{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T06:44:54Z","timestamp":1785912294384,"version":"3.56.0"},"posted":{"date-parts":[[2026,8,3]]},"group-title":"Microbiology","reference-count":57,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T00:00:00Z","timestamp":1785715200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,8,3]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  The continued emergence of genetically diverse high pathogenicity avian influenza (HPAI) H5N1 viruses with zoonotic potential highlights the urgent need for developing vaccines capable of providing broad protection against multiple circulating clades. Here, we developed a one-shot, multivalent, live-attenuated influenza vaccine (LAIV) based on the temperature-sensitive (ts), cold-adapted (ca), and attenuated (\n                  <jats:italic>att<\/jats:italic>\n                  ) influenza A\/Ann Arbor\/6\/1960 master donor virus (MDV) that incorporates the hemagglutinin (HA) and neuraminidase (NA) glycoproteins from representative clades 2.3.4.4b (A\/Louisiana\/12\/2024), 2.3.2.1a (A\/Victoria\/149\/2024), and 2.3.2.1e (A\/Cambodia\/2302009\/2023) H5N1 viruses. A single intranasal (IN) immunization of C57BL\/6 mice with the multivalent LAIV elicited robust humoral immune responses, with immune sera exhibiting broad cross-reactivity against antigens from all three H5N1 clades included in the vaccine. Following homologous viral challenge, vaccinated C57BL\/6 mice were completely protected from disease, demonstrating the immunogenicity and protective efficacy of the multivalent LAIV. By simultaneously targeting antigenically distinct H5N1 lineages with pandemic potential, this strategy expands antigenic coverage within a single LAIV to confirm pan-H5N1 protection. Together, these findings support the development and implementation of this multivalent LAIV as a broadly protective pan-H5N1 LAIV for pandemic preparedness.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Significance<\/jats:title>\n                  <jats:p>\n                    The increasing genetic diversity of zoonotic H5N1 viruses complicates vaccine design. We developed a multivalent live-attenuated influenza vaccine (LAIV) based on the temperature-sensitive, cold-adapted, and attenuated (\n                    <jats:italic>ts<\/jats:italic>\n                    ,\n                    <jats:italic>ca<\/jats:italic>\n                    ,\n                    <jats:italic>att<\/jats:italic>\n                    ) master donor virus (MDV) influenza A\/Ann Arbor\/6\/1960 backbone that expresses the hemagglutinin (HA) and neuraminidase (NA) glycoproteins of H5N1 clades 2.3.4.4b, 2.3.2.1a, and 2.3.2.1e. A single intranasal (IN) immunization with the multivalent LAIV induced broadly cross-reactive neutralizing antibody (NAb) responses and protected experimental vaccinated animals against homologous lethal viral challenge, demonstrating the feasibility of the multivalent LAIV to protect against H5N1 clades of highest concern to humans. These findings demonstrate the feasibility of developing and implementing this multivalent LAIV as a broad protective pan-H5N1 LAIV against divergent H5N1 viruses for human use.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.08.02.742385","type":"posted-content","created":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T06:20:33Z","timestamp":1785824433000},"source":"Crossref","is-referenced-by-count":0,"title":["A One-Shot Multivalent Live-Attenuated Candidate Influenza Vaccine against Divergent Zoonotic H5N1 Clades"],"prefix":"10.64898","author":[{"given":"Ahmed M.","family":"Elsayed","sequence":"first","affiliation":[{"name":"Host-pathogen interactions (HPI) and Disease Intervention and Prevention (DIP) programs, Texas Biomedical Research Institute, San Antonio, TX, USA"},{"name":"Center of Scientific Excellence for Influenza Viruses, National Research Centre, Giza, Egypt"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ramya S.","family":"Barre","sequence":"additional","affiliation":[{"name":"Host-pathogen interactions (HPI) and Disease Intervention and Prevention (DIP) programs, Texas Biomedical Research Institute, San Antonio, TX, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Arash","family":"Rahmani","sequence":"additional","affiliation":[{"name":"Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruby A.","family":"Escobedo","sequence":"additional","affiliation":[{"name":"Host-pathogen interactions (HPI) and Disease Intervention and Prevention (DIP) programs, Texas Biomedical Research Institute, San Antonio, TX, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Trushar","family":"Jeevan","sequence":"additional","affiliation":[{"name":"Department of Host-Microbe Interactions, St. Jude Children\u2019s Research Hospital, Memphis, TN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Himadri","family":"Nath","sequence":"additional","affiliation":[{"name":"Host-pathogen interactions (HPI) and Disease Intervention and Prevention (DIP) programs, Texas Biomedical Research Institute, San Antonio, TX, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Esteban M.","family":"Castro","sequence":"additional","affiliation":[{"name":"Host-pathogen interactions (HPI) and Disease Intervention and Prevention (DIP) programs, Texas Biomedical Research Institute, San Antonio, TX, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vinay","family":"Shivanna","sequence":"additional","affiliation":[{"name":"Host-pathogen interactions (HPI) and Disease Intervention and Prevention (DIP) programs, Texas Biomedical Research Institute, San Antonio, TX, 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