Multimodal mass spectrometry identifies a conserved protective epitope in S. pyogenes Streptolysin O

Introduction

Antibody-guided vaccine design leverages functional antibodies to identify protective epitopes in their native three-dimensional conformation, an essential process for effective vaccine development. Despite its huge potential, this approach faces significant analytical challenges in epitope mapping and managing the complexity of bacterial pathogens, which have impeded its success against bacterial infections. In this study, we present a novel multimodal mass spectrometry (MS) strategy to characterize antigen−antibody complexes in-depth, using the bacterial immunogen Streptolysin O (SLO) from Streptococcus pyogenes as a showcase.

Methods

Our approach began with the identification of a previously uncharacterized monoclonal antibody that neutralizes SLO-mediated haemolysis. Using MS-based de novo sequencing, we determined the primary structure of the antibody's light and heavy chains respectively. Next, chemical cross-linking mass spectrometry (XL-MS) was applied to generate informative distance constraints between the antibody's antigen-binding region and SLO. Integrative computational modelling subsequently identified a discontinuous epitope within domain 3 of SLO. Validation of this epitope was achieved through hydrogen-deuterium exchange mass spectrometry (HDX-MS) and reverse engineering of the targeted epitope.

Results and Conclusion

Our results demonstrate that the protective antibody inhibits SLO-induced cytolysis by binding to a conformational epitope in domain 3, which likely inhibits the SLO oligomerization and secondary structure transitions necessary for pore formation. Remarkably, this epitope is conserved in over 98% of S. pyogenes isolates analysed, making it an attractive target for therapeutic and vaccine strategies against severe streptococcal infections. This study underscores the importance of integrating advanced MS-based techniques in structural proteomics to elucidate immune protection mechanisms and further guide rational vaccine design.