The WHO report of Infectious Disease deaths for 1995 indicated that there were more than 13 million deaths world-wide during that year. The majority of those deaths were caused by organisms that first make contact with and then either colonize or cross mucosal surfaces to infect the host. A number of strategies have been developed to facilitate mucosal immunization to prevent these diseases, including addition of bacterial products with known adjuvant properties. The two bacterial products with the greatest potential to function as mucosal adjuvants are cholera toxin (T), produced by various strains of Vibrio cholerae, and the heat-labile enterotoxin (LT) produced by some enterotoxigenic strains of Escherichia coli. A number of mutants of CT and LT have been developed in an attempt to dissociate the desirable adjuvant properties of these molecules from their toxic effects. Both active-site and protease-site mutants have been constructed and evaluated in a variety of animal models with different antigens. Important questions regarding the adjuvanticity of CT and CT and mutants of these toxins remain to be answered. Some of these questions are practical and the answers will impact the immediate and short term use of these molecules in human vaccines. Other questions address the underlying mechanisms associated with adjuvanticity and the answers will have their greatest impact in the design of future adjuvants and vaccine strategies and in the development of a better understanding of vaccine induced immunity. The proposal includes a series of Specific Aims designed to directly address these issues. One of the most important aspects of the proposed study is a side-by-side comparison of CT, LT, active-site mutants, protease-site mutants, and recombinant B-subunits for the ability to induce specific, targeted immunologic outcomes as a function of route of immunization and nature of the co-administered antigen. With the information obtained in the proposed studies, future vaccine strategies can be designed employing the optimum adjuvant/antigen formulation and route of administration for a variety of bacterial and viral pathogens. This proposal also examines the underlying cellular and intracellular signaling pathways activated by these different molecules to better understand the mechanisms of adjuvanticity at the cellular level.
