key: cord-0711089-oa3145s3
authors: Bates, Timothy A.; Weinstein, Jules B.; Farley, Scotland; Leier, Hans C.; Messer, William B.; Tafesse, Fikadu G.
title: Cross-reactivity of SARS-CoV structural protein antibodies against SARS-CoV-2
date: 2021-01-26
journal: Cell Rep
DOI: 10.1016/j.celrep.2021.108737
sha: 3d194e2ee86457c3a712c467d7b083113892837e
doc_id: 711089
cord_uid: oa3145s3

In the ongoing COVID-19 pandemic, there remain unanswered questions regarding the nature and significance of the humoral immune response towards other coronavirus infections. Here, we investigate the cross-reactivity of antibodies raised against the first SARS-CoV for their reactivity towards SARS-CoV-2. We extensively characterize a selection of 10 antibodies covering all of the SARS-CoV structural proteins: spike, membrane, nucleocapsid, and envelope. While nearly all of the examined SARS-CoV antibodies displayed some level of reactivity to SARS-CoV-2, we found only partial cross-neutralization for the spike antibodies. The implications of our work are two-fold. Firstly, we have established a set of antibodies with known reactivity to both SARS-CoV and SARS-CoV-2, which will allow further study of both viruses. Secondly, we provide empirical evidence of the high propensity for antibody cross-reactivity between distinct strains of human coronaviruses, critical information for designing diagnostic and vaccine strategies for COVID-19.

Furthermore, several of these CoV structural proteins have been shown to have intracellular functions unrelated to their role as structural proteins (McBride et al., 2014) . There are limits to the utility of extrapolation; it is known, for example, that the topology of the CoV envelope protein varies dramatically between various viruses (Schoeman and Fielding, 2019) , and the differences between the receptor binding domains (RBDs) of the spike protein can be dramatic.

Therefore, tools to interrogate the specific functions of each of the SARS-CoV-2 structural proteins would be of immense and immediate use.

CoV specific antibodies are one type of tool used in such studies. Antibodies against the SARS-CoV-2 structural proteins could be used as reagents in microscopy and western blotting, as structural tools to probe functional epitopes, and even as antiviral therapies. The protein which produces the greatest SARS-CoV-2 specific antibody response in humans is the viral S protein (Ahmed et al., 2020) , but it is known that antibodies are produced against the N, M, and E proteins as well (Ahmed et al., 2020; Severance et al., 2008) . Since SARS-CoV and SARS-CoV-2 are such markedly similar viruses, as discussed below, it is reasonable to assume that there may be some cross-reactivity between SARS-CoV antibodies against their cognate SARS-CoV-2 structural proteins, and, indeed, there is already some evidence that this is the case Tian et al., 2020; Wrapp et al., 2020; Yuan et al., 2020) .

SARS-CoV and SARS-CoV-2 S proteins share 76% amino acid sequence homology and both rely on cellular angiotensin-converting enzyme 2 (ACE2) as an attachment receptor as well as the TMPRSS2 protease for priming . Recent reports have identified cross-reactive antibodies that bind to the S protein of both SARS-CoV and SARS-CoV-2, however no such cross-reactive antibodies have been identified for the remaining structural proteins Tian et al., 2020; Wrapp et al., 2020; Yuan et al., 2020) . A non-humanprimate model of SARS-CoV-2 DNA vaccination found that a polyclonal antibody response to S alone is sufficient to protect from SARS-CoV-2 challenge, similar to results from a human S-only vaccine trial for SARS-CoV (Martin et al., 2008; . Additionally, convalescent plasma from recovered COVID-19 cases has been broadly shown to reduce mortality of individuals with serious disease (Sullivan and Roback, 2020; Zhang et al., 2020) . The sequence similarity between SARS-CoV and CoV-2 N, M, and E proteins are high, at 91%, 90%, and 95% respectively, making it likely that any individual antibody may be cross-reactive. Indeed, there are reports of human antibodies against the S, N, and M proteins for which the epitopes are J o u r n a l P r e -p r o o f identical between SARS-CoV and SARS-CoV-2, further supporting the possibility of crossreactivity, though none have been experimentally verified (Ahmed et al., 2020) .

