{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,8]],"date-time":"2026-04-08T22:15:06Z","timestamp":1775686506480,"version":"3.50.1"},"reference-count":48,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2025,5,16]],"date-time":"2025-05-16T00:00:00Z","timestamp":1747353600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"CONACyT Fellowship","award":["398663"],"award-info":[{"award-number":["398663"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Antibodies"],"abstract":"<jats:p>Background\/Objetives: Nanobodies (VHH) have become an excellent tool for diagnosis, therapy, and research since VHH shows a high capability of recognizing and neutralizing antigens. VHHs are highly soluble and stable at high temperatures, and in the presence of chaotropic agents, they offer significant advantages over other biological therapeutic agents. This study aimed to identify and humanize VHH fragments with neutralizing potential against the influenza A\/H1N1 virus. Methods: A library of VHH antibody fragments was produced by phage display technique against an inactivated influenza A\/H1N1 vaccine. Three VHH sequences were selected and humanized. Specifically, the recognition capacity of the antibodies denominated 2-C10 and 2-C10H was confirmed by ELISA and western blot (WB), as well as their microneutralization capacity in a cellular model, suggesting their potential therapeutic use in patients infected with the influenza A\/H1N1 virus. Molecular docking assays were used to support the mechanism of viral inhibition. Results: The VHHs 2-C10 and 2-C10H showed specific recognition of influenza A\/H1N1 antigens by ELISA and Western Blot and demonstrated neutralizing activity in vitro. The optimal VHH, 2-C10H, showed 75% neutralization capacity at a concentration of 1.56 \u03bcg\/mL against the A\/H1N1 viral strain, potentially through the inactivation of hemagglutinin protein, a phenomenon supported by molecular docking assays. Conclusions: This study presents a strategic approach to identify VHH candidates that may be useful for diagnosing and potentially treating patients already infected by the A\/H1N1 virus, as it may reduce the severity of their symptoms.<\/jats:p>","DOI":"10.3390\/antib14020042","type":"journal-article","created":{"date-parts":[[2025,5,16]],"date-time":"2025-05-16T05:11:57Z","timestamp":1747372317000},"page":"42","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Neutralization of the Pandemic Influenza A\/H1N1 Virus with Lama glama Humanized Nanobodies (VHH)"],"prefix":"10.3390","volume":"14","author":[{"given":"Zeila Yazm\u00edn","family":"P\u00e1ez-Hern\u00e1ndez","sequence":"first","affiliation":[{"name":"Facultad de Ciencias Qu\u00edmicas e Ingenier\u00eda, Universidad Aut\u00f3noma de Baja California, Calzada Universidad 14418, Parque Industrial Internacional Tijuana, Tijuana 22390, Baja California, Mexico"},{"name":"Facultad de Ciencias, Universidad Aut\u00f3noma de Baja California, Carretera Transpeninsular Ensenada-Tijuana No. 3917, Colonia Playitas, Ensenada 22860, Baja California, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jose Luis","family":"Stephano-Hornedo","sequence":"additional","affiliation":[{"name":"Facultad de Ciencias, Universidad Aut\u00f3noma de Baja California, Carretera Transpeninsular Ensenada-Tijuana No. 3917, Colonia Playitas, Ensenada 22860, Baja California, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6169-9562","authenticated-orcid":false,"given":"Jose Alberto","family":"Bola\u00f1os-Prats","sequence":"additional","affiliation":[{"name":"Facultad de Ciencias, Universidad Aut\u00f3noma de Baja California, Carretera Transpeninsular Ensenada-Tijuana No. 3917, Colonia Playitas, Ensenada 22860, Baja California, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5528-400X","authenticated-orcid":false,"given":"Iv\u00e1n","family":"C\u00f3rdova-Guerrero","sequence":"additional","affiliation":[{"name":"Facultad de Ciencias Qu\u00edmicas e Ingenier\u00eda, Universidad Aut\u00f3noma de Baja California, Calzada Universidad 14418, Parque Industrial Internacional Tijuana, Tijuana 22390, Baja California, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6910-4583","authenticated-orcid":false,"given":"Mariana","family":"Mac\u00edas-Alonso","sequence":"additional","affiliation":[{"name":"Instituto Polit\u00e9cnico Nacional, UPIIG, Av. Mineral de Valenciana, No. 200, Col. Fracc, Industrial Puerto Interior, Silao de la Victoria 36275, Guanajuato, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3720-4528","authenticated-orcid":false,"given":"Joaqu\u00edn G.","family":"Marrero","sequence":"additional","affiliation":[{"name":"Instituto Polit\u00e9cnico Nacional, UPIIG, Av. Mineral de Valenciana, No. 200, Col. Fracc, Industrial Puerto Interior, Silao de la Victoria 36275, Guanajuato, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9650-2555","authenticated-orcid":false,"given":"Angel