key: cord-1049201-41n1xrxq
authors: Dieterle, M. Eugenia; Haslwanter, Denise; Bortz, Robert H.; Wirchnianski, Ariel S.; Lasso, Gorka; Vergnolle, Olivia; Abbasi, Shawn A.; Fels, J. Maximilian; Laudermilch, Ethan; Florez, Catalina; Mengotto, Amanda; Kimmel, Duncan; Malonis, Ryan J.; Georgiev, George; Quiroz, Jose; Barnhill, Jason; Pirofski, Liise-anne; Daily, Johanna P.; Dye, John M.; Lai, Jonathan R.; Herbert, Andrew S.; Chandran, Kartik; Jangra, Rohit K.
title: A replication-competent vesicular stomatitis virus for studies of SARS-CoV-2 spike-mediated cell entry and its inhibition
date: 2020-07-03
journal: Cell Host Microbe
DOI: 10.1016/j.chom.2020.06.020
sha: b9e8043f5b6c168cd2aea5641b546ea4623b8fd3
doc_id: 1049201
cord_uid: 41n1xrxq

Summary There is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. Rapid SARS-CoV-2 countermeasure development is contingent on the availability of robust, scalable, and readily deployable surrogate viral assays to screen antiviral humoral responses, define correlates of immune protection, and down-select candidate antivirals. Here, we generate a highly infectious recombinant vesicular stomatitis virus (VSV) bearing the SARS-CoV-2 spike glycoprotein S as its sole entry glycoprotein and show that this recombinant virus, rVSV-SARS-CoV-2 S, closely resembles SARS-CoV-2 in its entry-related properties. The neutralizing activities of a large panel of COVID-19 convalescent sera can be assessed in a high-throughput fluorescent reporter assay with rVSV-SARS-CoV-2 S, and neutralization of rVSV-SARS-CoV-2 S and authentic SARS-CoV-2 by spike-specific antibodies in these antisera is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific therapeutics and for mechanistic studies of viral entry and its inhibition.

A member of the family Coronaviridae, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 pandemic that emerged in Wuhan City, China in late 2019 . With more than 8 million confirmed cases and at least 435,000 deaths in over 216 countries/areas/territories as of June 15th, 2020, the global scale and impact of COVID-19 is unparalleled in living memory WHO Situation Report June 15th, 2020) . To date, mitigation strategies have relied largely on physical distancing and other public health measures.

Although treatments with some small molecule inhibitors and with convalescent plasma have received approvals for emergency use; and vaccines, antivirals, and monoclonal antibodies are being rapidly developed, no FDA-approved countermeasures are currently available.

The membrane-enveloped virions of SARS-CoV-2 are studded with homotrimers of the spike glycoprotein (S), which mediate viral entry into the host cell (Bosch et al., 2003; Walls et al., 2020; Wrapp et al., 2020) . S trimers are post-translationally cleaved in the secretory pathway by the proprotein convertase furin to yield N-and C-terminal S1 and S2 subunits, respectively. S1 is organized into an N-terminal domain (NTD), a central receptorbinding domain (RBD), and a C-terminal domain (CTD). S2 bears the hallmarks of a 'Class I' membrane fusion subunit, with an N-terminal hydrophobic fusion peptide, N-and Cterminal heptad repeat sequences, a transmembrane domain, and a cytoplasmic tail Wrapp et al., 2020) .

The S1 RBD engages the viral receptor, human angiotensin-converting enzyme 2 (hACE2) at the host cell surface (Hoffmann et al., 2020a; Wang et al., 2020; Zhou et al., 2020) . Receptor binding is proposed to prime further S protein cleavage at the S2' site by the transmembrane protease serine protease-2 (TMPRSS2) at the cell surface, and/or by host cysteine cathepsin(s) in endosomes. S2' cleavage activates S2 conformational rearrangements that catalyze the fusion of viral and cellular membranes and escape of the viral genome into the cytoplasm (Hoffmann et al., 2020a) .

