Hemagglutinin Trimer Research Articles

Recent H3N2 Viruses Have Evolved Specificity for Extended, Branched Human-type Receptors, Conferring Potential for Increased Avidity.

Human and avian influenza viruses recognize different sialic acid-containing receptors, referred to as human-type (NeuAcα2-6Gal) and avian-type (NeuAcα2-3Gal), respectively. This presents a species barrier for aerosol droplet transmission of avian viruses in humans and ferrets. Recent reports have suggested that current human H3N2 viruses no longer have strict specificity toward human-type receptors. Using an influenza receptor glycan microarray with extended airway glycans, we find that H3N2 viruses have in fact maintained human-type specificity, but they have evolved preference for a subset of receptors comprising branched glycans with extended poly-N-acetyl-lactosamine (poly-LacNAc) chains, a specificity shared with the 2009 pandemic H1N1 (Cal/04) hemagglutinin. Lipid-linked versions of extended sialoside receptors can restore susceptibility of sialidase-treated MDCK cells to infection byboth recent (A/Victoria/361/11) and historical (A/Hong Kong/8/1968) H3N2 viruses. Remarkably, these human-type receptors with elongated branches have the potential to increase avidity by simultaneously binding to two subunits of a single hemagglutinin trimer.

Generation and characterization of new monoclonal antibodies against swine origin 2009 influenza A (H1N1) virus and evaluation of their prophylactic and therapeutic efficacy in a mouse model

In 2009, a swine-origin influenza A virus – A(H1N1)pdm09 – emerged and has became a pandemic strain circulating worldwide. The hemagglutinin (HA) of influenza virus is a potential target for the development of anti-viral therapeutic agents. Here, we generated mAbs by immunization of baculovirus-insect expressing trimeric recombinant HA of the A(H1N1)pdm09 strain. Results indicated that the mAbs recognized two novel neutralizing and protective epitopes-“STAS” and “FRSK” which located near Cb and Ca1 antigenic regions respectively and were conserved in almost 2009–2016 influenza H1N1 stains. The mAb 12E11 demonstrated higher protective efficacy than mAb 8B10 in mice challenge assay. Both mAb pretreatments significantly reduced virus titers and pro-inflammatory cytokines in mice lung postinfection (p < 0.01), and showed prophylactic and therapeutic efficacies even 48 h postinfection (p < 0.05). Combination therapy using the mAbs with oseltamivir pre- and post-treatment showed synergistic therapeutic effect in mice model (p < 0.01). Further investigation for clinical application in humans is warranted.

Nanostructured glycan architecture is important in the inhibition of influenza A virus infection.

Rapid change and zoonotic transmission to humans have enhanced the virulence of the influenza A virus (IAV). Neutralizing antibodies fail to provide lasting protection from seasonal epidemics. Furthermore, the effectiveness of anti-influenza neuraminidase inhibitors has declined because of drug resistance. Drugs that can block viral attachment and cell entry independent of antigenic evolution or drug resistance might address these problems. We show that multivalent 6'-sialyllactose-polyamidoamine (6SL-PAMAM) conjugates, when designed to have well-defined ligand valencies and spacings, can effectively inhibit IAV infection. Generation 4 (G4) 6SL-PAMAM conjugates with a spacing of around 3 nm between 6SL ligands (S3-G4) showed the strongest binding to a hemagglutinin trimer (dissociation constant of 1.6 × 10-7 M) and afforded the best inhibition of H1N1 infection. S3-G4 conjugates were resistant to hydrolysis by H1N1 neuraminidase. These conjugates protected 75% of mice from a lethal challenge with H1N1 and prevented weight loss in infected animals. The structure-based design of multivalent nanomaterials, involving modulation of nanoscale backbone structures and number and spacing between ligands, resulted in optimal inhibition of IAV infection. This approach may be broadly applicable for designing effective and enduring therapeutic protection against human or avian influenza viruses.

