Characterization of the Salmonella Typhimurium core oligosaccharide and its reducing end 3-deoxy-D-manno-oct-2-ulosonic acid used for conjugate vaccine production

One of the strategies adopted for the development of a bivalent conjugate vaccine against invasive nontyphoidal Salmonella consists of linking the O-antigen component of S. Typhimurium and S. Entertidis lipopolysaccharides to the carrier protein CRM197, a non-toxic variant of diphtheria toxin. The conjugation reaction uses the reducing end residue 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) of the core to which the O-antigen chain is bound (OAg-core). OAg-core chains are cleaved from the lipid A directly in the fermentation broth by mild acid treatment. Kdo has been reported to undergo structural changes under these conditions and therefore the Kdo at the reducing end was thoroughly analysed to verify its structural integrity. For this purpose, low molecular mass OAg-core chains extracted from S. Typhimurium wild type bacteria and core oligosaccharides extracted from S. Typhimurium bacteria mutated not to produce O-antigen repeats were characterized by GLC-MS, MALDI-TOF-MS and NMR spectroscopy. Moreover, a combination of H-1-(1)Hand H-1-C-13 experiments confirmed the linkage positions, sequence and structure of the octasaccharide core with 5-linked Kdo present at the reducing end in its native structure: alpha-GlcpNAc-(1 -> 2)-alpha-Glcp-(1 -> 2)-alpha-Galp-(1 -> 3)-[alpha-Galp-(1 -> 6)]-alpha-Glcp-(1 -> 3)-[alpha-Hepp-(1 -> 7)]-alpha-Hepp- (1 -> 3)-alpha-Hepp-(1 -> 5)-Kdo.