If cross-reactivity with SARS-CoV-2 is a common feature of SARS-CoV antibodies, then many recovered SARS-CoV patients may still possess SARS-CoV-2 reactive antibodies; antibody responses were shown to remain at high levels for at least 12 years according to a recent preprint . While sequence conservation is lower for more common human coronaviruses, their high prevalence may lead to widespread antibodies with cross-reactivity to SARS-CoV-2. Furthermore, antibodies promoting antibody-dependent cellular phagocytosis have been shown to assist in elimination of SARS-CoV infection, showing that cross-reactive antibodies need not be neutralizing to play a productive role in resolution of coronavirus infection (Yasui et al., 2014) .

This report characterizes a series of SARS-CoV monoclonal antibodies for cross-reactivity, experimental utility, and neutralization of the live SARS-CoV-2 virus. Information about how antibodies from different coronavirus infections interact is critical for several reasons. It is an important factor to consider during the design of antibody-based coronavirus tests, particularly for those as closely related as SARS-CoV and SARS-CoV-2. New treatments for SARS-CoV-2 that interact with a patient's immune system will also need to take into account the prevalence of cross-reactive antibodies due to previous coronavirus infections. Further, information about the basic biology of this novel virus will be critical in developing such tailored treatments, and cross-reactive antibodies could be extremely useful in such studies.

To begin to evaluate structural potential for cross-reactivity, we compared the amino acid sequences of each SARS-CoV-2 structural protein with the homologous protein from the other HCoVs ( Figure 1A ). We first looked at the amino acid homology among the S proteins of the common human coronaviruses, and found that other human beta-CoVs (MERS-CoV, HCoV-HKU1 and HCoV-OC43) show only about 30% similarity to the SARS-CoV-2 S protein, and human alpha-CoVs (HCoV-229E and HCoV-NL63) show only about 24% similarity to SARS-CoV-2 S protein. The S protein of the original SARS-CoV, however, is much more closely related, showing 77% similarity between SARS-CoV and SARS-CoV-2, which lends support to J o u r n a l P r e -p r o o f the idea that anti-SARS-CoV S antibodies could be cross-reactive with the SARS-CoV-2 S protein. The E, M, and N protein sequences show striking similarity between SARS-CoV and SARS-CoV-2; they are 96%, 91%, and 91% similar, respectively ( Figure 1A ).

The Biodefense and Emerging Infections (BEI) Research Resources Repository has available several types of antibodies and immune sera against each of the structural SARS-CoV proteins as well as whole virus (summarized in Table 1 ). Eight of these are mouse monoclonal antibodies (240C, 341C, 540C, 154C, 472C, 19C, 283C, 42C) of either the IgM, IgG2a, or IgG1 class, recognizing either the SARS-CoV E, M, N, or S proteins. Of these, only two are neutralizing, 341C, and 540C (Tripp et al., 2005) . There are also polyclonal rabbit sera against the SARS-CoV S protein, and an anti-S monoclonal human IgG1 antibody (CR3022) isolated from a SARS-CoV patient (ter Meulen et al., 2006) , all of which are neutralizing.

While antibodies that recognize each of the structural proteins are of interest as experimental tools, antibodies that recognize the S protein are particularly so because of their potential to neutralize infectious virus. Structural information about the specific biochemical interactions between S-specific antibodies and the S protein is of great value. For the anti-S monoclonal antibodies (through BEI Resources) described in Table 1 , the epitopes can be traced to one of three regions of the RBD. While 240C, 341C, and 540C all bind within a region at the end of the RBD (epitope S A ) (Tripp et al., 2005) , the 154C antibody binds to a region at the beginning of the RBD (epitope S B ); and the human monoclonal antibody CR3022 binds to specific residues in a broad region in the middle of the RBD (epitope S C ) . These epitopes are indicated in figure 1B , along with the alignment of the SARS-CoV and SARS-CoV-2 RBDs.

While not identical, these regions do show some level of similarity between the two virus strains.

The three-dimensional structure of the spike protein in both monomeric and the functional trimeric form is displayed to illustrate the general accessibility of each portion of the protein ( Figure 1C ).

