Hemagglutinin Head Research Articles

Conserved sites on the influenza H1 and H3 hemagglutinin recognized by human antibodies.

Monoclonal antibodies (mAbs) targeting the influenza hemagglutinin (HA) have the potential to be used as prophylactics or templates for next-generation vaccines that provide broad protection. Here, we isolated broad, subtype-neutralizing mAbs from human B cells targeting the H1 or H3 HA head as well as a unique mAb targeting the stem. The H1 mAbs target the previously defined lateral patch epitope on H1 HAs and recognize HAs from 1933 to 2021 in addition to a swine H1N1 virus with pandemic potential. Using directed evolution, we improved the neutralization potency of these H1 mAbs towards a contemporary H1 strain. Using deep mutational scanning of four antigenically distinct H1N1 viruses, we identified potential viral escape pathways. For the H3 mAbs we used cryo-EM to define the targeted epitopes: one mAb recognizes the side of the H3 head, accommodating the N133 glycan and a pocket underneath the receptor binding site. The other H3 mAb recognizes an epitope in the HA stem that overlaps with previously characterized mAbs, but with distinct antibody variable genes and mode of recognition. Collectively, these mAbs identify common sites recognized by broad, subtype-specific mAbs that may be elicited by next-generation vaccines.

Headless hemagglutinin-containing influenza viral particles direct immune responses toward more conserved epitopes.

Seasonal influenza vaccines provide mostly strain-specific protection due to the elicitation of antibody responses focused on evolutionarily plastic antigenic sites in the hemagglutinin head domain. To direct the humoral response toward more conserved epitopes, we generated an influenza virus particle where the full-length hemagglutinin protein was replaced with a membrane-anchored, "headless" variant while retaining the normal complement of other viral structural proteins such as the neuraminidase as well as viral RNAs. We found that a single administration of a headless virus particle-based vaccine elicited high titers of antibodies that recognized more conserved epitopes on the major viral glycoproteins. Furthermore, the vaccine could elicit these responses even in the presence of pre-existing, hemagglutinin (HA) head-focused influenza immunity. Importantly, these antibody responses mediated protective, but non-neutralizing functions such as neuraminidase inhibition and antibody-dependent cellular cytotoxicity. Additionally, we show the vaccine can provide protection from homologous and heterologous challenges in mouse models of severe influenza without any measurable HA head-directed antibody responses. Thus, headless hemagglutinin containing viral particles may represent a tool to drive the types of antibody responses predicted to increase influenza vaccine breadth and durability.IMPORTANCECurrent seasonal influenza vaccines provide incomplete protection from disease. This is partially the result of the antibody response being directed toward parts of the virus that are tolerant of mutations. Redirecting the immune response to more conserved regions of the virus has been a central strategy of next-generation vaccine designs and approaches. Here, we develop and test a vaccine based on a modified influenza virus particle that expresses a partially deleted hemagglutinin protein along with the other viral structural proteins. We demonstrate this vaccine elicits antibodies that recognize the more conserved viral epitopes of the hemagglutinin stalk and neuraminidase protein to facilitate protection against influenza viruses despite a lack of classical viral neutralization activity.

Phase 1 dose-escalation trial evaluating a group 2 influenza hemagglutinin stabilized stem nanoparticle vaccine

The relative conservation of the influenza hemagglutinin (HA) stem compared to that of the immunodominant HA head makes the HA stem an attractive target for broadly protective influenza vaccines. Here we report the first-in-human, dose-escalation, open-label trial (NCT04579250) evaluating an unadjuvanted group 2 stabilized stem ferritin nanoparticle vaccine based on the H10 A/Jiangxi-Donghu/346/2013 influenza HA, H10ssF, in healthy adults. Participants received a single 20 mcg dose (n = 3) or two 60 mcg doses 16 weeks apart (n = 22). Vaccination with H10ssF was safe and well tolerated with only mild systemic and local reactogenicity reported. No serious adverse events occurred. Vaccination significantly increased homologous H10 HA stem binding and neutralizing antibodies at 2 weeks after both first and second vaccinations, and these responses remained above baseline at 40 weeks. Heterologous H3 and H7 binding antibodies also significantly increased after each vaccination and remained elevated throughout the study. These data indicate that the group 2 HA stem nanoparticle vaccine is safe and induces stem-directed binding and neutralizing antibodies.

