Staphylococcus aureus (S. aureus) remains a critical global public health threat, and the development of effective non-antibiotic prophylactic strategies is an urgent priority. To date, no licensed vaccine exists for the prevention of invasive S. aureus infections in humans. In the present study, a comprehensive multi-method pipeline was employed to design a bivalent self-amplifying mRNA (saRNA) vaccine cocktail targeting the ClfA, Hly, and SraP virulence antigens of S. aureus. An integrated framework combining immunoinformatics, structural bioinformatics, molecular simulations, and repository-curated experimental benchmark data was applied to delineate the immunodominant epitopic regions of each antigen, culminating in the rational design of two saRNA candidate vaccines, SaBVax807 and SaTVax876. Molecular docking and molecular dynamics simulations were subsequently performed to characterize and validate the binding interactions of both constructs with anti-S. aureus Fab fragment antibodies and human leukocyte antigen (HLA) alleles. Population coverage analysis for SaTVax876 was conducted across sixteen geographically diverse regions to evaluate global applicability. Each candidate vaccine was subjected to codon optimization to maximize translational efficiency in human host cells and rigorously evaluated for safety, stability, and immunogenic potential through a comprehensive assessment of allergenicity, antigenicity, autoimmune risk, physicochemical properties, toxicity profiles, and molecular interaction dynamics. Collectively, our findings demonstrate that the saRNA vaccine cocktail exhibits favorable safety, structural stability, and computationally predicted immunogenic profiles against S. aureus. This study establishes a comprehensive computational and in silico foundation supporting the capacity of these candidate vaccines to elicit broad anti-S. aureus immune responses, and provides a validated evidence base to guide subsequent preclinical and clinical investigations.
