Covalent attachment of saccharides, either polysaccharides or oligosaccharides of Gram-negative enteric pathogens to proteins, have been studied for optimum conditions of derivitization, conjugation and presentation as an immunogen to laboratory animals. The properties of the Vi were studied by chemically modifying the prosthetic groups and visualizing the change in structure in a three dimensional model and in bioassays. Conditions were established for removing the O-acetyl and carboxyl groups. The resultant chemically-modified Vi was then studied for its antigenicity and immunogenicity. The O-specific side-chain of Salmonella typhimurium was purified and derivitized by a variety of methods resulting in either site-specific or random activation. The glycolipids of deep rough Gram-negative enteric bacteria, responsible for Gram-negative shock following sepsis in hospitalized patients, were isolated and characterized. These LPS were detoxified with various methods suitable for human vaccines. The O-specific side-chains of V cholera were isolated and several carrier proteins were studied for their ability to form covalent conjugates with these saccharides. Pneumococcal capsular polysaccharides types 23F and 19F were conjugated in organic solvent to prevent decomposition. Conjugates are expected to increase immunogenicity in children and infants.
