Memory B Cells Induced by Sputnik V Vaccination Produce SARS-CoV-2 Neutralizing Antibodies Upon Ex Vivo Restimulation

Abstract

The development of effective vaccines against SARS-CoV-2 remains a global health priority. Despite extensive use, the effects of Sputnik V on B cell immunity need to be explored in detail. We performed comprehensive profiling of humoral and B cell responses in a cohort of vaccinated subjects (n = 22), and demonstrate that Sputnik vaccination results in robust B cell immunity. We show that B memory cell (MBC) and antibody responses to Sputnik V were heavily dependent on whether the vaccinee had a history of SARS-CoV-2 infection or not. 85 days after the first dose of the vaccine, ex vivo stimulated MBCs from the vast majority of Sputnik V vaccinees produced antibodies that robustly neutralized the Wuhan Spike-pseudotyped lentivirus. MBC-derived antibodies from all previously infected and some of the naïve vaccine recipients could also cross-neutralize Beta (B.1.351) variant of SARS-CoV-2. Virus-neutralizing activity of MBC-derived antibodies correlated well with that of the serum antibodies, suggesting the interplay between the MBC and long-lived plasma cell responses. Thus, our in-depth analysis of MBC responses in Sputnik V vaccinees complements traditional serological approaches and may provide important outlook into future B cell responses upon re-encounter with the emerging variants of SARS-CoV-2.

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

Figure 1

Virus-binding and virus-neutralizing activity of sera from Sputnik V-vaccinated individuals. (A) Study design. (B) Serum anti-RBD IgG (top row), IgA (middle row) or IgM (bottom row) levels for all Sputnik V-vaccinated individuals, measured by ELISA. IgA and IgM levels are shown as a relative units (RU) against a standard convalescent serum. (C) Representative neutralization curves of vaccine-induced sera from one recovered and one naïve individual at T3 time point. (D) Paired analysis of neutralization titers (ID₅₀) against WA1 strain and Beta variant at T3 time point. (E) Analysis of hillslopes of virus neutralization curves for sera from vaccinated individuals. (F) Spearman’s correlation between serum virus neutralization half-maximal inhibitory serum dilution (ID₅₀) values and the serum levels of anti-RBD IgG (left panel), IgA (middle panel) and IgM (right panel). Blue and red symbols indicate naïve (n = 17) and recovered (n = 5) participants. Symbols connected by solid lines represent time points considered for each individual. Data are presented as median ± IQR. The dotted lines indicate the threshold for positivity. Statistics were calculated using Mann-Whitney test (comparisons between WA1 strain and Beta variant) or the Kruskal–Wallis test (comparisons between time points and naïve and recovered, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant. GMT, geometric mean titer; ID₅₀, half-maximal inhibitory dilution; IQR, interquartile range; RBD, receptor-binding domain; RU, relative units.

Figure 2

Quantification of total and RBD-specific plasmablasts in blood samples from Sputnik V-vaccinated individuals. (A) Representative flow cytometry dot plots showing the gating strategy for measuring the percentage of total (left panel) and RBD⁺ (middle panel) plasmablasts. As a negative control, sample stained with an irrelevant protein Bet v 1 is shown (right panel). Numbers inside the plots indicate the percentage of events specific to respective gates. (B, C) Dynamic changes in total (B) and RBD⁺ (C) plasmablast frequencies in samples collected at different time points. (D) Representative ELISpot images for measuring the frequencies of circulating total (left), anti-RBD (middle) or anti-S (right) IgG ASCs. Numbers below the wells represent the frequencies of ASCs relative to the total number of cells in the well. (E) Frequencies of circulating IgG (left column), IgA (middle column) or IgM (right column) ASCs specific for RBD (upper row) or S (bottom row) antigens per 10⁶ PBMCs collected from vaccinated individuals at different time points. The dotted lines indicate the threshold for positive antigen-specific ASC responses. (F) Spearman’s correlation between RBD-specific (IgG + IgA + IgM) ASCs and the levels of RBD⁺ plasmablasts at T1 (left) and T2 (right) time points. Results are shown for individual samples (symbols) from naïve (n = 17) and recovered (n = 5) recipients. Data are presented as median ± IQR. Asterisks indicate significant difference between groups determined using the Kruskal–Wallis test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant. ACS, antibody-secreting cell; IQR, interquartile range; RBD, receptor-binding domain.

