Group B meningococci affect very young children. The group B capsule is not immunogenic, and protein vaccines provide only short-lived, serotype restricted, protection in older children. A vaccine for infants is needed. Protection is mediated by bactericidal antibodies that are induced by commensal organisms that share lipooligosaccharides (LOS). LOS are immunogenic at birth, and infants make bactericidal LOS antibodies during disease. By adolescence most children have LOS IgG that are bactericidal for most meningococcal strains. These antibodies bind to one or more conserved structures that have not been identified. It is these antibodies that prevent meningococcal disease in older children and adults. It is the goal of this project to develop a vaccine that reliably will induce bactericidal LOS IgG during infancy. To do this we first need to define the optimal LOS structure that can bind bactericidal IgG. We purified human LOS IgGs by passage of IVIG through Sepharose to which we coupled L1; L1,1a,8; L2,4; L8v; L2; and L7 LOS. These IgGs avidly bind one or more conserved structure within heterologous LOS and are bactericidal for strains that express heterologous LOS. We identified a consensus peptide that mimics the conserved LOS structure by panning a phage display library. Preliminary analyses suggest that: a) the LOS oligosaccharide must have at least one galactose residue for optimal binding; b) sialylation decreases binding; c) the pools contain both L-type and conserved specificities, and d) the number and location - cyclic or exocyclic - of basal phosphoethanolamine (PEA) influences the specificity and/or affinity of binding. We now propose to enrich for IgG that binds the conserved LOS structure by sequential passage of IgG over heterologous affinity gels and assess how well each IgG kills a representative sample of endemic case strains, and whether they can opsonize these strains for PMN killing. We will use High Performance Anion- Exchange Chromatography and Mass Spectrometry, each combined with genetic, chemical and/or enzymatic degradations, including O-deacylation of lipoidal moieties and dephosphorylation of glycose and lipoidal moieties, to identify the optimal conserved LOS structure that binds bactericidal LOS IgG. We will sequence the Igt genes that encode LOS glycosyl transferases and create mutants with sequential loss of glycoses from their LOS oligosaccharides and loss of cyclic and/or exocyclic PEA residues. We will assess the binding of IgG to these mutants by Flow cytometry and determine the affinity with which LOS IgG binds native and altered LOS by surface plasmon resonance. We will compare the ability of peptide mimics, identified by phage display panning with different LOS IgG, to inhibit the heterologous binding to, and killing of, the case strains, in order to determine whether more than one conserved structure is involved. The results should lead to the development of a vaccine against group B disease in infants. ? ? ?
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