Human monoclonal antibodies that target clade 2.3.4.4b H5N1 hemagglutinin

Sep 30, 2026

Highly pathogenic avian influenza H5N1 clade 2.3.4.4b has spread globally since 2022, infecting birds and spilling over into mammals, which highlights pandemic potential. The study generates a panel of anti-hemagglutinin human monoclonal antibodies against the H5 protein of this clade using humanized immunoglobulin mice and hybridoma technology. Fourteen of sixteen antibodies neutralize H5N1 in vitro, and top candidates show stronger in vivo protection under prophylactic or therapeutic dosing, supported by cryo-EM structural motif identification.

LanguageEnglish

CategoryHealthcare

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Pages11

Reading Time28 min

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Results

Generation and in vitro characterization of mAbs

Article [https://doi.org/10.1038/s41467-025-66829-y](https://doi.org/10.1038/s41467-025-66829-y) Human monoclonal antibodies that target clade [2.3.4.4](2.3.4.4)b H5N1 hemagglutinin Received: 7 July 2025 | Accepted: 17 November 2025 | | --- | | | | Check for updates | Garazi Peña Alzua 1,2, André Nicolás León 3, Temima Yellin 1,2, Disha Bhavsar 1,2, Madhumathi Loganathan 1,2, Kaitlyn Bushﬁeld1,2, Philip J. M. Brouwer3, Alesandra J. Rodriguez3, Trushar Jeevan4, Richard Webby 4, Christine Marizzi1,5,6, Julianna Han 3, Andrew B. Ward 3 , J. Andrew Duty1,7 & Florian Krammer 1,2,6,8,9 The highly pathogenic avian inﬂuenza H5N1 virus clade [2.3.4.4](2.3.4.4)b has been spreading globally since 2022, causing mortality and morbidity in domestic and wild birds, as well as in mammals, which underscores its potential to cause a pandemic. Here, we generate a panel of anti-hemagglutinin (HA) human monoclonal antibodies (mAbs) against the H5 protein of clade [2.3.4.4](2.3.4.4)b. To develop human chimeric antibodies, H2L2 Harbor Mice®, which express human immunoglobulin germline genes, were immunized with H5 and N1 recombinant proteins from A/mallard/New York/22-008760-007-original/ 2022 H5N1 virus. Through hybridoma technology, sixteen fully human mAbs are generated, most of which show cross-reactivity against H5 proteins from different clade [2.3.4.4](2.3.4.4) virus variants. Fourteen out of the sixteen mAbs neutralize the virus in vitro. The mAbs with the strongest hemagglutination inhibition activity also demonstrate greater neutralizing capacity and show increased protective effects in vivo when administered prophylactically or therapeutically in a murine H5N1 challenge model. Using cryo-electron microscopy, we identify a cross-clonotype conserved motif that bound a hydrophobic groove on the head domain of H5 HA. Akin to mAbs against severe acute respiratory syndrome coronavirus 2 during the coronavirus 2019 pandemic, these mAbs could serve as treatments in case ofa widespread H5N1 epidemic or pandemic. Highly pathogenic H5N1 avian inﬂuenza virus emerged as a human disease in 19971. After variable H5N1 virus activity in the years before the coronavirus disease 2019 (COVID-19) pandemic, a subclade of H5N1, clade [2.3.4.4](2.3.4.4)b, started to spread globally in 2022. This spread has resulted in signiﬁcant losses in the poultry industry and endangered millions of wild birds2,3. This virus clade has also spilled over into mammals, causing severe disease and high fatality rates4. In March 2024, the virus was detected in the United States (U.S.) in dairy cattle, 1Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 2Center for Vaccine Research and Pandemic Preparedness (CVaRPP), Icahn School of Medicine at Mount Sinai, New York, NY, USA. 3Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA. 4Department of Host Microbe Interactions, St Jude Children’s Research Hospital, Memphis, TN, USA. 5BioBus, New York, NY, USA. 6Ludwig Boltzmann Institute for Science Outreach and Pandemic Preparedness at the Medical University of Vienna, Vienna, Austria. 7Center for Therapeutic Antibody Development, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 8Department of Pathology, Molecular and Cell Based Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 9Ignaz Semmelweis Institute, Interuniversity Institute for Infection Research, Medical University of Vienna, Vienna, Austria. e-mail: [andrew.duty@mssm.edu](andrew.duty@mssm.edu); ﬂ[orian.krammer@mssm.edu](orian.krammer@mssm.edu) spreading among herds with high titers of virus present in the cows’milk, and in October 2024, the virus was detected in a pig on a backyard farm in Oregon5. Even more alarming is the reported transmission of the virus to humans, posing a risk to workers who are in contact with infected animals and raising concerns about the potential fo

FAQs

What is the main goal of generating these monoclonal antibodies?

To develop anti-hemagglutinin human monoclonal antibodies that target the H5 protein of H5N1 clade 2.3.4.4b and assess their neutralizing and protective performance.

How were the monoclonal antibodies developed in this study?

H2L2 humanized mice expressing human immunoglobulin germline genes were immunized with recombinant H5 and N1 proteins from an H5N1 virus, and hybridoma technology produced fully human mAbs.

What evidence supports that the antibodies could protect against infection?

Fourteen antibodies neutralized the virus in vitro, and the strongest hemagglutination inhibition candidates showed increased neutralizing capacity and enhanced in vivo protective effects in a murine H5N1 challenge model.

Sep 30, 2026

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