SARS-CoV-2 vaccination induces mucosal antibody responses in previously infected individuals – Nature Communications

Anti-SARS-CoV-2 spike SIgA antibodies are detected in human saliva following vaccination

We used longitudinal serum and saliva samples collected from 29 adult study participants over a period of 200–372 days. 18/29 of the study participants (62%) were infected with SARS-CoV-2 on average 295.5 days prior to the first vaccine dose and were seropositive prior to vaccination (seropositive group). All the 18 seropositive participants had mild COVID-19 at the beginning of the pandemic (from March to April 2020), a time when only ancestral wild-type SARS-CoV-2 was circulating in New York City. 11/29 study participants (38%) had no previous SARS-CoV-2 infection history and were seronegative for SARS-CoV-2 antibodies prior to vaccination (seronegative group). Participants received either the Moderna mRNA-1273 vaccine or the Pfizer-BioNTech BNT162b2 vaccine two times. There were no differences in the demographics of the participants in the seropositive and seronegative groups (Supplementary Table 1). Demographics of seropositive and seronegative study participants and sample collection timepoints from each individual are summarized in Supplementary Tables 2 and 3, respectively. Samples were collected at multiple time points prior to and after vaccination. We measured anti-SARS-CoV-2 spike binding IgG titers in serum samples and anti-SARS-CoV-2 spike IgG, SIgA, and nucleoprotein (NP) SIgA titers in saliva by enzyme-linked immunosorbent assay (ELISA). One of the challenges in the measurement of antigen-specific SIgA titers in saliva samples is the fact that monomeric IgA and IgG antibodies from serum leak into saliva via crevicular fluid³. Therefore, a detection antibody that only recognizes human SC-bound IgA antibodies was used to ensure specific detection of SIgA induced at the mucosa. Another challenge in the measurement of antigen-specific SIgA titers in the saliva is that the IgA concentration within saliva is variable not only between individuals but within samples collected from the same individual, depending on various factors such as circadian rhythm, stress, and collection method of saliva samples³. We measured, therefore, the total IgA concentration within each saliva sample and normalized the SARS-CoV-2-specific SIgA titers based on the total IgA content within each saliva sample. While this methodology may induce biases due to the presence of small amounts of serum IgA which leaks into the saliva via the crevicular fluid, it has been shown that this monomeric version of IgA amounts to only about 10% of IgA in saliva and we, therefore, think the bias is negligible³⁰.

First, we evaluated the changes in anti-SARS-CoV-2 serum and saliva antibody titers before and after vaccination in each individual. An evident peak in anti-SARS-CoV-2 spike serum and mucosal antibody titers was observed in the seropositive group (Fig. 1a, c, e). In the seronegative group, peaks in serum and saliva anti-spike IgG titers could be observed, though these titers were low compared to the seropositive group (Fig. 1b, d). Interestingly, some individuals in the seronegative group presented a peak in anti-spike SIgA titers in saliva (Fig. 1f). The peak that was observed in anti-SARS-CoV-2 spike reflected the mucosal IgA response to vaccination, as most individuals did not demonstrate an increase in anti-SARS-CoV-2 NP SIgA which would indicate potential concurrent infection (Supplementary Fig. 2).

Higher levels of anti-SARS-CoV-2 spike SIgA antibodies are induced in humans with prior virus infection

Next, we performed correlation analyses including all the samples tested to reveal the relationship between the different antibody titers. As expected, a positive correlation was observed between serum and saliva anti-spike IgG titers (Fig. 2a), reflecting the fact that the IgG antibodies measured in saliva were originally serum IgG antibodies that seeped into the mucosa from circulation. In contrast, titers of saliva anti-spike SIgA, which are not derived from the circulation but were produced at the mucosa, did not strongly correlate with serum IgG titers (Fig. 2b). Next, to evaluate the difference in antibody titers over time, we grouped the data based on the collection timepoints: before first vaccination, 1–100 days post first vaccination, and more than 100 days post first vaccination. As anticipated serum IgG and mucosal SIgA antibody titers were significantly higher in seropositive individuals than in seronegative individuals before the first vaccination (Supplementary Fig. 3a–d). The serum and mucosal antibody response to the SARS-CoV-2 spike following vaccination remained significantly higher in seropositive individuals than in seronegative individuals (Supplementary Fig. 3a–c). In contrast, the mucosal anti-NP SIgA antibody response was significantly higher in samples collected from previously infected individuals than those from previously naive individuals only before vaccination and 1–100 days post first vaccination. This difference was not observed in samples collected after 100 days post first vaccination (Supplementary Fig. 3d). This indicated that the anti-NP SIgA response observed in seropositive individuals was not boosted by vaccination, and waned over time. Comparison between the different timepoints revealed that serum and mucosal anti-spike antibody titers significantly increased after vaccination in both seropositive and seronegative individuals (Fig. 2c and Supplementary Fig. 3e, f). In contrast, an increase in mucosal anti-NP IgA titers over time was not observed in either group (Supplementary Fig. 3g).

