Influenza Virus Hemagglutinin Research Articles

The Effects of Viral Structural Proteins on Acidic Phospholipids in Host Membranes

Enveloped viruses rely on host membranes for trafficking and assembly. A substantial body of literature published over the years supports the involvement of cellular membrane lipids in the enveloped virus assembly processes. In particular, the knowledge regarding the relationship between viral structural proteins and acidic phospholipids has been steadily increasing in recent years. In this review, we will briefly review the cellular functions of plasma membrane-associated acidic phospholipids and the mechanisms that regulate their local distribution within this membrane. We will then explore the interplay between viruses and the plasma membrane acidic phospholipids in the context of the assembly process for two enveloped viruses, the influenza A virus (IAV) and the human immunodeficiency virus type 1 (HIV-1). Among the proteins encoded by these viruses, three viral structural proteins, IAV hemagglutinin (HA), IAV matrix protein-1 (M1), and HIV-1 Gag protein, are known to interact with acidic phospholipids, phosphatidylserine and/or phosphatidylinositol (4,5)-bisphosphate. These interactions regulate the localization of the viral proteins to and/or within the plasma membrane and likely facilitate the clustering of the proteins. On the other hand, these viral proteins, via their ability to multimerize, can also alter the distribution of the lipids and may induce acidic-lipid-enriched membrane domains. We will discuss the potential significance of these interactions in the virus assembly process and the property of the progeny virions. Finally, we will outline key outstanding questions that need to be answered for a better understanding of the relationships between enveloped virus assembly and acidic phospholipids.

Features of influenza virus hemagglutinin genes and their recoding possibilities

The world has already entered the stage of increasing odds for a new pandemic, which prompts to seek out for new flu vaccines, because existing vaccines demonstrate only suboptimal effectiveness. With the Covid-19 pandemic, the possibility of using mRNA vaccines has been opened up, and a prospect of finding hemagglutinin (HA) gene mRNA-based new influenza vaccines seems very attractive. As a rule, the mRNA vaccine is a product of recoding, which ensures the mRNA stability. However, the results of mRNA recoding can be ambiguous. The purpose of this report is to analyze the features of genes and proteins and to consider opportunities and limitations in their recoding. Primary structures of NA proteins and relevant genes were retrieved from Internet publicly available databases. The amino acid composition and frequency of dipeptides, nucleotide and dinucleotide compositions, %GC, translational code and compositions of neighboring di- and tricodones, distribution along primary structure for explicit and synonymous mutations were determined. H1N1 and H3N2 subtypes have both specific and general features (limitations) in their genes, differing not only in the number of protein substitutions, but also in the number and distribution of gene synonymous codons, which do not manifest in the protein primary structure, but appear, apparently, as a hidden factor, which causes the low effectiveness of classical influenza vaccines. The identification of several limitations in gene structure suggests that its any modification (in any gene) must not contradict each of the restrictions established by nature. The frequency of CpG dinucleotides in all studied strains is low, but a potential for optimizing it in H1N1 strains due to the prohibition of the quartet in the gene for arginine-encoding codons is especially limited and can be implemented through synonymous codons of other amino acids (alanine, proline, threonine or serine). Compared to the H1N1 subtype, the H3N2 subtype can be expected to have more possibilities in constructing stable NA gene mRNA.

Multivalent interactions between fully glycosylated influenza virus hemagglutinins mediated by glycans at distinct N-glycosylation sites

The hemagglutinin (HA) glycoprotein of influenza virus binds host cell receptors and mediates viral entry. Here we present cryo-EM structures of fully glycosylated HAs from H5N1 and H5N8 influenza viruses. We find that the H5N1 HA can form filaments that comprise two head-to-head HA trimers. Multivalent interactions between the two HA trimers are mediated by glycans attached to N158. The distal Sia1-Gal2-NAG3 sugar moiety of N158 interacts with the receptor binding site on the opposing HA trimer. Additional interactions are observed between NAG3 and residues K222 and K193. The H5N8 HA lacks the N158 glycosylation site and does not form the filamentous structure. However, the H5N8 HA exhibits an auto-inhibition conformation, where the receptor binding site is occupied by the glycan chain attached to residue N169 from a neighboring protomer. These structures represent native HA-glycan interactions, which may closely mimic the receptor-HA interactions on the cell surface.

