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Combination of molecular approaches to access homogeneous glycoconjugates for structure-immunogenicity relationship studies
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International audience. Glycoconjugate vaccines consist of the conjugation of bacterial surface polysaccharides (PS) to a carrier protein. The latter allows the induction of antibodies directed against the carbohydrate antigen thanks to the recruitment of CD4+ T lymphocytes to elicit a protective immune response. PS length, carrier protein/PS ratio and bioconjugation chemistry have been established as key factors that have a direct impact on the immune response. In addition, it has long been assumed that the sites to which the PS is attached to the carrier protein (connectivity) may also have an impact. Impact of connectivity on the immune response can now be addressed through a combination of molecular tools such as site-selective mutagenesis, incorporation of unnatural amino acids (UAA) and bioorthogonal conjugation strategies leading to fully-controlled, homogeneous glycoconjugates. In our study, pneumococcal surface adhesin A (PsaA), a highly conserved immunogenic protein expressed by Streptococcus pneumoniae (Pn) and a synthetic dodecasaccharide mimicking the capsule of Pn serotype 14 (Pn14DS) have been selected as model carrier protein and carbohydrate antigen, respectively.Taking advantage of the absence of cysteine in PsaA, up to four lysine residues have been mutated to cysteine for subsequent bioconjugation to Pn14DS using thiol-maleimide coupling chemistry. Site-specific UAA incorporation approach has also been envisaged as an alternative to prepare the glycoconjugates according to ‘click-chemistry’ reactionsThe conjugation sites were targeted to key locations within or outside T-helper epitopes to investigate how connectivity impacts the recruitment of CD4+ T lymphocytes and beyond, the level and quality of the humoral response induced against both the carbohydrate antigen and the PsaA.
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