There is a great need for a new generation of vaccines that can be delivered via the mucosal surface. The lack of relevant immunologic correlates to protection represents one of the major obstacles in the development of these vaccines. This problem is particularly acute in the case of mucosal pathogens that disseminate to systemic organs where humoral responses are incomplete indicators of protection. I propose to analyze the immunogenicity of oral vaccines expressing novel adjuvants. More generally, the proposed studies will improve our understanding of the mucosal immune system and its interaction with systemic immune compartments for the creation of long-term immunity to the mucosal pathogen Salmonella and toxigenic strains of Vibrio cholerae and Escherichia coli, which together are major contributors to diarrheal disease worldwide.Proposed research and summarized methodology. Over the past year, I have constructed the vaccine strain that constitutively expresses pagP (Aim 1). I have also derived two other constructs that place the regulation of pagP under SPI1 and SPI2 promoters. I have run one experimental trial using the constitutively expressed vaccine strain and over the next year I will repeat this trial and begin testing the new strains.I have developed a good number of the enterotoxin oral vaccine constructs that I originally proposed in Aim 2 and am presently moving those strains onto 1) an attenuated Salmonella enterica serovar Typhimurium aro/A-deficient background, 2) wild type 14028s virulent parent strain 3) aroA vaccine strain the co-expresses a stable OVA 257-264 SIINFEKL from the Salmonella chromosome. I am about to begin testinga number of these vaccines for their ability to synthesize the B subunits of E. coli LTIIb and V.cholerae CT toxins in vitro by ganglioside binding ELISA. After enterotoxin B subunit production has been established, the vaccines will be ready for testing in live animal models.
Aim 3 of the original proposal delineates the proposed study of long and short term immunity and T cell memory to the vaccines developed in aims 1&2. T cell memory will be explored after the vaccine trials identify which are the most effective vaccines. The first vaccine trial testing the PtacpagP-expressing aroA vaccine shows that mice immunized with constitutively expressed pagP are as susceptible as naTve mice to challenge with virulent Salmonella. This preliminary study gives us an indication of how this lipid A variant affects the host's immune response. Herein I propose that constitutively expressed pagP stimulates nitric oxide (NO) leading to the down-regulation of the PhoP/PhoQ signal transduction pathway required for the survival of bacteria in vivo. Ultimately, constitutively expressing pagP leads to the premature death of thevaccine. This hypothesis is in accord with my observations that NO is essential for the acquired immune response to Salmonella. This hypothesis will be tested in vitro in macrophages by comparing the capacity of, Salmonella constitutively expressing pagP and its isogenic wildtype background strain, to stimulate iNOS expression and synthesis. The involvement of NO in the acquired immune response and its impact on vaccine survival and persistence is a completely novel and exciting aspect of the proposed studies which only became evident as a result of the basic science conducted over the past year.
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