Molecular characterization and human T-cell responses to a member of a novel Mycobacterium tuberculosis mtb39 gene family

We have used expression screening of a genomic Mycobacterium tuberculosis l ibrary with tuberculosis (TB) patient sera to identify novel genes that may be used diagnostically or in the development of a TB vaccine. Using this s trategy, we have cloned a novel gene, termed mtb39a, that encodes a 39-kDa protein. Molecular characterization revealed that mtb39a is a member of a f amily of three highly related genes that are conserved among strains of M. tuberculosis and Mycobacterium bovis BCG but not in other mycobacterial spe cies tested. Immunoblot analysis demonstrated the presence of Mtb39A in M; tuberculosis lysate but not in culture filtrate proteins (CFP), indicating that it is not a secreted antigen. This conclusion is strengthened by the o bservation that a human T-cell clone specific for purified recombinant Mtb3 9A protein recognized autologous dendritic cells infected with TB or pulsed with purified protein derivative (PPD) but did not respond to M. tuberculo sis CFP. Purified recombinant Mtb39A elicited strong T-cell proliferative a nd gamma interferon responses in peripheral blood mononuclear cells from 9 of 12 PPD-positive individuals tested, and overlapping peptides were used t o identify a minimum of 10 distinct T-cell epitopes. Additionally, mice imm unized with mtb39a DNA have shown increased protection from M. tuberculosis challenge, as indicated by a reduction of bacterial load. The human T-cell responses and initial animal studies provide support for further evaluatio n of this antigen as a possible component of a subunit vaccine for M. tuber culosis.