Thin-film freeze-drying of an influenza virus hemagglutinin mRNA vaccine in unilamellar lipid nanoparticles with blebs

Messenger RNA (mRNA) vaccines have revolutionized the fight against infectious diseases and are poised to transform other therapeutic areas. Lipid nanoparticles (LNP) represent the most successful delivery system for mRNA. While the mRNA-LNP products currently in clinics are stored as frozen suspensions, there is evidence that freeze-drying mRNA-LNP into dry powders can potentially enable their storage and handling at non-freezing temperatures. Previously, we successfully applied thin-film freeze-drying (TFFD) to transform a polyadenylic acid [poly(A)]-LNP formulation from a liquid suspension to dry powders. The poly(A)-LNP were structurally multilamellar spheres without blebs, but the mRNA vaccines in clinics are comprised of mRNA-LNP that are structurally spheres surrounded by a unilamellar lipid bilayer, with some containing blebs, and it was reported that the presence of blebs increases the sensitivity of mRNA-LNP to freeze-drying-induced stress. In the present study, using an influenza A virus hemagglutinin (HA) mRNA in LNP that were structurally similar to that in the COVID-19 mRNA vaccines currently in clinic, we studied the effect of TFFD on the physical properties, internal structure, as well as immunogenicity of the HA mRNA-LNP vaccine. We concluded that TFFD can be utilized to prepare dry powders of the HA mRNA-LNP, but a sufficient amount of excipients were needed to minimize changes in the physical properties, structure, and immunogenicity of the HA mRNA-LNP vaccine.

Preparation of a thioxoimidazolidin-linked sialoside BSA conjugate for the inhibition of influenza virus

A novel thioxoimidazolidin-linked sialoside bovine serum albumin (WM-BSA) conjugate was synthesized and evaluated as an inhibitor of influenza virus hemagglutinin (HA) and neuraminidase (NA). The multivalent conjugate was prepared by the attachment of thioxoimidazolidin-sialoside monomer (WM) to BSA via adipate linker. Surface plasmon resonance analysis revealed that WM-BSA exhibited potent binding to recombinant influenza HA and NA proteins, with dissociation constants in the submicromolar range. WM-BSA also demonstrated inhibitory activities in the low micromolar range against HA and NA proteins from multiple influenza strains. Investigation of cytopathic effects in infected MDCK cells indicated that WM-BSA possessed antiviral activity with EC50 values of 35–55 μM. The multivalent presentation of sialosides on the BSA scaffold significantly enhanced both the binding affinity and degree of inhibition compared to the monomeric compound WM. These results demonstrate the potential of multivalent sialoside-protein conjugate as a platform for developing novel anti-influenza agent.

Antiviral activity of the water extract and ethanol extract of Sorbus commixta against influenza A virus in vitro

Although vaccines and antivirals are currently available, influenza virus infections continually present major threats to human health. Due to genetic diversity and variability of influenza viruses, the development of new antiviral agents against the virus remains as a considerable challenge. In this study, we evaluated the antiviral activity of the water extract and ethanol extract of Sorbus commixta Hedl. (SCH), widely used as a medical herb, against influenza A viruses and identified molecular mechanisms for the antiviral activity. The water extract and ethanol extract of SCH demonstrated virucidal activity against influenza A virus at the noncytotoxic concentrations. In addition, cytopathic effect reduction assay and GFP fluorescence image analysis suggest that SCH extracts have inhibitory activity on multiple stages during influenza virus life cycle. Mechanisms studies showed that SCH extracts inhibited the biological functions of influenza viral surface hemagglutinin and neuraminidase proteins that play critical roles in the viral entry and release steps. SCH extracts not only prevented the receptor binding of influenza viral HA to the cellular receptors but also inhibited the HA-mediated membrane fusion. In addition, SCH extracts suppressed the enzyme activity of the viral neuraminidase. The results together suggest that SCH extracts contain antiviral natural compounds that inhibit multiple influenza viral proteins including the viral surface proteins. Our findings suggest that SCH extracts could be promising resources for the development of novel antiviral agents against influenza A viruses.

Hemagglutinin and neuraminidase of a non-pathogenic H7N7 avian influenza virus coevolved during the acquisition of intranasal pathogenicity in chickens.