An explainable language model for antibody specificity prediction using curated influenza hemagglutinin antibodies

Despite decades of antibody research, it remains challenging to predict the specificity of an antibody solely based on its sequence. Two major obstacles are the lack of appropriate models and the inaccessibility of datasets for model training. In this study, we curated >5,000 influenza hemagglutinin (HA) antibodies by mining research publications and patents, which revealed many distinct sequence features between antibodies to HA head and stem domains. We then leveraged this dataset to develop a lightweight memory B cell language model (mBLM) for sequence-based antibody specificity prediction. Model explainability analysis showed that mBLM could identify key sequence features of HA stem antibodies. Additionally, by applying mBLM to HA antibodies with unknown epitopes, we discovered and experimentally validated many HA stem antibodies. Overall, this study not only advances our molecular understanding of the antibody response to the influenza virus but also provides a valuable resource for applying deep learning to antibody research.

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.

N-glycosylation on hemagglutinin head reveals inter-branch antigenic variability of avian influenza virus H5-subtypes

H5-subtype avian influenza virus (AIV) is globally prevalent and undergoes frequent antigenic drift, necessitating regular updates to vaccines. One of the many influencing elements that cause incompatibility between vaccinations and epidemic strains is the dynamic alteration of glycosylation sites. However, the biological significance of N-glycosylation in the viral evolution and antigenic changes is unclear. Here, we performed a systematic analysis of glycosylation sites on the HA1 subunit of H5N1, providing insights into the changes of primary glycosylation sites, including 140 N, 156 N, and 170 N within the antigenic epitopes of HA1 protein. Multiple recombinant viruses were then generated based on HA genes of historical vaccine strains and deactivated for immunizing SPF chickens. Inactivated recombinant strains showed relatively closer antigenicity compared to which has identical N-glycosylation patterns. The N-glycosylation modification discrepancy highlights the inter-branch antigenic diversity of H5-subtype viruses in avian influenza and serves as a vital foundation for improving vaccination tactics.

Vaccination with antigenically complex hemagglutinin mixtures confers broad protection from influenza disease.

Current seasonal influenza virus vaccines induce responses primarily against immunodominant but highly plastic epitopes in the globular head of the hemagglutinin (HA) glycoprotein. Because of viral antigenic drift at these sites, vaccines need to be updated and readministered annually. To increase the breadth of influenza vaccine-mediated protection, we developed an antigenically complex mixture of recombinant HAs designed to redirect immune responses to more conserved domains of the protein. Vaccine-induced antibodies were disproportionally redistributed to the more conserved stalk of the HA without hindering, and in some cases improving, antibody responses against the head domain. These improved responses led to increased protection against homologous and heterologous viral challenges in both mice and ferrets compared with conventional vaccine approaches. Thus, antigenically complex protein mixtures can at least partially overcome HA head domain antigenic immunodominance and may represent a step toward a more universal influenza vaccine.

Antibody Response to Influenza Hemagglutinin Conserved Stalk Domain after Sequential Immunization with Old Vaccine Strains.

Hemagglutinin (HA) is the major envelope glycoprotein and antigen on the surface of influenza virions. The glycoprotein comprises a globular head and a stalk region. While immunodominant epitopes on influenza HA head are highly variable, the stalk domain is conserved. The variability of the HA head causes the antigenic drift that made the requirement of annual update of vaccine strains. Induction of antibody against the stalk domain has been proposed as an approach for a broadly protective influenza vaccine strategy. Sequential exposure to influenza strains with highly diverse HA heads but conserved stalks have been shown to induce antibody to the low immunogenic stalk domain. Here, we tested this approach by using old influenza vaccine strains that are decades apart in evolution. Inactivated whole virion vaccine of influenza A/Puerto Rico/8/1934, A/USSR/92/1977, and A/Thailand/102/2009 (H1N1) was sequentially immunized into BALB/c mice in comparison to immunization using single strain (A/Thailand/102/2009 (H1N1)). The sequentially immunized mice developed higher levels of binding antibody to the stalk domain. These suggested that using old vaccine strains in sequential vaccination may be a possible approach to induce antibody to the conserved stalk domain.

