Capsid inclusion in a CHIKV mRNA vaccine impairs adaptive immune responses

Figures

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  Figure 1. V1 and V2 vaccinations induce distinct systemic immune-cell landscapes (A) A total of nine (n = 9) male rhesus macaques (aged 4–8 years) were randomly assigned to three groups: negative control (mock-treatment with phosphate-buffered saline), V1 (mRNA vaccine 1), and V2 (mRNA vaccine 2). The animals were vaccinated twice, with a 21-day interval between doses, and challenged with CHIKV on day 43. All animals were sacrificed on day 50. PBMCs and serum were collected before immunization (V1BG and V2BG) at 0 dpi, after immunization (V1 and V2) at 42 dpi, and post-challenge (CV1, CV2, and C) at 50 dpi for single-cell RNA sequencing (PBMC), BCR, and TCR repertoire sequencing (PBMC), as well as the Olink cytokine and chemokine panel (serum). Drawn by FigDraw 2.0 (copyright: STUYW53550). (B) PBMCs were clustered into cell types based on marker gene expression; UMAP shows the low-dimensional embedding (resolution = 0.8), where each point represents a single cell and proximity reflects transcriptomic similarity. (C and D) Distinct cell types are color-coded. Cell type annotation was performed by using marker genes for B cells (C) and T cells (D). (E and F) UMAP plots showing overall…

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  Figure 2. V2 vaccination preferentially promotes early B cell activation and pre-plasmablast differentiation (A) UMAP visualization of integrated B cells from all groups. B cells from all groups based on single-cell transcriptomic profiling were re-clustered to delineate subsets. Each point on the UMAP plot represents a single cell, and those with similar gene expression profiles are clustered in close proximity. Distinct B cell subsets are color-coded. (B) Bubble map showing the expression of representative marker genes across B cell subsets. The color gradient indicates the expression levels, and bubble size represents the percentage of cells expressing each gene. (C) B cell subsets were annotated on the basis of highly expressed marker genes in each cluster. (D) UMAP plots showing the spatial distribution of B cell subsets at baseline (V1BG, n = 3; V2BG, n = 3; top) and post-immunization (V1, n = 3; V2, n = 3; bottom). Green and red boxes highlight pre-plasmablasts in the V1 and V2 groups, respectively. The yellow box indicates increased abundance of early-activated B cells in the V2 group. (E) The proportions of B cell subsets before (n = 3) and after vaccination in V1 (n = 3) and V2…

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  Figure 3. V2 drives sustained B cell progression toward late differentiated and effector states (A) Cell trajectory analysis of B cell subtypes was performed using Monocle2. Each point in the trajectory plot represents a single cell, arranged along a pseudotime axis that reflects the progression from an early to a later developmental state. Pseudotime along the developmental trajectory is indicated by the color density bar (top), as is the distribution of each B cell subset along the trajectory (bottom). Black circles with numbers denote branching nodes. (B) Developmental trajectories of early-activated B cells (top) and pre-plasmablasts (bottom) are shown at baseline (V1BG and V2BG) and post-vaccination (V1 and V2). Each dot represents an individual cell. (C) Heatmap visualizing the top differentially expressed genes across the trajectory. Gene expression levels are indicated by the color scale (left). GO enrichment analysis of genes highly expressed at early (blue box) and late (green box) pseudotime stages was performed. Bubble size reflects the number of enriched genes, and color represents adjusted p values.

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  Figure 4. V2 establishes a differentiated, IgG-associated humoral immune repertoire with enhanced recall potential (A) KEGG pathway enrichment analysis of downregulated (left) and upregulated (right) DEGs in B cells of V2 (compared with V1). Bubble size indicates the number of genes enriched in each pathway, and color reflects the adjusted p value. (B) Gene set enrichment analysis (GSEA) revealed significantly upregulated pathways in V2 compared with V1 within early-activated B cells, including Fc gamma R-mediated phagocytosis (NES = 1.62, top) and inflammatory mediator regulation of TRP channels (NES = 1.55, bottom). (C) Comparison of antibody isotype abundance and BCR diversity (D50 index) between post-vaccination (V1, n = 3; V2, n = 3) and infection-only control (C) groups. (D) Comparison of antibody isotype abundance and BCR diversity (D50 index) between post-challenge (CV1, n = 3; CV2, n = 3) and infection-only control (C, n = 3) groups. (E) Calculated a plasmablast differentiation/secretory program score on the basis of genes associated with antibody secretion and plasma cell commitment through the AddModuleScore function across B cell subsets. (F) The VDJ usage frequencies of IgM…

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  Figure 5. V1 and V2 induce distinct T cell differentiation and activation trajectories (A) T cells were re-clustered to delineate finer subsets. UMAP showed dimensionality reduction for each subset (resolution = 0.8); cells with similar gene expression profiles clustered in close proximity. Distinct T cell subsets are color-coded. Expression maps of T cell marker genes CD3D/CD3E confirmed the accuracy of T cell identification. (B–E) Expression of CD4 and CD8 distinguished the location of corresponding T cell subsets (B and C), T cell subsets were annotated based on cluster-specific marker genes (D), and their spatial relationships were illustrated on UMAP (E). (F) Subset proportions across baseline (V1BG, n = 3; V2BG, n = 3), post-vaccination (V1, n = 3; V2, n = 3), post-challenge (CV1, n = 3; CV2, n = 3), and infection-only control (C, n = 3) groups were compared by using the Kruskal-Wallis H test (FDR-adjusted, Dunn’s test [∗q < 0.05, ∗∗q < 0.01, ∗∗∗q < 0.001]). (G) Pseudotime trajectory analysis of T cell subsets (upper) and the localization of pre-effector CD4 T cells along the inferred pseudotime trajectory (lower). Each dot represents a single cell, ordered along pseudotime to…

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  Figure 6. V2 elicits infection-like TCR clonal expansion and stronger Th1-associated T cell responses (A) KEGG pathway enrichment analysis of differentially expressed genes (DEGs) in T cells between CV1 and CV2. The inner bar plot represents the enriched pathways annotated from DEGs, with bar length indicating the number of genes enriched in each pathway and the bar color indicating the category for this pathway. The middle circle shows the number of upregulated and downregulated DEGs in CV1 compared with CV2 within each pathway. The outer ring represents the total number of genes in each pathway from the background gene set. (B and C) The TCR diversity index was compared across post-vaccination (V1, n = 3; V2, n = 3), vaccination-challenge (CV1, n = 3; CV2, n = 3), and infection groups (C, n = 3) by using Wilcoxon rank-sum test (FDR-adjusted, Dunn’s test [∗q < 0.05]). TCR similarity between each sample was assessed using Pearson sample-similarity matrix analysis. The red box highlights higher TCR similarity of CV2 (n = 3) and V2 (n = 3) to the C (n = 3) group, with similarity levels indicated by the colored bars (C). (D) The top 10 VDJ (TCR) frequencies of each clonotype were compared…

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  Figure 7. Capsid inclusion may impair recognition of immunodominant E2/E1 epitopes through antigenic competition or interference (A) Predicted structural diagrams and detailed epitope information for B cell linear epitopes of capsid protein and full-length E protein. (B) Predicted structural diagrams and detailed epitope information for MHC epitopes of capsid protein and full-length E protein. (C) Schematic diagram of all epitopes in CV1 and CV2 antigens (green, capsid protein; blue, E protein; red, B cell linear epitopes; orange, MHC class I epitopes; yellow, MHC class II epitopes).