Polybasic amino acid residues at the hemagglutinin (HA) cleavage site are insufficient to induce the highly pathogenic phenotype of avian influenza viruses in chickens. In our previous study, an H7N7 avian influenza virus named "Vac2sub-P0", which is nonpathogenic despite carrying polybasic amino acids at the HA cleavage site, was passaged in chick air sacs, and a virus with high intravenous pathogenicity, Vac2sub-P3, was obtained. Intranasal infection with Vac2sub-P3 resulted in limited lethality in chickens; therefore, in this study, this virus was further passaged in chicken lungs, and the resultant virus, Vac2sub-P3L4, acquired high intranasal pathogenicity. Experimental infection of chickens with recombinant viruses demonstrated that mutations in HA and neuraminidase (NA) found in consecutive passages were responsible for the increased pathogenicity. The HA and NA functions of Vac2sub-P3L4 were compared with those of the parental virus in vitro; the virus growth at 40 °C was faster, the binding affinity to a sialic acid receptor was lower, and the rate of release by NA from the cell surface was lower, suggesting that these changes enabled the virus to replicate efficiently in chickens with high intranasal pathogenicity. This study demonstrates that viruses that are highly pathogenic when administered intranasally require additional adaptations for increased pathogenicity to be highly lethal to intranasally infected chickens.

Co-production of hemagglutinin H9N2 influenza virus and fusion protein Newcastle virus in insect cell using baculovirus expression system.

Influenza and Newcastle disease are the most important poultry diseases that cause high annual damage to poultry farms worldwide. Newcastle virus fusion (F) gene and Influenza Virus Hemagglutinin (HA) gene are capable of encoding F and HA proteins that are the main factors in creating immunity, so this study aimed to clone and express these genes in Spodoptera frugiperda (Sf9) cells using baculovirus expression system. After isolating the Newcastle and Influenza virus genome, the HA gene of influenza virus and the F gene of Newcastle virus were amplified by reverse transcriptase PCR and specific primers and then cloned into pFastBacTM Dual plasmid. A recombinant sucker with these genes was produced in the DH10Bac host cell. By transfecting Sf9 cells with recombinant bacmid, expression was assessed by SDS-PAGE, western blotting, and Bradford methods. Cloning of genes into the bacmid was successful. By transfecting the recombinant bacmid into Spodoptera frugiperda cells, 218 µg/ml of the recombinant protein was obtained in the supernatant. In addition, the presence of protein was confirmed by western blotting. The PCR products of HA and F genes showed one band of 1.7 kb size using specific primers. The pFastHA1 vector was about 7 kb in size. Two bands of about 7 kb and 1.7 kb were created by ligation of the F gene and pFastHA1 vector based on enzymatic digestion, indicating the correct ligation of F gene under the P10 promoter. This is the first report on the cloning and Co-expression of two HA and F genes using baculovirus expression system and can be a candidate for dual influenza and Newcastle vaccine. Mixtures of these recombinant proteins can be used as vaccine candidates against both avian influenza and Newcastle disease.

The Hemagglutinin of Influenza A Virus Induces Ferroptosis to Facilitate Viral Replication.

Ferroptosis is a novel form of cell death caused by the accumulation of lipid peroxides in an iron-dependent manner. However, the precise mechanism underlying the exploitation of ferroptosis by influenza A viruses (IAV) remains unclear. The results demonstrate that IAV promotes its own replication through ferritinophagy by sensitizing cells to ferroptosis, with hemagglutinin identified as a key trigger in this process. Hemagglutinin interacts with autophagic receptors nuclear receptor coactivator 4 (NCOA4)and tax1-binding protein 1 (TAX1BP1), facilitating the formation of ferritin-NCOA4 condensates and inducing ferritinophagy. Further investigation shows that hemagglutinin-induced ferritinophagy causes cellular lipid peroxidation, inhibits aggregation of mitochondrial antiviral signaling protein (MAVS), and suppresses the type I interferon response, thereby contributing to viral replication. Collectively, a novel mechanism by which IAV hemagglutinin induces ferritinophagy resulting in cellular lipid peroxidation, consequently impairing MAVS-mediated antiviral immunity, is revealed.

Dual neutralization of influenza virus hemagglutinin and neuraminidase by a bispecific antibody leads to improved antiviral activity

Targeting multiple viral proteins is pivotal for sustained suppression of highly mutable viruses. In recent years, broadly neutralizing antibodies that target the influenza virus hemagglutinin and neuraminidase glycoproteins have been developed, and antibody monotherapy has been tested in preclinical and clinical studies to treat or prevent influenza virus infection. However, the impact of dual neutralization of the hemagglutinin and neuraminidase on the course of infection, as well as its therapeutic potential, has not been thoroughly tested. For this purpose, we generated a bispecific antibody that neutralizes both the hemagglutinin and the neuraminidase of influenza viruses. We demonstrated that this bispecific antibody has a dual antiviral activity as it blocks infection and prevents the release of progeny viruses from the infected cells. We show that dual neutralization of the hemagglutinin and the neuraminidase by a bispecific antibody is advantageous over monoclonal antibody combination as it resulted an improved neutralization capacity and augmented the antibody effector functions. Notably, the bispecific antibody showed enhanced antiviral activity in influenza virus-infected mice, reduced mice mortality and limited the virus mutation profile upon antibody administration. Thus, dual neutralization of the hemagglutinin and neuraminidase could be effective in controlling influenza virus infection.

