The long term objectives are to understand the immunochemistry of P. aeruginosa mucoid exopolysaccharide (MEP). It has been shown that cystic fibrosis (CF) patients are presistently infected with mucoid P. aeruginosa and colonization by this organism correlates with increasing severity of the disease. The main emphasis will be to identify and characterize different MEP epitopes. If the various MEP epitopes elicit antibody responses that differ with regards to function and protection, responding to the proper epitope may determine whether an individual with CF becomes colonized with P. aeruginosa. Monoclonal antibodies to MEP will be produced. The monoclonal antibodies will be characterized as to whether or not they are able to mediate killing in an opsonophagocytic assay. The MEP antigen will be fractionated by either mild acid hydrolysis or enzymatically with alginase. The oligosaccharides generated will be separated by high pressure liquid chromatography (HPLC) and analyzed for carbohydrate content by gas-liquid chromatography. The MEP fragments will be analyzed serologically for their antigenicity. An ELISA and hemagglutination assay (HA) will be used to test the reactivity of the oligosaccharides to antibody. The antibody source will be either sera or affinity purified antibody to MEP from colonized and noncolonized CF patients, normal volunteers, rabbits immunized with MEP, and the monoclonal antibodies. These serological analyses should allow us to identify epitopes on MEP that bind to either killing of nonkilling antibodies. Several MEP oligosaccharides will be chosen to study for their immunogenicity. The MEP epitopes will be conjugated to a protein and then used to immunize a rabbit. The antibody response to thse conjugates will be assessed by the ELISA and HA using the MEP as the antigen. These sera will also be tested for their ability to mediate killing of mucoid P. aeruginosa in the opsonophagocytic assay. These investigations should improve our understanding of the immunochemistry of the P. aeruginosa MEP antigen which could form the basis for the development of a vaccine.