Coated protein nanoclusters from influenza H7N9 HA are highly immunogenic and induce robust protective immunity

Recurring influenza viruses pose an annual threat to public health. A time-saving, cost-effective and egg-independent influenza vaccine approach is important particularly when responding to an emerging pandemic. We fabricated coated, two-layer protein nanoclusters from recombinant trimeric hemagglutinin from an avian-origin H7N9 influenza A virus as an approach for vaccine development in response to an emerging pandemic. Assessment of the virus-specific immune responses and protective efficacy in mice immunized with the nanoclusters demonstrated that the vaccine candidates were highly immunogenic, able to induce protective immunity and long-lasting humoral antibody responses to this virus without the use of adjuvants. Because the advantages of the highly immunogenic coated nanoclusters also include rapid productions in an egg-independent system, this approach has great potential for influenza vaccine production not only in response to an emerging pandemic, but also as a replacement for conventional seasonal influenza vaccines.

Visualization and Sequencing of Membrane Remodeling Leading to Influenza Virus Fusion.

Protein-mediated membrane fusion is an essential step in many fundamental biological events, including enveloped virus infection. The nature of protein and membrane intermediates and the sequence of membrane remodeling during these essential processes remain poorly understood. Here we used cryo-electron tomography (cryo-ET) to image the interplay between influenza virus and vesicles with a range of lipid compositions. By following the population kinetics of membrane fusion intermediates imaged by cryo-ET, we found that membrane remodeling commenced with the hemagglutinin fusion protein spikes grappling onto the target membrane, followed by localized target membrane dimpling as local clusters of hemagglutinin started to undergo conformational refolding. The local dimples then transitioned to extended, tightly apposed contact zones where the two proximal membrane leaflets were in most cases indistinguishable from each other, suggesting significant dehydration and possible intermingling of the lipid head groups. Increasing the content of fusion-enhancing cholesterol or bis-monoacylglycerophosphate in the target membrane led to an increase in extended contact zone formation. Interestingly, hemifused intermediates were found to be extremely rare in the influenza virus fusion system studied here, most likely reflecting the instability of this state and its rapid conversion to postfusion complexes, which increased in population over time. By tracking the populations of fusion complexes over time, the architecture and sequence of membrane reorganization leading to efficient enveloped virus fusion were thus resolved. Enveloped viruses employ specialized surface proteins to mediate fusion of cellular and viral membranes that results in the formation of pores through which the viral genetic material is delivered to the cell. For influenza virus, the trimeric hemagglutinin (HA) glycoprotein spike mediates host cell attachment and membrane fusion. While structures of a subset of conformations and parts of the fusion machinery have been characterized, the nature and sequence of membrane deformations during fusion have largely eluded characterization. Building upon studies that focused on early stages of HA-mediated membrane remodeling, here cryo-electron tomography (cryo-ET) was used to image the three-dimensional organization of intact influenza virions at different stages of fusion with liposomes, leading all the way to completion of the fusion reaction. By monitoring the evolution of fusion intermediate populations over the course of acid-induced fusion, we identified the progression of membrane reorganization that leads to efficient fusion by an enveloped virus.

Antiviral Agents from Multivalent Presentation of Sialyl Oligosaccharides on Brush Polymers.

Bioinspired brush polymers containing α-2,6-linked sialic acids at the side chain termini were synthesized by protection-group-free, ring-opening metathesis polymerization. Polymers showed strain-selective antiviral activity through multivalent presentation of the sialosides. The multivalent effect was further controlled by independently varying the degree of polymerization, the number density of sialic acids, and the length of side chains in the brush polymers. Optimizing the three-dimensional sialoside spacing for better binding to hemagglutinin trimers was of critical importance to enhance the multivalent effect and the antiviral activity determined by hemagglutination inhibition assays and in vitro infection assays. By taking advantage of their structural similarities with native mucins, these brush polymers can be used as model systems to dissect the intricate design principles in natural mucins.

Combination Nanovaccine Demonstrates Synergistic Enhancement in Efficacy against Influenza.

