Germinal centre-driven maturation of B cell response to SARS-CoV-2 vaccination

Abstract

Germinal centres (GC) are lymphoid structures where vaccine-responding B cells acquire affinity-enhancing somatic hypermutations (SHM), with surviving clones differentiating into memory B cells (MBCs) and long-lived bone marrow plasma cells (BMPCs) ^1-4 . Induction of the latter is a hallmark of durable immunity after vaccination ⁵ . SARS-CoV-2 mRNA vaccination induces a robust GC response in humans ^6-8 , but the maturation dynamics of GC B cells and propagation of their progeny throughout the B cell diaspora have not been elucidated. Here we show that anti-SARS-CoV-2 spike (S)-binding GC B cells were detectable in draining lymph nodes for at least six months in 10 out of 15 individuals who had received two doses of BNT162b2, a SARS-CoV-2 mRNA vaccine. Six months after vaccination, circulating S-binding MBCs were detected in all participants (n=42) and S-specific IgG-secreting BMPCs were detected in 9 out of 11 participants. Using a combined approach of single-cell RNA sequencing of responding blood and lymph node B cells from eight participants and expression of the corresponding monoclonal antibodies, we tracked the evolution of 1540 S-specific B cell clones. SHM accumulated along the B cell differentiation trajectory, with early blood plasmablasts showing the lowest frequencies, followed by MBCs and lymph node plasma cells whose SHM largely overlapped with GC B cells. By three months after vaccination, the frequency of SHM within GC B cells had doubled. Strikingly, S ⁺ BMPCs detected six months after vaccination accumulated the highest level of SHM, corresponding with significantly enhanced anti-S polyclonal antibody avidity in blood at that time point. This study documents the induction of affinity-matured BMPCs after two doses of SARS-CoV-2 mRNA vaccination in humans, providing a foundation for the sustained high efficacy observed with these vaccines.

Figure 1.. Persistence of humoral immune responses to SARS-CoV-2 mRNA vaccination.

a, Study design. Forty-three healthy adult volunteers (13 with a history of SARS-CoV-2 infection) were enrolled, followed by BNT162b2 mRNA SARS-CoV-2 vaccination. Blood (n=42) was collected before immunization, and at 3, 4, 5, 7, 15, and 29 weeks after primary immunization. For 15 participants without a history of SARS-CoV-2 infection, aspirates of ipsilateral axillary lymph nodes were collected at 3, 4, 5, 7, 15, and 29 weeks after primary vaccination. For 11 participants without a history of SARS-CoV-2 infection, aspirates of bone marrow were collected at 29 weeks after primary vaccination. b, Representative flow cytometry plots of GC B cells (CD19⁺ CD3⁻ IgD^low BCL6⁺ CD38^int live singlet lymphocytes) and SARS-CoV-2 S staining on GC B cells in draining lymph nodes 29 weeks post-vaccination. c, Kinetics of total (left) and S⁺ GC B cells (right) as gated in b. d, Representative ELISpot wells coated with the indicated antigens or anti-immunoglobulin and developed in blue and red for IgG and IgA, respectively, after plating the indicated numbers of magnetically enriched BMPCs. e, Frequencies of BMPCs secreting IgG antibodies specific for the indicated antigens 29 weeks after vaccination. f, Plasma IgG titers against SARS-CoV-2 S measured by ELISA in participants without (red) and with (black) a history of SARS-CoV-2 infection. Horizontal lines indicate geometric means, also shown above time points. Results are from one experiment performed in duplicate. g, Representative flow cytometry plot of SARS-CoV-2 S staining on MBCs (CD20⁺ CD38⁻ IgD^low CD19⁺ CD3⁻ live singlet lymphocytes) in blood 29 weeks after primary vaccination. h, Frequencies of S⁺ MBCs in participants without (red) and with (black) a history of SARS-CoV-2 infection as gated in g. Horizontal lines indicate median values in e and h. Dotted lines indicate limits of detection in e and f. Symbols at each time point represent one sample in c (n=15), e (n=11), f (n=38), and h (n=42).

