{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T06:02:59Z","timestamp":1782280979254,"version":"3.54.5"},"posted":{"date-parts":[[2025,9,21]]},"group-title":"Immunology","reference-count":44,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,9,21]],"date-time":"2025-09-21T00:00:00Z","timestamp":1758412800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"DOI":"10.13039\/100005825","name":"National Institute of Food and Agriculture","doi-asserted-by":"crossref","award":["2020-06448"],"award-info":[{"award-number":["2020-06448"]}],"id":[{"id":"10.13039\/100005825","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100005825","name":"National Institute of Food and Agriculture","doi-asserted-by":"crossref","award":["2024-08723"],"award-info":[{"award-number":["2024-08723"]}],"id":[{"id":"10.13039\/100005825","id-type":"DOI","asserted-by":"crossref"}]},{"name":"NIH - NIAID","award":["R01AI147109"],"award-info":[{"award-number":["R01AI147109"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,9,21]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>Since its discovery in U.S. dairy cattle in early 2024, the highly pathogenic H5N1 avian influenza (clade 2.3.4.4b) has spread widely among herds, causing major economic losses. This zoonotic event emphasizes the urgent need for H5 vaccines that elicit strong, durable, cross-reactive immune responses in cattle, especially young calves. To address this, we immunized mice and cattle with a centralized consensus H5 vaccine, designed to localize to the central node of the human H5 phylogenetic tree. The vaccine was delivered using serotype-switched adenoviral vectors in a prime\u2013boost regimen, combined with intramuscular and intranasal coadministration to target systemic and mucosal immunity and elicit strong humoral and cellular immune responses. This approach strategically integrates multiple innovative features: centralized consensus immunogens, mucosal targeting, and vector serotype switching that are aimed at maximizing immune protection against H5N1 viruses. Our results show that vaccination elicited strong humoral and cellular immunity in both mice and calves. In challenge experiments, vaccinated mice were fully protected against lethal infection with multiple divergent H5N1 strains, including the 2024 bovine isolate (A\/bovine\/Ohio\/B24OSU-439\/2024). Given that vaccine induced immunity was consistent across species, these results support the translatability of the mouse model findings to cattle. Overall, our findings represent a promising approach for immunizing cattle and other key livestock against HPAI H5N1, mitigating agricultural losses, and reducing the risk of zoonotic transmission.<\/jats:p>\n                <jats:sec>\n                  <jats:title>Significance Statement<\/jats:title>\n                  <jats:p>H5N1 influenza A virus is a serious pathogen recently detected in many mammals, including cattle. It transmits sporadically to humans and causes major economic losses in dairy and poultry industries, raising global concern. No H5N1 vaccines are currently licensed for cattle. To address this gap, we tested a centralized consensus H5 (H5CC) vaccine, previously effective in mice and swine. Using multiple Adenovirus vectors, we delivered H5CC intramuscularly and intranasally to one-week-old calves. The vaccines induced strong humoral and cellular immune responses, essential for preventing infection and limiting transmission. These findings highlight the vaccine\u2019s potential to reduce agricultural losses and remove cattle as a newly established reservoir for zoonotic spread, providing a promising strategy for mitigating pandemic risk.<\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.09.21.677614","type":"posted-content","created":{"date-parts":[[2025,9,22]],"date-time":"2025-09-22T03:05:15Z","timestamp":1758510315000},"source":"Crossref","is-referenced-by-count":1,"title":["Dual-Route H5N1 Vaccination Induces Systemic and Mucosal Immunity in Murine and Bovine Models"],"prefix":"10.64898","author":[{"given":"Joshua","family":"Wiggins","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Adthakorn","family":"Madapong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eric A","family":"Weaver","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025092504350696000_2025.09.21.677614v1.1","doi-asserted-by":"crossref","first-page":"e0042425","DOI":"10.1128\/jvi.00424-25","article-title":"Clade 2.3.4.4b highly pathogenic avian influenza H5N1 viruses: knowns, unknowns, and challenges","volume":"99","year":"2025","journal-title":"J