H5N1 influenza virus has the potential to become a significant global health threat, and next generation vaccine technologies are needed. In this work, the combined efficacy of two nanoadjuvant platforms (polyanhydride nanoparticles and pentablock copolymer-based hydrogels) to induce protective immunity against H5N1 influenza virus was examined. Mice received two subcutaneous vaccinations (day 0 and 21) containing 10 μg of H5 hemagglutinin trimer alone or in combination with the nanovaccine platforms. Nanovaccine immunization induced high neutralizing antibody titers that were sustained through 70 days postimmunization. Finally, mice were intranasally challenged with A/H5N1 VNH5N1-PR8CDC-RG virus and monitored for 14 days. Animals receiving the combination nanovaccine had lower viral loads in the lung and weight loss after challenge in comparison to animals vaccinated with each platform alone. These data demonstrate the synergy between polyanhydride nanoparticles and pentablock copolymer-based hydrogels as adjuvants in the design of a more efficacious influenza vaccine.

The role of fusion activity of influenza A viruses in their biological properties.

Influenza A viruses (IAVs) cause acute respiratory infections of humans, which are repeated yearly. Human IAV infections are associated with significant morbidity and mortality and therefore they represent a serious health problem. All human IAV strains are originally derived from avian IAVs, which, after their adaptation to humans, can spread in the human population and cause pandemics with more or less severe course of the disease. Presently, however, the potential of avian IAV to infect humans and to cause the disease cannot be predicted. Many studies are therefore focused on factors influencing the virulence and pathogenicity of IAV viruses in a given host. The virus-host interaction starts by virus attachment via the envelope glycoprotein hemagglutinin (HA) to the receptors on the cell surface. In addition to receptor binding, HA mediates also the fusion of viral and endosomal membranes, which follows the virus endocytosis. The fusion potential of HA trimer, primed by proteolytic cleavage, is activated by low pH in endosomes, resulting in HA refolding into the fusion-active form. The HA conformation change is predetermined by its 3-D structure, is pH-dependent, irreversible and strain-specific. The process of fusion activation of IAV hemagglutinin is crucial for virus entry into the cell and for the ability of the virus to replicate in the host. Here we discuss the known data about the characteristics of fusion activation of HA in relation to IAV virulence and pathogenicity.

Combination nanovaccine enhances influenza vaccine efficacy in aged animals

Event Abstract Back to Event Combination nanovaccine enhances influenza vaccine efficacy in aged animals Kathleen Ross1, Jonathan Goodman1, Matthew Jefferson2, Hyelee Loyd3, Metin Uz1, Michael Wannemuehler4, Surya Mallapragada1, Susan Carpenter3, Marian Kohut5 and Balaji Narasimhan1 1 Iowa State University, Chemical & Biological Engineering, United States 2 Iowa State University, Neuroscience, United States 3 Iowa State University, Animal Science, United States 4 Iowa State University, Veterinary Microbiology & Preventive Medicine, United States 5 Iowa State University, Kinesiology, United States Elderly populations have suboptimal responses to influenza vaccination due to age-associated deficiencies in immune responses[1],[2]. Due to poor antibody responses, measures of cell-mediated immunity may better predict protection in aged individuals[1]. Thus, influenza vaccines that stimulate both antibody and cell-mediated immunity would benefit aged populations. Polyanhydride nanoparticles are capable of both adjuvanting and sustaining the release of influenza antigens[3],[4]. Pentablock copolymer hydrogels form depots of antigen and/or nanoparticles that promote rapid development of antibody titers[5]. In this work, the ability of a combination nanovaccine composed of nanoparticles and hydrogels to enhance neutralizing antibody titers and cell-mediated immunity was studied in aged mice. Aged (17 mo.) and young (2 mo.) mice were immunized thrice (days 0, 21, and 56) with 10 µg of H5 hemagglutinin trimer (sH53), or a combination nanovaccine (2 µg H53 encapsulated into 200 µg nanoparticles + 8 µg H53 entrapped in 25 mg hydrogel). Serum samples were collected at 35, 56, and 66 days post-immunization and the neutralizing antibody titer was determined. To assess T cell induction, both mice received 100 µg of ovalbumin (OVA) either solubly or within the combination nanovaccine at day 66. After 6 days, draining lymph nodes were collected and homogenized. Cells were stimulated with PMA/ionomycin and subsequently labelled with anti-CD8β, anti-CD4, anti-IFNγ, and anti-TNFα. In young mice, robust neutralizing antibody titers were detected at each time point. In contrast, no titers were detected in aged mice receiving either the sH53 or the combination nanovaccine until after administration of a 3rd dose. However, the average titer in mice receiving the combination nanovaccine was five-fold higher than that in the sH53-immunized mice. The combination nanovaccine induced greater numbers of CD4+ and CD8+ T cells in draining lymph nodes when compared to that induced by soluble OVA in both age groups; however, this effect was only statistically significant in young mice. Additionally, the aged mice had greater numbers of CD4+ and CD8+ cells producing IFNγ and TNFα in comparison to young mice (Fig. 1). Fig. 1. Combination nanovaccine immunization enhanced T cell response in aged mice. Mice administered the combination nanovaccine demonstrated greater numbers of CD8+ IFNγ+ TNFα+ T cells than mice administered soluble antigen in both age groups. In this work, a combination nanovaccine was demonstrated to enhance both antibody- and cell-mediated immunity in aged mice. While the neutralizing antibody responses in aged mice were impaired, the combination nanovaccine induced enhanced titers in comparison to that induced by sH53. In addition, the combination nanovaccine induced greater numbers of functional T cells after immunization in both age groups. These data indicate the potential of the combination nanovaccine to increase vaccine efficacy in aged populations.