To assess SARS-CoV antibodies against SARS-CoV-2, we first performed immunofluorescence (IF) staining of Vero E6 cells infected with live SARS-CoV-2 virus ( Figure 2 ). The S-specific antibodies NRC-772, CR3022, and 240C all showed strong staining, while 540C and 154C showed weak staining. Antibody 341C showed no staining. The E-specific (472C), M-specific J o u r n a l P r e -p r o o f (19C & 283C) , and N-specific (42C) all displayed robust staining. We confirmed the presence of SARS-CoV-2 infected cells by co-staining with human convalescent serum, which demonstrates that negative 341C staining is not due to a lack of infection. To further validate the utility of these antibodies for IF, we performed staining of 293T cells transiently transfected with Strep-tagged constructs of each of the individual SARS-CoV-2 structural proteins (Gordon et al., 2020) . We compared the staining of the strep-tag within each structural protein in immunofluorescence against that of the experimental antibodies, finding that the staining pattern of a majority of these antibodies as detectable, with some being highly similar to the strep-tag antibody ( Figure   S2 ). These results match our findings for the live SARS-CoV-2 infection, however 42C (Nspecific) showed markedly reduced staining of transiently transfected cells. Together, these antibodies provide complete coverage of SARS-CoV-2 structural proteins, showing their utility for SARS-CoV-2 experiments involving microscopy.

We next evaluated these antibodies by western blot. His 6 -tagged RBD from SARS-CoV-2 was produced in HEK 293 cells and purified by Ni-NTA chromatography ( Figure S3A ). The purified RBD was then used for a western blot with each of the mouse monoclonal antibodies ( Figure   3A -B). Anti-His6 antibody demonstrates high purity of the RBD protein. The staining produced by each experimental antibody was compared to lysate from untransfected 293T cells to assess background. Of these antibodies, 240C and NR-772 produced strong signal with little background, whereas the other monoclonal antibodies (CR3022, 154C, 341C, and 540C) did not produce detectable signal.

We also performed western blots on SARS-CoV-2 (Isolate USA-WA1/2020) infected and uninfected Vero E6 cell lysates. Probing with human convalescent serum revealed bands at the expected size for each of the structural SARS-CoV-2 proteins: S, N, M, and E ( Figure S3D ). The 42C, 540C, NRC-772, 240C, and 283C antibodies each developed bands unique to the SARS-CoV-2 infected samples, however not all of the bands were at the expected molecular weight.

Previous reports have shown that SARS-CoV-2 proteins including S, N, M, and E all produce bands at several different molecular weights when expressed exogenously in HEK 293T cells, and it is not surprising that these bands also exist in our blots (Gordon et al., 2020) . What is unexpected, is that for S monoclonal antibodies 240C, 540C, and 283C, we detect the lower molecular weight band (~50 kDa) and not the band at the expected molecular weight ( Figure   J o u r n a l P r e -p r o o f S3). This could be due to masking of the epitope by glycosylation absent from the truncated protein, or specific recognition of proteolytically cleaved peptides. For instance, proteolytic cleavage of the S protein is known to be important for proper maturation of SARS-CoV-2 particles Walls et al., 2020) . The N monoclonal antibody 42C is able to detect a band at the correct molecular weight (~46 kDa), however there is a moderate level of background ( Figure 3D ). The M monoclonal antibody 283C also detects a band at the expected molecular weight (~25 kDa), but also detects a lower molecular weight band similar what has been shown in previous reports which showed that the M protein is particularly prone to proteolytic degradation in western blots as well as a high molecular weight smear (Gordon et al., 2020) . The M monoclonal antibody 19C shows similar staining, but weaker, and both display a high level of background staining. The E monoclonal antibody 472C displays a weak band at the expected molecular weight (~8 kDa), but the intensity is similar to that of background so a positive determination cannot be made.

The fact that the S glycoprotein is responsible for virus binding and entry into host cells makes it an attractive target for antibody generation as some of these antibodies may be neutralizing.

Because of the potential functional role for these antibodies, and because of the number of different antibody clones, we decided to examine the S-protein-specific antibodies more thoroughly.

