SARS-CoV-2 mRNA vaccines induce persistent human germinal centre responses

Abstract

SARS-CoV-2 mRNA-based vaccines are about 95% effective in preventing COVID-19^1-5. The dynamics of antibody-secreting plasmablasts and germinal centre B cells induced by these vaccines in humans remain unclear. Here we examined antigen-specific B cell responses in peripheral blood (n = 41) and draining lymph nodes in 14 individuals who had received 2 doses of BNT162b2, an mRNA-based vaccine that encodes the full-length SARS-CoV-2 spike (S) gene¹. Circulating IgG- and IgA-secreting plasmablasts that target the S protein peaked one week after the second immunization and then declined, becoming undetectable three weeks later. These plasmablast responses preceded maximal levels of serum anti-S binding and neutralizing antibodies to an early circulating SARS-CoV-2 strain as well as emerging variants, especially in individuals who had previously been infected with SARS-CoV-2 (who produced the most robust serological responses). By examining fine needle aspirates of draining axillary lymph nodes, we identified germinal centre B cells that bound S protein in all participants who were sampled after primary immunization. High frequencies of S-binding germinal centre B cells and plasmablasts were sustained in these draining lymph nodes for at least 12 weeks after the booster immunization. S-binding monoclonal antibodies derived from germinal centre B cells predominantly targeted the receptor-binding domain of the S protein, and fewer clones bound to the N-terminal domain or to epitopes shared with the S proteins of the human betacoronaviruses OC43 and HKU1. These latter cross-reactive B cell clones had higher levels of somatic hypermutation as compared to those that recognized only the SARS-CoV-2 S protein, which suggests a memory B cell origin. Our studies demonstrate that SARS-CoV-2 mRNA-based vaccination of humans induces a persistent germinal centre B cell response, which enables the generation of robust humoral immunity.

Extended Data Fig. 1|. Antibody response to SARS-CoV-2 immunization.

a, Plasma IgA (left) and IgM (right) titres against SARS-CoV-2 S measured by ELISA in participants without (red) and with (black) a history of SARS-CoV-2 infection at baseline, and 3, 4, 5, 7 and 15 weeks after immunization. b, Neutralizing activity of serum against WA1/2020 D614G (left), B.1.1.7 (middle) and a chimeric virus expressing B.1.351 S (right) in Vero-TMPRSS2 cells at baseline, 3, and 5 or 7 weeks after immunization in participants without (red) and with (black) a history of SARS-CoV-2 infection. P values from two-sided Mann-Whitney tests. Dotted lines indicate limits of detection. Horizontal lines indicate the geometric mean. Symbols at each time point represent one sample (n = 41). Results are from one experiment performed in duplicate.

Extended Data Fig. 2|. Gating strategies for analysis of germinal centre response to SARS-CoV-2 immunization.

a, c, Sorting gating strategies for S-binding germinal centre B cells from FNAs (a) and total plasmablasts from PBMCs (c). b, Representative plot of germinal centre B cells in tonsil.

Extended Data Fig. 3|. Clonal analysis of germinal centre response to SARS-CoV-2 immunization.

a, Distance-to-nearest-neighbour plots for choosing a distance threshold for inferring clones via hierarchical clustering. After partitioning sequences based on common V and J genes and CDR3 length, the nucleotide Hamming distance of a CDR3 to its nearest nonidentical neighbour from the same participant within its partition was calculated and normalized by CDR3 length (blue histogram). For reference, the distance to the nearest nonidentical neighbour from other participants was calculated (green histogram). A clustering threshold of 0.15 (dashed black line) was chosen via manual inspection and kernel density estimate (dashed purple line) to separate the two modes of the within-participant distance distribution representing, respectively, sequences that were probably clonally related and unrelated. b, Clonal relationship of sequences from S-binding germinal centre-derived monoclonal antibodies (cyan) to sequences from bulk repertoire analysis of plasmablasts sorted from PBMCs (red) and germinal centre B cells (blue) 4 weeks after immunization. Each clone is visualized as a network in which each node represents a sequence and sequences are linked as a minimum spanning tree of the network. Symbol shape indicates sequence isotype: IgG (circle), IgA (star) and IgM (square); symbol size corresponds to sequence count.

