Mapping of Th1-Cell Epitope Regions of Mycobacterium tuberculosis Protein MPT64 (Rv1980c) Using Synthetic Peptides and T-Cell Lines from M. tuberculosis-Infected Healthy Humans

Objective: To identify T helper 1 (Th1)-cell stimulating and HLA-promiscuous peptides of MPT64 (Rv1980c), a major secreted antigen of Mycobacterium tuberculosis. Materials and Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from 35 healthy subjects and typed for HLA-DR molecules using genomic methods. To identify subjects infected with M. tuberculosis, PBMCs were tested in antigen-induced proliferation assays with whole cells and culture filtrate antigens of M. tuberculosis, M. tuberculosis-specific antigens ESAT-6 and CFP10, and MPT64. Culture filtrate-induced T-cell lines were established in vitro from 12 M. tuberculosis-infected and HLA-heterogeneous healthy subjects, and tested with 20 overlapping synthetic peptides covering the sequence of MPT64 in Th1-cell assays, i.e. antigen-induced proliferation and/or IFN-γ secretion. In addition, T-cell lines from three HLA-heterogeneous subjects were tested for cytotoxic activity against peptide-pulsed antigen-presenting cells. Results: PBMCs from 12 of 35 subjects responded to M. tuberculosis-specific antigens ESAT-6 and CFP10 as well as to MPT64, which suggested that they were infected with M. tuberculosis. Ten of twelve T-cell lines established from these donors responded to MPT64, and nine T-cell lines responded to 1 or more of the peptides of MPT64 in anti- gen-induced proliferation assays. Furthermore, 18 of the 20 peptides of MPT64 were recognized by the T-cell lines in 1 or more assay systems, and at least 5 peptides were recognized by T-cell lines from HLA-DR-heterogeneous subjects. Conclusion: Th1-cell-reactive epitopes are scattered throughout the sequence of MPT64, and at least 5 of its peptides are presented to Th1-cells in a HLA-promiscuous manner.