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Isolation and Characterization of Cross-Neutralizing Coronavirus Antibodies from COVID-19+ Subjects
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Madeleine F. Jennewein
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, Anna J. MacCamy
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, Nicholas R. Akins
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, Junli Feng
1
, Leah J. Homad
1
, Nicholas K.
Hurlburt
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, Emily Seydoux
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, Yu-Hsin Wan
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, Andrew B. Stuart
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, Venkata Viswanadh Edara
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, Katharine Floyd
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,
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Abigail Vanderheiden
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, John R. Mascola
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, Nicole Doria-Rose
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, Lingshu Wang
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, Eun Sung Yang
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, Helen Y. Chu
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,
Jonathan L. Torres
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, Gabriel Ozorowski
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, Andrew B. Ward
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, Rachael E. Whaley
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, Kristen W. Cohen
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, Marie
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Pancera
1,3
, M. Juliana McElrath
1,4,5
, Janet A. Englund
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, Andrés Finzi
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, Mehul S. Suthar
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*, Andrew T. McGuire,
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*, Leonidas Stamatatos
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*
#
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1
Fred Hutchinson Cancer Research Center, Vaccines and Infectious Disease Division, Seattle, WA, 98109, USA
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Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA,
92037, USA
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Vaccine Research Center, NAID, NIH, Bethesda, MD 20892, USA
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University of Washington, Department of Medicine, Seattle, WA 98109, USA
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University of Washington, Department of Global Health, Seattle, WA 98109, USA
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Department of Pediatrics, University of Washington and Seattle Children’s Research Institute, Seattle, WA
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98109, USA
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Université de Montréal, Montreal, QC, Canada
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Center for Childhood Infections and Vaccines of Children's Healthcare of Atlanta, Department of Pediatrics,
Emory University School of Medicine, Emory Vaccine Center, Yerkes National Primate Research Center,
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Atlanta, GA 30322, USA
* Correspondence: lstamata@fredhutch.org; amcguire@fredhutch.org; mehul.s.suthar@emory.edu
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#
Lead Contact: lstamata@fredhutch.org
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SUMMARY
SARS-CoV-2 is one of three coronaviruses that have crossed the animal-to-human barrier in the past two
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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
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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
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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
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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
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blueprints for the design of immunogens capable of eliciting cross-neutralizing coronavirus antibodies.
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Keywords: SARS-CoV-2, SARS-CoV-1, S2 subunit, RBD, NTD, neutralization, monoclonal antibodies, B.1.351
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INTRODUCTION
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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
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2019/early 2020, infecting over 120 million people and killing over 2.6 million people by March 2021 (Dong et
al., 2020; Patel et al., 2020). Studies conducted in mice, hamsters and non-human primates strongly suggest
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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.,
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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
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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
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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).
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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
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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).
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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
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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
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binding to RBD (Chi et al., 2020; Liu et al., 2020), and their mechanisms of action are not fully understood (Gavor
et al., 2020).
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Plasma from SARS-CoV-1 and SARS-CoV-2 infected people contains cross-reactive binding antibodies (Ju et al.,
2020; Lv et al., 2020), and a small number of monoclonal antibodies that can neutralize both viruses have been
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isolated from SARS-CoV-2 (Brouwer et al., 2020; Rogers et al., 2020a; Wec et al., 2020) or SARS-CoV-1-infected
subjects (Tortorici et al., 2020). Overall, it appears that most of the cross-reactive antibodies do not cross-
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neutralize and that cross-neutralizing antibodies are infrequently generated during SARS-CoV-2 or SARS-CoV-1
infections. Antibodies capable of neutralizing SARS-CoV-1, SARS-CoV-2 and endemic human coronaviruses, such
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as the betacoronaviruses OC43 and HKU1 or the alphacoronaviruses 229E and NL63 have not yet been
identified.
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Here, we report on the isolation and full characterization of 198 S-specific mAbs from four SARS-CoV-2-infected
individuals. Although a number of these mAbs recognized both SARS-CoV-2 and SARS-CoV-1, we observed
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minimal cross-reactivity with MERS-CoV, betacoronaviruses (OC43 and HKU1) or alphacoronaviruses (NL63 and
229E). A significant fraction of cross-reactive antibodies bound the SARS-CoV-2 S2 domain of the Spike protein.
