A long term objective of this research is to understand the immunochemical properties of the mucoid exopolysaccharide (MEP) produced by strains of Pseudomonas aeruginosa that chronically colonize the respiratory tract of cystic fibrosis patients. These objectives include characterization of the immune response to this material in the colonized CF patient, characterization of the functional activity of this antibody in an opsonophagocytosis assay, and determination of the vaccine potential of MEP by looking at the immunologic status of older, non-colonized CF patients. These determinations should document whether or not human antibody to MEP is able to mediate opsonic killing of P. aeruginosa. If it is not able to do this we will determine if it can block opsonic killing mediated by rabbit antibody raised to MEP. If non-killing, blocking actibity is documented in colonized CF patient's sera and sputa, this could be a major reason why they are unable to eliminate their colonizing P. aeruginosa strains. Furthermore, if we can document opsonic killing activity in the sera and sputa of older non-colonized CF patients who have remained free from P. aerginosa disease, this would suggest a protective immunity to mucoid P. aeruginosa with a MEP vaccine. Killing and blocking antibodies to MEP will be characterized after isolation by affinity chromatography, wherein the MEP has been covalently linked to epoxy activated Sepharose. Immunoglobulin isotypes will be determined using the techniques of immunodiffusion and ELISA analysis. Polyacrylamide gel electrophoresis will be used to characterize the physical state of these antibodies. We will test the vaccine potential, in animals, of a protein polysaccharide conjugate prepared by coupling carbodiimide activated MEP with the exotoxin A protein of P. aeruginosa. We will also test the hypotheses that a reason why CF patients develop blocking antibody to MEP is because this antigen is delivered across a mucosal barrier. To do this we will colonize mice or rabbits with mucoid P. aeruginosa in the presence of streptomycin and analyze their serum immune responses for the development of antibody to MEP. These data are being generated to see what effects the MEP antigen has on the immune system and if defective immune responses can be documented that will explain why CF patients are unable to eliminate their colonizing strains of mucoid P. aeruginosa.
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