Identification and HLA restriction of naturally derived Th1-cell epitopes from the secreted Mycobacterium tuberculosis antigen 85B recognized by antigen-specific human CD4(+) T-cell lines

Antigen 85B (Ag85B/MPT59) is a major secreted protein from Mycobacterium tu berculosis which is a promising candidate antigen for inclusion in novel su bunit vaccines against tuberculosis (TB), The present study was undertaken to map naturally derived T-cell epitopes from M. tuberculosis Ag85B in rela tion to major histocompatibility complex (MHC) class II restriction. Antige n-specific CD4(+) T-cell lines were established from HLA-typed TB patients and Mycobacterium bovis BCG vaccinees by stimulation of peripheral blood mo nonuclear cells with purified Ag85B in vitro. The established T-cell lines were then tested for proliferation and gamma interferon (IFN-gamma) secreti on in response to 31 overlapping synthetic peptides (18-mers) covering the entire sequence of the mature protein, The results showed that the epitopes recognized by T-cell lines from TB patients were scattered throughout the Ag85B sequence whereas the epitopes recognized by T-cell lines from BCC vac cinees were located toward the N-terminal part of the antigen. The T-cell e pitopes represented by peptides p2 (amino acids [aa] 10 to 27), p3 (aa 19 t o 36), and pll (aa 91 to 108) were frequently recognized by antigen-specifi c T-cell lines from BCG vaccinees in both proliferation and IFN-gamma assay s. MHC restriction analysis demonstrated that individual T-cell lines speci fically recognized the complete Ag85B either in association with one of the self HLA-DRB1, DRB3, or DRB4 gene products or nonspecifically in a promisc uous manner. At the epitope level, panel studies showed that peptides p2, p 3, and p11 were presented to T cells by HLA-DR-matched as well as mismatche d allogeneic antigen-presenting cells, thus representing promiscuous epitop es, The identification of naturally derived peptide epitopes from the M, tu berculosis Ag85B presented to Th1 cells in the context of multiple HLA-DR m olecules strongly supports the relevance of this antigen to subunit vaccine design.