Figure 3

Analysis of the MBC response in Sputnik V-vaccinated individuals. (A) Representative flow cytometry dot plots showing double discrimination of RBD⁺ MBCs. Numbers inside the plots indicate the percentage of events specific to the respective gates. (B) RBD⁺ MBCs as a percentage of all memory B cells (CD19⁺CD27⁺CD38^-IgD^-). (C) Representative ELISpot showing SARS-CoV-2-specific MBC-derived ASCs. Purified B cells were stimulated with IL-21/CD40L for 7 days and then incubated in ELISpot plates for 16 h to detect ASCs secreting total (left column), RBD-(middle column) or S-specific (right column) IgG at T2 time point (upper row), T3 (middle row) or in historic control samples (bottom row). The numbers indicated below the wells represent positive dots and the total number of cells in the well. (D) RBD- (upper row) and S-specific (bottom row) MBC-derived ASCs per 10⁶ B cells from blood samples of naïve (n = 17) or recovered (n = 5) vaccinated individuals at different time points. Data for IgG (left column), IgM (middle column) and IgA (right column) ASCs are presented. The dotted lines indicate the threshold for a positive antigen-specific ASC response calculated with pre-pandemic samples per 10⁶ B cells. Results are shown for individual samples (symbols) from naïve (n = 17) and recovered (n = 5) recipients. Data are presented as median ± IQR. Asterisks indicate significant difference between groups determined using the Kruskal–Wallis test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant. ASC, antibody-secreting cell; IQR, interquartile range; RBD, receptor-binding domain.

Figure 4

Analysis of MBC-derived antibody response in supernatants of CD40L/IL-21 stimulated B cells from Sputnik V-vaccinated individuals. (A) Production of RBD-specific IgG (left panel), IgA (middle panel) or IgM (right panel) in cultures of IL-21/CD40L-stimulated B cells from Sputnik V-vaccinated individuals evaluated using ELISA. (B) Virus-neutralizing activity of MBC-derived antibodies against WA1 strain (left panel) and Beta variant (right panel) at T2 and T3 time points. (C) Paired analysis of virus-neutralizing activity of MBC-derived antibodies against WA1 strain and Beta variant at T3 time point. (D) Spearman’s correlation between virus neutralization (%) and the levels of anti-RBD IgG in supernatants of IL-21/CD40L-stimulated B cells obtained from Sputnik V-vaccinated individuals at T3 time point. (E) Spearman’s correlation between the virus-neutralizing activity of plasma and MBC-derived antibodies. Each symbol represents half-maximal inhibitory plasma dilution (ID₅₀) values and % of virus neutralization by supernatant of IL-21/CD40L-stimulated B cells. (F) Heatmap and hierarchical clustering of Sputnik V recipients. Columns denote Sputnik V recipients. Rows correspond to immune response variables. Dendrograms on the top illustrate the clustering of Sputnik V recipients. Immune response measurement values are color-coded according to the key shown in the upper left. (G) Principal component analysis of Sputnik V recipients. Recipient IDs are shown. Two distinct clusters are indicated by the ovals. Results are shown for individual samples (symbols) from naïve (n = 17) and recovered (n = 5) recipients. Data are presented as median ± IQR. Asterisks indicate significant difference between groups determined using the Kruskal–Wallis test, statistics in (C) panel were calculated using Mann-Whitney test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant. IQR, interquartile range; RBD, receptor-binding domain.