Impact of acetylation of carbohydrate epitopes on the immunogenicity of glycoconjugate vaccines

Glycoconjugate vaccines are among the safest and most successful vaccines developed during the last 40 years. They are composed by poly- or oligosaccharides covalently linked to a carrier protein, which provides T-cell epitopes that are necessary for the affinity maturation of polysaccharide specific B-cells. The immunogenicity of glycoconjugates depends on several factors regarding both the protein (size, site of glycosylation, glycodensity), and the saccharide (sugar to protein ratio, length, charges). Traditional glycoconjugate vaccines are formed by large oligo- or polysaccharides extracted from bacteria for which it is difficult to establish a correlation between chemical structure and immunogenicity. In the last few years, structural approaches for the definition of the polysaccharide portion responsible for the immunological activity have shown promise for the rational design of more efficacious and safer vaccines. The aim of my PhD was to explore the impact of saccharide acetylation on the immunogenicity of glycoconjugate vaccines, focusing on three saccharide antigens where acetylation plays distinct roles. The role of N-acetylation in influencing the immunogenicity of Poly-β-(1→6)-N-acetylglucosamine (PNAG) was investigated, while an in-depth study was conducted on the role of O-acetylation for Staphylococcus aureus capsular polysaccharides serotype 5 (CP5) and serotype 8 (CP8). PNAG is a surface polysaccharide involved in biofilm formation highly conserved across species and considered a potential universal polysaccharide antigen. Natural antibodies to PNAG are poorly effective in mediating in vitro microbial killing or in vivo protection. Conversely, deacetylated PNAG (dPNAG) conjugated to carrier proteins was shown to elicit immune response towards several microorganisms. Our objective was to identify the properties of polyclonal sera raised against dPNAG glycoconjugates. To this end, dPNAG glycoconjugates were prepared and mice immunized to evaluate elicited IgG titers. We assessed the ability of the triggered IgGs to bind de-N-acetylated PNAG compared to partially and fully N-acetylated PNAG, which are immunodominant antigens. The generated polyclonal sera were used for the evaluation of PNAG expression in Klebsiella pneumoniae and to identify growth conditions suitable for analysis of the antibody functional activity. This comprehensive approach aims to enhance our understanding of protective immune responses to PNAG and design improved glycoconjugate vaccines. Studies on S. aureus have focused on evaluating how O-acetylation can influence the immunogenicity of a glycoconjugate vaccine. First, evaluated immune responses to glycoconjugate vaccines with varying O-acetylation levels in CP5 and CP8 polysaccharides conjugated to CRM carrier protein. Further investigation using High Affinity ELISA screening, an essential tool in immunology for measuring antibody-antigen interaction strength, revealed that varying acetylation levels in CP5 had little effect on its immunogenicity or recognition. Conversely, CP8 showed a positive correlation between increased O-acetylation and enhanced antibody affinity. The study and characterization of structural features that influence antigenicity represent a key step in the development of effective next-generation glycoconjugate vaccines.