Virol"},{"key":"2025092504350696000_2025.09.21.677614v1.2","doi-asserted-by":"crossref","unstructured":"G. Graziosi , C. Lupini , E. Catelli , S. Carnaccini , Highly Pathogenic Avian Influenza (HPAI) H5 Clade 2.3.4.4b Virus Infection in Birds and Mammals. Animals (Basel) 14 (2024).","DOI":"10.20944\/preprints202404.1123.v1"},{"key":"2025092504350696000_2025.09.21.677614v1.3","doi-asserted-by":"crossref","first-page":"2270068","DOI":"10.1080\/22221751.2023.2270068","article-title":"High number of HPAI H5 virus infections and antibodies in wild carnivores in the Netherlands, 2020-2022","volume":"12","year":"2023","journal-title":"Emerg Microbes Infect"},{"key":"2025092504350696000_2025.09.21.677614v1.4","doi-asserted-by":"publisher","DOI":"10.3201\/eid2912.230464"},{"key":"2025092504350696000_2025.09.21.677614v1.5","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-024-07849-4"},{"key":"2025092504350696000_2025.09.21.677614v1.6","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-024-08166-6"},{"key":"2025092504350696000_2025.09.21.677614v1.7","unstructured":"USDA, HPAI Confirmed Cases in Livestock | Animal and Plant Health Inspection Service. (2025)."},{"key":"2025092504350696000_2025.09.21.677614v1.8","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMc2405495"},{"key":"2025092504350696000_2025.09.21.677614v1.9","doi-asserted-by":"crossref","first-page":"e0213824","DOI":"10.1128\/jvi.02138-24","article-title":"Detection of antibodies against influenza A viruses in cattle","volume":"99","year":"2025","journal-title":"J Virol"},{"key":"2025092504350696000_2025.09.21.677614v1.10","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-024-08063-y"},{"key":"2025092504350696000_2025.09.21.677614v1.11","doi-asserted-by":"crossref","first-page":"6520","DOI":"10.1038\/s41467-025-61553-z","article-title":"The impact of highly pathogenic avian influenza H5N1 virus infection on dairy cows","volume":"16","year":"2025","journal-title":"Nat Commun"},{"key":"2025092504350696000_2025.09.21.677614v1.12","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMc2405371"},{"key":"2025092504350696000_2025.09.21.677614v1.13","doi-asserted-by":"publisher","DOI":"10.15585\/mmwr.mm7321e1"},{"key":"2025092504350696000_2025.09.21.677614v1.14","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-024-07766-6"},{"key":"2025092504350696000_2025.09.21.677614v1.15","doi-asserted-by":"crossref","first-page":"4637","DOI":"10.1038\/s41598-025-87831-w","article-title":"Evaluation of humoral immune response and milk antibody transfer in calves and lactating cows vaccinated with inactivated H5 avian influenza vaccine","volume":"15","year":"2025","journal-title":"Sci Rep"},{"key":"2025092504350696000_2025.09.21.677614v1.16","doi-asserted-by":"crossref","first-page":"197","DOI":"10.1038\/s41541-025-01238-2","article-title":"Immunogenicity and safety of a rabies-based highly pathogenic influenza A virus H5 vaccine in cattle","volume":"10","year":"2025","journal-title":"NPJ Vaccines"},{"key":"2025092504350696000_2025.09.21.677614v1.17","doi-asserted-by":"publisher","DOI":"10.1101\/2025.05.01.651548"},{"key":"2025092504350696000_2025.09.21.677614v1.18","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1038\/s41541-022-00503-y","article-title":"The use of viral vectors in vaccine development","volume":"7","year":"2022","journal-title":"NPJ Vaccines"},{"key":"2025092504350696000_2025.09.21.677614v1.19","doi-asserted-by":"crossref","first-page":"1535328","DOI":"10.3389\/fvets.2025.1535328","article-title":"Research advances in replication-deficient viral vector vaccines","volume":"12","year":"2025","journal-title":"Front Vet Sci"},{"key":"2025092504350696000_2025.09.21.677614v1.20","doi-asserted-by":"crossref","first-page":"544","DOI":"10.4161\/hv.27238","article-title":"Vaccines within vaccines: the use of adenovirus types 4 and 7 as influenza vaccine vectors","volume":"10","year":"2014","journal-title":"Human vaccines & immunotherapeutics"},{"key":"2025092504350696000_2025.09.21.677614v1.21","doi-asserted-by":"crossref","first-page":"e0241320","DOI":"10.1128\/JVI.02413-20","article-title":"Infection of Bronchial Epithelial Cells by the Human Adenoviruses A12, B3, and C2 Differently Regulates the Innate Antiviral Effector APOBEC3B","volume":"95","year":"2021","journal-title":"J Virol"},{"key":"2025092504350696000_2025.09.21.677614v1.22","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.74.20.9617-9628.2000"},{"key":"2025092504350696000_2025.09.21.677614v1.23","doi-asserted-by":"crossref","first-page":"e0140702","DOI":"10.1371\/journal.pone.0140702","article-title":"Centralized Consensus Hemagglutinin Genes Induce Protective Immunity against H1, H3 