Evidence of SARS-CoV-2-Specific Memory B Cells Six Months After Vaccination With the BNT162b2 mRNA Vaccine

Abstract

SARS-CoV-2 mRNA vaccines have demonstrated high efficacy and immunogenicity, but limited information is currently available on memory B cell generation and long-term persistence. Here, we investigated spike-specific memory B cells and humoral responses in 145 subjects, up to 6 months after the BNT162b2 vaccine (Comirnaty) administration. Spike-specific antibodies peaked 7 days after the second dose and significant antibody titers and ACE2/RBD binding inhibiting activity were still observed after 6 months, despite a progressive decline over time. Concomitant to antibody reduction, spike-specific memory B cells, mostly IgG class-switched, increased in the blood of vaccinees and persisted 6 months after vaccination. Following the in vitro restimulation, circulating memory B cells reactivated and produced spike-specific antibodies. A high frequency of spike-specific IgG⁺ plasmablasts, identified by computational analysis 7 days after boost, positively correlated with the generation of IgG⁺ memory B cells at 6 months. These data demonstrate that mRNA BNT162b2 vaccine elicits strong B cell immunity with spike-specific memory B cells that still persist 6 months after vaccination, playing a crucial role for a rapid response to SARS-CoV-2 virus encounter.

Keywords: BNT162b2 vaccine; COVID-19; SARS-CoV-2; memory B cells; vaccination.

Figure 1

Study design. HCWs (145 subjects) vaccinated with two doses of the BNT162b2 mRNA (Pfizer-BioNTech; Comirnaty) vaccine 3 weeks apart were enrolled in the study. Blood samples were collected at the baseline (day 0), 7, 21, 28 (7 days post second dose), 90 (3 months after the first vaccination dose), and 160–180 (5–6 months after the first vaccination dose) days after vaccination. Plasma and peripheral blood mononuclear cells (PBMCs) were assessed for spike-specific antibodies and memory B cells, respectively.

Figure 2

Spike-specific antibody response following BNT162b2 mRNA vaccination. (A) Spike-specific IgG analyzed by ELISA in plasma collected 0, 7, 21, 28, 90, 160–180 days after the first dose of the BNT162b2 mRNA vaccine (arrows). Antibody titers are expressed as the reciprocal of the dilution of sample reporting an OD value double respect to the background. Data are shown as box and whiskers diagram showing the minimum and maximum of all the data. (B) Titers of IgG anti wild type Spike and RBD, or anti mutated Spike protein (Alpha B.1.1.7, Beta B.1.351, Gamma P.1 and Delta B1.617) in plasma collected at day 28 (7 days after the second vaccination dose). (C) Spike-specific IgG1, IgG2, IgG3, IgG4, IgM, and IgA titers in plasma collected at day 28 (7 days after the second vaccination dose). (D) Surrogate virus neutralization test performed at days 21, 28, 90, and 160–180 after the first vaccination dose of the BNT162b2 mRNA vaccine. Data are reported as ACE2/RBD binding inhibition percentage with box and whiskers diagram showing the minimum and maximum of all the data. A threshold (dotted red line) was placed at 30% inhibition percentage to discriminate between positive and negative samples. Kruskal-Wallis test, followed by Dunn’s post-test for multiple comparisons, was used for assessing statistical differences between groups. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Figure 3

Spike-specific memory B cell response following BNT162b2 mRNA vaccination. Identification of spike-specific B cells by flow cytometry within PBMCs collected at different time points following vaccine administration. (A) Gating strategy for identifying CD19⁺ spike-specific B cells (named S⁺ B cells) by multiparametric flow cytometry. (B) Percentages of S⁺ B cells in each subjects assessed immediately before (day 21) and after (days 28, 90, and 160–180) the second dose. (C) Dot plot analysis of CD38 vs. IgD within S⁺ B cells, for identifying IgD^-CD38 ^high plasmablasts (PB) and Ig-switched memory B cells (swMBC). (D, E) Percentages of PB (D) and swMBC (E) in single subjects assessed at days 21, 28, 90, and 160–180. (F) Dot plot analysis of IgA, IgG, and IgM expression within swMBC. (G). Mean value (± SEM) of IgM, IgA and IgG swMBC percentages at 28, 90 and 180 days after vaccination. (H) Percentages of IgM, IgA, and IgG swMBC in single subjects assessed at days 160–180. Dot plots in (A, C, F) are representative from a single subject; values in (B, D, E, G, H) are reported as box and whiskers diagram showing the minimum and maximum of all of the data; percentages are reported respect to the parent population (in brackets). Statistical difference was assessed by Kruskal-Wallis test; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Figure 4

Computational analysis of spike-specific B cells following BNT162b2 mRNA vaccination. (A) Heatmap of metaclusters from the FlowSOM analysis of CD19⁺Spike⁺ B cells. Each marker is reported in column, while the different metaclusters (M) are reported in rows. The percentage of cells positive for each marker is reported inside the heatmap boxes, and visualized with a color scale from blue (0%) to red (100%). (B) Heatmap reporting the normalized frequency of each metacluster (rows) assessed in each subject (columns) in a color scale from blue (low) to red (high). Columns were grouped by the sampling day, as reported above the heatmap. (C) Box and whiskers plots showing the frequency of each metacluster at different time points. Values from individual samples were reported as circles. Mann–Whitney test corrected for multiple tests (Benjamini-Hochberg method) was used for assessing the statistical differences between different time points; statistical significance was defined as FDR < 10^-2. (D) Correlation analysis between significantly modulated metaclusters at day 28 (M4, M5, M6, and M7) and at day 180 (M1, M2, and M8). Multiple correlations were visualized as matrix with the Spearman’s correlation coefficient values reported. Significant values FDR < 0.05; correlation coefficient between 1 [red] and –1 [blue]. Only significant correlation were colored.

Figure 5

Spike-specific memory B cell response following BNT162b2 mRNA vaccination evaluated by B-cell ELISPOT. (A) Representative images of ELISpot wells coated with spike (left), an unrelated antigen (center), or anti-immunoglobulin (Ig, right) and developed in blue and red for IgG and IgM, respectively, after incubation of PBMCs. Cells were collected 160–180 days following the first dose of mRNA BTN162b2 vaccination, and restimulated in vitro with B-Poly-S for 4 days to induce resting MBC differentiation into antibody-secreting cells. (B, C) The frequencies of spike-specific MBCs secreting IgG (B) or IgM (C) antibodies are reported as percentages of total MBCs producing antibodies of the respective isotype. Bars indicate mean ± SEM. Mann–Whitney test, followed by Dunn’s post-test for multiple comparisons, was used for assessing the statistical difference between Spike-specific and unrelated antigen-specific B cells. **P ≤ 0.01.