Background: Two of the most challenging viruses for vaccine development are SARS-CoV-2, causing the current COVID-19 pandemic, and influenza virus (H1N1), which spreads annually, causing seasonal epidemics or increasing the pandemic risk. Materials and Methods: In this study, we analyzed the immunodominant epitope regions in fusion peptides consisting of the Spike_S1_ N-terminal domain from SARS-CoV-2 in frame to hemagglutinin H2 (HA2) gene from influenza A virus (H1N1) and also human IFNɣ gene by two (G4S)3 linkers. A comprehensive analysis based on immunoinformatic has been conducted on prediction servers to predict T- and B-cell epitopes. In silico cloning and expression in pET-28(+) expression vector and vaccine optimization were assessed. The overall model quality was accessed, and the docking or binding affinity of the designed vaccine to the Toll-like receptor 3 was analyzed. Results: The efficiency of the constructed vaccine confirmed by appropriate expression of the designed vaccine candidate tested by in silico cloning in pET–28(+) vector and codon optimization might increase the production of vaccine candidate into Escherichia coli strain K-12. Conclusion: In conclusion, we suggest that this fusion peptide would be an attractive design strategy for developing a bivalent vaccine against COVID-19 and influenza as promising vaccine candidates without the need to reformulate or vaccinate each year
