Preparation and physicochemical characterization of glycoconjugate vaccines

The project of my PhD course has been focused on the characterization of glycoconjugate vaccines to develop a largely applicable methodology to identify their glycosylation sites. The saccharide antigens are covalently attached to the carrier protein by a spacer or using specific functionalities available in both components. The general analytical strategy has involved a tryptic digestion and an enzymatic or acid hydrolysis of carbohydrate antigens in glycoconjugates. The resulting mixture of peptides and glycopeptides with well-defined mass increment has been analyzed by liquid chromatography interfaced with a mass spectrometer (LC-MS) technique to qualitatively understand which lysine residues have been involved in the glycosylation process. Several conjugate vaccines against Candida albicans, Group A Streptococcus (GAS), Group B Streptococcus (GBS) and Neisseria meningitidis group A (MenA) have been analyzed to estimate the distribution of glycosylated lysine residues. Among those, GBS resulted the less glycosylated in comparison to Candida and MenA ones. This aspect it is probably due to the different conjugation chemistries and GBS polysaccharide size. The second part of my PhD project has been the synthesis of two β(1,3)-glucans antigens, a trisaccharide and a hexasaccharide and their conjugation to CRM197. The hexasaccharide has also been linked on a multimeric structure PAMAM4 and then conjugated to CRM197. The last part of this work has been the conjugation to CRM197 of a modified MenB LPS, provided from Institute of Biological Science (IBS) laboratory (National Research Council - Ottawa Canada). The target sites of the carrier protein (CRM197) and LPS have been activated by two different reactans, and the final glycoconjugate has been obtained by the reaction of these two. The resulting conjugate has been characterized (saccharide and protein content, MALDI-TOF mass spectrometry and NMR) using an accurate analytical panel.