticles underwent particle sizing before nebulisation. following nebulisation using a vibrating mesh nebuliser (vmn) and passage through the tsi, the sirna nanoparticles were collected from the throat and stage a and b of the tsi and resized. it was found that the vibrating mesh nebulisation of sirna nanoparticles did not result in significant changes to the nanoparticle size in the respirable fraction ( figure 2 ). in the case of the pei sirna nanoparticles, the greatest increases in nanoparticle size and polydispersity were found in stage a, which corresponds to the non-respirable fraction. however, the nanoparticle size was found to be unchanged in samples collected from stage b of the tsi, corresponding to the respirable fraction. similarly, the pei-lpeg sirna nanoparticles collected in the throat and upper chamber were found to have significant increases in size and polydispersity compared to the un-nebulised samples. however, the nanoparticles found in stage b demonstrated no significant changes. on comparing the pei and pei-lpeg sirna nanoparticles, the pegylated particles were significantly larger than the unmodified pei in the lower stage (stage b) of the tsi. however, the polydispersity indices for both did not demonstrate any significant differences. the aerogen pro vibrating mesh nebuliser was used to nebulise the pei and pei-lpeg sirna nanoparticles, and the vmd and %fpf was determined and compared to the nebulisation of pbs using the same device (table 1) . on the examination of the vmd and %fpf of each sirna nanoparticle suspension, it was found that there were no significant changes when compared to the pbs controls. the vmd of the droplets containing sirna nanoparticles remained around ~5 μm in all cases, which is within the required size range for successful delivery to the deep lungs [36] . in addition, the %fpf of each nebulised sirna nanoparticle sample remained between 55% and 60%. however, on examining the changes in output rate, the pei-lpeg sirna n/p = 15 nanoparticle samples demonstrated a 33% drop in output efficiency compared to the pbs controls. the surface tension of the pei and pei-lpeg sirna nanoparticle samples at the concentrations tested was measured, as this has been found to impact on the nebulisation of liquids. the pei-lpeg-sirna nanoparticle system had a decreased surface tension, although this was found not to be statistically significant compared to the other groups. table 1 . effect of pei and pei-lpeg sirna nanoparticles on the vibrating mesh nebuliser output rate (ml/min), % fine particle fraction (%fpf) and by volumetric median diameter (dv (50)). surface the aerogen pro vibrating mesh nebuliser was used to nebulise the pei and pei-lpeg sirna nanoparticles, and the vmd and %fpf was determined and compared to the nebulisation of pbs using the same device (table 1) . on the examination of the vmd and %fpf of each sirna nanoparticle suspension, it was found that there were no significant changes when compared to the pbs controls. the vmd of the droplets containing sirna nanoparticles remained around~5 µm in all cases, which is within the required size range for successful delivery to the deep lungs [36] . in addition, the %fpf of each nebulised sirna nanoparticle sample remained between 55% and 60%. however, on examining the changes in output rate, the pei-lpeg sirna n/p = 15 nanoparticle samples demonstrated a 33% drop in output efficiency compared to the pbs controls. the surface tension of the pei and pei-lpeg sirna nanoparticle samples at the concentrations tested was measured, as this has been found to impact on the nebulisation of liquids. the pei-lpeg-sirna nanoparticle system had a decreased surface tension, although this was found not to be statistically significant compared to the other groups. table 1 . effect of pei and pei-lpeg sirna nanoparticles on the vibrating mesh nebuliser output rate (ml/min), % fine particle fraction (%fpf) and by volumetric median diameter (dv (50)). to examine the suitability of pei/pei-peg sirna nanoparticles for the delivery of a potentially therapeutic anti-inflammatory sequence, anti-il-8 sirna was directly delivered to fully polarised calu-3 monolayers (n/p = 15). for a more meaningful assessment of the potential therapeutic effect, the il-8 expression in calu-3 cells was investigated at the protein level using an il-8-specific elisa. this also allowed for the examination of protein expression in both the apical and basal secretions of the calu-3 cells grown on transwell™ inserts. on the analysis of the pei anti-il8 sirna nanoparticle-treated samples, it was found that there were no significant changes in il-8 secretion in any of the apical samples. however, there were significant changes in the il-8 secretion in the basal samples ( figure 3a ) detected for pei anti-il-8 sirna nanoparticles (780 pg/ml ± 85.08 pg/ml) in comparison to the pbs samples (1131 pg/ml ± 8.95 pg/ml). the administration of the anti-gapdh sirna nanoparticles or an equivalent concentration of free pei (as controls) did not result in any significant changes in the il-8 secretion. on the analysis of the pei-lpeg sirna nanoparticle-treated cells, decreases in basal secretions of il-8 were also observed ( figure 3b ). however, these were not found to be statistically significant. similar to the pei data, the administration of pei-lpeg anti-gapdh sirna control nanoparticles did not cause any significant changes in the il-8 expression. finally, the administration of pei-lpeg in solution at concentrations equivalent to n/p = 15 nanoparticles to calu-3 cells did not result in a significant increase in il-8 secretion, as was also seen with the equivalent pei treated samples. as a means of investigating the effect that the administered sirna nanoparticles had on the calu-3 cell monolayer tight junctions, the teer values of the treated cells were taken at regular intervals and compared against pbs-treated cells ( figure 3c ). compared to the pbs-treated samples, the administration of pei and pei-lpeg sirna nanoparticle solutions did not significantly alter the teer integrity for the first 4 h post-treatment. however, a comparative treatment with pei-lpeg sirna nanoparticles resulted in a greater, but non-significant, decrease (26% decrease at 4 h) in the teer values compared to the pbs (8% decrease at 4 h) or pei sirna nanoparticle (7% decrease at 4 h)-treated samples. interestingly, the greatest drop in teer in the pei sirna nanoparticle-treated samples occurred at 30 min post-treatment, whereas this point was not reached until 2 h post-treatment in the pei-lpeg sirna nanoparticle-treated groups. finally, the teer values of both the pei and pei-lpeg nanoparticles were also found to recover and were significantly higher than the pbs controls at 24 h post treatment. this represents a recovery of tight junction integrity in all the samples and an ability to withstand the stresses involved in sirna nanoparticle delivery. in order to comprehensively validate the tsi system, pei and pei-lpeg sirna nanoparticles at n/p = 15 containing 0.6, 1.2 or 2.4 µg/well of anti-gapdh sirna were nebulised in five independent experiments. for comparison, 0.6µg/well of sirna was the standard amount used in previous non-nebulised transfections in section 3.3. following the real-time rt-pcr analysis of gapdh inhibition, there was a significant difference in transfection efficiencies between the pei and pei-lpeg-treated samples (figure 4) . the pei sirna nanoparticle-treated samples displayed highly variable levels of gapdh knockdown. even at the highest doses, the nebulised pei-mediated gapdh expression was 1.69-fold (±1.62) that of the pbs-treated cells. in contrast, the nebulised pei-lpeg sirna nanoparticles demonstrated significantly greater levels of gapdh knockdown versus the pbs-treated controls at higher doses. in 2.4 µg/well sirna nanoparticle-treated samples, the gapdh expression was found to be 0.46 (±0.58) that of pbs-treated cells. this was significantly lower than both the pbs controls and the corresponding pei sirna nanoparticle-treated sample (* p < 0.05) when analysed via a 2-way anova. following the initial validation of the anti-il-8 sirna sequence specificity in standard transfection experiments and the validation of the tsi set-up using anti-gapdh sirna, the il-8 sirna nanoparticles were administered via the tsi ( figure 5 ). in the case of pei anti-il-8 sirna nanoparticle-treated cells, protein inhibition was again observed in the basal secretions ( figure 5a ). using 1.2 and 2.4 µg sirna doses, the il-8 expression was reduced by up to 45% compared to the pbs-treated controls (824 pg/ml ± 152 sd vs. 1513 pg/ml ± 201 sd, respectively) but were not found to be statistically significant. in contrast, the nebulised doses of pei-lpeg sirna demonstrated il-8 reduction in both the apical and basal samples ( figure 5b ). significant decreases in il-8 secretion of up to 50% were seen in apical secretions as well as decreases of up to 40% in basal secretions (non-significant). this demonstrated that only the nebulised pei-lpeg anti-il-8 sirna particles were capable of eliciting a significant inhibitory effect at the protein level. 1.69-fold (±1.62) that of the pbs-treated cells. in contrast, the nebulised pei-lpeg sirna nanoparticles demonstrated significantly greater levels of gapdh knockdown versus the pbstreated controls at higher doses. in 2.4 μg/well sirna nanoparticle-treated samples, the gapdh expression was found to be 0.46 (±0.58) that of pbs-treated cells. this was significantly lower than both the pbs controls and the corresponding pei sirna nanoparticle-treated sample (* p < 0.05) when analysed via a 2-way anova. following the initial validation of the anti-il-8 sirna sequence specificity in standard transfection experiments and the validation of the tsi set-up using anti-gapdh sirna, the il-8 sirna nanoparticles were administered via the tsi ( figure 5 ). in the case of pei anti-il-8 sirna nanoparticle-treated cells, protein inhibition was again observed in the basal secretions ( figure 5a ). using 1.2 and 2.4 μg sirna doses, the il-8 expression was reduced by up to 45% compared to the pbs-treated controls (824 pg/ml ± 152 sd vs. 1513 pg/ml ± 201 sd, respectively) but were not found to be statistically significant. in contrast, the nebulised doses of pei-lpeg sirna demonstrated il-8 reduction in both the apical and basal samples ( figure 5b ). significant decreases in il-8 secretion of up to 50% were seen in apical secretions as well as decreases of up to 40% in basal secretions (non-significant). this demonstrated that only the nebulised pei-lpeg anti-il-8 sirna particles were capable of eliciting a significant inhibitory effect at the protein level. the intra-tracheal instillation of sirna nanoparticles in a lps-stimulated rat model of acute inflammation was investigated in a pilot study. based on the promising data obtained from in vitro monolayer culture experiments, n/p = 15 was chosen as the n/p ratio, with a sirna dose of 75 μg extrapolated from a previous in-house study [37] . however, the n/p ratio was reduced due to the intra-tracheal instillation of sirna nanoparticles in a lps-stimulated rat model of acute inflammation was investigated in a pilot study. based on the promising data obtained from in vitro monolayer culture experiments, n/p = 15 was chosen as the n/p ratio, with a sirna dose of 75 µg extrapolated from a previous in-house study [37] . however, the n/p ratio was reduced due to practical considerations and n/p = 7 was chosen, as it was more comparable with the prior literature [38] [39] [40] . on analysis, it was found that there was a significant increase in the overall bal cell population following lps stimulation, going from 19.5 (±12.26) × 10 4 cells/ml to 197.4 (±50.82) ×10 4 cell/ml ( figure 7a ). for groups treated with the pei and pei-lpeg anti-il8 sirna nanoparticles, no significant decrease in the overall cell number was evident. a similar level of inflammation across all the lps-treated groups was also evident upon histological analysis ( figure s6 ). using the differential cell staining of bal samples with eosin y and azur/methylene blue, it was in the case of the pei-lpeg sirna nanoparticle-treated groups, both the non-targeting (nt) and anti-cxcl-1 sirna-treated groups demonstrated 10-fold decreases in the cxcl-1 gene expression compared to the pbs-lps samples (4-vs. 40-fold respectively). while the gene expression was significantly decreased versus the lps-treated controls, there were no significant differences between the targeted and non-targeted sirna-treated animals. this indicated that the effect was not solely related to sirna-mediated protein inhibition. this was unexpected, considering the pei-lpeg polymer has been previously validated in vitro as causing no off-target effects in previous work [32] as well as through the results in this study. the cxcl-1 protein expression in the bal fluid of treated rats was then examined as part of the 7-plex multi-analyte array ( figure 6b ). the results found that, at the early timepoint of 3 h post lps challenge, only the pei-lpeg nt sirna-treated groups demonstrated significantly higher levels of cxcl-1 secretion when compared to the pbs-pbs samples. however, there were no significant differences compared to the pbs-lps-treated control animals. the results for each remaining cytokine of the multi-plex array were analysed and the test samples were compared for cytokine expression against the pbs-lps-treated control animals ( figure s4 ). overall, it was found that the administration of sirna nanoparticles did not lead to statistically relevant changes in cytokine levels compared to the pbs-lps-treated animals. when compared with the pbs-pbs-treated animals, there were similarly few significant changes in cytokine expression. the pei-lpeg nt sirna-treated animals were found to have a significantly higher il-5 expression compared to the pbs-pbs control. the pei-lpeg nt and anti-cxcl-1 sirna-treated animals had a higher il-4 secretion compared to the pbs-pbs-treated group. finally, it was noted that the cxcl-1 levels were significantly higher compared to several other cytokines in the pbs-pbs-treated animals ( figure s5 ). on analysis, it was found that there was a significant increase in the overall bal cell population following lps stimulation, going from 19.5 (±12.26) × 10 4 cells/ml to 197.4 (±50.82) ×10 4 cell/ml ( figure 7a ). for groups treated with the pei and pei-lpeg anti-il8 sirna nanoparticles, no significant decrease in the overall cell number was evident. a similar level of inflammation across all the lps-treated groups was also evident upon histological analysis ( figure s6 ). using the differential cell staining of bal samples with eosin y and azur/methylene blue, it was also possible to identify the separate cell populations in each treatment group ( figure 7b-h) . overall, macrophages were the predominant cell types identified in bal. the effect of lps stimulation on immune cell infiltration was evident, with the macrophage levels increasing from 34.8 (±12.41) macrophages per field of view in the pbs-pbs-treated group to 153.5 (±73.38) in the pbs-lps-treated group. however, for the animals treated with the pei anti-cxcl-1 sirna nanoparticles, there was a significant (p < 0.05) decrease in macrophage infiltration compared to those treated with the pei nt sirna nanoparticles (93.95 ± 55.21 vs. 258.5 ± 131.9 