Hemagglutinin Spatial Distribution Shifts in Response to Cholesterol in the Influenza Viral Envelope

Influenza virus delivers its genome to the host cytoplasm via a process of membrane fusion mediated by the viral hemagglutinin protein. Optimal fusion likely requires multiple hemagglutinin trimers, so the spatial distribution of hemagglutinin on the viral envelope may influence fusion mechanism. We have previously shown that moderate depletion of cholesterol from the influenza viral envelope accelerates fusion kinetics even though it decreases fusion efficiency, both in a reversible manner. Here, we use electron cryo-microscopy to measure how the hemagglutinin lateral density in the viral envelope changes with cholesterol extraction. We extract this information by measuring the radial distribution function of electron density in >4000 viral images per sample, assigning hemagglutinin density by comparing images with and without anti-HA Fab bound. On average, hemagglutinin trimers move closer together: we estimate that the typical trimer-trimer spacing reduces from 94 to 84 Å when ∼90% of cholesterol is removed from the viral membrane. Upon restoration of viral envelope cholesterol, this spacing once again expands. This finding can qualitatively explain the observed changes to fusion kinetics: contemporary models from single-virus microscopy are that fusion requires the engagement of several hemagglutinin trimers in close proximity. If removing cholesterol increases the lateral density of hemagglutinin, this should result in an increase in the rate of fusion.

Intermonomer Interactions in Hemagglutinin Subunits HA1 and HA2 Affecting Hemagglutinin Stability and Influenza Virus Infectivity.

Influenza virus hemagglutinin (HA) mediates virus entry by binding to cell surface receptors and fusing the viral and endosomal membranes following uptake by endocytosis. The acidic environment of endosomes triggers a large-scale conformational change in the transmembrane subunit of HA (HA2) involving a loop (B loop)-to-helix transition, which releases the fusion peptide at the HA2 N terminus from an interior pocket within the HA trimer. Subsequent insertion of the fusion peptide into the endosomal membrane initiates fusion. The acid stability of HA is influenced by residues in the fusion peptide, fusion peptide pocket, coiled-coil regions of HA2, and interactions between the surface (HA1) and HA2 subunits, but details are not fully understood and vary among strains. Current evidence suggests that the HA from the circulating pandemic 2009 H1N1 influenza A virus [A(H1N1)pdm09] is less stable than the HAs from other seasonal influenza virus strains. Here we show that residue 205 in HA1 and residue 399 in the B loop of HA2 (residue 72, HA2 numbering) in different monomers of the trimeric A(H1N1)pdm09 HA are involved in functionally important intermolecular interactions and that a conserved histidine in this pair helps regulate HA stability. An arginine-lysine pair at this location destabilizes HA at acidic pH and mediates fusion at a higher pH, while a glutamate-lysine pair enhances HA stability and requires a lower pH to induce fusion. Our findings identify key residues in HA1 and HA2 that interact to help regulate H1N1 HA stability and virus infectivity. Influenza virus hemagglutinin (HA) is the principal antigen in inactivated influenza vaccines and the target of protective antibodies. However, the influenza A virus HA is highly variable, necessitating frequent vaccine changes to match circulating strains. Sequence changes in HA affect not only antigenicity but also HA stability, which has important implications for vaccine production, as well as viral adaptation to hosts. HA from the pandemic 2009 H1N1 influenza A virus is less stable than other recent seasonal influenza virus HAs, but the molecular interactions that contribute to HA stability are not fully understood. Here we identify molecular interactions between specific residues in the surface and transmembrane subunits of HA that help regulate the HA conformational changes needed for HA stability and virus entry. These findings contribute to our understanding of the molecular mechanisms controlling HA function and antigen stability.

