Immunotherapeutic Approach for Improving the Efficacy of a Novel Subunit Vaccine Against SARS‐CoV‐2 by Cytotoxic T‐Lymphocytes (CTL) Epitopes

The present study aimed to employ a diverse range of immunoinformatics and in vitro techniques to construct and validate a potentially active multiepitope subunit vaccine against SARS-CoV-2 using cytotoxic T-lymphocyte epitopes. To design the vaccine, a library of antigenic, nonallergic, and immunogenic epitopes of the spike protein was prepared. To improve the immunogenicity and safety of the final subunit vaccine, a sequence comprising three antigenic and nontoxic CTL epitopes was selected. To predict the tertiary structure of the vaccine, docking studies manipulating human major histocompatibility complex 1 (MHC-1) and Toll-like receptor-4 and Toll-like receptor-8 (TLR-4 and TLR-8) receptors were carried out. The consistency of the vaccine's binding to the selected receptors was confirmed by molecular dynamics (MD) simulations. In addition, the cloned vaccine was introduced into a bacterial culture, and its expression and antigenicity were assessed using SDS-PAGE and Western blotting, respectively. The vaccine design revealed a strong affinity for the TLR-8 and MHC-1 receptors, as evidenced by molecular docking analysis. The MD simulations conducted in specific systems yielded further data supporting the robust and enduring binding of TLR-8 and MHC-1 receptors to CTL epitopes. The bacterial cells harboring the vaccine sequence demonstrated robust production of the vaccine protein upon induction with IPTG. In addition, Western blotting demonstrated the antigenic properties of the vaccine protein. Computational and in vitro analyses suggested that the designed multiepitope subunit vaccine is stable and can induce specific immune responses against SARS-CoV-2.