This proposal is aimed at identifying and characterizing T cell antigens of M. tuberculosis which may be important vaccine candidates. To identify novel Mtb antigens which may be relevant in protective immunity against tuberculosis we have used a variety of antigen discovery methods which emphasize expression cloning with anti-M. tuberculosis antisera and human T cells. Eight vaccine candidates (Mtb 8.4, Mtb 9.8, Mtb 9.9, Mtb 11, Mtb 19, Mtb 32, Mtb 39, Mtb 40) have so far been selected based on their ability to stimulate PBMC from donors infected with M. tuberculosis. These eight antigen genes will be evaluated as recombinant antigens or as naked DNA, alone or in combination, for immunogenicity and protection against tuberculosis in three animal models including cynomolgus monkeys, mice, and guinea pigs. These models will be used to assess T cell responses as well as protection. In addition, we will emphasize the use of human T cell expression cloning to identify other vaccine candidate antigens. These will be characterized in the in vitro and animal models as above.