Design of a multi-epitope Staphylococcus aureus vaccine integrating a protein A variant, α-hemolysin, and fibronectin-binding protein A through reverse vaccinology.

Staphylococcus aureus has become a major public health concern because of its increasing antibiotic resistance. Given limited clinical success of previous S. aureus vaccine candidates, this study aimed to design and computationally evaluate a novel multi-epitope construct integrating sequences derived from fibronectin-binding protein A, α-hemolysin, and a detoxified protein A variant. Candidate epitopes were computationally predicted and evaluated to determine immunogenicity, toxicity, and cross-reactivity. The epitopes were then joined using linkers with the addition of an adjuvant and an enhancer sequence to improve immunological response. The final construct showed favorable physicochemical properties, predicted solubility and high structural quality after refinement with 97.115% of amino acids in favored regions of the Ramachandran plot. Docking analysis supported plausible binding of selected epitopes to their representative HLA class I and II molecules. The complete vaccine construct showed stable in silico interaction with the TLR4/MD-2 heterodimer and a persistent receptor-vaccine interface throughout the 100 ns of the molecular dynamics simulation. The immune simulation predicted a predominantly humoral and CD4+ T helper response, with increments of B-cells, IgM and IgG1 production, memory CD4+ T-cells, macrophages, IFN-γ, IL-2, and IL-12. Codon optimization and in silico cloning supported the recombinant expression of the vaccine construct in Escherichia coli. Together, these findings identify this multi-epitope construct as a promising candidate for further experimental evaluation against S. aureus infection.