The purpose of this proposal is to continue and extend the investigations initiated in the previous period of support, focusing on the GP46/M-2 gene family of Leishmania. The objectives should determine the immunological mechanisms involved in protective immunity elicited by the GP46/M-2 membrane surface glycoprotein. Based on the fact that immunization with GP46/M-2 appears to effect an early phase(s) of infection (resulting in either a delay in onset or complete protection), these studies will focus on the early immunological events occurring in response to infection in immunized/non-immunized mice. Further, this proposal seeks to employ recent genetic as well as biochemical approaches to determine the biological role of the GP46/M-2 family in vivo. Specifically the experimental approaches proposed are: 1. To determine the effector cells, mediators and mechanisms responsible for protection against infection induced by immunization with GP46/M-2 and the adjuvant, Corynebacterium parvum. Experimental approaches will employ cellular reconstitution (using cloned T cell lines) and immunohistochemical analyses at the site of infection. Epitope mapping will determine protective epitopes for potential use in a peptide subunit vaccine. 2. To determine the mechanism(s) (cell subpopulations, cytokines) involved in protection due to immunization using GP46/M-2-vaccinia recombinant viruses. The ability of recombinant GP46-vaccinia virus to induce protection against infection with L. major and L. donovani will also be evaluated. In addition, the potential of recombinant non-pathogenic vaccinia and canary pox viruses in the induction of a protective immune response against infection with Leishmania species will be investigated. 3. To investigate the structure (biochemical), subcellular distribution, gene regulation (at the mRNA level) and biological function of various members of the GP46/M-2 gene family. Functional studies, employing a molecular genetic approach, will focus on development of the parasite within the phlebotomine sand fly vector.
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