key: cord-0803840-zhrsm3jh
authors: Jennewein, Madeleine F.; MacCamy, Anna J.; Akins, Nicholas R.; Feng, Junli; Homad, Leah J.; Hurlburt, Nicholas K.; Seydoux, Emily; Wan, Yu-Hsin; Stuart, Andrew B.; Edara, Venkata Viswanadh; Floyd, Katharine; Vanderheiden, Abigail; Mascola, John R.; Doria-Rose, Nicole; Wang, Lingshu; Yang, Eun Sung; Chu, Helen Y.; Torres, Jonathan L.; Ozorowski, Gabriel; Ward, Andrew B.; Whaley, Rachael E.; Cohen, Kristen W.; Pancera, Marie; McElrath, M. Juliana; Englund, Janet A.; Finzi, Andrés; Suthar, Mehul S.; McGuire, Andrew T.; Stamatatos, Leonidas
title: Isolation and Characterization of Cross-Neutralizing Coronavirus Antibodies from COVID-19+ Subjects
date: 2021-03-24
journal: bioRxiv
DOI: 10.1101/2021.03.23.436684
sha: 40ddfb9d5c13fca8e07db3bfa78660102eae8d05
doc_id: 803840
cord_uid: zhrsm3jh

SARS-CoV-2 is one of three coronaviruses that have crossed the animal-to-human barrier in the past two decades. The development of a universal human coronavirus vaccine could prevent future pandemics. We characterized 198 antibodies isolated from four COVID19+ subjects and identified 14 SARS-CoV-2 neutralizing antibodies. One targeted the NTD, one recognized an epitope in S2 and twelve bound the RBD. Three anti-RBD neutralizing antibodies cross-neutralized SARS-CoV-1 by effectively blocking binding of both the SARS-CoV-1 and SARS-CoV-2 RBDs to the ACE2 receptor. Using the K18-hACE transgenic mouse model, we demonstrate that the neutralization potency rather than the antibody epitope specificity regulates the in vivo protective potential of anti-SARS-CoV-2 antibodies. The anti-S2 antibody also neutralized SARS-CoV-1 and all four cross-neutralizing antibodies neutralized the B.1.351 mutant strain. Thus, our study reveals that epitopes in S2 can serve as blueprints for the design of immunogens capable of eliciting cross-neutralizing coronavirus antibodies.

In the past 2 decades there have been 3 zoonotic transmissions of highly pathogenic coronaviruses. SARS-CoV-1, MERS-CoV and SARS-CoV-2. The most recent one, SARS-CoV-2, has been rapidly spreading globally since late 52 2019/early 2020, infecting over 120 million people and killing over 2.6 million people by March 2021 Patel et al., 2020) . Studies conducted in mice, hamsters and non-human primates strongly suggest 54 that neutralizing antibodies (nAbs) isolated from infected patients can protect from infection, and in the case of established infection, can reduce viremia and mitigate the development of clinical symptoms (Baum et al., 56 2020b; Cao et al., 2020b; Mercado et al., 2020; Rogers et al., 2020b; Schafer et al., 2021; Shi et al., 2020; Tortorici et al., 2020; Wu et al., 2020; Yu et al., 2020) . Cocktails of neutralizing monoclonal antibodies have been approved 58 by the FDA for the treatment of infection (Baum et al., 2020a; Weinreich et al., 2020) . Thus, nAbs are believed to be an important component of the protective immune responses elicited by effective vaccines. Indeed, both 60 the mRNA-based Pfizer and Moderna vaccines elicit potent serum neutralizing antibody responses against SARS-CoV-2 (Jackson et al., 2020; Walsh et al., 2020) . 62

Monoclonal antibodies (mAbs) with neutralizing activities have been isolated from infected patients and their characterization led to the identification of vulnerable sites on the viral spike protein (S) (Cao et al., 2020a; Ju 64 et al., 2020; Kreer et al., 2020; Liu et al., 2020; Nielsen et al., 2020; Robbiani et al., 2020; Seydoux et al., 2020; Wan et al., 2020a; Zost et al., 2020) . 66

Many known SARS-CoV-2 nAbs bind the receptor-binding domain (RBD) and block its interaction with its cellular receptor, Angiotensin converting enzyme 2 (ACE2), thus preventing viral attachment and cell fusion (Hoffmann 68 et al., 2020; Yan et al., 2020) . However, some RBD-binding mAbs prevent infection without interfering with the RBD-ACE2 interaction (Pinto et al., 2020; Tai et al., 2020; Wang et al., 2020a) . Other mAbs neutralize without 70 binding to RBD (Chi et al., 2020; Liu et al., 2020) , and their mechanisms of action are not fully understood (Gavor Monoclonal antibodies (mAbs) have been isolated and characterized previously by us and others (Cao et al., 112 2020a; Ju et al., 2020; Kreer et al., 2020; Nielsen et al., 2020; Robbiani et al., 2020; Seydoux et al., 2020) . We isolated individual S-2P+ and RBD+ IgG+ B cells (Supplemental Table 1 ) from all four subjects. The percentage 114 of S-2P+ cells in the four patients ranged from 0.23%-1.84% of IgG+ B cells and out of which 5-12.7% targeted the RBD. In agreement with the above-discussed serum antibody observations, the frequency of S-2P+ IgG+ B 116 cells in PCV1 was 3-8-fold higher than those in the other patients while no major differences were observed in the frequencies of RBD+ IgG+ B cells among the four patients. As expected, the frequency of S-2P+ cells in a 118 healthy (pre-pandemic) control individual was lower than those found in the four patients (0.104% and 0.128%), as were the frequency of RBD+ IgG+ B cells (first sort: 0.015% and second sort: 0.019%). A total of 341 HC, 353 120

LCs and 303 LCs were successfully sequenced from the four SARS-CoV-2-positive donors (Supplemental Table   1 , Supplemental Figure 1 ), from which 228 paired HC/LCs were generated, and 198 antibodies were successfully 122 produced and characterized. 59 mAbs were generated from the healthy individual. As discussed above we performed an initial characterization of the 45 mAbs from CV1 (Seydoux et al., 2020) , here we performed a more 124

in-depth characterization of these mAbs.