The S glycoprotein is the major antigenic target on the virus for protective antibodies (Rogers et al., 2020; Wec et al., 2020; Wu et al., 2020a) , and is thus of high significance for the development of vaccines and therapeutic antibodies. Plasma derived from human convalescents and replete with such antibodies has shown early promise as a COVID-19 treatment, and it is currently being evaluated in clinical trials of antiviral prophylaxis and therapy (Casadevall and Pirofski, 2020) . Considerable efforts are also being aimed at the identification and deployment of S glycoprotein-specific neutralizing monoclonal antibodies (mAbs) Pinto et al., 2020; Rogers et al., 2020; Wec et al., 2020; Wu et al., 2020b; Zost et al., 2020) . A key requirement for the rapid development of such vaccines and treatments with convalescent plasma, small-molecule inhibitors, and recombinant biologics is the availability of safe, robust, and faithful platforms to study S-glycoprotein inhibition with high assay throughput. Given limited access to biosafety level-3 (BSL-3) containment facilities required to safely handle SARS-CoV-2, researchers have turned to surrogate viral systems that afford studies of cell entry at biosafety level-2 (BSL-2) and facilitate rapid inhibitor screening through the use of fluorescence or luminescence-based reporters. These include retroviruses, lentiviruses, or vesiculoviruses 'pseudotyped' with SARS-CoV-2 S and competent for a single round of viral entry and infection Nie et al., 2020; Ou et al., 2020; Pu et al., 2020; Tan et al., 2020; Xiong et al., 2020) . However, these single-cycle pseudotyped viruses are typically laborious to produce and challenging to scale up, yield poorly infectious preparations, and suffer background issues in some cases due to contamination with viral particles bearing the orthologous entry glycoprotein (e.g., low levels of vesicular stomatitis virus (VSV) pseudotypes bearing VSV G).

In contrast to the single-cycle pseudotypes, replication-competent recombinant VSVs (rVSVs) encoding the heterologous virus entry glycoprotein gene(s) in cis as their only entry protein(s) are easier to produce at high yields and also afford forward-genetic studies of viral entry. We and others have generated and used such rVSVs to safely and effectively study entry by lethal viruses that require high biocontainment (Caì et al., 2019; Jae et al., 2013; Jangra et al., 2018; Kleinfelter et al., 2015; Maier et al., 2016; Raaben et al., 2017; Whelan et al., 1995; Wong et al., 2010) Although rVSVs bearing the S glycoprotein from SARS-CoV (Fukushi et al., 2006; Kapadia et al., 2005 Kapadia et al., , 2008 and the Middle East respiratory syndrome coronavirus (MERS-CoV) (Liu et al., 2018) have been developed, no such systems have been described to date for SARS-CoV-2.

Here, we generate a rVSV encoding SARS-CoV-2 S and identify key passageacquired mutations in the S glycoprotein that facilitate robust rVSV replication. We show that the entry-related properties of rVSV-SARS-CoV-2 S closely resemble those of the authentic agent and use a large panel of COVID-19 convalescent sera to demonstrate that the neutralization of the rVSV and authentic SARS-CoV-2 by spike-specific antibodies is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific antivirals and for mechanistic studies of viral entry and its inhibition.

To generate a replication-competent rVSV expressing SARS-CoV-2 S, we replaced the openreading frame of the native VSV entry glycoprotein gene, G, with that of the SARS-CoV-2 S (Wuhan-Hu-1 isolate) (Fig. 1A) . We also introduced a sequence encoding the enhanced green fluorescent protein (eGFP) as an independent transcriptional unit at the first position of the VSV genome. Plasmid-based rescue of rVSV-SARS-CoV-2 S generated a slowly replicating virus bearing the wild-type S sequence. Five serial passages yielded viral populations that displayed enhanced spread. This was associated with a dramatic increase in the formation of syncytia ( Fig. 1B and Fig. S1 ) driven by S-mediated membrane fusion (Fig. S1 ). Sequencing of this viral population identified nonsense mutations that introduced stop codons in the S glycoprotein gene (amino acid position C1250* and C1253*), causing 24-and 21-amino acid deletions in the S cytoplasmic tail, respectively. S∆24 and S∆21 were maintained in the viral populations upon further passage, and S∆21 in all plaque-purified isolates, highlighting their likely importance as adaptations for viral growth. Viral population sequencing after four more passages identified two additional mutations, L517S and P812R in S1 and S2, respectively, whose emergence coincided with more rapid viral spread and the appearance of non-syncytium-forming infectious centers (Fig. 1B, passage 5) . Pelleted viral particles from clarified infected-cell supernatants incorporated the S glycoprotein, as determined by an Sspecific ELISA ( Fig 1C) .

We next sequenced six plaque-purified viral isolates derived from the passage 9 (P9) population. All of these viral clones bore the S∆21 deletion in the S cytoplasmic tail and spread without much syncytia formation (Fig. 1D) . Interestingly, all of these isolates contained three amino acid changes at S-glycoprotein positions other than 517 or 812-W64R, G261R, and A372T-in addition to the C-terminal S∆21 deletion (Table S1 and Fig.   S2 ). Five of the six isolates also contained mutations H655Y or R685G. Importantly, peak titers of all these viral isolates ranged between 1-3×10 7 infectious units per mL (Fig. 1E) , suggesting that the mutations they share (or a subset of these mutations) drive rVSV-SARS-CoV-2 S adaptation for efficient spread in tissue culture with little or no syncytium formation.

