HLA-DR Binding Prediction and Experimental Evaluation of T-Cell Epitopes of Mycolyl Transferase 85B (Ag85B), a Major Secreted Antigen of Mycobacterium tuberculosis

Objective: To identify T-cell epitopes of Ag85B by analysis of its sequence for prediction to bind HLA-DR alleles and evaluate the predicted peptides for recognition by T cells in antigen-induced proliferation assays. Materials/ Subjects and Methods: The complete sequence of Ag85B was analyzed for HLA-DR binding prediction to 51 HLA-DR alleles by using a virtual matrix-based prediction program (ProPred). Synthetic peptides covering the sequence of mature Ag85B were also analyzed for binding to HLA-DR alleles, and evaluated for recognition in anti-gen-induced proliferation assays with Ag85B-specific Tcell lines established from the peripheral blood mononuclear cells of 10 HLA-DR-heterogeneous tuberculosis patients. Results: The ProPred analysis of the full-length Ag85B (325 aa), signal peptide (40 aa) and the mature protein (285 aa) predicted their binding to 100, 76 and 98% of the 51 HLA-DR alleles, respectively. The analysis of 31 synthetic peptides for binding to HLA-DR alleles showed that 4 of them could bind 150% HLA-DR alleles, and were considered promiscuous. Testing of Ag85B-specific T-cell lines with synthetic peptides showed that all of the T-cell lines responded to one or more peptides of Ag85B, and 9 of the 10 cell lines responded to one or more of the four peptides considered promiscuous for binding to HLA-DR alleles. Conclusion: The ProPred program was useful in predicting the HLA-DR alleles binding regions of Ag85B and identifying the promiscuous peptides recognized by T cells.