In agreement with previous reports, the antibodies isolated from the patients utilized diverse V regions (Cao et 126 al., 2020a; Nielsen et al., 2020; Robbiani et al., 2020; Seydoux et al., 2020) ( Table 1 ), their V genes sequenced, and their relative frequencies were again compared to those found in total B cells from healthy donors (Figure 2 G) . Although IGHV3-30 was 144 present in higher frequency in B cells sorted with S-2P from CN than in the total B cell population, the difference was not as large as in the infected patients. Similarly, no differences were observed for the other IGHV, 18 and no instances of the IGKV or IGLV genes that were predominant in the anti-S response after infection appeared in CN. Thus, it appears that the anti-spike B cell response that predominates at 3-7 weeks post 148 infection is dissimilar from the naïve B cells that preferentially bind to S-2P.

The length distribution for the CDRH3 and CDRL3 of antibodies isolated after infection were comparable to those 150 present in the pre-infection, healthy B cell repertoires (Figure 2H, I) . Interestingly, the IGHVs and IGLVs sequences derived from samples collected at 6 (CV3) and 7 (PCV1) weeks after infection had significantly more 152 amino acid mutations than those derived from samples collected at 3 (CV1) or 3.5 (CV2) weeks after symptomdevelopment ( Figure 2J , K). These observations are suggestive of a continuous B cell evolution during SARS-CoV-154 2 infection as others recently reported (Gaebler et al., 2021) .

The binding specificities of the 198 monoclonal antibodies (mAbs) to S-subdomains were determined using recombinant proteins including, S1, RBD, N-terminal domain (NTD) and S2 ectodomain (ECD) subunits ( Figure  158 3A, Supplemental Figure 3 A, B) . Only a small percentage of mAbs bound RBD, irrespective of the time of B cell isolation following the development of symptoms. However, the relative proportion of anti-S2 antibodies was 160 higher in samples collected at 3 and 3.5 weeks (51% in CV1 and 70% in CV2, respectively) than in samples collected 6-and 7-weeks post symptom onset (35% in CV3 and 27% in PCV1, respectively). PCV1 had a high 162

proportion (32%) of antibodies whose epitopes could not be mapped to S1 or S2, while such antibodies were rarer in the other three patients examined here (15% in CV1, 7% in CV2 and 0% in CV3). 164 We also determined the abilities of these antibodies to recognize SARS-CoV-1, MERS, the two endemic human beta coronaviruses, OC43 and HKU1, and the two endemic human alpha coronaviruses, NL63 and 229E ( Figure  166 3B). 81 mAbs (41%) displayed SARS-CoV-1 reactivity (to varying degrees), approximately half of which recognized the SARS-CoV-1 RBD. In contrast, only 4 mAbs (2.3%) displayed cross reactivity towards MERS (and 168 none to the MERS RBD), 13 bound OC43 (7.1%), 12 bound HKU1 (6.6%), 2 bound NL63 (1.1%) and only 1 bound 229E (0.56%). There was no association between the number of amino acid mutations in the antibody V regions 170

and cross-reactivity with divergent HCoVs (Supplemental Figure 3C -I).

Only 14 mAbs (7%) neutralized SARS-CoV-2 ( Figure 4A) , with IC50s ranging from 0.007 g/ml to 15.1 g/ml (although, as we discuss below, we were unable to assign an IC50 to CV2-74) ( Figure 4B , Supplemental Table 2 , 174

Supplemental Figure 4) . 11 of 14 neutralizing mAbs bound RBD, in agreement with our (Seydoux et al., 2020) and other reports that RBD is the major target of anti-SARS-CoV-2 nAbs (Barnes et al., 2020; Cao et al., 2020a; 176 Ju et al., 2020; Liu et al., 2020; Rogers et al., 2020a) . Three of the nAbs, CV1-1 (from patient CV1), CV2-74 (from patient CV2) and CV3-25 (from patient CV3) bound epitopes outside the RBD. CV1-1 binds the S1 NTD, binds the S2 subunit, CV2-74 bound neither the recombinant S1 or S2 proteins used here, and we were unable to define its specificity ( Figure 5B and Supplemental Figure 4A , B).