SARS-CoV-2 entry in cells has been shown to be dependent on the proteolytic activity of acid-dependent endosomal cysteine cathepsins, including cathepsin L (Hoffmann et al., 2020a; Wang et al., 2020) . Accordingly, we tested the effects of chemical inhibitors of cysteine cathepsins on rVSV-SARS-CoV-2 S infection. Pretreatment of cells with NH 4 Cl, an inhibitor of endosomal acidification, reduced entry by rVSVs bearing SARS-CoV-2 S or the Ebola virus glycoprotein (EBOV GP) in a dose-dependent manner ( Fig. 2A) . However, Smediated entry was comparatively less sensitive to NH 4 Cl than that by EBOV GP (Fig. 2A ).

Next, we tested cysteine cathepsin inhibitors E-64 (Fig. 2B) and FYdmk ( Fig 2C) . Pretreatment of cells with both of these compounds also inhibited S-mediated entry, albeit with reduced sensitivity relative to that observed for EBOV GP-dependent entry ( Fig. 2B-C) .

Together, these findings confirm that rVSV-SARS-CoV-2 S resembles the authentic agent in its requirements for endosomal acid pH and cysteine cathepsins. They also suggest a reduced dependence on these host factors for entry by SARS-CoV-2 S relative to EBOV GP, a model glycoprotein known to fuse in late endo/lysosomal compartments following extensive endosomal proteolytic processing.

The Type II transmembrane serine protease TMPRSS2 plays a key role in the infection and spread of a number of enveloped viruses in cells of the human airway (Choi et al., 2009 ) (Shen et al., 2017) . TMPRSS2 cleavage of the hemagglutinin spike precursors (HA0) of some influenza A and B viruses at a monobasic site generates HA1 and HA2 subunits, thereby priming HA for viral membrane fusion (Böttcher-Friebertshäuser et al., 2014; Limburg et al., 2019; Böttcher-Friebertshäuser et al., 2010; Chaipan et al., 2009) . TMPRSS2 can also activate membrane fusion by the spike glycoproteins of human coronaviruses, including those of SARS-CoV and MERS-CoV, by cleaving the spike at the monobasic S2' site during entry (Kawase et al., 2012; Zhou et al., 2015) . Recent work indicates that TMPRSS2 may play a similar role in SARS-CoV-2 entry into human airway and intestinal cells (Bestle et al., 2020; Zang et al., 2020; Hoffmann et al., 2020b) . Accordingly, we evaluated the effect of the trypsin-like serine protease inhibitor camostat mesylate (camostat), previously shown to block TMPRSS2 catalytic activity and inhibit viral glycoprotein activation (Zhou et al., 2015; Kawase et al., 2012; Nimishakavi et al., 2015) , on rVSV-SARS-CoV-2 S infection.

Pretreatment of Vero grivet monkey cells with camostat had little effect, consistent with their low expression levels of TMPRSS2 (Hoffmann et al., 2020b) . By contrast, camostat treatment significantly reduced VSV-SARS-CoV-2 S infection in Vero cells transduced to express human TMPRSS2 (Vero-TMPRSS2), as reported previously (Hoffmann et al., 2020a) (Fig. 2D ). These findings suggest that TMPRSS2 can promote cell entry by rVSV-SARS-CoV-2 S.

SARS-CoV-2 uses hACE2 as its entry receptor (Letko et al., 2020; Shang et al., 2020b Shang et al., , 2020a . Baby hamster kidney (BHK21) cells do not express detectable levels of ACE2 protein and are resistant to SARS-CoV-2 entry (Chu et al., 2020; Hoffmann et al., 2020a; Wang et al., 2020) . Concordantly, we observed no detectable infection by rVSV-SARS-CoV- To directly establish an entry-relevant interaction between rVSV-SARS-CoV-2 S and hACE2, we expressed and purified the spike RBD ( Fig. 3C ) and pre-incubated it with target cells. RBD pre-treatment inhibited rVSV-SARS-CoV-2 S entry in a specific and dosedependent manner (Fig. 3D ). Moreover, pre-incubation of cells with an hACE2-specific mAb, but not an isotype-matched control mAb, potently abolished rVSV-SARS-CoV-2 S entry (Fig. 3E ). These findings provide evidence that rVSV-SARS-CoV-2 S entry and infection, like that of the authentic agent, requires spike RBD-hACE2 engagement.

