Multimodal Mass Spectrometry Identifies a Conserved Protective Epitope in S. pyogenes Streptolysin O.
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Tang D, Gueto-Tettay C, Hjortswang E, Ströbaek J, Ekström S, Happonen L, Malmström L, Malmström J
Anal. Chem. 96 (22) 9060-9068 [2024-06-04; online 2024-05-03]
An important element of antibody-guided vaccine design is the use of neutralizing or opsonic monoclonal antibodies to define protective epitopes in their native three-dimensional conformation. Here, we demonstrate a multimodal 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 cross-linking 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 transitions 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.
PubMed 38701337
DOI 10.1021/acs.analchem.4c00596
Crossref 10.1021/acs.analchem.4c00596