macrophages/field, respectively). for the groups treated with pei-lpeg sirna nanoparticles, no significant differences in macrophage infiltration were seen between the nt and anti-cxcl-1 groups (301 ± 363 cells and 395 ± 284 cells/field, respectively) as well as when compared to the pbs-lps-treated control group. the successful development of any pulmonary sirna nanoparticle therapy is dependent on both the efficient delivery of these nanoparticles through a suitable device and the bioactivity of the delivered sirna nanoparticles. the effect of the vibrating mesh nebulisation on the sirna nanoparticle size was first examined. an analysis of the effect of nebulisation on the sirna nanoparticle size determined that the most significant changes in nanoparticle size occurred in the fractions of the tsi corresponding to the non-respirable fractions. in contrast, the nanoparticles collected in the lower (respirable) fraction of the tsi demonstrated no significant changes. while the nanoparticle size and polydispersity were high, this is most likely due to the use of pbs as a suspension buffer to facilitate nebulisation. similarly, the use of pbs in the subsequent recovery of the particles from the tsi may have affected the size of the particles. for instance, the high ionic strength of pbs can cause electrostatically formed nanoparticles to swell considerably [23] . however, as the fractions were treated in the same manner, the relative changes in size and pdi are thus comparable. therefore, it was encouraging to note that the pei-lpeg particles maintained their integrity in an undiluted and physiologically relevant buffer. furthermore, the pei-lpeg sirna nanoparticles demonstrated higher particle sizes than the unmodified pei sirna nanoparticles. this was expected since grafting the peg molecules to pei can result in a loss of charge density [41] and thus become more sensitive to the effects of pbs dilution. overall, these results indicated the relatively low impact of vibrating mesh nebulisers on the integrity of the sirna nanoparticles for pulmonary delivery. the vibrating mesh nebuliser (vmn) performance was similarly unaffected when used for the nebulisation of the pei/pei-lpeg sirna nanoparticles. the emitted droplets were all approximately 5 µm, which is in the desired size range for delivery to the lower airways [36] . however, there was a slight decrease in the output efficiency apparent when the vmn were used to nebulise the pei-lpeg sirna samples. this can be explained by the ability of peg to decrease the surface tension in a solution, which can cause a reduction in the output rate. this has previously been related to the fact that samples with a low surface tension more readily wet the aperture surface and therefore decrease the efficiency of droplet formation, a phenomenon known as the "loxy effect" [17, 42] . prior to assessing the nebulised sirna delivery and efficacy, the il-8 knockdown using the nanoparticles was first assessed by direct delivery onto calu-3 monolayers to establish the sequence specificity as well as investigating any underlying inflammatory effects. when examined for changes in il-8 protein levels, it was found that both the pei and pei-lpeg anti-il8 sirna nanoparticles were capable of eliciting decreases in il-8 expression, but only pei anti-il8 sirna significantly so and only on the basal side. previous work with polarised epithelial cells has found that cells preferentially secrete apically [43, 44] . therefore, it is likely that apical secretion is prioritised and kept at homeostatic levels as much as possible with basal secretion bearing the decrease in il-8 production following anti-il-8 sirna treatment. furthermore, while cationic polymers can stimulate il-8 secretion in their own right [44] , the persistent presence of the pei/pei-lpeg free polymer in solution on the apical cell surface did not trigger any significant increases versus the pbs-treated wells. recent studies have also highlighted that the delivery of exogenous sirna can mediate inflammation [45, 46] . however, using anti-gapdh as a non-relevant negative control, it was also found that there were no non-specific changes in il-8 production using either polymer. an analysis of fluctuations in the teer values following direct anti-il8 sirna nanoparticle administration revealed that treatment with both pei and pei-lpeg sirna nanoparticles resulted in slight, but non-significant, decreases in teer following transfection, with significant increases in teer values 24 h post-transfection. this slightly greater teer decrease seen for the pegylated constructs has also been previously reported using pegylated chitosan. in that study, cassettari et al. postulated that the greater decrease in teer values mediated by pegylated constructs was due to the higher masses used for pegylated polymers to reach equivalent concentrations of cationic polymer [47] . this is also in keeping with findings regarding a temporary disruption recovery in epithelial cell tight junctions following transfection [48] . once the utility of the vibrating mesh nebuliser and sequence specificity of the anti-il-8 sirna nanoparticles were established, it was then possible to investigate the impact of nebulisation on the bioactivity of the sirna nanoparticles. previously, our lab had investigated post-nebulisation sirna transfection using simple non-polarized calu-3 cell cultures [23] . in this study, to better recapitulate the clinical environment, a twin-stage impinger deposition instrument was coupled with a polarised cell monolayer grown on a transwell insert (figure 1 ) for transfection studies. in the initial gapdh validation of the system, experiments found that there were significant differences in sirna nanoparticle behaviour going from non-nebulised to nebulised transfection. the pei sirna nanoparticle transfection efficiency became highly variable following nebulisation, whereas the pei-lpeg sirna nanoparticles were capable of consistent and significant decreases in gapdh gene expression. while dispersing sirna nanoparticles through a twin-stage impinger has been previously recorded [49, 50] , to date this has not been applied to monitor the post-nebulisation transfection efficiency. the positive role of pegylation in nebulisation and mucus trafficking was further demonstrated in tsi anti-il-8 sirna transfections of polarised calu-3 monolayers. the pei-lpeg sirna-transfected cells displayed decreased il-8 cytokine levels both apically and basally, with significant decreases in the apical secretion. in relation to the strong post-nebulisation efficiency of the pei-lpeg samples, this is most likely as pegylation is known to enhance colloidal stability as well as enhance the mucus penetration and trafficking of delivered particles [51] [52] [53] . in addition to an enhanced ability to traverse the mucus barrier, it has also been found that the aerosol administration of drugs can result in a faster trafficking time compared to solution administration. specifically, the lower the apical fluid volume, the faster the transport [54, 55] . this would explain the more obvious apical and basal il-8 inhibition in nebulised sirna nanoparticle-treated samples compared to the un-nebulised samples, with the mucus trafficking abilities of pegylation becoming more quickly apparent. following these positive results, the sirna nanoparticles were tested in a rat model of acute lps-mediated inflammation. the effects of this were then examined at the genetic, protein and cellular levels. at a genetic level, the use of lps to drive cxcl-1 up-regulation was validated with significant increases observed in gene expression. furthermore, pei anti-cxcl-1 sirna nanoparticles were also found to demonstrate reduced gene expression compared to their non-targeting controls although this was not found to be significant due to the variability in the nt-sirna-treated animals. interestingly, it was seen that there was some disparity in the non-targeting sirna treatment groups. the cxcl-1 gene expression in pei nt-sirna-lps-treated rats was found to be strongly up-regulated, while the pei-lpeg nt-sirna-treated animals demonstrated cxcl-1 upregulation to a much lower degree. considering that the same non-targeting sequence was used in both test groups, it is unlikely that this was nucleic acid sequence dependent. the in vitro work described here using non-targeted sirna also demonstrated that neither pei nor pei-lpeg elicited any significant changes in the il-8 expression. however, it is also known that pegylation can modulate the inflammatory effect of nanoparticles. previous work has shown that pei caused higher levels of complement activation, whereas conjugation with high molecular weight peg reduced the inflammatory reactions in vivo [56] [57] [58] . however, it is possible that the lps is interacting with the pei-lpeg to some degree. previous work has demonstrated that peg chains with cationic moieties can neutralise lps [59] . combined with the increased residency time in the lungs of high molecular weight pegylated compounds [60] , this could explain why the diminished effect of lps at a genetic level may be more pronounced in the pei-lpeg-treated animals. the multi-parameter cytokine screen revealed that there were few significant changes in protein expression in treated groups compared to pbs-pbs sham-treated animals. this was likely due to the very short time between lps challenge and euthanasia of the animals (3 h). previous work has demonstrated that it can take between 24-72 h for cytokine levels to peak in pulmonary instilled animals [39, 61] . this was the case for cxcl-1 expression in all animals, with the exception of the pei-lpeg nt sirna-treated animals. these rats demonstrated significantly higher levels of cxcl-1 secretion when compared to the pbs-pbs-treated animals. however, the cxcl-1 levels in all the experimental groups were not significantly different from the pbs-lps-treated animals. however, it was also noted that there were some very high levels of expression in two out of four animals treated with pei-lpeg sirna. while it was not possible to say definitively what is causing this spike in cxcl-1 secretion in these animals, it is possible that animals may be reacting to the peg moieties. there is a growing body of literature (reviewed in [62] ) highlighting the risks of pegylation in stimulating the immune system. however, the complexities of this are far from certain, with previous studies demonstrating immune responses are highly dependent on peg chain length and grafting density. specifically, higher levels of peg-grafting resulted in less pro-inflammatory effects without depleting macrophages [63] . thus, care must be taken in the future in developing low intensity dosing regimens and delivery routes such as nebulisation that minimise this outcome. finally, intratracheally delivered pbs-pbs negative controls were also found to elicit~40 pg/ml of cxcl-1 secretion, which is roughly equivalent to levels recorded in asthma patients (30 pg/ml) [64] . this would indicate that the procedure itself may be immune-stimulatory. when the ifn-γ levels were examined, it was found that the administration of lps resulted in significant increases in secretion. interestingly, similar significant increases in ifn-γ protein expression in the pei or pei-lpeg-treated groups were not observed following lps administration, regardless of the sirna type. this was in line with studies which have found that pei has a lower stimulatory effect on ifn-γ expression than other polymers in mice [39, 65] . in addition to cytokine content, the cellular composition in the extracted bal fluid was investigated. here, it was found that the administration of lps resulted in a rapid and significant influx of a variety of immune cells. from the total cell population counts of the extracted bal fluid, there were no significant differences between all the lps-treated sample groups. however, differential staining for macrophages revealed that the delivery of anti-cxcl-1 sirna could elicit a significant reduction using pei nanoparticles. this further validates the in vitro evidence from this study demonstrating the higher rate of transfection with pei when directly administered to cells. this may also be aided by the fact that pei has also been documented as depleting bal macrophages when administered to the lungs [63] (although no evidence of this was observed in nt-sirna-treated rats). given the large influx of innate immune cells, an important consideration for future sirna-based inflammatory therapies in the lungs would be to enable targeting of both the epithelial cells and macrophages. this would allow for a more complete inhibition of the inflammatory response. this is currently being investigated in our own lab and elsewhere [66] [67] [68] and must be partnered with advanced 3d cell culture models for enhanced in vitro assessment [69] . considering the in vitro/in vivo disparity in efficacy of pei-lpeg nanoparticles in significantly decreasing il-8/cxcl-1 at the genetic level and indications of high levels of cytokine secretion in vivo, it is important to examine the differences between the nebulised and intratracheal delivery of sirna nanoparticles. specifically, in terms of invasiveness and direct delivery, intratracheal delivery was deemed necessary due to the high levels of sirna that would be required for in vivo nebulisation (a previous study of nebulised mrna at 0.5 mg/ml [21] ) combined with the technical challenges associated with nebulised delivery to small animals. as a result of this, the n/p ratios that were optimised for nebulisation and had demonstrated efficient knockdown were now delivered directly to the lungs in a much more concentrated fashion. this concentrated dose would further exacerbate any underlying inflammation associated with sirna nanoparticle delivery. this is in agreement with a recent study by ng et al. which reported no differences between the bolus and intra-tracheal delivery of sirna in mice [45] . therefore, the benefits of pegylation observed in the study were very much dependent on their means of delivery and may have been obscured by intra-tracheal/bolus administration in vivo. there is growing evidence now demonstrating delivery-related inflammatory responses to the bolus delivery of sirna [56, 70] (indications of which were present in bal fluid). complement activation related pseudo-allergy (carpa) has triggered a move towards lower density/more controlled delivery methods such as i.v. perfusion for continued clinical development [5] . similarly, this study has demonstrated that nebulisation represents an analogous low-density, aerosol approach to achieving high levels of transfection efficiency while avoiding inflammatory events associated with bolus delivery. future work for pulmonary delivery of sirna (and all oligonucleotide therapeutics) must now focus on not reverting to bolus delivery methods when progressing to pre-clinical studies. the continued development and refinement of existing pre-clinical models of nebulisation is a priority in this case. in this study, convergent device-nanoparticle development was addressed through the in vitro testing of knockdown efficiency of nebulised sirna nanoparticles in successively more complex cell assays. in parallel, the effect of sirna nanoparticle cargo on the nebuliser output and performance was also assessed and vice versa. finally, these