"STRUCTURAL BIOLOGY APPROACHES FOR CHARACTERIZATION OF SHIGELLA O-ANTIGEN SPECIFIC MONOCLONAL ANTIBODIES"

Shigella is leading cause of diarrheal infections, especially in developing countries1. Antibiotic resistance of Shigella is increasing and common therapeutic antibiotics against shigellosis have become progressively less efficient. The World Health Organization (WHO) has listed Shigella as a priority pathogen for the development of new therapeutic solutions2. Currently there are no licensed vaccines available against Shigella but several candidates, based on the O-antigen (O-Ag) moiety of lipopolysaccharides (LPSs) of the outer membrane of these bacteria, are in development3. Recently, monoclonal antibodies (mAbs) against Shigella O-Ag have been isolated from subjects vaccinated with a Shigella 4-component vaccine4 in development or after exposure to Shigella challenge. In this study we have developed innovative tools to characterize the O-Ag epitopes responsible for protection and cross-reactivity, avoiding the use of synthetic oligosaccharides. The O-Ag was extracted from Generalized Modules for Membrane Antigens (GMMA), exosomes released from different Shigella strains engineered to reduce the O-Ag size5, and next subjected to different purification steps. Chemical and structural characterization of the O-Ag was performed via compositional, MS and NMR analysis. Finally, ligand-based NMR approaches were successfully applied to the characterization of O-Ag specific human mAbs, to map recognition and binding process and define epitope mapping ligands bioactive conformation. The methodology developed opens the possibility to generate many O-Ag oligomers with various structural features to better characterize anti-O-Ag specific mAbs, providing useful information on epitope structural characteristics essential for protection, which can be relevant for Shigella vaccine design or refinement. This work also supports the use of mAbs as alternative intervention to fight Shigella disease.