mRNA-based influenza vaccine expands breadth of B cell response in humans

Abstract

Eliciting broad and durable antibody responses against rapidly evolving pathogens like influenza viruses remains a formidable challenge^1,2. The germinal center (GC) reaction enables the immune system to generate broad, high-affinity, and durable antibody responses to vaccination^3-5. mRNA-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines induce persistent GC B cell responses in humans^6-9. Whether an mRNA-based influenza vaccine could induce a superior GC response in humans compared to the conventional inactivated influenza virus vaccine remains unclear. We assessed B cell responses in peripheral blood and draining lymph nodes in cohorts receiving the inactivated or mRNA-based quadrivalent seasonal influenza vaccine. Participants receiving the mRNA-based vaccine produced more robust plasmablast responses and higher antibody titers to H1N1 and H3N2 influenza A viruses and comparable antibody titers against influenza B virus strains. Importantly, mRNA-based vaccination stimulated robust recall B cell responses characterized by sustained GC reactions that lasted at least 26 weeks post-vaccination in three of six participants analyzed. In addition to promoting the maturation of responding B cell clones, these sustained GC reactions resulted in enhanced engagement of low-frequency pre-existing memory B cells, expanding the landscape of vaccine-elicited B cell clones. This translated to expansion of the serological repertoire and increased breadth of serum antibody responses. These findings reveal an important role for the induction of persistent GC responses to influenza vaccination in humans to broaden the repertoire of vaccine-induced antibodies.

Extended Data Fig. 1.. Humoral response to mRNA-1010.

a, HA proteins encoded by mRNA-1010 for the 2022-2023 Northern Hemisphere influenza vaccine strains. Fluarix vaccine included the same strains, except H3N2 was egg-based (A/Darwin/9/2021). b, ELISpot quantification of mean HA-binding IgM-secreting PBs in blood at baseline, 1, and 2 weeks post vaccination in mRNA-1010 (red) and Fluarix (black) participants. Numbers of HA-specific PBs were quantified against the four vaccine HAs and averaged. c, ELISpot quantification of HA-binding IgM-secreting PBs at 1 week post vaccination in mRNA-1010 (red) and Fluarix (black) participants. Horizontal bars represent geometric means. P values determined by Mann-Whitney U test. d, Flow cytometry gating strategy for HA-specific MBCs from PBMCs e, Quantification of HA-specific MBCs as a percentage of CD19+ cells in blood by flow cytometry at baseline, 4, and 17/26 weeks post vaccination in mRNA-1010 (red) and Fluarix (black) participants. Numbers represent geometric mean frequencies of MBCs for each time point. f, Fold change in HA-specific MBCs as a percentage of CD19+ cells in blood by flow cytometry at indicated time points over week 0 for mRNA-1010 (red) and Fluarix (black) participants. Numbers represent mean values. In e and f, P values determined by Mann-Whitney U test. For participants that did not complete a blood collection at week 26 or PBMCs were not available, samples from 17 weeks post vaccination were used for analysis (mRNA-1010, n=3; Fluarix, n=2).

Extended Data Fig. 2.. Flow cytometry gating strategies for analysis of B cell responses in vaccinees.

a, Flow cytometry gating strategy for HA-specific GC B cells from FNAs. b, Gating strategy for sorting PBs and MBCs from peripheral blood. PBs were sorted as CD3⁻CD19⁺IgD^loCD20^loCD38⁺ live singlet lymphocytes; MBCs were sorted as CD3⁻CD19⁺IgD^lo live singlet lymphocytes.

Extended Data Fig. 3.. Identification of HA-specific B cell clones in lymph nodes and blood.

a and c, UMAPs showing scRNA-seq transcriptional clusters of total cells (a) and of B cells (c) from PBs and MBCs sorted from blood and FNA of draining axillary lymph nodes combined. b and d, Dot plots for the marker genes used for identifying annotated clusters in a and c, respectively. e, UMAP plots of transcriptional clusters of B cells for all samples from mRNA-1010 (06, 17) and Fluarix (22) participant with HA-specific clones as determined by mAb ELISA mapped onto transcriptional clusters as in Fig. 2c. Numbers of HA-specific cells (red) are shown at the bottom right. Numbers of HA-specific clones for all participants are shown in Extended Data Table 1.

