Characterization of neutralizing antibodies from a SARS-CoV-2 infected individual

Emilie Seydoux, Leah J Homad, Anna J MacCamy, K Rachael Parks, Nicholas K Hurlburt, Madeleine F Jennewein, Nicholas R Akins, Andrew B Stuart, Yu-Hsin Wan, Junli Feng, Rachael E Nelson, Suruchi Singh, Kristen W Cohen, M Juliana McElrath, Janet A Englund, Helen Y Chu, Marie Pancera, Andrew T McGuire, Leonidas Stamatatos, Emilie Seydoux, Leah J Homad, Anna J MacCamy, K Rachael Parks, Nicholas K Hurlburt, Madeleine F Jennewein, Nicholas R Akins, Andrew B Stuart, Yu-Hsin Wan, Junli Feng, Rachael E Nelson, Suruchi Singh, Kristen W Cohen, M Juliana McElrath, Janet A Englund, Helen Y Chu, Marie Pancera, Andrew T McGuire, Leonidas Stamatatos

Abstract

B cells specific for the SARS-CoV-2 S envelope glycoprotein spike were isolated from a COVID-19-infected subject using a stabilized spike-derived ectodomain (S2P) twenty-one days post-infection. Forty-four S2P-specific monoclonal antibodies were generated, three of which bound to the receptor binding domain (RBD). The antibodies were minimally mutated from germline and were derived from different B cell lineages. Only two antibodies displayed neutralizing activity against SARS-CoV-2 pseudo-virus. The most potent antibody bound the RBD in a manner that prevented binding to the ACE2 receptor, while the other bound outside the RBD. Our study indicates that the majority of antibodies against the viral envelope spike that were generated during the first weeks of COVID-19 infection are non-neutralizing and target epitopes outside the RBD. Antibodies that disrupt the SARS-CoV-2 spike-ACE2 interaction can potently neutralize the virus without undergoing extensive maturation. Such antibodies have potential preventive/therapeutic potential and can serve as templates for vaccine-design.

Keywords: B cells; COVID-19; SARS; SARS-CoV-2; antibody; neutralization; receptor binding domain; spike protein.

Conflict of interest statement

DECLARATION OF INTERESTS The authors declare no competing financial interests. A provisional patent application on the antibodies discussed here has been filed. HYC: Merck, Sanofi-Pasteur, GSK

