“Structural biology approaches for the characterization of Shigella O-antigen specific monoclonal antibodies”

Shigella is leading cause of diarrheal infections, especially in developing countries1. Recent estimates report approximately 270 million diarrhea episodes due to Shigella, with around 212,000 deaths of which 64,000 are in children under 5 years in underdeveloped countries2. Antibiotic resistance of Shigella is in-creasing and common therapeutic antibiotics against shigellosis have become progressively less effi-cient. The World Health Organization (WHO) has listed Shigella as a priority pathogen for the develop-ment of new therapeutic solutions3. 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 out-er membrane of these bacteria, are in development4. In fact, the O-Ag is involved in many interactions between pathogen and host and has been recognized as a key protective antigen5. Multi-component vaccines are needed to cover Shigella O-Ag diversity and have good coverage. Recently, monoclonal antibodies (mAbs) against Shigella O-Ag have been isolated from subjects vaccinated with a Shigella 4-component vaccine6 in development or after exposure to Shigella challenge. Importantly functional po-tent mAbs were identified that demonstrated ability to protect mice from challenge in a dose dependent manner. 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 ex-tracted from Generalized Modules for Membrane Antigens (GMMA), exosomes released from different Shigella strains7 engineered to reduce the O-Ag size6, 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 oli-gomers 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 Shi-gella vaccine design or refinement. This work also supports the use of mAbs as alternative intervention to fight Shigella disease.