Isolation of broadly neutralizing antibodies from single human memory B cell against human influenza A viruses (TECH2P.922)

Abstract During the past five years, broadly neutralizing antibodies (BnAbs) represent promising prophylactic and therapeutic treatments against influenza. Here, we have developed a novel method to isolate broadly neutralizing antibodies against influenza A virus from human memory B cells. Antigen-specific memory B cells were isolated from human PBMCs using tetramerized H3 (A/Brisbane/10/2007) hemagglutinin (HA) trimers. The H3-reactive single memory B cells were sorted into plates and stimulated in vitro. More than 40% sorted B cells produced on average 200 ng/ml IgG in the supernatant after 14 days. Supernatants from the expanded B cells were measured for their heterosubtypic binding specificity and neutralizing activity by MSD or a highly sensitive neutralization assay. The selective clones were recovered by single cell RT-PCR. Through screening 2688 memory B clones from 7 individuals, 11% clonable memory B cells were reactive with H3 hemagglutinin. Among them, H3/H7, H3/H7/H1 and H3/H7/H1/influenza B heterosubtypic binding population were 16%, 6.9% and 0.35%, respectively. A new broadly neutralizing Ab, 3I14 encoded by the IGHV3-30 germline gene, was further characterized and shown to possess cross-reactive binding and neutralization activity against both group 1 and group 2 influenza A viruses. Epitope mapping and competition assay revealed a highly conserved epitope located in the HA stalk. Passive transfer of this antibody also conferred in vivo protection.

Infection-permissive immunity provided by NA- and M2e-based vaccines protects against influenza A virus challenge and allows the induction of heterosubtypic immunity during subsequent infections. (VAC5P.1127)

Abstract Licensed influenza vaccines protect by inducing neutralizing antibodies against the main viral surface antigens hemagglutinin (HA) and neuraminidase (NA). Influenza infection induces an antiviral T-cell response that correlates with protection against a subsequent infection with a virus of a different subtype (heterosubtypic immunity, HSI). Sterilizing immunity provided by classical vaccines however, may impair the induction of cellular HSI. We previously demonstrated in mice that vaccination with virus like particles containing the ectodomain of Matrix 2 protein (M2e-VLP) induce infection-permissive immunity that not only protects against influenza A viruses of different subtypes, but also allows the induction of cellular HSI. We could recently show that infection-permissive immunity provided by vaccination with recombinantly produced soluble tetrameric N1 NA, alone or combined with M2e-VLP, not only is protective against homologous challenge with pandemic 2009 H1N1 virus but also allows establishment of cellular HSI that protects against challenge with H3N2 virus. NA-immune animals also have an enhanced frequency of circulating functional antiviral memory CD8+ T-cells upon rechallenge with heterologous virus. In contrast, vaccination with recombinant soluble trimeric H1 HA derived from pandemic 2009 H1N1 virus induces strain-specific sterilizing immunity without infection-induced cellular HSI and fails to protect against challenge with H3N2 virus.

Design and Structure of an Engineered Disulfide-Stabilized Influenza Virus Hemagglutinin Trimer.

We engineered a disulfide-stabilized influenza virus hemagglutinin (HA) trimer, termed HA3-SS, by introducing cysteine residues into the HA stem to covalently bridge the three protomers. HA3-SS has increased thermostability compared to wild-type HA, and binding of head- and stem-targeted antibodies (Abs) is preserved; only minor structural changes are found in the vicinity of the additional disulfide. This platform has been applied to H1 and H3 HAs and provides prospects for design of intact, stabilized influenza virus HA immunogens.