Figures

Figure 1.. Serum antibody reactivity to the…
Figure 1.. Serum antibody reactivity to the SARS-CoV-2 ecto- and receptor binding domain.
Total antibody binding in serum from a donor with confirmed SARS-CoV-2 infection (COVID-19+), from two donors collected prior to the COVID-19 pandemic with an unknown history of coronavirus infection (pre-pandemic), and from nine donors with confirmed infection by endemic corona viruses (endemic), was tested for binding to the SARS-CoV-2 S2P ectodomain (A) and the RBD (B) by ELISA. Serum from the donor in SARS-CoV-2 infection in A was tested for binding to the SARS-CoV-2 S2P ectodomain (C) and the RBD (D) using isotype-specific secondary antibodies by ELISA. (E) Serum from donor with confirmed SARS-CoV-2 infection, and serum from a pre-pandemic donor were evaluated for their ability to neutralize a SARS-CoV-2 pseudovirus.
Figure 2.. Early B cells response to…
Figure 2.. Early B cells response to SARS-CoV-2 is diverse and largely unmutated.
(A) Class switched (IgM− IgG+) B cells were stained with SARS-CoV-2 S2P labeled with BV710 or PE. (B) SARS-CoV-2 S2P+ IgG+ B cells were further analyzed for binding to Alexafluor647-labeled SARS-CoV-2 RBD. (C, D, E) Individual SARS-CoV-2 S2P+ IgG+ B cells were sorted into separate wells of a 96 well plate and sequenced using RT-PCR. VH (C), VK (D), and VL (E) gene usage of successfully sequenced S2P-specific B cells. CDRH3 (F) and CDRL3 (G) length distributions of successfully sequenced S2P-specific B cells. Number of amino acid substitutions from germline in S2P-specific heavy and light chains (H).
Figure 3.. Sorted mAbs bind to SARS-CoV-2…
Figure 3.. Sorted mAbs bind to SARS-CoV-2 and a subset cross-react with SARS-CoV S.
mAbs isolated from SARS-CoV-2 S2P-specific B cells were tested for binding to SARS-CoV-2 S2P (A) and to SARS-CoV-2 RBD (B) using BLI. (C) mAbs were labeled with phycoerythrin (PE) and used to stain 293 cells transfected with wildtype SARS-Cov-2 S by flow cytometry. Heatmap shows mean fluorescence intensity of PE+ cells at 2.5μg/ml. Titration curves are shown in Fig. S2. (D) mAbs were tested for binding to SARS-CoV S2P by BLI (D). (E) Heatmap shows maximum binding response (average nm shift of the last 5 seconds of association phase) of binding data in A, B and D.
Figure 4.. The RBD-specific mAb CV30 neutralizes…
Figure 4.. The RBD-specific mAb CV30 neutralizes SARS CoV-2 by blocking the ACE2- SARS-CoV-2 S interaction.
(A) CV1 and CV30 were serially diluted and tested for their ability to neutralize SARS-CoV-2 pseudovirus infection of 293T cells stably expressing ACE2. An ACE2-FC fusion and the anti-EBV mAb AMMO1 were included as positive and negative controls. Data are representative of 6 independent experiments (see Table S1 for details). (B) The same mAbs were tested for neutralization of an MLV pseudovirus. (C) Biotinlyated ACE2-Fc was immobilized on streptavidin biosensors and then tested for binding to SARS-CoV-2 RBD in the absence and presence of the indicated mAbs using BLI. (D) ACE2-Fc was immobilized Protein A biosensors and binding to the indicated serial dilutions of SARS-CoV-2 RBD were measured by BLI and used to determine the binding constant (kD). Red lines represent the measured data and black lines indicate the theoretical fit. (E) CV30 was immobilized onto anti-human Fc biosensors and binding to the indicated serial dilutions of SARS-CoV-2 RBD were measured by BLI and used to determine the binding constant (kD). Blue lines represent the measured data and black lines indicate the theoretical fit. Kinetic measurements from D and E are summarized in Table S2.

