Hemagglutinin glycosylation pattern-specific effects: implications for the fitness of H9.4.2.5-branched H9N2 avian influenza viruses.

Since 2007, h9.4.2.5 has emerged as the most predominant branch of H9N2 avian influenza viruses (AIVs) that affects the majority of the global poultry population. The spread of this viral branch in vaccinated chicken flocks has not been considerably curbed despite numerous efforts. The evolutionary fitness of h9.4.2.5-branched AIVs must consequently be taken into consideration. The glycosylation modifications of hemagglutinin (HA) play a pivotal role in regulating the balance between receptor affinity and immune evasion for influenza viruses. Sequence alignment showed that five major HA glycosylation patterns have evolved over time in h9.4.2.5-branched AIVs. Here, we compared the adaptive phenotypes of five virus mutants with different HA glycosylation patterns. According to the results, the mutant with 6 N-linked glycans displayed the best acid and thermal stability and a better capacity for multiplication, although having a relatively lower receptor affinity than 7 glycans. The antigenic profile between the five mutants revealed a distinct antigenic distance, indicating that variations in glycosylation level have an impact on antigenic drift. These findings suggest that changes in the number of glycans on HA can not only modulate the receptor affinity and antigenicity of H9N2 AIVs, but also affect their stability and multiplication. These adaptive phenotypes may underlie the biological basis for the dominant strain switchover of h9.4.2.5-branched AIVs. Overall, our study provides a systematic insight into how changes in HA glycosylation patterns regulate the evolutionary fitness and epidemiological dominance drift of h9.4.2.5-branched H9N2 AIVs, which will be of great benefit for the glycosylation-dependent vaccine design.

Epistasis mediates the evolution of the receptor binding mode in recent human H3N2 hemagglutinin.

The receptor-binding site of influenza A virus hemagglutinin partially overlaps with major antigenic sites and constantly evolves. In this study, we observe that mutations G186D and D190N in the hemagglutinin receptor-binding site have coevolved in two recent human H3N2 clades. X-ray crystallography results show that these mutations coordinately drive the evolution of the hemagglutinin receptor binding mode. Epistasis between G186D and D190N is further demonstrated by glycan binding and thermostability analyses. Immunization and neutralization experiments using mouse and human samples indicate that the evolution of receptor binding mode is accompanied by a change in antigenicity. Besides, combinatorial mutagenesis reveals that G186D and D190N, along with other natural mutations in recent H3N2 strains, alter the compatibility with a common egg-adaptive mutation in seasonal influenza vaccines. Overall, our findings elucidate the role of epistasis in shaping the recent evolution of human H3N2 hemagglutinin and substantiate the high evolvability of its receptor-binding mode.

Epitopes in the HA and NA of H5 and H7 avian influenza viruses that are important for antigenic drift.

Avian influenza viruses evolve antigenically to evade host immunity. Two influenza A virus surface glycoproteins, the haemagglutinin and neuraminidase, are the major targets of host immunity and undergo antigenic drift in response to host pre-existing humoral and cellular immune responses. Specific sites have been identified as important epitopes in prominent subtypes such as H5 and H7, which are of animal and public health significance due to their panzootic and pandemic potential. The haemagglutinin is the immunodominant immunogen, it has been extensively studied, and the antigenic reactivity is closely monitored to ensure candidate vaccine viruses are protective. More recently, the neuraminidase has received increasing attention for its role as a protective immunogen. The neuraminidase is expressed at a lower abundance than the haemagglutinin on the virus surface but does elicit a robust antibody response. This review aims to compile the current information on haemagglutinin and neuraminidase epitopes and immune escape mutants of H5 and H7 highly pathogenic avian influenza viruses. Understanding the evolution of immune escape mutants and the location of epitopes is critical for identification of vaccine strains and development of broadly reactive vaccines that can be utilized in humans and animals.

Ultrapotent influenza hemagglutinin fusion inhibitors developed through SuFEx-enabled high-throughput medicinal chemistry.

Seasonal and pandemic-associated influenza strains cause highly contagious viral respiratory infections that can lead to severe illness and excess mortality. Here, we report on the optimization of our small-molecule inhibitor F0045(S) targeting the influenza hemagglutinin (HA) stem with our Sulfur-Fluoride Exchange (SuFEx) click chemistry-based high-throughput medicinal chemistry (HTMC) strategy. A combination of SuFEx- and amide-based lead molecule diversification and structure-guided design led to identification and validation of ultrapotent influenza fusion inhibitors with subnanomolar EC50 cellular antiviral activity against several influenza A group 1 strains. X-ray structures of six of these compounds with HA indicate that the appended moieties occupy additional pockets on the HA surface and increase the binding interaction, where the accumulation of several polar interactions also contributes to the improved affinity. The compounds here represent the most potent HA small-molecule inhibitors to date. Our divergent HTMC platform is therefore a powerful, rapid, and cost-effective approach to develop bioactive chemical probes and drug-like candidates against viral targets.

Self-assembled nanonization of fatty acid-conjugated vaccine antigen for enhanced thermal stability.