sirna nanoparticles were progressed to in vivo testing in an lps-induced rat model of acute pulmonary inflammation. through these experiments, it was determined that sirna nanoparticles are capable of being efficiently nebulised through a vibrating mesh nebuliser without any impact on the integrity of the nanoparticles or the device performance. twin-stage impinger cell studies highlighted the significantly enhanced ability of pei-peg sirna nanoparticles to be nebulised, traverse the mucus lining of airway epithelial cells and reduce gene and protein expression compared to pei sirna nanoparticles. to support in vivo testing, a rat model of acute pulmonary inflammation was established. the cxcl-1 gene expression could be reduced using some of the sirna nanoparticle treatments to a level comparable to negative (healthy) controls. furthermore, in the case of pei anti-cxcl-1 sirna nanoparticle-treated animals, significant reductions in macrophage infiltration were also observed. however, there was significant variability and inconsistency in the effects seen for the treatment groups on cxcl-1 protein expression. in this study we have noted initial evidence of a relationship between the efficacy of the pegylated pei and the means of delivery. specifically, issues were identified when switching to direct intubation and bolus delivery instead of nebulisation, as was used in the in vitro studies. the genetic knockdown elicited by some of the treatments may well be realised at a protein level with further refinement in dosing, dose timing and the application of a less invasive means of delivery in future studies. supplementary materials: the following are available online at http://www.mdpi.com/2079-4991/10/7/1248/s1, figure s1 : gpc size and purity analysis of synthesized pei-lpeg overlaid with its respective starting materials., figure s2 : pei-lpeg carboxylic bonding presence was shown in 13 c nmr at 164 ppm, figure s3 : 1 h nmr of synthesized pei-lpeg polymer with pei present at 2.5 ppm and peg present at 3.5 ppm, figure s4 figure s5 : bal cytokine expression levels in pbs-pbs treated rats (minimum n = 3 ±sd, kruskal-wallis test and dunn's post-hoc test, * p < 0.05), figure s6 : pulmonary histopathology semi quantitative scoring of neutrophil-rich inflammation. haemotoxylin and eosin stained lung sections from top pbs, pbs-lps and sirna nanoparticle treated rats were scored based on the degree of neutrophil-rich inflammation observed (− absent, + mild, ++ moderate and +++ highly inflamed). images were acquired at 40×, 200× and 600× magnification with arrows indicating evidence of inflammation and of blood and protein in the alveoli and loss of the alveolar lining at higher levels of severity. therapeutic oligonucleotides: state of the art co-delivery of free vancomycin and transcription factor decoy-nanostructured lipid carriers can enhance inhibition of methicillin resistant staphylococcus aureus (mrsa) rna-based therapeutics: from 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evidence, mechanisms and approaches to inhibition. in handbook of immunological properties of engineered nanomaterials this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank lorraine nolan and awadh yadav, school of pharmacy, rcsi for assistance in preliminary experiments and drafting of animal ethics application. sincere thanks to anita umerska and lidja tajber of trinity college dublin for kind use of the ring tensiometer.conflicts of interest: r.m. is an employee of aerogen. key: cord-292862-ezrkg0dc authors: myerson, jacob w.; patel, priyal n.; habibi, nahal; walsh, landis r.; lee, yi-wei; luther, david c.; ferguson, laura t.; zaleski, michael h.; zamora, marco e.; marcos-contreras, oscar a.; glassman, patrick m.; johnston, ian; hood, elizabeth d.; shuvaeva, tea; gregory, jason v.; kiseleva, raisa y.; nong, jia; rubey, kathryn m.; greineder, colin f.; mitragotri, samir; worthen, george s.; rotello, vincent m.; lahann, joerg; muzykantov, vladimir r.; brenner, jacob s. title: supramolecular organization predicts protein nanoparticle delivery to neutrophils for acute lung inflammation diagnosis and treatment date: 2020-04-18 journal: biorxiv doi: 10.1101/2020.04.15.037564 sha: doc_id: 292862 cord_uid: ezrkg0dc acute lung inflammation has severe morbidity, as seen in covid-19 patients. lung inflammation is accompanied or led by massive accumulation of neutrophils in pulmonary capillaries (“margination”). we sought to identify nanostructural properties that predispose nanoparticles to accumulate in pulmonary marginated neutrophils, and therefore to target severely inflamed lungs. we designed a library of nanoparticles and conducted an in vivo screen of biodistributions in naive mice and mice treated with lipopolysaccharides. we found that supramolecular organization of protein in nanoparticles predicts uptake in inflamed lungs. specifically, nanoparticles with agglutinated protein (naps) efficiently home to pulmonary neutrophils, while protein nanoparticles with symmetric structure (e.g. viral capsids) are ignored by pulmonary neutrophils. we validated this finding by engineering protein-conjugated liposomes that recapitulate nap targeting to neutrophils in inflamed lungs. we show that naps can diagnose acute lung injury in spect imaging and that nap-like liposomes can mitigate neutrophil extravasation and pulmonary edema arising in lung inflammation. finally, we demonstrate that ischemic ex vivo human lungs selectively take up naps, illustrating translational potential. this work demonstrates that structure-dependent interactions with neutrophils can dramatically alter the biodistribution of nanoparticles, and naps have significant potential in detecting and treating respiratory conditions arising from injury or infections. the covid-19 pandemic tragically illustrates the dangers of acute inflammation and infection of the lungs, for both individuals and societies. acute alveolar inflammation causes the clinical syndrome known as acute respiratory distress syndrome (ards), in which inflammation prevents the lungs from oxygenating the blood. severe ards is the cause of death in most covid-19 mortality and was a major cause of death in the 1918 influenza epidemic, but ards is common even outside of epidemics, affecting ~200,000 american patients per year with a ~35-50% mortality rate. 1-6 ards is caused not just by viral infections, but also by sepsis, pneumonia (viral and bacterial), aspiration, and trauma. 3, 4 largely because ards patients have poor tolerance of drug side effects, no pharmacological strategy has succeeded as an ards treatment. 5, [7] [8] [9] therefore, there is an urgent need to develop drug delivery strategies that specifically target inflamed alveoli in ards and minimize systemic side effects. neutrophils are "first responder" cells in acute inflammation, rapidly adhering and activating in large numbers in inflamed vessels and forming populations of "marginated" neutrophils along the vascular lumen. [10] [11] [12] [13] [14] [15] [16] neutrophils can be activated by a variety of initiating factors, including pathogen-and damage-associated molecular patterns such as bacterial lipopolysaccharides (lps). 17, 18 after acute inflammatory insults, neutrophils marginate in most organs, but by far most avidly in the lung capillaries. 6, 14, 15, 19, 20 neutrophils are therefore key cell types in most forms of ards. in ards, marginated neutrophils can secrete tissue-damaging substances (proteases, reactive oxygen species) and extravasate into the alveoli, leading to disruption of the endothelial barrier and accumulation of neutrophils and edematous fluid in the air space of the lungs ( figure 1a ). 6, 15, 16, [19] [20] [21] targeted nanoparticle delivery to marginated neutrophils could provide an ards treatment with minimal side effects, but specific delivery to marginated neutrophils remains an open challenge. antibodies against markers such as ly6g have achieved targeting to neutrophils in mice, but also deplete populations of circulating neutrophils. [22] [23] [24] [25] additionally, while ly6g readily marks neutrophils in mice, there is no analogous specific and ubiquitous marker on human neutrophils. 22 therefore, antibody targeting strategies have not been widely adopted for targeted drug delivery to these cells. 24 as another route to neutrophil targeting, two previous studies noted that activated neutrophils take up denatured and agglutinated bovine albumin, concluding that denatured protein was critical in neutrophil-particle interactions. 26, 27 nanoparticle structural properties such as shape, size, and deformability can define unique targeting behaviors. [28] [29] [30] [31] [32] here, we screened a diverse panel of nanoparticles to determine the nanostructural properties that predict uptake in pulmonary marginated neutrophils during acute inflammation. as a high-throughput animal model for ards, we administered lps to mice, causing a massive increase in pulmonary marginated neutrophils. we show that two initial leads in our screen, lysozyme-dextran nanogels (ldngs) and crosslinked albumin nanoparticles (anps), selectively home to marginated neutrophils in inflamed lungs, but not naïve lungs. in our subsequent screen of over 20 diverse nanoparticles, we find that 13 protein nanoparticles, all defined by agglutination of protein in amorphous nanostructures (nanoparticles with agglutinated proteins, naps), but not by denatured protein, have specificity for lps-inflamed lungs. in contrast to naps, we demonstrate that three symmetric protein nanostructures (viruses/nanocages) have biodistributions unaffected by lps injury. we show that polystyrene nanoparticles and five liposome formulations do not accumulate in injured lungs, indicating that nanostructures that are not based on protein are not intrinsically drawn to marginated neutrophils in acute inflammation. we then engineered liposomes (the most clinically relevant nanoparticle drug carriers) as naps, through conjugation to protein modified with hydrophobic cyclooctynes, encouraging protein agglutination on the liposome surface by hydrophobic interactions. we thus show that supramolecular organization of proteins, rather than chemical composition, best predicts uptake in marginated neutrophils in acutely inflamed lungs. we then demonstrate proof of concept for naps as diagnostic and therapeutic tools for ards. we show; a) 111 in-labeled naps provide diagnostic imaging contrast that distinguishes inflammatory lung injury from cardiogenic pulmonary edema; b) napliposomes can significantly ameliorate edema in a mouse model of severe ards; c) naps, but not crystalline protein nanostructures, accumulate in ex vivo human lungs rejected for transplant due to ards-like conditions. collectively, our results will demonstrate that supramolecular organization of protein, namely protein agglutination, predicts strong, intrinsic nanoparticle tropism for marginated neutrophils. this finding indicates that naps, encompassing a wide range of nanoparticles based on or incorporating protein, have biodistributions that are responsive to inflammation. naps could be useful beyond ards, since marginated neutrophils play a pathogenic role in a diverse array of inflammatory diseases, including infections, heart attack, and stroke. [10] [11] [12] [13] [14] but our findings provide a clear path forward for using naps to improve diagnosis and treatment of ards. to quantify the increase in pulmonary marginated neutrophils after inflammatory lung injury, radiolabeled clone 1a8 anti-ly6g antibody (specific for mouse neutrophils) was administered intravenously (iv) to determine the location and concentration of neutrophils in mice. iv injection of lps subjected mice to a model of mild ards. accumulation of anti-ly6g antibody in the lungs was dramatically affected by iv lps, with 20.81% of injected antibody adhering in lps-injured lungs, compared to 2.82% of injected antibody in naïve control lungs ( figure 1b) . agreeing with previous studies addressing the role of neutrophils in systemic inflammation, biodistributions of anti-ly6g antibody indicated that systemic lps injury profoundly increased the concentration of neutrophils in the lungs. 6, 10, 14, 18 single cell suspensions prepared from mouse lungs were probed by flow cytometry to further characterize pulmonary neutrophils in naïve mice and in mice following lps-induced inflammation. to identify intravascular populations of leukocytes, mice received iv fluorescent cd45 antibody five minutes prior to sacrifice. single cell suspensions prepared from iv cd45-stained lungs were then stained with anti-ly6g antibody to identify neutrophils. a second stain of single cell suspensions with cd45 antibody indicated the total population of leukocytes in the lungs, distinct from the intravascular population indicated by iv cd45. flow cytometry showed greater concentrations of neutrophils in lps-injured lungs, compared to naïve lungs ( figure 1c , counts above horizontal threshold indicate positive staining for neutrophils, figure 1d , rightmost peak indicates positive staining for neutrophils). comparison of ly6g stain to total cd45-positive cells indicated 53.5% of leukocytes in the lungs were ly6g-positive after lps injury, compared to 5.6% in the naïve control ( figure 1d , center panel). comparison of ly6g stain to iv cd45 stain indicated that the majority of neutrophils were intravascular, in both naïve and lpsinjured mice. in naïve mice, 85.3% of neutrophils were intravascular and in lps-injured mice, 96.2% of neutrophils were intravascular ( figure 1d , right panel). the presence of large populations of intravascular neutrophils following inflammatory injury is consistent with previously published observations. 6, 10, 14, 18, 33 histological analysis confirmed results obtained with flow cytometry and radiolabeled anti-ly6g biodistributions. staining of lung sections indicated increased concentration of neutrophils in the lungs following iv lps injury ( figure 1e , left panels). co-registration of neutrophil staining with tissue autofluorescence (indicating tissue architecture) broadly supported the finding that pulmonary neutrophils reside in the vasculature ( figure 1e , right panels). previous work has traced the neutrophil response to bacteria in the lungs, determining that pulmonary neutrophils pursue and engulf active bacteria following either intravenous infection or infection of the airspace in the lungs. 