Polysaccharides, from capsule or from the LPS of Gram-negative enteric pathogens, are covalently conjugated to carrier proteins to increase their immunogenicity. Conditions for optimal derivatization of the saccharides, proteins and conjugation of the two are investigated. A new approach to synthesize Vi conjugate without disulfide linkages was devised. The carboxylic groups on Vi polysaccharide was derivatized with adipic dihydrazide through carbodiimide. The derivatized Vi could then bind to proteins through carbodiimide. The LPS of Vibro cholerae is a virulence factor and potential protective antigen. The toxicity of the LPS was greatly reduced by treatment in an organic base. The treatment retained most of the LPS structure. Conjugates made with the detoxified LPS and cholera toxin induced vibrocidal antibodies in laboratory animals. Similar techniques were devised for preparing polysaccharide of Salmonella typhimurium, E. coli 0111 for diarrhea diseases, mutant J5 for endotoxin shock and 0157 for gastroenteritis.
