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-gamma 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.