References

    1. Briney B., Inderbitzin A., Joyce C., and Burton D.R. (2019). Commonality despite exceptional diversity in the baseline human antibody repertoire. Nature.
    1. Brochet X., Lefranc M.P., and Giudicelli V. (2008). IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis. Nucleic acids research 36, W503–508.
    1. Cao Z., Liu L., Du L., Zhang C., Jiang S., Li T., and He Y. (2010). Potent and persistent antibody responses against the receptor-binding domain of SARS-CoV spike protein in recovered patients. Virology journal 7, 299.
    1. Charif D, a. L.J. (2007). SeqinR 1.0–2: a contributed package to the R Project for statistical computing devoted to biological sequences retrieval and analysis (New York: Springer Verlag; ).
    1. Corti D., Voss J., Gamblin S.J., Codoni G., Macagno A., Jarrossay D., Vachieri S.G., Pinna D., Minola A., Vanzetta F., et al. (2011). A neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins. Science 333, 850–856.
    1. Crawford K.H.D., Eguia R., Dingens A.S., Loes A.N., Malone K.D., Wolf C.R., Chu H.Y., Tortorici M.A., Veesler D., Murphy M., et al. (2020). Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 Spike protein for neutralization assays. bioRxiv, 2020.2004.2020.051219.
    1. DeKosky B.J., Lungu O.I., Park D., Johnson E.L., Charab W., Chrysostomou C., Kuroda D., Ellington A.D., Ippolito G.C., Gray J.J., et al. (2016). Large-scale sequence and structural comparisons of human naive and antigen-experienced antibody repertoires. Proceedings of the National Academy of Sciences of the United States of America 113, E2636–2645.
    1. Dong E., Du H., and Gardner L. (2020). An interactive web-based dashboard to track COVID-19 in real time. The Lancet infectious diseases 20, 533–534.
    1. Gui M., Song W., Zhou H., Xu J., Chen S., Xiang Y., and Wang X. (2017). Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein reveal a prerequisite conformational state for receptor binding. Cell research 27, 119–129.
    1. Hoffmann M., Kleine-Weber H., Schroeder S., Kruger N., Herrler T., Erichsen S., Schiergens T.S., Herrler G., Wu N.H., Nitsche A., et al. (2020). SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 181, 271–280.e278.
    1. Hwang W.C., Lin Y., Santelli E., Sui J., Jaroszewski L., Stec B., Farzan M., Marasco W.A., and Liddington R.C. (2006). Structural basis of neutralization by a human anti-severe acute respiratory syndrome spike protein antibody, 80R. The Journal of biological chemistry 281, 34610–34616.
    1. Jiang L., Wang N., Zuo T., Shi X., Poon K.M., Wu Y., Gao F., Li D., Wang R., Guo J., et al. (2014). Potent neutralization of MERS-CoV by human neutralizing monoclonal antibodies to the viral spike glycoprotein. Science translational medicine 6, 234ra259.
    1. Kirchdoerfer R.N., Wang N., Pallesen J., Wrapp D., Turner H.L., Cottrell C.A., Corbett K.S., Graham B.S., McLellan J.S., and Ward A.B. (2018). Stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis. Scientific reports 8, 15701.
    1. Letko M., Marzi A., and Munster V. (2020). Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat Microbiol 5, 562–569.
    1. Li W., Moore M.J., Vasilieva N., Sui J., Wong S.K., Berne M.A., Somasundaran M., Sullivan J.L., Luzuriaga K., Greenough T.C., et al. (2003). Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426, 450–454.
    1. Mouquet H., Scheid J.F., Zoller M.J., Krogsgaard M., Ott R.G., Shukair S., Artyomov M.N., Pietzsch J., Connors M., Pereyra F., et al. (2010). Polyreactivity increases the apparent affinity of anti-HIV antibodies by heteroligation. Nature 467, 591–595.
    1. Ni L., Ye F., Cheng M.-L., Feng Y., Deng Y.-Q., Zhao H., Wei P., Ge J., Gou M., Li X., et al. Detection of SARS-CoV-2-specific humoral and cellular immunity in COVID-19 convalescent individuals. Immunity.
    1. Niu P., Zhang S., Zhou P., Huang B., Deng Y., Qin K., Wang P., Wang W., Wang X., Zhou J., et al. (2018). Ultrapotent Human Neutralizing Antibody Repertoires Against Middle East Respiratory Syndrome Coronavirus From a Recovered Patient. The Journal of infectious diseases 218, 1249–1260.
    1. Okba N.M.A., Muller M.A., Li W., Wang C., GeurtsvanKessel C.H., Corman V.M., Lamers M.M., Sikkema R.S., de Bruin E., Chandler F.D., et al. (2020). Severe Acute Respiratory Syndrome Coronavirus 2-Specific Antibody Responses in Coronavirus Disease 2019 Patients. Emerging infectious diseases 26.
    1. Ou X., Liu Y., Lei X., Li P., Mi D., Ren L., Guo L., Guo R., Chen T., Hu J., et al. (2020). Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nature communications 11, 1620.
    1. Pages H, A. P., Gentleman R, and DebRoy S (2018). Biostrings: efficient manipulation of biological strings.
    1. Pallesen J., Wang N., Corbett K.S., Wrapp D., Kirchdoerfer R.N., Turner H.L., Cottrell C.A., Becker M.M., Wang L., Shi W., et al. (2017). Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen. Proceedings of the National Academy of Sciences of the United States of America 114, E7348–e7357.
