Enhancement of the humoral immune response to Pseudomonas aeruginosa flagellin

The goal of this project was to develop a vaccination strategy that would enhance the protective humoral immune response against the flagellin protein of Pseudomonas aeruginosa. DNA vaccine constructs encoding the type A flagellin gene were tested for protective efficacy in animal studies. Even though high titers of anti-flagellar antibodies were produced in mice injected with these constructs, no protection was seen. Further analyses suggested that type A flagellin expressed by eukaryotic cells was not glycosylated, while type A flagellin expressed by P. aeruginosa is, and that antibodies against these glycosyl groups may be needed for protection.; The ability of a T cell binding ligand to promote a Th2 response and enhance the production of IgGI antibodies upon intramuscular injection of DNA was tested. The DNA encoding amino acids 135-149 of the beta2 domain of MHCII was cloned in front of the type B flagellin gene. After injection into mice, no enhancement or directing of the humoral immune response was seen, suggesting that this T cell binding ligand did not direct a Th2 response when it was fused to the whole flagellin protein in this DNA vaccine.; The mechanism behind the enhancement of the humoral immune response seen with dendritic cell targeting was also examined. Purified flagellin cross-linked to N418, a hamster monoclonal antibody specific for CD11c on dendritic cells, induced high titers of anti-flagellar antibodies 7 days after injection into mice. This rapid response depended on the presence of the N418 antibody. The optimum boost schedule that resulted in the highest titers of antibody was determined to be two injections of N418 conjugate one week apart. Using this schedule, mice were significantly protected after challenge. It was determined that binding of the N418 conjugate to dendritic cells activated them causing an increase in the number of cells expressing MHCII and CD86. This correlated with the ability of the dendritic cells to stimulate primed T cells to secrete IL-2. These experiments also indicated that conjugate immunized mice produced primed T cells one week after injection.