The three most potent nAbs, all anti-RBD, were CV1-30 (IC50=0.044 g/ml) (Seydoux et al., 2020) , CV3-1 (IC50=0.007 g/ml) and PCV19 (IC50=0.072 g/ml). The anti-NTD mAb (CV1-1) had lower neutralizing potency 182 (IC50=8.2 g/ml) and as we previously reported (Seydoux et al., 2020) , its maximum level of neutralization was lower than 100% (Supplemental Figure 4C ), similar to other anti-NTD mAbs . CV1-1 displayed 184 decreased binding against more stable SARS-2-CoV S engineered proteins (S-6P), as shown by lower overall unit response and faster off-rate by BLI and does not bind like other published NTD-targeting antibodies by negative 186 stain EM (Supplemental Figure 5A ,B) . The IC50 of anti-S2 mAb, CV3-25, was 0.34 g/ml, which is comparable to most anti-RBD nAbs with the exception of CV1-30, CV3-1 and PCV19. Table 2 ). Interestingly, while the IC50s of CV2-71, CV2-75 and CV3-25 against SARS-CoV-1 and 192 SARS-CoV-2 were not significantly different, CV3-17 neutralized SARS-CoV-1 more potently than SARS-CoV-2 (Supplemental Figure 4E) . Furthermore, the two most potent anti-SARS-CoV-2 mAbs (CV1-30 and CV3-1) did 194 not neutralize SARS-CoV-1.

CV1-30 has only 2 non-silent somatic mutations (both in VH) that we previously reported are important for potent neutralization of SARS-CoV-2 (Hurlburt et al., 2020) . To examine if this is a general phenomenon among 198

anti-RBD SARS-CoV-2 nAbs, we generated the inferred-germline (iGL) versions of 6 anti-RBD Abs (CV2-20, CV2-71, CV2-75, CV3-1, CV3-7, and CV3-43) and measured their neutralizing potencies ( Figure 4D and Supplemental 200 Fig 6A) . Three of six anti-RBD iGL-nAbs, CV2-20 (3 amino acid mutations), CV2-75 (3 amino acid mutations) and

CV3-43 (9 amino acid mutations) were non-neutralizing. However, no differences in neutralizing potency 202 Neutralization by non-RBD binding Abs 234

As mentioned above CV2-74 binds to an undefined epitope on S, that is present on S-2P but absent or not properly presented on the recombinant S1 or S2 proteins used here ( Figure 5B ). We identified several mAbs 236

sharing this binding property (especially in PCV1) and the majority (75%) of these mAbs did compete the binding of CV2-74 to S-2P ( Figure 5D ,E). The fact that among these mAbs only CV2-74 displayed neutralizing activity 238

suggests that either the other mAbs bind distinct epitopes on S-2P and indirectly affect the binding of CV2-74 to S-2P or that CV2-74 binds a unique but overlapping epitope. It is note-worthy that CV2-74 displays an unusual 240 neutralization curve, where the mAb neutralizes only 50% of the virus across a thousand-fold concentration range ( Figure 5A ). For that reason, we did not assign an IC50 value to Out of the 14 anti-NTD mAbs we identified, 8 (57%) competed the binding of CV1-1 to S-2P ( Figure 5D ,E) and yet, CV-1-1 was the only neutralizing anti-NTD mAb ( Figure 4B ). Interestingly, CV1-1 displayed decreased binding 244

to more stable SARS-CoV-2 engineered soluble proteins (Supplemental Figure 5) . While BLI revealed binding of CV1-1 to recombinant NTD, the on-rate and maximal binding signal was lower than to the entire S1 domain, 246

suggesting that secondary (or quaternary) contacts are important ( Figure 5B ). Indeed, negative-stain EM analysis indicates that it recognizes NTD differently than other anti-NTD mAbs (such as COVA1-22 (Brouwer et 248 al., 2020) , with a footprint that might also include an area just above the S1/S2 cleavage site (Supplemental Figure 5B ).

Out of 87 anti-S2 mAbs, CV3-25 was the only one capable of neutralizing SARS-CoV-2 and SARS-CoV-1 ( Figure   4B and C) and of binding the S proteins of the OC43 and HKU1 betacoronaviruses ( Figure 5C , Supplemental 252 Table 2 ). As none of the other 86 anti-S2 mAbs competed the binding of CV3-25 to S2-P ( Figure 5D ,E) we expect that CV3-25 binds a unique epitope on the S2 subunit which is present not only on SARS-CoV-1 but also on the 254 other coronaviruses tested here.

To assess whether nAbs with different epitope specificities offer the same level of protection in vivo, we compared the protective abilities of CV1-1, CV1-30 and CV2-75 in the K18-hACE2 mouse model (Winkler et al., 258 2020). As discussed above, CV1-1 binds NTD and has an IC50 of 8.2 g/ml, while CV1-30 and CV2-75 bind the RBD and have IC50s of 0.044 and 1.7 g/ml, respectively. Thus, CV1-1 and CV2-75 have similar neutralizing 260 potentials but recognize different regions of the viral spike.

SARS-CoV-2 ( Figure 6A ). Two days post challenge, half of the animals were euthanized to assess viral loads in 264 the lung, and the remaining 5 animals were monitored for survival for up to 14 days. Two days post-challenge, mice receiving AMMO1, CV1-1 and CV2-75 had high levels (1 x 10 8 PFU) of infectious virus and viral RNA in the 266 lung ( Figure 6B and C). 3 of 5 remaining animals in the CV1-1-and CV2-75 groups did not survive beyond 6 days post-challenge ( Figure 6C,D) . In contrast, CV1-30 significantly limited viral replication in the lungs at 2 268 days post-challenge ( Figure 6B ,C) and all remaining mice survived ( Figure 6D ).

