# An Ad5‐Based COVID‐19 Vaccine Encoding SARS‐CoV‐2 Spike Glycoprotein Induces Measurable Antibody and Cytokine Responses in Mice

## TL;DR

This study shows that an adenovirus-based vaccine encoding the SARS-CoV-2 Spike protein induces both antibody and immune cell responses in mice, supporting its potential as a vaccine platform.

## Contribution

The study introduces and evaluates a new adenovirus-based vaccine candidate for SARS-CoV-2 that elicits measurable immune responses in mice.

## Key findings

- Ad5Spike vaccination induced anti-Spike IgG antibodies that persisted for 90 days.
- The vaccine triggered cytokine secretion (IFN-γ, TNF-α, IL-2) and neutralizing antibody activity in a dose-dependent manner.
- Immune responses were detectable at both 30 and 90 days post-immunization, indicating sustained immunity.

## 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 108, 1010, or 101
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 101
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.

A recombinant adenovirus type 5–based vaccine encoding the full‐length SARS‐CoV‐2 Spike glycoprotein (Ad5Spike) was evaluated for its ability to induce cellular and humoral immune responses in BALB/c mice. Immunization with Ad5Spike resulted in the generation of antigen‐specific cellular immunity, characterized by increased secretion of IFN‐γ, IL‐2, and TNF‐α, as well as humoral responses, including anti‐Spike IgG production and neutralizing antibody activity. Immune responses were assessed at early (day 30) and long‐term (day 90) time points following immunization, demonstrating measurable and sustained vaccine‐induced immunity.