In silico Binding Predictions for Identification of HLA-DR-Promiscuous Regions and Epitopes of Mycobacterium tuberculosis Protein MPT64 (Rv1980c) and Their Recognition by Human Th1 Cells

Objective: To identify HLA-promiscuous regions and epitopes of MPT64 (Rv1980c), a major secreted antigen of Mycobacterium tuberculosis, by in silico analysis for binding to HLA-DR molecules. Materials and Methods: The sequence of mature MPT64 protein (aa 1–205) was analyzed in silico for HLA-DR binding regions and T cell epitopes using ProPred, a web-based prediction server. The prediction results were experimentally verified by testing 20-mer synthetic peptides corresponding to the predicted HLA-DR binding regions and epitopes with T cell lines established from peripheral blood mononuclear cells of PPD-positive and HLA-heterogeneous healthy subjects in Th1 cell assays (antigen-induced proliferation and IFN-γ secretion). Results: The in silico analysis for binding of the mature MPT64 sequence to HLA-DR molecules suggested that it could bind to molecules expressed from all HLA-DR alleles (n = 51) included in ProPred. Furthermore, ProPred identified 26 epitopes and 8 nonoverlapping HLA-DR binding regions (9–35 aa in length) in the Rv1980c sequence, with 5 regions (aa 20–44, aa 68–102, aa 132–146, aa 164–186 and aa 194–202) being HLA-DR-promiscuous. By using synthetic peptides and T cell lines in Th1 cell assays, 4 peptides of MPT64 (aa 21–40, aa 81–100, aa 171–190 and aa 191–20), from 4 of the 5 HLA-DR-promiscuous regions predicted by ProPred, were experimentally verified to be HLA-DR-promiscuous and to have immunodominant epitopes. Conclusion: The in silico method (ProPred) suggested promiscuous HLA-DR-binding of mature MPT64 and identified HLA-promiscuous and immunodominant regions and epitopes of this protein.