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Clade 2.3.4.4b H5N1 is causing an unprecedented outbreak in dairy cows in the United States. To understand if recent H5N1 viruses are changing their receptor use, we screened recombinant hemagglutinin (HA) from historical and recent 2.3.4.4b H5N1 viruses for binding to distinct glycans bearing terminal sialic acids using a glycan microarray. We find that H5 from A/Texas/37/2024, an isolate from the dairy cow outbreak, has increased binding breadth to core glycans bearing terminal α2,3 sialic acids, the avian receptor, compared to historical and recent 2.3.4.4b H5N1 viruses. We do not observe any binding to α2,6 sialic acids, the receptor used by human seasonal influenza viruses. Using molecular dynamics and a cryo-EM structure of A/Texas/37/2024 H5, we show A/Texas/37/2024 H5 is more flexible within the receptor-binding site compared to a 2.3.4.4b H5 from 2022. We identify a single mutation outside of the receptor binding site, T199I, is responsible for increased binding breadth, as it increases receptor binding site flexibility. Together, these data show recent H5N1 viruses are evolving increased receptor binding breadth which could impact the host range and cell types infected with H5N1. Highly pathogenic avian influenza clade 2.3.4.4b H5N1 viruses are causing a multistate outbreak in dairy cows. In this study, the authors report that recent H5N1 viruses have acquired a single mutation that increases their receptor binding range, which may result in increased host range of H5N1.

The highly pathogenic avian influenza H5N1 virus clade 2.3.4.4b 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 2.3.4.4b. 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 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 of a widespread H5N1 epidemic or pandemic. Clade 2.3.4.4b highly pathogenic H5N1 is currently causing a panzootic and has the potential to become a pandemic. Here, Peña Alzua and colleagues develop specific monoclonal antibodies against this virus that could be used to prevent or treat human infections.

Influenza viruses evolve rapidly, driving seasonal epidemics and posing global pandemic threats. While neuraminidase (NA) has emerged as a vaccine target, shared molecular features of NA antibody responses are still not well understood. Here, we describe cryo-electron microscopy structures of the broadly protective human antibody DA03E17, which was previously identified from an H1N1-infected donor, in complex with NA from A/H1N1, A/H3N2, and B/Victoria-lineage viruses. DA03E17 targets the highly conserved NA active site using its long CDR H3, which features a DR (Asp–Arg) motif that engages catalytic residues and mimics sialic acid interactions. We further demonstrate that this motif is conserved among several NA active site-targeting antibodies, indicating a common receptor mimicry strategy. We also identified BCR sequences containing this DR motif across all donors in a healthy human repertoire database, suggesting that such precursors may be relatively common and have vaccine targeting potential. Our findings reveal shared molecular features in NA active site-targeting antibodies that can be harnessed to design broad, immune-focused influenza vaccines. Recurrent features in human antibodies targeting the influenza neuraminidase active site reveal a convergent strategy of receptor mimicry, providing structural insights that could guide the design of broad and effective influenza vaccines.

The continuous evolution and global spread of highly pathogenic avian influenza (HPAI) H5N1 viruses, particularly clade 2.3.4.4b, pose major challenges for pandemic preparedness. This study evaluates a low-dose inactivated split-virus vaccine derived from H5N1 clade 2.3.4.4b, formulated with an Alum/CpG adjuvant, in a preclinical female mouse model. The vaccine induces strong humoral and cellular immunity, generating high titers of cross-reactive antibodies against diverse H5 hemagglutinin (HA) and across different N1 neuraminidase (NA) glycoproteins. The Alum/CpG adjuvant supports substantial antigen dose sparing and promotes a balanced Th1/Th2 profile. Functional assays show potent virus neutralization, neuraminidase inhibition, and antibody-dependent cellular cytotoxicity, alongside robust antigen-specific CD4 + and CD8 + T cell responses, efficient control of lung viral replication, and reduced lung inflammation. Vaccinated mice are fully protected from lethal challenge with both homologous H5N1 clade 2.3.4.4b and heterologous clade 1 viruses, despite low hemagglutination inhibition (HAI) titers. Electron microscopy polyclonal epitope mapping shows serum antibodies recognizing multiple epitopes on homologous HA and NA, with cross-reactivity to conserved epitopes on heterologous proteins, indicating broad recognition. Together, these findings support this vaccine candidate as a promising strategy to provide broad, multifunctional, and durable immunity against current and emerging H5N1 threats. Highly pathogenic avian influenza H5N1 viruses are of global concern. This study shows that a low-dose H5N1 clade 2.3.4.4b Alum/CpG-adjuvanted vaccine elicits broad, durable antibody and T cell responses and protects female mice against lethal homologous and heterologous H5N1 challenges.

Seasonal influenza viruses cause hundreds of thousands of deaths each year and up to a billion infections; under the proper circumstances, influenza A viruses with pandemic potential could threaten the lives of millions more. The variable efficacies of traditional influenza virus vaccines and the desire to prevent pandemic influenzas have motivated work toward finding a universal flu vaccine. Many promising universal flu vaccine candidates currently focus on guiding immune responses to highly conserved epitopes on the central stem of the influenza hemagglutinin viral fusion protein. To support the further development of these stem-targeting vaccine candidates, in this study, we use negative stain electron microscopy to assess the prevalence of central stem-targeting antibodies in individuals who were exposed to influenza antigens through traditional vaccination and/or natural infection during the 2018–2019 flu season.