a, b Correlation between anti-spike serum IgG antibody titers and saliva IgG antibody titers (a) or mucosal SIgA antibody titers (b) of samples collected from both seropositive and seronegative individuals at all time points. A total of 264 data points (6–13 data points per individual) were plotted for each panel. X and Y-axis values represent antibody titers (AUC: area under the curve). Spearman correlation coefficient and p values (two-tailed) are labeled above graphs. c Comparison of anti-SARS-CoV-2 spike SIgA titers in saliva between samples collected during different timepoints: before first vaccination (Pre, 91 samples in seropositive group and 62 samples in seronegative group), 1–100 days post first vaccination (1–100 DPV, 50 samples in seropositive group and 32 samples in seronegative group), and over 100 days post first vaccination (>100 DPV, 28 samples in seropositive group and 16 samples in seronegative group). Antibody titers (adjusted AUC) are plotted for each group. (statistical test: one-way ANOVA with Kruskal–Wallis test, p values above brackets). d Comparison of peak anti-SARS-CoV-2 spike mucosal SIgA titers between seropositive and seronegative individuals. The highest anti-spike SIgA titer among all timepoints (the peak saliva SIgA adjusted AUC) was plotted per individual (18 in the seropositive group and 11 in the seronegative group). Red dots (16 in the seropositive group and 6 in the seronegative group) indicate individuals with peak SIgA titers above the cut-off value (SIgA detected). e Comparison of baseline pre-vaccination anti-SARS-CoV-2 spikes SIgA titers in saliva between SIgA detected (16 in seropositive group and 6 in seronegative group) and SIgA non-detected (2 in seropositive group and 5 in seronegative group) seropositive and seronegative individuals. f Comparison of peak anti-SARS-CoV-2 spike IgG titers in serum of SIgA detected or SIgA non-detected seropositive and seronegative individuals. Mann–Whitney test was used in (d–f) (p-value (two-tailed) above bracket). All scatter plots in this figure are presented with geometric mean with geometric SD and a dotted line indicating the cut-off value (mean+3SD of the AUC of all samples collected from pre-vaccinated seronegative individuals). Source data are provided as a Source Data file.

SIgA is efficiently induced in seropositive individuals with higher pre-exisiting SIgA levels

In order to identify study participants who were able to successfully induce SIgA responses by vaccination, peak mucosal SIgA titers (the highest SIgA titer observed after vaccination) were assessed. Peak saliva SIgA titers were significantly higher in the seropositive individuals than the seronegative individuals (Fig. 2d). The same was observed for serum IgG titers (Supplementary Fig. 4a), which was consistent with a previous report¹³. 16/18 seropositive individuals presented peak SIgA titers above the cut-off value (mean+3 SD of the AUC for all samples collected from pre-vaccinated seronegative individuals), whereas only 6/11 seronegative individuals did (Fig. 2d). To identify whether pre-vaccination antibody titers were associated with induction of SIgA by vaccination, baseline antibody titers (measured using samples collected on the nearest possible date before vaccination), were evaluated. In order to assess this, we separated the seropositive and seronegative groups each into two categories: SIgA detected (those with peak saliva SIgA titers above the cut-off value) and SIgA non-detected (those with peak saliva SIgA titers below the cut-off value) individuals (Fig. 2d). In the seropositive group, baseline anti-SARS-CoV-2 spike saliva SIgA titers were significantly higher in SIgA detected individuals than SIgA non-detected individuals before vaccination (Fig. 2e). Significant differences were not observed in baseline anti-SARS-CoV-2 spike serum IgG and anti-SARS-CoV-2 NP saliva SIgA titers between SIgA detected and non-detected individuals (Supplementary Fig. 4b, c). However, for seronegative individuals, no significant difference was observed in baseline saliva anti-SARS-CoV-2 spike SIgA titers (Fig. 2e). Next, in order to assess whether pre-existing antibodies affect the magnitude of the antibody response induced by vaccination, correlation analyses between fold-increases in antibody titers by vaccination and baseline antibody titers were conducted. As a result, baseline saliva SIgA and serum IgG levels did not correlate with fold-increase in saliva SIgA levels, whereas baseline serum IgG antibody titers negatively correlated with fold-increase in serum IgG levels by vaccination (Supplementary Fig. 4d–f).

It may be possible that mucosal immunity induced by seasonal coronaviruses (HCoVs) is boosted by vaccination and that this cross-reactive immunity (which mostly targets S2^14,15, with cross-reactivity being common between betacoronaviruses OC43, HKU1, and SARS-CoV-2) is responsible for the weak induction of SIgA found in the previously seronegative individuals. To evaluate this, SIgA binding activity against the spike proteins of betacoronaviruses HCoV-OC43 and HCoV-HKU1, and the antigenically conserved SARS-CoV-2 S2 domain were assessed. However, no significant differences in baseline anti-HCoV-OC43, HCoV-HKU1, and SARS-CoV-2 S2 saliva SIgA titers between SIgA detected and non-detected groups were observed in seronegative individuals (Supplementary Fig. 5a). In addition, the S2/RBD ratio of peak saliva SIgA was not significantly different between SIgA-induced seropositive and seronegative individuals, suggesting no skewing towards cross-reactive S2 antibodies (Supplementary Fig. 5b). In addition, the fold-increase of anti-SARS-CoV-2 spike SIgA levels by vaccination was significantly higher than that of anti-HCoV-OC43 and HCoV-HKU1 spike SIgA titers in SIgA-induced seropositive and seronegative individuals (Supplementary Fig. 5c). These data indicate that the saliva SIgA antibodies induced by vaccination in seronegative individuals were not back-boosted antibodies against HCoVs, but rather SARS-CoV-2 spike-specific antibodies that were newly elicited by vaccination. To assess whether the level of systemic immune response induced was associated with successful SIgA induction, peak serum anti-SARS-CoV-2 spike IgG antibodies were assessed. As a result, SIgA detected individuals presented significantly higher peak serum anti-SARS-CoV-2 spike IgG titers than SIgA non-detected individuals in the seropositive group but not in the seronegative group (Fig. 2f). Interestingly, there were no significant difference between the peak serum anti-SARS-CoV-2 spike IgG titers observed in SIgA non-detected seropositive individuals and SIgA detected seronegative individuals (p = 0.0714, Mann–Whitney test). Of note, neither the type of vaccine administered, sex, nor age influenced the induction of anti-SARS-CoV-2 spike saliva SIgA antibodies upon immunization (Supplementary Fig. 6a–c).