We assessed the binding of the S-protein-specific antibodies to both the full-length SARS-CoV-2 S protein and the purified Receptor Binding Domain (RBD) by ELISA ( Figure 3F -G and Figure   S4 , summarized in Figure 3E ). The CR3022 and 240C antibodies showed strong binding to both the full-length spike and the RBD (EC 50 75 ng/mL and 127 ng/mL, respectively, to the RBD).

154C and 341C showed weak but detectable binding (6.046 µg/mL and 10.03 µg/mL, respectively, to the RBD), while 540C did not demonstrate binding at all. The trend for these antibodies is generally similar to what was seen in the previous studies where the antibodies were tested against recombinant SARS-CoV S protein (Tripp et al., 2005) . The original report of CR3022 did not perform a direct ELISA for us to compare our results to, however our data agree with studies of CR3022 on SARS-CoV-2 showing that it binds strongly to both full-length spike and the RBD . While CR3022 appears to be the strongest binder to RBD, 240C is marginally better on the full-length spike protein.

To assess the binding kinetics of the antibody-RBD interaction in more detail, we measured the antibody-epitope interactions using biolayer interferometry (BLI). The three monoclonal antibodies that showed strongest-binding with the ELISA displayed high affinity for the SARS-CoV-2 RBD, CR3022 showed the strongest binding with a calculated K D of 758 pM ( Figure 4A ), while 240C demonstrated a 1.36 nM K D ( Figure 4B ), and 154C a 481 nM K D ( Figure 4C ). As summarized in figure 4D , these antibodies showed fast-on/slow-off kinetics in agreement with a previous report of CR3022 binding kinetics on RBD . The other antibodies we tested displayed no measurable binding at the highest concentration used ( Figure S5 ). Importantly, BLI does not account for the avidity of these antibodies, and it is likely that the interaction of each epitope/paratope pair is substantially lower than that of the intact antibody; however, the intact antibody more closely resembles the interaction that is likely to occur in most in vitro assays, or indeed in vivo. Our K D is substantially lower than reported in Tian et al., however this is likely due to differences in the reagents used (Tian et al., 2020) . Tian et al. expressed their RBD in E. coli, preventing glycosylation, while our RBD was produced in mammalian cells. Additionally, their CR3022 was produced as a single chain variable fragment (scFv) in E. coli, which would contain only a single paratope and may fold differently than our full CR3022 antibody, which was produced in a plant expression system, is bivalent, and contains intact constant domains.

Finally, we assessed the neutralizing capabilities of these S protein-specific monoclonal antibodies. We set up a neutralization assay using a Lentivirus GFP-reporter pseudotyped with the SARS-CoV-2 S protein (Crawford et al., 2020) . Neutralization was assessed by quantitative fluorescent microscopy, using the area of GFP expression compared to that of an antibodyuntreated control. Serial dilutions of antibodies were used to generate neutralization curves and estimate the antibody concentration necessary for 50% neutralization. This readout was used because the monoclonal antibodies displayed only partial neutralization at the highest concentration used in our assay. To validate our assay, we used human convalescent serum from a SARS-CoV-2 positive patient. This anti-serum demonstrated 50% neutralization at a dilution of 1:270 ( Figure 4E ). Consistent with a previous report, CR3022 failed to show any neutralization at 100 µg/mL despite its potent binding in every other assay .

154C and 240C both showed partial neutralization, with a 50% reduction in GFP area at 57.8 µg/mL and 61.3 µg/mL respectively. Consistent with the BLI results, 341C and 540C did not show substantial neutralization. We were surprised to see 154C perform the best in this assay, J o u r n a l P r e -p r o o f particularly because the original report of these antibodies on SARS-CoV showed 341 and 540 as the only antibodies with neutralizing capabilities. One unique aspect of 154C is that it is the only IgM antibody from this selection of Spike-specific antibodies, however it is not clear how this might affect neutralization .