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14 mAbs neutralized SARS-CoV-2. One neutralizing mAb bound NTD, another bound the S2 subunit, one bound
an unidentified site on S and the remaining 11 bound RBD. Some competed with the RBD-ACE-2 interaction
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while others did not. Although 7 of the SARS-CoV-2 neutralizing mAbs bound SARS-CoV-1, only 4 mAbs
neutralized both viruses. Three targeted the RBD and one targeted the S2 subunit. Using the K18-hACE
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transgenic mouse model, therapeutic treatment with CV-30, a potent RBD-binding antibody, reduced lung viral
loads and protected mice from SARS-CoV-2 infection. In contrast, a weaker anti-RBD neutralizing mAbs, CV2-75,
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and the anti-NTD neutralizing mAb, CV1-1, displayed minimal protective efficacies. These observations strongly
suggest that neutralization potency rather than antibody epitope-specificity regulates the in vivo protective
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potential of anti-SARS-CoV-2 antibodies. Interestingly, the anti-S2 mAb, CV3-25, was the only one that was
unaffected by mutations found in the recently emerged B.1.351 variant. These mAbs, especially CV3-25, can
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serve as blueprints for the development of immunogens to elicit protective neutralizing antibody responses
against multiple coronaviruses.
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RESULTS
Serum antibody titers and neutralizing activities against SARS-CoV-2
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Peripheral blood mononuclear cells (PBMCs) and serum or plasma were collected from four SARS-CoV-2-
infected adults (CV1 – previously discussed in Seydoux et al. 2020, CV2, CV3 and PCV1) at 3, 3.5, 6 and 7 weeks
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after the onset of symptoms, respectively (Supplemental Table 1). Sera from PCV1 had the highest anti-
stabilized spike (S-2P) IgG and IgM titers, while the anti-S-2P IgA titers were higher in CV1 (Figure 1 A-C). In
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contrast, to the higher anti-S-2P IgG titers in the PCV1 sera, all four sera displayed similar anti-receptor binding
domain (RBD) IgG titers (Figure 1 D-F). PCV1 and CV1 had higher levels of anti-RBD IgA than the other two donors
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and CV1 showed slightly lower anti-RBD IgM than the three other sera.
While all sera neutralized SARS-CoV-2 (Figure 1G), serum PCV1 was significantly more potent (Figure 1H). The
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serum neutralizing differences do track with timepoint in infection, with the samples collected at later
timepoints show greater potency, potentially indicating maturation of the humoral response. Thus, though all
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four patients had similar anti-RBD binding antibody titers, PCV1 developed higher anti-S-2P binding antibody
titers and higher neutralizing titers than the other three patients examined here.
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Specific VH and VL genes give rise to anti-S antibodies during SARS-CoV-2 infection
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Monoclonal antibodies (mAbs) have been isolated and characterized previously by us and others (Cao et al.,
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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
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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
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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
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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
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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
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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
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in-depth characterization of these mAbs.
In agreement with previous reports, the antibodies isolated from the patients utilized diverse V regions (Cao et
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al., 2020a; Nielsen et al., 2020; Robbiani et al., 2020; Seydoux et al., 2020) (Figure 2A-C, Supplemental Figure
1). Similarly, the S-specific mAbs isolated from the healthy donor originate from diverse V regions. To determine
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whether anti-S-2P+ B cells that express certain VH and VL genes preferentially expand during infection, we
compared the relative frequencies of each VH and VL sequence to those present in healthy individuals. For this,
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we performed a 10x-based sequence analysis of total circulating B cells (i.e., not S-2P specific) from 5 SARS-CoV-
2-unexposed adults (Figure 2D-F, Supplemental Figure 2). Significantly higher frequencies of S-2P+ IGHV3-30
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and IGHV1-18 antibody sequences were observed in the patients as compared to the relative frequencies of
these two genes present in healthy adults (Figure 2D). Interestingly, lower frequencies of S-2P+ IGHV3-33 usage
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was observed in the patients than in healthy donors. Differences were also observed in kappa (Figure 2E) and
lambda (Figure 2F) gene usage between patients and healthy donors. Specifically, IGKV3-15, IGKV1-33/1D-33
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and IGKV1-17 were significantly elevated in patients as compared to healthy donors while the expression of
IGKV1-39/1D-39 was reduced. IGLV1-51 was significantly elevated in the patients as compared to healthy
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donors, as was IGLV2-23, though this appears to be driven by a greatly elevated usage in patient CV3.
The above observations suggest that naïve B cell clones expressing the above IGHV, IGKV or IGLV genes
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preferentially recognize the viral S protein at the initial stages of infection. To address this point IgD+ IgM+ S-
2P+ and RBD+ B cells were isolated from a healthy donor, CN1 (following two independent B cell sorting
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