Thirty-six HLA-E-restricted peptides on SARS-CoV-2 were predicted and synthesized
HLA-E*0101 or HLA-E*0103-restricted peptides on SARS-CoV-2 structure protein S protein, E protein, M protein and N protein were predicted. According to the binding affinity, thirty-six predicted nonapeptides on SARS-CoV-2 with strong binding or weak binding ability in either of the two databases were selected. A total of 36 SARS-CoV-2-derived HLA-E-restricted nonapeptides with the detail information including 25 peptides on S protein, one peptide on E protein, six peptides on M protein and four peptides on N protein were listed in Table S1.
Four nonapeptides of SARS-CoV-2 exhibited high binding affinity to bothHLA-E*0103 and HLA-E*0101 molecules
Then we used the K562/HLA-E cell binding assay to further screen the high binding affinity SARS-CoV-2 nonapeptides to HLA-E*0103 or HLA-E*0101 molecules, respectively. Among the 36 predicted HLA-E restricted SARS-CoV-2 peptides, six peptides including S7, S10, S13, S15, S19 and S25 could increase the expression of HLA-E*0101 on K562/HLA-E cell surface characterized by fluorescence index (FI) ≥ 1, indicating a high binding affinity to HLA-E*0101 molecule (Figure 1A). Moreover, four of the 23 predicted peptides including S7, S13, S19 and S25 could also increase the HLA-E*0103 expression on K562/HLA-E cell surface with FI ≥ 1, suggesting a high binding affinity to HLA-A*0103 molecule as well (Figure 1B). Therefore, the four nonapeptides S7, S13, S19 and S25 of SARS-CoV-2 exhibited high binding affinity to both HLA-E*0103 and HLA-E*0101 molecules. However, there may be slight difference of the binding affinity among these peptides according to the FI value. Specifically, S19 showed higher binding affinity to HLA-E*0103 molecule, while S7 and S15 exhibited higher binding affinity to HLA-E*0101 molecule. Detailed information for the screened peptides with high binding affinity were summarized in Table S2.
HLA-E restricted epitopes ofSARS-CoV-2 were identified by inducing specific IFN-γ-producing CD8+ T cell responses
We next assessed the capacity of each peptide to elicit epitope-specific CD8+ T cell responses by detecting the secretion of IFN-γ in vitro in SARS-CoV-2 vaccinated subjects. The results showed that four peptides (S7, S13, S19, S25) could effectively elicit epitope-specific IFN-γ-secreting CD8+ T cell responses in peripheral blood of vaccinated subjects (Figure 2A), indicating that the four SARS-CoV-2 peptides could be defined as HLA-E restricted CD8+ T cell epitopes. Specifically, the median (range) was 108 (13-685) SFC/106 PBMCs for peptide S7, 87 (10–516) SFC/106 PBMCs for peptide S13, 102 (15–588) SFC/106 PBMCs for peptide S19 and 104 (8-591) SFC/106 PBMCs for peptide S25. However, there was only 11 out of 56 samples collected showed the HLA-E restricted epitopes-specific CD8+ T cell responses. Moreover, the frequencies of IFN-γ-secreting CD8+ T cell responses showed no difference among the four HLA-E restricted SARS-CoV-2 epitopes (Figure 2B).
The frequencies of HLA-E restricted SARS-CoV-2 epitopes-specific CD8+ T cells could be detected in peripheral blood of vaccinated individuals
The SARS-CoV-2 epitopes S7 and S19 were selected to construct HLA-E/peptide tetramer to accurately detect the frequencies of epitope-specific CD8+ T cells in the peripheral blood of SARS-CoV-2 vaccinated subjects. The frequencies of both epitope S7 and epitope S19-specific CD8+ T cells could be detected (Figure 3A). The frequency of epitope S7-specific CD8+ T cells in 56 samples ranged from 0.020% to 1.120% (median values: 0.11%, CI95%: 0.1234%-0.2398%), while the frequency of epitope S19-specific CD8+ T cells ranged from 0.020% to 0.74% (median value: 0.10%, CI95%: 0.1280%-0.2156%). Notably, there was no difference of the frequencies of epitope-specific CD8+ T cells between the two epitopes in each SARS-CoV-2 vaccination group (Figure 3B). When compared the frequencies of epitope-specific CD8+ T cells among three different of SARS-CoV-2 vaccines immunization groups for each epitope, the frequencies of epitope-specific CD8+ T cells in individuals receiving adenovirus-vectored vaccine group seemed higher than that in inactivated vaccine group and recombinant protein subunit vaccine group (Figure 3C).