Broadly protective bispecific antibodies that simultaneously target influenza virus hemagglutinin and neuraminidase.

Infection by the influenza virus remains a global health burden. The approaches utilized here to augment the activity of broadly protective influenza virus antibodies may lead to a new class of immunotherapies with enhanced activity.

A small molecule compound targeting hemagglutinin inhibits influenza A virus and exhibits broad-spectrum antiviral activity.

Influenza A virus (IAV) is a widespread pathogen that poses a significant threat to human health, causing pandemics with high mortality and pathogenicity. Given the emergence of increasingly drug-resistant strains of IAV, currently available antiviral drugs have been reported to be inadequate to meet clinical demands. Therefore, continuous exploration of safe, effective and broad-spectrum antiviral medications is urgently required. Here, we found that the small molecule compound J1 exhibited low toxicity both in vitro and in vivo. Moreover, J1 exhibits broad-spectrum antiviral activity against enveloped viruses, including IAV, respiratory syncytial virus (RSV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human coronavirus OC43 (HCoV-OC43), herpes simplex virus type 1 (HSV-1) and HSV-2. In this study, we explored the inhibitory effects and mechanism of action of J1 on IAV in vivo and in vitro. The results showed that J1 inhibited infection by IAV strains, including H1N1, H7N9, H5N1 and H3N2, as well as by oseltamivir-resistant strains. Mechanistic studies have shown that J1 blocks IAV infection mainly through specific interactions with the influenza virus hemagglutinin HA2 subunit, thereby blocking membrane fusion. BALB/c mice were used to establish a model of acute lung injury (ALI) induced by IAV. Treatment with J1 increased survival rates and reduced viral titers, lung index and lung inflammatory damage in virus-infected mice. In conclusion, J1 possesses significant anti-IAV effects in vitro and in vivo, providing insights into the development of broad-spectrum antivirals against future pandemics.

Occurrence of Circulating Antibodies against the Hemagglutinins of Influenza Viruses in the 2022/2023 Epidemic Season in Poland.

The aim of this study was to determine the level of anti-hemagglutinin antibodies in blood sera collected from patients during the 2022/2023 epidemic season in Poland. A total of 700 sera samples from patients across the country were tested. The samples were divided into seven groups according to the age of the patients, with 100 samples from each age group. The hemagglutination inhibition test (OZHA) was used to determine the level of anti-hemagglutinin antibodies. The test results have confirmed the presence of anti-hemagglutinin antibodies for antigens A/Victoria/2570/2019 (H1N1)pdm09, A/Darwin/9/2021 (H3N2), B/Austria/1359417/2021 (B/Yamagata lineage) and B/ Phuket/3073/2013 (B/Victoria lineage) present in the influenza vaccine recommended by the World Health Organization (WHO) for the 2022/2023 epidemic season. The highest geometric mean antibody titres (GMT) and protection rate values (%) were recorded for hemagglutinin A/H3N2. In Poland, in the 2022/2023 epidemic season, the percentage of the population vaccinated against influenza was 5.7%. Therefore, the test results can be interpreted as the response of the immune system in patients who have been previously infected with an influenza virus.

Identification of broad-spectrum B-cell and T-cell epitopes of H9 subtype avian influenza virus HA protein using polypeptide scanning1

