Immunogenicity of different nanoparticle adjuvants containing recombinant RBD coronavirus antigen in animal model

Abstract Ongoing mutations of SARS‐CoV‐2 present challenges for vaccine development, promising renewed global efforts to create more effective vaccines against coronavirus disease (COVID‐19). One approach is to target highly immunogenic viral proteins, such as the spike receptor binding domain (RBD), which can stimulate the production of potent neutralizing antibodies. This study aimed to design and test a subunit vaccine candidate based on the RBD. Bioinformatics analysis identified antigenic regions of the RBD for recombinant protein design. In silico analysis identified the RBD region as a feasible target for designing a recombinant vaccine. Bioinformatics tools predicted the stability and antigenicity of epitopes, and a 3D model of the RBD‐angiotensin‐converting enzyme 2 complex was constructed using molecular docking and codon optimization. The resulting construct was cloned into the pET‐28a (+) vector and successfully expressed in Escherichia coli BL21DE3. As evidenced by sodium dodecyl‐polyacrylamide gel electrophoresis and Western blotting analyses, the affinity purification of RBD antigens produced high‐quality products. Mice were immunized with the RBD antigen alone or combined with aluminum hydroxide (AlOH), calcium phosphate (CaP), or zinc oxide (ZnO) nanoparticles (NPs) as adjuvants. Enzyme‐linked immunosorbent assay assays were used to evaluate immune responses in mice. In‐silico analysis confirmed the stability and antigenicity of the designed protein structure. RBD with CaP NPs generated the highest immunoglobulin G titer compared to AlOH and ZnO after three doses, indicating its effectiveness as a vaccine platform. In conclusion, the recombinant RBD antigen administered with CaP adjuvant NPs induces potent humoral immunity in mice, supporting further vaccine development. These results contribute to ongoing efforts to develop more effective COVID‐19 vaccines.