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(448-477) and Spike(489-513(N501Y))-specific CD4(+) Th cell lines were generated by repetitive stimulation of healthy donor-derived CD4(+)T-cells with each peptide. Their HLA-restrictions were addressed by using blocking antibodies against HLA and HLA-transfected L-cells. The epitopes of Spike(448-477)-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(448-477) peptide was found to be a promiscuous peptide presented by HLA- DRB1*08:02, DR53, and DPB1*02:02. Although HLA-DPB1*02:02-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-DRB1*08:02/Spike(449-463)- and Spike(449-463)(L452R/Y453F)-recognizing CD4(+) Th cells. Spike(489-513(N501Y)) peptide was also a promiscuously presented to HLA-DRB1*09:01 and DRB1*15:02. The T-cell responses specific to both peptides Spike(448-477) and Spike(489-513) were detected in PBMCs after vaccinations. In addition, we observed that the Spike(448-477) peptide activated both CD8(+) T-cells and CD4(+) Th cells in individuals receiving mRNA vaccines. SARS-CoV-2 spike protein-derived peptides, Spike(448-477) and Spike(489-513), 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.