The continuous evolution and widespread dissemination of highly pathogenic avian influenza (HPAI) H5N1 viruses, particularly clade 2.3.4.4b, pose critical challenges to global pandemic preparedness. In this study, we assessed a low-dose inactivated split virus vaccine derived from clade 2.3.4.4b H5N1, formulated with an Alum/CpG adjuvant, using a preclinical mouse model. This vaccine induced potent humoral and cellular immune responses, generating high titers of cross-reactive antibodies targeting both hemagglutinin (HA) and neuraminidase (NA) glycoproteins across homologous and heterologous H5 clades. The Alum/CpG adjuvant enabled significant antigen dose-sparing while promoting a balanced Th1/Th2 immune profile. Functional analyses demonstrated strong virus neutralization, neuraminidase inhibition, and potent antibody-dependent cellular cytotoxicity activity. Additionally, the vaccine elicited robust antigen-specific CD4 and CD8 T cell responses and effectively controlled viral replication in the lungs, accompanied by reduced lung inflammation. Importantly, vaccinated mice were fully protected against lethal challenges with both the homologous clade 2.3.4.4b and heterologous clade 1 H5N1 viruses, despite low hemagglutination inhibition titers. Electron microscopy polyclonal epitope mapping revealed serum antibodies targeting multiple epitopes on homologous HA and NA, with some cross-reacting to conserved epitopes on heterologous proteins, underscoring broad immune recognition. Collectively, these results highlight the potential of this vaccine candidate to provide broad, multifunctional, and durable immunity against both current and emerging H5N1 threats, supporting its further development for pandemic preparedness.

The regular emergence of influenza strains with pandemic potential creates a strong incentive to develop vaccines that stimulate protective responses across all human populations. A critical consideration is how variation in the human immunoglobulin (IG) loci influences B cell recognition of viral epitopes and elicitation of neutralizing antibodies. Here, we applied personalized IG germline genotyping and high-throughput sequencing of paired antibody chains from influenza A virus hemagglutinin (HA)-binding B cells to demonstrate that the response to HA is highly individual. We show that a germline-encoded polymorphism in IGHV2-70 alters the functionality of the LPAF-a class of neutralizing antibodies, and we describe HA stem-targeting broadly neutralizing antibodies (bNAbs) that use germline IGHV genes other than the population-restricted IGHV genes used by many previously known stem bNAbs. Our results demonstrate that the approach used here can be used to discover and avert population vulnerabilities arising from IG gene variation when designing HA-based influenza vaccines aimed for the global human population.

Human B cell immunity to the influenza hemagglutinin (HA) stem region, a universal influenza vaccine target, is often stereotyped and immunogenetically restricted, posing challenges for study outside humans. Here, we show that macaques vaccinated with a HA stem immunogen elicit human-like public B cell lineages targeting two major conserved sites of vulnerability, the central stem and anchor epitopes. Central stem antibodies were predominantly derived from V 1-138, the macaque homolog of human V 1-69, a V -gene preferentially used in human central stem broadly neutralizing antibodies (bnAbs). Similarly, macaques produced anchor bnAbs with the human-like NWP motif. Both bnAb lineages were functionally and structurally analogous to their human counterparts, with recognition mediated largely by germline-encoded motifs. Thus the macaque immunoglobulin repertoire supports human-like public bnAb responses to influenza HA. Moreover, this underscores the utility of homologous germline-encoded immunity, suggesting that immune repertoires of macaques and humans may have been similarly shaped during evolution.

Cellular and molecular characterization of immune responses elicited by influenza virus infection and seasonal vaccination have informed efforts to improve vaccine efficacy, breadth, and longevity. Here, we use negative stain electron microscopy polyclonal epitope mapping (nsEMPEM) to structurally characterize the humoral IgG antibody responses to hemagglutinin (HA) from human patients vaccinated with a seasonal quadrivalent flu vaccine or infected with influenza A viruses. Our data show that both vaccinated and infected patients had humoral IgGs targeting highly conserved regions on both H1 and H3 subtype HAs, including the stem and anchor, which are targets for universal influenza vaccine design. Responses against H1 predominantly targeted the central stem epitope in infected patients and vaccinated donors, whereas head epitopes were more prominently targeted on H3. Responses against H3 were less abundant, but a greater diversity of H3 epitopes were targeted relative to H1. While our analysis is limited by sample size, on average, vaccinated donors responded to a greater diversity of epitopes on both H1 and H3 than infected patients. These data establish a baseline for assessing polyclonal antibody responses in vaccination and infection, providing context for future vaccine trials and emphasizing the importance of carefully designing vaccines to boost protective responses towards conserved epitopes.

Influenza A virus subtype H2N2, which caused the 1957 influenza pandemic, remains a global threat. A recent phase I clinical trial investigating a ferritin nanoparticle displaying H2 hemagglutinin in H2-naïve and H2-exposed adults. Therefore, we could perform comprehensive structural and biochemical characterization of immune memory on the breadth and diversity of the polyclonal serum antibody response elicited after H2 vaccination. We temporally map the epitopes targeted by serum antibodies after first and second vaccinations and show previous H2 exposure results in higher responses to the variable head domain of hemagglutinin while initial responses in H2-naïve participants are dominated by antibodies targeting conserved epitopes. We use cryo-EM and monoclonal B cell isolation to describe the molecular details of cross-reactive antibodies targeting conserved epitopes on the hemagglutinin head including the receptor binding site and a new site of vulnerability deemed the medial junction. Our findings accentuate the impact of pre-existing influenza exposure on serum antibody responses.