Chimeric hemagglutinin split vaccines elicit broadly cross-reactive antibodies and protection against group 2 influenza viruses in mice.

Seasonal influenza virus vaccines are effective when they are well matched to circulating strains. Because of antigenic drift/change in the immunodominant hemagglutinin (HA) head domain, annual vaccine reformulations are necessary to maintain a match with circulating strains. In addition, seasonal vaccines provide little to no protection against newly emerging pandemic strains. Sequential vaccination with chimeric HA (cHA) constructs has been proven to direct the immune response toward the immunosubdominant but more conserved HA stalk domain. In this study, we show that immunization with group 2 cHA split vaccines in combination with the CpG 1018 adjuvant elicits broadly cross-reactive antibodies against all group 2 HAs, as well as systemic and local antigen-specific T cell responses. Antibodies elicited after sequential vaccination are directed to conserved regions of the HA such as the stalk and the trimer interface and also to the N2 neuraminidase (NA). Immunized mice were fully protected from challenge with a broad panel of influenza A viruses.

Influenza H3 hemagglutinin vaccine with scrambled immunodominant epitopes elicits antibodies directed toward immunosubdominant head epitopes.

Vaccination is the most effective countermeasure to reduce the severity of influenza. Current seasonal influenza vaccines mainly elicit humoral immunity targeting hemagglutinin (HA). In particular, the amino acid residues around the receptor-binding site in the HA head domain are predominantly targeted by humoral immunity as "immunodominant" epitopes. However, mutations readily accumulate in the head domain due to high plasticity, resulting in antigenic drift and vaccine mismatch, particularly with influenza A (H3N2) viruses. A vaccine strategy that targets more conserved immunosubdominant epitopes is required to attain a universal vaccine. Here, we designed an H3 HA vaccine antigen with various amino acids at immunodominant epitopes of the HA head domain, termed scrambled HA (scrHA). In ferrets, scrHA vaccination induced lower serum neutralizing antibody levels against homologous virus compared with wild-type (WT) HA vaccination; however, similar levels of moderately neutralizing titers against antigenically distinct H3N2 viruses were observed. Ferrets vaccinated with scrHA twice and then challenged with homologous or heterologous virus showed the same level of reduced virus shedding in nasal swabs as WT HA-vaccinated animals but reduced body temperature increase, whereas WT HA-vaccinated ferrets exhibited body temperature increases similar to those of mock-vaccinated animals. scrHA elicited antibodies against HA immunodominant and -subdominant epitopes at lower and higher levels, respectively, than WT HA vaccination, whereas antistalk antibodies were induced at the same level for both groups, suggesting scrHA-induced redirection from immunodominant to immunosubdominant head epitopes. scrHA vaccination thus induced broader coverage than WT HA vaccination by diluting out the immunodominancy of HA head epitopes. IMPORTANCE Current influenza vaccines mainly elicit antibodies that target the immunodominant head domain, where strain-specific mutations rapidly accumulate, resulting in frequent antigenic drift and vaccine mismatch. Targeting conserved immunosubdominant epitopes is essential to attain a universal vaccine. Our findings with the scrHA developed in this study suggest that designing vaccine antigens that "dilute out" the immunodominancy of the head epitopes may be an effective strategy to induce conserved immunosubdominant epitope-based immune responses.

Bioprocess development for universal influenza vaccines based on inactivated split chimeric and mosaic hemagglutinin viruses.

Seasonal influenza viruses account for 1 billion infections worldwide every year, including 3-5 million cases of severe illness and up to 650,000 deaths. The effectiveness of current influenza virus vaccines is variable and relies on the immunodominant hemagglutinin (HA) and to a lesser extent on the neuraminidase (NA), the viral surface glycoproteins. Efficient vaccines that refocus the immune response to conserved epitopes on the HA are needed to tackle infections by influenza virus variants. Sequential vaccination with chimeric HA (cHA) and mosaic HA (mHA) constructs has proven to induce immune responses to the HA stalk domain and conserved epitopes on the HA head. In this study, we developed a bioprocess to manufacture cHA and mHA inactivated split vaccines and a method to quantify HA with a prefusion stalk based on a sandwich enzyme-linked immunosorbent assay. Virus inactivation with beta-propiolactone (βPL) and splitting with Triton X-100 yielded the highest amount of prefusion HA and enzymatically active NA. In addition, the quantity of residual Triton X-100 and ovalbumin (OVA) was reduced to very low levels in the final vaccine preparations. The bioprocess shown here provides the basis to manufacture inactivated split cHA and mHA vaccines for pre-clinical research and future clinical trials in humans, and can also be applied to produce vaccines based on other influenza viruses.

