Detection of Antibody Responses Against SARS-CoV-2 in Plasma and Saliva From Vaccinated and Infected Individuals

Abstract

Antibodies (Abs) are essential for the host immune response against SARS-CoV-2, and all the vaccines developed so far have been designed to induce Abs targeting the SARS-CoV-2 spike. Many studies have examined Ab responses in the blood from vaccinated and infected individuals. However, since SARS-CoV-2 is a respiratory virus, it is also critical to understand the mucosal Ab responses at the sites of initial virus exposure. Here, we examined plasma versus saliva Ab responses in vaccinated and convalescent patients. Although saliva levels were significantly lower, a strong correlation was observed between plasma and saliva total Ig levels against all SARS-CoV-2 antigens tested. Virus-specific IgG1 responses predominated in both saliva and plasma, while a lower prevalence of IgM and IgA1 Abs was observed in saliva. Antiviral activities of plasma Abs were also studied. Neutralization titers against the initial WA1 (D614G), B.1.1.7 (alpha) and B.1.617.2 (delta) strains were similar but lower against the B.1.351 (beta) strain. Spike-specific antibody-dependent cellular phagocytosis (ADCP) activities were also detected and the levels correlated with spike-binding Ig titers. Interestingly, while neutralization and ADCP potencies of vaccinated and convalescent groups were comparable, enhanced complement deposition to spike-specific Abs was noted in vaccinated versus convalescent groups and corresponded with higher levels of IgG1 plus IgG3 among the vaccinated individuals. Altogether, this study demonstrates the detection of Ab responses after vaccination or infection in plasma and saliva that correlate significantly, although Ig isotypic differences were noted. The induced plasma Abs displayed Fab-mediated and Fc-dependent functions with comparable neutralization and ADCP potencies, but a greater capacity to activate complement was elicited upon vaccination.

Keywords: ADCP; COVID-19; SARS-CoV-2; antibody isotypes; complement fixation; neutralization; saliva; vaccination.

Figure 1

Levels of SARS-CoV-2-specific total Ig in plasma and saliva. (A) Titers of antigen-specific total Ig in plasma versus saliva specimens from vaccinated donors and convalescent COVID-19 patients. End-point titers were calculated from reciprocal dilutions that reached the cut-off values (mean + 3SD of negative controls at the lowest dilution). Data points below the cut-off are shown at the lowest reciprocal dilutions (100 for plasma, 2 for saliva) as gray circles (vaccinated) or gray diamonds (convalescent). (B) The percentages of responders above cut-off for each antigen based on plasma versus saliva total Ig from seven vaccinated subjects (left panel) and seven convalescent COVID-19 patients (right panel). (C) Spearman correlation of spike- and S1-specific total Ig levels in plasma versus saliva from vaccinated and convalescent subjects. Areas under the curves (AUC) were calculated from the titration curves in Supplemental Figure 1 . The dotted line indicates the cut-off value.

Figure 2

Ig isotypes against SARS-CoV-2 spike and RBD in plasma versus saliva after vaccination and infection. (A) Total Ig, IgM, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2 levels against spike were measured in plasma (left) and saliva (right) specimens from vaccinated (top panels) and convalescent COVID-19 patients (lower panels). For controls, samples from four COVID-19-negative individuals (open symbols) were tested in parallel. The dotted line represents the cut-off calculated as mean of the four control specimens + 3SD for each isotype. (B) The percentages of responders among vaccinated (top panels) and convalescent subjects (lower panels) for each spike- or RBD-specific Ig isotype on the basis of plasma (left) and saliva (right) reactivity. (C) Spearman correlation between spike- and RBD-specific IgG1, IgA1 and IgM levels in plasma versus saliva from vaccinated and convalescent subjects.

Figure 3

Plasma neutralization activities against WT versus variants. (A) Neutralization of recombinant VSV viruses bearing the spike proteins of SARS-CoV-2 WT, B.1.531, B.1.1.7 or B.1.617.2 by plasma specimens from vaccinated and convalescent COVID-19 donors. Plasma samples from three COVID-19-negative individuals were tested in parallel; these negative control data are shown as mean + SD of replicates from all three samples. The dotted line indicates 50% neutralization. (B) Comparison of neutralization IC50 titers against WT versus B.1.351, B.1.1.7 and B.1.617.2. The specimens that did not reach 50% neutralization were shown as gray symbols at the lowest reciprocal dilution. Statistical analysis was performed using a Kruskal-Wallis test. *p <0.05; ns, non-significant. Red line: median.

Figure 4

ADCP activities in plasma of vaccinated and convalescent individuals. Spike-specific ADCP activities in plasma specimens from (A) vaccinated and (B) convalescent donors were tested along with two control plasma samples from COVID-19-negative individuals. ADCP was measured by flow cytometry after incubation of plasma-treated spike-coated fluorescent beads with THP-1 phagocytes. ADCP scores were calculated as % bead⁺ cells × MFI of bead⁺ cells. The dotted line indicates the background. (C) Correlation between spike-specific ADCP AUC and spike-specific total Ig AUC from the seven vaccinated individuals, seven convalescent patients and two negative controls. (D) Ratio of spike-specific ADCP AUC to spike-specific total Ig AUC from the seven vaccinated individuals and seven convalescent patients. ns, not significant (p > 0.05).

Figure 5

Complement-binding activities in the plasma of vaccinated and convalescent individuals. (A, B) The binding of C1q (A) and C3d (B) to spike-specific Abs in plasma specimens from vaccinated (left) and convalescent (right) donors was assessed together with four COVID-19-negative controls in multiplex bead assays. Specimens were diluted 2-fold from 1:100 to 1:6,400 or 12,800. The dotted line represents the 100x dilution cut-off values calculated as mean + 3SD of the four control specimens. ΔC1q and ΔC3d MFI values were calculated by subtracting background MFI from each assay. (C) Spearman correlation between C1q AUC and C3d AUC values for spike- or RBD-specific Abs in plasma specimens from vaccinated and convalescent donors. (D, E) Ratio of C1q and C3d binding AUC to total Ig AUC of specimens from 7 vaccinated and 7 convalescent donors (D) and from additional 20 recipients of Pfizer or Moderna mRNA vaccines and 13 convalescent donors (E). Statistical significance was assessed using a Kruskal-Wallis test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 6

Heatmap to show the relative levels of neutralization (IC₅₀) and Fc-mediated activities (ratios to total Ig) in plasma specimens from vaccinated and convalescent donors. Z-scores calculated for each Ab activity were used to generate the heatmap.