Fulya Erendor, Fatih Üzer, Salih Şanlıoğlu
ABSTRACT The global SARS‐CoV‐2 pandemic has underlined the urgent need for effective vaccine platforms. Adenoviral vectors have gained attention due to their high transgene capacity, broad tissue tropism, and innate immunostimulatory properties. This study aimed to develop and evaluate a recombinant adenoviral vaccine, Ad5Spike, encoding the full‐length SARS‐CoV‐2 Spike glycoprotein. The Ad5Spike vector was generated using Gateway Cloning Technology and produced by transient calcium phosphate‐mediated transfection of 293A cells. Viral particles (VP) were purified via CsCl density gradient ultracentrifugation. Female BALB/c mice (6–8 weeks old, n = 5 per group per timepoint) were immunized intraperitoneally with 10 8 , 10 10 , or 10 1 2 viral particles. Humoral and cellular immune responses were evaluated at 30‐ and 90‐days post‐immunization using ELISA, ELISpot, and pseudovirus neutralization assays. Ad5Spike vaccination induced measurable anti‐Spike IgG responses, with persistent antibody levels observed up to 90 days. Splenocyte analysis revealed elevated IFN‐γ, TNF‐α, and IL‐2 secretion, consistent with initial humoral and cellular activation. Neutralizing antibody activity against a lentiviral pseudovirus bearing the SARS‐CoV‐2 Spike (Wuhan‐1) was dose‐dependent and highest in the 10 1 2 group. In conclusion, this early preclinical study demonstrates that the Ad5Spike vaccine elicited detectable humoral and cellular immune responses, providing a proof‐of‐concept for the immunogenicity of this adenoviral‐based platform.