Efficient recall of Omicron-reactive B cell memory after a third dose of SARS-CoV-2 mRNA vaccine

Abstract

Despite a clear role in protective immunity, the durability and quality of antibody and memory B cell responses induced by mRNA vaccination, particularly by a 3 ^rd dose of vaccine, remains unclear. Here, we examined antibody and memory B cell responses in a cohort of individuals sampled longitudinally for ∼9-10 months after the primary 2-dose mRNA vaccine series, as well as for ∼3 months after a 3 ^rd mRNA vaccine dose. Notably, antibody decay slowed significantly between 6- and 9-months post-primary vaccination, essentially stabilizing at the time of the 3 ^rd dose. Antibody quality also continued to improve for at least 9 months after primary 2-dose vaccination. Spike- and RBD-specific memory B cells were stable through 9 months post-vaccination with no evidence of decline over time, and ∼40-50% of RBD-specific memory B cells were capable of simultaneously recognizing the Alpha, Beta, Delta, and Omicron variants. Omicron-binding memory B cells induced by the first 2 doses of mRNA vaccine were boosted significantly by a 3rd dose and the magnitude of this boosting was similar to memory B cells specific for other variants. Pre-3 ^rd dose memory B cell frequencies correlated with the increase in neutralizing antibody titers after the 3 ^rd dose. In contrast, pre-3 ^rd dose antibody titers inversely correlated with the fold-change of antibody boosting, suggesting that high levels of circulating antibodies may limit reactivation of immunological memory and constrain further antibody boosting by mRNA vaccines. These data provide a deeper understanding of how the quantity and quality of antibody and memory B cell responses change over time and number of antigen exposures. These data also provide insight into potential immune dynamics following recall responses to additional vaccine doses or post-vaccination infections.

Figure 1.. Antibody responses after 2 and 3 doses of mRNA vaccine.

A) Study design and cohort characteristics. B) anti-Spike and C) anti-RBD IgG concentrations over time in plasma samples from vaccinated individuals. D) Pseudovirus (PSV) neutralization titers against wild-type D614G Spike protein over time in plasma samples from vaccinated individuals. Data are represented as focus reduction neutralization titer 50% (FRNT50) values. E) Antibody neutralization potency against D614G over time. Potency was calculated as neutralizing titer (FRNT50) divided by the paired concentration of anti-RBD IgG. F and G) Plasma neutralizing activity against D614G and Omicron before and after booster vaccination. Dotted lines indicate limit of detection for the assay. Green boxes and lines indicate interquartile range (IQR) and median of D614G neutralizing titers ~1 week following the second vaccine dose in SARS-CoV-2 naïve subjects. H and I) Comparison of antibody potency against D614G, Delta, and Omicron between SARS-CoV-2 naïve and previously infected vaccinees. For I, bars indicate mean with 95% confidence intervals. Statistics were calculated using unpaired non-parametric Wilcoxon test with BH correction for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

Figure 2.. Memory B cell responses after 2 and 3 doses of mRNA vaccine.

A) Flow cytometry gating strategy for SARS-CoV-2-specific plasmablasts. B) Frequency of Spike+ plasmablasts ~1 week after booster vaccination or post-vaccine breakthrough infection. Data are represented as a percentage of total B cells. C) Flow cytometry gating strategy for SARS-CoV-2-specific memory B cells. D and E) Frequency of Spike+ and F and G) Spike+ RBD+ memory B cells over time in PBMCs from vaccinated individuals. Data are represented as a percentage of total B cells. H) Isotype composition of Spike+ memory B cells in vaccinated individuals pre- and post-boost. I) Activation status of Spike+ memory B cells over time in vaccinated individuals following booster vaccination. Statistics were calculated using unpaired non-parametric Wilcoxon test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

Figure 3.. Variant-reactive memory B cell responses after 2 and 3 doses of mRNA vaccine.

A) Experimental design and B) flow cytometry gating strategy for SARS-CoV-2 variant-reactive memory B cells. C) Frequency of NTD+, WT RBD+, All Variant RBD+, and S2+ memory B cells in vaccinated individuals pre- and post-boost. D) Fold change in the frequency of antigen-specific memory B cells after booster vaccination relative to paired pre-boost samples. Dotted lines indicate no change in frequency compared to pre-boost samples. E) Variant cross-binding of RBD-specific memory B cells in vaccinated individuals. Data are represented as a percentage of WT RBD+ cells. F) Boolean analysis of variant cross-binding memory B cell populations in vaccinated individuals ~2 weeks after 3^rd vaccination or at a cross-sectional timepoint in individuals with a post-vaccine breakthrough infection. Pie charts indicate the fraction of WT RBD+ memory B cells that cross-bind zero, one, two, three, or four variant RBDs. Colored arcs indicate cross-binding to specific variants. G) Comparison of RBD variant cross-binding between SARS-CoV-2 naïve and previously infected vaccinees before and ~2 weeks after 3rd vaccination. For G, bars indicate mean with 95% confidence intervals. Statistics were calculated using unpaired non-parametric Wilcoxon test with BH correction for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

Figure 4.. Activation of Omicron-specific B cell memory after a 3^rd dose of mRNA vaccine.

A) Heatmap and hierarchal clustering of memory B cell activation status by antigen specificity at pre- and post-3^rd dose timepoints. Prior COVID infection and/or post-vaccine breakthrough infection are indicated. B) Fold change in the frequency of Omicron RBD-binding versus non-binding memory B cells after booster vaccination relative to paired pre-boost samples. Dotted lines indicate no change in frequency compared to pre-boost samples. C) Representative flow cytometry plots for activation phenotype of Omicron RBD-binding versus non-binding memory B cells. D) Frequency of activated memory (AM), resting memory (RM), or double negative (DN) subsets in Omicron RBD-binding versus non-binding memory B cells before and ~2 weeks after 3rd vaccination. For B-D, analysis was restricted to SARS-CoV-2 naïve vaccinees. Statistics were calculated using paired non-parametric Wilcoxon test with BH correction for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

Figure 5.. Immune relationships after 2 and 3 doses of mRNA vaccine.

(A) UMAP of antibody and memory B cell responses to mRNA vaccination. Data points represent individual participants and are colored by timepoint relative to primary vaccine. B) UMAP coordinates of SARS-CoV-2–naïve and –recovered subjects over time. Labels indicate centroids for each group at the indicated timepoint. C) Correlation matrix of antibody and memory B cell responses over time in SARS-CoV-2–naïve subjects. D) Correlation of RBD+ memory B cell frequencies with neutralizing antibody recall responses to D614G and Omicron. Recall responses were calculated as the difference between pre- and post-boost titers ~2 weeks after the 3^rd vaccine dose. E) Peak binding and neutralizing antibody responses after 3 versus 4 exposures to SARS-CoV-2 antigen (~2 weeks post 3^rd dose in SARS-CoV-2 naïve and SARS-CoV-2 recovered individuals). F) Correlation of pre-boost binding antibody responses with peak post-boost antibody responses. G) Fold change in antibody responses after 3 versus 4 exposures to SARS-CoV-2 antigen. H) Correlation of fold-change in antibody responses after boosting with pre-3^rd dose antibody levels. Statistics were calculated using unpaired non-parametric Wilcoxon test with BH correction for multiple comparisons. All correlations were calculated using nonparametric Spearman rank correlation. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.