Collectively these data indicate that neutralizing potency rather than epitope specificity is the most important 270 factor in defining the prophylactic efficacy of anti-SARS-CoV-2 antibodies. Consistent with this, CV3-25 also showed partial protection in a K18-hACE2 animal model ( Brazil (P.1) that harbor specific mutations in their S proteins that may be associated with increased transmissibility (Davies et al., 2020; Faria et al., 2021; Rambaut et al., 2020; Sabino et al., 2021; Tegally et al., 278 2020; Volz et al., 2021) . The B.1.351 lineage appears to be more resistant to convalescent sera and mAbs (Edara et al., 2021; Liu et al., 2021; Stamatatos et al., 2021; Wang et al., 2020b; Wibmer et al., 2021; Wu et al., 2021) . deletion 242-244, and S2 A701V, but at lower frequencies.

We recently reported that these mutations abrogated the neutralizing activity of CV1-1 and reduced the 284 neutralizing activities of the two most potent nAbs CV1-30 and CV3-1 (Stamatatos et al., 2021 them, only CV3-7 was derived from VH3-30. In fact, the 11 anti-RBD nAbs were derived from distinct B cell clones, that cross-competed for binding, and 4 prevented the RBD-ACE2 interaction. These observations, 294

combined with the fact that anti-RBD nAbs can neutralize the virus with no, or minimal somatic mutation, may explain why potent anti-SARS-CoV-2 neutralizing antibody responses are rapidly generated within a few weeks 296 of infection, or shortly following 2 immunizations with vaccines that express the viral spike (Jackson et al., 2020; Walsh et al., 2020) . The observation that 7 of 11 anti-RBD nAbs do not prevent the RBD-ACE2 interaction, 298

indicates different mechanisms of neutralization by anti-RBD antibodies. The former nAbs may prevent RBDheparin interactions (Clausen et al., 2020) , stabilize the RBDs in their 'up' conformation and thus prematurely 300 activate the fusion machinery (Koenig et al., 2021; Wrapp et al., 2020a) , or limit the conformational changes, and particularly the RBD movement, that are required for cell fusion, allowing them to neutralize without 302

directly blocking ACE2 binding.

The two most potent anti-SARS-CoV-2 nAbs, CV1-30 and CV3-1, which both bind SARS-CoV-2 RBD but not SARS-304

CoV-1 RBD, did not neutralize SARS-CoV-1 while CV2-75 and CV3-17, which bind not only SARS-CoV-2 RBD but also SARS-CoV-1 RBD and displayed weaker anti-SARS-CoV-2 neutralizing activities, were able to efficiently 306 neutralize SARS-CoV-1. A comparison of the CV2-75-RBD and CV1-30-RBD (Hurlburt et al., 2020) structures reveal that CV2-75 binds an area of SARS-CoV-2 RBD with higher sequence homology with SARS-CoV-1 RBD. In 308

contrast, CV1-30 binds directly to the receptor binding motif which only has 50% sequence homology among SARS-CoV-1 and SARS-CoV-2 (Finkelstein et al., 2021; Hurlburt et al., 2020; Wan et al., 2020b) .

The mechanisms of neutralization of the three non-RBD binding nAbs characterized here (CV1-1, CV2-74 and CV3-25) are presently unknown. As CV1-1, CV2-74 and CV3-25 do not interfere with the binding of ACE-2 to S-312 2P, we anticipate that they mediate neutralization by interfering with a step in the fusion process that follows attachment. The viral spike undergoes conformational changes, specifically in the S2 region, during virus-cell 314 binding and fusion (Cai et al., 2020; Gavor et al., 2020; Walls et al., 2020) . Potentially, these three mAbs may prevent these conformational changes from occurring, either by locking the spike in an intermediate 316 conformation, preventing cleavage or by stabilizing its pre-fusion conformation.

The fact that the binding of CV1-1 to S-2P was competed by the other anti-NTD mAbs (14 total), all of which 318

were non-neutralizing, suggests that CV1-1 recognizes the NTD in a distinct manner from the non-neutralizing anti-NTD mAbs. Similarly, the binding of CV2-74 to S-2P was competed by the other non-neutralizing non-S1/S2 320 mAbs (23 total), which strongly suggests that these mAbs all recognize the same immunogenic region, but that CV2-74 recognizes it in a unique manner. In contrast, none of the anti-S2 mAbs isolated here (65 total) competed 322 the binding of CV3-25 to S-2P. These observations and the fact that CV3-25 potently neutralizes both SARS-CoV-1 (IC50 2.1 g/mL) and SARS-CoV-2 (IC50 0.34 g/mL) and the B.1.351 mutant strain and binds the S proteins of 324 HKU1 and OC43, strongly suggests that it recognizes a conserved epitope among diverse coronaviruses. As only two other anti-S2 antibodies that neutralize both SARS-CoV-1 and SARS-CoV-2, but with weaker neutralizing 326 activities than CV3-25, were reported so far Wang et al., 2020b) we expect the epitope of CV3-25 to be less immunogenic than those recognize by non-neutralizing anti-S2 antibodies.

We propose that because of its cross-neutralizing activity, its ability to neutralize the B.1.351 and because it binds the OC43 and HKU1 spikes, CV3-25 represents an antibody type that a pan-coronavirus vaccine should 330 elicit. We expect that the protective potentials of such antibodies will improve through the accumulation of amino acid mutations in their VH and VLs through sequential immunizations. As a first step, the epitope of CV3-332 25 must be identified, and immunogens should be designed expressing it in the most immunogenic form.

