Poster presentato alla conferenza Instruct-ERIC Biennial Structural Biology (IBSB) 2026

Growing ethical concerns about animal testing and the widespread adoption of the 3Rs (Replacement, Reduction, Refinement) have increased the need for validated in vitro assays that reliably demonstrate product potency and quality in pharmaceutical research. In this context, antibody-based immunoassays offer the possibility of correlating an antigen’s in vitro potency with its in vivo immunogenicity. Developing a fit-for-purpose in vitro potency assay (IVRP) therefore requires detailed characterization of antibody binding sites and their interactions with vaccine antigens under reference and subpotent conditions. This project combines development of an IVRP for a Streptococcus A vaccine with the structural characterization and epitope mapping of antibody–antigen interactions. Specific mouse monoclonal antibodies (mAbs) were preselected to identify and quantify the three recombinant proteins in our candidate vaccine (SLO, SpyCEP, and SpyAD) directly in the final formulation. Antigens and corresponding antibodies were expressed, and multiple structural techniques were applied to characterize complexes and define epitopes, including HDX–MS, X-ray crystallography, and cryo-EM. Fab fragments of each selected antibody were produced to facilitate the performance of such structural methods. Furthermore, binding affinity and stability assessments were conducted using competitive ELISA (cELISA) and bio-layer interferometry (BLI) on antigens at reference conditions and after thermal stress to evaluate mAb suitability for monitoring vaccine stability. Antigen-specific functional assays were also performed to measure the inhibitory potential of the mAbs. Together, the structural, binding affinity, and functional data will support the development and refinement of the IVRP, validating its use as a reliable tool to determine Streptococcus A vaccine potency.