Preparation and immunogenicity of a bivalent cell-surface protein-polysaccharide conjugate of Vibrio cholerae

Alkali-treated lipopolysaccharides (LPS) from Ogawa and Inaba serotypes of Vibrio cholerae were chemically coupled to cell-surface proteins of V. cholerae. The reaction product was eluted in the void volume when fractionated on a column of Sephacryl S-300. The material did not enter the gel when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE). The bivalent protein-polysaccharide conjugate was nonpyrogenic, as determined by the Limulus lysate assay. It was immunogenic and elicited, in rabbits, antibodies against both intact LPS and cell surface proteins, as determined by enzyme linked immunosorbent assay. LPS from Ogawa serotype was resolved into two major bands by SDS-PAGE and that from the Inaba serotype into one major band. Immunoblotting studies indicated that antisera to the protein-polysaccharide conjugate contained antibodies to the major LPS fractions from both serotypes. Antisera to the protein-polysaccharide conjugate tested by crossed-immunoelectrophoresis produced immunoprecipitation with whole-cell sonicates of both biotypes and serotypes of V. cholerae. Such antisera also possessed agglutinating and complement-mediated bactericidal activities towards V. cholerae strains of both biotypes and serotypes. These results suggest that a bivalent cell-surface protein-polysaccharide conjugate of V. cholerae could be developed as a nonpyrogenic vaccine against cholera.