Pulido","family":"Capiz","sequence":"additional","affiliation":[{"name":"Departamento de Bioqu\u00edmica, Facultad de Medicina Mexicali, Universidad Aut\u00f3noma de Baja California, Mexicali 21000, Baja California, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9421-3730","authenticated-orcid":false,"given":"Victor Garc\u00eda","family":"Gonz\u00e1lez","sequence":"additional","affiliation":[{"name":"Departamento de Bioqu\u00edmica, Facultad de Medicina Mexicali, Universidad Aut\u00f3noma de Baja California, Mexicali 21000, Baja California, Mexico"},{"name":"Centro de Innovaci\u00f3n e Investigaci\u00f3n en Salud (CIIS), Facultad de Medicina Mexicali, Universidad Aut\u00f3noma de Baja California, Mexicali 21000, Baja California, Mexico"},{"name":"Laboratorio Multidisciplinario de Estudios Metab\u00f3licos y C\u00e1ncer, Facultad de Medicina Mexicali, Universidad Aut\u00f3noma de Baja California, Mexicali 21000, Baja California, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,5,16]]},"reference":[{"key":"ref_1","unstructured":"WHO (2025, May 10). Influenza Seasonal Influenza and Influenza A(H1N1), Available online: https:\/\/www.who.int\/news-room\/fact-sheets\/detail\/influenza-(seasonal)."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Lee, C.C., Yang, C.Y., Lin, L.L., Ko, T.P., Chang, A.H., Chang, S.S., and Wang, A.H. (2019). An Effective Neutralizing Antibody Against Influenza Virus H1N1 from Human B Cells. Sci. Rep., 9.","DOI":"10.1038\/s41598-019-40937-4"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"10704","DOI":"10.1128\/JVI.01166-12","article-title":"Entry of influenza A Virus with a \u03b12,6-linked sialic acid binding preference requires host fibronectin","volume":"86","author":"Leung","year":"2012","journal-title":"J. Virol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"6852","DOI":"10.1016\/j.vaccine.2007.07.027","article-title":"Influenza vaccine: The challenge of antigenic drift","volume":"25","author":"Carrat","year":"2007","journal-title":"Vaccine"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"645","DOI":"10.1016\/j.aogh.2015.08.014","article-title":"Treatment and Prevention of Pandemic H1N1 Influenza","volume":"81","author":"Rewar","year":"2015","journal-title":"Ann. Glob. Health"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"318","DOI":"10.2174\/187152607783018736","article-title":"Influenza virus transmission: Basic science and implications for the use of antiviral drugs during a pandemic","volume":"7","author":"Lowen","year":"2007","journal-title":"Infect. Disord. Drug Targets"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"785","DOI":"10.1056\/NEJMp068030","article-title":"Antiviral Resistance in Influenza Viruses\u2014Implications for Management and Pandemic Response","volume":"354","author":"Hayden","year":"2006","journal-title":"N. Engl. J. Med."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"4406","DOI":"10.1002\/jmv.27870","article-title":"Frequency and distribution of H1N1 influenza A viruses with oseltamivir-resistant mutations worldwide before and after the 2009 pandemic","volume":"94","author":"Zhang","year":"2022","journal-title":"J. Med. Virol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"S41","DOI":"10.1016\/j.jaci.2009.09.046","article-title":"Structure and function of immunoglobulins","volume":"125","author":"Schroeder","year":"2010","journal-title":"J. Allergy Clin. Immunol."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Muramatsu, M., Yoshida, R., Yokoyama, A., Miyamoto, H., Kajihara, M., Maruyama, J., Nao, N., Manzoor, R., and Takada, A. (2014). Comparison of antiviral activity between IgA and IgG specific to influenza virus hemagglutinin: Increased potential of IgA for heterosubtypic immunity. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0085582"},{"key":"ref_11","unstructured":"Baron, S. (1996). Immune Defenses. Medical Microbiology, University of Texas Medical Branch at Galveston. [4th ed.]. Chapter 50."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Liu, Y., Li, H., Xue, Y., Zhao, S., Li, C., Qu, L., Zhang, Y., and Liu, M. (2017). Characterization of Monoclonal Antibodies against HA Protein of H1N1 Swine Influenza Virus and Protective Efficacy against H1 Viruses in Mice. Viruses, 9.","DOI":"10.3390\/v9080209"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Ito, M., Yamayoshi, S., Murakami, K., Saito, K., Motojima, A., Nakaishi, K., and Kawaoka, Y. (2019). Characterization of Mouse Monoclonal Antibodies Against the HA of A(H7N9) Influenza Virus. Viruses, 11.","DOI":"10.3390\/v11020149"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"10114","DOI":"10.1128\/JVI.01348-13","article-title":"An eight-segment swine influenza virus harboring H1 and H3 hemagglutinins is attenuated and protective against H1N1 and H3N2 subtypes in pigs","volume":"87","author":"Masic","year":"2013","journal-title":"J. Virol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"319","DOI":"10.1016\/j.imbio.2017.10.040","article-title":"Human