Of the average 57 million annual deaths over the past ten years, about 14.8 million are directly due to infectious diseases with millions more due to secondary effects of infections (65). The numbers of annual deaths due to infections by Salmonella Typhi and S. Paratyphi A, hepatitis B virus (HBV), Streptococcus pneumoniae and Mycobacterium tuberculosis (Mtb) vary widely but may account for about 30 percent of the total deaths with an even greater cost being due to the severe morbidity associated with these diseases (232). In the belief that improving health, nutrition and economic well being (the latter dependent on the first two) provides the best means to enhance the quality of life globally and thus reduces conditions that result in warlike and terrorist behavior, we propose a vaccine development program based on our recent technical developments in using recombinant attenuated Salmonella vaccines (RASV). Our Specific Aims are (i) to enhance induction of mucosal and systemic antibody responses to RASV-expressed protective pneumococcal antigens (as a model antigen) by increased production and release of membrane vesicles and by altering RASV cell adherence attributes, (ii) to enhance biological containment of live RASV strains by ensuring inability to persist and/or to completely lyse in vivo and especially in the intestinal tracts of birds and animals, (iii) to design and construct S. Paratyphi A as an antigen and DNA vaccine delivery vector with a diversity of genetically-specified features to ensure safety and efficacy when used to immunize infants and adults to protect against enteric fever and additional infectious diseases, and (iv) to design, construct and evaluate recombinant attenuated S. Typhi and S. Paratyphi A vaccines to prevent infections by Mtb using delivery of protective antigens by Type II and III secretion and/or by regulated delayed lysis in vivo with lysis occurring in cell compartments to enhance antigen presentation either by MHC Class I or Class II and by regulated delayed lysis in vivo in the cytoplasmic compartment to release a DNA vaccine optimized for nuclear targeting leading to synthesis and modification of protective antigens by the immunized individual. We will also add to our Master File (filed with FDA), prepare and fully characterize candidate vaccine Master Seeds for stability and safety, prepare and submit protocols for IRB approvals, submit information necessary to obtain INDs, and perform any other work needed to arrange that the best candidate vaccines be clinically evaluated in human volunteers. PUBLIC HEALTH RELEVANCE: The research proposed will further develop and refine technologies for design and construction of live recombinant attenuated Salmonella vaccine (RASV) vectors to deliver after needle-free oral inoculation protective antigens and/or DNA vaccines to stimulate mucosal, systemic and cellular immunities against bacterial, viral and parasite infectious disease agents. More specifically, we will develop a safe, efficacious S. Paratyphi A vaccine vector for oral immunization of newborns and infants. We will use this S. Paratyphi A vector in addition to an S. Typhi vector to construct RASVs to prevent transmission and infection by Mycobacterium tuberculosis.