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Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2026,5,5]]},"abstract":"<jats:p>Integrating complementary vaccine modalities is essential for combating emerging pathogens. Although the recent mRNA-VLP hybrids enable spontaneous virus-like particles (VLPs) self-assembly, thereby enhancing immunogenicity, they fail to elicit robust pulmonary mucosal immunity against respiratory pathogens. Here, we developed Ad5-Envp-VLP, a chimeric adenoviral platform enabling spontaneous in vivo assembly of envelope protein-displaying VLPs using advanced technology that recruits ESCRT (endosomal sorting complex required for transport) via the EABR (ESCRT and ALIX-binding region). Compared with the intramuscular route, intranasal administration of a single-dose Ad5-HA-VLP confers long-lasting protection against both homologous and heterologous influenza A strains. Integrated single-cell RNA sequencing and flow cytometry analyses reveal that intranasal delivery of Ad5-HA-VLP recruits and functionally reprograms lung innate immune cells, promoting antigen presentation and driving robust mucosal secretory IgA (sIgA) secretion and cytotoxic T lymphocyte responses. Similarly, intranasal delivery of Ad5-S-VLP elicits potent cross-neutralizing antibody titers against SARS-CoV-2 variants. Importantly, intranasal immunization with Ad5-S-HA-VLP (coexpressing S- and HA-VLPs) generates dual influenza and SARS-CoV-2 neutralizing antibodies, alongside pulmonary antigen-specific sIgA, confirming Ad5-Envp-VLP as a promising \u201csingle-dose multiplexed mucosal vaccine\u201d against respiratory pathogens. Further extended applications show that Ad5-RVDG-VLP also induces broad protective immunity in mouse, dog, and cat models, verifying its feasibility as an efficient rabies vaccine. Collectively, the Ad5-Envp-VLP platform represents a universal and versatile mucosal vaccine strategy, leveraging pulmonary delivery of vectors that encode in vivo-assembling VLPs to concurrently elicit robust mucosal and systemic immunity against a wide spectrum of pathogens.<\/jats:p>","DOI":"10.1073\/pnas.2519857123","type":"journal-article","created":{"date-parts":[[2026,4,29]],"date-time":"2026-04-29T17:37:36Z","timestamp":1777484256000},"update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":2,"title":["An Ad5-vectored platform generating self-assembling VLPs elicits potent mucosal immunity against influenza A virus and SARS-CoV-2"],"prefix":"10.1073","volume":"123","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-5526-7928","authenticated-orcid":false,"given":"Yuan","family":"Zhang","sequence":"first","affiliation":[{"name":"National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University","place":["Wuhan, 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