GENETICALLY ENGINEERED ORAL VACCINES AND CARIES IMMUNITY

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Dental caries is an infectious disease in which the major etiologic agents in humans are Streptococcus mutans and Streptococcus sobrinus. Studies aimed at inducing immunity against infectious diseases, including dental caries, have provided valuable information on microbial antigens important in inducing protective responses; on the role of IgA antibodies in defense against infections which invade or colonize surfaces bathed by external secretions; and on the mechanisms involved in inducing immune responses. Concurrent with these studies, the advancements in recombinant DNA technology and gene cloning have led to additional information on microbial gene products important in virulence and to the development of novel vaccines. The overall goal of this project is to define mechanisms by which mucosal vaccines consisting of recombinant, avirulent Salmonella strains expressing cloned genes of mutans streptococci, with and without adjuvant, induce specific immune responses. Specifically, we will: l) Determine the effectiveness of Salmonella expressed cloned B subunit of cholera toxin (CTB) in promoting immune responses to co-expressed cloned antigens of mutans streptococci. The levels and isotype of antibodies to the cloned antigens in serum and external secretions of mice and rats immunized orally with Salmonella expressing chimeric proteins will be measured by ELISA to determine the immunogenic and adjuvant properties of the different types of fusions between antigen and CTB in vivo. Analysis of caries activity in a rat model will assess the protective effect of the responses. These studies will determine which structural type of chimeric protein, when expressed by Salmonella in vivo, is most effective in promoting salivary IgA antibody responses protective against mutans streptococci. 2) Determine the effect of the Salmonella vaccine strain, vector, and promoter on the expression of cloned antigen of mutans streptococci and on the induction of immune responses. The amount of cloned antigen of mutans streptococci produced by Salmonella mutant strains containing expression vectors with different promoters and replicons will be measured by ELISA. Stability of the expression vectors will be assessed by microbiologic detection of Salmonella in tissues of mice orally-immunized with the Salmonella vaccine. The level and isotype of antibodies to the cloned antigen in serum and external secretions of animals orally immunized with a Salmonella strain containing different vectors will be measured to determine if differences exist in the magnitude and duration of the mucosal immune responses and in protection against infection by mutans streptococci. These studies will determine the effect of the vector replicon on the in vivo expression of cloned protein and on the induction of immune responses, and whether immune responses induced by the in vivo expression of cloned protein under the control of a constitutive promoter (trc) versus one induced in an anaerobic environment (nirB) are of greater magnitude and longer duration. 3) Determine the effect of route of immunization with a recombinant Salmonella vaccine on the magnitude and protective capability of the salivary IgA antibody response. The level of IgA antibodies to the cloned antigen in saliva in animals immunized by the oral or intranasal route will be measured by ELISA to determine which route of immunization induces the highest salivary IgA antibody response protective against infection by mutans streptococci. The results of these studies will be relevant in establishing the practicability of Salmonella vaccine delivery systems for the induction of protective immune responses against mucosal pathogens including those associated with the oral cavity.