monoclonal antibodies neutralizing influenza virus A\/H1N1pdm09 and seasonal A\/H1N1 strains\u2014Distinct Ig gene repertoires with a similar action mechanism","volume":"223","author":"Hiroi","year":"2018","journal-title":"Immunobiology"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"446","DOI":"10.1038\/363446a0","article-title":"Naturally occurring antibodies devoid of light chains","volume":"363","author":"Atarhouch","year":"1993","journal-title":"Nature"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"66","DOI":"10.1134\/S0026893311010134","article-title":"\u201cCamel nanoantibody\u201d is an efficient tool for research diagnostics and therapy","volume":"45","author":"Tillib","year":"2010","journal-title":"Mol. Biol."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Richard, G., Meyers, A.J., McLean, M.D., Arbabi-Ghahroudi, M., MacKenzie, R., and Hall, J.C. (2013). In vivo neutralization of \u03b1-cobratoxin with high-affinity llama single-domain antibodies (VHHs) and a VHH-Fc antibody. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0069495"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1016\/S0167-4838(99)00030-8","article-title":"Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies","volume":"1431","author":"Frenken","year":"1999","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"5985","DOI":"10.1007\/s00216-016-9585-x","article-title":"VHH antibodies: Emerging reagents for the analysis of environmental chemicals","volume":"408","author":"Bever","year":"2016","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"33","DOI":"10.2310\/7290.2011.00025","article-title":"Rapid visualization of human tumor xenografts through optical imaging with a near-infrared fluorescent anti-epidermal growth factor receptor nanobody","volume":"11","author":"Oliveira","year":"2012","journal-title":"Mol. Imaging"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Saeed, A.F., Wang, R., Ling, S., and Wang, S. (2017). Antibody Engineering for Pursuing a Healthier Future. Front. Microb., 8.","DOI":"10.3389\/fmicb.2017.00495"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1266","DOI":"10.1016\/j.imbio.2012.07.009","article-title":"Novel applications of nanobodies for in vivo bio-imaging of inflamed tissues in inflammatory diseases and cancer","volume":"217","author":"Schoonooghe","year":"2012","journal-title":"Immunobiology"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"175","DOI":"10.1080\/02648725.2013.801235","article-title":"Antibody humanization methods\u2014A review and update","volume":"29","author":"Safdari","year":"2013","journal-title":"Biotechnol. Genet. Eng. Rev."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Bannas, P., Hambach, J., and Koch-Nolte, F. (2017). Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics. Front. Immunol., 8.","DOI":"10.3389\/fimmu.2017.01603"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"3273","DOI":"10.1074\/jbc.M806889200","article-title":"General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold","volume":"284","author":"Vincke","year":"2009","journal-title":"J. Biol. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"4304","DOI":"10.1111\/febs.15809","article-title":"Immunogenicity and humanization of single-domain antibodies","volume":"289","author":"Rossotti","year":"2022","journal-title":"FEBS J."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1093\/jb\/mvv038","article-title":"Isolation and characterization of antigen-specific alpaca (Lama pacos) VHH antibodies by biopanning followed by high-throughput sequencing","volume":"158","author":"Miyazaki","year":"2015","journal-title":"J. Biochem."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"e4450","DOI":"10.1002\/pro.4450","article-title":"Molecular basis for thermal stability and affinity in a VHH: Contribution of the framework region and its influence in the conformation of the CDR3","volume":"31","author":"Kinoshita","year":"2022","journal-title":"Protein Sci."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"717","DOI":"10.1002\/ijc.1523","article-title":"Effects of humanization and gene shuffling on immunogenicity and antigen binding of anti-TAG-72 single-chain Fvs","volume":"94","author":"Pavlinkova","year":"2001","journal-title":"Int. J. Cancer"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"982","DOI":"10.1128\/JVI.06322-11","article-title":"Structural characterization of the hemagglutinin receptor specificity from the 2009 H1N1 influenza pandemic","volume":"86","author":"Xu","year":"2012","journal-title":"J. Virol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1016\/j.amsu.2014.09.001","article-title":"The history of monoclonal antibody development\u2014Progress, remaining challenges and future innovations","volume":"3","author":"Liu","year":"2014","journal-title":"Ann. Med. Surg."