Group 1 and group 2 hemagglutinin stalk antibody response according to age.

Antibodies elicited by seasonal influenza vaccines mainly target the head of hemagglutinin (HA). However, antibodies against the stalk domain are cross-reactive and have been proven to play a role in reducing influenza disease severity. We investigated the induction of HA stalk-specific antibodies after seasonal influenza vaccination, considering the age of the cohorts. A total of 166 individuals were recruited during the 2018 influenza vaccine campaign (IVC) and divided into groups: <50 (n = 14), 50-64 (n = 34), 65-79 (n = 61), and ≥80 (n = 57) years old. Stalk-specific antibodies were quantified by ELISA at day 0 and day 28 using recombinant viruses (cH6/1 and cH14/3) containing an HA head domain (H6 or H14) from wild bird origin with a stalk domain from human H1 or H3, respectively. The geometric mean titer (GMT) and the fold rise (GMFR) were calculated, and differences were assessed using ANOVA adjusted by the false discovery rate (FDR) and the Wilcoxon tests (p <0.05). All age groups elicited some level of increase in anti-stalk antibodies after receiving the influenza vaccine, except for the ≥80-year-old cohort. Additionally, <65-year-old vaccinees had higher group 1 antibody titers versus group 2 before and after vaccination. Similarly, vaccinees within the <50-year-old group showed a higher increase in anti-stalk antibody titers when compared to older individuals (≥80 years old), especially for group 1 anti-stalk antibodies. Seasonal influenza vaccines can the induction of cross-reactive anti-stalk antibodies against group 1 and group 2 HAs. However, low responses were observed in older groups, highlighting the impact of immunosenescence in adequate humoral immune responses.

An influenza H1 hemagglutinin stem-only immunogen elicits a broadly cross-reactive B cell response in humans.

Current yearly seasonal influenza vaccines primarily induce an antibody response directed against the immunodominant but continually diversifying hemagglutinin (HA) head region. These antibody responses provide protection against the vaccinating strain but little cross-protection against other influenza strains or subtypes. To focus the immune response on subdominant but more conserved epitopes on the HA stem that might protect against a broad range of influenza strains, we developed a stabilized H1 stem immunogen lacking the immunodominant head displayed on a ferritin nanoparticle (H1ssF). Here, we evaluated the B cell response to H1ssF in healthy adults ages 18 to 70 in a phase 1 clinical trial (NCT03814720). We observed both a strong plasmablast response and sustained elicitation of cross-reactive HA stem-specific memory B cells after vaccination with H1ssF in individuals of all ages. The B cell response was focused on two conserved epitopes on the H1 stem, with a highly restricted immunoglobulin repertoire unique to each epitope. On average, two-thirds of the B cell and serological antibody response recognized a central epitope on the H1 stem and exhibited broad neutralization across group 1 influenza virus subtypes. The remaining third recognized an epitope near the viral membrane anchor and was largely limited to H1 strains. Together, we demonstrate that an H1 HA immunogen lacking the immunodominant HA head produces a robust and broadly neutralizing HA stem-directed B cell response.