Extended Data Fig. 4.. Analysis of germinal center antibody clones from mRNA-1010 participants

a, Equilibrium dissociation constant (K_D) of Fabs (n=21) derived from paired week 1 PB and week 26 GC B cell clones of mRNA-1010 participants interacting with immobilized HA protein measured by BLI. Clones were tested against HA strains based on predetermined binding specificity by mAb ELISA; clones with multiple binding specificities were tested individually against each HA strain. b, Area under the curve (AUC) values for mAbs (n=21) derived from paired week 1 PB and week 26 GC B cell clones of mRNA-1010 participants as measured by binding ELISA against HA protein. Clones were tested against HA strains based on predetermined binding specificity by mAb ELISA; clones with multiple binding specificities were tested individually against each HA strain. c, Phylogenetic trees of representative HA-specific IgA GC B cell clones from scRNA-seq data of mRNA-1010 participants. Left (397-29) represents a clone likely derived from an IgA+ MBC; right (397-06) represents a clone likely derived from an IgG+ MBC that class switched to IgA. IGHA, immunoglobulin heavy chain alpha constant region; IGHG, immunoglobulin heavy chain gamma constant region. PB, plasmablast; MBC, memory B cell; GC, germinal center B cell; LNPC, lymph node plasma cell.

Extended Data Fig. 5.. Antibody clonotypes comprising the H3-specific serological repertoire.

a, Heat maps showing the relative amounts of IgG clonotypes comprising the serological repertoire against H3 at different time points from all participants. Each column represents a unique clonotype, with its relative amount determined through proteomic analysis. b, Persistence of post-vaccination serological repertoire between week 4 and week 17/26. Each data point represents the relative abundance of the serum IgG clonotype, quantified through Ig-seq, detected at week 17/26 which originate from week 4.

Extended Data Fig. 6.. The B cell lineage tree of representative clonotypes identified from mRNA-1010 and Fluarix participants.

a and b, B cell lineage trees of clonotypes abundantly present in serum identified from mRNA-1010 participants 06 and 28 (a) and Fluarix participants 05, 09, and 22 (b) are shown. Each node is colored based on whether its CDRH3 sequence is categorized as pre-existing (turquoise) or vaccine-elicited (indigo) as determined by Ig-seq. Black color indicates CDRH3 sequences not detected in circulation. Branch lengths correspond to SHM per site, according to the scale bar. GC B cells are highlighted with the time points at which they were detected based on the scRNA-seq data.

Extended Data Fig. 7.. Breadth of binding of H1-specific serological repertoire.

a, Phylogenetic tree of influenza strains. H1N1 strains used for bead assay are highlighted in orange; H3N2 strains are highlighted in blue. b, Fold change in MFI of binding of plasma samples at 4 weeks over baseline for mRNA-1010 (red) and Fluarix (black) participants to beads coated in A/H1 glycoproteins. c, Fold change in MFI of binding of plasma samples at 17/26 weeks over baseline for mRNA-1010 (red) and Fluarix (black) participants to beads coated in A/H1 glycoproteins. Samples collected at 17 weeks post vaccination were used for patients that did not complete a blood draw at 26 weeks (mRNA-1010, n=2, Fluarix, n=2). For b and c, P values were determined by Mann-Whitney U test; bars represent median values.

