The proposed research project will investigate both the outer membrane proteins and lipooligosaccharide (LOS) of Haemophilus influenzae type beta (Hib) as potential vaccine candidates and as possible virulence factors. (i) Molecular genetic methods will be used to clone the genes encoding selected Hib surface proteins into Escherichia coli. Both native Hib cells and recombinant E. coli cells expressing these Hib surface proteins will be used as sources of these surface antigens which will be purified by the use of monoclonal antibody-based immunoaffinity systems. The ability of these purified Hib proteins to induce protective immunity against systemic Hib disease will be evaluated in an infant rat model. The availability of these cloned genes encoding major outer membrane proteins of Hib will also permit experimental investigation of the possible involvement of these surface proteins in the expression of virulence by Hib. (ii) Hib LOS will be investigated as a possible source of vaccinogen components. Oligosaccharides derived from Hib LOS will be covalently coupled to carrier proteins to generate non-toxic immunogens. These oligosaccharide-protein conjugates will be tested for their ability to induce the synthesis of LOS-directed antibodies which protect against experimental Hib disease. (iii) The association between changes in LOS phenotype and changes in the virulence of Hib will be investigated by the use of mutant analysis. Chemically-induced mutants of Hib unreactive with LOS-directed monoclonal antibodies will be identified in a colony blot-radioimmunoassay. These mutations will be genetically transformed into the wild-type parental strain to construct isogenic sets of LOS mutants. The effect of specific LOS mutations on the virulence of Hib will be studied in a number of different systems.
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