To further validate our pseudotyped lentivirus neutralization data, we set up a focus forming assay (FFA)-based neutralization studies using live SARS-CoV-2 (Isolate USA-WA1/2020) as previously described (Case et al., 2020) . The virus was titrated such that each well received 30 pfu/well which was pre-incubated for 1 hour with antibody dilutions starting at 1:10 down to 1:1280. The results from this assay were broadly similar with those seen in the pseudovirus neutralization assay, with 240C and 154C showing partial neutralization and 341C, 540C, and CR3022 showing minimal neutralization ( Figure 4E and S6). The human convalescent serum from a SARS-CoV-2 patient (1v6) performed better in the FFA, while the monoclonal antibodies each performed slightly less well than in the pseudotype neutralization assay. The reasons for this variation may be due to the substantial differences between the design of these two assays including the cell type, virus type and quantity, and detection method. Despite these differences, the similar neutralizing trends in both assays show limited cross-neutralization of SARS-CoV-2 by the spike monoclonal antibodies of SARS-CoV.

The utility of each of the antibodies used in this study has been summarized in Table 2 . In particular, the S-protein-specific 240C performed well in every assay we performed, excluding neutralization. In contrast, 540C showed no detectable binding in any of our assays. The other S-protein-specific monoclonal antibodies 154C, 341C, and CR3022 showed mixed utility in different assays ( Table 2 ). The rabbit polyclonal antibody NRC-772 also worked in every assay in which it was tested, however, polyclonal sera is limited to experiments where structural information about particular epitopes is not important due to the unknown admixture of the contained antibody clones. The antibodies against E, M, and N demonstrated utility in immunofluorescence and showed some success in western blots in the case of the 42C, and 283C antibodies (Table 2) . Further studies could explore these antibodies in greater detail by producing purified E, M, and N proteins for use in biochemical assays such as the ones we used to characterize the S-protein-specific antibodies in this report.

J o u r n a l P r e -p r o o f DISCUSSION Our results demonstrate measurable cross-reactivity from a majority of the SARS-CoV structural-protein-targeted antibodies that we evaluated against SARS-CoV-2 S, N, M and E proteins. These tools can be readily obtained from BEI Resources and utilized by labs to study the properties of untagged SARS-CoV-2 structural proteins. These antibodies can serve the unmet need for more resources enabling the study of the SARS-CoV-2. It is critical to understand the basic biology of SARS-CoV-2 in order to inform efforts towards improved diagnostics and treatments. Further, information about cross-reactivity of antibodies between SARS-CoV and SARS-CoV-2 may assist bioinformaticians in developing computational tools for predicting cross-reactivity of other antibodies, or even guiding rational design of improved coronavirus antibodies and small-molecule therapeutics.

We have shown that these publicly available antibodies are of potential use in several different types of assays with SARS-CoV-2 proteins. We found that several of these SARS-CoV structural protein antibodies demonstrated good staining in immunofluorescence of SARS-CoV-2 infected cells and in an overexpression system (240C, NRC-772, and CR3022 against S; 42C against N; 283C against M; 472C against E). The anti-S antibodies, 240C and NRC-772, also give clear signal in western blot with minimal background. Several S antibodies show potent binding to full-length S and the RBD by ELISA, as well as binding to the RBD by biolayer interferometry (240C, CR3022, and NRC-772). This wide range of uses substantially broadens our ability to investigate the biochemical properties of SARS-CoV-2 structural proteins.

The neutralization experiments we performed showed that antibodies which were previously shown to be neutralizing against SARS-CoV were actually less likely to be strongly crossreactive with SARS-CoV-2. This may be due to a phenomenon well described among rapidly evolving viruses such as HIV and influenza wherein neutralizing antibodies are more likely to bind to highly variable epitopes lying on the host-interacting surfaces of the viral proteins (Pauthner and Hangartner, 2020; Sicca et al., 2018) . It is likely that the specific amino acid substitutions present in the RBD of the SARS-CoV-2 spike protein compared to that of SARS-CoV were selected for, in part, due to their ability to avoid binding by existing SARS-CoV antibodies among the wild animal populations from whence SARS-CoV-2 emerged. It is then, perhaps, unsurprising that 240C and 154C retained partial neutralizing ability whereas 341C

and 540C seem to have lost their capacity to neutralize when faced with SARS-CoV-2. There are ongoing efforts to determine the evolutionary forces that are shaping the continued change J o u r n a l P r e -p r o o f of the SARS-CoV-2 spike protein in response to more widespread antibody-based immunity in the global population; it may even be possible to anticipate mutations that could give rise to more virulent strains (Starr et al., 2020) .

