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

Abstract

We examined antibody and memory B cell responses longitudinally for ∼9-10 months after primary 2-dose SARS-CoV-2 mRNA vaccination and 3 months after a 3rd dose. Antibody decay stabilized between 6 and 9 months, and antibody quality continued to improve for at least 9 months after 2-dose vaccination. Spike- and RBD-specific memory B cells remained durable over time, and 40%-50% of RBD-specific memory B cells simultaneously bound the Alpha, Beta, Delta, and Omicron variants. Omicron-binding memory B cells were efficiently reactivated by a 3rd dose of wild-type vaccine and correlated with the corresponding increase in neutralizing antibody titers. In contrast, pre-3rd dose antibody titers inversely correlated with the fold-change of antibody boosting, suggesting that high levels of circulating antibodies may limit the added protection afforded by repeat short interval boosting. These data provide insight into the quantity and quality of mRNA-vaccine-induced immunity over time through 3 or more antigen exposures.

Keywords: COVID-19; Omicron; SARS-CoV-2; antibody; booster; immune memory; mRNA; memory B cell; vaccine; variants of concern.

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 Benjamini-Hochberg correction for multiple comparisons. Breakthrough infection samples were excluded from statistical comparisons. Median fold changes for selected comparisons are indicated. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Binding antibody and D614G pseudovirus neutralization data from pre-vaccine baseline through six months post-primary vaccination were described previously (Goel et al., 2021a).

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) Frequency of Spike+ and E) Spike+ RBD+ memory B cells over time in PBMCs from vaccinated individuals. Data are represented as a percentage of total B cells. F) Fold change in the frequency of Spike+ and G) Spike+ RBD+ memory B cells after booster vaccination relative to paired pre-boost samples. Median fold change is indicated in dashed blue or red lines. Dashed black lines at fold change = 1 indicate no change in frequency compared to pre-boost samples. 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. See also Figure S1. Memory B cell responses from pre-vaccine baseline through six months post-primary vaccination were re-analyzed from a previous dataset (Goel et al., 2021a).

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. Median fold change for each variable is indicated in dashed blue or red lines. Dashed black lines at fold change = 1 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 Benjamini-Hochberg correction for multiple comparisons. Breakthrough infection samples were excluded from statistical comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. See also Figure S2.

Figure 4.. Activation of Omicron-reactive 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) Median fold change in the frequency of Omicron RBD-binding versus non-binding memory B cells after booster vaccination relative to paired pre-boost samples. Dashed black lines at fold change = 1 indicate no change in frequency compared to pre-boost samples. C) Representative flow cytometry plots for activation phenotype of Omicron RBD-binding versus Omicron RBD non-binding (but still wild-type RBD 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 a 3rd vaccination. For B-D, analysis was restricted to SARS-CoV-2 naïve vaccinees. Statistics were calculated using paired non-parametric Wilcoxon test with Benjamini-Hochberg correction for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. See also Figure S2.

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. Breakthrough infection samples were excluded from calculations of group centroids. C) Correlation matrix of antibody and memory B cell responses over time in SARS-CoV-2–naïve subjects. D) Correlation of pre-boost 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) Change in binding and neutralizing antibody responses after a 3^rd vaccine dose in individuals with 3 versus 4 exposures to SARS-CoV-2 antigen, calculated as in D. Dotted lines indicate no change in antibodies. F) Correlation of pre-boost binding antibody responses with change in antibody responses after boost, calculated as in D. G) 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). Dotted lines indicate the limit of detection for the assay. H) Correlation of pre-boost binding antibody responses with peak post-boost antibody responses. I) Fold change in antibody responses after 3 versus 4 exposures to SARS-CoV-2 antigen. Dotted lines indicate no change in antibodies. J) 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 Benjamini-Hochberg 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. See also Figure S3.