Modified Project Summary/Abstract Section Glycoconjugate vaccines provide enormous health benefits globally, although they have been less successful in some populations at high risk for developing disease, such as the elderly. In most cases, conjugation of the sugar antigen to the carrier protein has been done empirically with little attention paid to variables critical for the immune response. Recent findings from our lab offer a new and rational explanation for how conjugates work. The new model suggests that carbohydrate presentation to T cells by antigen-presenting cells may strongly enhance the efficacy of antibody responses. Application of this principle in mouse models of group B streptococcal disease and Francisella tularensis infection by using a carrier peptide rather than a protein, resulted in vaccines more protective than standard glycoconjugate vaccines. We have also demonstrated the critical role of the peptide linker and the conjugation site in enhancing both the conjugation efficiency and the immune response to vaccines. The results support the notion that peptide glycoconjugate vaccines provide superior protection against bacterial challenges. Remarkably, the use of a peptide instead of a protein as a carrier confers better protection at much lower levels of carbohydrate specific IgG. This observation challenges the paradigm of a direct correlation between the amount of IgG induced by a glycoconjugate and protection. A comprehensive evaluation of antibodies and immune cells generated by protein vs. peptide glycoconjugates will clarify the features that make peptide vaccines extremely potent. We will evaluate the antibody response to glycopeptide vaccines in terms of IgG subtypes, affinity, avidity and functional activity, and elucidate the costimulatory/coinhibitory molecules being expressed on memory B cells and relate this to the functionality of the antibodies being made. Finally, we will define the interactions between the MHCII antigen presentation molecule and the helper T cell receptor using a proteomic approach. Recombinant TCRs will be used to structurally define the interactions with glycopeptide epitopes. Our approach will identify the characteristics that make glycopeptide-induced antibodies so potent. We will apply our studies of these peptide/polysaccharide conjugates to create more efficacious and longer lasting immunity to glycoconjugate vaccines.