Multimodal Mass Spectrometry Identifies a Conserved Protective Epitope in S. pyogenes Streptolysin O

• Published in: Analytical chemistry (Washington) Vol. 96; no. 22; pp. 9060 - 9068
• Main Authors: Tang, Di, Gueto-Tettay, Carlos, Hjortswang, Elisabeth, Ströbaek, Joel, Ekström, Simon, Happonen, Lotta, Malmström, Lars, Malmström, Johan
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 04.06.2024
• Subjects: Amino Acid Sequence; Analytical Chemistry; Analytisk kemi; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - immunology; Bacterial Proteins - chemistry; Bacterial Proteins - immunology; Basic Medicine; Biologi; Biological Sciences; Chemical Sciences; Epitopes - chemistry; Epitopes - immunology; Immunologi inom det medicinska området; Immunology in the medical area; Kemi; Mass Spectrometry; Medical and Health Sciences; Medicin och hälsovetenskap; Medicinska och farmaceutiska grundvetenskaper; Models, Molecular; Natural Sciences; Naturvetenskap; Streptococcus pyogenes - chemistry; Streptococcus pyogenes - immunology; Streptolysins - chemistry; Streptolysins - immunology; Streptolysins - metabolism; Structural Biology; Strukturbiologi
• ISSN: 0003-2700; 1520-6882; 1520-6882
• DOI: 10.1021/acs.analchem.4c00596

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.