Although these antibodies only partially neutralized a SARS-CoV-2 model infection, they are still of interest for their potential to elucidate the structure and function of their protein targets.

Antibodies have been critical tools in structure determination and in the mapping of proteins' functional regions. Having a wide array of antibodies that recognize varying epitopes is of great help in this endeavor. Additionally, with the current dearth of knowledge regarding the life cycle and pathogenesis of SARS-CoV-2, particularly regarding the understudied M, N, and E proteins, we believe that these antibodies could be used in experiments to better understand the nuances of their functions beyond their obvious structural roles.

Our results also speak to the high proportion of SARS-CoV antibodies that display substantial cross-reactivity to SARS-CoV-2 structural proteins. Anecdotal evidence supports the efficacy of convalescent plasma treatment for COVID-19, indicating that cross-reactive antibodies generated during previous coronavirus infections may prove beneficial for emerging coronavirus infections . Another recent study found that following recovery from infection with SARS-CoV-2, patients expressed increased levels of antibodies capable of binding to peptides from more distantly related, human coronaviruses such as HCoV-OC43 and HCoV-229E (Shrock et al., 2020) . Conversely, studies of COVID-19 patients have found neutralizing antibody titers to be directly proportional to disease severity, suggesting a more complicated relationship between antibodies and COVID-19 (Long et al., 2020; Wu et al., 202(Long et al., 2020; Wu et al., 2020) 0). Some have hypothesized that this may be due to high concentrations of virus and neutralizing antibodies acting together to drive greater immune pathology (Jacobs, 2020; Zohar and Alter, 202(Jacobs, 2020; Zohar and Alter, 2020) 0). A better understanding of the functions of individual antibody isotypes against different antigenic targets will be critical to predicting the utility of a particular antibody against SARS-CoV-2.

Further studies could also investigate possible cooperation between antibodies recognizing different epitopes, especially as CR3022 neutralization was shown to have synergy with another anti-S antibody which recognized a different epitope on the protein (ter Meulen et al., 2006) . A recent study (Chi et al., 2020) , for example, characterized a neutralizing monoclonal antibody that did not bind the RBD at all, and instead recognized an epitope in the NTD of the S protein.

Knowledge about the variety of vulnerable epitopes, and possible synergy between antibodies that target them, brings us ever closer to being able to design and deploy effective therapeutics and vaccines in this time of urgent need. 

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Fikadu G. Tafesse (tafesse@ohsu.edu).

No unique reagents were generated during the course of this study.

This study did not generate any unique datasets or code. 

293T stable cell lines expressing Ace2 receptor (293T-Ace2) were a kind gift from Dr. Jesse D.

Bloom from University of Washington, and described previously (Crawford et al., 2020 

Protein sequences were obtained from uniprot and aligned using the T-Coffee multiple sequence alignment server.

Transfections were carried out in 293T cells seeded at 70-90% cell density using Lipofectamine plate. Structural SARS-CoV-2 protein plasmids were a kind gift from the Krogan Lab at UCSF and are described previously (Gordon et al., 2020) . For pseudotyped lentivirus production, lentivirus packaging plasmids, HDM_Hgpm2, HDM_tat1b, PRC_CMV_Rev1b, SARS_CoV-2 S plasmid HDM_IDTSpike_fixK, and LzGreen reporter plasmid pHAGE2_CMV_ZsGreen_W were transfected using 0.44µg for packaging, 0.68µg for S, and 2µg for reporter plasmids per 6 cm dish. Packaging, SARS-CoV-2 S, and reporter plasmids were a kind gift from Jesse D. Bloom from University of Washington, and are described previously (Crawford et al., 2020) .

Transfection media was carefully removed 6 hours post transfection, and replaced with DMEM.

293T cells were seeded at 2 million cells/dish in 6cm TC-treated dishes. The following day, cells were transfected as described above with lentivirus packaging plasmids, SARS-CoV-2 S plasmid, and lzGreen reporter plasmid (Crawford et al., 2020) . After transfection, cells were incubated at 37C for 60 hours. Viral media was harvested, filtered with 0.45µm filter, then frozen before use. Virus transduction capability was then tittered on 293T-Ace2 cells treated with 50µl of 5µg/ml polybrene (Sigma-Aldritch LLC). LzGreen titer was determined by fluorescence using BZ-X700 all-in-one fluorescent microscope (Keyence), a 1:16 dilution was decided as optimal for following neutralization assays due to broad transduced foci distribution.