SARS-CoV-2 can infect multiple cell types in the human airway, including ciliated epithelial cells of the bronchial and bronchiolar mucosae and type I and II pneumocytes of the lung (Rockx et al., 2020; Hui et al., 2020) . We assessed rVSV-SARS-CoV-2 entry and infection in epithelial cell lines that serve as models of human respiratory function. Specifically, we found that the human lung adenocarcinoma cell line Calu3 was highly susceptible to infection ( Fig. 4A ) in a manner that was sensitive to treatment with a hACE2-specific mAb ( Fig. 4B -C). By contrast, the human lung adenocarcinoma cell line A549 was refractory to infection ( Fig. 4D ), as previously documented with authentic SARS-CoV-2 and a single-cycle VSV vector bearing SARS-CoV-2 S (Chu et al., 2020; Harcourt et al., 2020; Hoffmann et al., 2020a; Hui et al., 2020) . Because poorly differentiated A549 cells express little ACE2 (Jia et al., 2005; Mossel et al., 2005) (Fig. 4E ), we transduced these cells to express hACE2 and then exposed them to virus. The A549-hACE2 cells were more susceptible than their parental cells by a factor of ≈10 4 (Fig. 4E) . Thus, rVSV-SARS-CoV-2 S can enter and infect cells of human airway origin in an ACE2-dependent fashion.

S protein-targeting antibodies in COVID-19 convalescent sera specifically account for rVSV-SARS-CoV-2 S neutralization.

Prior to examining the performance of rVSV-SARS-CoV-2 S in neutralization assays with human antisera, we sought to establish a specific role for interaction between anti-spike antibodies in these sera and the VSV-borne S protein. Accordingly, we first evaluated the reactivity of two sera with rVSV-neutralizing activity (Fig. S3 ) against viral particles by ELISA. Both sera specifically recognized rVSV-SARS-CoV-2 S particles ( Fig. 5A ) and were also shown to be reactive against a purified, trimeric preparation of the spike glycoprotein Bortz et al., manuscript in preparation) . Further, serial pre-incubation of each serum with purified S immobilized on a high-binding plate depleted its capacity to inhibit rVSV-SARS-CoV-2 S infection to a degree that was commensurate with its content of S-specific antibodies ( Fig. 5B -C). By contrast, parallel pre-incubations with blocked plates had little or no effect (Fig. 5C ). These results indicate that S glycoprotein-targeting antibodies in COVID-19 convalescent sera specifically mediate rVSV-SARS-CoV-2 S neutralization.

We compared the capacities of human antisera derived from 40 COVID-19 convalescent donors to block infection by rVSV-SARS-CoV-2 S and authentic SARS-CoV-2 in a microneutralization format. Briefly, pre-titrated amounts of viral particles were incubated with serial dilutions of each antiserum, and target cells were then exposed to the virusantiserum mixtures. Viral infection was determined by enumerating eGFP-positive cells (rVSV) as above ( Fig. 1 ) or cells immunoreactive with a SARS-CoV-2 nucleocapsid proteinspecific antibody (authentic virus) (Fig. 6A ). Heatmaps of viral infectivity revealed similar antiserum donor-and dose-dependent neutralization patterns for rVSV-SARS-CoV-2 S and authentic SARS-CoV-2 (Fig. 6B) . Comparison of the serum dilutions at half-maximal neutralization derived from logistic curve fits (neutralization IC 50 ) revealed a 3-10-fold shift towards enhanced neutralization with rVSV-SARS-CoV-2 S (Fig. 6C) . The origin of this difference is unclear but does not appear to arise from viral passage-dependent changes in the rVSV-encoded spike gene sequence (Fig. S4) . Rather, it may reflect assay-specific differences in the rVSV and authentic virus microneutralization formats employed herein.

Nevertheless, the relative potencies of the antisera against rVSV-SARS-CoV-2 S and authentic SARS-CoV-2 were well correlated (R 2 =0.76) (Fig. 6D ). In sum, these findings demonstrate the suitability of rVSV-SARS-CoV-2 S for rapid, high-throughout, reporterbased assays of spike glycoprotein-dependent entry and its inhibition.

There is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. The rapid development of SARS-CoV-2 countermeasures is contingent on the availability of robust, scalable, and readily deployable surrogate viral systems to screen antiviral humoral responses and define correlates of immune protection. Such tools would also facilitate the efficient down-selection of candidate antivirals and studies of their mechanisms of action.

Here, we describe a highly infectious recombinant vesicular stomatitis virus bearing the SARS-CoV-2 spike glycoprotein S that closely resembles the authentic agent in its entryrelated properties. We show that rVSV-SARS-CoV-2 S affords the high-throughput, reporterbased screening of small-molecule and antibody-based inhibitors targeting the viral spike glycoprotein with performance characteristics comparable to those of SARS-CoV-2.

rVSV-SARS-CoV-2 S initially replicated poorly in cell culture following its rescue from plasmids, but we noted accelerated viral growth at passage 5 ( Fig. 1 ). This coincided with the emergence of viral variants bearing S glycoproteins with 21-or 24-amino acid truncations of their cytoplasmic tails, as also observed by Case and co-workers . The cytoplasmic tails of the S glycoproteins of SARS-CoV and SARS-CoV-2 are highly similar and carry signals for their retention in the endoplasmic reticulum (ER), including a conserved K×H×× motif located near the C-terminus (McBride et al. 2007; Ujike et al. 2016) . 18-19-amino acid deletions in the cytoplasmic tails of SARS-CoV S (Fukushi et al., 2006; Fukushi et al., 2005) and SARS-CoV-2 S (Ou et al., 2020) have been shown to increase the infectivity of single-cycle VSV-S pseudotypes. As previously observed for ER/Golgi-localizing hantavirus glycoproteins (Slough et al., 2019) , these deletions likely redistribute S glycoproteins to the cell surface, thereby relieving the spatial mismatch in budding between VSV and SARS-CoV2 (plasma membrane vs. ER, respectively) and enhancing S incorporation into VSV particles.

