Five billion people suffer from dental disease. Broad-based public health anti-caries measures are necessary before this disease is conquered. Dental caries, especially in childhood, is strongly correlated with levels of mutans streptococcal colonization, particularly S. mutans and S. sobrinus. Despite intensive investigation, a major gap exists regarding whether the initial colonization and accumulation of mutans streptococci in the oral biofilm of the one-two year old child can be influenced by the presence of antibody to virulence antigens of mutans streptococci, for example through """"""""passive"""""""" immunotherapy. The goal of this continuation of DE- 04733 is to develop up to 3 high affinity human anti-glucosyltransferase (GIF) monoclonal reagents for pediatric immunotherapeutic use. To do so we will merge our experience in identifying protective epitopes on GTF and our successful application of active and passive immune approaches to reduce experimental dental caries, with the proven ability of the Marasco/Dana Farber laboratories to successfully develop human monoclonal reagents (HMRs) for human diseases. To this end we propose to develop high-affinity, inhibitory HMRs directed against S. mutans GTF epitopes through isolation from a validated 27 billion member human single-chain variable region antibody fragment (scFv) phage display library, subsequent bivalent scFvFc antibody (minibody) construction, epitope mapping and affinity determination to maximize inhibition potential, followed by full human monoclonal antibody (HMR) construction. Inhibition of GTF enzymatic activity and interference with accumulation of mutans streptococci in artificial biofilms will be used to measure in vitro effects on GTF functional activity by scFv phage, scFvFc during HMR development. We will then evaluate the protective potential of the immunotherapeutic candidates in vivo, by using these HMRs, individually and collectively, to block colonization, accumulation and dental caries-forming ability of both mutans streptococcal species that infect man. This protocol will be performed under 3 aims: To isolate a panel of GTF-inhibitory HMR; to determine the ability of HMR to block mutans streptococcal entry into biofilm and to determine the ability of HMR to interfere with dental caries in rats. Once developed and vetted through our experimental animal model, we will be poised to use these tools to pursue pediatric immunotherapeutic approaches to block or delay the oral colonization/accumulation of cariogenic mutans streptococci.
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