The long term goal of this application is to understand the molecular and cellular basis for the ineffective human immune response to Pseudomonas aeruginosa alginate and to develop immunologic interventions that will provide effectors that mediate alginate-specific resistance to infection. Alginate is over-expressed by strains of P. aeruginosa isolated from chronically infected cystic fibrosis (CF) patients. Results generated from current funding indicate that alginate is expressed at sufficient levels on many clinical non-mucoid isolates, particularly during in vivo infection, to allow for targeting of alginate as a vaccine for these strains. Alginate is an effective antigenic target for immune resistance and clearance but virtually all natural human immune responses result in antibody that fails to mediate opsonic killing and immunity. Vaccine induced antibody is opsonic and protective but unconjugated alginate is poorly immunogenic for humans.
The aims of this application include further development of alginate-conjugate vaccines, focusing on use of P. aeruginosa flagella as a carrier protein and improvement of the affinity of human monoclonal antibodies (Mab) to alginate for evaluation of their protective potential. Flagella will be conjugated to seaweed polymannuronic acid or P. aeruginosa alginate using thiol-bonds, vaccines evaluated for immunogenicity in mice and rabbits and for protective activity using non-mucoid P. aeruginosa strains in a murine acute pneumonia model and a model of chronic lung infection in transgenic CF mice. Currently available human Mabs to alginate can mediate opsonic activity against both mucoid and non-mucoid strains, and protection in mice against pneumonia due to non-mucoid strains. Their affinities will be further improved using phage display and error-prone PCR. The resultant Mabs will be evaluated for opsonic activity against both mucoid and non-mucoid strains of P. aeruginosa in vitro. In vivo studies will encompass prophylactic, passive protection against acute pneumonia due to non-mucoid strains and chronic oropharyngeal colonization and lung infections in transgenic CF mice using both non-mucoid strains. The Mabs will also be evaluated for their ability to modulate infection and pathology from established mucoid P. aeruginosa infection in transgenic CF mice.
These aims will lead to further development of immunotherapeutics for P. aeruginosa infection in CF and evaluate alginate as an antigenic target for non-mucoid, as well as mucoid, P. aeruginosa infections.
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