Accelerated growth by rVSV-SARS-CoV-2 S around passage 5 was accompanied by a marked increase in the occurrence of syncytia (see Fig. 1B ) due to S-mediated cell-cell fusion (Fig. S1 ). This may reflect a functional property of the cytoplasmic tail-deleted S variants, including perturbations in their subcellular localization, as discussed above.

Strikingly, passage 9 stocks and highly infectious viral plaque isolates from these stocks displayed a pattern of spreading infection more typical for rVSVs, with few large syncytia in evidence (Fig. 1B) . In this regard, it is tempting to speculate that one or more additional S glycoprotein mutations detected in the passage 5-9 viral populations and in the six plaque isolates (Table S1 ) arose as compensatory changes to suppress the syncytiogenic propensity of the rVSV-encoded S∆21 glycoprotein spikes. Indeed, several mutations in the S1 NTD and RBD may serve to modulate spike glycoprotein fusogenicity, as also may mutations near or at the S1-S2 cleavage site (H655Y and R685G, respectively) and/or at the S2' cleavage site (P812R) (Table S1 and Fig. S2) . Further, at least one mutation (H655Y), present in five of six rVSV-SARS-CoV-2 S plaque isolates, has arisen during natural SARS-CoV-2 evolution in humans , during transmission studies in a hamster model , and possibly during SARS-CoV-2 passage in tissue culture (this report). Our current efforts are aimed at understanding what role(s) these mutations in the S glycoprotein ectodomain play in the maintenance of high levels of rVSV infectivity without the formation of large numbers of syncytia. These findings also highlight a feature of rVSV-SARS-CoV-2 S not shared by any of the viral entry surrogates described to date-its utility for forward genetics. This can be used to dissect structure-function relationships in the SARS-CoV-2 spike glycoprotein and to elucidate the mechanisms of action of spike-or entry-targeted antivirals.

We demonstrate that rVSV-SARS-CoV-2 S can be used to rapidly and faithfully assess the neutralizing activities of large panels of COVID-19 convalescent sera (this report, Fig. 6 ) and spike-directed mAbs (Wec et al., 2020) . We have exploited the fidelity and high throughput of our rVSV-based 384-well plate microneutralization assay to rapidly pre-screen >300 COVID-19 convalescents and identify potential convalescent plasma donors first for the expanded access program and now for an ongoing randomized controlled trial of convalescent plasma therapy (Casadevall and Pirofski, 2020; Chandran & Pirofski, manuscript in preparation) . The utility and reliability of this approach is further enhanced by its synergy with the new SARS-CoV-2 microneutralization assay also described herein ( Fig.   6A-B) , which provides a rapid and non-subjective alternative to classical PRNT assays.

When used in combination, these assays should afford the rapid mechanistic interrogation of cellular factors and antivirals that act at any step of the viral multiplication cycle-entry hits should affect both the rVSV and the authentic virus, whereas post-entry hits should affect only the latter.

As the COVID-19 pandemic continues apace and the development of plasma-, hyperimmune globulin-, mAb-, and small molecule-based countermeasures accelerates, the need for highly scalable viral assays continues to mount. The availability of highly infectious rVSV surrogates that can be scaled up with relative ease for antiviral screening and readily deployed in reporter-based microneutralization assays will facilitate these efforts.

rVSV-SARS-CoV-2 S has only been tested in cell culture. Thus, we cannot conclusively rule out that it may not be well tolerated in vivo. Given this theoretical safety concern, lab workers should exercise due caution in handling an agent that is potentially infectious in humans.

Until the safety of rVSV-SARS-CoV-2 S has been evaluated and established in appropriate animal model(s), we will only distribute it to researchers for in vitro work to be performed at biosafety level 2 or higher, as approved by their institutional biosafety committee. were immunostained for hACE2 expression as described in Fig 3A. using an anti-ACE2 antibody. Cells were imaged by fluorescence microscopy. The hACE2 signal is pseudocolored green and representative images are shown (scale bar = 20 µm). * p<0.033, ** p<0.002, *** p<0.001. In panels C-D, the depletion #4 and depletion control were compared. * p<0.033, ** p<0.002, *** p<0.001. 

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Kartik Chandran (kartik.chandran@einsteinmed.org).

