key: cord-286390-ytgw3j4s
authors: Case, James Brett; Rothlauf, Paul W.; Chen, Rita E.; Liu, Zhuoming; Zhao, Haiyan; Kim, Arthur S.; Bloyet, Louis-Marie; Zeng, Qiru; Tahan, Stephen; Droit, Lindsay; Ilagan, Ma. Xenia G.; Tartell, Michael A.; Amarasinghe, Gaya; Henderson, Jeffrey P.; Miersch, Shane; Ustav, Mart; Sidhu, Sachdev; Virgin, Herbert W.; Wang, David; Ding, Siyuan; Corti, Davide; Theel, Elitza S.; Fremont, Daved H.; Diamond, Michael S.; Whelan, Sean P.J.
title: Neutralizing antibody and soluble ACE2 inhibition of a replication-competent VSV-SARS-CoV-2 and a clinical isolate of SARS-CoV-2.
date: 2020-07-03
journal: Cell Host Microbe
DOI: 10.1016/j.chom.2020.06.021
sha: 
doc_id: 286390
cord_uid: ytgw3j4s

Abstract Antibody-based interventions against SARS-CoV-2 could limit morbidity, mortality, and possibly transmission. An anticipated correlate of such countermeasures is the level of neutralizing antibodies against the SARS-CoV-2 spike protein, which engages with host ACE2 receptor for entry. Using an infectious molecular clone of vesicular stomatitis virus (VSV) expressing eGFP as a marker of infection, we replaced the glycoprotein gene (G) with the spike protein of SARS-CoV-2 (VSV-eGFP-SARS-CoV-2) and developed a high-throughput imaging-based neutralization assay at biosafety level 2. We also developed a focus-reduction neutralization test with a clinical isolate of SARS-CoV-2 at biosafety level 3. Comparing the neutralizing activities of various antibodies and ACE2-Fc soluble decoy protein in both assays revealed a high degree of concordance. These assays will help define correlates of protection for antibody-based countermeasures and vaccines against SARS-CoV-2. Additionally, replication-competent VSV-eGFP-SARS-CoV-2 provides a tool for testing inhibitors of SARS-CoV-2 mediated entry under reduced biosafety containment.

Here, we developed a simple and robust BSL2 assay for evaluating SARS-CoV-2 entry 82 and its inhibition by antibodies. We engineered an infectious molecular clone of vesicular 83 stomatitis virus (VSV) to encode the SARS-CoV-2 S protein in place of the native envelope 84 glycoprotein (G) and rescued an autonomously replication-competent virus bearing the spike.

Through passage of VSV-eGFP-SARS-CoV-2, we selected a gain-of-function mutation in S that 86 allowed more efficient viral propagation yielding titers of > 

S AA as determined by expression of the virus-encoded eGFP reporter (Fig 1A, right panel) .

However, VSV-eGFP-SARS-CoV-2-S AA propagation was inefficient on Vero CCL81 cells. This 106 result prompted us to test additional modifications of the cytoplasmic tail of S, which also were 107 defective in autonomous amplification (Fig S1B) . To overcome this limitation, we used a forward 108 genetic approach to isolate two adaptive variants of VSV-eGFP-SARS-CoV-2-S AA (Fig S1C) . 

which truncates the cytoplasmic tail of SARS-CoV-2 S by 21 residues (Fig 1A) . This virus,

hereafter referred to as VSV-SARS-CoV-2-S Δ21 , was passed 12 times in total to assess genetic 6 stability by next generation sequencing, which revealed no additional mutations in the spike 118 (SRA: SRR11878607; BioProject: PRJNA635934). Comparison of plaque morphology of VSV-

SARS-CoV-2-S Δ21 and VSV-eGFP-SARS-CoV-2-S AA on three Vero cell subtypes and MA104 120 cells demonstrates that the selected variant spreads more efficiently (Fig 1B) . Screening of a 121 larger panel of cell types (Fig 1C) identified MA104 and Vero E6 cells as supporting the highest 122 levels of virus production. Ectopic expression of TMPRSS2 led to a further ~10-fold increase in 123 viral titer and larger plaque size (Fig 1D) . VSV-SARS-CoV-2-S Δ21 also was capable of infecting 124 Calu-3 cells, a human epithelial lung adenocarcinoma cell line (Fig S2) . particles but not in the parental VSV (Fig 1E) . The protein detected migrated at ~100 133 kilodaltons, a band that corresponds to the cleaved S1 subunit of the glycoprotein (Watanabe et 134 al., 2020). To examine whether the S Δ21 incorporated into VSV particles is processed to S1 and 135 S2, we performed [ 35 S] cysteine-methionine metabolic labeling in BSRT7 cells, which support 136 robust VSV replication, and analyzed released particles by SDS-PAGE and phosphorimaging.

In addition to the VSV structural proteins (N, P, M and L), two additional bands were observed 138 for VSV-SARS-CoV-2-S Δ21 that correspond in size to glycosylated S1 (107 kDa) and S2 Δ21 (85 139 kDa) (Fig 1F) . Negative-stain electron microscopy of sucrose-gradient purified virus particles 140 revealed that the membrane protein projecting from VSV-SARS-CoV-2-S Δ21 is larger than 141 observed on wild-type VSV particles (Fig 1G) , which reflects the larger size of the coronavirus 142 spike. 

