Lembo, Antonio (2024) Development and analysis of glycoprotein based antiviral vaccines using Mass Spectrometry. [Tesi di dottorato]

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Abstract

Glycosylation is the most prominent post-translational modification occurring on protein antigens and its specific pattern depends both on the cells used for their expression (e.g. mammalian cells, insect cells, yeast) and the cells growth conditions. Oligosaccharide moieties on protein antigen surface play an important role in the modulation of its immunological properties; indeed, glycans can affect these features in different ways, like protein epitope shielding, formation of oligosaccharide epitopes and presence of non-self monosaccharides. Mass spectrometry (MS) represents a powerful tool for the in-depth analysis of proteins glycosylation pattern; indeed, it allows to describe the microheterogeneity of each glycosylation site in a reproducible manner, unlike X-ray crystallography, which fails to describe highly flexible structures like glycans. Two recombinant glycoprotein antigens from Human Cytomegalovirus (HCMV) envelope have been considered as case study for the development of MS-based methods for glycans analysis: glycoprotein B (gB) and Pentamer (gH/gL/UL128/UL130/UL131); these two antigens are the most promising candidates for the development of a vaccine against HCMV. Furthermore, different expression conditions have been considered in order to compare antigens with different glycan patterns. In this thesis the glycoprotein antigens have been characterized by using different hyphenated MS techniques (LC-MS/MS, LC-FLR-MS, LC-UV-MS, GC-MS), depending on the class of analytes originating from the antigen: glycopeptides, glycans and monosaccharides. The analysis of the first category of molecules relies on a bottom-up proteomic approach in which the N- and O-glycosites heterogeneity and occupancy have been investigated by using tandem mass-spectrometry; this study allowed to see that both gB and Pentamer N-glycosylation sequons possess glycans with different maturation degrees, depending on the accessibility of the processing enzymes to each site. In addition, it has been proved that, in cell culture conditions that arrest glycans maturation to oligomannose structures, site-specific maturation is almost identical for each site. Released N-glycans analysis has been performed by enzymatic glycans shaving followed by liquid chromatography with fluorescence detection and mass-spectrometry characterization; this analysis allowed to confirm the identities of the glycans found by site-specific analysis, also providing further information about the presence of highly processed tetrantennary complex glycans and Neu5Gc-containing oligosaccharides. Antigens absolute monosaccharide composition has been assessed by gas chromatography-mass spectrometry, degrading the oligosaccharides in acetylated methyl glycosides and using the internal standard method for quantification; furthermore, a comparison with monosaccharide analysis by LC-UV-MS has been done, which showed to be a highly sensitive method. Immunogenicity of gB and Pentamer with different glycosylation pattern have been measured by neutralizing antibodies titer elicited in mice after vaccination; both gB and Pentamer showed higher immunogenicity when bringing only high-mannose structures. To assess the shielding that glycans play on gB and Pentamer epitopes, tridimensional models of the oligosaccharides on these proteins have been built; therefore, it has been seen that antigens bringing only M5 glycans have more exposed epitopes than once bringing M9 glycans. To conclude, glycoprotein oligosaccharide pattern can be broadly explored by MS techniques, providing a lot of information about the nature and the stoichiometry of the glycans on the antigens surface. The combination of the MS data with a glycoprotein modeling software is a strong tool to predict how glycans can affect the recognition of antigen epitopes.

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