Fig. 3. Memory B cells induced by mRNA vaccination or infection are cross-reactive to SARS-CoV-2 VOCs and increase in frequency over time.

(A and B) Experimental design (A) and gating strategy (B) for quantifying the frequency and phenotype of spike subunit and variant-specific memory B cells by flow cytometry. Specific mutations in B.1.1.7, B.1.351, or B.1.617.2 variant RBDs are indicated. (C) Frequencies of spike⁺ NTD⁺, spike⁺ WT RBD⁺, spike⁺ all variant⁺ (all variant RBD binding), and spike⁺ S2⁺ memory B cells over time in PBMC samples from vaccinated or convalescent individuals. Data are represented as a percentage of total B cells. (D) Percent NTD⁺, RBD⁺, or S2⁺ of total spike⁺ memory B cells over time. (E) Representative plots of variant RBD cross-binding gated on spike⁺ WT RBD⁺ cells in vaccinated or convalescent individuals. Mean and standard error values at the 6-month time point are indicated. (F) Percent B.1.1.7⁺, B.1.351⁺, B.1.617.2⁺, or all variant⁺ of WT RBD⁺ memory B cells over time. (G) Boolean analysis of variant cross-binding memory B cell populations in vaccinated, infected then vaccinated, or infected-only individuals at 6 months after vaccination or seropositivity. Pie charts indicate the fraction of WT RBD⁺ memory B cells that cross-bind zero, one, two, or three variant RBDs. Colored arcs indicate cross-binding to specific variants. (H) Cross-sectional analysis of variant binding as a percentage of WT RBD⁺ memory B cells at 6 months after vaccination or seropositivity. For (C), (D), and (F), thick lines indicate mean values, and thin lines represent individual subjects. Statistics were calculated using paired [(C), (D), and (F)] or unpaired (H) nonparametric Wilcoxon test with BH correction for multiple comparisons. Blue, red, and purple values indicate comparisons within naïve, recovered, or infection-only groups, respectively. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.