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.
D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis*; D001428:Bacterial Vaccines / Q000276:immunology*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D007074:Immunoglobulin G / Q000096:biosynthesis; D007158:Immunologic Techniques; D008070:Lipopolysaccharides / Q000032:analysis / Q000276:immunology* / Q000302:isolation & purification; D051379:Mice; D011135:Polysaccharides, Bacterial / Q000032:analysis / Q000276:immunology* / Q000302:isolation & purification; D054406:Pseudomonas Vaccines; D011550:Pseudomonas aeruginosa / Q000276:immunology*; D013745:Tetanus Toxoid / Q000032:analysis / Q000276:immunology*
D000907:Antibodies, Bacterial; D001428:Bacterial Vaccines; D000939:Epitopes; D007074:Immunoglobulin G; D008070:Lipopolysaccharides; D011135:Polysaccharides, Bacterial; D054406:Pseudomonas Vaccines; C052223:Pseudomonas aeruginosa O-polysaccharide-tetanus toxoid conjugate; D013745:Tetanus Toxoid