Ströbaek, J. | bioRxiv Science

bioRxiv2025

Design of a Streptolysin O Epitope-Centric Nanoparticle Vaccine Against Streptococcus pyogenes

Streptococcus pyogenes (Group A Streptococcus, GAS) is a significant human pathogen for which no licensed vaccine is currently available. Here, we report a de novo designed epitope-centric protein-based nanoparticle vaccine against GAS. By integrating structural mass spectrometry techniques and deep learning approaches, we re-engineered a protective epitope (D3m) present in domain 3 of streptolysin O, a prominent pore-forming toxin produced by GAS. D3m was displayed on the surface of a self-assembling icosahedral nanoparticle (D3m-NP) to enhance epitope presentation and immunogenicity. Mice immunised with D3m-NP mounted haemolysis-neutralising titres and displayed a more uniform, epitope-centric antibody response than those receiving the community-standard detoxified full-length streptolysin O. Our findings highlight a promising strategy for GAS vaccine development by combining multimodal protein mass spectrometry, protein design and a versatile protein-based nanoparticle vaccine platform.

immunology↗

bioRxiv2023

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

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.

biochemistry↗