Tuberculosis is once again becoming a major health problem within the United States. The causative agent is Mycobacterium tuberculosis (Mtb). Presently, the only vaccine available is an attenuated strain of Mycobacterium bovis known as Bacille Calmette Gurin (BCG). The reported efficacy of this vaccine varies widely, and it is not in general use in the United States To develop a better vaccine it is necessary to identify the mycobacterial antigens that are responsible for eliciting the protective immune response and the corresponding T cells subsets responsible for protective immunity. The role of alpha/beta and gamma/delta T cells in primary vs secondary resistance to Mtb infection will be analyzed by defining the activity of different classes of alpha/beta and gamma/delta T cells during infection. Changes in T cell expression at sites of initial infection with Mtb will be measured and the activity of alpha/beta and gamma/delta T cells will be specifically modulated by using anti-TCR antibody in vivo. The Mtb-antigen-specific T cell repertoire of infected and immune mice will be immortalized by producing T cell hybridomas. These will supply precise and convenient cellular probes for defining the Mtb-specific T cell epitopes. To identify specific mycobacterial genes responsible for eliciting the immune response, the panel of hybridomas will be screened for activity towards bacteria expressing cloned mycobacterial genes. Libraries of expressed mycobacterial genes will be made either by cloning mycobacterial DNA into expression vectors, or by using a new approach to identify bacterial genes expressed in infected macrophages. This latter technique will be designed to test the idea that important protective epitopes may be expressed by Mtb genes specifically regulated during infection in macrophages. This new approach will combine cDNA cloning with subtractive hybridization and PCR amplification. Once genes whose products elicit a response are identified, their T cell epitopes will be mapped using synthetic peptides. Both the immortal T cell hybridomas and the cloned Mtb genes will not only provide important tools for defining effective vaccines, but they will also supply reagents for the scientific community to potential use in diagnosis and monitoring of disease progression.
