A majority of uninfected adults show preexisting antibody reactivity against SARS-CoV-2

Abstract

Preexisting cross-reactivity to SARS-CoV-2 occurs in the absence of prior viral exposure. However, this has been difficult to quantify at the population level due to a lack of reliably defined seroreactivity thresholds. Using an orthogonal antibody testing approach, we estimated that about 0.6% of nontriaged adults from the greater Vancouver, Canada, area between May 17 and June 19, 2020, showed clear evidence of a prior SARS-CoV-2 infection, after adjusting for false-positive and false-negative test results. Using a highly sensitive multiplex assay and positive/negative thresholds established in infants in whom maternal antibodies have waned, we determined that more than 90% of uninfected adults showed antibody reactivity against the spike protein, receptor-binding domain (RBD), N-terminal domain (NTD), or the nucleocapsid (N) protein from SARS-CoV-2. This seroreactivity was evenly distributed across age and sex, correlated with circulating coronaviruses' reactivity, and was partially outcompeted by soluble circulating coronaviruses' spike. Using a custom SARS-CoV-2 peptide mapping array, we found that this antibody reactivity broadly mapped to spike and to conserved nonstructural viral proteins. We conclude that most adults display preexisting antibody cross-reactivity against SARS-CoV-2, which further supports investigation of how this may impact the clinical severity of COVID-19 or SARS-CoV-2 vaccine responses.

Keywords: Adaptive immunity; COVID-19; Immunology.

Figure 1. Hierarchical clustering of individual based on serum SARS-CoV-2 antibody reactivity profiles.

COVID-19 diagnosis identifies convalescing individuals who had a positive viral test by PCR. This figure combines data from 276 study participants plus the 5 COVID-19 convalescent control sera. Color scale represents antibody reactivity as a Z score.

Figure 2. Specificity of SARS-CoV-2 antibody reactivity.

(A and B) Competition of SARS-CoV-2 spike and RBD antibody reactivity by SARS-CoV-2 spike and RBD proteins (A) or by circulating coronaviruses (cCoVs) spike proteins (B). (C and D) Competition of cCoVs spike antibody reactivity by SARS-CoV-2 spike and RBD proteins (C) or cCoVs spike proteins (D). (E) Competition of SARS-CoV-2 spike antibody reactivity by SARS-CoV-2 spike and RBD proteins or cCoVs spike proteins, in COVID-19 convalescent sera (seropositive, n = 5), or sera from uninfected individuals who showed highest SARS-CoV-2 spike (n = 10) and high RBD (n = 9), or lowest SARS-CoV-2 spike and RBD antibody reactivity (all low; n = 10). All values represent the ratios of antibody reactivity in competed samples over the antibody reactivity measured in absence of competing proteins (dash line). One sample in the RBD-high group failed, and these data are not shown. In E, data are represented as boxes (25th to 75th percentile, line at median) and whiskers (minimum to maximum); comparisons were made using 2-tailed paired t tests.

Figure 3. Thresholds of antibody reactivity based on infants’ sera.

Comparison of antibody reactivity (AU/mL) in infants sampled before 6 months of age (darker blue) and again about 8 months later (lighter blue; n = 45), in SARS-CoV-2–uninfected (orange; n = 273), in SARS-CoV-2–infected (convalescent) adults (red; n = 8), and in prepandemic sera (yellow; n = 99). Infants sampled before the pandemic (January 1, 2020) are represented by the larger circle symbols, whereas infants sampled after January 1, 2020, are shown using the small circle symbols. Boxes represent median with 25th and 75th percentiles with positive/negative antibody reactivity thresholds for SARS-CoV-2 spike calculated at the 99th percentile for value distribution (10.00 AU/mL), RBD (10.00 AU/mL), and N protein (10.00 AU/mL) as 10^{(mean log[antibody reactivity] + SD log [antibody reactivity] × 2.33)} in infants’ sera. Antibody detection for NTD was low and inconsistent between experiments; therefore, the data are not presented and reactivity thresholds were not calculated.

Figure 4. Mapping of SARS-CoV-2 antibody reactivity in sera from uninfected individuals.

Serum antibody binding to 15-mer peptides distributed across the SARS-CoV-2 proteome or an IgG-binding peptide (positive control), from 5 randomly selected prepandemic samples, adults showing high level of spike or RBD reactivity (n = 20 each), or infants (n = 5). Values represent signals on a scale from 0 to 10, after subtracting background. The column labeled C shows the immunoreactivity signal in the absence of sera, but with the addition of anti–human IgA, IgM, and IgG horse-radish peroxidase–coupled secondary antibody. In the spike protein, labels indicate the N-terminal (NTD), C-terminal (CTD), or receptor-binding (RBD) domains, receptor-binding motif (RBM), heptad repeat sequence (HR1), central helix (CH), or connector domain (CD); N, nucleocapsid protein; M, membrane protein; ORF, open-reading frame polypeptide proteins; nsp, nonstructural proteins.