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

ABSTRACT

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. 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-gamma secretion]. 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. 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