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"2486390","DOI":"10.1080\/19420862.2025.2486390","article-title":"Nanoscale warriors against viral invaders: A comprehensive review of Nanobodies as potential antiviral therapeutics","volume":"17","author":"Verma","year":"2025","journal-title":"mAbs"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Wu, Y., Jiang, S., and Ying, T. (2017). Single-Domain Antibodies As Therapeutics against Human Viral Diseases. Front. Immunol., 8.","DOI":"10.3389\/fimmu.2017.01802"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1076","DOI":"10.1016\/j.drudis.2016.04.003","article-title":"Nanobodies as therapeutics: Big opportunities for small antibodies","volume":"21","author":"Steeland","year":"2016","journal-title":"Drug Discov. Today"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"17193","DOI":"10.3390\/ijms160817193","article-title":"Protein Folding and Mechanisms of Proteostasis","volume":"16","year":"2015","journal-title":"Int. J. Mol. Sci."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1304","DOI":"10.1016\/j.bbagen.2006.03.027","article-title":"Structural analysis and classification of native proteins from E. coli commonly co-purified by immobilised metal affinity chromatography","volume":"1760","author":"Davies","year":"2006","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1007\/s11010-009-0105-6","article-title":"Disorder-to-order conformational transitions in protein structure and its relationship to disease","volume":"330","author":"Moreno","year":"2009","journal-title":"Mol. Cell. Biochem."},{"key":"ref_39","first-page":"133","article-title":"Development of Neutralizing Nanobodies to the Hemagglutinin Stem Domain of Influenza A Viruses","volume":"3","author":"Voronina","year":"2021","journal-title":"Acta Naturae"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Barbieri, E.S., Sosa-Holt, C., Iba\u00f1ez, L.I., Baztarrica, J., Garaicoechea, L., Gay, C.L., Caceres, C.J., Aduriz, M., Baumeister, E., and Escribano, J.A. (2024). Anti-hemagglutinin monomeric nanobody provides prophylactic immunity against H1 subtype influenza A viruses. PLoS ONE, 19.","DOI":"10.1371\/journal.pone.0301664"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1128\/AAC.01802-15","article-title":"Generation and Characterization of ALX-0171, a Potent Novel Therapeutic Nanobody for the Treatment of Respiratory Syncytial Virus Infection","volume":"60","author":"Detalle","year":"2015","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1639","DOI":"10.1007\/s40265-018-0989-0","article-title":"Caplacizumab: First Global Approval","volume":"78","author":"Duggan","year":"2018","journal-title":"Drugs"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"458","DOI":"10.1038\/s41419-024-06802-7","article-title":"A broad neutralizing nanobody against SARS-CoV-2 engineered from an approved drug","volume":"15","author":"Liu","year":"2024","journal-title":"Cell Death Dis."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"eadg9459","DOI":"10.1126\/sciimmunol.adg9459","article-title":"An armed anti-immunoglobulin light chain nanobody protects mice against influenza A and B infections","volume":"8","author":"Liu","year":"2023","journal-title":"Sci. Immunol."},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Nosenko, M., Mkhonov, V., Kruglov, A., Drutskaya, M., Atretkhany, K., Tillib, S., and Nedospasov, S. (2017). VHH-Based Bispecific Antibodies Targeting Cytokine Production. Front. Immunol., 8.","DOI":"10.3389\/fimmu.2017.01073"},{"key":"ref_46","unstructured":"Barbas, C.F., Burton, D.R., Scott, J.K., and Silverman, G.J. (2001). Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press. Chapter 1\u20136."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1093\/oxfordjournals.aje.a118408","article-title":"A simple method of estimating fifty percent endpoints","volume":"27","author":"Reed","year":"1938","journal-title":"Am. J. Epidemiol."},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Pulido-Capiz, A., Chimal-Vega, B., Avila-Barrientos, L.P., Campos-Valenzuela, A., D\u00edaz-Molina, R., Mu\u00f1iz-Salazar, R., Galindo-Hern\u00e1ndez, O., and Garc\u00eda-Gonz\u00e1lez, V. (2024). Auraptene Boosts the Efficacy of the Tamoxifen Metabolites Endoxifen and 4-OH-Tamoxifen in a Chemoresistant ER+ Breast Cancer Model. Pharmaceutics, 16.","DOI":"10.3390\/pharmaceutics16091179"}],"container-title":["Antibodies"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4468\/14\/2\/42\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T17:33:43Z","timestamp":1760031223000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4468\/14\/2\/42"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,5,16]]},"references-count":48,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2025,6]]}},"alternative-id":["antib14020042"],"URL":"https:\/\/doi.org\/10.3390\/antib14020042","relation":{},"ISSN":["2073-4468"],"issn-type":[{"value":"2073-4468","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,5,16]]}}}