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The landscape of antibody binding in SARS-CoV-2 infection

Anna S. Heffron, Sean J. McIlwain, Maya F. Amjadi, David A. Baker, Saniya Khullar, Tammy Armbrust, Peter J. Halfmann, Yoshihiro Kawaoka, Ajay K. Sethi, Ann C. Palmenberg, Miriam A. Shelef, David H. O’Connor, Irene M. Ong


The search for potential antibody-based diagnostics, vaccines, and therapeutics for pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has focused almost exclusively on the spike (S) and nucleocapsid (N) proteins. Coronavirus membrane (M), ORF3a, and ORF8 proteins are humoral immunogens in other coronaviruses (CoVs) but remain largely uninvestigated for SARS-CoV-2. Here, we use ultradense peptide microarray mapping to show that SARS-CoV-2 infection induces robust antibody responses to epitopes throughout the SARS-CoV-2 proteome, particularly in M, in which 1 epitope achieved excellent diagnostic accuracy. We map 79 B cell epitopes throughout the SARS-CoV-2 proteome and demonstrate that antibodies that develop in response to SARS-CoV-2 infection bind homologous peptide sequences in the 6 other known human CoVs. We also confirm reactivity against 4 of our top-ranking epitopes by enzyme-linked immunosorbent assay (ELISA). Illness severity correlated with increased reactivity to 9 SARS-CoV-2 epitopes in S, M, N, and ORF3a in our population. Our results demonstrate previously unknown, highly reactive B cell epitopes throughout the full proteome of SARS-CoV-2 and other CoV proteins.


Antibodies correlate with protection from coronaviruses (CoVs) including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1–8], severe acute respiratory syndrome coronavirus (SARS-CoV) [8–12], and Middle Eastern respiratory syndrome coronavirus (MERS-CoV) [8,13–16]. All CoVs encode 4 main structural proteins, spike (S), envelope (E), membrane (M), and nucleocapsid (N), as well as multiple nonstructural proteins and accessory proteins [17]. In SARS-CoV-2, anti-S and anti-N antibodies have received the most attention to date [1–8], including in serology-based diagnostic tests [1–5] and vaccine candidates [6–8]. The immunogenicity of S-based vaccines is variable [18,19], so better representation of the breadth of antibody reactivity in vaccines, therapeutics, and diagnostics will be important as the pandemic continues especially as new variants emerge. Prior reports observed that not all individuals infected with SARS-CoV-2 produce detectable antibodies against S or N [1–5], indicating a need for expanded antibody-based options.


Peptide microarray design and synthesis:

Viral protein sequences were selected and submitted to Nimble Therapeutics (Madison, Wisconsin, USA) for development into a peptide microarray [73]. Sequences represented include proteomes of all 7 coronaviruses known to infect humans, proteomes of closely related coronaviruses found in bats and pangolins, and spike proteins from other coronaviruses (accession numbers and replicates per peptide shown in S5 Data). A number of proteins were included as controls, including poliovirus, 7 strains of human rhinovirus, and human cytomegalovirus 65kDa phosphoprotein. We chose these controls given that we expect most human adults will have antibody reactivity to at least one of these proteins and proteomes.

Human patients and controls:

The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of the University of Wisconsin-Madison.


In our analysis of antibody binding to the full proteome of SARS-CoV-2, the highest magnitude binding of anti-SARS-CoV-2 antibodies from human sera occurred for an epitope in the N-terminus of M protein, with high specificity and sensitivity. Antibodies produced after infection with SARS-CoV-2 reacted with epitopes throughout the proteomes of other human and nonhuman CoVs, recognizing homologous regions across all CoVs.


The authors are grateful to Mr. Eric Sullivan, Dr. Richard Pinapati, Dr. John Tan, Dr. Daniel Agnew, Dr. Brad Garcia, and Dr. Jigar Patel, all of Nimble Therapeutics for access to their unique peptide array technology. The authors are also grateful to Dr. Christina Newman, Dr. Nathan Sherer, Dr. Thomas Friedrich, Dr. Amelia Haj, Dr. James Gern, Dr. Christine Seroogy, and Gage Moreno for their thoughtful comments and helpful discussions in preparing this manuscript. The authors also thank Dr. Robert Kirchdoerfer for generously providing the chimeric pdb file used for structural representations of SARS-CoV-2 spike protein in this work (see Fig 4 and “Protein structures” in the Methods).

Citation: Heffron AS, McIlwain SJ, Amjadi MF, Baker DA, Khullar S, Armbrust T, et al. (2021) The landscape of antibody binding in SARS-CoV-2 infection. PLoS Biol 19(6): e3001265.

Editor: Galit Alter, Massachusetts General Hospital, UNITED STATES

Received: January 6, 2021; Accepted: May 6, 2021; Published: June 18, 2021.

Copyright: © 2021 Heffron et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All peptide microarray datasets and code used in these analyses can be downloaded from

Funding: I.M.O. acknowledges support by the Clinical and Translational Science Award (CTSA) program (, through the National Institutes of Health National Center for Advancing Translational Sciences (NCATS), grants UL1TR002373 and KL2TR002374.

Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: The authors declare the following competing interests: A.S.H., S.J.M., D.A.B., M.F.A., S.K., M.A.S., D.H.O., and I.M.O are listed as the inventors on a patent filed that is related to findings in this study.

Abbreviations: ACE2, angiotensin converting enzyme 2; β-CoV, betacoronavirus; BH, Benjamini–Hochberg; CCCoVs, “common cold” CoVs; CoV, coronavirus; COVID-19, coronavirus disease 2019; E, envelope; ELISA, enzyme-linked immunosorbent assay; EMR, electronic medical record; IgG, immunoglobulin G; M, membrane; MAS, Maskless Array Synthesizer; MERS-CoV, Middle Eastern respiratory syndrome coronavirus; N, nucleocapsid; NPPOC, 2-(2-nitrophenyl) propyloxycarbonyl; S, spike; PBS, phosphate-buffered saline; RBD, receptor-binding domain; SARS-CoV, severe acute respiratory syndrome coronavirus; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TBS, tris-buffered saline; UW, University of Wisconsin; VOC, variant of concern.

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