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Influenza viruses that are adapted to transmission in birds and a variety of mammals tend to have a less stable hemagglutinin (HA) than seasonal influenza viruses, enabling membrane fusion at comparatively higher pH levels. Here, we combined five mutations in the H5 HA that increased its melting temperature and promoted stable closure of the HA trimer. Structural analysis by cryo\u2013electron microscopy revealed that the stabilizing mutations create several new hydrophobic interactions while maintaining the local HA structure. We found that vaccinating mice with stabilized H5 HA immunogens resulted in higher hemagglutination inhibition and neutralization titers than nonstabilized comparators. Epitope mapping of vaccine-elicited polyclonal antibody responses using negative-stain electron microscopy and deep mutational scanning showed that site E on the side of the HA receptor binding domain was immunodominant across all groups; however, the stabilized immunogens shifted responses toward the receptor binding site, which elicited a higher proportion of neutralizing antibodies. Consistent with these findings, stabilized H5 HA immunogens delivered as messenger RNA\u2013lipid nanoparticle (mRNA-LNP) vaccines protected mice against H5N1 challenge. These findings highlight that H5 HA\u2013stabilizing mutations enhance the quality of antibody responses across different vaccine formats, underscoring their potential to improve pandemic preparedness vaccines targeting viruses from this widely circulating clade.<\/jats:p>","DOI":"10.1126\/scitranslmed.aea8770","type":"journal-article","created":{"date-parts":[[2026,3,4]],"date-time":"2026-03-04T18:58:28Z","timestamp":1772650708000},"update-policy":"https:\/\/doi.org\/10.34133\/aaas_crossmark","source":"Crossref","is-referenced-by-count":8,"title":["Stabilization of the H5 clade 2.3.4.4b hemagglutinin improves vaccine-elicited neutralizing antibody responses in mice"],"prefix":"10.1126","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6858-4725","authenticated-orcid":true,"given":"Annie","family":"Dosey","sequence":"first","affiliation":[{"name":"Department of Biochemistry, University of Washington, Seattle, WA 98195, USA."},{"name":"Institute for Protein Design, University of Washington, Seattle, WA 98195, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0908-6982","authenticated-orcid":true,"given":"Bernadeta","family":"Dadonaite","sequence":"additional","affiliation":[{"name":"Basic Sciences Division and Computational Biology Program, Fred Hutchinson Cancer Center, Seattle, WA 98109, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5741-3699","authenticated-orcid":true,"given":"Rebecca A.","family":"Gillespie","sequence":"additional","affiliation":[{"name":"Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5354-6010","authenticated-orcid":true,"given":"Elizabeth M.","family":"Leaf","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, University of Washington, Seattle, WA 98195, USA."},{"name":"Institute for Protein Design, University of Washington, Seattle, WA 98195, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7110-9623","authenticated-orcid":true,"given":"Matthew J.","family":"Vukovich","sequence":"additional","affiliation":[{"name":"Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-6418-3371","authenticated-orcid":true,"given":"Jackson","family":"McGowan","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, University of Washington, Seattle, WA 98195, USA."},{"name":"Institute for Protein Design, University of Washington, Seattle, WA 98195, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1153-7471","authenticated-orcid":true,"given":"Emily","family":"Grey","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, University of Washington, Seattle, WA 98195, USA."},{"name":"Institute for Protein Design, University of Washington, Seattle, WA 98195, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1544-1493","authenticated-orcid":true,"given":"Hiromi","family":"Muramatsu","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-9733-4715","authenticated-orcid":true,"given":"Rachel H. 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Kesavardhana Avian influenza virus neuraminidase stalk length and haemagglutinin glycosylation patterns reveal molecularly directed reassortment promoting the emergence of highly pathogenic clade 2.3.4.4b A (H5N1) viruses. bioRxiv 595329 [Preprint] (2024); https:\/\/doi.org\/10.1101\/2024.05.22.595329.","DOI":"10.1101\/2024.05.22.595329"},{"key":"e_1_3_2_11_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-54934-3"},{"key":"e_1_3_2_12_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pbio.3002916"},{"key":"e_1_3_2_13_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.adt0180"},{"key":"e_1_3_2_14_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-55546-7"},{"key":"e_1_3_2_15_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-med-042420-112725"},{"key":"e_1_3_2_16_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-48555-z"},{"key":"e_1_3_2_17_2","doi-asserted-by":"publisher","DOI":"10.1126\/scitranslmed.ads1273"},{"key":"e_1_3_2_18_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41541-024-00932-x"},{"key":"e_1_3_2_19_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41541-024-00988-9"},{"key":"e_1_3_2_20_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.abm0271"},{"key":"e_1_3_2_21_2","unstructured":"Arcturus Therapeutics Inc. \u201cSafety and immunogenicity study of self-amplifying RNA pandemic influenza vaccine in adults\u201d (ClinicalTrials.gov 2026); https:\/\/clinicaltrials.gov\/study\/NCT06602531."