Multi-Modal Mass Spectrometry Identifies a Conserved Protective Epitope in S. pyogenes Streptolysin O

Tang, D.; Gueto-Tettay, C. A.; Hjortswang, E.; Ströbaek, J.; Ekström, S.; Happonen, L.; Malmström, L.; Malmström, J.

doi:10.1101/2023.12.02.569700

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bioRxiv · 10.1101/2023.12.02.569700

Multi-Modal Mass Spectrometry Identifies a Conserved Protective Epitope in S. pyogenes Streptolysin O

Tang, D.·Gueto-Tettay, C. A.·Hjortswang, E.·Ströbaek, J.·Ekström, S.·Happonen, L.·Malmström, L.·Malmström, J.

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Abstract

An important element of antibody-guided vaccine design is the use of neutralizing/opsonic monoclonal antibodies to define protective epitopes in their native three-dimensional conformation. Here, we demonstrate a multi-modal mass spectrometry-based strategy for in-depth characterization of antigen-antibody complexes to enable the identification of protective epitopes using the cytolytic exotoxin Streptolysin O (SLO) from Streptococcus pyogenes as a showcase. We first discovered a monoclonal antibody with an undisclosed sequence capable of neutralizing SLO-mediated cytolysis. The amino acid sequence of both the antibody light and the heavy chain was determined using mass spectrometry-based de novo sequencing, followed by chemical crosslinking mass spectrometry to generate distance constraints between the antibody fragment antigen-binding region and SLO. Subsequent integrative computational modeling revealed a discontinuous epitope located in Domain 3 of SLO that was experimentally validated by hydrogen-deuterium exchange mass spectrometry and reverse-engineering of the targeted epitope. The results show that the antibody inhibits SLO-mediated cytolysis by binding to a discontinuous epitope in Domain 3, likely preventing oligomerization and subsequent secondary structure changes critical for pore-formation. The epitope is highly conserved across >98% of the characterized S. pyogenes isolates, making it an attractive target for antibody-based therapy and vaccine design against severe streptococcal infections.

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Tang, D., Gueto-Tettay, C. A., Hjortswang, E., Ströbaek, J., Ekström, S., Happonen, L., Malmström, L., Malmström, J.. 2023-12-02. Multi-Modal Mass Spectrometry Identifies a Conserved Protective Epitope in S. pyogenes Streptolysin O. https://doi.org/10.1101/2023.12.02.569700

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