In summary, our study indicates that neutralization of SARS-CoV-2 and SARS-CoV-1 does not necessitate the 334 expansion of B cell lineages that express particular VH/VL pairings and that even the unmutated forms of some antibodies can potently neutralize SARS-CoV-2 and SARS-CoV-1. As these viruses are capable of tolerating 336 mutations in distinct regions of its viral spike, they will be able to escape the neutralizing activities of most nAbs. Ventures outside the submitted work, and non-financial support from Cepheid and Ellume. Serum from four patients infected with SARS-CoV-2 (Supplemental Table 1 ) was assessed for binding and 376 neutralization capacity. (A-F) Serum antibody binding titers to S-2P and the RBD were measured by ELISA in the four participants using the indicated isotype specific secondary antibodies. CV1=Patient 1, CV2=Patient 2, 378

CV3=Patient 3, PCV1=Patient 4. Negative sera were collected prior to the SARS-CoV-2 pandemic. (G) Serum from the indicated donors were evaluated for their capacity to neutralize SARS-CoV-2 pseudovirus. (H) ID50 of serum 380

neutralization. Values are shown for two independent replicates. Statistics evaluated as one-way ANOVA with

Tukey's multiple comparison test. Significance indicated for select comparisons. *p<0.05, **p<0.01, 382 ***p<0.001, ****p<0.0001.

Sequences for the 198 mAbs elicited from the SARS-CoV-2 infected patients were compared for VH and VL gene ANOVA with Tukey's multiple comparison test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

The percentage of mAbs from each donor specific for the SARS-CoV-2 spike subdomains and their crossreactivity was determined by BLI. (A) mAbs were grouped into the antibodies that bound RBD in the S1 subunit 400

(S1: RBD, blue), mAbs that bound S1 outside of RBD (S1: non-RBD, teal), mAbs that bound the S2 ECD (S2 ECD, yellow) or those that bound S2P but did not bind either S1 or S2 (S2P: Non-S1/Non-S2. (B) The percentage of Significant differences were determined using two-way ANOVA with Tukey's multiple comparison test, *p<0.05, 404 **p<0.01, ***p<0.001, ****p<0.0001. Additional BLI data and comparison to number of amino acid mutations indicates what is considered true competition, dots below the line are considered competitive. For CV1-1, S1

CV3-25 all S2-binding mAbs in all four sorts were tested. Median of plot is indicated as solid line with quartiles indicated as dashed lines. (E) Pie charts show percentage of mAbs in each set that effectively compete with each 436 tested mAb. mAbs that competed are indicated in the purple section while non-competitive mAbs are in blue. Versions of Nabs reverted to their germline forms were created and tested for neutralization potential and 500 ability to bind their epitope. (A) Neutralization curves for mature (blue) and inferred germline (teal) mAbs. (B)

The binding of the mature (solid lines) and inferred germline versions of nAbs (dotted lines) to the indicated 502 antigens was measured by BLI. Binding to SARS-CoV-2 S2P (blue) was compared. For CV3-25, binding to SARS- neutralizing mAbs isolated and their binding epitopes, neutralization IC50, the VH and VL genes they are derived from, the number of mutations in these genes, the length of their CDRH3 and CDRL3, and whether 542 they bind to SARS-CoV-2, SARS-CoV-1 and the other endemic human coronaviruses. 

IGKV3-20*01 2 0 12 9

IGLV3-21*02 1 2 19 13 

Non-S1/S2 N/Aa

IGLV3-21*02 2 1 15 11 

a. An IC50 was not able to be assigned to CV2-74, as discussed in the text. b. These mAbs did not neutralize SARS-CoV-1 546 Supplemental Data Table 3 . Data collection and refinement statistics for crystal structure. Related to Figure 4 and Supplemental Figure 5 . 

Blood and peripheral blood mononuclear cells (PBMCs) were isolated from COVID19+ patients using protocols approved by Institutional Review Boards at Fred Hutch Cancer Research Center, University of Washington and 556

Seattle Children's Research Institute.

Peripheral blood mononuclear cells (PBMCs) and serum from pre-pandemic controls were blindly selected at 558 random from the study "Establishing Immunologic Assays for Determining HIV-1 Prevention and Control", with no considerations made for age, or sex, participants were recruited at the Seattle Vaccine Trials Unit (Seattle, 560

Washington, USA). Informed consent was obtained from all participants and the University of Washington and/or Fred Hutchinson Cancer Research Center and CHUM Institutional Review Boards approved the entire 562 study and procedures. were a kind gift from Dr. Jason McLellan (Pallesen et al., 2017; Wrapp et al., 2020b) .

Proteins were produced as described in (Seydoux et al., 2020) . Briefly, 1L of 293 EBNA cells at 1 x 10 6 cells/mL 570 were transfected with 500 mg of pαH-SARS-CoV-2 S2P, pαH-SARS-CoV S2P, pαH-SARS-CoV-2 RBD-Fc, pαH-SARS-CoV RBD-Fc, pαH-MERS S2P, pαH-MERS RBD-Fc using 2 mg of polyethyleneimine (Polyscience, Cat# 24765). 572

After 6 days of growth, supernatants were harvested and filtered through a 0.22 mM filter. S2P supernatants were passed over a HisTrap FF affinity column (GE Healthcare, Cat# 17-5255-01) and further purified using a 2 574 mL StrepTactin sepharose column (IBA Lifesciences Cat# 2-1201-002) and a Strep-Tactin Purification Buffer Set (IBA Lifesciences Cat#2-1002-001). The S-2P variants were further purified using a Superose 6 10/300 GL column.

