Research ArticleCORONAVIRUS

The receptor-binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in SARS-CoV-2 patients

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Science Immunology  11 Jun 2020:
Vol. 5, Issue 48, eabc8413
DOI: 10.1126/sciimmunol.abc8413
  • Fig. 1 Production and characterization of the RBD of the coronavirus spike antigens.

    (A) The spike protein on the virion surface engages its cognate receptor via the RBD. (B) RBD of the spike protein is the main human antibody target in SARS-CoV-1. (C) The amino acid sequence corresponding to RBD of the spike protein is poorly conserved between SARS-CoV-2 and common human coronaviruses. (D) Coomassie-stained SDS-PAGE of purified spike RBD antigens from different CoVs. (E) Binding characterization of the spike RBD antigens with immune sera and a monoclonal antibody (mAb). SARS-CoV-1 monoclonal antibody (240C), serum from a mouse immunized with VRP-expressing SARS-CoV-2 or SARS-CoV-1 spike protein, serum from a rabbit immunized with SARS-CoV-1 spike protein, and an archived human sample collected before SARS-CoV-2 were tested for binding against RBD spike antigens from SARS-CoV-2, SARS-CoV-1, HCoVα (NL63), and HCoVβ (HKU-1).

  • Fig. 2 Evaluation of SARS-CoV-2 spike RBD antigen specificity using blood samples collected before the emergence of SARS-CoV-2.

    Spike RBD antigen binding was assessed by in-house ELISA assay against a panel of de-identified archived serum specimens obtained from (A) healthy American adults; (B) convalescent sera from dengue/Zika patients in South Asia, Caribbean, and Central America; (C) people who had recently recovered from viral respiratory illnesses; and (D) guinea pigs immunized with respiratory viruses or SARS-CoV-1 spike protein. The cutoff values determined by the ROC curve analysis (fig. S3) for the ELISA assay are indicated by the broken line. RSV, respiratory syncytial virus.

  • Fig. 3 Evaluation of SARS-CoV-2 spike RBD antigen specificity against common human CoV and animal CoV sera.

    (A) Phylogenetic tree of the spike protein from representative coronaviruses. Coronavirus genera are grouped by classic subgroup designations (α, βa–d, γ, and δ). SADS-CoV is a distinctive member of the α subgroup (indicated by *). Numbers following the underscores in each sequence correspond to the GenBank accession number. Spike RBD antigen binding was assessed by in-house ELISA assay using (B) human convalescent samples obtained from PCR-confirmed HCoVα (NL63, black) and HCoVβ [OC43 (red) and HKU-1 (blue)] infections and (C) sera from guinea pigs or pigs immunized with spike antigen from SARS-CoV-1 or indicated animal CoV. The cutoff values for the ELISA assay are indicated by the broken line. Feline infectious peritonitis virus, 79-1146 (Feline CoV, pink); respiratory coronavirus strain ISU-1 (Porcine CoV, green); porcine transmissible gastroenteritis virus (TGEV; orange); bovine coronavirus strain mebus (Bovine CoV, cyan); avian infectious bronchitis virus, Massachusetts (Avian CoV, violet); turkey coronavirus, Indiana (Turkey CoV, yellow); canine coronavirus strain UCD1 (Canine CoV, hot pink); SARS-CoV-2 (SARS, brown).

  • Fig. 4 Evaluation of SARS-CoV-2 spike RBD antigen sensitivity.

    (A) Overall SARS-CoV-2 spike RBD antigen sensitivity as assessed by the in-house Ig and IgM ELISA assays using clinical specimens obtained from PCR-confirmed SARS-CoV-2 individuals. For comparison, binding results of the RBD spike antigens from a representative HCoVβ (HKU-1) with the same specimens are also presented. The changes of the levels of (B) total Ig and (C) IgG, IgA, and IgM antibodies binding to RBD of the SARS-CoV-2 spike antigen. The binding of the spike RBD antigen from SARS-CoV-2 to 49 de-identified serum samples obtained from SARS-CoV-2–positive individuals at different time points since onset of symptoms is presented. The cutoff values for the ELISA assay are indicated by the broken line. The dashed blue box in (B) indicates a single PCR-positive and seronegative individual. Seroconversion of (D) total Ig and (E) IgM antibodies against RBD of the SARS-CoV-2 spike antigen among 14 representative SARS-CoV-2 patients during the acute phase since onset of symptoms. The first sample (green) and follow-up sample (red) are connected by black arrows. The time intervals between the first and follow-up samples are provided on the x axis. The binding signals below the broken line are denoted as seronegative.

  • Fig. 5 Correlation between spike RBD antigen binding and SARS-CoV-2 neutralizing antibody titers.

    Correlations between (A) total Ig and (B) IgM RBD-binding and the SARS-CoV-2 neutralizing antibody titers. Scatter plots were generated using individual serum binding to RBD antigen (y axis) versus SARS-CoV-2 neutralizing antibody titers (x axis). The nonparametric Spearman correlation coefficient (rs) and the associated two-tailed P values were calculated (GraphPad Prism, version 5.0). (C) Relationship between SARS-CoV-2 neutralizing antibody titer and days after onset of symptoms. (D) Total Ig antibody binding to RBD as a surrogate for identifying people with high SARS-CoV-2 neutralizing antibodies. A total of 50 serum samples collected between 1 and 39 days after onset of symptoms from PCR-confirmed SARS-CoV-2 individuals were measured for Ig and IgM binding to spike RBD antigen and SARS-CoV-2 neutralization assay. The FDA-recommended neutralizing antibody titer for plasma therapy (1:160) is indicated by the broken green line.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/5/48/eabc8413/DC1

    Fig. S1. Titration curves of sera from SARS-CoV-2–positive patients.

    Fig. S2. Seroconversion of SARS-CoV-2 neutralizing antibodies.

    Fig. S3. Estimation of RBD ELISA assay cutoff.

    Table S1. Summary of samples tested and associated characteristics (Excel spreadsheet).

    Table S2. Raw data file (Excel spreadsheet).

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Titration curves of sera from SARS-CoV-2 positive patients.
    • Fig. S2. Seroconversion of SARS-CoV-2 neutralizing antibodies.
    • Fig. S3. Estimation of RBD ELISA assay cutoff.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1. Summary of samples tested and associated characteristics (Excel spreadsheet).
    • Table S2. Raw data file (Excel spreadsheet).

    Files in this Data Supplement:

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