Pangenome-based design of a multi-epitope vaccine candidate against Streptococcus pasteurianus with computational evaluation of TLR1–TLR2 interaction

Abstract Multidrug-resistant (MDR) Streptococcus pasteurianus is an emerging pathogen causing severe hospital and community-acquired infections. However, there is currently no vaccine, prompting the need for new vaccine strategies. Here, we used a systematic computational workflow combining pangenome analysis, subtractive proteomics, immunoinformatics and molecular dynamics to develop a multi-epitope vaccine (MEV) targeting common antigenic epitopes among different strains of S. pasteurianus . Pangenome analysis of 11 strains identified 1,436 core gene clusters (34.2%) conserved within the 11 genomes analyzed. Subtractive proteomics analysis yielded three predicted candidate proteins, DisA, FtsX and FtsW, based on essentiality, limited human sequence similarity and predicted antigenicity; their native surface accessibility remains unconfirmed. Computational epitope prediction identified six cytotoxic T-lymphocyte (CTL), four helper T-lymphocyte (HTL), and three linear B-cell (LBL) epitopes from these proteins with high antigenicity and immunogenicity. The 344-amino acid multi-epitope vaccine (MEV) design, comprising the Mycobacterium tuberculosis L7/L12 adjuvant and peptide linkers, exhibited enhanced stability and physicochemical characteristics. Docking and molecular dynamics simulations a putative interaction between the MEV and a TLR1–TLR2 ectodomain model derived from PDB 2Z7X; the contact-based predicted was 1.3 × 10⁻²⁴ M, and MM/PBSA binding free energies were − 32.15 and − 16.83 kcal/mol for Replicates 1 and 2, respectively, consistent with a favorable predicted interaction. These computational findings provide a structural basis for further experimental investigation of the proposed vaccine construct. Structural stability was assessed using two independent 100 ns molecular dynamics replicates (200 ns aggregate), and the energetics of the predicted complex were further evaluated using MM/PBSA binding free-energy calculations. In silico population coverage analysis estimated 76.87% global population coverage across various geographical and ethnic groups. In silico immune analysis predicted humoral and cellular immune-response patterns, with increased IgG antibody response, prolonged cytokine secretion (IFN-γ and IL-2) and generation of memory cells. Optimized gene sequence and in silico cloning into the pET28a(+) vector provided a proposed construct for experimental expression testing in Escherichia coli. This in silico study presents a computationally designed multi-epitope vaccine candidate against S. pasteurianus that requires experimental evaluation before any conclusions about immunogenicity or protection.