Mapping SARS-CoV-2 Antibody Epitopes in COVID-19 Patients with a Multi-Coronavirus Protein Microarray

Microbiol Spectr. 2021 Oct 31;9(2):e0141621. doi: 10.1128/Spectrum.01416-21. Epub 2021 Oct 27.


The rapid worldwide spread of SARS-CoV-2 has accelerated research and development for controlling the COVID-19 pandemic. A multi-coronavirus protein microarray was created containing full-length proteins, overlapping protein fragments of various lengths, and peptide libraries from SARS-CoV-2 and four other human coronaviruses. Sera from confirmed COVID-19 patients as well as unexposed individuals were applied to multicoronavirus arrays to identify specific antibody reactivity. High-level IgG, IgM, and IgA reactivity to structural proteins S, M, and N of SARS-CoV-2, as well as accessory proteins such as ORF3a and ORF7a, were observed that were specific to COVID-19 patients. Antibody reactivity against overlapping 100-, 50-, and 30-amino acid fragments of SARS-CoV-2 proteins was used to identify antigenic regions. Numerous proteins of SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), and the endemic human coronaviruses HCoV-NL63 and HCoV-OC43 were also more reactive with IgG, IgM, and IgA in COVID-19 patient sera than in unexposed control sera, providing further evidence of immunologic cross-reactivity between these viruses. Whereas unexposed individuals had minimal reactivity against SARS-CoV-2 proteins that poorly correlated with reactivity against HCoV-NL63 and HCoV-OC43 S2 and N proteins, COVID-19 patient sera had higher correlation between SARS-CoV-2 and HCoV responses, suggesting that de novo antibodies against SARS-CoV-2 cross-react with HCoV epitopes. Array responses were compared with validated spike protein-specific IgG enzyme-linked immunosorbent assays (ELISAs), showing agreement between orthologous methods. SARS-CoV-2 microneutralization titers were low in the COVID-19 patient sera but correlated with array responses against S and N proteins. The multi-coronavirus protein microarray is a useful tool for mapping antibody reactivity in COVID-19 patients. IMPORTANCE With novel mutant SARS-CoV-2 variants of concern on the rise, knowledge of immune specificities against SARS-CoV-2 proteins is increasingly important for understanding the impact of structural changes in antibody-reactive protein epitopes on naturally acquired and vaccine-induced immunity, as well as broader topics of cross-reactivity and viral evolution. A multi-coronavirus protein microarray used to map the binding of COVID-19 patient antibodies to SARS-CoV-2 proteins and protein fragments as well as to the proteins of four other coronaviruses that infect humans has shown specific regions of SARS-CoV-2 proteins that are highly reactive with patient antibodies and revealed cross-reactivity of these antibodies with other human coronaviruses. These data and the multi-coronavirus protein microarray tool will help guide further studies of the antibody response to COVID-19 and to vaccination against this worldwide pandemic.

Keywords: COVID-19; HCoV; SARS-CoV-2; antibody binding sites.

MeSH terms

  • Antibodies, Viral / blood
  • Antibodies, Viral / immunology*
  • Binding Sites, Antibody / immunology
  • COVID-19 / immunology
  • Coronavirus NL63, Human / immunology*
  • Coronavirus Nucleocapsid Proteins / immunology
  • Coronavirus OC43, Human / immunology*
  • Cross Reactions / immunology
  • Enzyme-Linked Immunosorbent Assay
  • Epitopes / immunology*
  • Humans
  • Immunoglobulin A / immunology
  • Immunoglobulin G / immunology
  • Immunoglobulin M / immunology
  • Middle East Respiratory Syndrome Coronavirus / immunology*
  • Phosphoproteins / immunology
  • Protein Array Analysis
  • SARS-CoV-2 / immunology*
  • Spike Glycoprotein, Coronavirus / immunology
  • Viral Proteins / immunology
  • Viroporin Proteins / immunology


  • Antibodies, Viral
  • Coronavirus Nucleocapsid Proteins
  • Epitopes
  • Immunoglobulin A
  • Immunoglobulin G
  • Immunoglobulin M
  • ORF3a protein, SARS-CoV-2
  • ORF7a protein, SARS-CoV-2
  • Phosphoproteins
  • Spike Glycoprotein, Coronavirus
  • Viral Proteins
  • Viroporin Proteins
  • nucleocapsid phosphoprotein, SARS-CoV-2
  • spike protein, SARS-CoV-2