18, 34, 35 we injected heat-inactivated, oxidized, and fixed e. coli in naïve and iv-lps-injured mice. with the bacteria stripped of their functional behavior, e. coli did not accumulate in the lungs of naïve control mice (1.47% of initial dose in the lungs, blue bars in figure 1f ). however, pre-treatment with lps to recapitulate the inflammatory response to infection led to enhanced accumulation of the deactivated e. coli in the lungs (7.69% of initial dose in the lungs, red bars in figure 1f ). with e. coli structure maintained but e. coli function removed, the inactivated bacteria were taken up more avidly in lungs primed by an inflammatory injury. in order to identify nanostructural parameters that correlate with nanoparticle uptake in inflamed lungs, we conducted an in vivo screen of a diverse array of nanoparticle drug carriers. the screen was based on the method used above for tracing inactivated bacteria: inject radiolabeled nanoparticles into mice and measure biodistributions, comparing naïve with iv-lps mice. to validate that the radiotracing screen would measure uptake in pulmonary marginated neutrophils, we more fully characterized the in vivo behavior of two early hits in the screen. lysozyme-dextran nanogels (ldngs, ngs) and poly(ethylene)glycol (peg)crosslinked albumin nps have been characterized as targeted drug delivery agents in previous work. [36] [37] [38] here, ldngs (136.4±3.6 nm diameter, 0.10±0.02 pdi, supplementary figure 1a ) and peg-crosslinked human albumin nps (317.8±3.6 nm diameter, 0.14±0.05 pdi, supplementary figure 1b) were administered in naïve and iv-lps-injured mice. neither np was functionalized with antibodies or other affinity tags. the protein component of each particle was labeled with 125 i for tracing in biodistributions, and assessed 30 minutes after iv administration of nps. both absolute ldng lung uptake and ratio of lung uptake to liver uptake registered a ~25-fold increase between naïve control and lps-injured animals (figure 2a , supplementary table 1) . specificity for lps-injured lungs was recapitulated with peg-crosslinked human albumin nps. albumin nps accumulated in naïve lungs at 6.34% injected dose per gram organ weight (%id/g), and in lps-injured lungs at 87.62 %id/g, accounting for a 14-fold increase in lung uptake after intravenous lps insult ( figure 2b , supplementary table 1) . single cell suspensions were prepared from lungs after administration of fluorescent ldngs or peg-crosslinked albumin nps. flow cytometric analysis of cells prepared from lungs after np administration enabled identification of cell types with which nps associated. firstly, the total number of cells containing ldngs or albumin nps increased between naïve and lps-injured lungs. in naïve control lungs, 2. ly6g stain for neutrophils indicated that the bulk of ldng and albumin np accumulation in lps-injured lungs could be accounted for by uptake in neutrophils. in figure 2c and 2d, counts above the horizontal threshold indicate neutrophils and counts to the right of the vertical threshold indicate cells containing ldngs ( figure 2c ) or albumin nps ( figure 2d ). in iv-lps-injured lungs, ldng and albumin np uptake was dominated by neutrophils ( figure 2c , figure 2d , upper right quadrants indicate nppositive neutrophils). in lps-injured lungs, the majority of neutrophils, >70% of cells, contained significant quantities of nanoparticles, compared to <20% in naïve lungs. likewise, the majority of nanoparticle uptake in the lungs (>70%) was accounted for by nanoparticle uptake in neutrophils ( figure 2e , 2f, 2g, 2h, supplementary table 2) . for np uptake not accounted for by neutrophils, cd45 staining indicated that the remaining np uptake was attributable to other leukocytes. co-localization of albumin np fluorescence with cd45 stain showed that 91.9% of albumin np uptake was localized to leukocytes in naïve lungs and 97.8% of albumin np uptake was localized to leukocytes in injured lungs (supplementary figure 3c, supplementary figure 3d ). for ldngs, localization to neutrophils in injured lungs was confirmed via histology. ly6g staining of lps-injured lung sections confirmed colocalization of fluorescent nanogels with neutrophils in the lung vasculature ( figure 2i ). slices in confocal images of lung sections indicated that ldngs were inside neutrophils ( figure 2j ). intravital imaging of injured lungs allowed real-time visualization of ldng uptake in leukocytes in injured lungs. ldng fluorescent signal accumulated over 30 minutes and reliably colocalized with cd45 staining for leukocytes ( figure 2k , supplementary movie 1). ldng pharmacokinetics were evaluated in naïve and iv-lps-injured mice (supplementary figure 4) . in both naïve and injured mice, bare ldngs were rapidly cleared from the blood with a distribution half-life of ~3 minutes. in naïve mice, transient retention of ldngs in the lungs (25.91 %id/g at five minutes after injection) leveled off over one hour. in iv-lps-treated mice, ldng concentration in the lungs reached a peak value at 30 minutes after injection, as measured either by absolute levels of lung uptake or by lungs:blood localization ratio. ldng biodistributions were also assessed in mice undergoing alternative forms of lps-induced inflammation. intratracheal (it) instillation of lps led to concentration of ldngs in the lungs at 81.31 %id/g. liver and spleen ldng uptake was also reduced following it lps injury, leading to a 45-fold increase in the lungs:liver ldng localization ratio induced by it lps injury (supplementary figure 5) . as with iv lps injury, it lps administration leads to neutrophil-mediated vascular injury focused in the lungs. 14 mice were administered lps via footpad injection to provide a model of systemic inflammation originating in lymphatic drainage. 39 ldng uptake in the lungs and in the legs was enhanced by footpad lps administration. at 6 hours after footpad lps administration, ldngs concentrated in the lungs at 59.29 %id/g, an 11-fold increase over naïve. at 24 hours, ldngs concentrated in the lungs at 202.64 %id/g (supplementary figure 6a) . total ldng accumulation in the legs accounted for 0.85% of initial dose (%id) in naïve mice, 2.65 %id in mice 6 hours after footpad lps injection, and 8.34 %id at 24 hours after footpad injection (supplementary figure 6b) , indicating ldngs can concentrate in inflamed vasculature outside the lungs. previous work has indicated that nps based on denatured albumin accumulate in neutrophils in inflamed lungs and at sites of acute vascular injury, whereas nps coated with native albumin do not. 26, 27 we have characterized lysozyme-dextran nanogels and crosslinked human albumin nps with circular dichroism (cd) spectroscopy to compare secondary structure of proteins in the nps to secondary structure of the native component proteins (supplementary figure 7a-b) . identical cd spectra were recorded for ldngs vs. lysozyme and for albumin nps vs. human albumin. deconvolution of the cd spectra via neural network algorithm trained against a library of cd spectra for known structures verified that secondary structure composition of lysozyme and albumin was unchanged by incorporation of the proteins in the nps. 40 free protein and protein nps were also probed with 8-anilino-1naphthalenesulfonic acid (ansa), previously established as a tool for determining the extent to which hydrophobic domains are exposed on proteins. 41 consistent with known structures of the two proteins, ansa staining indicated few available hydrophobic domains on lysozyme and substantial hydrophobic exposure on albumin (supplementary figure 7c -d, blue curves). ldngs had increased hydrophobic accessibility vs. native lysozyme whereas albumin nps had reduced hydrophobic accessibility compared with native albumin. therefore, our data indicate that lysozyme and albumin are not denatured in ldngs and albumin nps, but the nps composed of the two proteins present a balance of hydrophobic and hydrophilic surfaces differing from the native proteins. the previous section demonstrates that two different nanoparticles based on protein, shown not to be denatured in cd spectroscopy studies, have uptake in lpsinflamed lungs driven by uptake in marginated neutrophils. we next undertook a broader study considering how aspects of np structure including size, composition, surface chemistry, and structural organization impact np uptake in lps-injured lungs. as examples of different types of protein nps, variants of ldngs (representing nps based on hydrophobic interactions between proteins), crosslinked protein nps, and nps based on electrostatic interactions between proteins were traced in naïve control and iv-lps-injured mice. as examples of nps based on site-specific protein interactions (rather than site-indiscriminate interactions leading to crosslinking, gelation, or chargebased protein nps), we also traced viruses and ferritin nanocages in naïve and lpstreated mice. liposomes and polystyrene nps were studied as examples of lipid and polymeric nanostructures. nanoparticles based on hydrophobic protein interactions ldng size was varied by modifying lysozyme-dextran composition of the nps and ph at which particles were formed. 42 figure 2 , all sizes of ldngs accumulated in lps-injured lungs at higher concentrations than in naïve lungs, with accumulation in injured lungs reaching ~20% of initial dose for all types of ldngs (supplementary table 3 ). variations in size and composition of ldngs therefore did not affect ldng specificity for lps-injured lungs. expanding on data with peg-nhs ester-crosslinked human serum albumin particles, we varied the geometry and protein composition of nps based on peg-nhs protein crosslinking. human serum albumin nanorods (aspect ratio 3:1), bovine serum albumin nps (317. table 4 ). lysozyme nps accumulated in naïve lungs at a uniquely high concentration of 137.47 %id/g, compared to 170.92 %id/g in inflamed lungs. degree of uptake in injured lungs, along with injured vs. naïve contrast, did vary with protein np composition. however, acute inflammatory injury resulted in a minimum three-fold increase in lung uptake for all examined crosslinked protein nps, excluding crosslinked lysozyme, which still accumulated in injured lungs at a high concentration (25.64% of initial dose). we traced recently-developed poly(glutamate) tagged green fluorescent protein (e-gfp) nps, representing a third class of protein np based on electrostatic interactions between proteins and carrier polymer or metallic particles. 43 negatively-charged e-gfp was paired to arginine-presenting gold nanoparticles (89.0±1.6 nm diameter, pdi 0.14±0.04) or to poly(oxanorborneneimide) (poni) functionalized with guanidino and tyrosyl side chains (158.9±6.2 nm diameter, pdi 0.17±0.03) (supplementary figure 1d) . for biodistribution experiments with poni/e-gfp hybrid nps, tyrosine-bearing poni was labeled with 131 i and e-gfp was labeled with 125 i, allowing simultaneous tracing of each component of the hybrid nps. the two e-gfp nps, with structure based on charge interactions, had specificity for iv lps-injured lungs. comparing uptake in lps-injured lungs to naïve lungs, we observe an lps:naïve ratio of 2.37 for poni/e-gfp nps as traced by the poni component, 2.57 for poni/e-gfp nps as traced by the e-gfp component, and 2.79 for au/e-gfp nps ( figure 3c , supplementary figure 10 ). poni/e-gfp particles, specifically, accumulated in lpsinjured lungs at 26.77% initial dose as measured by poni tracing and 27.24% initial dose as measured by gfp tracing, indicating effective co-delivery in the inflamed organ. acute inflammatory injury therefore resulted in a two-to three-fold increase in pulmonary uptake of nps constructed via electrostatic protein interactions. nanoparticles based on symmetric protein organization adeno-associated virus (aav), adenovirus, and horse spleen ferritin nanocages were employed as examples of protein-based nps with highly symmetrical structure (see supplementary figure 1d for dls confirmation of structure). [44] [45] [46] for each of these highly ordered protein nps, iv lps injury had no significant effect on biodistribution and levels of uptake in the injured lungs were minimal ( figure 3d table 5 ). therefore, highly ordered protein nps traced in our studies did not have tropism for the lungs after acute inflammatory injury. liposomes and polystyrene nps were studied as example nps that are not structurally based on proteins. dota chelate-containing lipids were incorporated into bare liposomes, allowing labeling with 111 in tracer for biodistribution studies. carboxylate polystyrene nps were coupled to trace amounts of 125 i-labeled igg via edci-mediated carboxy-amine coupling. liposomes had a diameter of 103.6±8.7 nm (pdi 0.09±0.01) and igg-polystyrene nps had a diameter of 230.5±2.8 nm (pdi 0.14±0.01) (supplementary figure 1c -d). liposomes accumulated in inflamed lungs at a concentration of 16.89 %id/g, accounting for no significant change against naïve lungs. lps injury actually induced a reduction in the lungs:liver metric, from 0.20 for naïve mice to 0.15 for lps-injured mice. polystyrene nps accumulated in inflamed lungs at 11.67 %id/g (1.75% initial dose), so iv lps injury did in fact induce increased levels of np uptake in the lungs, from a concentration of 2.40 %id/g in the naïve lungs ( figure 3e, supplementary figure 12 ). however, neither bare liposomes nor polystyrene nps were drawn to lps-injured lungs in significant concentrations. significantly, isolated proteins did not home to lps-inflamed lungs themselves. we traced radiolabeled albumin, lysozyme, and transferrin in naïve control and iv lpsinjured mice (supplementary figure 13 , supplementary table 6 ). in injured mice, albumin, lysozyme, and transferrin localized to the lungs at low concentrations and no significant differences were recorded when comparing naïve to lps-injured lung uptake. the data presented in figure 3 and supplementary figures 8-13 indicate that a variety of protein-based nanostructures have tropism for acute inflammatory injury in the lungs. nps based on agglutination of proteins in non-site-specific interactions (naps, figure 3a -c, supplementary figures 8-10 ) all exhibited either significant increases in lung uptake after lps injury or high levels of lung uptake in both naïve control and lpsinjured animals. nanostructures based on highly symmetrical protein organization had no specific tropism for inflamed lungs ( figure 3d ). representative nanostructures not based on proteins, bare liposomes and polystyrene beads, did not home to inflamed lungs ( figure 3e ). we next engineered naps from liposomes, a nanoparticle shown above to have no intrinsic neutrophil tropism. our methods for engineering nap-like liposomes serve to validate the finding that supramolecular organization of protein in nanoparticles predicts neutrophil tropism. liposomes were functionalized with rat igg conjugated via sata-maleimide chemistry (sata-igg liposomes) or via recently demonstrated copper-free click chemistry methods. 