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.


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.


CONCLUSIONS
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.
Mustafa
Abu Salim
AS
Department of Microbiology, Faculty of Medicine, Health Sciences Centre, Kuwait University, Safat 13110, Kuwait. abusalim@hsc.edu.kw
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000276:immunology*; D001426:Bacterial Proteins / Q000276:immunology*; D001665:Binding Sites; D002460:Cell Line; D000939:Epitopes; D006684:HLA-DR Antigens / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D011485:Protein Binding; D020539:Sequence Analysis, Protein; D018417:Th1 Cells / Q000276:immunology*
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D000939:Epitopes; D006684:HLA-DR Antigens; C080865:MPB64 protein, Mycobacterium