The major goals of this project are to understand basic aspects of virulence and host immunity to mucoid strains of P. aeruginosa that are major causes of morbidity and mortality in cystic fibrosis (CF) patients. About 2500 new babies with CF are born each year worldwide, and by adolescence 80 per cent of them will become chronically infected with mucoid P. aeruginosa. Clinical data clearly show that the decline in pulmonary function and ultimate early morality that occurs in P. aeruginosa-infected CF patients is due to mucoid P. aeruginosa infection and not to any other pathogen, including non-mucoid P. aeruginosa. Therefore, preventing new infections or treating established infections with immunotherapeutic agents that target mucoid P. aeruginosa should have a major impact on the health of CF patients. The primary hypotheses to be evaluated are that chronic mucoid P. aeruginosa infection is dependent on elaboration of an acetylated version of the mucoid exopolysaccharide (MEP) surface antigen, and that antibodies to the acetylated epitopes on MEP confer protection from chronic infection. A key component of this work will use our ability to initiate and establish a chronic mucoid P. aeruginosa infection in transgenic CF mice, a tool that, to date has eluded other researchers using these mice. The first hypothesis will be evaluated using in vitro studies to ascertain the ability of the acetylated MEP to confer resistance on mucoid P. aeruginosa to opsonic killing as well as in in vivo studies in CF mice. In the mice we will evaluate the ability of strains with differing abilities to acetylate MEP to establish and maintain a chronic colonization. The second hypothesis will be evaluated by production of conjugate vaccines composed of MEP with differing levels of acetylation, prepared by chemical manipulations. The conjugate vaccines will be evaluated for their immunogenicity in mice and rabbits and for their in vitro characteristics in regard to their ability to elicit antibodies that mediate opsonic killing of a multitude of mucoid P. aeruginosa strains. Next the vaccine(s) with the best ability to elicit high titers of broadly-reactive opsonic antibody will be tested in transgenic CF mice, using both active vaccination to prevent9 infection and passive therapy of established infection. In addition, we will test our proposed hypothesis that the presence of pre-existing, non-opsonic antibodies to non-acetylated epitopes on MEP in CF patients prior to the onset of mucoid P. aeruginosa infection interferes with the ability to mount a protective, opsonic response to the acetylated epitopes. Finally, we will evaluate the potential of some recently prepared human monoclonal antibodies to MEP to reduce the levels of mucoid P. aeruginosa in the lungs of infected CF mice. The results of this work should further our insights into the pathogenesis of mucoid P. aeruginosa infection, and promote development of effective vaccines and passive therapeutic reagents to prevent and treat mucoid P. aeruginosa infections in CF patients.
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