RBD proteins were purified using protein A agarose resin (Goldbio CAT# P-400), followed by on-column cleavage with HRV3C protease (made in-house) to release the RBD from the Fc domain. The RBD containing flow through 578 was further purified by SEC using a HiLoad 16/600 Superdex 200 pg column (GE healthcare). Proteins were flash frozen and stored at -80°C until use. 580

HCoV-OC43, HCoV-HKU1, HCoV-NL63 and HCoV-229E S1+S2 ECTs 

B cell sorting was performed as described in (Seydoux et al., 2020) . Briefly, fluorescent probes were made from SARS-CoV-2 S-2P and RBD. S-2P and RBD were biotinylated protein at a theoretical 1:1 ration using the Easylink 602 NHS-biotin kit (Thermo Fisher Scientific) according to manufacturer's instructions. Excess biotin was removed via size exclusion chromatography using an Enrich SEC 650 10 x 300 mm column (Bio-Rad). The S-2P probes were 604 made at a ratio of 2 moles of trimer to 1 mole streptavidin, one labeled with phycoerythrin (PE) (Invitrogen), and one with brilliant violent (BV) 711 (Biolegend), both probes were used in order to increase the specificity of 606 detection and reduce identification of non-specific B cells. The RBD probe was prepared at a molar ratio of 4 moles of protein to 1 mole of Alexa Fluor 647-labeled streptavidin (Invitrogen). PBMCs from the five participants 608

were thawed and stained for SARS-CoV-2-specific IgG+ memory B cells. First, cells were stained with the three FBS/RPMI containing 7AAD. Cells were sorted on a FACS Aria II (BD Biosciences) by gating on singlets, 614 lymphocytes, live, CD3-, CD14-, CD4-, CD19+, IgD-, IgG+, S-2P-PE+ and S-2P-BV711+. for 30 minutes at 37C followed by 5 minutes at 95C. Purified samples were Sanger sequenced (Genewiz, Seattle, WA). IMGT/V-QUEST was used to assign V, D, J gene identity, and CDRL3 length to the sequences (Brochet et 630 al., 2008) . Sequences were included in analysis if V and J gene identity could be assigned and the CDR3 was inframe.

For sorts CV1, CV2 and CN, paired VH and VL sequences were optimized for human expression using the 634

Integrated DNA Technologies (IDT) codon optimization tool. Sequences were ordered as eBlocks (IDT) and

cloned into full-length pTT3 derived IgL and IgK expression vectors (Snijder et al., 2018) or subcloned into the 636 pT4-341 HC vector (Mouquet et al., 2010) using InFusion cloning (InFusion HD Cloning Kit, Cat#: 639649).

Sorts CV3 and PCV1 were directly cloned using Gibson Assembly. Second round PCR primers were adapted to 638 include homology regions that corresponding to the leader sequence and constant regions on the expression vector. Cycling parameters and post-PCR clean-up remained the same. The backbone expression plasmid was 640 amplified using primers specific for the leader sequence and constant regions in 25 l reactions containing 2x

Platinum SuperFi II DNA polymerase (Invitrogen, Cat# 12358010), 100 nM 5' and 3' primers, 10 ng template 642 DNA, and 21.5 l Nuclease-free water. The reaction was cycled at 98C for 30 seconds, 30 cycles of 98C for 10 seconds, 60C for 10 seconds, and 72C for 3 minutes and 30 seconds, followed by 72C for 5 minutes. The reaction was treated with 20 units of dpnI (NEB, Cat#:R0176S) and incubated at 37C for 60 minutes. The reaction was purified using a PCR clean-up kit according to manufacturer's directions (NEB, Cat#: T1030S) or using ExoSAP-646

IT. The cloning reaction was performed using 100 ng of second round PCR product, 25 ng of backbone, 1 l 5x

InFusion HD Enzyme and nuclease-free water for a total reaction volume of 3 ul and incubated at 50C for 15 648 minutes. We recently reported an initial characterization of the anti-S antibody responses generated by CV1 (Seydoux et al., 2020) . Fred Hutch Genomics core performed the sequencing and the Fred Hutch Bioinformatics core performed 672

processing of the raw sequence data.

All BLI experiments were performed on an Octet Red instrument at 30°C with shaking at 500-1000 rpm. All loading steps were 300s, followed by a 60s baseline in KB buffer (1X PBS, 0.01% Tween 20, 001% BSA, and 676 0.005% NaN3, pH 7.4), and then a 300s association phase and a 300s dissociation phase in KB. For the binding BLI experiments, mAbs were loaded at a concentration of 20 mg/mL in PBS onto Anti-Human IgG Fc capture 678 (AHC) biosensors (Fortebio). After baseline, probes were dipped in either SARS-CoV2 proteins; SARS-CoV-2 RBD, S-2P, S1, S1 NTD orS2; SARS-CoV proteins; SARS-CoV-RBD or S-2P, or human coronavirus spike proteins; HCoV2-680 OC43, HKU1, NL63 or 229, at a concentration of 2-0.5 mM for the association phase. The binding of mature VRC01 was used as negative control to subtract the baseline binding in all of these experiments.

