SARS-CoV-2 breakthrough infections enhance T cell response magnitude, breadth, and epitope repertoire

Abstract

Little is known about the effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or SARS2) vaccine breakthrough infections (BTIs) on the magnitude and breadth of the T cell repertoire after exposure to different variants. We studied samples from individuals who experienced symptomatic BTIs during Delta or Omicron waves. In the pre-BTI samples, 30% of the donors exhibited substantial immune memory against non-S (spike) SARS2 antigens, consistent with previous undiagnosed asymptomatic SARS2 infections. Following symptomatic BTI, we observed (1) enhanced S-specific CD4 and CD8 T cell responses in donors without previous asymptomatic infection, (2) expansion of CD4 and CD8 T cell responses to non-S targets (M, N, and nsps) independent of SARS2 variant, and (3) generation of novel epitopes recognizing variant-specific mutations. These variant-specific T cell responses accounted for 9%-15% of the total epitope repertoire. Overall, BTIs boost vaccine-induced immune responses by increasing the magnitude and by broadening the repertoire of T cell antigens and epitopes recognized.

Keywords: B cells; COVID-19 vaccination; Delta; Omicron; SARS-CoV-2; T cells; breakthrough infection; coronavirus; de novo responses.

Graphical abstract

Figure 1

Impact of BTIs on T cell, RBD, and neutralizing antibody responses T cell responses and neutralizing responses assessed in a longitudinal cohort (cohort 1, Table S1) of COVID-19-vaccinated individuals (n = 27), sampled before and after BTI (median 89 days). For T cells, ancestral S responses are shown as circles, non-S (CD4-RE) responses as triangles, and the sum of S and non-S reactivity as squares. For antibodies, S-RBD responses are shown as rhombuses. (A and B) CD4⁺ T cell reactivity to (A) CD4-RE and (B) S protein for pre-BTI samples, with CD4-RE+ highlighted in red and non-reactive samples in blue. (C) PSV neutralization titers. (D–F) Pre- and post-BTI CD4⁺ T cell responses for (D) total responses and separately for (E) S and (F) non-S proteins. (G–I) CD8⁺ T cell responses for (G) total responses and separately for (H) S and (I) non-S proteins. (J and K) The ancestral (J) S-RBD IgG and (K) PSV neutralization titers in pre- and post-BTI samples. Bars represent geometric mean. Pre-post comparisons are performed by paired Wilcoxon T test and p values are listed at the top of each graph for the entire cohort (black), CD4-RE+ (red), and CD4-RE- (blue) donor samples. The y-axis starts at the LOD, and the dotted line indicates the LOS. See also Figures S1, S2, and Table S1.

Figure 2

CD4⁺ T cell antigen-specific immunophenotyping CD4+ T cell responses evaluated in the longitudinal cohort (cohort 1, Table S1) of COVID-19-vaccinated individuals (n = 27) sampled before and after BTI (median 89 days). The T cell responses are shown for ancestral S (circles), non-S (CD4-RE) (triangles), and the sum of S and non-S reactivity (squares). (A–C) Immunophenotyping of AIM-positive S-specific CD4⁺ T cell responses considering (A) memory subsets (CD45RA and CCR7), (B) cT_FH fraction (CXCR5 expression on OX40⁺CD40L⁺ cells), and (C) exhaustion (CTLA-4, PD-1, TIGIT, and TIM3). (D and E) Antigen-specific cytokine and granzyme B responses in CD40L⁺ cells. (D) total, S, and non-S-specific responses and (E) sum of S and non-S IFNγ, IL-2, TNFα, and granzyme B responses. (F) Pie chart of single or combined cytokine/granzyme B production (function count: red = 4, orange = 3, yellow = 2, white = 1). Pre-post comparisons are performed by paired Wilcoxon T test; p values are listed at the top of each graph for the entire cohort (black), CD4-RE+ (red), and CD4-RE- (blue) donor samples. Bars represent the geometric mean, except for the memory and cT_FH graphs which show the median to account for zero values. The y-axis begins at the LOD, and the dotted line represents the LOS. See also Figures S1, S2, and Table S1.

