Strategies for generating high affinity antibodies against Gram negative bacteria

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Project Summary Sugars cover the outer surfaces of bacteria and other pathogens and are usually very effective at shielding these organisms from the immune system. Lacking T cell help, anti-glycan responses are often of low affinity. Yet the structural complexity of oligosaccharides make them, in principle, excellent candidates for species-specific markers should specific immune recognition occur. The proposed research will use an approach for the generation of such immune responses against unique tri- and tetrasaccharide motifs found on the outer surfaces of dominant pathogenic strains of the Gram-negative bacteria Pseudomonas aeruginosa and Burkholderia cepacia. The method involves the chemical synthesis of the glycan motifs of interest in forms amenable to bioconjugation, the attachment of those molecules to immunogenic virus-like particles with precise control of the type and placement of the linkages used, and the use of a highly active activator of natural killer T (NKT) cells as adjuvant, these cells being central to the elicitation of robust immune responses against sugars in both natural infections and vaccines. This approach has been previously shown to generate ultra-high-affinity and protective antibody responses in mouse against Gram-positive Streptococcus pneumoniae. Gram-negative organisms remain a significantly tougher challenge; a potent immune response against pathogen- specific cell-surface glycan motifs could represent an effective response. The studies proposed here involve the creation and testing of eight candidate vaccine formulations against four carbohydrate motifs, two for each species. Screening of primary immune response will allow for optimization of the design and selection of final immunogens that will be carried into detailed studies. High-affinity antibodies generated from these agents will be further explored to determine their structures, binding affinities and kinetics, mutations from germ line, and abilities to protect against pathogen challenge in a relevant mouse model. Success in this endeavor would identify great potential for the development of human vaccines against these organisms and would provide confidence that the overall approach could be useful for other glycan-based vaccines.