To measure competition between mAb and RBD for ACE2 binding, ACE2-Fc was biotinylated with EZ-Link NHS-684 PEG4-Biotin (Thermo Fisher Scientific) at a molar ratio of 1:2. Biotinylated protein was purified using a Zeba spin desalting column (Thermo Fisher Scientific). ACE2-Fc was then diluted to 20-83.3 mg/mL in PBS and loaded onto 686 streptavidin biosensors (Forte Bio). Following the baseline phase, association was recorded by dipping into a 0.5 mM solution of either SARS-CoV-2 RBD or 0.5 mM solution of SARS-CoV-2 RBD plus mAb. The binding of RBD 688 and mAb to uncoated sensors was used as background binding and was subtracted from each sample. The area under the curve (AUC) of competition was compared to the AUC of the RBD-alone condition. Samples that 690

showed reduced binding are considered competition. Some samples appear to show enhanced binding in the presence of ACE2, perhaps because ACE2 binding stabilizes and exposes their binding sites, these antibodies are 692

considered not competitive with ACE2.

To measure competition between individual mAbs for binding to SARS-CoV-2 S-2P and RBD, S-2P and RBD were biotinylated using EZ-Link NHS-PEG4 Biotin at a molar ratio of 1:2/ Biotinylated protein was purified using a Zeba 696 spin desalting column. RBD was loaded onto streptavidin biosensors. For these experiments, following the baseline in KB, the probe was dipped in the first mAb for a first association phase, with this mAb at a saturating 698 concentration of 2 mM. This was followed by a second baseline in KB. The probe was then dipped into the secondary mAb, at a concentration of 0.5 mM for a second association phase, followed by the standard 700 dissociation phase. For a background control, one sample was run with the second mAb identical to the first mAb, to show the residual binding capacity, and this was subtracted from all samples. 702

To calculate the competition percentage, the binding of the secondary antibodies to RBD or S-2P was also assessed. Here, streptavidin probes were loaded with biotinylated S-2P or RBD, probes were then dipped in the control, the binding of mature VRC01 to the RBD or S-2P was assessed and subtracted from all samples. To 706 calculate competition percentage, the area under the curve (AUC) of this binding curve was calculated, along with the AUC of the competition curve. Percent competition was calculated as: AUC binding -AUC competition 708

x 100. Full competition was considered when less than 15% binding capacity remained.

HIV-1 derived viral particles were pseudotyped with full length wild-type SARS CoV-2 S (Crawford et al., 2020; Seydoux et al., 2020) . An equal volume of viral supernatant was added to each well and incubated for 60 min at 37C. Meanwhile 50 720 µl of cDMEM containing 6 µg/ml polybrene was added to each well of 293T-ACE2 cells (2 µg/ml final concentration) and incubated for 30 min. The media was aspirated from 293T-ACE2 cells and 100 µl of the virus-722

antibody mixture was added. The plates were incubated at 37˚C for 72 hours. The supernatant was aspirated, and cells were lysed with 100 µl of Steadyglo luciferase reagent (Promega), and luminescence was read on a 724

Fluoroskan Ascent Fluorimeter. CV1-30 was used as a positive control and AMMO 1 (Snijder et al., 2018) and average RLU between wells containing cells alone, multiplied by 100. The antibody concentration that 730 neutralized 50% of infectivity (IC50) , or serum dilution that neutralized 50% infectivity (ID50)was interpolated from the neutralization curves determined using the log(-inhibitor) versus response-variable slope (four 732 parameters) fit using automatic outlier detection in GraphPad Prism software.

The neutralizing activities of CV1-1 and CV1-30 mAbs were also determined with a slightly different pseudovirus-

8 pmol of biotinylated S-2P with strep tag peptide sequence on C terminus were mixed with 10 pmol of mAb 

The CV2-75 Fab and SARS-CoV-2 RBD complex was obtained my mixing Fab with a 2-fold molar excess of RBD and incubated for 90 min at RT with nutation followed by SEC. The complex was verified by SDS-PAGE analysis.

The complex was concentrated to 19 mg/mL for initial crystal screening by sitting-drop vapor-diffusion in the MCSG Suite (Anatrace) using a NT8 drop setter (Formulatrix). Initial crystal conditions were optimized using the 756

Additive Screen (Hampton Research, HR2-138) Diffracting crystals were obtained in a mother liquor (ML) containing 0.1 M Tris, pH 7.5, 0.1 M Calcium Acetate, 15% (w/v) PEG 3350, and 4mM glutathione. The crystals 758

were cryoprotected by soaking in ML supplemented with 30% (v/v) ethylene glycol. Diffraction data was collected at Advanced Photon Source (APS) SBC 19-ID at a 12.662 keV. The data set was processed using XDS 760 (Kabsch, 2010) to a resolution of 2.80Å. The structure of the complex was solved by molecular replacement using Phaser (McCoy et al., 2007 ) with a search model of SARS-CoV-2 RBD (PDBid: 6xe1) (Hurlburt et al., 2020) 762 and the Fab structure (PDBid: 4fqq) (Mouquet et al., 2012) divided into Fv and Fc portions. Remaining model building was completed using COOT (Emsley and Cowtan, 2004) and refinement was performed in Phenix

SARS-2 CoV 6P S protein was incubated with a three-fold molar excess of CV1-1 Fab for 30 minutes at room 768 temperature. The complex was diluted to 0.03 mg/ml in 1X TBS pH 7.4 and negatively stained with Nano-W on 400 mesh copper grids. For data collection, a Thermo Fisher Tecnai Spirit (120 kV) and an FEI Eagle (4k x4k) CCD 770 camera were used to produce 296 raw micrographs. Leginon (Suloway et al., 2005) was used for automated data collection and resulting micrographs were stored in Appion (Lander et al., 2009) . Particles were picked 772

with DogPicker ) and processed in RELION 3.0 (Scheres, 2012) .

