Meningococcal meningitis remains a relatively common disease in all parts of the world and not infrequently serious epidemics occur. The current meningococcal polysaccharide vaccines are of great value in aborting group A and C epidemic meningitis, but are not suitable or endemic control of the disease and provide no protection against group B strains. In order to devise the optimal strategy and to promote development of vaccines based on outer membrane proteins the molecular biology and function of these antigens need to be understood in much greater detail. The proposal aims to obtain: 1. Molecular and functional characterization of porin proteins of class 2 and 3 2. Molecular and functional characteriztion of class 4 protein 3. Molecular characterization of clas 1 protein 4. Characterization of peptidoglycan associated antigens The characterization of proteins 1, 2, 3 and 4 includes optimizing purification protocols, obtaining N-terminal sequences for proteins 1 and 3 (those of proteins 2 and 4 are known), obtaining N-terminal sequences of CNBr fragments, and cloning and sequencing the genes from lambda gtll banks. Clones will be selected by hybridization with DNA probes and by immunological means. The purified proteins will be used to immunize mice and select monoclonal antibodies. With these antibodies (and additional ones from other investigators) the surface exposed domains of the proteins will be defined by immunological, biochemical and rDNA techniques. The ability of monoclonal antibodies to mediate (or block) serum bactericidal activity and to protect in vivo in a mouse bacteremia model will be determined. The functional properties of the proteins will be studied by creating deletion and site-specific mutations in the cloned genes, re-introducing the mutated genes into meningococci organisms and observing the alterations in phenotype. Changes in growth under various conditions, alterations in outer membrane anatomy and composition, pathogenicity in the mouse bacteremia model, behavior in organ cultures and, in the case of the porins, alterations in their biophysical properties will be examined. To explore whether other surface proteins should be considered as vaccine candidates peptidoglycan associated proteins will be characterized.
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