MOLECULAR-CLONING AND IMMUNOLOGICAL REACTIVITY OF A NOVEL LOW-MOLECULAR-MASS ANTIGEN OF MYCOBACTERIUM-TUBERCULOSIS

Polypeptide Ags present in the culture filtrate of Mycobacterium tuber culosis were purified and evaluated for their ability to stimulate PBM C from purified protein derivative (PPD)-positive healthy donors. One such Ag, which elicited strong proliferation and IFN-gamma production, was further characterized, The N-terminal amino acid sequence of this polypeptide was determined and used to design oligonucleotides for sc reening a recombinant M. tuberculosis genomic DNA library, The gene (M tb 8.4) corresponding to the identified polypeptide was cloned, sequen ced, and expressed in Escherichia coli. The predicted m.w. of the reco mbinant protein without its signal peptide was 8.4 kDa, By Southern an alysis, the DNA encoding this mycobacterial protein was found in the M , tuberculosis substrains H37Rv, H37Ra, Erdman, and ''C'' strain, as w ell as in certain other mycobacterial species, including Mycobacterium avium and Mycobacterium bovis BCG (bacillus Calmette-Guerin, Pasteur) . The Mtb 8.4 gene appears to be absent from the environmental mycobac terial species examined thus far, including Mycobacterium smegmatis, M ycobacterium gordonae, Mycobacterium chelonae, Mycobacterium fortuitum , and Mycobacterium scrofulaceum. Recombinant Mtb 8.4 Ag induced signi ficant proliferation as well as production of IFN-gamma, IL-10, and TN F-alpha, but not IL-5, from human PBMC isolated from PPD-positive heal thy donors. Mtb 8.4 did not stimulate PBMC from PPD-negative donors. F urthermore, immunogenicity studies in mice indicate that Mtb 8.4 elici ts a Th1 cytokine profile, which is considered important for protectiv e immunity to tuberculosis. Collectively, these results demonstrate th at Mtb 8.4 is an immunodominant T cell Ag of M. tuberculosis.