47 briefly, click chemistry methods entailed nhs-ester conjugation of an excess of strained alkyne (dibenzocyclooctyne, dbco) to igg, followed by reaction of the dbco-functionalized igg with liposomes containing peg-azide-terminated lipids (dbco-igg liposomes, figure 4a ). dbco-igg liposomes had a diameter of 128.3±4.3 nm and a pdi of 0.17±0.03 and sata-igg liposomes had a diameter of 178.8±6.9 nm and a pdi of 0.23±0.03 (supplementary figure 1c) . in mice subjected to iv-lps, sata-igg liposomes accumulated in the lungs at a concentration of 22.26 %id/g ( figure 4b , yellow bars). dbco-igg liposomes, by contrast, concentrated in the lungs at 117.16 %id/g, corresponding to 17.57% of initial dose and roughly matching the accumulation of 130nm ldngs in the inflamed lungs ( figure 4b , brown bars). for comparison, bare liposomes, as in figure 3e , concentrated in the inflamed lungs at 16.89 %id/g ( figure 4b , green bars). for dbco-igg liposomes, the inflamed vs. naïve lung uptake accounted for a twelve-fold change. dbco-igg liposomes specifically accumulated in injured lungs, whereas sata-igg liposomes and bare liposomes did not (supplementary figure 14 , supplementary table 7) . it lps instillation also led to elevated concentrations of dbco-igg liposomes in the lungs. biodistributions of the dbco-igg liposomes indicated a pulmonary concentration of 145.89 %id/g at 1 hour after it lps, 160.13 %id/g at 2 hours after it lps, and 127.78 %id/g at 6 hours after it lps (supplementary figure 15 ). even at early time points after direct pulmonary lps insult, dbco-igg liposomes accumulated in the inflamed lungs. results in figure 4b were obtained by introducing a 20-fold molar excess of nhs-ester-dbco to rat igg before dbco-igg conjugation to liposomes (dbco(20x)-igg liposomes). optical density quantification of dbco indicated ~14 dbco per igg following reaction of dbco and igg at 20:1 molar ratio (supplementary figure 16) . to test the hypothesis that dbco functions as a tag that modifies dbco-igg liposomes for neutrophil affinity in settings of inflammation, we varied the concentration of dbco on igg prepared for conjugation to azide liposomes. dbco was added to igg at 10-fold, five-fold, and 2.5-fold molar excesses. a 10-fold molar excess resulted in ~6 dbco per igg, a 5-fold molar excess resulted in ~3 dbco per igg, and a 2.5-fold molar excess resulted in ~2 dbco per igg (supplementary figure 16) . igg with different dbco loading concentrations was conjugated to azide liposomes. dbco-igg liposomes had similar sizes across all dbco concentrations (supplementary figure 1c) , with diameters of ~130 nm and pdis < 0.20. the different types of dbco-igg liposomes were each traced in iv-lps injured mice. titrating the quantity of dbco on dbco-igg liposomes indicated that liposome accumulation in the lungs of injured mice was dependent on dbco concentration on the liposome surface. concentration of dbco-igg liposomes in inflamed lungs attenuated with decreasing dbco concentration on igg (supplementary table 8 , figure 4c ). therefore, only igg with high concentrations of dbco served as a tag for modifying the surface of liposomes for specificity to pulmonary injury. flow cytometry verified the specificity of dbco-igg liposomes for neutrophils in injured lungs ( figure 4d -e). as with ldngs and albumin nps in figure 2c -h, single cell suspensions were prepared from lps-inflamed and naïve control lungs after circulation of fluorescent dbco-igg liposomes. confirming the results of biodistribution studies, 4.90% of cells were liposome-positive in naïve lungs, compared to 33.92% of all cells in lps-inflamed lungs (supplementary figure 17a-b) . dbco-igg liposomes predominantly accumulated in pulmonary neutrophils after iv lps. there were more neutrophils in the injured lungs and a greater fraction of neutrophils took up dbco-igg liposomes in the injured lungs, as compared to the naïve control ( figure 4d -e). approximately one half of neutrophils in iv lps-injured lungs contained liposomes. dbco-igg liposomes were also highly specific for neutrophils in inflamed lungs, with ~90% of liposome-positive cells in the injured lungs being neutrophils (supplementary table 9 ). the remaining dbco-igg liposome uptake in the lungs was accounted for by other cd45-positive cells (supplementary figure 17c -e). 99.0% of liposome uptake colocalized with cd45-positive cells in lps-injured lungs and 98.7% of liposome uptake in the naïve lungs was associated with cd45-positive cells. accordingly, less than 1% of liposome uptake was associated with endothelial cells (supplementary figure 17f -g). dbco(20x)-igg itself did not have specificity for inflamed lungs (supplementary figure 18 ). uptake of dbco(20x)-igg in naïve and injured lungs was statistically identical and the biodistribution of the modified igg resembled published results with unmodified igg. 48 these results verify that dbco-igg modifies the structure of immunoliposomes, but does not function as a standard affinity tag by acting as a surface motif with intrinsic affinity for neutrophils. indeed, cd spectroscopic and ansa structural characterization of dbcomodified igg and dbco-igg liposomes resembled results obtained for ldngs and crosslinked albumin nps. igg secondary structure, as assessed by cd spectroscopy, was unchanged by dbco modification (supplementary figure 19a) . deconvolution of cd spectra via neural network algorithm indicated identical structural compositions for dbco(20x)-igg, dbco(10x)-igg, dbco(5x)-igg, dbco(2.5x)-igg, and unmodified igg, showing that igg was not denatured by conjugation to dbco. ansa was used to probe accessible hydrophobic domains on dbco(20x)-igg and dbco(20x)-igg liposomes (supplementary figure 19b) . ansa fluorescence indicated more hydrophobic domains available on dbco(20x)-igg liposomes than on dbco(20x)-igg itself, resembling results for lysozyme and ldngs. therefore, addition of a hydrophobic moiety to protein on the surface of liposomes led to uptake of the liposomes in pulmonary marginated neutrophils after inflammatory insult. this result indicates that hydrophobic interactions between proteins on the surface of functionalized liposomes, like the protein interactions in naps, predict liposome tropism for marginated neutrophils in inflamed lungs. including nps from our four classes of protein-based nps, two non-protein nps (bare liposomes and polystyrene nps), and five types of igg-coated liposomes, we traced 23 nanoparticles in naïve and inflamed mice. direct assessment of naïve-toinflamed shifts in lung uptake led us to identify 13 naps with specificity for inflamed lungs. to verify this assessment and derive additional patterns in the broader data set, we undertook linear discriminant and principal components analyses of the biodistribution data for our 23 nanoparticles, along with three isolated proteins. grouping the 23 nanoparticles and three proteins according to the classes defined in figure 3 and supplementary figures 8-13 , we completed a linear discriminant analysis of the naïve-to-inflamed shift for particle retention in the lungs, blood, liver, and spleen (supplementary figure 20a) . data for particle uptake in each organ was normalized by subtracting and then dividing by the mean uptake over all particles. the first two eigenvectors, dominated by splenic uptake and a combination of liver and lung uptake, respectively, accounted for 96% of variation in the data. the resulting projection of the data along the first two linear discriminant analysis eigenvectors was analyzed by k-means clustering to confirm the classes of nanoparticle with specificity for the inflamed lungs (supplementary figure 20b) . indeed, division of the data into two clusters supported the delineation of the 13 nanoparticles with specificity for inflamed lungs. naps, nanoparticles based on protein gelation, crosslinking, and charge association, all aligned in one cluster. as an exception, dbco(20x)-igg liposomes were considered as a unique class of particle and the linear discriminant analysis indicated that the inflammation-specific liposomes had in vivo behavior resembling that of ldngs or poni-gfp nanoparticles. this analysis of the liposome biodistributions supports the classification of dbco(20x)-igg liposomes as naps. igg-coated polystyrene nanoparticles and dbco(10x)-igg liposomes were part of the k-means cluster without inflammation specificity, but data for these two particles resided close to the voronoi boundary distinguishing the two clusters. principal component analysis comparing normalized nanoparticle uptake in inflamed lungs to normalized retention in liver, spleen, and blood provided a reductive metric to compare the distinct in vivo behavior of nanoparticles in the classes identified by linear discriminant analysis. most variation in the biodistribution data was accounted for by an eigenvector closely aligned to variation in pulmonary uptake (supplementary figure 21a) . data was projected along that first eigenvector and magnitude of the projection was determined for each nanoparticle (supplementary figure 21b) . first eigenvector projection values were then grouped according to the classes examined above via linear discriminant analysis. only the classes in the inflammation-specific kmeans cluster had positive average first eigenvector projections. all other particle classes had average first eigenvector projections indistinguishable from isolated protein (supplementary figure 21b) . principal component and linear discriminant analyses of our compiled biodistributions confirmed; a) identification of naps as nanoparticles with distinct tropism for inflamed lungs and; b) alignment of dbco(20x)-igg liposome in vivo behavior with that of other naps. computerized tomography (ct) imaging is a standard diagnostic tool for ards. ct images can identify the presence of edematous fluid in the lungs, but ct cannot distinguish between the two major types of pulmonary edema: non-inflammatory cardiogenic pulmonary edema (cpe) and ards-associated edema. 49 we sought to use naps to distinguish inflammatory lung injury from cpe in diagnostic imaging experiments. we induced cpe in mice via prolonged iv propranolol infusion. 50 edema was confirmed via ct imaging of inflated lungs ex vivo and in situ. three-dimensional reconstructions of chest ct images were partitioned to distinguish airspace and lowdensity tissue, as in normal lungs (white, yellow, and light orange signal in figure 5a ), from high-density tissue and edema (red and black/transparent signal in figure 5a ). quantification of ct attenuation and gaps in the reconstructed three-dimensional lung images indicated profuse edema in lungs afflicted with model cardiogenic pulmonary edema ( figure 5a 200 nm ldngs were traced in mice with induced cardiogenic pulmonary edema. ldngs accumulated in the edematous lungs at 14.52 %id/g concentration, statistically indistinguishable from lung uptake in naïve mice and an order of magnitude lower than the level of lung uptake in mice treated with iv lps ( figure 5c ). naïve and iv lps-injured mice were dosed with ldngs labeled with 111 in via chelate conjugation to lysozyme. 111 in uptake in naïve and lps-injured lungs was visualized with ex vivo spect-ct imaging to indicate capacity of ldngs for imagingbased diagnosis of inflammatory lung injury ( figure 5d ). 111 in signa was colocalized with anatomical ct images for reconstructions in figure 5d . 111 in spect signal was detectable in lps-injured lungs, but 111 in spect signal was at background level in naïve lungs (supplementary movies 4 and 5). reduced spect signal in the liver of lps-injured mice, in agreement with biodistribution data, was also evident in coregistration of spect imaging with full body skeletal ct imaging (supplementary movies 6 and 7). therefore, naps with tropism for marginated neutrophils have the ability to detect and assess ards-like inflammation via spect-ct imaging. since those same naps do not accumulate in lungs afflicted with cpe, naps have potential for differential diagnosis of acute lung inflammation against cpe. in recent work, we demonstrated that human donor lungs rejected for transplant due to ards-like phenotypes can be perfused with nanoparticle solutions. 51 these perfusion experiments evaluate the tendency of nanoparticles to distribute to human lungs ex vivo. we used this perfusion method to evaluate nap retention in inflamed human lungs. first, fluorescent ldngs were added to single cell suspensions prepared from human lungs. 5 µg, 10 µg, or 50 µg of ldngs were incubated with 6x10 5 cells in suspension for 1 hour at room temperature. after three washes to remove unbound ldngs, cells were stained for cd45 and analyzed with flow cytometry ( figure 7a -b). the majority of ldng uptake in the single cell suspensions was attributable to cd45positive cells. ldngs accumulated in the human leukocytes, extracted from inflamed lungs, in a dose-dependent manner, with 35.1% of leukocytes containing ldngs at a loading dose of 50 µg. therefore, our prototype nap was retained in leukocytes from human lungs. to test ldng tropism for inflamed intact human lungs, fluorescent or 125 i-labeled ldngs were infused via arterial catheter into ex vivo human lungs excluded from transplant. immediately prior to ldng administration, tissue dye was infused via the same arterial catheter to stain regions of the lungs directly perfused by the catheterized branch of the pulmonary artery ( figure 7c ). after infusion of ldngs, phosphate buffered saline infusion was used to rinse away unbound particles. perfused regions of the lungs were dissected and divided into ~1g segments, then sorted into regions deemed to have high, medium, or low levels of tissue dye staining. for lungs receiving fluorescent ldngs, well-perfused and poorly-perfused regions were selected for sectioning and fluorescent imaging. fluorescent signal from ldngs was clearly detectable in sections of well-perfused tissue, but not poorly-perfused tissue ( figure 7d ). in experiments with 125 i-labeled ldngs, 131 i-labeled ferritin was concurrently infused (i.e. a mix of ferritin and ldngs was infused) as an internal control particle shown to have no tropism for injured mouse lungs. with ldngs and ferritin infused into the same lungs via the same branch of the pulmonary artery, ldngs retained in the lungs at 52.15% initial dose and ferritin retained at 9.27% initial dose ( figure 7e ). ldng accumulation in human lungs was focused in regions of the lungs with high levels of perfusion stain, with concentrations of 4.66 %id/g in the "high" perfusion regions, compared to 0.44 %id/g in the "medium" perfusion regions. ferritin accumulation was more diffuse, with 0.47 %id/g in the "high" perfusion regions, compared to 0.35 %id/g in the "medium" perfusion regions (supplementary figure 21) . ldngs, a prototype nap shown to home to neutrophils in acutely inflamed mouse lungs, specifically accumulated in perfused regions of inflamed human lungs, but ferritin nanocages, a particle with no tropism for neutrophils, concentrated at much lower levels in injured human lungs. our data thus indicate that nap tropism for neutrophils in inflamed mouse lungs may be recapitulated in human lungs. previous studies indicate that nanoparticles can interfere with neutrophil adhesion in inflamed vasculature. 