American trypanosomiasis (Chagas' disease) is caused by Trypanosoma cruzi, a protozoan parasite transmitted by reduviid bugs. This illness is a major cause of morbidity and mortality in Latin America, and little progress has been made in controlling transmission of the causative agent to humans. The-term goal of the proposed project is to identify and produce candidate parasite proteins for vaccine development. To this end, a T. cruzi cDNA library will be screened with monoclonal and polyclonal antibody reagents prepared in preliminary work, with the purpose of cloning inserts that encode proteins present on the infective insect-derived metacyclic form of the parasite (IMT). After determination of restriction sites and nucleotide sequencing, cloned inserts will be subcloned into a high level expression vector. Synthesis of immunoreactive recombinant T. cruzi proteins produced in E. coli will be assayed by immunoblot analysis using the antibody reagent employed for the original immunoselection of the cDNA insert. Amounts of the immunoreactive protein sufficient for vaccination studies will be produced in the recombinant expression vectors in E. coli or by chemical methods as synthetic peptides. In initial experiments 3 cDNA inserts have been immunoselected using a polyclonal rabbit directed against a 72 kDa glycoprotein of T. cruzi, and prelinminary characterization and sequencing of these inserts has been done. Mice and rabbits will be vaccinated with recombinant proteins using varying dosages and routes of administration, as well as Freund's and aluminum hydroxide as adjuvants, in an effort to establish procedures that elicit an optimal immune response. Procedures will be developed for challenge of vaccinated animals with IMT that closely simulate natural transmission of the parasite. These efforts will be focused on topical application of IMT to the conjunctivae or oral mucosa, infusion of IMT into the gastrointestinal tract, and allowing the animals to eat infected reduviids. Mice and rabbits then will be immunized with cloned T. cruzi gene products and challenged with IMT to assess the degree of protection afforded by the vaccination. The efficacy of the immunoprophylaxis will be determined by searching for parasites in the animals' blood by direct examination, culture and xenodiagnosis, and also by assaying the sera of challenged animals for the presence of antibodies that bind to T. cruzi antigens other than those contained in the vaccines.