Antigen Format Determines Immunogenicity of AAV-Based SARS-CoV-2 Vaccines: Full-Length Spike Versus Truncated Subunits.

BACKGROUND: Antigen format strongly influences the immunogenicity of gene-based vaccines. Full-length Spike is widely used in licensed COVID-19 vaccines, while truncated subunits such as S1 or the receptor-binding domain (RBD) may simplify vector design but risk reduced potency. We aimed to compare these antigen formats in an AAV9 delivery platform. METHODS: BALB/c mice were immunized intramuscularly with recombinant AAV9 encoding full-length Spike, S1, or RBD at doses of 1 × 1010 or 1 × 1011 viral genomes. Immune responses were assessed by serology, virus neutralization, T-cell profiling, and histopathology. RESULTS: All constructs expressed antigen in vitro and in vivo. Only full-length Spike elicited robust neutralizing antibodies at both doses, with titers rising significantly by week 12. High-dose RBD induced neutralization in a minority of animals, whereas S1 failed to do so. Antigen-specific IgG responses scaled with insert length (Spike > S1 > RBD). Cellular immunity was dominated by CD8+ effector memory T cells, strongest in the Spike group, which also induced measurable CD4+ responses. Local transient myositis was observed at the injection site but resolved by week 24, with no systemic pathology. CONCLUSIONS: Full-length Spike outperforms truncated subunits in the AAV context, highlighting antigen structure as a critical factor for next-generation coronavirus vaccine design.