Figure 3

T and B cell responses in BTI donors as a function of Delta or Omicron SARS-CoV-2 variant wave T and B cell responses assessed in a cross-sectional cohort (cohort 2, Table S1) of BTI donors infected during Delta (n = 24 at 3 months post symptoms onset [PSO]), Omicron BA.1 (n = 20 at 1 month PSO, n = 32 at 3 months PSO), or Omicron BA.5 (n = 19) SARS2 variant waves. (A) AIM+CD4⁺ T cell responses to S and CD4-RE are displayed on a two-dimensional plot, categorized by response type (none, S only, non-S only, non-S > S, non-S < S) with color-coded variant groups (Delta, orange; Omicron BA.1, dark blue; Omicron BA.5, purple). p values are shown for the χ² to assess response distribution across categories T cell reactivity. The percentage of each donor sample cohort in the different categories is shown as a bar graph. (B and C) AIM+CD4⁺ and CD8⁺ T cell response to ancestral S comparing (B) Delta versus Omicron BA.1 or (C) BA.1 versus BA.5 BTI. Bars represent the geometric mean and p values are shown for Mann-Whitney tests. (D and E) (D) Frequency of ancestral (Anc) RBD-binding B_Mem (among total B cells) for Delta or BA.1 donors and (E) composition of B_Mem subsets among ancestral, Delta or BA.1 RBD-binding B_Mem in Delta or Omicron BA.1 BTI donors. (F and G) (F) Ancestral RBD IgG binding endpoint titers and (G) neutralization of SARS2 ancestral S PSV in Delta or Omicron donors. p values are shown for paired Wilcoxon t tests for the magnitude graphs. The y-axis begins at the LOD, and the LOS is indicated by the dotted line. See also Figures S1, S3, and Table S1.

Figure 4

SARS-CoV-2 antigen-specific CD4⁺ and CD8⁺ T cell responses in vaccinated-only or post-BTI donor samples AIM⁺ T cell responses in COVID-19 vaccinated only (n = 20, light blue; pre-samples of cohort 1 Table S1) and BTI (n = 15, black) individuals (cohort 3 Table S1). Both cohorts were tested for S and non-S (CD4-RE); the BTI cohort was additionally tested for all the protein antigens. (A and B) (A) CD4⁺ T cell responses to total (S + non-S CD4-RE) S and non-S (CD4-RE) and (B) individual antigens. (C and D) (C) CD8⁺ T cell responses to total (S + non-S CD4-RE) S and non-S (CD4-RE) and (D) individual antigens. Statistical comparisons across cohorts are done by the Mann-Whitney test. p values above the individual antigens are calculated by using one-sample Wilcoxon signed-rank test compared to the limit of sensitivity (LOS). The y axes start at the limit of detection (LOD), and the dotted lines represent the LOS. See also Figure S1, and Table S1.

Figure 5

Impact of variant sequences on CD4⁺ and CD8⁺ T cell epitopes in Delta and Omicron BTI donors SARS-CoV-2-specific T cell epitopes in Delta (n = 5; orange) and Omicron BA.1 (n = 5; dark blue) BTI individuals (cohort 3 Table S1). (A–D) CD4⁺ T cell epitopes include conserved (A and C) and mutation-affected (B and D). Mutation impacts are categorized by response changes: increased, unchanged, decreased, or novel. (E–H) CD8⁺ T cell epitopes include conserved (E and G) and mutation affected (F and H) and categorized responses. The number of epitopes is indicated at the top of each graph. The dotted line represents the LOS, and the y-axis begins at the LOD. See also Figure S1 and Tables S1, S2, and S3.

Figure 6

Impact of variant sequences on the total T and B cell responses in Delta and Omicron BTI donors SARS2-specifc T cell responses in the BTI cohorts collected post infection from this study (Table S1; Delta n = 24; Omicron BA.1 n = 68) measuring S and CD4-RE MPs. Responses to variant-specific mutations are analyzed using ancestral and variant peptide pools. (A and C) SARS-CoV-2-specific T cells in total (S + non-S) responses (light gray) compared to peptides affected by Delta (A) or Omicron BA.1 (C) mutations, using ancestral (A in dark gray; C in brown) and variant-specific peptides (Delta in orange, A; Omicron in blue, C). (B and D) The bar graph displays the percentage of preserved response per donor, calculated by subtracting the response to the ancestral pool affected by Delta (B) or Omicron BA.1 (D) mutations from the total (S + CD4-RE). The pie chart illustrates the percentage of individuals showing no change (± 0.005%), increase, novel, or decreased responses when comparing the ancestral and variant-specific peptide pools. (E) Ancestral and variant RBD-binding B_Mem cells are plotted for their cross-reactive and non-cross-reactive binding in switched B cells (IgD−IgM−) for Delta (n = 19) and Omicron BA.1 (n = 20) BTI donors of cohort 2 (Table S1). p values result from the paired Wilcoxon test and the comparison between ancestral and variant sequences is performed only if at least one value is above the LOS. Bars represent the geometric mean. Graphs start the LOD and the dotted line represents the LOS. See also Figure S1 and Table S1.