Identification and HLA Restriction of Naturally Derived Th1-Cell Epitopes from the SecretedMycobacterium tuberculosisAntigen 85B Recognized by Antigen-Specific Human CD4+T-Cell Lines

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Abstract
Antigen 85B (Ag85B/MPT59) is a major secreted protein fromMycobacterium tuberculosiswhich is a promising candidate antigen for inclusion in novel subunit vaccines against tuberculosis (TB). The present study was undertaken to map naturally derived T-cell epitopes fromM. tuberculosisAg85B in relation to major histocompatibility complex (MHC) class II restriction. Antigen-specific CD4+T-cell lines were established from HLA-typed TB patients andMycobacterium bovisBCG vaccinees by stimulation of peripheral blood mononuclear cells with purified Ag85B in vitro. The established T-cell lines were then tested for proliferation and gamma interferon (IFN-γ) secretion 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 BCG vaccinees were located toward the N-terminal part of the antigen. The T-cell epitopes represented by peptides p2 (amino acids [aa] 10 to 27), p3 (aa 19 to 36), and p11 (aa 91 to 108) were frequently recognized by antigen-specific T-cell lines from BCG vaccinees in both proliferation and IFN-γ assays. MHC restriction analysis demonstrated that individual T-cell lines specifically recognized the complete Ag85B either in association with one of the selfHLA-DRB1,DRB3, orDRB4gene products or nonspecifically in a promiscuous manner. At the epitope level, panel studies showed that peptides p2, p3, and p11 were presented to T cells by HLA-DR-matched as well as mismatched allogeneic antigen-presenting cells, thus representing promiscuous epitopes. The identification of naturally derived peptide epitopes from theM. tuberculosisAg85B presented to Th1 cells in the context of multiple HLA-DR molecules strongly supports the relevance of this antigen to subunit vaccine design.

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