High-resolution analysis of individual spike peptide-specific CD4 T-cell responses in vaccine recipients and COVID-19 patients.

Objectives: Potential differences in the breadth, distribution and magnitude of CD4 T-cell responses directed against the SARS-CoV-2 spike glycoprotein between vaccinees, COVID-19 patients and subjects who experienced both ways of immunisation have not been comprehensively compared on a peptide level.

Methods: Following virus-specific cultivation, we determined the T-cell responses directed against 253 individual overlapping 15-mer peptides covering the entire SARS-CoV-2 spike glycoprotein using IFN-γ ELISpot and intracellular cytokine staining. HLA binding was determined for selected peptides.

Results: We mapped 955 single peptide-specific CD4 T-cell responses in a cohort of COVID-19 patients ( = 8), uninfected vaccinees ( = 16) and individuals who experienced both infection and vaccination ( = 11). Patients and vaccinees (two-time and three-time vaccinees alike) had a comparable number of CD4 T-cell responses (median 26 vs. 29,  = 0.7289). Most of these specificities were conserved in B.1.1.529 and the BA.4 and BA.5 sublineages. The highest magnitude of these IFN-γ CD4 T-cell responses was observed in COVID-19 patients (median 0.35%), and three-time vaccinees showed a higher magnitude than two-time vaccinees (median 0.091% vs. 0.175%,  < 0.0001). Twelve peptide specificities were each detected in at least 40% of subjects. HLA binding showed promiscuous presentation by DRB1 molecules for several peptides.

Conclusion: Both SARS-CoV-2 infection and vaccination prime broadly directed T-cell responses directed against the SARS-CoV-2 spike glycoprotein. This comprehensive high-resolution analysis of spike peptide specificities will be a useful resource for further investigation of spike-specific T-cell responses.