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.


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-gamma 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.


CONCLUSIONS
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.
Department of Microbiology, Faculty of Medicine, Kuwait University, Kuwait. abusalim@hsc.edu.kw
Abu Salim
AS
Mustafa
Fatema
F
Shaban
D000942:Antigens, Bacterial / Q000276:immunology*; D001426:Bacterial Proteins / Q000276:immunology; D001771:Blood Banks; D018604:Epitope Mapping; D006650:Histocompatibility Testing; D006801:Humans; D007730:Kuwait; D007963:Leukocytes, Mononuclear; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D010446:Peptide Fragments / Q000276:immunology; D013601:T-Lymphocytes; D018417:Th1 Cells / Q000276:immunology*; D014376:Tuberculosis
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C489634:CFP-10 protein (71-85), Mycobacterium tuberculosis; C093276:ESAT-6 protein, Mycobacterium tuberculosis; C510537:MPT64 protein, Mycobacterium tuberculosis; D010446:Peptide Fragments