One tube of frozen SARS-CoV-2 (BEI Resources) was thawed and diluted 1:10 for inoculation in minimal volume onto 70% confluent Vero E6 cells. The cells were incubated for 1 hour at 37°C, rocking every 15 minutes to ensure even coverage. Additional media was added up to the manufacturer's recommended culture volume, and the cells were incubated for 72 hours at 37°C. Supernatant was collected and spun at 3,000×g for 5 minutes, then aliquoted for storage at -80°C.

A 96-well plate of 50% confluent Vero cells was inoculated with 50 µL frozen SARS-CoV-2 virus stock for 1 hour at 37°C with rocking every 15 minutes. Added an additional 50 µL of fresh media and incubated for 24 hours at 37°C. Fixed plate by submerging in 4% PFA in PBS for 1 hour, then brought into BSL-1 for immunofluorescence staining. 

Neutralization protocol was based on previously reported neutralization research utilizing SARS-CoV-2 S pseudotyped lentivirus (Crawford et al., 2020) . 293T-Ace2 cells were seeded on tissue culture treated, poly-lysine treated 96-well plates at a density of 10,000 cells per well. Cells were allowed to grow overnight at 37°C. LzGreen SARS-COV-2 S pseudotyped lentivirus were mixed with 2-fold dilutions of the following monoclonal or polyclonal anti-SARS-CoV-2 S antibodies: mouse anti-SARS-CoV S monoclonal IgM 154C, mouse anti-SARS-CoV S monoclonal IgG2a

240C, mouse anti-SARS-CoV S monoclonal IgG2a 341C, mouse anti-SARS-CoV S monoclonal IgG2a 540C, rabbit anti-SARS-CoV S polyclonal sera, Guinea pig anti-SARS-CoV S polyclonal sera, human monoclonal anti-SARS-CoV S CR3022 (BEI Resources). Human patient sera from a SARS-CoV-2 patient was used as positive neutralization control, while virus alone was used as negative control. Sera and antibody dilutions ranged from 1:10 to 1:1048. Virus-antibody J o u r n a l P r e -p r o o f mixture was incubated at 37C for 1 hour after which virus was added to 293T-Ace2 treated with 5µg/ml polybrene. Cells were incubated with neutralized virus for 44 hours before imaging. Cells were fixed with 4% PFA for 1 hour at RT, incubated with DAPI for 10 minutes at RT, and imaged with BZ-X700 all-in-one fluorescent microscope (Keyence). Estimated area of DAPI and GFP fluorescent pixels was calculated with built in BZ-X software (Keyence).

The FFA was performed as previously described (Case et al., 2020) . In brief, Vero E6 cells were 

Purified SARS-CoV-2 S-RDB protein was prepared as described previously (Stadlbauer et al., 2020) . Breifly, codon optimized His-tagged RBD in pInducer-20 was used to make lentivirus in HEK 293T cells which was then used to infect HEK 293-F suspension cells. The suspension cells were allowed to grow for 3 days with shaking at 37°C at 8% CO 2 . Cell supernatant was collected, sterile filtered, and purified by Ni-NTA chromatography. The purified protein was then buffer exchanged into PBS and concentrated. For use in BLI, purified RBD was biotinylated using the ChromaLINK biotin protein labeling kit according to the manufacturer's instructions with 5x molar equivalents of labeling reagent to achieve 1.92 biotins/protein.

