{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,13]],"date-time":"2026-03-13T01:47:16Z","timestamp":1773366436456,"version":"3.50.1"},"reference-count":30,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2026,3,12]],"date-time":"2026-03-12T00:00:00Z","timestamp":1773273600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Major Talent Project of Taijiao Jiang for Guangzhou Laboratory","award":["MP-GZNL2025C03010-01"],"award-info":[{"award-number":["MP-GZNL2025C03010-01"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Background: The H1N1 influenza A virus evades host immunity through continuous antigenic drift, posing a significant challenge to broad-spectrum neutralizing antibody therapies. This study aims to systematically evaluate the neutralizing capacity of the broad-spectrum antibody C12H5 against H1N1 strains from different eras and identify key immune escape mutation sites. Methods: Three representative H1N1 virus strains from 2009, 2018, and 2023 were selected. An antigen\u2013antibody binding prediction model based on the ESM-2 large language model was constructed by integrating 48,762 GISAID sequence data and deep mutation scanning data from the Bloom laboratory. Candidate escape sites were screened using SHAP (SHapley Additive exPlanations) value analysis. Mutant viruses were constructed via reverse genetics, and their neutralizing capacity and replication fitness were validated through hemagglutination inhibition assays, microneutralization assays, and viral growth kinetics analysis. Results: Machine learning scoring identified five potential escape sites, with K147 exhibiting the highest overall score (0.92). SHAP analysis revealed that the K147 site within the HA protein\u2019s 130-loop region received the highest importance score (0.28), significantly surpassing other candidate sites. Experimental validation revealed that the K147N mutation reduced neutralizing potency against C12H5 by 8-fold (from 1:1024 to 1:128) and approximately 6-fold in microneutralization assays (from 8.3 log2 to 5.7 log2), while exhibiting a replication advantage in MDCK cells. Microneutralization assays further confirmed an approximately 6-fold reduction in neutralization sensitivity. Structural analysis indicated that K147 is located at the periphery of the HA receptor-binding domain, immediately adjacent to the receptor-binding site. Conclusions: K147N is identified as the critical mutation mediating C12H5 immune escape, and this mutation has emerged in 2023 circulating strains. This study provides important molecular targets and early warning mechanisms for broad-spectrum antibody optimization and influenza vaccine updates.<\/jats:p>","DOI":"10.3390\/v18030349","type":"journal-article","created":{"date-parts":[[2026,3,12]],"date-time":"2026-03-12T13:12:24Z","timestamp":1773321144000},"page":"349","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Identification of a Key Hemagglutinin Mutation Mediating Antibody Escape in Influenza A(H1N1)pdm09 Viruses"],"prefix":"10.3390","volume":"18","author":[{"given":"Weili","family":"Song","sequence":"first","affiliation":[{"name":"Guangzhou Institute of Respiratory Health, Guangzhou Medical University, Guangzhou 511436, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chuan","family":"Wang","sequence":"additional","affiliation":[{"name":"Guangzhou National Laboratory, Guangzhou 510005, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wenping","family":"Xie","sequence":"additional","affiliation":[{"name":"College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yiqing","family":"Li","sequence":"additional","affiliation":[{"name":"Guangzhou Institute of Respiratory Health, Guangzhou Medical University, Guangzhou 511436, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kaiyun","family":"Chen","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Vaccines for Infectious Disease, Xiamen University, Xiamen 361102, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0165-7894","authenticated-orcid":false,"given":"Wenjun","family":"Song","sequence":"additional","affiliation":[{"name":"Guangzhou National Laboratory, Guangzhou 510005, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Taijiao","family":"Jiang","sequence":"additional","affiliation":[{"name":"Guangzhou Institute of Respiratory Health, Guangzhou Medical University, Guangzhou 511436, China"},{"name":"Guangzhou National Laboratory, Guangzhou 