Extended Data Fig. 4|. Lymph node plasmablast response to SARS-CoV-2 immunization.

a, c, Representative flow cytometry plots showing gating of CD20^lowCD38⁺ CD71⁺BLIMP1⁺S⁺ plasmablasts from IgD^lowCD19⁺CD3⁻ live singlet lymphocytes (a) and IgA and IgM staining on S⁺ plasmablasts (c) in FNA samples. b, Kinetics of S⁺ plasmablasts gated as in a from FNA of draining lymph nodes. Symbols at each time point represent one FNA sample; square symbols denote second lymph node sampled (n = 14). Horizontal lines indicate the median. d, Percentages of IgM⁺ (teal), IgA⁺ (yellow) or IgM⁻IgA⁻ (purple) S⁺ plasmablasts gated as in c in FNA of draining lymph nodes 4 weeks after primary immunization. Each bar represents one sample (n = 14).

Fig. 1|. Plasmablast and antibody response to SARS-CoV-2 immunization.

a, Study design. Forty-one healthy adult volunteers (ages 28–73, 8 with a history of SARS-CoV-2 infection) were enrolled and received the BNT162b2 mRNA SARS-CoV-2 vaccine. Blood was collected before immunization, and at 3, 4, 5, 7 and 15 weeks after immunization. For 14 participants (ages 28–52, none with a history of SARS-CoV-2 infection), FNAs of ipsilateral axillary lymph nodes (LNs) were collected at 3, 4, 5, 7 and 15 weeks after immunization. b, c, ELISpot quantification of S-binding IgG- (b) and IgA- (c) secreting plasmablasts (PBs) in blood at baseline, and at 3, 4, 5 and 7 weeks after immunization in participants without (red) and with (black) a history of SARS-CoV-2 infection. d, Plasma IgG titres against SARS-CoV-2 S (left) and the RBD of S (right) measured by ELISA in participants without (red) and with (black) a history of SARS-CoV-2 infection at baseline, and at 3, 4, 5, 7 and 15 weeks after immunization. Dotted lines indicate limits of detection. Symbols at each time point in b–d represent one sample (n = 41). Results are from one experiment performed in duplicate.

Fig. 2|. Germinal centre B cell response to SARS-CoV-2 immunization.

a, Representative colour Doppler ultrasound image of two draining lymph nodes (‘1’ and ‘2’) adjacent to the axillary vein ‘LAX V’ 5 weeks after immunization. b, c, Representative flow cytometry plots of BCL6 and CD38 staining on IgD^lowCD19⁺CD3⁻ live singlet lymphocytes in FNA samples (b; LN1, top row; LN2, bottom row) and S staining on BCL6⁺CD38^int germinal centre B cells in tonsil and FNA samples (c) at the indicated times after immunization. d, e, Kinetics of total (blue) and S⁺ (white) germinal centre (GC) B cells as gated in b and c (d) and S-binding per cent of germinal centre B cells (e) from FNA of draining lymph nodes. Symbols at each time point represent one FNA sample; square symbols denote the second lymph node sampled (n = 14). Horizontal lines indicate the median.

Fig. 3|. Clonal analysis of germinal centre response to SARS-CoV-2 immunization.

a, Binding of monoclonal antibodies (mAbs) generated from germinal centre B cells to SARS-CoV-2 S, N-terminal domain (NTD) of S, RBD, or S proteins of betacoronavirus OC43 or HKU1, measured by ELISA. Results are from one experiment performed in duplicate. Baseline for area under the curve was set to the mean + three times the s.d. of background binding to bovine serum albumin. b, Clonal relationship of sequences from S-binding germinal centre-derived monoclonal antibodies (cyan) to sequences from bulk repertoire analysis of plasmablasts from PBMCs (red) and germinal centre B cells (blue) sorted 4 weeks after immunization. Each clone is visualized as a network in which each node represents a sequence and sequences are linked as a minimum spanning tree of the network. Symbol shape indicates sequence isotype: IgG (circle), IgA (star) and IgM (square); symbol size corresponds to sequence count. c, d, Comparison of nucleotide mutation frequency in IGHV genes of naive B cells sorted from individuals vaccinated with influenza virus vaccine (grey) to clonal relatives of S-binding monoclonal antibodies among plasmablasts sorted from PBMCs and germinal centre B cells 4 weeks after immunization (green) in indicated participants (c) and between clonal relatives of S-binding monoclonal antibodies cross-reactive (purple) or not (teal) to seasonal coronavirus S proteins among plasmablasts sorted from PBMCs and germinal centre B cells 4 weeks after immunization (d). Horizontal lines and error bars indicate the median and interquartile range. Sequence counts were 2,553 (naive), 199 (participant 07), 6 (participant 20), 240 (participant 22), 54 (cross-reactive) and 391 (not cross-reactive). P values from two-sided Kruskal–Wallis test with Dunn’s post-test between naive B cells and S-binding clones (c) or two-sided Mann–Whitney U test (d).