Effect of Cholesterol Depletion on HA Distribution in the Viral Membrane of Influenza

Influenza virus enters cells via a process of membrane fusion mediated by the hemagglutinin coat protein. We have previously shown that depletion of cholesterol from the viral envelope increases the rate of fusion despite decreasing fusion efficiency. Furthermore, this increased rate of fusion is only observed when cholesterol is removed from the viral envelope, not in the target membrane. Here we use cryo-electron microscopy to measure how the lateral distribution of hemagglutinin in influenza virions changes upon cholesterol extraction. Following removal of cholesterol from the viral envelope, hemagglutinin trimers move closer together. We estimate that HA trimer-trimer spacing decreases from 92.6 angstroms to 84.9 angstroms. Upon re-addition of cholesterol, hemagglutinin trimer spacing expands once again. Current models from single-virus microscopy suggest that fusion requires the engagement of several hemagglutinin trimers in close proximity. Our findings are in agreement with these models. If hemagglutinin trimers indeed shift closer together in the viral envelope upon cholesterol extraction, this would result in an increase in the rate of fusion.

Viral Membrane Fusion at Single Pore Resolution

Fusion between membrane-enveloped viruses and their hosts is a critical step in infection, enabling the virus to release its genome and subsequently hijack the host cell's replication machinery. The genomes of enveloped viruses are packaged into multiple bulky ribonucleoprotein (RNP) particles whose release requires the dilation of the fusion pore - the initial, narrow, connection between fusing membranes. Electrophysiological studies of influenza A hemagglutinin (HA)-mediated fusion between HA-expressing fibroblasts and red blood cells and between HA-fibroblasts and voltage-clamped suspended bilayers comprise the most complete information to date about the dynamics of viral fusion pores. Small pores (∼2-5nm) were found to flicker repetitively and/or fluctuate in size prior to terminal dilation. A quantitative understanding of the biophysical driving forces of viral membrane fusion, including membrane contact area, membrane tension and protein fusogen cooperativity, however, remains elusive, and a number of questions - regarding the physical basis of pore flickering and the number of HA trimers required for successful pore dilation - remain open. Here we describe a novel electrophysiological approach, which has been successfully applied to study SNARE-mediated fusion pores, to probe the HA-mediated fusion pore. We voltage-clamped HA-expressing fibroblasts in the cell-attached configuration and included nanodiscs (NDs) in the patch pipette as fusion partners to probe fusion pore nucleation and evolution at single pore resolution. NDs are flat, bilayer discs whose composition and size can be varied. Current through voltage-clamped fusion pores reports pore size with sub-millisecond time resolution. Preliminary data shows that isolated fusion pores flicker, are long-lived (∼10s) and are of diameter ∼1.5nm.

Hemagglutinin-based polyanhydride nanovaccines against H5N1 influenza elicit protective virus neutralizing titers and cell-mediated immunity

H5N1 avian influenza is a significant global concern with the potential to become the next pandemic threat. Recombinant subunit vaccines are an attractive alternative for pandemic vaccines compared to traditional vaccine technologies. In particular, polyanhydride nanoparticles encapsulating subunit proteins have been shown to enhance humoral and cell-mediated immunity and provide protection upon lethal challenge. In this work, a recombinant H5 hemagglutinin trimer (H53) was produced and encapsulated into polyanhydride nanoparticles. The studies performed indicated that the recombinant H53 antigen was a robust immunogen. Immunizing mice with H53 encapsulated into polyanhydride nanoparticles induced high neutralizing antibody titers and enhanced CD4+ T cell recall responses in mice. Finally, the H53-based polyanhydride nanovaccine induced protective immunity against a low-pathogenic H5N1 viral challenge. Informatics analyses indicated that mice receiving the nanovaccine formulations and subsequently challenged with virus were similar to naïve mice that were not challenged. The current studies provide a basis to further exploit the advantages of polyanhydride nanovaccines in pandemic scenarios.