Streptavidin biosensors (ForteBio) were soaked in PBS for at least 30 minutes prior to starting the experiment. Biosensors were prepared with the following steps: equilibration in kinetics buffer (10 mM HEPES, 150 mM NaCl, 3mM EDTA, 0.005% Tween-20, 0.1% BSA, pH 7.5) for 300 seconds, loading of biotinylated RBD protein (10ug/mL) in kinetics buffer for 200 seconds, and blocking in 1 µM D-Biotin in kinetics buffer for 50 seconds. Binding was measured for seven 3-fold serial dilutions of each monoclonal antibody using the following cycle sequence: baseline for 300 seconds in kinetics buffer, association for 300 seconds with antibody diluted in kinetics buffer, dissociation for 750 seconds in kinetics buffer, and regeneration by 3 cycles of 20 seconds in 10 mM glycine pH 1.7, then 20 seconds in kinetics buffer. All antibodies were run against an isotype control antibody at the same concentration. Data analysis was performed using the ForteBio data analysis HT 10.0 software. Curves were reference subtracted using the isotype control and each cycle was aligned according to its baseline step. KDs were calculated using a 1:1 binding model using global fitting of association and dissociation of all antibody concentrations, excluding dilutions with response below 0.005 nm.

293T cells were seeded in 10 cm dishes at a density of 3.5 million cells per dish. After overnight growth, cells were transfected using lipofectamine 3000 as described above. Plasmids pTwist- 

Around 10 

The authors declare no competing interests. 9.0 × 10 4 (± 2.8 × 10 1 ) 6.8 × 10 -5 (± 3.0 × 10 -7 ) 3.7 × 10 3 (± 2.6 × 10 1 ) 1.8 × 10 -3 (± 6.8 × 10 -6 ) 5.4 × 10 4 (± 2.0 × 10 2 ) 7.3 × 10 -5 (± 3.3 × 10 -6 ) 

SARS-CoV-2 Spike Pseudovirus

Preliminary Identification of Potential Vaccine Targets for the COVID-19 Coronavirus (SARS-CoV-2) Based on SARS-CoV Immunological Studies

Growth, detection, quantification, and inactivation of SARS-CoV-2

A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2

2020. Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV

Spike Protein for Neutralization Assays

An interactive web-based dashboard to track COVID-19 in real time

Effectiveness of convalescent plasma therapy in severe COVID-19 patients

Molecular Evolution of Human Coronavirus Genomes

Coronaviridae Study Group of the International Committee on Taxonomy of Viruses, 2020. The species Severe acute respiratory syndrome-related coronavirus : classifying 2019-nCoV and naming it SARS-CoV-2

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing

Long-Term Persistence of IgG Antibodies in SARS-CoV Infected Healthcare Workers

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Neutralizing antibodies mediate virus-immune pathology of COVID-19

Genomic characterization of a novel SARS-CoV-2

Human coronavirus circulation in the United States

Structure, Function, and Evolution of Coronavirus Spike Proteins

Cross-reactive Antibody Response between SARS-CoV-2 and SARS-CoV Infections

A SARS DNA vaccine induces neutralizing antibody and cellular immune responses in healthy adults in a Phase I clinical trial

The Coronavirus Nucleocapsid Is a Multifunctional Protein

A structural analysis of M protein in coronavirus assembly and morphology

T-Coffee: A novel method for fast and accurate multiple sequence alignment

Broadly Neutralizing Antibodies to Highly Antigenically Variable Viruses as Templates for Vaccine Design

Coronavirus envelope protein: current knowledge

Development of a Nucleocapsid-Based Human Coronavirus Immunoassay and Estimates of Individuals Exposed to Coronavirus in a

Viral epitope profiling of COVID-19 patients reveals cross-reactivity and correlates of severity

Effector mechanisms of influenza-specific antibodies: neutralization and beyond

SARS-CoV-2 Seroconversion in Humans: A Detailed Protocol for a Serological Assay, Antigen Production, and Test Setup

Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding

Convalescent Plasma: Therapeutic Hope or Hopeless Strategy in the SARS-CoV-2 Pandemic

Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirusspecific human monoclonal antibody

Monoclonal antibodies to SARS-associated coronavirus

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

Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies

Neutralizing antibody responses to SARS-CoV-2 in a COVID-19 recovered patient cohort and their implications. medRxiv 2020.03.30

Phagocytic cells contribute to the antibodymediated elimination of pulmonary-infected SARS coronavirus

DNA vaccine protection against SARS-CoV-2 in rhesus macaques

A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV

Anti-SARS-CoV-2 virus antibody levels in convalescent plasma of six donors who have recovered from COVID-19

A Novel Coronavirus from Patients with Pneumonia in China

Dissecting antibody-mediated protection against SARS-CoV-2