O-polysaccharide-protein conjugates induce high levels of specific antibodies to Pseudomonas aeruginosa immunotype 3 lipopolysaccharide.

A semi-synthetic vaccine against Pseudomonas aeruginosa immunotype 3 was prepared by chemical coupling of P. aeruginosa immunotype 3 O-polysaccharide to tetanus toxoid. The O-polysaccharide was obtained by mild acid hydrolysis of immunotype 3 lipopolysaccharide, and purified by gel permeation chromatography. The purification was evaluated by high performance liquid chromatography. Additional analyses revealed a high grade of purity of the O-polysaccharide, and an at least 1000-fold reduction of endotoxic activity as compared to homologous lipopolysaccharide. O-Polysaccharide was conjugated to tetanus toxoid, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as coupling reagent. Antigenic determinants of both O-polysaccharide and tetanus toxoid were retained after conjugation, as tested in a sandwich enzyme-linked immunosorbent assay. Immunization of mice revealed that O-polysaccharide was nonimmunogenic in mice, while the O-specific part of the conjugate was able to induce high levels of IgG antibodies reacting with immunotype 3 lipopolysaccharide in an enzyme-linked immunosorbent assay. By immunoblotting it was shown that the antibodies were directed to high molecular weight lipopolysaccharide only, demonstrating specificity for its O-polysaccharide moiety.