RATIONAL DESIGN AND SYNTHESIS OF A GLYCOCONJUGATE VACCINE AGAINST GROUP A STREPTOCOCCUS

Glycoconjugate vaccines are one of the most effective ways of preventing bacterial and fungal infections. They are generally composed of a saccharide antigen covalently linked to a carrier protein to convert the saccharide from a T-independent to a T-dependent antigen, resulting in increased immunogenicity from infancy and memory response. Currently, most of the conjugate vaccines are produced from polysaccharides extracted and purified from bacterial cultures. However, use of synthetic oligosaccharides constitutes a promising alternative approach. Synthetic saccharides have well-defined structure and can be equipped with a linker at their reducing end for selective conjugation allowing sugar epitopes preservation. Moreover, synthetic conjugates allow to avoid the handling of pathogens and presence of bacterial impurities and are characterized by minimal batch-to-batch variability and higher quality control standards compared to conjugates with native polysaccharides. New techniques such as solid phase automated synthesis and chemo-enzymatic approaches reduce reaction time and use of protective groups during synthetic steps and favor oligosaccharides production at large scale. In addition, nanoparticles have been recently proposed as potential multivalent delivery systems also for carbohydrate-based vaccines. Gold nanoparticles (AuNPs) have attracted great attention in this field due to their unique characteristics of biocompatibility and easy production. In the last years, AuNPs have been tested as carrier for short synthetic oligosaccharides in animal models. Natural nanoparticles can be obtained by the methodology called Generalized Modules for Membrane Antigens (GMMA). GMMA are Outer Membrane Vescicles (OMV) naturally released from Gram-negative bacteria genetically manipulated to increase blebbing and reduce toxicity. Similar to other nanoparticle systems, GMMA combine the multivalent display of carbohydrates, favoring B-cell activation and in a conformation that resembles on native bacteria, with optimal size for immune stimulation. GMMA also work as self-adjuvants, due to the presence of structures acting as agonist of Toll-like receptors 2 and 4, two receptors expressed on antigen presenting cells surface playing a crucial role in the innate immune response. Here the chemical synthesis to obtain oligosaccharide fragments of Group A Carbohydrate (GAC) and the preparation of the corresponding glycoconjugates with CRM197 (cross-reacting material 197, a non-toxic mutant of diphtheria toxin) carrier protein have been reported. The main aim was to explore such synthetic structures for the development of a vaccine against Group A Streptococcus (GAS) disease for which no vaccine is currently available. The use of well-defined synthetic structures will help to better understand the impact of GAC structural features on the immune response induced. Synthetic oligomers have been conjugated to GMMA and AuNPs, with the aim to i) compare GMMA and CRM197 as carrier systems, and ii) to explore multivalency on AuNPs to increase antibodies binding affinity.