The long term objectives of this proposal are to understand the ineffectual nature of the immune response of cystic fibrosis (CF) patients to chronic mucoid Pseudomonas aeruginosa infections and to develop immunotherapeutic strategies to alleviate the pathologic effects of chronic infection. Results to date suggest that the mucoid exopolysaccharide (MEP or alginate) capsular antigen is a critical bacterial component needed for pathogenesis, and this antigen functions principally by allowing bacterial cells to avoid host defenses. One important property of MEP involved in avoiding host defenses is that the antibodies it provokes are nonopsonic. A few CF patients have been identified that lack chronic infection and have MEP- specific opsonic antibodies, suggesting that provision of these opsonic antibodies might provide protection against chronic mucoid infections. This proposal will focus on the structural and molecular characteristics of MEP, particularly the acetate substituents on the mannuronic acid residues, as critical to its immunopathogenic properties. One hypothesis to be investigated is that acetate substituents serve several critical functions: to protect mucoid P. aeruginosa cells from phagocytic killing by complement (C') with or without nonopsonic antibodies to MEP; as components of the epitopes that bind opsonic antibodies; and the acetate level determines the ability of MEP to provoke opsonic antibodies. These studies will use recombinant strains of mucoid P. aeruginosa that produce partially or completely acetylated versions of MEP, obtained by controlling the level of the acetylase enzyme, algF. Strains with different levels of acetylated MEP will be evaluated for their C' activating and C'3 binding capacity, ability to resist host phagocytic defenses, and ability to bind monoclonal and polyclonal MEP- specific opsonic and nonopsonic antibodies. Purified MEP antigen will be chemically modified to contain variable amounts of acetate or other short chain fatty acid substituents, then evaluated in mice for their immunogenic properties. A second hypothesis under investigation is that the inability to produce opsonic antibody to MEP correlates with the presence of preexisting nonopsonic antibody to MEP. Immunosuppression may be mediated by Fc-receptor- positive cytotoxic-T-cells, which have been shown in an in vitro assay to bind immune complexes containing MEP antigen and kill hybridoma cells that secrete MEP-specific antibodies. To determine how the properties of antigen size, which partially overcomes the inability to produce opsonic antibodies, and acetylation level impact upon this interaction leading to suppression, variants of MEP will be used to identify those with reduced capacity to induce cytotoxic T-cell mediated immunosuppression. This will be evaluated using in vivo adoptive transfer and in vitro cytotoxicity assays. These assays will also be used to examine the role of cytokines in promoting and inhibiting the production of MEP-specific opsonic antibodies and to identify cytokines that could be used as adjuvants to elicit MEP- specific opsonic antibody in humans. These studies should result in improved methods for inducing MEP-specific opsonic antibodies in humans and a better understanding of the mechanisms whereby mucoid P. aeruginosa can persist for decades in the lungs of Cf patients.
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