Oral Presentation Lancefield International Symposium for Streptococci and Streptococcal Diseases 2025

Deciphering protective immunity: A multimodal mass spectrometry approach to targeting conformational epitopes in streptococcal antigen (117177)

Di Tang 1 , Carlos Gueto-Tettay 1 , Elisabeth Hjortswang 1 , Joel Ströbaek 1 , Simon Ekström 2 , Lotta Happonen 1 , Lars Malmström 1 , Johan Malmstrom 1 2
  1. Lund University, Lund, Sweden
  2. SciLifeLab, Lund, Sweden

Introduction

Structure-based reverse vaccinology uses functional antibodies to identify critical epitopes in their native 3-D conformations, essential for effective vaccine development. However, challenges such as conformational epitope mapping and the immunodominance of diverse bacterial antigens have limited its success against complex bacterial pathogens. Our study introduces a novel multimodal protein mass spectrometry (MS) approach to characterise antigen-antibody complexes under near-native conditions, using streptolysin O (SLO) from Group A Streptococcus as a showcase.

Methods

We identified an uncharacterised monoclonal antibody that neutralises SLO-induced haemolysis. Using de novo MS sequencing, we determined the full amino acid sequences of the antibody light and heavy chains. Chemical cross-linking MS provided informative distance constraints within SLO-antibody complexes, and integrative computational modelling revealed a conformational epitope in domain 3 of SLO. This was further validated through hydrogen/deuterium exchange MS and reverse engineering of the targeted epitope.

Results

Our findings show that protective antibodies are able to neutralise SLO-induced cytolysis by targeting a conserved conformational epitope in domain 3, likely preventing SLO oligomerisation and subsequent pore formation on the cellular membrane. This epitope, present in over 98% of sequenced Group A Streptococcus isolates, provides a foundation for therapeutic and vaccine development against severe streptococcal infections.

Conclusion

Taken together, this result with the extended application of this methodology to another significant pneumococcal antigen underscores the importance of identifying and distinguishing structural epitopes for immune protection. Multimodal MS holds great potential in accelerating the design of next-generation epitope-targeting therapies and vaccines.

  1. (1) Tang, D.; Gueto-Tettay, C.; Hjortswang, E.; Ströbaek, J.; Ekström, S.; Happonen, L.; Malmström, L.; Malmström, J. Multimodal Mass Spectrometry Identifies a Conserved Protective Epitope in S. Pyogenes Streptolysin O. Anal. Chem. 2024, 96 (22), 9060–9068. https://doi.org/10.1021/acs.analchem.4c00596.
  2. (2) Gueto-Tettay, C.; Tang, D.; Happonen, L.; Heusel, M.; Khakzad, H.; Malmström, J.; Malmström, L. Multienzyme Deep Learning Models Improve Peptide de Novo Sequencing by Mass Spectrometry Proteomics. Plos Comput Biol 2023, 19 (1), e1010457. https://doi.org/10.1371/journal.pcbi.1010457.
  3. (3) Bahnan, W.; Happonen, L.; Khakzad, H.; Ahnlide, V. K.; Neergaard, T.; Wrighton, S.; André, O.; Bratanis, E.; Tang, D.; Hellmark, T.; Björck, L.; Shannon, O.; Malmström, L.; Malmström, J.; Nordenfelt, P. A Human Monoclonal Antibody Bivalently Binding Two Different Epitopes in Streptococcal M Protein Mediates Immune Function. Embo Mol Med 2022. https://doi.org/10.15252/emmm.202216208.
  4. 4) Hauri, S.; Khakzad, H.; Happonen, L.; Teleman, J.; Malmström, J.; Malmström, L. Rapid Determination of Quaternary Protein Structures in Complex Biological Samples. Nat Commun 2019, 10 (1), 192. https://doi.org/10.1038/s41467-018-07986-1.