The H9N2 subtype avian influenza virus (AIV) hemagglutinin (HA) protein is a major immunogen in which HA1 is a genetic variant and HA2 is relatively conserved. Identifying broad-spectrum antigen epitopes targeting HA1 is crucial for vaccine design and detection. Based on the phylogenetic and serological analyses, we identified 2 antigenic groups and 3 representative viruses: A/chicken/Jiangsu/JY040218C/2019, A/pigeon/Jiangsu/JY020616/2019, and A/chicken/Jiangsu/WX090312/2018. An overlapping peptide library was synthesized using HA1 amino acid sequences of the viruses as templates. Through peptide scanning of the sera against different strains of H9N2 subtype AIV, we identified peptides from 4 regions (H9-2/3, H9-20/21, H9-26, and H9-29/30/31) that demonstrated broad-spectrum reactivity. Immunological assay results demonstrated that H9-21 (219RIFKPLIGPRPLVNGLMGRI239), H9-26 (269SGESHGRILKTDLKMGSCTV289), and H9-30 (309YAFGNCPKYI GVKSLKLAVG329) effectively induced antibody generation and conferred partial protective efficacy against the parent virus JY040218C. The results of lymphocyte proliferation and ELISpot assays indicated that peptides H9-15 (159MRWLTQKNNAYPTQDAQYTN179), H9-22 (229PLVNGLMGRINYYWSVLKP G249), and H9-23 (239NYYWSVLKPGQTLRIKSDGN259) could effectively stimulate the expression of interferon-gamma in peripheral blood lymphocytes of chickens immunized against different strains of H9N2 AIV. Collectively, 5 novel cell epitopes H9-15, H9-22, H9-23, H9-26, and H9-30, including the best B cell epitope H9-26 and the best T cells epitope H9-22, were identified that could be targeted for vaccine design or detection approaches against H9N2 AIVs.

A protective and broadly binding antibody class engages the influenza virus hemagglutinin head at its stem interface.

Influenza infection and vaccination impart strain-specific immunity that protects against neither seasonal antigenic variants nor the next pandemic. However, antibodies directed to conserved sites can confer broad protection. Here we identify and characterize a class of human antibodies that engage a previously undescribed, conserved epitope on the influenza hemagglutinin (HA) protein. Prototype antibody S8V1-157 binds at the normally occluded interface between the HA head and stem. Antibodies to this HA head-stem interface epitope are non-neutralizing in vitro but protect against lethal influenza infection in mice. Antibody isotypes that direct clearance of infected cells enhance this protection. Head-stem interface antibodies bind to most influenza A serotypes and seasonal human variants, and are present at low frequencies in the memory B cell populations of multiple human donors. Vaccines designed to elicit these antibodies might contribute to "universal" influenza immunity.

Molecular epidemiology and genetic evolution of avian influenza H5N1 subtype in Nigeria, 2006 to 2021.

Nigeria recorded one of the earliest outbreaks of the Highly Pathogenic Avian Influenza (HPAI) virus H5N1 in 2006, which spread to other African countries. In 2023, 18 countries reported outbreaks of H5N1 in poultry, with human cases documented in Egypt, Nigeria, and Djibouti. There is limited information on the molecular epidemiology of HPAI H5N1 in Nigeria. We determined the molecular epidemiology and genetic evolution of the virus from 2006 to 2021. We investigated the trend and geographical distribution across Nigeria. The evolutionary history of 61 full-length genomes was performed from 13 countries worldwide, and compared with sequences obtained from the early outbreaks in Nigeria up to 2021. MEGA 11 was used to determine the phylogenetic relationships of H5N1 strains, which revealed close ancestry between sequences in Nigeria and those from other African countries. Clade classification was performed using the subspecies classification tool for Bacterial and Viral Bioinformatics Research Center (BV-BRC) version 3.35.5. H5N1 Clade 2.2 was observed in 2006, with 2.3.2, 2.3.2.1f clades observed afterwards and 2.3.4.4b in 2021. Our findings underscore the need for genomics surveillance to track antigenic variation and clades switching to monitor the epidemiological of the virus and safeguard human and animal health.Impacts Specific variations in the hemagglutinin (HA) and neuraminidase (NA) genes of Avian influenza virus are consistent in different geographical regions. H5N1 Clade 2.2 was reported in 2006, with 2.3.2, 2.3.2.1f afterwards and 2.3.4.4b in 2021. Nigeria is an epicentre for avian influenza with three major migratory routes for wild birds transversing the country. It is plausible that the Avian influenza in Northern Nigeria may be linked to wild bird sanctuaries in the region.

H19 influenza A virus exhibits species-specific MHC class II receptor usage

Avian influenza A virus (IAV) surveillance in Northern California, USA, revealed unique IAV hemagglutinin (HA) genome sequences in cloacal swabs from lesser scaups. We found two closely related HA sequences in the same duck species in 2010 and 2013. Phylogenetic analyses suggest that both sequences belong to the recently discovered H19 subtype, which thus far has remained uncharacterized. We demonstrate that H19 does not bind the canonical IAV receptor sialic acid (Sia). Instead, H19 binds to the major histocompatibility complex class II (MHC class II), which facilitates viral entry. Unlike the broad MHC class II specificity of H17 and H18 from bat IAV, H19 exhibits a species-specific MHC class II usage that suggests a limited host range and zoonotic potential. Using cell lines overexpressing MHC class II, we rescued recombinant H19 IAV. We solved the H19 crystal structure and identified residues within the putative Sia receptor binding site (RBS) that impede Sia-dependent entry.

