The goal of this project is the construction and preclinical evaluation of bivalent Salmonella -vectored live oral vaccines that will provide protection against the human diseases, tetanus and diphtheria. This work will also yield information that could lead to the improvement of vaccines for typhoid fever; and to the development of multivalent vaccines that contain antigens for infectious diseases that require the development of cell mediated immunity, such as malaria and the acquired immunodeficiency syndrome. Preliminary work has resulted in the development of a vector delivery system that allows the construction of live Salmonella vaccines that contain genes encoding heterologous antigens inserted within the pagC virulence locus. This allows the construction of safer, stable multivalent vaccines that have acquired genes that express heterologous antigens without acquisition of antibiotic resistance. Preliminary results also indicate that PhoP regulated expression of a heterologous antigen (HA) fused to the amino-terminus of the PagC protein results in the development of antibody titers of 1:10,000 against the HA. Therefore experiments are proposed to construct and test Salmonella vaccine strains that express important antigenic determinants of tetanus toxin (TT)and diphtheria toxin (DT) fused to the PagC amino-terminus. The importance of the PagC portion of the immunogen will be tested by constructing and testing the immunogenicity of strains that have PhoP regulated expression of independent DT and TT antigens. The optimal promoters for expression of heterologous antigens will be determined by comparison of the pagC promoter with other in vivo expressed promoters identified in the laboratory of Dr. John Mekalanos by use of in vivo expression technology (IVET). Based on these results multivalent Salmonella typhimurium vaccines will be constructed and evaluated in mice. In anticipation of Phase l testing in human volunteers, bivalent Salmonella typhi vaccines will be constructed and evaluated for expression of TT and DT antigens on infection of cultured human cells.