Conserved Neutralizing Epitope at Globular Head of Hemagglutinin in H3N2 Influenza Viruses

Neutralizing antibodies that target the hemagglutinin of influenza virus either inhibit binding of hemagglutinin to cellular receptors or prevent the low-pH-induced conformational change in hemagglutinin required for membrane fusion. In general, the former type of antibody binds to the globular head formed by HA1 and has narrow strain specificity, while the latter type binds to the stem mainly formed by HA2 and has broad strain specificity. In the present study, we analyzed the epitope and function of a broadly neutralizing human antibody against H3N2 viruses, F005-126. The crystal structure of F005-126 Fab in complex with hemagglutinin revealed that the antibody binds to the globular head, spans a cleft formed by two hemagglutinin monomers in a hemagglutinin trimer, and cross-links them. It recognizes two peptide portions (sites L and R) and a glycan linked to asparagine at residue 285 using three complementarity-determining regions and framework 3 in the heavy chain. Binding of the antibody to sites L (residues 171 to 173, 239, and 240) and R (residues 91, 92, 270 to 273, 284, and 285) is mediated mainly by van der Waals contacts with the main chains of the peptides in these sites and secondarily by hydrogen bonds with a few side chains of conserved sequences in HA1. Furthermore, the glycan recognized by F005-126 is conserved among H3N2 viruses. F005-126 has the ability to prevent low-pH-induced conformational changes in hemagglutinin. The newly identified conserved epitope, including the glycan, should be immunogenic in humans and may induce production of broadly neutralizing antibodies against H3 viruses. Antibodies play an important role in protection against influenza virus, and hemagglutinin is the major target for virus neutralizing antibodies. It has long been believed that all effective neutralizing antibodies bind to the surrounding regions of the sialic acid-binding pocket and inhibit the binding of hemagglutinin to the cellular receptor. Since mutations are readily introduced into such epitopes, this type of antibody shows narrow strain specificity. Recently, however, broadly neutralizing antibodies have been isolated. Most of these bind either to conserved sites in the stem region or to the sialic acid-binding pocket itself. In the present study, we identified a new neutralizing epitope in the head region recognized by a broadly neutralizing human antibody against H3N2. This epitope may be useful for design of vaccines.

The Mechanism of Poly-Galloyl-Glucoses Preventing Influenza A Virus Entry into Host Cells

Hemagglutinin (HA) is essential for Influenza A virus infection, but its diversity of subtypes presents an obstacle to developing broad-spectrum HA inhibitors. In this study, we investigated the molecular mechanisms by which poly-galloyl glucose (pGG) analogs inhibit influenza hemagglutinin (HA) in vitro and in silico. We found that (1) star-shaped pGG analogs exhibit HA-inhibition activity by interacting with the conserved structural elements of the receptor binding domain (RBD); (2) HA inhibition depends on the number of galloyl substituents in a pGG analog; the best number is four; and when PGG binds with two HA trimers at their conserved receptor binding domains (loop 130, loop 220, and 190-α-helix), PGG acts as a molecular glue by aggregating viral particles so as to prevent viral entry into host cells (this was revealed via an in silico simulation on the binding of penta-galloyl-glucose (PGG) with HA). pGGs are also effective on a broad-spectrum influenza A subtypes (including H1, H3, H5, H7); this suggests that pGG analogs can be applied to most influenza A subtypes as a prophylactic against influenza viral infections.

A novel neutralizing antibody against diverse clades of H5N1 influenza virus and its mutants capable of airborne transmission

Highly pathogenic avian influenza A virus H5N1 continues to spread among poultry and has frequently broken the species barrier to humans. Recent studies have shown that a laboratory-mutated or reassortant H5N1 virus bearing hemagglutinin (HA) with as few as four or five mutations was capable of transmitting more efficiently via respiratory droplets between ferrets, posing a serious threat to public health and underscoring the priority of effective vaccines and therapeutics. In this study, we identified a novel monoclonal antibody (mAb) named HAb21, that has a broadly neutralizing activity against all tested strains of H5N1 covering clades 0, 1, 2.2, 2.3.4, and 2.3.2.1. Importantly, HAb21 efficiently neutralized diverse H5N1 variants with single or combination forms of mutations capable of airborne transmission. We demonstrated that HAb21 blocked viral entry during the receptor-binding step by targeting a previously uncharacterized epitope at the tip of the HA head. This novel epitope closely neighbors the receptor-binding site (RBS) and the interface of HA trimer and is highly conserved among divergent H5N1 strains. Our studies provide a new tool for use either for therapeutic purposes or as a basis of vaccine development.