Figure 2.. Identification of SARS-CoV-2 S-binding B cell clones in draining lymph nodes.

a, Uniform manifold approximation and projection (UMAP) of scRNA-seq data from PBs sorted from PBMC (upper) and whole FNA of draining axillary lymph nodes (lower), and UMAP of B cell scRNA-seq clusters from each compartment (right). Each dot represents a cell, colored by phenotype as defined by the gene expression profile. Total numbers of cells are at the top right corner. FDC, follicular dendritic cell; GC, germinal center B cell; Mo, monocyte; NK, natural killer cell; LNPC, lymph node plasma cell; PB, plasmablast; pDC, plasmacytoid dendritic cell; RMB, resting memory B cell. b, Positive binding of recombinant monoclonal antibodies (mAbs) derived from GC B cells (blue) or LNPCs (green) to SARS-CoV-2 S measured by ELISA. Areas under the curve were calculated by setting the mean + three times the s.d. of background binding to bovine serum albumin (BSA) as a baseline. Results are from one experiment performed in duplicate. c, SARS-CoV-2 S-binding clones visualized on UMAP of B cell clusters in each participant. Percentages are of SARS-CoV-2 S-binding clones within GC B cells (blue), LNPCs (green), PBs (red), RMBs (pink) or naive B cells (yellow). Total numbers of cells are at the bottom right corner.

Figure 3.. Maturation of SARS-CoV-2 S-binding B cells in the lymph node.

a, Clonal overlap of SARS-CoV-2 S-binding sequences from bulk and single-cell BCR repertoire analysis between PBs sorted from blood 4 week post-vaccination and GC B cells at indicated time points. Arc length corresponds to the number of BCR sequences and chord width corresponds to clonal group size. Purple chords correspond to clones spanning both the PB and GC compartments. Grey chords correspond to clones spanning only the GC compartment. Percentages are of GC B cell clones related to PBs at each time point. b, Comparison of immunoglobulin heavy chain variable (IGHV) region nucleotide mutation frequency of clonally related, SARS-CoV-2 S-binding PBs and GC B cell clones that are clonally linked at the indicated time points. Median values are presented on the top of each data set. c, Comparison of IGHV nucleotide mutation frequency of SARS-CoV-2 S-binding GC B cells at the indicated time points. Horizontal lines and colored numbers represent median values. P values were determined by Kruskal-Wallis test followed by Dunn’s multiple comparison test. d, Percentages of GC B cells expressing IGHG (blue), IGHA (red), IGHM (green) or IGHD (pink) at the early (E) or the late (L) time point. The early time point represents 4, 5 or 7 weeks after immunization. The late time point represents 15 weeks post-immunization. Total cell numbers are on the top of each column. e, Circos diagram showing overlap of S-binding clonal groups between GC B cells and LNPCs over combined time points. Arc length corresponds to the number of BCR sequences and chord width corresponds to clonal group size. Purple chords link GC B cells to clonally overlapping LNPCs. Percentages are of GC B cell clones overlapping with LNPCs or vice versa in each participant. f, Comparison of IGHV nucleotide mutation frequency of clonally related, S-binding GC B cell and LNPCat the indicated time points. Median values are presented on the top of each data set. Each dot represents the median SHM of a clonal group within the indicated compartment in b and f. P values were determined by paired two-sided non-parametric Mann-Whitney test corrected for multiple testing using Benjamini and Hochberg’s method in b and f.

Figure 4.. Evolution of B cell clones induced by SARS-CoV-2 vaccination.

a, Comparison of avidity indices of plasma IgG against SARS-CoV-2 S between the indicated time points in participants without (red) and with (black) a history of SARS-CoV-2 infection. Avidity index was defined as the ratio of optical density values obtained in the presence and absence of 8M urea. Data are P values were determined by Wilcoxon matched-pairs signed rank test. Results are from one experiment performed in duplicate. b, IGHV nucleotide mutation frequency of S-binding PB, LNPC, and BMPC at the indicated time points. Horizontal lines and colored numbers represent median values. P values were determined by Kruskal-Wallis test followed by Dunn’s multiple comparison test. c, Phylogenetic trees of each clonal group showing inferred relations between PB (squares), LNPC (triangles), and BMPC (diamonds). ELISA-verified S-binding mAb IDs and clonal group IDs are indicated in the diagram. Horizontal branch length represents the expected number of substitutions per codon in V-region genes, corresponding to the key.