Zhao, L.; Zhang, Y.; Wang, C.; Zheng, Y.; Chen, F.; Feng, Y.; Fang, L.; Wang, Z.; Fu, Z. F.; Zhou, M.

Integrating complementary vaccine modalities is essential for combating emerging infectious threats. Here, we developed Ad5-Envp-VLP, a chimeric adenoviral platform synergizing adenoviral delivery efficiency with virus-like particle (VLP) structural mimicry. This system stably produces self-assembling VLPs in suspension HEK293 cultures, exhibiting enhanced immunogenicity over soluble antigens. Following intramuscular immunization, the platform induces early B cell expansion and sustains germinal center reactions, driven by the upregulation of B cell cycle-related genes (Cdc6, Cdc45, Cdc20, Cdc25C, Aurka, Aurkb, and Ccnb1/2) and robust T follicular helper (Tfh) cell differentiation, generating durable neutralizing antibodies against both influenza virus and rabies virus. These effects are conserved across mouse, canine, and feline models. Crucially, integrated flow cytometry and scRNA-seq demonstrate that intranasal delivery recruits and functionally reprograms lung innate immune cells (notably alveolar macrophages and dendritic cells), driving mucosal sIgA secretion and CTL responses. A single nasal dose confers lasting protection against homologous and heterologous influenza A strains. The platform also elicits cross-neutralizing antibodies against SARS-CoV-2 variants. Together, Ad5-Envp-VLP thus establishes a modular vaccine platform for antigenically plastic pathogens by combining in vivo self-assembly with dual pulmonary-muscular delivery.