Epistasis reduces fitness costs of influenza A virus escape from stem-binding antibodies

The hemagglutinin (HA) stem region is a major target of universal influenza vaccine efforts owing to the presence of highly conserved epitopes across multiple influenza A virus (IAV) strains and subtypes. To explore the potential impact of vaccine-induced immunity targeting the HA stem, we examined the fitness effects of viral escape from stem-binding broadly neutralizing antibodies (stem-bnAbs). Recombinant viruses containing each individual antibody escape substitution showed diminished replication compared to wild-type virus, indicating that stem-bnAb escape incurred fitness costs. A second-site mutation in the HA head domain (N129D; H1 numbering) reduced the fitness effects observed in primary cell cultures and likely enabled the selection of escape mutations. Functionally, this putative permissive mutation increased HA avidity for its receptor. These results suggest a mechanism of epistasis in IAV, wherein modulating the efficiency of attachment eases evolutionary constraints imposed by the requirement for membrane fusion. Taken together, the data indicate that viral escape from stem-bnAbs is costly but highlights the potential for epistatic interactions to enable evolution within the functionally constrained HA stem domain.

Vinyl Sulfone-functionalized Acetalated Dextran Microparticles as a Subunit Broadly Acting Influenza Vaccine.

Influenza is a global health concern with millions of infections occurring yearly. Seasonal flu vaccines are one way to combat this virus; however, they are poorly protective against influenza as the virus is constantly mutating, particularly at the immunodominant hemagglutinin (HA) head group. A more broadly acting approach involves Computationally Optimized Broadly Reactive Antigen (COBRA). COBRA HA generates a broad immune response that is capable of protecting against mutating strains. Unfortunately, protein-based vaccines are often weekly immunogenic, so to help boost the immune response, we employed the use of acetalated dextran (Ace-DEX) microparticles (MPs) two ways: one to conjugate COBRA HA to the surface and a second to encapsulate cGAMP. To conjugate the COBRA HA to the surface of the Ace-DEX MPs, a poly(L-lactide)-polyethylene glycol co-polymer with a vinyl sulfone terminal group (PLLA-PEG-VS) was used. MPs encapsulating the STING agonist cGAMP were co-delivered with the antigen to form a broadly active influenza vaccine. This vaccine approach was evaluated in vivo with a prime-boost-boost vaccination schedule and illustrated generation of a humoral and cellular response that could protect against a lethal challenge of A/California/07/2009 in BALB/c mice.

Influenza vaccination-induced H3 stalk-reactive memory B-cell clone expansion

Current vaccine formulations elicit a recall immune response against viruses by targeting epitopes on the globular head of hemagglutinin (HA), and stalk-reactive antibodies are rarely found. However, stalk-specific memory B-cell expansion after influenza vaccination is poorly understood. In this study, B cells were isolated from individuals immunized with seasonal tetravalent influenza vaccines at days 0 and 28 for H7N9 stimulation in vitro. Plasma and supernatants were collected for the analysis of anti-HA IgG using ELISA and a Luminex assay. Memory B cells were positively enriched, and total RNA was extracted for B cell receptor (BCR) H-CDR3 sequencing. All subjects displayed increased anti-H3 antibody secretion after vaccination, whereas no increase in cH5/3-reactive IgG levels was detected. The number of shared memory B-cell clones among individuals dropped dramatically from 593 to 37. Four out of 5 subjects displayed enhanced frequencies of the VH3-23 and VH3-30 genes, and one exhibited an increase in the frequency of VH1-18, which are associated with the stalk of HA. An increase in H3 stalk-specific antibodies produced by B cells stimulated with H7N9 viruses was detected after vaccination. These results demonstrated that H3 stalk-specific memory B cells can expand and secrete antibodies that bind to the stalk in vitro, although no increase in serum H3 stalk-reactive antibodies was found after vaccination, indicating potential for developing a universal vaccine strategy.

Breathing and Tilting: Mesoscale Simulations Illuminate Influenza Glycoprotein Vulnerabilities.

Influenza virus has resurfaced recently from inactivity during the early stages of the COVID-19 pandemic, raising serious concerns about the nature and magnitude of future epidemics. The main antigenic targets of influenza virus are two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Whereas the structural and dynamical properties of both glycoproteins have been studied previously, the understanding of their plasticity in the whole-virion context is fragmented. Here, we investigate the dynamics of influenza glycoproteins in a crowded protein environment through mesoscale all-atom molecular dynamics simulations of two evolutionary-linked glycosylated influenza A whole-virion models. Our simulations reveal and kinetically characterize three main molecular motions of influenza glycoproteins: NA head tilting, HA ectodomain tilting, and HA head breathing. The flexibility of HA and NA highlights antigenically relevant conformational states, as well as facilitates the characterization of a novel monoclonal antibody, derived from convalescent human donor, that binds to the underside of the NA head. Our work provides previously unappreciated views on the dynamics of HA and NA, advancing the understanding of their interplay and suggesting possible strategies for the design of future vaccines and antivirals against influenza.