510005, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,3,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"693","DOI":"10.1016\/S0140-6736(22)00982-5","article-title":"Influenza","volume":"400","author":"Uyeki","year":"2022","journal-title":"Lancet"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"D49","DOI":"10.1016\/j.vaccine.2008.07.039","article-title":"The biology of influenza viruses","volume":"26","author":"Bouvier","year":"2008","journal-title":"Vaccine"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1038\/nrmicro.2017.146","article-title":"The evolution of seasonal influenza viruses","volume":"16","author":"Petrova","year":"2018","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2605","DOI":"10.1056\/NEJMoa0903810","article-title":"Emergence of a novel swine-origin influenza A (H1N1) virus in humans","volume":"360","author":"Dawood","year":"2009","journal-title":"N. Engl. J. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"e16777","DOI":"10.7554\/eLife.16777","article-title":"Origins of the 2009 H1N1 influenza pandemic in swine in Mexico","volume":"5","author":"Mena","year":"2016","journal-title":"eLife"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e01206-18","DOI":"10.1128\/JVI.01206-18","article-title":"Functional Evolution of the 2009 Pandemic H1N1 Influenza Virus NS1 and PA in Humans","volume":"92","author":"Nogales","year":"2018","journal-title":"J. Virol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"e12217","DOI":"10.7554\/eLife.12217","article-title":"The global antigenic diversity of swine influenza A viruses","volume":"5","author":"Lewis","year":"2016","journal-title":"eLife"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Scarpa, F., Sernicola, L., Farcomeni, S., Ciccozzi, A., Sanna, D., Casu, M., Vitale, M., Cicenia, A., Giovanetti, M., and Romano, C. (2024). Phylodynamic and Evolution of the Hemagglutinin (HA) and Neuraminidase (NA) Genes of Influenza A(H1N1) pdm09 Viruses Circulating in the 2009 and 2023 Seasons in Italy. Pathogens, 13.","DOI":"10.3390\/pathogens13040334"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"417","DOI":"10.1016\/0092-8674(82)90135-0","article-title":"The antigenic structure of the influenza virus A\/PR\/8\/34 hemagglutinin (H1 subtype)","volume":"31","author":"Caton","year":"1982","journal-title":"Cell"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"12364","DOI":"10.1128\/JVI.01381-14","article-title":"Epitope mapping of the hemagglutinin molecule of A\/(H1N1)pdm09 influenza virus by using monoclonal antibody escape mutants","volume":"88","author":"Matsuzaki","year":"2014","journal-title":"J. Virol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"5721","DOI":"10.1128\/jvi.67.10.5721-5723.1993","article-title":"Dissociation of influenza virus hemagglutinin and neuraminidase eliminates their intravirionic antigenic competition","volume":"67","author":"Johansson","year":"1993","journal-title":"J. Virol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1016\/0168-1702(93)90056-S","article-title":"Influenza virus strains selectively recognize sialyloligosaccharides on human respiratory epithelium; the role of the host cell in selection of hemagglutinin receptor specificity","volume":"29","author":"Couceiro","year":"1993","journal-title":"Virus Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1239","DOI":"10.1128\/jvi.63.3.1239-1246.1989","article-title":"Purified influenza virus hemagglutinin and neuraminidase are equivalent in stimulation of antibody response but induce contrasting types of immunity to infection","volume":"63","author":"Johansson","year":"1989","journal-title":"J. Virol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Gao, J., Couzens, L., Burke, D.F., Wan, H., Wilson, P., Memoli, M.J., Xu, X., Harvey, R., Wrammert, J., and Ahmed, R. (2019). Antigenic Drift of the Influenza A(H1N1)pdm09 Virus Neuraminidase Results in Reduced Effectiveness of A\/California\/7\/2009 (H1N1pdm09)-Specific Antibodies. mBio, 10.","DOI":"10.1128\/mBio.00307-19"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"541","DOI":"10.1016\/j.virs.2023.05.008","article-title":"Development of PREDAC-H1pdm to model the antigenic evolution of influenza A\/(H1N1) pdm09 viruses","volume":"38","author":"Liu","year":"2023","journal-title":"Virol. Sin."