Fig. 1.. Robust antibody response to mRNA-based seasonal influenza vaccination.

a, WU397 study design. We enrolled 29 healthy adults (ages 24-51) who received Fluarix (n=15) or mRNA-1010 (n=14) intramuscularly. Blood was collected before vaccination and at 1, 2, 4, 8, 17, and 26 weeks after vaccination. FNAs of ipsilateral axillary lymph nodes were collected before vaccination and at 2, 8, 17, and 26 weeks after vaccination. b, ELISpot quantification of mean HA-binding IgG- and IgA-secreting PBs in blood at baseline, 1, and 2 weeks after vaccination in mRNA-1010 (red) and Fluarix (black) participants. Numbers of HA-binding PBs were quantified against the four vaccine HAs and averaged. c, ELISpot quantification of HA-binding IgG- and IgA-secreting PBs at 1 week post vaccination in mRNA-1010 (red) and Fluarix (black) participants. Horizontal bars represent geometric means. P values determined by Mann-Whitney U test. d, Plasma IgG titers at baseline, 4, and 17/26 weeks post vaccination (left) and fold change in plasma IgG titers at 4 and 17/26 weeks over baseline (right) against the four vaccine HAs in mRNA-1010 (red) and Fluarix (black) participants. Numbers on left panels represent geometric mean titers for each time point; numbers on right panels represent mean fold changes. P values determined by Mann-Whitney U test. e, HAI titers for the four vaccine virus strains in mRNA-1010 (red) and Fluarix (black) participants at baseline, 4, and 17/26 weeks. Bars represent geometric mean with 95% confidence interval. P values determined by Wilcoxon matched pairs signed rank test. In d-e, for participants that did not complete a blood collection at week 26, samples from 17 weeks post vaccination were used for analysis (mRNA-1010, n=2; Fluarix, n=2).

Fig. 2.. Characterizing the germinal center response to mRNA-1010.

a, Representative flow cytometry plots of Bcl6 and HA-probe staining on CD3⁻CD19⁺CD20⁺IgD^loBcl6⁺CD38^int live singlet lymphocytes in FNA samples at baseline, 2, 8, 17, and 26 weeks post vaccination. Top, representative mRNA-1010 participant (red, 06); bottom, representative Fluarix participant (black, 05). b, Frequencies of HA-specific GC B cells determined by flow cytometry in FNA samples from mRNA-1010 (red) and Fluarix (black) participants. P values determined by Mann-Whitney U test (P=0.0365, *P<0.05). c, Representative uniform manifold approximation and projection (UMAP) plots of transcriptional clusters of B cells from baseline (sorted MBCs, FNA), 1 (sorted PBs), 2 (FNA), 8 (FNA), 17 (FNA), and 26 (sorted MBCs, FNA) weeks post vaccination (mRNA-1010 participant 29). Each dot represents a cell, colored by phenotype as defined by transcriptomic profile. Total numbers of cells are shown at the bottom right. PB, plasmablast; ABC, activated B cell; MBC, memory B cell; GCB, GC B cell; LNPC, lymph node plasma cell. d, UMAP plots of transcriptional clusters of B cells for all samples from mRNA-1010 (28, 29) and Fluarix (05, 09) participants as in c, with HA-specific clones as determined by mAb ELISA mapped onto transcriptional clusters. Numbers of HA-specific cells (red) are shown at the bottom right. Numbers of HA-specific clones for all participants are shown in Extended Data Table 1.

Fig. 3.. Germinal centers induced by mRNA-1010 recruit low frequency memory B cells and increase somatic hypermutation.

