Recombinant attenuated Salmonella vaccines for induction of cross-protective immunity and antigen delivery

We have designed strains of Salmonella typhimurium that optimize expression of wild-type attributes that facilitate successful colonization and invasion of the GALT and other lymphoid effect or organs before undergoing changes that cause their attenuation. Modifications have also been included to minimize expression of serotype-specific antigens and cause antigens that display immunological relatedness among diverse serotypes of S. enterica and closely related E. coli pathovars to be over expressed. These strains possess a Δpmi mutation for a mannose dependent synthesis of O-antigen side chains that ceases to occur in vivo, a Δ(gmd-fcl) mutation to block conversion of GDP-Mannose to GDP-Fucose to block colanic acid synthesis, a ΔPfur::TTaraC PBAD fur deletion-insertion mutation that causes constitutive high-level expression of all gene products needed for iron acquisition, and ΔfliC and ΔfljB mutations to abolish synthesis of variable flagellar antigen epitopes. In other vaccine constructions designed for delivery of antigens or DNA vaccine vectors, we engineered strains to exhibit a regulated delayed cell lysis in vivo. By combination of chromosomal deletion-insertion mutations and vector construction gene sequences, we designed strains that are arabinose-dependent and after 5 to 10 generations of growth in vivo cease to synthesize the rigid layer of the bacterial cell wall because of an inability to synthesize muramic acid and diaminopimelic acid. The strains possess additional mutations to uncouple any requirement of protein synthesis and growth from display of the cell wall-less death phenotype. Other mutations ensure complete biological containment to the vaccine constructs to ensure their ability to lyse completely in vivo to release recombinant protective antigens from diverse pathogens or DNA vaccine vectors encoding protective antigens to be synthesized within the immunized animal host.