Plasmids and a passage 9 stock of rVSV-SARS-CoV-2 S generated in this study, together with documenting information, will be made available upon request and following execution of a UBMTA between Albert Einstein College of Medicine and the recipient institution.

rVSV-SARS-CoV-2 S will be made available for in vitro research only (please see the 'Limitations of the Study' section).

Primary data are available on request. This study did not generate any new software code.

Cells. Human hepatoma Huh7.5.1 (received from Dr. Jan Carette; originally from Dr. Frank Chisari) and 293FT (ThermoFisher) cells were cultured in Dulbecco's Modified Eagle Medium (DMEM high glucose, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Atlanta Biologicals), 1% Penicillin/Streptomycin (P/S, Gibco) and 1% Gluta-MAX (Gibco). The African vervet monkey kidney Vero cells and baby hamster kidney BHK21 cells were maintained in DMEM (high glucose) supplemented with 2% heatinactivated FBS, 1% P/S and 1% Gluta-MAX. Vero-E6 cells were grown in Minimum Essential Medium (MEM) supplemented in 10% FBS and Gentamicin (all from Sigma).

A549 cells were maintained in DMEM (high glucose) supplemented with 10% heatinactivated FBS, 1% P/S and 1% Gluta-MAX. These cell lines were passaged every 2-3 days using 0.05% Trypsin/EDTA solution (Gibco). Calu3 cells were cultured in Eagle's Minimum Essential Medium (EMEM, ATCC) supplemented with 10% heat-inactivated FBS, 1% P/S, and 1% Gluta-MAX and passaged weekly using 0.05% Trypsin/EDTA solution.

coding sequences were PCR-amplified from the hACE2 plasmid Addgene #1786 (a generous gift from Hyeryun Choe) and TMPRSS2 plasmid Addgene #53887 (a generous gift from Roger Reeves), respectively and cloned into a retroviral pBabe-puro vector. Retroviruses were produced by transfecting 293FT cells with the hACE2 and TMPRSS2 expressing pBabe-puro plasmids along with those expressing the Moloney murine leukemia virus (MMLV) gag-pol and VSV G proteins. Retroviral supernatants passed through a 0.45-µm filter were used to transduce BHK21, Vero cells or A549 cells. Transfected cells were selected with puromycin (2 µg/ml).

Serum samples were collected from healthy adult volunteers residing in Westchester County, NY who had recovered from COVID-19 in April 2020.

Patients had reported a positive nasopharyngeal swab by PCR for SARS-CoV-2 during illness and had been asymptomatic for at least 14 days prior to sample collection. After obtaining informed consent, serum was obtained by venipuncture (BD Vacutainer, serum), centrifuged, aliquoted and stored at -80°C prior to use. The sera were heat-inactivated at 56°C for 30 minutes and stored at 4°C prior to analysis. Protocol approval was obtained by the Institutional Review Board (IRB) of the Albert Einstein College of Medicine.

A plasmid encoding the VSV antigenome was modified to replace its native glycoprotein, G, with the full-length wild-type S glycoprotein gene of the Wuhan-Hu-1 isolate of SARS-CoV-2 (GenBank MN908947.3). The VSV antigenome also encodes for an eGFP reporter gene as a separate transcriptional unit. Plasmid-based rescue of the rVSV was carried out as described previously (Kleinfelter et al., 2015; Whelan et al., 1995; Wong et al., 2010) . Briefly, 293FT cells were transfected with the VSV antigenome plasmid along with plasmids expressing T7 polymerase and VSV N, P, M, G and L proteins by using polyethylenimine. Supernatants from the transfected cells were transferred to Huh7.5.1 cells every day (day 2-7 post-transfection) till the appearance of eGFP-positive cells. The poorly spreading virus was initially propagated by cell subculture. RNA was isolated from viral supernatants at different passages and Sanger sequencing was used to verify S gene sequences. A passage #9 viral stock was plaque-purified on Vero cells.

Supernatants were aliquoted and stored at -80°C. The generation of rVSV-SARS-CoV-2 S and its use in tissue culture at biosafety level 2 was approved by the Environmental Health and Safety Department and the Institutional Biosafety Committee at Albert Einstein College of Medicine.

All work with authentic SARS-CoV-2 was completed in BSL-3 laboratories at USAMRIID in accordance with federal and institutional biosafety standards and regulations. Vero-76 cells were inoculated with SARS-CoV-2 (GenBank MT020880.1) at a MOI=0.01 and incubated at 37°C with 5% CO2 and 80% humidity. At 50 hours post-infection, cells were frozen at -80°C for 1 hour, allowed to thaw at room temperature, and supernatants were collected and clarified by centrifugation at ~2,500 ×g for 10 min. Clarified supernatant was aliquoted and stored at -80°C. Sequencing data from this virus stock indicated a single mutation in the spike glycoprotein (H655Y) relative to Washington state isolate MT020880.1.