a, Clonal overlap of sequences between PBs sorted from PBMCs 1 week after vaccination and GC B cells from all FNA time points among HA-specific clones for mRNA-1010 participants (06, 17, 28, 29, top) and Fluarix participants (05, 09, 22, bottom). Chord width corresponds to clonal population size; numbers of HA-specific clones are in Extended Data Table 2. Percentages are of GC B cell clones overlapping with PBs and PB clones overlapping with GC B cells. b, Nucleotide mutation frequency in the immunoglobulin heavy chain variable gene (IGHV) and light chain variable gene (IGLV) region for clonally related week 1 PBs and week 26 GC B cells (n=21) from mRNA-1010 participants. P values determined by Wilcoxon matched pairs signed rank test. c, Representative flow cytometry plots of IgG and IgA staining on CD3−CD19⁺CD20⁺IgD^loBcl6⁺CD38^intHA⁺ live singlet lymphocytes in FNA samples at 8 weeks post vaccination. Left, representative mRNA-1010 participant (red, 06); right, representative Fluarix participant (black, 05). d, Frequencies of IgG+ (top) and IgA+ (bottom) HA-specific GC B cells determined by flow cytometry in FNA samples from mRNA-1010 (red) and Fluarix (black) participants. e, Proportions of isotypes of HA-specific cells from scRNA-seq; BCR specificity determined by mAb ELISA and cells identified in transcriptional scRNA-seq clusters from mRNA-1010 (06, 17, 28, and 29) and Fluarix (05, 09, and 22) participants. Left, all B cells in scRNA-seq data; right, only GC B cells in scRNA-seq data.

Fig. 4.. Delineation of H3-specific serological repertoires following vaccination.

a, A schematic illustration of the proteomic analysis of serum immunoglobulins (Ig-seq) combined with high-throughput sequencing of B cell transcripts (BCR-seq) to identify the serological antibody repertoire to H3. b, Heat maps showing the relative amounts of IgG clonotypes comprising the serological repertoire against H3 at different time points from representative subjects (06, mRNA-1010; 05, Fluarix). Each column represents a unique serum IgG clonotype, with its relative amount determined through proteomic analysis. c, Relative abundance of pre-existing antibody clonotypes in serum at peak responses. Each data point represents an individual participant, quantified via Ig-seq. d, Number of serum vaccine-elicited H3-specific IgG clonotypes in serum at peak responses. Each data point represents an individual participant. e, Diversification of CDRH3 peptides detected sequences from representative clonotypes of the serological repertoire against H3 at baseline and peak time point. New amino acid replacement mutations are highlighted in red. Asterisks (*) indicate clones identified in scRNA-seq data in the week 1 PB compartment. f, Number of unique CDRH3 peptides that constitute individual IgG clonotypes. g, Relative abundance of newly elicited IgG following vaccination detected via Ig-seq. h, Change in diversity indices from baseline to peak response. Each line represents the change in diversity indices for an individual participant. i, Predominance of H3-specific GC cells determined by scRNA-seq data. Each data point indicates the fraction of H3-specific GC cells or GC clones for a specific participant. j, Correlation between serum titer at peak response and the proportion of vaccine-elicited IgG at peak response. ρ represents Spearman’s correlation coefficient. For c, d, g, and i, individual data points and the mean values are shown. For c, d, and h, statistical analyses were performed using two-tailed Mann-Whitney U test (*P<0.05). For j, two-tailed Spearman rank correlation test was performed.

Fig. 5.. Increase in breadth of the H3-specific serological repertoire.

a, B cell lineage trees of selected clonotypes. Each node is colored based on whether its CDRH3 sequence is categorized as pre-existing (turquoise) or vaccine-elicited (indigo) as determined by Ig-seq. Black color indicates CDRH3 sequences not detected in circulation. Branch lengths correspond to SHM per site, according to the scale bar. GC B cells are highlighted with the time points at which they were detected based on the scRNA-seq data. A sub-branch of BCRs with CDRH3 sequences matching to vaccine-elicited IgG and GC B cells is highlighted in a box. Top, mRNA-1010 participant (06); bottom, Fluarix partiicpant (05). b, Fold change in MFI of binding of plasma samples at 4 weeks post vaccination over baseline for mRNA-1010 (red) and Fluarix (black) participants to beads coated in a panel of A/H3 glycoproteins. c, Fold change in MFI of binding of plasma samples at 17/26 weeks over baseline for mRNA-1010 (red) and Fluarix (black) participants to beads coated in a panel of A/H3 glycoproteins. Samples collected at 17 weeks post vaccination were used for patients that did not complete a blood draw at 26 weeks (mRNA-1010, n=2, Fluarix, n=2). For b and c, P values were determined by Mann-Whitney U test. Bars represent median values.