Molecular basis of the interactions between carbohydrate antigens and monoclonal antibodies

Carbohydrate-based vaccines induce an immune response against surface glycans found on pathogenic bacteria and are used to protect children from meningitis and other bacterial diseases. The definition of the epitope targeted by protective antibodies is a crucial information for the design, description and even registration of glycoconjugate vaccines. The aim of this project is the characterization at atomic level of the interaction between Group B Streptococcus (GBS) oligosaccharides with functional antibodies mediating bacterial phagocytic killing. The specificity of anti-GBS type III polysaccharide (GBS PSIII) antibodies has been ascribed to a conformational epitope with an extended helical structure which would be formed by multiple repeating units. Here fragments of GBS PSIII were obtained by chemical synthesis and by sizing of the native polysaccharide from bacterial source. The oligosaccharides (OSs) were purified by ion exchange chromatography and were characterized by mass spectrometry, HPLC and NMR analysis. First, the obtained OSs were used to characterize the interaction to protective anti PSIII rabbit monoclonal antibody (mAb) by competitive ELISA and surface plasmon resonance analysis. The dependence of binding affinity on oligosaccharide length was confirmed and the minimal structures showing comparable binding to full-length PS III were identified. Next, a combination of saturation transfer difference NMR (STD-NMR) and X-ray crystallography was used to map at atomic level the interactions in the rabbit mAb-OS complex. The challenging crystallization of the carbohydrate-protein complex was achieved and the molecular bases of the antigen-antibody interaction were unveiled. The obtained results were used to optimize a vaccine based on short GBS PSIII fragments.