Immunodominant T-cell epitopes from the SARS-CoV-2 spike antigen reveal robust pre-existing T-cell immunity in unexposed individuals

Abstract

The COVID-19 pandemic has revealed a range of disease phenotypes in infected patients with asymptomatic, mild, or severe clinical outcomes, but the mechanisms that determine such variable outcomes remain unresolved. In this study, we identified immunodominant CD8 T-cell epitopes in the spike antigen using a novel TCR-binding algorithm. The predicted epitopes induced robust T-cell activation in unexposed donors demonstrating pre-existing CD4 and CD8 T-cell immunity to SARS-CoV-2 antigen. The T-cell reactivity to the predicted epitopes was higher than the Spike-S1 and S2 peptide pools in the unexposed donors. A key finding of our study is that pre-existing T-cell immunity to SARS-CoV-2 is contributed by TCRs that recognize common viral antigens such as Influenza and CMV, even though the viral epitopes lack sequence identity to the SARS-CoV-2 epitopes. This finding is in contrast to multiple published studies in which pre-existing T-cell immunity is suggested to arise from shared epitopes between SARS-CoV-2 and other common cold-causing coronaviruses. However, our findings suggest that SARS-CoV-2 reactive T-cells are likely to be present in many individuals because of prior exposure to flu and CMV viruses.

Figure 1

Identification of immunogenic epitopes from SARS-CoV-2 by OncoPeptVAC. (A) ROC curves of OncoPeptVAC TCR-binding and netMHCpan-4.1 HLA binding algorithms. A blind dataset of non-immunogenic or immunogenic HLA class-I binding T-cell epitopes from IEDB was used to assess the performance of OncoPeptVAC (cyan). The HLA-binding affinity of the epitopes expressed as percentile rank < 1% was used to assess the performance of netMHCpan-4.1 in predicting true immunogenic epitopes (orange). (B) Separation of immunogenic from non-immunogenic epitopes by OncoPeptVAC score. (C) Separation of immunogenic from non-immunogenic epitopes by HLA-binding percentile rank. (D) Schematic showing the steps used to identify immunogenic epitopes from SARS-CoV-2 proteome. (E) Number of immunogenic epitopes identified in different SARS-CoV-2 antigens. (F) HLA-A, B and C-restricted epitopes from SARS-CoV-2 proteome.

Figure 2

T-cell reactivity to SARS-CoV-2 Spike peptide pools and OncoPeptVAC prioritized peptides. Reactivity was determined by intracellular IFN-γ staining and surface expression of 4-1BB by FACS after stimulation of PBMCs from unexposed donors (n = 14) using separate pools of Spike-S1 and S2 peptides and the 11-Peptide-mix predicted by OncoPeptVAC (see “Methods”). (A) Structure of Spike—ACE2 receptor complex showing the location of the 11-peptides predicted by OncoPeptVAC. (B-C) T-cell activation after 48 h incubation with the peptides. (D-E) T-cell activation after a 7-day incubation with the peptides. (F) Kinetics and magnitude of CD8 T-cell activation in unexposed donor PBMCs. (G,H) CD8 T-cell response of donors D167 and D089 to individual peptides from the 11-peptide-mix. Statistical significance determined by Wilcoxon matched pairs signed rank test in Figures (B-E).

Figure 3

Bulk TCR repertoire analysis after in unexposed donors following in vitro stimulation of PBMCs at different time points with the indicated peptides. (A) Expanded public CDR3-βs recognizing shared antigens in the D089 (upper panel) and D225 (lower panel). (B) Expanded private CDR3-βs in D089 (upper panel) and D225 (lower panel). (C) V–J gene usage in D225. (D) V–J gene usage in D089.

Figure 4

UMAP projection of different cell types identified in unexposed donor D225 after 14 days of in vitro stimulation assay with different antigens. (A) Clusters of different cell types and their relative proportions present in the assay mixture (left panel). Clusters expressing IFN-γ (middle panel) and the top-3 amplified clonotypes (right panel). (B) Heat map showing the expression of cell-type and cell-phenotype-specific markers in the top-10 amplified TCR-β clones. (C) Frequency of CDR3-β recognizing public and private antigens in the top 20 clonotypes. (D) Amplified V and J-genes in the top-20 clonotypes.

Figure 5

T-cell reactivity to Spike-S1, S2 pools, and 11-peptide-mix in asymptomatic, mild-moderate, and severe disease patients after in vitro stimulation for 48 h. (A) Convalescent patient PBMC collected between 45–60 days after PCR confirmation of COVID-19 infection was incubated with spike-S1 or S2 peptide pools or the All-peptide mix. T-cell activation was quantitated by intracellular IFN-γ and 4-1BB expression by FACS. T-cell activation for individual donors is plotted. (A,B) IFN-γ and 4-1BB expression in activated CD8 T-cells in asymptomatic (AS, 9 patients), mild/moderate (M/M, 6 patients) and severe (5 patients). (C,D) IFN-γ and 4-1BB expression in activated CD4 T-cells in the three patient groups. Differences in T-cell activation between groups were not statistically significant by Students T-test. Group-wise aggregated data is shown in Figure S13.