N-terminal pyroglutamylation of an HLA-A24-restricted immunodominant epitope enhances SARS-CoV-2-specific T-cell responses.

T-cell responses play a critical role in the control of SARS-CoV-2 infections. Here, we examined the T-cell response to a conserved, immunodominant HLA-A*24:02-restricted SARS-CoV-2 spike protein epitope, QI9/A24 (QYIKWPWYI; residues 1208-1216). TCR repertoire analysis revealed that QI9/A24-specific T cells are highly diverse, with 219 clonotypes isolated and 169 αβ TCR pairs identified in six donors. To further characterize this response, we evaluated T-cell recognition of alanine-substituted and homologous coronavirus-derived peptides, using a TCR reconstitution system, and found that QI9/A24 TCRs tolerated numerous substitutions at the N-terminus and position 3. During peptide synthesis, we unexpectedly discovered that an N-terminally pyroglutamylated QI9/A24 peptide (pyrQI9) exhibited increased protease resistance, compared with the unmodified peptide. Further analysis revealed that the pyrQI9 peptide was recognized with higher sensitivity than the wild-type sequence and efficiently induced antigen-specific T-cell responses at low concentrations in both vaccinated and convalescent HLA-A*24:02-positive individuals. Moreover, immunization of HLA-A24 transgenic mice with the pyrQI9 peptide elicited antigen-specific T-cell responses more efficiently than the wild-type peptide and conferred superior T-cell-mediated protection. Together, our findings suggest that subtle modifications of antigenic peptides at a tolerable site in the N-terminus with pyroglutamate can enhance T-cell immunity in SARS-CoV-2 infection, providing critical insights into the design of next-generation T-cell-based vaccines against viral infections.