Evolution of SARS-CoV-2 T cell responses as a function of multiple COVID-19 boosters

Abstract

We investigate the long-term impact of repeated COVID-19 vaccinations on adaptive immunity through a 3-year study of 78 individuals without reported symptomatic infections. We observe distinct dynamics in spike-specific responses across multiple vaccine doses. While antibody levels increase and stabilize with each booster, T cell responses quickly plateau and remain stable. Notably, approximately 30% of participants show evidence suggestive of asymptomatic infections. Single-cell RNA sequencing reveals a diverse and stable landscape of spike-specific T cell phenotypes without signs of exhaustion or functional impairment. Individuals with evidence of asymptomatic infection display increased frequencies of Th17-like CD4⁺ T cells and GZMKhi/IFNR⁺ CD8⁺ T cell subsets. In this group, repeated vaccinations correlate with an increase in regulatory T cells, potentially indicating a balanced immune response that may mitigate immunopathology. By regularly stimulating T cell memory, boosters contribute to a stable and enhanced immune response, which may provide better protection against symptomatic infections.

Keywords: COVID-19; CP: Immunology; SARS-CoV-2; T cell responses; antibody responses; asymptomatic infection; booster vaccination; exhaustion; mRNA vaccines; scRNA-seq.

Figure 1.. Study design and prevalence of asymptomatic infections.

(A) Overall cohort surveillance for selection of 78 participants without COVID-19 symptoms or previous evidence of infection. (B) Participants categorization by the number of COVID-19 vaccine doses administered (color-coded). (C) Multiple blood donations were collected from each subject at various timepoints (T) post-vaccination. The y axis represents each individual participant, and the x axis represents different time points at which their blood was collected in a longitudinal follow-up throughout the study timeline (Jan 2021-Jan 2024). Samples within the period of each vaccine dose are color-coded. (D) CD4+ T cell responses to Spike and CD4RE MPs were measured as percentage of AIM+ (OX40+CD137+) CD4+ T cells and plotted in 2 dimensions as stimulation index (SI). Each dot represents a sample of a donor for a given timepoint. Percentages denote the frequency of samples that are either negative (CD4RE^NEG) or positive (CD4RE^POS) for CD4RE reactivity using a cut-off of SI=10 (dotted line).

Figure 2.. Evolution of antibody responses over repeated vaccinations and potential exposures.

(A) Longitudinal monitoring of RDB IgG levels across multiple vaccinations and timepoints. Each donor is represented by a colored dot and longitudinal samples for each individual donor connected by a gray line. Black bold line represents the geometric mean. Geometric mean value and % of donors with positive response for each timepoint is indicated. (B) Representation of paired RDB IgG levels before and after vaccination for each dose cohort for all samples (upper panel), or CD4RE^NEG (middle panel) and CD4RE^POS (lower panel) donors. Fold change (FC) is indicated in each graphic. (C) Cross-sectional RBD IgG responses from time of vaccination (days) for the different dose cohorts in donors associated with CD4RE^NEG (upper panel) or CD4RE^POS (lower panel) reactivity. t_1/2 is shown as the median half-life calculated based on linear mixed effects model. (A-C) Dotted lines indicate threshold of positivity. Data were analyzed for statistical significance using the paired Wilcoxon’s (B) or Spearman’s (C) test. p values are shown.

Figure 3.. Evolution of memory Spike-specific CD4+ T cell responses over repeated vaccinations and potential exposures.

(A) Longitudinal monitoring of AIM+ (OX40+CD137+) CD4+ T cell responses across multiple vaccinations and timepoints. Each donor is represented by a colored dot and longitudinal samples for each individual donor connected by a gray line. Black bold line represents the geometric mean. Geometric mean value and % of donors with positive response for each timepoint is indicated. (B) Representation of paired AIM+ CD4+ T cell responses before and after vaccination for each dose cohort for all samples (upper panel), or CD4RE^NEG (middle panel) and CD4RE^POS (lower panel) donors. Fold change (FC) is indicated in each graphic. (C) Cross-sectional AIM+ CD4+ T cell responses from time of vaccination (days) for the different dose cohorts in donors associated with CD4RE^NEG (upper panel) or CD4RE^POS (lower panel) reactivity. t_1/2 is shown as the median half-life calculated based on linear mixed effects model. (A-C) Dotted lines indicate threshold of positivity. Data were analyzed for statistical significance using the paired Wilcoxon’s (B) or Spearman’s (C) test. p values are shown.

