Expression cloning of an immunodominant family of Mycobacterium tuberculosis antigens using human CD4(+) T cells

Development of a subunit vaccine for Mycobacterium tuberculosis (Mtb) is li kely to be dependent on the identification of T cell antigens that induce s trong proliferation and interferon gamma production from healthy purified p rotein derivative (PPD)(+) donors. We have developed a sensitive and rapid technique for screening an Mtb genomic library expressed in Escherichia col i using Mtb-specific CD4(+) T cells. Using this technique, we identified a family of highly related Mtb antigens. The gene of one family member encode s a 9.9-kD antigen, termed Mtb9.9A. Recombinant Mtb9.9A protein, expressed and purified from E. coli, elicited strong T cell proliferation and IFN-gam ma production by peripheral blood mononuclear cells from PPD+ but not PPD- individuals. Southern blot analysis and examination of the Mtb genome seque nce revealed a family of highly related genes. A T cell line from a PPD+ do nor that failed to react with recombinant Mtb9.9A recognized one of the oth er family members, Mtb9.9C. Synthetic peptides were used to map the T cell epitope recognized by this line, and revealed a single amino acid substitut ion in this region when compared with Mtb9.9A. The direct identification of antigens using T cells from immune donors will undoubtedly be critical for the development of vaccines to several intracellular pathogens.