    1. Prabakaran P., Gan J., Feng Y., Zhu Z., Choudhry V., Xiao X., Ji X., and Dimitrov D.S. (2006). Structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody. The Journal of biological chemistry 281, 15829–15836.
    1. R Core Team (2017). R: A Language and Environment for Statistical Computing (Vienna, Austria: ).
    1. Rockx B., Corti D., Donaldson E., Sheahan T., Stadler K., Lanzavecchia A., and Baric R. (2008). Structural basis for potent cross-neutralizing human monoclonal antibody protection against lethal human and zoonotic severe acute respiratory syndrome coronavirus challenge. Journal of virology 82, 3220–3235.
    1. Snijder J., Ortego M.S., Weidle C., Stuart A.B., Gray M.D., McElrath M.J., Pancera M., Veesler D., and McGuire A.T. (2018). An Antibody Targeting the Fusion Machinery Neutralizes Dual-Tropic Infection and Defines a Site of Vulnerability on Epstein-Barr Virus. Immunity 48, 799–811 e799.
    1. Song W., Gui M., Wang X., and Xiang Y. (2018). Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2. PLoS pathogens 14, e1007236.
    1. Soto C., Bombardi R.G., Branchizio A., Kose N., Matta P., Sevy A.M., Sinkovits R.S., Gilchuk P., Finn J.A., and Crowe J.E. Jr. (2019). High frequency of shared clonotypes in human B cell receptor repertoires. Nature 566, 398–402.
    1. Sui J., Li W., Murakami A., Tamin A., Matthews L.J., Wong S.K., Moore M.J., Tallarico A.S., Olurinde M., Choe H., et al. (2004). Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S1 protein that blocks receptor association. Proceedings of the National Academy of Sciences of the United States of America 101, 2536–2541.
    1. Tang X.C., Agnihothram S.S., Jiao Y., Stanhope J., Graham R.L., Peterson E.C., Avnir Y., Tallarico A.S., Sheehan J., Zhu Q., et al. (2014). Identification of human neutralizing antibodies against MERS-CoV and their role in virus adaptive evolution. Proceedings of the National Academy of Sciences of the United States of America 111, E2018–2026.
    1. Tiller T., Meffre E., Yurasov S., Tsuiji M., Nussenzweig M.C., and Wardemann H. (2008). Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. Journal of immunological methods 329, 112–124.
    1. Traggiai E., Becker S., Subbarao K., Kolesnikova L., Uematsu Y., Gismondo M.R., Murphy B.R., Rappuoli R., and Lanzavecchia A. (2004). An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nature medicine 10, 871–875.
    1. Vazquez Bernat N., Corcoran M., Hardt U., Kaduk M., Phad G.E., Martin M., and Karlsson Hedestam G.B. (2019). High-Quality Library Preparation for NGS-Based Immunoglobulin Germline Gene Inference and Repertoire Expression Analysis. Frontiers in immunology 10, 660.
    1. Walls A.C., Park Y.J., Tortorici M.A., Wall A., McGuire A.T., and Veesler D. (2020). Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell 181, 281–292.e286.
    1. Walls A.C., Xiong X., Park Y.J., Tortorici M.A., Snijder J., Quispe J., Cameroni E., Gopal R., Dai M., Lanzavecchia A., et al. (2019). Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus Fusion. Cell 176, 1026–1039.e1015.
    1. Wan Y., Shang J., Graham R., Baric R.S., and Li F. (2020). Receptor Recognition by the Novel Coronavirus from Wuhan: an Analysis Based on Decade-Long Structural Studies of SARS Coronavirus. Journal of virology 94.
    1. Wang C., Li W., Drabek D., Okba N.M.A., van Haperen R., Osterhaus A., van Kuppeveld F.J.M., Haagmans B.L., Grosveld F., and Bosch B.J. (2020). A human monoclonal antibody blocking SARS-CoV-2 infection. Nature communications 11, 2251.
    1. World Health Organization (2020). WHO announces COVID-19 outbreak a pandemic.
    1. Wrapp D., Wang N., Corbett K.S., Goldsmith J.A., Hsieh C.L., Abiona O., Graham B.S., and McLellan J.S. (2020). Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 367, 1260–1263.
    1. Ying T., Du L., Ju T.W., Prabakaran P., Lau C.C., Lu L., Liu Q., Wang L., Feng Y., Wang Y., et al. (2014). Exceptionally potent neutralization of Middle East respiratory syndrome coronavirus by human monoclonal antibodies. Journal of virology 88, 7796–7805.
    1. Ying T., Prabakaran P., Du L., Shi W., Feng Y., Wang Y., Wang L., Li W., Jiang S., Dimitrov D.S., et al. (2015). Junctional and allele-specific residues are critical for MERS-CoV neutralization by an exceptionally potent germline-like antibody. Nature communications 6, 8223.
    1. Yuan M., Wu N.C., Zhu X., Lee C.D., So R.T.Y., Lv H., Mok C.K.P., and Wilson I.A. (2020). A highly conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS-CoV. Science.
    1. Yuan Y., Cao D., Zhang Y., Ma J., Qi J., Wang Q., Lu G., Wu Y., Yan J., Shi Y., et al. (2017). Cryo-EM structures of MERS-CoV and SARS-CoV spike glycoproteins reveal the dynamic receptor binding domains. Nature communications 8, 15092.

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