Co-immunization with hemagglutinin stem immunogens elicits cross-group neutralizing antibodies and broad protection against influenza A viruses

Current influenza vaccines predominantly induce immunity to the hypervariable hemagglutinin (HA) head, requiring frequent vaccine reformulation. Conversely, the immunosubdominant yet conserved HA stem harbors a supersite that is targeted by broadly neutralizing antibodies (bnAbs), representing a prime target for universal vaccines. Here, we showed that the co-immunization of two HA stem immunogens derived from group 1 and 2 influenza A viruses elicits cross-group protective immunity and neutralizing antibody responses in mice, ferrets, and nonhuman primates (NHPs). Immunized mice were protected from multiple group 1 and 2 viruses, and all animal models showed broad serum-neutralizing activity. A bnAb isolated from an immunized NHP broadly neutralized and protected against diverse viruses, including H5N1 and H7N9. Genetic and structural analyses revealed strong homology between macaque and human bnAbs, illustrating common biophysical constraints for acquiring cross-group specificity. Vaccine elicitation of stem-directed cross-group-protective immunity represents a step toward the development of broadly protective influenza vaccines.

Primary antibody response after influenza virus infection is first dominated by low-mutated HA-stem antibodies followed by higher-mutated HA-head antibodies.

Several studies have shown that the first encounter with influenza virus shapes the immune response to future infections or vaccinations. However, a detailed analysis of the primary antibody response is lacking as this is difficult to study in humans. It is therefore not known what the frequency and dynamics of the strain-specific hemagglutinin (HA) head- and stem-directed antibody responses are directly after primary influenza virus infection. Here, sera of twelve H1N1pdm2009 influenza virus-infected cynomolgus macaques were evaluated for HA-head and HA-stem domain antibody responses. We observed an early induction of HA-stem antibody responses, which was already decreased by day 56. In contrast, responses against the HA-head domain were low early after infection and increased at later timepoint. The HA-specific B cell repertoires in each animal showed diverse VH-gene usage with preferred VH-gene and JH-gene family usage for HA-head or HA-stem B cells but a highly diverse allelic variation within the VH-usage. HA-head B cells had shorter CDRH3s and higher VH-gene somatic hyper mutation levels relative to HA-stem B cells. In conclusion, our data suggest that HA-stem antibodies are the first to react to the infection while HA-head antibodies show a delayed response, but a greater propensity to enter the germinal center and undergo affinity maturation.

Broadly neutralizing antibodies recognizing different antigenic epitopes act synergistically against the influenza B virus.

The discovery of broadly neutralizing monoclonal antibodies against influenza viruses has raised hope for the successful development of new antiviral drugs. However, due to the speed and variety of mutations in influenza viruses, single-component antibodies that recognize specific epitopes are susceptible to viral escape and have limited efficacy when administration is delayed. Hence, it is necessary to develop alternative strategies with better antiviral activity. Influenza B virus infection can cause severe illness in children and the elderly. Commonly used anti-influenza drugs have low clinical efficacy against influenza B virus. In this study, we investigated the antiviral efficacy of combinations of representative monoclonal antibodies targeting different antigenic epitopes against the influenza B virus. We found that combinations of antibodies recognizing the hemagglutinin (HA) head and stem regions showed a stronger neutralizing activity than single antibodies and other antibody combinations in vitro. In addition, we found that pair-wise combinations of antibodies recognizing the HA head region, HA stem region, and neuraminidase enzyme-activated region showed superior antiviral activity than single antibodies in both mouse and ferret in vivo protection assays. Notably, these antibody combinations still displayed good antiviral efficacy when treatment was delayed. Mechanistic studies further revealed that combining antibodies recognizing different epitope regions resulted in extremely strong antibody-dependent cell-mediated cytotoxicity, which may partly explain their superior antiviral effects. Together, the findings of this study provide new avenues for the development of better antiviral drugs and vaccines against influenza viruses.