52 we designed studies to evaluate whether or not naps mitigate the neutrophil-mediated effects of lung inflammation. namely, we administered ldngs, dbco(20x)-igg liposomes, or bare liposomes in mice subjected to model ards and determined whether or not the nanoparticles prevented lung edema induced by inflammation. mice were treated with nebulized lps as a high-throughput model for severe ards. to evaluate physiological effects of the model injury, bronchoalveolar lavage (bal) fluid was harvested from mice at 24 hours after exposure to lps. in three separate experiments, nebulized lps induced elevated concentrations of neutrophils, cd45-positive cells, and protein in the bal fluid. in naïve mice, cd45-positive cells concentrated at 1.42x10 4 cells per ml bal and neutrophils concentrated at 1.11x10 4 cells per ml bal. after lps injury, cd45-positive cells and neutrophils concentrated at 6.97x10 5 and 6.96x10 5 cells per ml bal, respectively. in naïve mice, protein concentrated in the bal fluid at 0.12 mg/ml and in lps-injured mice, protein concentrated in the bal at 0.36 mg/ml ( figure 6 , white and grey bars). vascular disruption after nebulized lps treatment thus led to accumulation of protein-rich edema in the alveolar space. dbco(20x)-igg liposomes, ldngs, and bare liposomes were compared for effects on vascular permeability in model ards. nps were administered as an iv bolus (20 mg per kg body weight) two hours after nebulized lps administration. as in untreated mice, bal fluid was harvested and analyzed at 24 hours after exposure to nebulized lps. bare liposomes or ldngs did not have significant effects on vascular injury induced by nebulized lps, as measured by either leukocyte or protein concentration in bal fluid ( figure 6 , red and green bars). dbco(20x)-igg liposomes, however, had a significant salient effect on both protein leakage and cellular infiltration in the bal ( figure 6 , brown bars). with dbco(20x)-igg liposomes administered two hours after nebulized lps, cd45-positive cells and neutrophils in bal were reduced to concentrations of 3.04x10 5 and 3.48x10 5 cells per ml, respectively. protein concentration in the bal was reduced to 0.21 mg/ml by dbco(20x)-igg liposome treatment. as measured by protection against cellular or protein leakage, relative to untreated mice, dbco(20x)-igg liposomes provided 59.6% protection against leukocyte leakage, 49.7% protection against neutrophil leakage, and 67.4% protection against protein leakage. dbco(20x)-igg liposomes, without any drug, altered the course of inflammatory lung injury to limit protein and leukocyte edema in the alveoli. our results with dbco(20x)-igg liposomes indicate that some naps can interfere with neutrophil extravasation into the alveoli and thus limit edema following inflammatory injury. however, our results with ldngs show that tropism for marginated neutrophils is not alone sufficient to limit the neutrophil-mediated effects of inflammatory lung injury. neutrophils concentrate in the pulmonary vasculature during either systemic or pulmonary inflammation. 10, 11, 14, 17, 18 these marginated neutrophils can recognize and engulf bacteria. 17, 18, 35 therefore, neutrophils surveil the vasculature for potentially pathogenic foreign species, with the pulmonary vasculature serving as a "surveillance hub" in the case of systemic or pulmonary infection and inflammation. 10, 17, 18, 35 our results with e. coli are noteworthy in this context: when e. coli are stripped of functional properties by heat treatment, oxidation, and fixation, but maintain their structure, uptake of the bacteria in the lungs only occurs after systemically prompting neutrophils with an inflammatory signal, lps. inflammation thus leads to pulmonary uptake of the e. colishaped particles. in large part, the overall outcome of this study is an accounting of nanoparticle structural properties that lead to recognition by "surveilling" neutrophils in the inflamed lungs, analogously to e. coli recognition by pulmonary neutrophils. including different liposomal formulations, 23 nanoparticles were screened in our biodistribution studies comparing pulmonary nanoparticle uptake in naïve and lps-inflamed mice. thirteen different nanoparticles exhibited specificity for inflamed lungs over naïve lungs, with flow cytometry data indicating that at least three of those nanoparticle species specifically and avidly gather in neutrophils. the thirteen nanoparticles with specificity for the inflamed lungs have a range of properties. seven different proteins were used in the inflammation-specific particles. the particles have sizes ranging from ~75 nm to ~350 nm, include both spheres and rods, and have a range of zeta potentials. however, our analyses classify the inflammation-specific nanoparticles as; 1) nanoparticles with structure based on hydrophobic interactions between proteins; 2) nanoparticles with structure based on non-site-specific protein crosslinking; 3) nanoparticles based on charge interactions between proteins. put broadly, these three classes can all be grouped as structures based on protein agglutination, without regard for site-specific interactions or symmetry in the resulting protein superstructure. we define the term nanoparticles with agglutinated proteins (naps) to indicate that particles with tropism for pulmonary marginated neutrophils during inflammation share commonalities in supramolecular organization. we identify naps as a broad class, rather than a single particle type. accordingly, we have presented diverse nap designs, implying a diversity of potential nap-based strategies for targeted treatment and diagnosis of ards and other inflammatory disorders in which marginated neutrophils play a role (e.g. local infections or thrombotic disorders). 10, 12, 13, 17, 18 the diversity of naps will allow versatile options for engineering neutrophil-specific drug delivery strategies to accommodate different pathologies. in contrast to naps, three particles (adenovirus, aavs, and ferritin) characterized by highly symmetric arrangement of protein subunits into a protein superstructure [44] [45] [46] did not accumulate in the inflamed neutrophil-rich lungs. these three particles have evolved structures that lead to prolonged circulation or evasion of innate immunity in mammals. [53] [54] [55] [56] it is conceivable that neutrophils more effectively recognize less patterned and more variable protein arrangements that may better parallel the wide variety of structures presented by the staggering diversity of microbes against which neutrophils defend. 20, 35 to support our conclusions regarding supramolecular organization and neutrophil tropism, we re-engineered liposomes, particles with no intrinsic neutrophil tropism, to behave like naps. protein arrangement on the surface of dbco-igg liposomes was predicted to recapitulate protein agglutination seen in naps based on hydrophobic interactions. introduction of dbco to igg entails conjugation of a highly hydrophobic moiety 57 to hydrophilic residues on the igg. replacing dbco with the less hydrophobic modifying group used in sata-maleimide conjugation 58 abrogates the inflammation specificity observed with dbco-igg liposomes. likewise, titrating down the amount of dbco on the igg, thus limiting the hydrophobic groups on the protein, also ratchets down the targeting behavior of the dbco-igg liposomes. our data therefore points towards hydrophobic interactions between proteins on the liposome surface being a determinant in liposome uptake in neutrophils in the inflamed lungs. essentially, the dbco-igg liposomes may reproduce the hydrophobic interaction structural motif seen in naps produced by protein gelation (i.e. ldngs). nap-liposomes may be particularly attractive for future clinical translatability. liposomes are prominent among fda-approved nanoparticle drug carriers. 59 further, even without cargo drugs, nap-liposomes conferred significant therapeutic effects in a mouse model of severe ards. ldngs, despite high levels of uptake in inflamed lungs, did not have the same therapeutic effect as the nap-liposomes. this result suggests that the composition of the liposomes may be important for their therapeutic effect. among possible mechanisms for the therapeutic effect, we note that lipid rafts are major signaling hubs in neutrophils. 60, 61 the lipid content of the nap-liposomes (particularly the cholesterol content) may modulate neutrophil lipid rafts dependent on cholesterol. we have also observed that neutrophil content in the inflamed alveoli is markedly reduced by nap-liposomes. in this context, we note published work demonstrating that certain nanoparticles, in a still undetermined manner dependent on particle composition, can drive redistribution of neutrophils from the lungs to the liver. 52 as a major corollary, our findings indicate many protein-based or proteinincorporating nanoparticles developed for therapeutic applications may accumulate in inflamed lungs, even when those nanoparticles were designed to accumulate elsewhere. the variety of protein nanostructures accumulating in inflamed lungs in our data includes particles that have been investigated as targeted drug delivery vehicles where marginated neutrophils are not the intended site of accumulation. 36, 38, 47, 48, 62 the patterns in our data indicate that future studies may reveal additional nanoparticles that accumulate in the lungs following inflammatory insult. this study therefore serves as evidence that inflammatory challenges may prompt profound off-target changes in the biodistributions of nanomaterials, including dramatic shunting of nanoparticles and any associated drug payload to the lungs. the nanoparticle targeting profiles documented in naïve or, for instance, tumor model studies may be overturned by, for instance, bacterial infection in a patient receiving the nanoparticle. in conclusion, supramolecular organization in nanoparticle structure predicts nanoparticle uptake in pulmonary marginated neutrophils during acute inflammation. specifically, nanoparticles with agglutinated protein (naps) accumulate in marginated neutrophils, while nanoparticles with more symmetric protein organization do not. nap tropism for neutrophils allowed us to develop naps as diagnostics and therapeutics for ards, and even to demonstrate nap uptake in inflamed human lungs. future work may more deeply explore therapeutic effects of naps in ards and other diseases in which neutrophils play key roles. this study also obviates future testing of supramolecular organization as a variable in in vivo behavior of nanoparticles, including screens of tropism for other pathologies and cell types. these studies could in turn guide engineering of new particles with intrinsic cell tropisms, as with our engineering of nap-liposomes with neutrophil tropism. these "targeting" behaviors, requiring no affinity moieties, may apply to a wide variety of nanomaterials. but our current findings with neutrophil-tropic naps indicate that many protein-based and protein-coated nanoparticles could be untapped resources for treatment and diagnosis of devastating inflammatory disorders like ards. lysozyme-dextran nanogels (ldngs) were synthesized as previously described. 37 ,42 70 kda rhodamine-dextran or fitc-dextran (sigma) and lysozyme from hen egg white (sigma) were dissolved in deionized and filtered water at a 1:1 or 2:1 mol:mol ratio, and ph was adjusted to 7.1 before lyophilizing the solution. for maillard reaction between lysozyme and dextran, the lyophilized product was heated for 18 hours at 60°c, with 80% humidity maintained via saturated kbr solution in the heating vessel. dextran-lysozyme conjugates were dissolved in deionized and filtered water to a concentration of 5 mg/ml, and ph was adjusted to 10.70 or 11.35. solutions were stirred at 80°c for 30 minutes. diameter of ldngs was evaluated with dynamic light scattering (dls, malvern) after heat gelation. particle suspensions were stored at 4°c. crosslinked protein nanoparticles and nanorods were prepared using previously reported electrohydrodynamic jetting techniques. 63 the protein nanoparticles were prepared using bovine serum albumin, human serum albumin, human lysozyme, human transferrin, or human hemoglobin (all proteins were purchased from sigma). protein nanorods were prepared using chemically modified human serum albumin. for electrohydrodynamic jetting, protein solutions were prepared by dissolving the protein of interest at a 7.5 w/v% (or 2.5 w/v% for protein nanorods) concentration in a solvent mixture of di water and ethylene glycol with 4:1 (v/v) ratio. the homobifunctional amine-reactive crosslinker, o,o′-bis[2-(n-succinimidylsuccinylamino)ethyl]polyethylene glycol with molecular weight of 2kda (nhs-peg-nhs, sigma) was mixed with the protein solution at 10 w/w%. protein nanoparticles were kept at 37°c for 7 days for completion of the crosslinking reaction. the as-prepared protein nanoparticles were collected in pbs buffer and their size distribution was analyzed using dynamic light scatting (dls, malvern). glutamic acid residues (e20-tag) were inserted at the c-terminus of enhanced green fluorescent protein (egfp) through restriction cloning and site-directed mutagenesis as previously reported. 64 proteins were expressed in an e. coli bl21 strain using standard protein expression protocol. briefly, protein expression was carried out in 2xyt media with an induction condition of 1 mm iptg and 18 °c for 16 h. at this point, the cells were harvested, and the pellets were lysed using 1% triton-x-100 (30 min, 37 °c)/dnase-i treatment (10 minutes). proteins were purified using hispur cobalt columns. after elution, proteins were preserved in pbs buffer. the purity of native proteins was determined using 8% sds−page gel. polymers (poni) were synthesized by ring-opening metathesis polymerization using third generation grubbs' catalyst as previously described. 65 in brief, solutions in dichloromethane of guanidium functionalized monomer and grubbs' catalyst were placed under freeze thawing cycles for degassing. after warming the solutions to room temperature, the degassed monomer solutions were administrated to degassed catalyst solutions and allowed to stir for 30 minutes. the polymerization reaction was terminated by the addition of excess ethyl vinyl ether. the reaction mixture was further stirred for another 30 min. the resultant polymers were precipitated from excess hexane or diethyl ether anhydrous, filtered, washed and dried under vacuum to yield a light-yellow powder. polymers were characterized by 1 h nmr and gel permeation chromatography (gpc) to assess chemical compositions and molecular weight distributions, respectively. subsequent to deprotection of boc functionalities, polymer was dissolved in the dcm with the addition of tfa at 1:1 ratio. the reaction was allowed to stir for 4 hours and dried under vacuum. excess tfa was removed by azeotropic distillation with methanol. afterwards, the resultant polymers were re-dissolved in dcm and precipitated in anhydrous diethyl ether, filtered, washed and dried. polymers were then dissolved in water and transferred to biotech ce dialysis tubing membranes with a 3000 g/mol cutoff and dialyzed against ro water (2−3 days). the polymers were then lyophilized dried to yield a light white powder. poni polymer/e-tag protein nanocomposites (ppncs) were prepared in polypropylene microcentrifuge tubes (fisher) through a simple mixing procedure. 0.5625 nmol of 54 kda poni was incubated with 0.45 nmol of egfp at room temperature for 10 minutes prior to dilution to 200 µl in sterile pbs and subsequent injection. similarly, 0.9 nmol of arginine-tagged gold nanoparticles, prepared as described, 43 were combined with 0.45 nmol of egfp to prepare egfp/gold nanoparticle complexes. azide-functionalized liposomes were prepared by thin film hydration techniques, as previously described. 47 the lipid film was composed of 58 mol% dppc (1,2dipalmitoyl-sn-glycero-3-phosphocholine), 40 mol% cholesterol, and 2 mol% azide-peg2000-dspe (all lipids from avanti). 0.5 mol% top fluor pc (1-palmitoyl-2-(dipyrrometheneboron difluoride) undecanoyl-sn-glycero-3-phosphocholine) was added to prepare fluorescent liposomes. 