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"5182","DOI":"10.1038\/s41467-022-32926-5","article-title":"Identification of a cross-neutralizing antibody that targets the receptor binding site of H1N1 and H5N1 influenza viruses","volume":"13","author":"Li","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"6108","DOI":"10.1073\/pnas.100133697","article-title":"A DNA transfection system for generation of influenza A virus from eight plasmids","volume":"97","author":"Hoffmann","year":"2000","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"e01070-14","DOI":"10.1128\/mBio.01070-14","article-title":"Antigenic variation of clade 2.1 H5N1 virus is determined by a few amino acid substitutions immediately adjacent to the receptor binding site","volume":"5","author":"Koel","year":"2014","journal-title":"mBio"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"12550","DOI":"10.3390\/cimb46110745","article-title":"Benchmark Investigation of SARS-CoV-2 Mutants\u2019 Immune Escape with 2B04 Murine Antibody: A Step Towards Unraveling a Larger Picture","volume":"46","author":"Kapusta","year":"2024","journal-title":"Curr. Issues Mol. Biol."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Doud, M.B., Hensley, S.E., and Bloom, J.D. (2017). Complete mapping of viral escape from neutralizing antibodies. PLoS Pathog., 13.","DOI":"10.1371\/journal.ppat.1006271"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"583","DOI":"10.1038\/s41586-021-03819-2","article-title":"Highly accurate protein structure prediction with AlphaFold","volume":"596","author":"Jumper","year":"2021","journal-title":"Nature"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"843","DOI":"10.1126\/science.1204839","article-title":"A highly conserved neutralizing epitope on group 2 influenza A viruses","volume":"333","author":"Ekiert","year":"2011","journal-title":"Science"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Kim, S.J., Nguyen, V.G., Park, Y.H., Park, B.K., and Chung, H.C. (2020). A Novel Synonymous Mutation of SARS-CoV-2: Is This Possible to Affect Their Antigenicity and Immunogenicity?. Vaccines, 8.","DOI":"10.3390\/vaccines8020220"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Starr, T.N., Greaney, A.J., Dingens, A.S., and Bloom, J.D. (2021). Complete map of SARS-CoV-2 RBD mutations that escape the monoclonal antibody LY-CoV555 and its cocktail with LY-CoV016. bioRxiv.","DOI":"10.1101\/2021.02.17.431683"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"4196","DOI":"10.1038\/s41467-021-24435-8","article-title":"Mapping mutations to the SARS-CoV-2 RBD that escape binding by different classes of antibodies","volume":"12","author":"Greaney","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"294","DOI":"10.1111\/lam.13513","article-title":"Comparison of hemagglutination inhibition and hemagglutinin pseudovirus neutralization titres in relation to protection against influenza in a mouse model","volume":"73","author":"Huang","year":"2021","journal-title":"Lett. Appl. Microbiol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"e2208718120","DOI":"10.1073\/pnas.2208718120","article-title":"Epistasis reduces fitness costs of influenza A virus escape from stem-binding antibodies","volume":"120","author":"Lee","year":"2023","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2484330","DOI":"10.1080\/22221751.2025.2484330","article-title":"Unique immune and other responses of human nasal epithelial cells infected with H5N1 avian influenza virus compared to seasonal human influenza A and B viruses","volume":"14","author":"Tan","year":"2025","journal-title":"Emerg. Microbes Infect."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Russell, C.J. (2021). Hemagglutinin Stability and Its Impact on Influenza A Virus Infectivity, Pathogenicity, and Transmissibility in Avians, Mice, Swine, Seals, Ferrets, and Humans. Viruses, 13.","DOI":"10.3390\/v13050746"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1081","DOI":"10.1016\/j.csbj.2025.03.010","article-title":"VirusWarn: A mutation-based early warning system to prioritize concerning SARS-CoV-2 and influenza virus variants from sequencing data","volume":"27","author":"Kirschbaum","year":"2025","journal-title":"Comput. Struct. Biotechnol. J."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/3\/349\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,3,12]],"date-time":"2026-03-12T13:31:30Z","timestamp":1773322290000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/3\/349"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,12]]},"references-count":30,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2026,3]]}},"alternative-id":["v18030349"],"URL":"https:\/\/doi.org\/10.3390\/v18030349","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,3,12]]}}}