In Silico identification of highly conserved CTL epitopes from Streptococcus pyogenes exoenzymes for epitope-based vaccine development

Background: Streptococcus pyogenes represents a major global health concern attributable to its extensive repertoire of virulence factors, which contribute to severe diseases, most notably streptococcal pharyngitis. To facilitate the development of a safer, more targeted vaccine, this study employed a comprehensive in silico immunoinformatics approach to identify highly conserved cytotoxic T-lymphocyte (CTL) epitopes from five critical exoenzymes: SpeB, mitogenic factor (MF), streptolysin O (SLO), streptodornase (DNase), and NAD-glycohydrolase (NADase). Methods: Conserved sequences were screened using the Protein Variability Server. CTL epitopes were predicted using Immune Epitope Database (IEDB) tools based on MHC class I binding affinity (IC50 500 nM), proteasomal processing, and TAP transport efficiency. Prioritization was given to non-allergenic, non-toxic, and non-cross-reactive epitopes to ensure safety. The selected epitopes were assembled into a multi-epitope vaccine construct, followed by the prediction, refinement, and validation of its secondary and tertiary structures. Results: Population coverage analysis revealed high global HLA allele representation, with SLO-derived epitopes achieving the broadest worldwide population coverage (91.07%). Molecular docking and thermodynamic analysis validated strong, stable HLA-epitope interactions, with Delta G values ranging from -8.6 to -12. kcal/mol and dissociation constants (KD) ≤ 8.8×10-7M. Secondary structure prediction revealed a stable arrangement with balanced alpha-helices and beta-strands, while tertiary structure modeling and refinement confirmed proper folding and surface accessibility of the epitopes. Conclusion: These findings highlight SLO, SpeB, and NADase as premier antigenic sources for epitope-based vaccine design. The resulting epitope set demonstrates promising immunogenic potential, safety, and population coverage, necessitating further in vitro and in vivo validation for streptococcal vaccine development.