CoV2 RBD plasmid (a generous gift from Florian Krammer) was used for the expression of recombinant RBD as previously described Stadlbauer et al., 2020) . 

plasmid encoding stabilized S glycoprotein gene (a generous gift from Jason McLellan) was used for the expression of recombinant S protein as described previously with several modifications. ExpiCHO-S cells (ThermoFisher) were transiently transfected with plasmid DNA diluted in OptiPRO Serum-Free Medium (0.8 μg total DNA per ml of culture) using ExpiFectamine (ThermoFisher) at a DNA-to-ExpiFectamine ratio of 1:4. At 8 days post-transfection, cultures were harvested by centrifugation at 4,000 x g for 20 min.

Clarified supernatant was dialyzed in 50 mM Tris HCl pH 8.0, 250 mM NaCl at a clarified supernatant to dialysis buffer ratio of 1:25 prior affinity chromatography. Dialyzed supernatant was incubated with Ni-NTA resin (GoldBio) for 2 h at 4°C. Resin was collected in columns by gravity flow, washed with wash buffer (50 mM Tris HCl pH 8.0, 250 mM NaCl, 20 mM Imidazole) and eluted with elution buffer (50 mM Tris HCl pH 8.0, 250 mM NaCl, 250 mM Imidazole). Eluate was concentrated in Amicon centrifugal units (EMD Millipore) and exchanged into a storage buffer (50 mM Tris HCl pH 8.0, 250 mM NaCl).

Protein was analyzed by SDS-PAGE, aliquoted, and stored at -80°C.

High-protein binding 96-well ELISA plates (Corning) were coated with 25 µl of concentrated rVSV-SARS-CoV-2 S or rVSV-EBOV (2.73 µg/ml) overnight at 4˚C, and blocked with 3% nonfat dry milk in PBS (PBS-milk) for 1 h at 25˚C. Plates were extensively washed and incubated with serum 18, serum 39 or negative serum diluted to 1:100 first then with serial 2-fold dilutions in PBS milk 1% -Tween 0.1% for 1 h at 25˚C. Plates were washed three times and incubated with Goat anti-human IgG-HRP (#31410 Invitrogen) diluted 1:3000 (PBS milk 1% -Tween 0.1%) for 1 h at 25˚C and detected using 1-Step™ Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific, Waltham, MA). Plates were read using a Cytation 5 imager (BioTek) at 450 nm. NH 4 Cl inhibition experiments. Huh7.5.1 cell monolayers were incubated for 1hour with 20-100 mM NH 4 Cl in DMEM. Next, pre-titrated rVSVs expressing EBOV GP or SARS-CoV-2 S were used to infect cells. Infection was scored 16-18 h later as described above.

with E-64 (37.5 or 75 µM), Z-Phe-tyr-dmk (FYdmk, 18.75 or 37.5 µM), or 1.5% DMSO (vehicle control) were infected with pre-titrated amounts of rVSVs carrying SARS-CoV 2 S, EBOV GP or VSV G. At 1 h post-infection, 20 mM NH 4 Cl was added. Infected cells were fixed 16-18 h later and scored for infection as described above.

for 2 h at 37℃ with camostat mesylate (Tocris) or 1% DMSO (vehicle control) were infected with pre-titrated amounts of rVSV-SARS-CoV 2 S. At 1 h post-infection, 20 mM NH 4 Cl was added to terminate viral entry. Infected cells were fixed 7 h later and scored for infection as described above.

To stain for surface-expressed hACE2, BHK21, A549, BHK21-hACE2 and A549-hACE2 cells or the control cells were seeded onto fibronectin-coated glass coverslips were incubated with 0.4 μg/mL of hACE2specific goat antibody (#AF933, R&D systems) at 4ºC in media containing 25 mM HEPES.

Next, cells were washed with cold PBS, fixed with 4% paraformaldehyde, and blocked with buffer (2% (w/v) bovine serum albumin, 5% (v/v) glycerol, 0.2% (v/v) Tween20 in Ca 2+ /Mg 2+ -free PBS). Secondary donkey AlexaFluor 594-conjugated anti-goat IgG (#A32758 Invitrogen) was used to detect the hACE2 signal. Coverslips were mounted in Prolong with DAPI (Invitrogen) and imaged on an Axio Observer inverted microscope (Zeiss). Hoechst-33342 (Invitrogen) at a dilution of 1:2,000 in. Viral infectivity was measured by automated enumeration of GFP-positive cells from captured images using a Cytation5 automated fluorescence microscope (BioTek) and analyzed using the Gen5 data analysis software (BioTek). The half-maximal inhibitory concentration (IC 50 ) of the mAbs was calculated using a nonlinear regression analysis with GraphPad Prism software.

