SARS-CoV-2 spike protein-derived immunogenic peptides that are promiscuously presented by several HLA-class II molecules and their potential for inducing acquired immunity.

The current coronavirus disease 2019 (COVID-19) pandemic that is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a significant threat to public health. Although vaccines based on the mRNA of the SARS-CoV-2 spike protein have been developed to induce both cellular and humoral immunity against SARS-CoV-2, there have been some concerns raised about their high cost, particularly in developing countries. In the present study, we aim to identify an immunogenic peptide in the SARS-CoV-2 spike protein to activate cellular immunity, particularly CD4 helper T lymphocytes (Th cells), which are a commander of immune system. SARS-CoV-2 spike protein-derived peptides Spike and Spike-specific CD4 Th cell lines were generated by repetitive stimulation of healthy donor-derived CD4T-cells with each peptide. Their HLA-restrictions were addressed by using blocking antibodies against HLA and HLA-transfected L-cells. The epitopes of Spike-specific CD4 Th cell lines were defined using a series of 7-14-mer overlapping truncated peptides and alanine-substituted epitope peptides. To address responsiveness of these CD4 Th cell lines to several SARS-CoV-2 variants, we stimulated the CD4 Th cell lines with mutated peptides. We addressed whether these identified peptides were useful for monitoring T-cell-based immune responses in vaccinated donors using the IFN-γ ELISpot assay. The Spike peptide was found to be a promiscuous peptide presented by HLA- DRB108:02, DR53, and DPB102:02. Although HLA-DPB102:02-restricted CD4 Th cells did not response to some peptides with the L452R and L452Q mutations, the other CD4 Th cells were not affected by any mutant peptides. We developed two tetramers to detect HLA-DRB108:02/Spike- and Spike(L452R/Y453F)-recognizing CD4 Th cells. Spike peptide was also a promiscuously presented to HLA-DRB109:01 and DRB115:02. The T-cell responses specific to both peptides Spike and Spike were detected in PBMCs after vaccinations. In addition, we observed that the Spike peptide activated both CD8 T-cells and CD4 Th cells in individuals receiving mRNA vaccines. SARS-CoV-2 spike protein-derived peptides, Spike and Spike, include several epitopes that are presented by multiple HLA-class II alleles to activate CD4 Th cells, which are considered useful for monitoring the establishment of acquired immunity after vaccination.