Influenza virus uses mGluR2 as an endocytic receptor to enter cells

Influenza virus infection is initiated by the attachment of the viral haemagglutinin (HA) protein to sialic acid receptors on the host cell surface. Most virus particles enter cells through clathrin-mediated endocytosis (CME). However, it is unclear how viral binding signals are transmitted through the plasma membrane triggering CME. Here we found that metabotropic glutamate receptor subtype 2 (mGluR2) and potassium calcium-activated channel subfamily M alpha 1 (KCa1.1) are involved in the initiation and completion of CME of influenza virus using an siRNA screen approach. Influenza virus HA directly interacted with mGluR2 and used it as an endocytic receptor to initiate CME. mGluR2 interacted and activated KCa1.1, leading to polymerization of F-actin, maturation of clathrin-coated pits and completion of the CME of influenza virus. Importantly, mGluR2-knockout mice were significantly more resistant to different influenza subtypes than the wild type. Therefore, blocking HA and mGluR2 interaction could be a promising host-directed antiviral strategy.

Virion-surface display of a chimeric immunoglobulin Fc domain facilitating uptake by antigen-presenting cells

Antigen-presenting cells (APCs) play an important role in virus infection control by bridging innate and adaptive immune responses. Macrophages and dendritic cells (DCs) possess various surface receptors to recognize/internalize antigens, and antibody binding can enhance pathogen-opsonizing uptake by these APCs via interaction of antibody fragment crystallizable (Fc) domains with Fc receptors, evoking profound pathogen control in certain settings. Here, we examined phagocytosis-enhancing potential of Fc domains directly oriented on a retroviral virion/virus-like particle (VLP) surface. We generated an expression vector coding a murine Fc fragment fused to the transmembrane region (TM) of a retroviral envelope protein, deriving expression of the Fc-TM fusion protein on the transfected cell surface and production of virions incorporating the chimeric Fc upon co-transfection. Incubation of Fc-displaying simian immunodeficiency virus (SIV) with murine J774 macrophages and bone marrow-derived DCs derived Fc receptor-dependent enhanced uptake, being visualized by imaging cytometry. Alternative preparation of a murine leukemia virus (MLV) backbone-based Fc-displaying VLP loading an influenza virus hemagglutinin (HA) antigen resulted in enhanced HA internalization by macrophages, stating antigen compatibility of the design. Results show that the Fc-TM fusion molecule can be displayed on certain viruses/VLPs and may be utilized as a molecular adjuvant to facilitate APC antigen uptake.

Preparation and characterization of mouse-derived monoclonal antibodies against the hemagglutinin of the H1N1 influenza virus

H1N1 influenza virus is a significant global public health concern. Monoclonal antibodies (mAbs) targeting specific viral proteins such as hemagglutinin (HA) have become an important therapeutic strategy, offering highly specific targeting to block viral transmission and infection. This study focused on the development of mAbs targeting HA of the A/Victoria/2570/2019 (H1N1pdm09, VIC-19) strain by utilizing hybridoma technology to produce two mAbs with high binding capacity. Notably, mAb 2B2 has demonstrated a strong affinity for HA proteins in recent H1N1 influenza vaccine strains. In vitro assessments showed that both mAbs exhibited broad-spectrum hemagglutination inhibition and potent neutralizing effects against various vaccine strains of H1N1pdm09 viruses. 2B2 was also effective in animal models, offering both preventive and therapeutic protection against infections caused by recent H1N1 strains, highlighting its potential for clinical application. By individually co-cultivating each of the aforementioned mAbs with the virus in chicken embryos, four amino acid substitution sites in HA (H138Q, G140R, A141E/V, and D187E) were identified in escape mutants, three in the antigenic site Ca2, and one in Sb. The identification of such mutations is pivotal, as it compels further investigation into how these alterations could undermine the binding efficacy and neutralization capacity of antibodies, thereby impacting the design and optimization of mAb therapies and influenza vaccines. This research highlights the necessity for continuous exploration into the dynamic interaction between viral evolution and antibody response, which is vital for the formulation of robust therapeutic and preventive strategies against influenza.