Anti-hACE2 antibody blocking assay. Huh7.5.1 cells were seeded into a 384-well plate and Calu3 cells in a 96-well plate pre-coated with 1% gelatin/PBS, respectively. Nex day, goat anti-human ACE2 antibody (#AF933, R&D Systems) was serially diluted and applied to the cells. After 1 h incubation at 37°C and 5% CO2, cells were infected with rVSV-SARS-CoV-2 S. At 16-18 h (Huh-7.5.1) or 8 h (Calu3) post-infection, cells were fixed and scored for infection as described above. Human gamma globulin (009-000-002) purchased from Jackson ImmunoResearch was used as negative control.

RBD competition assay. Monolayers of Huh7.5.1 cells in a 384-well plate were incubated with serial dilutions of recombinant RBD domain for 1 hour at 37°C and 5% CO2. Cells were then infected with pre-titrated amounts of rVSV-SARS-CoV-2 or rVSV-EBOV GP and scored for infection 16-18 h later.

S-mediated antibody depletion assay. High-protein binding 96-well ELISA plates (Corning) coated with PBS alone or with 2 µg/ml of SARS-CoV-2 S protein in PBS overnight at 4˚C were blocked for 1 hour with 3% nonfat dry milk (Biorad) in PBS. Serum samples diluted in DMEM (1:50 dilution) were serially incubated 4 times for 1 h each at 37°C on S protein-coated or control wells. The depleted sera were tested for their neutralization capacity as described above.

SARS-CoV-2 neutralization assay. Serially diluted serum samples were mixed with prediluted SARS-CoV-2 in infection media (EMEM/2% FBS/Gentamicin) and incubated for 1 hour at 37°C/5% CO2/80% humidity. Virus/serum inoculum was added to Vero-E6 cells, seeded in 96 well plates, at a MOI of 0.4 and incubated for 1 hour at 37°C/5% CO2/80% humidity. Virus/serum inoculum was removed and cells were washed with PBS prior to addition of culture media (MEM/10% FBS/Gentamicin). Following 24 h incubation at 37°C/5% CO2/80% humidity, media was removed and cells were washed with PBS. PBS was removed and cells were submerged in 10% formalin for 24 h. Formalin was removed and cells were washed with PBS prior to permeabilization with 0.2% Triton-X for 10 minutes at room temperature. Cells were blocked for 2 h, then immunostained with SARS-1 nucleocapsid protein-specific antibody (Sino Biologic; 40143-V08B) and AlexaFluor 488 labeled secondary antibody. Cells were imaged using an Operetta (Perkin Elmer) high content imaging instrument and infected cells were determined using Harmony Software (Perkin Elmer).

Vero cells were infected with pre-titrated amounts of rVSV-SARS-CoV-2 S for 2 h at 37°C and 5% CO2. Following the removal of virus inocula, cells were washed with PBS to remove any residual virus and indicated dilutions of convalescent sera were applied to the infected cells. Cells were fixed, their nuclei were counterstained, and syncytia formation was imaged by eGFP expression at 16 h post-infection.

The n number associated with each dataset in the figures indicates the number of biologically independent samples. The number of independent experiments and the measures of central tendency and dispersion used in each case are indicated in the figure legends. Dose-response neutralization curves were fit to a logistic equation by nonlinear regression analysis. Unless otherwise indicated in the figure legends, statistical comparisons were carried out by two-way ANOVA with a post hoc correction for family-wise error rate (Dunnett test for comparison of an untreated sample mean to treated sample means, Tukey test for all possible comparisons of sample means). Testing level (alpha) was 0.05 for all statistical tests. All analyses were carried out in GraphPad Prism 8.

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We thank I. Gutierrez, E. Valencia, and L. Polanco for laboratory management. We thank J.McLellan for his generous gifts of wild-type and recombinant SARS-CoV-2 spike constructs.We also thank F. Krammer, H. Choe and R. Reeves for their generous gifts of SARS-CoV-2 RBD, hACE2, and TMPRSS2 constructs, respectively. We are grateful to J. Carette for his generous gift of Huh-7.5.1 cells and S. Anthony, E. Choi, B. Gomperts, B. Maniccasamy, K.Stapleford, and C. Sen for their generous gifts of airway epithelial cell lines. This work was supported in part by National Institutes of Health (NIH) grants U19AI142777 and R01AI132633 (to K.C.), R01AI143453 and R01AI123654 (to L.P.), R01AI125462 (to J.R.L.) and R21AI141367 (to J.P.D). M.E.D. is a Latin American Fellow in the Biomedical

• Highly infectious recombinant VSV expressing SARS-CoV-2 spike (S) was generated• rVSV-SARS-CoV-2 S resembles SARS-CoV-2 in entry and inhibitor/antibody sensitivity • rVSV-SARS-CoV-2 S affords rapid screens and forward-genetic analyses of antivirals