},{"key":"e_1_3_2_22_2","doi-asserted-by":"publisher","DOI":"10.1126\/scitranslmed.adu2085"},{"key":"e_1_3_2_23_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.chom.2021.02.002"},{"key":"e_1_3_2_24_2","doi-asserted-by":"publisher","DOI":"10.3390\/vaccines13050523"},{"key":"e_1_3_2_25_2","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms9143"},{"key":"e_1_3_2_26_2","doi-asserted-by":"publisher","DOI":"10.1038\/nsmb.3267"},{"key":"e_1_3_2_27_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-022-28931-3"},{"key":"e_1_3_2_28_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-44534-y"},{"key":"e_1_3_2_29_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2115379119"},{"key":"e_1_3_2_30_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.02790-15"},{"key":"e_1_3_2_31_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1003151"},{"key":"e_1_3_2_32_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41541-017-0017-5"},{"key":"e_1_3_2_33_2","doi-asserted-by":"publisher","DOI":"10.1038\/nm.3927"},{"key":"e_1_3_2_34_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0112302"},{"key":"e_1_3_2_35_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.adf6591"},{"key":"e_1_3_2_36_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.03422-13"},{"key":"e_1_3_2_37_2","doi-asserted-by":"publisher","DOI":"10.1006\/viro.1997.8958"},{"key":"e_1_3_2_38_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-025-02137-x"},{"key":"e_1_3_2_39_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1222908"},{"key":"e_1_3_2_40_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2019.04.011"},{"key":"e_1_3_2_41_2","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.4347"},{"key":"e_1_3_2_42_2","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.57659"},{"key":"e_1_3_2_43_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-021-03365-x"},{"key":"e_1_3_2_44_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.01100-18"},{"key":"e_1_3_2_45_2","doi-asserted-by":"crossref","unstructured":"A. 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Du S. M. Ansell \u201cLipids and lipid nanoparticle formulations for delivery of nucleic acids \u201d world patent WO\/2017\/004143 (2017); https:\/\/patentscope.wipo.int\/search\/en\/WO2017004143."},{"key":"e_1_3_2_66_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-21954-2"},{"key":"e_1_3_2_67_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2023.02.001"},{"key":"e_1_3_2_68_2","unstructured":"B. Dadonaite J. Bloom Escape from vaccine-elicited serum antibody neutralization (2025); https:\/\/dms-vep.org\/Flu_H5_American-Wigeon_South-Carolina_2021-H5N1_DMS\/htmls\/Stabilized_HA_sera_escape_faceted.html."},{"key":"e_1_3_2_69_2","unstructured":"B. Dadonaite J. Bloom W. Hannon jennyahn dms-vep\/Flu_H5_American-Wigeon_South-Carolina_2021-H5N1_DMS: VRC_mAb_publication Zenodo (2025); http:\/\/dx.doi.org\/10.5281\/ZENODO.16740862."},{"key":"e_1_3_2_70_2","unstructured":"B. Dadonaite J. Bloom W. Hannon J. Ahn Flu_H5_American-Wigeon_South-Carolina_2021-H5N1_DMS: Deep mutational scanning of H5 VEP Github (2025); https:\/\/github.com\/dms-vep\/Flu_H5_American-Wigeon_South-Carolina_2021-H5N1_DMS."}],"container-title":["Science Translational Medicine"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/scitranslmed.aea8770","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,3,19]],"date-time":"2026-03-19T17:58:53Z","timestamp":1773943133000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/scitranslmed.aea8770"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,4]]},"references-count":69,"journal-issue":{"issue":"839","published-print":{"date-parts":[[2026,3,4]]}},"alternative-id":["10.1126\/scitranslmed.aea8770"],"URL":"https:\/\/doi.org\/10.1126\/scitranslmed.aea8770","relation":{},"ISSN":["1946-6234","1946-6242"],"issn-type":[{"value":"1946-6234","type":"print"},{"value":"1946-6242","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,3,4]]},"assertion":[{"value":"2025-07-23","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-12-24","order":1,"name":"revised","label":"Revised","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-02-05","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-03-04","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"eaea8770"}}