HLA class II binding analysis for SARS-CoV-2 Spike through cell-surface MHC density assay

Abstract

Abstract Introduction: We developed MHC-peptide interaction assay that measures cell-surface expression of covalently linked peptide-MHC II complex. The utility of the assay has been validated for known CD4+ T-cell epitopes of Mycobacterium tuberculosis antigens (Miyadera et al. submitted). Using this assay (named “delta MHC (ΔMHC) assay”), we screened HLA II-binding regions in SARS-CoV-2 Spike protein to identify potential T-cell epitopes. Method: ΔMHC assay uses engineered fibroblast cell line that stably expresses DPA1. The cell line is transduced with pMXs-IG (Kitamura et al. 2003 Exp Hematol) that carries DPB1*05:01-peptide fusion. The assay measures cell-surface HLA expression through flow cytometry and calculates the expression levels normalized to the internal control GFP and control peptide. The peptides (15-mer) that covered the entire Spike protein were analyzed in this study. Results: We performed the assay for HLA-DP5 (DPA1*02:02-DPB1*05:01), which is present at high frequency in East Asian populations. Among the 211 Spike peptides analyzed, we found > 8 peptides that showed strong binding to DP5, indicating that these regions might act as T-cell epitopes. Additional >10 regions showed intermediate binding. Part of binding regions were overlapped with DR-restricted T-cell epitopes reported for SARS-CoV (Yang et al. 2009, Int Imm). Some of the DP5-binding regions were also overlapped with viral mutation sites of contagious variants (VOC 2020 12/01 (UK), 501Y.V2 (South Africa), B1.1.24B (Brazil)). The binding analyses of the variants for DP5 and DP0401 are underway.