We designed a high-throughput assay for titrating SARS-CoV-2 that could be applied to 

Coronaviruses possess a roughly 30 kb RNA genome, which requires that they encode 

Asymptomatic Carrier Transmission of COVID-19

SPAdes: a new genome assembly 591 algorithm and its applications to single-cell sequencing

Deployment of convalescent plasma 596 for the prevention and treatment of COVID-19

Vesicular 599 stomatitis virus-based vaccine protects hamsters against lethal challenge with Andes virus

Generation of bovine respiratory 603 syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue 604 culture, and the human RSV leader region acts as a functional BRSV genome promoter

BBMerge -Accurate paired shotgun read merging 608 via overlap

Ebola virus entry requires the 612 cholesterol transporter Niemann-Pick C1

Endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection

HIV-1 ENVELOPE. Effect of the cytoplasmic domain on antigenic 620 characteristics of HIV-1 envelope glycoprotein

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fastp: an ultra-fast all-in-one FASTQ 626 preprocessor

Fast algorithms for large-629 scale genome alignment and comparison

Coronaviruses: an RNA proofreading machine regulates replication fidelity and diversity

Eukaryotic transient-expression 636 system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA 637 polymerase

Evaluation of a novel vesicular stomatitis virus pseudotype-based assay for detection of 641 neutralizing antibody responses to SARS-CoV

Vesicular stomatitis virus pseudotyped with severe acute 645 respiratory syndrome coronavirus spike protein

A single dose of a vesicular stomatitis virus-based 649 influenza vaccine confers rapid protection against H5 viruses from different clades

Properties of replication-competent vesicular 654 stomatitis virus vectors expressing glycoproteins of filoviruses and arenaviruses

Development of a new vaccine for the 659 prevention of Lassa fever

Retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus 663 S protein

Clinical Characteristics of Coronavirus Disease 2019 in China

Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Virus entry. Lassa virus entry requires a 676 trigger-induced receptor switch

Live attenuated recombinant vaccine 680 protects nonhuman primates against Ebola and Marburg viruses

Human immunodeficiency viral vector pseudotyped with the spike envelope of severe acute 684 respiratory syndrome coronavirus transduces human airway epithelial cells and dendritic cells

Therapeutic strategies in an outbreak scenario to treat the novel coronavirus 687 originating in Wuhan, China

CoV-2 spike pseudotyped virus by recombinant ACE2-Ig

Functional assessment of cell entry and receptor 693 usage for SARS-CoV-2 and other lineage B betacoronaviruses

Structure of SARS coronavirus spike 696 receptor-binding domain complexed with receptor

The Sequence Alignment/Map format and SAMtools

A unique strategy for mRNA cap methylation used 703 by vesicular stomatitis virus

Angiotensin-converting enzyme 2 is 707 a functional receptor for the SARS coronavirus

Intracellular targeting signals contribute to 710 localization of coronavirus spike proteins near the virus assembly site

The cytoplasmic tail of the severe acute 713 respiratory syndrome coronavirus spike protein contains a novel endoplasmic reticulum retrieval 714 signal that binds COPI and promotes interaction with membrane protein

Synthetic antibodies neutralize SARS-CoV-2 718 infection of mammalian cells

Retroviruses pseudotyped with the severe acute respiratory 722 syndrome coronavirus spike protein efficiently infect cells expressing angiotensin-converting 723 enzyme 2

Enhancing dengue virus maturation using a stable furin over-expressing cell line

Establishment and validation of a pseudovirus neutralization assay for

Emerging microbes & infections 9

Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-735 reactivity with SARS-CoV

Cross-neutralization of SARS-CoV-2 by a human 742 monoclonal SARS-CoV antibody

Dipeptidyl peptidase 4 is a functional receptor for the 746 emerging human coronavirus-EMC

The coronavirus E protein: assembly and beyond. 749 Viruses

Treatment of 5 Critically Ill Patients With COVID-19 With Convalescent Plasma

A recombinant vesicular stomatitis virus 756 bearing a lethal mutation in the glycoprotein gene uncovers a second site suppressor that 757 restores fusion

Specificity, cross-reactivity, and function of 761 antibodies elicited by Zika virus infection

Identification of protective epitopes on ebola virus glycoprotein at the 765 single amino acid level by using recombinant vesicular stomatitis viruses

Tailored tetravalent antibodies potently and specifically 769 activate Wnt/Frizzled pathways in cells, organoids and mice

Human monoclonal antibody 773 combination against SARS coronavirus: synergy and coverage of escape mutants

Structural insights into coronavirus entry

An efficient method to make human 780 monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus

Chimeric Viruses with Vesicular Stomatitis 785 Virus: Actions in the Brain

Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

Site-specific 791 glycan analysis of the SARS-CoV-2 spike

Efficient recovery of infectious 794 vesicular stomatitis virus entirely from cDNA clones

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• Neutralization can be assessed by BSL2 and BSL3 high-throughput assays • SARS-CoV-2 and VSV-SARS-CoV-2-based neutralization assays correlate eTOC Blurb

generate a replication-competent vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike and compare the neutralizing activity of antibodies with VSV-SARS-CoV-2 to fully infectious SARS-CoV-2. They show that VSV-SARS-CoV-2 is a useful BSL2 surrogate virus, as neutralization profiles strongly correlate with focus-reduction neutralization tests using SARS-CoV-2


