The overall aim of this proposal is to develop a subunit dental caries vaccine that elicits enhanced immunity to significant peptide and polysaccharide epitopes of the glucosylfransferase(GTF)-glucan virulence pathway that can be delivered in a manner to stimulate protective mucosal immune responses. We have demonstrated that two synthetic peptides, representing areas of GTF function, when injected in a multiple antigenic peptide (MAP) format, protect rats from experimental caries. The research described in this proposal will, in the first specific aim, evaluate the immunogenicity in rats and protective potential of synthetic peptides associated with a putative second catalytic site of GTF administered in a MAP format. Antibody will be determined by ELISA and T cell responses will be evaluated by proliferation assays. The antigenicity in humans and the presence of B and T cell epitopes will also be investigated using overlapping truncated peptides. Also, we will seek a surrogate universal T cell epitope or GTF T cell epitope in the outbred Sprague-Dawley rat. In the second aim, these peptide sequences, together with those already shown to be effective, will permit the design of multiple sequence, multiple epitopic peptide vaccines in a MAP format. These multiple sequence MAPs may by themselves, or by coimmunization with MAP mixtures, achieve enhanced functional inhibition of GTF as judged by 14C glucose incorporation into glucan and protection in the rodent dental caries model. In the third specific aim the conjugation of protein to polysaccharide which can significantly intensify immune responses to both components will be used to evaluate antibody to glucan and to enhance antibody to multiple epitopic sequences. To explore the potential amplification in immunity achieved by this technique, we will conjugate these multiple epitopic peptide sequences directiy to glucan, or to glucan through tetanus toxoid, and evaluate the enhancement in immunological and dental caries-protective effect of the individual and combined components in animal studies. Since we hypothesize that immunity in saliva is likely to result in the most direct protection, in the fourth specific aim we will explore the effect of mucosal application of the subunit vaccine on salivary antibody formation and protection. Subunit vaccines will be delivered to inductive mucosal sites directly in Al(OH)3 Also, attenuated Salmonella constructs will be developed to express tetanus toxin fragment C-GTF peptide chimeric proteins. These attenuated Salmonella encoding GTF peptides to be expressed as protein fusions will also be administered to inductive mucosal sites and evaluated immunologically and for the ability to elicit protection. Our goal is to design a vaccine that contains an array of functional and immunologically relevant epitopes, in a format that will result in a sustained and high level of protection against dental caries and is acceptable for human use.
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