and H5 Influenza Viruses","volume":"10","year":"2015","journal-title":"PloS one"},{"key":"2025092504350696000_2025.09.21.677614v1.24","doi-asserted-by":"crossref","first-page":"14912","DOI":"10.1038\/s41598-017-14891-y","article-title":"Efficacy of an Adenoviral Vectored Multivalent Centralized Influenza Vaccine","volume":"7","year":"2017","journal-title":"Sci Rep"},{"key":"2025092504350696000_2025.09.21.677614v1.25","doi-asserted-by":"crossref","first-page":"928","DOI":"10.3390\/vaccines13090928","article-title":"Systemic and Mucosal Immune Responses Induced by Adenoviral-Vectored Consensus H5 Influenza A Vaccines in Mice and Swine","volume":"13","year":"2025","journal-title":"Vaccines"},{"key":"2025092504350696000_2025.09.21.677614v1.26","doi-asserted-by":"crossref","unstructured":"C. Sreenivasan , M. Thomas , R. S. Kaushik , D. Wang , F. Li , Influenza A in Bovine Species: A Narrative Literature Review. Viruses 11 (2019).","DOI":"10.3390\/v11060561"},{"key":"2025092504350696000_2025.09.21.677614v1.27","doi-asserted-by":"publisher","DOI":"10.1080\/21645515.2015.1016674"},{"key":"2025092504350696000_2025.09.21.677614v1.28","doi-asserted-by":"crossref","first-page":"102594","DOI":"10.1016\/j.omtn.2025.102594","article-title":"A bovine adenoviral-vector-based universal influenza vaccine confers protection against influenza A and B viruses in mice and ferrets","volume":"36","year":"2025","journal-title":"Mol Ther Nucleic Acids"},{"key":"2025092504350696000_2025.09.21.677614v1.29","doi-asserted-by":"publisher","DOI":"10.1006\/viro.2000.0661"},{"key":"2025092504350696000_2025.09.21.677614v1.30","doi-asserted-by":"publisher","DOI":"10.1016\/S0264-410X(01)00458-3"},{"key":"2025092504350696000_2025.09.21.677614v1.31","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.00979-13"},{"key":"2025092504350696000_2025.09.21.677614v1.32","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.173.3.1978"},{"key":"2025092504350696000_2025.09.21.677614v1.33","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.168.6.2930"},{"key":"2025092504350696000_2025.09.21.677614v1.34","doi-asserted-by":"crossref","unstructured":"B. L. Bullard , E. A. Weaver , Strategies Targeting Hemagglutinin as a Universal Influenza Vaccine. Vaccines (Basel) 9 (2021).","DOI":"10.3390\/vaccines9030257"},{"key":"2025092504350696000_2025.09.21.677614v1.35","doi-asserted-by":"crossref","first-page":"14912","DOI":"10.1038\/s41598-017-14891-y","article-title":"Efficacy of an Adenoviral Vectored Multivalent Centralized Influenza Vaccine","volume":"7","year":"2017","journal-title":"Scientific Reports"},{"key":"2025092504350696000_2025.09.21.677614v1.36","doi-asserted-by":"crossref","unstructured":"A. Weaver et al., Comparison of Systemic and Mucosal Immunization with Helper-Dependent Adenoviruses for Vaccination against Mucosal Challenge with SHIV. PloS one in press (2013).","DOI":"10.1371\/journal.pone.0067574"},{"key":"2025092504350696000_2025.09.21.677614v1.37","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0073313"},{"key":"2025092504350696000_2025.09.21.677614v1.38","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.172.10.6290"},{"key":"2025092504350696000_2025.09.21.677614v1.39","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.172.9.5598"},{"key":"2025092504350696000_2025.09.21.677614v1.40","doi-asserted-by":"publisher","DOI":"10.1093\/infdis\/148.5.785"},{"key":"2025092504350696000_2025.09.21.677614v1.41","doi-asserted-by":"publisher","DOI":"10.1016\/j.coviro.2016.12.002"},{"key":"2025092504350696000_2025.09.21.677614v1.42","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.100133697"},{"key":"2025092504350696000_2025.09.21.677614v1.43","doi-asserted-by":"publisher","DOI":"10.1084\/jem.118.2.295"},{"key":"2025092504350696000_2025.09.21.677614v1.44","doi-asserted-by":"crossref","unstructured":"M. J. Pekarek , A. Madapong , J. Wiggins , E. A. Weaver , Adenoviral-Vectored Multivalent Vaccine Provides Durable Protection Against Influenza B Viruses from Victoria-like and Yamagata-like Lineages. Int J Mol Sci 26 (2025).","DOI":"10.3390\/ijms26041538"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.09.21.677614","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T05:12:19Z","timestamp":1782277939000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.09.21.677614"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,9,21]]},"references-count":44,"URL":"https:\/\/doi.org\/10.1101\/2025.09.21.677614","relation":{"is-preprint-of":[{"id-type":"doi","id":"10.1038\/s41541-026-01460-6","asserted-by":"subject"}]},"subject":[],"published":{"date-parts":[[2025,9,21]]},"subtype":"preprint"}}