0.2 mol% dtpa-pe (1,2-distearoyl-sn-glycero-3phosphoethanolamine-n-diethylenetriaminepentaacetic acid) was added to prepare liposomes with capacity for radiolabeling with 111 in. lipid solutions in chloroform, at a total lipid concentration of 20 mm, were dried under nitrogen gas, then lyophilized for 2 hours to remove residual solvent. dried lipid films were hydrated with dulbecco's phosphate buffered saline (pbs). lipid suspensions were passed through 3 freezethaw cycles using liquid n2/50°c water bath then extruded through 200 nm cutoff tracketched polycarbonate filters in 10 cycles. dls assessed particle size after extrusion and after each subsequent particle modification. liposome concentration following extrusion was assessed with nanosight nanoparticle tracking analysis (malvern). for conjugation to liposomes, rat igg was modified with dibenzylcyclooctyne-peg4-nhs ester (dbco, jena bioscience). igg solutions (pbs) were adjusted to ph 8.3 with 1 m nahco3 buffer and reacted with dbco for 1 hour at room temperature at molar ratios of 2.5:1, 5:1, 10:1, or 20:1 dbco:igg. unreacted dbco was removed after reaction via centrifugal filtration against 10 kda cutoff filters (amicon [def] . dbcomodified igg was incubated with azide liposomes at 200 igg per liposome overnight at room temperature. unreacted antibody was removed via size exclusion chromatography, and purified liposomes were concentrated to original volume against centrifugal filters (amicon). maleimide liposomes were also prepared via lipid film hydration. 66 lipid films comprised 54% dppc, 40% cholesterol, and 6% mpb-pe (1,2-dioleoyl-sn-glycero-3phosphoethanolamine-n-[4-(p-maleimidophenyl) butyramide]), with lipids prepared, dried, resuspended, and extruded as described above for azide liposomes. igg was prepared for conjugation to maleimide liposomes by one-hour reaction of 10 sata (n-succinimidyl s-acetylthioacetate) per igg at room temperature in 0.5 mm edta in pbs. unreacted sata was removed from igg by passage through 7 kda cutoff gel filtration columns. sata-conjugated igg was deprotected by one-hour room temperature incubation in 0.05 m hydroxylamine in 2.5 mm edta in pbs. excess hydroxylamine was removed and buffer was exchanged for 0.5 mm edta in pbs via 7 kda cutoff gel filtration column. sata-conjugated and deprotected igg was added to liposomes at 200 igg per liposomes for overnight reaction at 4°c. excess igg was removed by size exclusion column purification, as above for azide liposomes. 150 nm carboxylate nanoparticles (phosphorex) were exchanged into 50 mm mes buffer at ph 5.2 via gel filtration column. n-hydroxysulfosuccinimide (sulfo-nhs) was added to the particles at 0.275 mg/ml, prior to incubation for 3 minutes at room temperature. edci was then added to the particles at 0.1 mg/ml, prior to incubation for 15 minutes at room temperature. igg was added to the particle mixture at 200 igg per nanoparticle, prior to incubation for 3 hours at room temperature while vortexing. for radiotracing, 125 i-labeled igg was added to the reaction at 5% of total igg mass. the igg/particle mixture was diluted with 10-fold volume excess of ph 5.2 mes buffer and the diluted mixture was centrifuged at 12000xg for 3 minutes. supernatant was discarded and pbs with 0.05% bsa was added at desired volume before resuspending the particles via sonication probe sonication (three pulses, 30% amplitude). particle size was assessed via dls after resuspension, and particles were used immediately after dls assessment. top10 e. coli were grown overnight in terrific broth with ampicillin. bacteria were heat-inactivated by 20-minute incubation at 60°c, then fixed by overnight incubation in 4% paraformaldehyde. after fixation, bacteria were pelleted by centrifugation at 1000xg for 10 minutes. pelleted bacteria were washed three times in pbs, prior to resuspension by pipetting. bacterial concentration was verified by optical density at 600 nm, prior to radiolabeling as described for nanoparticles below. bacteria were administered in mice (7.5x10 7 colony forming units in a 100 µl suspension per mouse). protein, horse spleen ferritin nanocages (sigma), or adeno-associated virus (empty capsids, serotype 8) were prepared in pbs at concentrations between 1 and 2 mg/ml in volumes between 100 and 200 µl. films of oxidizing agent were prepared in borosilicate tubes by drying 300 µl of 0.5 mg/ml iodogen (perkin-elmer, chloroform solution) under nitrogen gas. alternatively, iodobeads (perkin-elmer) were added to borosilicate tubes (one per reaction). protein solutions were added to coated or beadcontaining tubes, before addition of na 125/131 i at 25 µci per 100 µg of protein. protein was incubated with radioiodine at room temperature for 5 minutes under parafilm in a ventilated hood. iodide-protein reacottions were terminated by purifying protein solutions through a 7 kda cutoff gel column (zeba). additional passages through gel filtration columns or against centrifugal filters (amicon, 10 kda cutoff) were employed to remove free iodine, assuring that >95% of radioactivity was associated with protein. lysozyme-dextran nanogels, crosslinked protein nanoparticles, e. coli, or adenovirus were similarly iodinated. at least 100 µl of particle suspension was added to a borosilicate tube containing two iodobeads, prior to addition of 100 µci of na 125 i per 100 µl of suspension. particles were incubated with radioiodine and iodobeads for 30 minutes at room temperature, with gentle shaking every 10 minutes. to remove free iodine, particle suspensions were moved to a centrifuge tube, diluted in ~1 ml of buffer and centrifuged to pellet the particles (16000xg/30 minutes for nanogels, 16000xg/30 minutes for crosslinked protein particles, 10000xg/30 minutes for adenovirus, and 1000xg/10 minutes for e. coli). supernatant was removed and wash/centrifugation cycles were repeated to assure >95% of radioactivity was associated with particles. particles were resuspended by probe sonication (three pulses, 30% amplitude) for nanogels or crosslinked protein nanoparticles or pipetting for adenovirus or e. coli. nanoparticle labeling with 111 in 111 in labeling of nanoparticles followed previously described methods, with adaptation for new particles. 47 all radiolabeling chelation reactions were performed using metal free conditions to prevent contaminating metals from interfering with chelation of 111 in by dtpa or dota. metals were removed from buffers using chelex 100 metal affinity resin (biorad, laboratories, hercules ca). lysozyme-dextran nanogels were prepared for chelation to 111 in by conjugation to s-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-scn-bn-dota, macrocyclics). nanogels were moved to metal free ph 8.3 1 m nahco3 buffer by three-fold centrifugation (16000xg for 15 minutes) and pellet washing with metal free buffer. p-scn-bn-dota was added to nanogels at 1:25 mass:mass ratio, prior to reaction for 30 minutes at room temperature. free p-scn-bn-dota was removed by three-fold centrifugal filtration against 10 kda cutoff centrifugal filters, with resuspension of nanogels in metal-free ph 4 citrate buffer after each centrifugation. dota-conjugated nanogels or dtpa-containing liposomes in ph 4 citrate buffer were combined with 111 incl3 for one-hour chelation at 37°c. nanoparticle/ 111 incl3 mixtures were treated with free dtpa (1 mm final concentration) to remove 111 in not incorporated in nanoparticles. efficiency of 111 in incorporation in nanoparticles was assessed by thin film chromatography (aluminum/silica strips, sigma) with 10 µm edta mobile phase. chromatography strips were divided between origin and mobile front and the two portions of the strip were analyzed in a gamma counter to assess nanoparticleassociated (origin) vs. free (mobile front) 111 in. free 111 in was separated from nanoparticles by centrifugal filtration and nanoparticles were resuspended in pbs (liposomes) or saline (nanogels). for spect/ct imaging experiments (see spect/ct imaging methods below) with nanogels, 80 µci of 111 in-labeled nanogels, used within one day 111 in labeling as described above, were administered to each mouse. for tracing 111 in-labeled liposomes in biodistribution studies, liposomes were labeled with 50 µci 111 in per µmol of lipid. nanoparticle or protein biodistributions were tested by injecting radiolabeled nanoparticles or protein (suspended to 100 µl in pbs or 0.9% saline at a dose of 2.5 mg/kg with tracer quantities of radiolabeled material) in c57bl/6 male mice from jackson laboratories. biodistributions in naïve mice were compared to biodistributions in several injury models. biodistribution data were collected at 30 minutes after nanoparticle or protein injection, unless otherwise stated, as in pharmacokinetics studies. briefly, blood was collected by vena cava draw and mice were sacrificed via terminal exsanguination and cervical dislocation. organs were harvested and rinsed in saline, and blood and organs were examined for nanoparticle or protein retention in a gamma counter (perkin-elmer). nanoparticle or protein retention in harvested organs was compared to measured radioactivity in injected doses. for calculations of nanoparticle or protein concentration in organs, quantity of retained radioactivity was normalized to organ weights. mice subject to intravenous lps injury were anesthetized with 3% isoflurane before administration of lps from e. coli strain b4 at 2 mg/kg in 100 µl pbs via retroorbital injection. after five hours, mice were anesthetized with ketamine-xylazine (10 mg/kg ketamine, 100 mg/kg xylazine, intramuscular administration) and administered radiolabeled nanoparticles or protein via jugular vein injection to determine biodistributions as described above. for mice subject to intratracheal (it) lps injury, b4 lps was administered to mice (anesthetized with ketamine/xylazine) at 1 mg/kg in 50 µl of pbs via tracheal catheter, followed by 100 µl of air. 66 biodistributions of lysozyme-dextran nanogels in it-lps-injured mice were assessed as above 16 hours after lps administration. biodistributions of liposomes in it-lps-injured mice were assessed at 1, 2, or 6 hours after lps administration. mice subject to footpad lps administration were provided b4 lps at 1 mg/kg in 50 µl pbs via footpad injection. biodistributions of lysozyme-dextran nanogels were obtained at 6 or 24 hours after footpad lps administration. lysozyme-dextran nanogel biodistributions were also traced in a mouse model of cardiogenic pulmonary edema. 50 to establish edema, mice were anesthetized with ketamine/xylazine and administered propranolol in saline (3 µg/ml) via jugular vein catheter at 83 µl/min over 120 minutes. lysozyme-dextran nanogel biodistributions were subsequently assessed as above. single cell suspensions were prepared from lungs for flow cytometric analysis of cell type composition of the lungs and/or nanoparticle distribution among different cell types in the lungs. c57bl/6 male mice were anesthetized with ketamine/xylazine (10 mg/kg ketamine, 100 mg/kg xylazine, intramuscular administration) prior to installation of tracheal catheter secured by suture. after sacrifice by terminal exsanguination via the vena cava, lungs were perfused by right ventricle injection of ~10 ml of cold pbs. the lungs were then infused via the tracheal catheter with 1 ml of a digestive enzyme solution consisting of 5 u/ml dispase, 2.5 mg/ml collagenase type i, and 1 mg/ml of dnase i in cold pbs. immediately after infusion, the trachea was sutured shut while removing the tracheal catheter. the lungs with intact trachea were removed via thoracotomy and kept on ice prior to manual disaggregation. disaggregated lung tissue was aspirated in 2 ml of digestive enzyme solution and incubated at 37°c for 45 minutes, with vortexing every 10 minutes. after addition of 1 ml of fetal calf serum, tissue suspensions were strained through 100 µm filters and centrifuged at 400xg for 5 minutes. after removal of supernatant, the pelleted material was resuspended in 10 ml of cold ack lysing buffer. the resulting suspensions were strained through 40 µm filter and incubated for 10 minutes on ice. the suspensions were centrifuged at 400xg for 5 minutes and the resulting pellets were rinsed in 10 ml of facs buffer (2% fetal calf serum and 1 mm edta in pbs). after centrifugation at 400xg for 5 minutes, the rinsed cell pellets were resuspended in 2% pfa in 1 ml facs buffer for 10 minutes incubation. the fixed cell suspensions were centrifuged at 400xg for 5 minutes and resuspended in 1 ml of facs buffer. for analysis of intravascular leukocyte populations in naïve and inflamed lungs, mice received an intravenous injection of fitc-conjugated anti-cd45 antibody five minutes prior to sacrifice and preparation of single cell suspensions as described above. populations of intravascular vs. extravascular leukocytes were assessed by subsequent stain of fixed cell suspensions with percp-conjugated anti-cd45 antibody and/or apcconjugated clone 1a8 anti-ly6g antibody. to accomplish staining of fixed cells, 100 µl aliquots of the cell suspensions described above were pelleted at 400xg for 5 minutes, then resuspended in labeled antibody diluted in facs buffer (1:150 dilution for apcconjugated anti-ly6g antibody and 1:500 dilution for percp-conjugated anti-cd45 antibody). samples were incubated with staining antibodies for 20 minutes at room temperature in the dark, diluted with 1 ml of facs buffer, and pelleted at 400xg for 5 minutes. stained pellets were resuspended in 200 µl of facs buffer prior to immediate flow cytometric analysis on a bd accuri flow cytometer. all flow cytometry data was gated to remove debris and exclude doublets. control samples with no stain, obtained from naïve and iv-lps-injured mice, established gates for negative/positive staining with fitc, percp, and apc. single stain controls allowed automatic generation of compensation matrices in fcs express software. comparison of percp anti-cd45 signal with fitc anti-cd45 signal indicated intravascular vs. extravascular leukocytes. comparison of apc anti-ly6g signal with fitc anti-cd45 signal indicated intravascular vs. extravascular neutrophils, with percp and apc co-staining verifying identification of cells as neutrophils. similar staining and analysis protocols enabled identification of nanoparticle distribution among different cell types in the lungs. to enable fluorescent tracing, lysozyme-dextran nanogels contained fitc-dextran, dbco-igg liposomes contained green fluorescent top fluor pc lipid, and crosslinked albumin nanoparticles were labeled with nhs ester alexa fluor 488. alexa fluor 488 labeling of albumin nanoparticles was accomplished by incubation of the nhs ester fluorophore with nanoparticles at 1:25 mass:mass fluorophore:nanoparticle ratio for two hours on ice. excess fluorophore was removed from nanoparticles by 3-fold centrifugation at 16000xg for 15 minutes followed by washing with pbs. nanoparticles were administered at 2.5 mg/kg via jugular vein injection and circulated for 30 minutes, prior to preparation of single cell suspensions from lungs as above. fixed single cell suspensions were stained with apc-conjugated anti-ly6g or percp-conjugated anti-cd45 as above. additional suspensions were stained with 1:150 dilution of apc-conjugated anti-cd31, in lieu of anti-ly6g, to identify endothelial cells. association of nanoparticles with cell types was identified by coincidence of green fluorescent signal with anti-cd45, anti-ly6g, or anti-cd31 signal. as described previously, 47 thirty minutes after injection of 80 µci of 111 in-labeled nanogels, anesthetized mice were sacrificed by cervical dislocation. mice were placed into a milabs u-spect (utrecht, netherlands) scanner bed. a region covering the entire body was scanned for 90 min using listmode acquisition. the animal was then moved, while maintaining position, to a milabs u-ct (utrecht, netherlands) for a fullbody ct scan using default acquisition parameters (240 µa, 50 kvp, 75 ms exposure, 0.75° step with 480 projections). for naïve mice and mice imaged after cardiogenic pulmonary edema, ct data was acquired as