B6.Cg-Tg(K18-ACE2)2Prlmn/J mice were purchased from Jackson Laboratories. All mice used in these experiments were females between 8 -12 weeks of age. icSARS-CoV-2 virus (Xie et al., 2020) was diluted in PBS 776

to a working concentration of 2 x 10 5 pfu/mL. Mice were anesthetized with isoflurane and infected intranasally with icSARS-CoV-2 (50 uL, 1 x 10 4 pfu/ mouse) in a ABSL-3 facility. Mice were monitored daily for weight loss. converted to cDNA using the High-capacity Reverse Transcriptase cDNA Kit (Thermo Fisher Scientific, #4368813).

RNA levels were quantified using the IDT Prime Time Gene Expression Master Mix, and Taqman gene expression 790

Primer/Probe sets (IDT). All qPCR was performed in 384-well plates and run on a QuantStudio5 qPCR system. SARS-CoV-2 viral RNA-dependent RNA polymerase levels were measured as previously described (Vanderheiden 792 et al., 2020) . The following Taqman Primer/Probe sets (Thermo Fisher Scientific) were used in this study: Gapdh (Mm99999915_g1). 794

Sequences were analyzed using Geneious software (Version 8.1.9). Identification and alignments to VH/VL 796 genes, quantification of mutations and CDRH3 length were done using V Quest (Brochet et al., 2008) . Mutations were counted beginning at the 5' end of the V-gene to the 3' end of the 428 FW3. 798

All graphs were completed using GraphPad Prism. For column analysis of multiple independent groups one-800

way ANOVA with Tukey's multiple comparison test or with Dunnett's multiple comparison test was used. For grouped analysis two-way ANOVA with Tukey's or Šídák's multiple comparison test. Correlations were 802 determined using nonparametric spearmen correlation and p values and nonlinear fit R squared values are reported. . *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. 804 806

Serum from four patients infected with SARS-CoV-2 (Supplemental Table 1 IGLV3-25  IGLV3-21  IGLV7-43  IGLV7-46   IGHV4-39  IGHV1-18  IGHV4-59  IGHV1-69  IGHV3-23  IGHV4-61  IGHV1-24  IGHV1-2  IGHV3- The percentage of mAbs from each donor specific for the SARS-CoV-2 spike subdomains and their cross-reactivity was determined by BLI. (A) mAbs were grouped into the antibodies that bound RBD in the S1 subunit (S1: RBD, blue), mAbs that bound S1 outside of RBD (S1: non-RBD, teal), mAbs that bound the S2 ECD (S2 ECD, yellow) or those that bound S2P but did not bind either S1

or S2 (S2P: Non-S1/Non-S2. Full sequencing data for all VH and VL genes isolated from the four SARS-CoV-2 positive patients.

(A-C) Full gene analysis for all paired heavy (A), Kappa (B), and Lambda (C) sequences from all four sorts displayed as above as percentage of total sequences from each sort. Significance calculated using two-way-ANOVA. Statistics between different samples evaluated as two-way-ANOVA with Šídák's multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. IGHV3-23  IGHV3-33  IGHV4-59  IGHV4-39  IGHV1-18  IGHV4-34  IGHV4-61  IGHV1-2  IGHV3-48  IGHV1-69  IGHV5-51  IGHV3-21  IGHV3-7  IGHV1-24 IGHV3-15 IGHV3-11 IGHV1-46 IGHV4-31 IGHV2-5 IGHV3-49 IGHV3-53 IGHV4-4 IGHV1-3 IGHV3-43 IGHV3-64 IGHV3-74 IGHV4-38 IGHV5-10 IGHV3-66 IGHV7-4-1 IGHV1-58 IGHV2-70 IGHV6-1 IGHV3-9 IGHV2-26 IGHV3-18 IGHV7-81 IGHV3-73 IGHV3-20 IGHV3-13 IGHV4-30 IGHV3- -30 IGHV3-23 IGHV3-33 IGHV4-59 IGHV4-39 IGHV1-18 IGHV4-34 IGHV4-61 IGHV1-2 IGHV3-48 IGHV1-69 IGHV5-51 IGHV3-21 IGHV3-7 IGHV1-24 IGHV3-15 IGHV3-11 IGHV1-46 IGHV4-31 IGHV2-5 IGHV3-49 IGHV3-53 IGHV4-4 IGHV1-3 IGHV3-43 IGHV3-64 IGHV3-74 IGHV4-38 IGHV5-10 IGHV3-66 IGHV7-4-1 IGHV1-58 IGHV2-70 IGHV6-1 IGHV3-9 IGHV2-26 IGHV3-18 IGHV7-81 IGHV3-73 IGHV3-20 IGHV3-13 IGHV4-30 IGHV3- IGLV1-51  IGLV2-14  IGLV2-23  IGLV1-40  IGLV3-1 IGLV1-44 IGLV3-25 IGLV3-21 IGLV3-10 IGLV2-11 IGLV4-69 IGLV1-47 IGLV6-57 IGLV2-8 IGLV2-18 IGLV3-9 IGLV3-27 IGLV9-49 IGLV1-36 IGLV3-12 IGLV3-16 IGLV3-19 IGLV3-22 IGLV4-3 IGLV4-60 IGLV5-37 IGLV5-45 IGLV5-52 IGLV7-43 IGLV7-46 IGLV8-61 IGLV10- 

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