Integrated In Silico and In Vivo Evaluation of a Tetravalent SARS-CoV-2 RBD-Fc Fusion Vaccine with Broad...

Background/Objectives: SARS-CoV-2 continues to generate antigenically divergent variants that reduce the breadth of existing vaccine-induced antibody responses. Fc-fusion subunit vaccines offer advantages in stability, antigen display, and Fc-mediated immune engagement. This study aimed to design and evaluate a tetravalent RBD-Fc fusion construct incorporating RBDs from Wuhan-Hu-1 and Omicron BA.4/BA.5 and to determine whether this configuration can induce broad antibody recognition across SARS-CoV-2 variants. The objective was to assess its feasibility, biochemical properties, and initial immunogenicity. Methods: Immune responses to the construct were first assessed using the C-ImmSim simulation platform. The full-length fusion was synthesized, subcloned into pcDNA3.1(+), expressed in HEK293 cells, and purified by Protein G affinity chromatography. Protein integrity was evaluated by reducing SDS-PAGE. BALB/c mice (female, 8 weeks) were immunized with a prime-boost-boost schedule, and sera were analyzed by ELISA, considering binding to Wuhan-Hu-1, Omicron BA.4/BA.5, and a panel of RBD variants. Results: In silico analysis predicted coordinated antigen clearance, class switching, memory B- and CD4⁺ T-cell formation, and transient cytokine induction. The recombinant protein was expressed efficiently, yielding a major ~56 kDa band and a ~23 kDa RBD fragment. Vaccinated mice generated strong IgG responses to Wuhan-Hu-1 and BA.4/BA.5 RBDs and showed broad binding to major variant RBDs. Conclusions: The tetravalent RBD-Fc fusion vaccine was successfully produced and elicited broad antibody binding across SARS-CoV-2 variants, supporting its potential as a versatile protein-based vaccine platform.