Figure 4.. Evolution of memory Spike-specific CD8+ T cell responses over repeated vaccinations and potential exposures.

(A) Longitudinal monitoring of AIM+ (CD69+CD137+) CD8+ T cell responses across multiple vaccinations and timepoints. Each donor is represented by a colored dot and longitudinal samples for each individual donor connected by a gray line. Black bold line represents the geometric mean. Geometric mean value and % of donors with positive response for each timepoint is indicated. (B) Representation of paired AIM+ CD8+ T cell responses before and after vaccination for each dose cohort for all samples (upper panel), or CD4RE^NEG (middle panel) and CD4RE^POS (lower panel) donors. Fold change (FC) is indicated in each graphic. (C) Cross-sectional AIM+ CD8+ T cell responses from time of vaccination (days) for the different dose cohorts in donors associated with CD4RE^NEG (upper panel) and CD4RE^POS (lower panel) reactivity. t_1/2 is shown as the median half-life calculated based on linear mixed effects model. (A-C) Dotted lines indicate threshold of positivity. Data were analyzed for statistical significance using the paired Wilcoxon’s (B) or Spearman’s (C) test. p values are shown.

Figure 5.. Spike-Specific CD4+ and CD8+ T Cell subsets are highly diverse and phenotypically stable upon multiple vaccinations.

Single-cell transcriptomes of sorted AIM+ (A) CD4+ or (B) CD8+ T cells displayed by uniform manifold approximation and projection (UMAP). Cells are colored according to cluster identity as defined by unbiased clustering of 608,720 CD4+ T cells and 460,937 CD8+ T cells. Annotations of cell populations are shown for each cluster. (C) Plots show normalized proportion of spike-specific CD4+ T cells in each cluster as function of different vaccine doses. Proportions were normalized according to minimum cell totals across vaccine doses to ensure comparability. (D) Pie charts represent the relative frequency of spike-specific CD4+ T cell clusters within each dose cohort in CD4RE^NEG (upper panel) or CD4RE^POS (lower panel) samples. (E) Proportion of spike-specific CD8+ T cells in each cluster as function of different vaccine doses. Proportions were normalized as in (C). (F) Pie charts represent the relative frequency of spike-specific CD8+ T cell clusters within each dose cohort in CD4RE^NEG (upper panel) or CD4RE^POS (lower panel) samples. See also Figure S1 and S2.

Figure 6.. T cell functionality and exhaustion profiles remain stable after booster vaccination.

Graphs show relative expression of relevant genes cross-sectionally across different vaccine doses for (A) CD4+ Th1 cells (cluster 2; top panel) and CD4+ Tfh cells (cluster 3; lower panel), (B) CD8+ GZMKhi cells (cluster 0; upper panel), and CD8+ TEFF cells (cluster 3; lower panel) or (C) CD8+ TEX cells (cluster 1). Each color-coded gene is indicated. (A-C) Data were analyzed for statistical significance using the Kruskal-Wallis test followed by Dunn’s post-hoc test with FDR correction. (D) GSEA for exhaustion signature genes (Exhaustion consensus) across vaccine doses in spike-specific TEX CD8+ T cells. P-value and normalized enrichment score (NES) are indicated. (E-F) Graphs represent the relative frequency of CD8+ TEX (cluster 1) cells across timepoints for the indicated vaccine doses in all samples (E) or in samples associated with negative or positive CD4RE reactivity (F). Data were analyzed for statistical significance using the unpaired Mann-Whitney test. p values are shown.

Figure 7.. Asymptomatic infections are associated with increased Th17-like and GZMKhi/IFNR cell phenotypes concurrent with Tregs expansion over repeated vaccinations.

(A) Graphs represent the relative frequency of CD4+ Th17 cells in CD4RE negative and positive samples across doses and timepoints. (B) Graphs represent the relative frequency of CD8+ GZMKhi, LTBhi and IFNR cells in CD4RE negative and positive samples across doses and timepoints. (C, D) Graph represents the relative frequency of CD4+ Tregs in different vaccine doses across samples associated with (C) positive or (D) negative CD4RE reactivity. Data were analyzed for statistical significance using the unpaired Mann-Whitney test. p values are shown. See also Figure S4 and S5.