above without spect data. the spect data was reconstructed using reconstruction software provided by the manufacturer, with 400 µm voxels. the ct data were reconstructed using reconstruction software provided by the manufacturer, with 100 µm voxels. spect and ct data, in nifti format, were opened with imagej software (fiji package). background signal was removed from spect images by thresholding limits determined by applying renyi entropic filtering, as implemented in imagej, to a spect image slice containing ngassociated 111 in in the liver. background-subtracted pseudo-color spect images were overlayed on ct images and axial slices depicting lungs were selected for display, with ct thresholding set to emphasize negative contrast in the airspace of the lungs. imagej's built-in 3d modeling plugin was used to co-register background-subtracted pseudo-color spect images with ct images in three-dimensional reconstructions. ct image thresholding was set in the 3d modeling tool to depict skeletal structure alongside spect signal. for three-dimensional reconstructions of lung ct images, thresholding was set, as above, for contrast emphasizing the airspace of the lungs, with thresholding values standardized between different ct images (i.e. identical values were used for naïve and edematous lungs). images were cropped in a cylinder to exclude the airspace outside of the animal, then contrast was inverted, allowing airspace to register bright ct signal and denser tissue to register as dark background. three-dimensional reconstructions of the lung ct data, and co-registrations of spect data with lung ct data, were generated as above with imagej's 3d plugin applied to ct data cropped and partitioned for lung contrast. quantification of ct attenuation employed imagej's measurement tool iteratively over axial slices, with measurement fields of view manually set to contain lungs and exclude surrounding tissue. mice were exposed to nebulized lps in a 'whole-body' exposure chamber, with separate compartments for each mouse (mpc-3 aero; braintree scientific). to maintain adequate hydration, mice were injected with 1ml of sterile saline warmed to 37°c, intraperitoneally, immediately before exposure to lps. lps (l2630-100mg, sigma aldrich) was reconstituted in pbs to 10 mg/ml and stored at -80°c until use. immediately before nebulization, lps was thawed and diluted to 5 mg/ml with pbs. lps was aerosolized via a jet nebulizer connected to the exposure chamber (neb-med h, braintree scientific, inc.). 5 ml of 5 mg/ml lps was used induce the injury. nebulization was performed until all liquid was nebulized (~20 minutes). liposomes or saline sham were administered via retro-orbital injections of 100 µl of suspension (25 mg/kg liposome dose) at 2 hours after lps exposure. mice were anesthetized with 3% isoflurane to facilitate injections. bronchoalveolar lavage (bal) fluid was collected 24 hours after lps exposure, as previously described. 66 briefly, mice were anesthetized with ketamine-xylazine (10 mg/kg ketamine, 100 mg/kg xylazine, intramuscular administration). the trachea was isolated and a tracheostomy was performed with a 22-gauge catheter. the mice were euthanized via exsanguination. 0.8 ml of cold bal buffer (0.5 mm edta in pbs) was injected into the lungs over ~1min via the tracheostomy and then aspirated from the lungs over ~1min. injections/aspirations were performed three times for a total of 2.4ml of fluid added to the lungs. recovery bal fluid typically amounted to ~2.0ml. bal samples were centrifuged at 300xg for 4 minutes. the supernatant was collected and stored at -80°c for further analysis. protein concentration was measured using bio-rad dc protein assay, per manufacturer's instructions. the cell pellet was fixed for flow cytometry as follows. 333 µl of 1.6% pfa in pbs was added to each sample. samples were incubated in the dark at room temperature for 10 minutes, then 1 ml of bal buffer was added. samples were centrifuged at 400xg for 3min, the supernatant was aspirated, and 1 ml of facs buffer (2% fetal calf serum and 1 mm edta in pbs) was added. at this point, samples were stored at 4°c for up to 1 week prior to flow cytometry analysis. to stain for flow cytometry, samples were centrifuged at 300xg for 4 min, the supernatant was aspirated, and 100 µl of staining buffer was added. staining buffer used was a 1:1000 dilution of stock antibody solution (apc anti-mouse cd45; alexa fluor 488 anti-mouse ly6g, biolegend) into facs buffer. samples were incubated with staining antibody for 30 minutes at room temperature in the dark. to terminate staining, 1 ml of facs buffer was added, samples were centrifuged at 300xg for 4 minutes, and supernatant was aspirated. cells were resuspended in 900 µl of facs buffer and immediately analyzed via flow cytometry. flow cytometric analysis was completed with a bd accuri flow cytometer as follows: sample volume was set to 100 µl and flow rate was set to 'fast'. unstained and single-stained controls were used to set gates. forward scatter (pulse area) vs. side scatter (pulse area) plots were used to gate out non-cellular debris. forward scatter (pulse area) vs. forward scatter (pulse height) plots were used to gate out doublets. the appropriate fluorescent channels were used to determine stained vs. unstained cells. the gates were placed using unstained control samples. single-stain controls were tested and showed there was no overlap/bleed-through between the fluorophores. final analysis indicated the quantity of leukocytes (cd45-positive cells) and neutrophils (ly6g-positive cells) in bal samples. human lungs were obtained after organ harvest from transplant donors whose lungs were in advance deemed unsuitable for transplantation. the lungs were harvested by the organ procurement team and kept at 4°c until the experiment, which was done within 24 hours of organ harvest. the lungs were inflated with low pressure oxygen and oxygen flow was maintained at 0.8 l/min to maintain gentle inflation. pulmonary artery subsegmental branches were endovascularly cannulated, then tested for retrograde flow by perfusing for 5 minutes with steen solution containing a small amount of green tissue dye at 25 cm h2o pressure. the pulmonary veins through which efflux of perfusate emerged were noted, allowing collection of solutions after passage through the lungs. a 2 ml mixture of 125 i-labeled lysozyme-dextran nanogels and 131 ilabeled ferritin nanocages were injected through the arterial catheter. ~100ml of 3% bsa in pbs was passed through the same catheter to rinse unbound nanoparticles. a solution of green tissue dye was subsequently injected through the same catheter. the cannulated lung lobe was dissected into ~1 g segments, which were evaluated for density of tissue dye staining. segments were weighed, divided into 'high', 'medium', 'low', and 'null' levels of dye staining, and measured for 131 i and 125 i signal in a gamma counter. for experiments with cell suspensions derived from human lungs (chosen for research use according to the above standards), single cell suspensions were generously provided by the laboratory of edward e. morrisey at the university of pennsylvania. aliquots of 600,000 cells were pelleted at 400xg for 5 minutes and resuspended in 100 µl pbs containing different quantities of lysozyme-dextran nanogels synthesized with fitc-labeled dextran. cells and nanogels were incubated at room temperature for 30 minutes before two-fold pelleting at 400xg with 1 ml pbs washes. cells were re-suspended in 200 µl facs buffer for staining with apcconjugated anti-human cd45, applied by 20-minute incubation with a 1:500 dilution of the antibody stock. cells were pelleted at 400xg for 5 minutes and resuspended in 200 µl pbs for immediate analysis with flow cytometry (bd accuri). negative/positive nanogel or anti-cd45 signal was established by comparison to unstained cells. singlestained controls indicated no spectral overlap between fitc-nanogel fluorescence and anti-cd45 apc fluorescence. proteins were prepared in deionized and filtered water at concentrations of 0.155 mg/ml for human albumin, 0.2 mg/ml for hen lysozyme, and 0.48 mg/ml for igg. crosslinked albumin nanoparticles, lysozyme-dextran nanogels, and igg-coated liposome suspensions were prepared such that albumin, lysozyme, and igg concentrations in the suspensions matched the concentrations of the corresponding protein solutions. protein and nanoparticle solutions were analyzed in quartz cuvettes with 10 mm path length in an aviv circular dichroism spectrometer. the instrument was equilibrated in nitrogen at 25°c for 30 minutes prior to use and samples were analyzed with sweeps between 185 and 285 nm in 1 nm increments. each data point was obtained after a 0.333 s settling time, with a 2 s averaging time. cdnn 40 software deconvolved cd data (expressed in millidegrees) via neural network algorithm assessing alignment of spectra with library-determined spectra for helices, antiparallel sheets, parallel sheets, beta turns, and random coil. 8-anilino-1-naphthalenesulfonic acid (ansa) at 0.06 mg/ml was mixed with lysozyme, human albumin, or igg at 1.5 mg/ml in pbs. for nanoparticle analysis, nanoparticle solutions were prepared such that albumin, lysozyme, and igg concentrations in the suspensions matched the 1.5 mg/ml concentration of the protein solutions. protein or nanoparticles and ansa were reacted at room temperature for 30 minutes. excess ansa was removed from solutions by 3 centrifugations against 3 kda cutoff centrifugal filters (amicon). after resuspension to original volume, ansa-stained protein/nanoparticle solutions/suspension were examined for fluorescence (excitation 375 nm, emission 400-600 nm) and absorbance (240-540 nm) maxima corresponding to ansa. for imaging neutrophil content in naïve and iv-lps-injured lungs, mice were intravenously injected with rat anti-mouse anti-ly6g antibody (clone 1a8) and sacrificed 30 minutes later. lungs were embedded in m1 medium, flash frozen, and sectioned in 10 µm slices. sections were stained with percp-conjugated anti-rat secondary antibody and neutrophil-associated fluorescence was observed with epifluorescence microscopy. similar procedures enabled histological imaging of lysozyme-dextran nanogels in iv-lps-injured lungs. nanogels synthesized with rhodamine-dextran were administered intravenously in injured mice 30 minutes prior to sacrifice. lungs were sectioned as above and stained with clone 1a8 anti-ly6g antibody, followed by briliant violetconjugated anti-rat secondary antibody. sections of human lungs were obtained after ex vivo administration (see nanoparticle administration in human lungs above) of lysozyme-dextran nanogels synthesized with rhodamine dextran. regions of tissue delineated as perfused and nonperfused, as determined by arterial administration of tissue dye as above, were harvested, embedded in m1 medium, flash frozen, and sectioned in 10 µm slices. epifluorescence imaging indicated rhodamine fluorescence from nanogels, coregistered to autofluorescence indicating tissue architecture. a mouse was anesthetized with ketamine/xylazine five hours after intravenous administration of 2 mg/kg b4 lps. a jugular vein catheter was fixed in place for injection of lysozyme-dextran nanogels, anti-cd45 antibody, and fluorescent dextran during imaging. in preparation for exposure of the lungs, a patch of skin on the back of the mouse, around the juncture between the ribcage and the diaphragm, was denuded. while the mouse was maintained on mechanical ventilation, an incision at the juncture between the ribs and the diaphragm, towards the posterior, exposed a portion of the lungs. a coverslip affixed to a rubber o-ring was sealed to the incision by vacuum. the exposed portion of the mouse lung was placed in focus under the objective by locating autofluorescence signal in the "fitc" channel. with 100 ms exposure, fluorescent images from channels corresponding to violet, green, near red, and far red fluorescence were sequentially acquired. a mixture of rhodamine-dextran nanogels (2.5 mg/kg), brilliant violet-conjugated anti-cd45 antibody (0.8 mg/kg), and alexa fluor 647 labeled 70 kda dextran (40 mg/kg) for vascular contrast was administered via jugular vein catheter and images were recorded for 30 minutes. images were recorded in slidebook software and opened in imagej (fiji distribution) for composition in movies with coregistration of the four fluorescent channels. all animal studies were carried out in strict accordance with guide for the care and use of laboratory animals as adopted by national institute of health and approved by university of pennsylvania institutional animal care and use committee (iacuc). male c57bl/6j mice, 6-8 weeks old, were purchased from jackson laboratories. mice were maintained at 22-26°c and on a 12/12 hour dark/light cycle with food and water ad libitum. ex vivo human lungs were donated from an organ procurement agency, gift of life, after determination the lungs were not suitable for transplantation into a recipient, and therefore would have been discarded if they were not used for our study. gift of life obtained the relevant permissions for research use of the discarded lungs, and in conjunction with the university of pennsylvania's institutional review board ensured that all relevant ethical standards were met. error bars indicate standard error of the mean throughout. significance was determined through paired t-test for comparison of two samples and anova for group comparisons. linear discriminant analysis and principal components analysis were completed in gnu octave scripts (adapted from https://www.bytefish.de/blog/pca_lda_with_gnu_octave/, and made available in full in the supplementary materials). findings in this study contributed to united states provisional patent application number 62/943469. raw imaging, flow cytometry, gamma counter, and spectroscopy data supporting the findings of this study are available from the corresponding author upon reasonable request. all other data supporting the findings of this study are available within the paper and its supplementary information files. covid-19 in critically ill patients in the seattle region -case series the 1918 influenza pandemic: insights for the 21 st century lung safe investigators; esicm trials group. epidemiology, patterns of care, and mortality for patients with acute respiratory distress syndrome in intensive care units in 50 countries incidence and outcomes of acute lung injury. b. engl nanomedicine for the 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(f) biodistributions of heat-inactivated, fixed, and 125 ilabeled e. coli in naïve (n=4) and iv-lps-injured (n=4) mice tissue autofluorescence). (k) single frame from real-time intravital imaging of ldng (red) uptake in leukocytes (green) in iv-lps-inflamed pulmonary vasculature (blue, alexa fluor 647-dextran) biodistributions in iv-lps-injured mice for azide-functionalized liposomes conjugated to igg loaded with 2.5, 5, 10, and 20 dbco molecules per igg (bars further to the right correspond to more dbco per igg). (d) mouse lungs flow cytometry data indicating ly6g anti-neutrophil staining density vs. levels of dbco(20x)-igg liposome uptake. (e) flow cytometry data verifying increased dbco(20x)-igg liposome uptake in and specificity for neutrophils following lps insult (inset: verification of increased concentration of neutrophils in the lungs following lps 