The aim of this study was to evaluate the enhanced thermal stability and physicochemical properties of fattigated vaccine antigens. High molecular weight influenza hemagglutinin (Heg) was used as a model antigen because of low heat stability requiring cold chamber. Heg was conjugated with long-chain oleic acid (C18) and short-chain 3-decenoic acid (C10) to prepare fattigated Heg. Circular dichroism analysis revealed no significant changes in the three-dimensional structure post-conjugation. In the liquid state, the fattigated Heg was self-assembled into nanoparticles (NPs) due to its amphiphilic nature, with sizes of 136.27 ± 12.78 nm for oleic acid-conjugated Heg (HOC) and 88.73 ± 3.27 nm for 3-decenoic acid-conjugated Heg (HDC). Accelerated thermal stability studies at 60 °C for 7 days demonstrated that fattigated Heg exhibited higher thermal stability than Heg in various liquid or solid states. The longer-chained HOC showed better thermal stability than HDC in the liquid state, attributed to increased hydrophobic interactions during self-assembly. In bio-mimicking liquid states at 37 °C, HOC exhibited higher thermal stability than Heg. Furthermore, solid-state HOC with cryoprotectants (trehalose, mannitol, and Tween® 80) had significantly increased thermal stability due to reduced exposure of protein surface area via nanonization behavior. The current fattigation platform could be a promising strategy for developing thermostable nano vaccines of heat-labile vaccine antigens.

Recent H9N2 avian influenza virus lost hemagglutination activity due to a K141N substitution in hemagglutinin.

The H9N2 avian influenza virus (AIV) represents a significant risk to both the poultry industry and public health. Our surveillance efforts in China have revealed a growing trend of recent H9N2 AIV strains exhibiting a loss of hemagglutination activity at 37°C, posing challenges to detection and monitoring protocols. This study identified a single K141N substitution in the hemagglutinin (HA) glycoprotein as the culprit behind this diminished hemagglutination activity. The study evaluated the evolutionary dynamics of residue HA141 and studied the impact of the N141K substitution on aspects such as virus growth, thermostability, receptor-binding properties, and antigenic properties. Our findings indicate a polymorphism at residue 141, with the N variant becoming increasingly prevalent in recent Chinese H9N2 isolates. Although both wild-type and N141K mutant strains exclusively target α,2-6 sialic acid receptors, the N141K mutation notably impedes the virus's ability to bind to these receptors. Despite the mutation exerting minimal influence on viral titers, antigenicity, and pathogenicity in chicken embryos, it significantly enhances viral thermostability and reduces plaque size on Madin-Darby canine kidney (MDCK) cells. Additionally, the N141K mutation leads to decreased expression levels of HA protein in both MDCK cells and eggs. These findings highlight the critical role of the K141N substitution in altering the hemagglutination characteristics of recent H9N2 AIV strains under elevated temperatures. This emphasizes the need for ongoing surveillance and genetic analysis of circulating H9N2 AIV strains to develop effective control and prevention measures.IMPORTANCEThe H9N2 subtype of avian influenza virus (AIV) is currently the most prevalent low-pathogenicity AIV circulating in domestic poultry globally. Recently, there has been an emerging trend of H9N2 AIV strains acquiring increased affinity for human-type receptors and even losing their ability to bind to avian-type receptors, which raises concerns about their pandemic potential. In China, there has been a growing number of H9N2 AIV strains that have lost their ability to agglutinate chicken red blood cells, leading to false-negative results during surveillance efforts. In this study, we identified a K141N mutation in the HA protein of H9N2 AIV to be responsible for the loss of hemagglutination activity. This finding provides insight into the development of effective surveillance, prevention, and control strategies to mitigate the threat posed by H9N2 AIV to both animal and human health.

Complex N-glycans are important for interspecies transmission of H7 influenza A viruses.

Influenza A viruses (IAVs) can overcome species barriers by adaptation of the receptor-binding site of the hemagglutinin (HA). To initiate infection, HAs bind to glycan receptors with terminal sialic acids, which are either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc); the latter is mainly found in horses and pigs but not in birds and humans. We investigated the influence of previously identified equine NeuGc-adapting mutations (S128T, I130V, A135E, T189A, and K193R) in avian H7 IAVs in vitro and in vivo. We observed that these mutations negatively affected viral replication in chicken cells but not in duck cells and positively affected replication in horse cells. In vivo, the mutations reduced virus virulence and mortality in chickens. Ducks excreted high viral loads longer than chickens, although they appeared clinically healthy. To elucidate why these viruses infected chickens and ducks despite the absence of NeuGc, we re-evaluated the receptor binding of H7 HAs using glycan microarray and flow cytometry studies. This re-evaluation demonstrated that mutated avian H7 HAs also bound to α2,3-linked NeuAc and sialyl-LewisX, which have an additional fucose moiety in their terminal epitope, explaining why infection of ducks and chickens was possible. Interestingly, the α2,3-linked NeuAc and sialyl-LewisX epitopes were only bound when presented on tri-antennary N-glycans, emphasizing the importance of investigating the fine receptor specificities of IAVs. In conclusion, the binding of NeuGc-adapted H7 IAV to tri-antennary N-glycans enables viral replication and shedding by chickens and ducks, potentially facilitating interspecies transmission of equine-adapted H7 IAVs.IMPORTANCEInfluenza A viruses (IAVs) cause millions of deaths and illnesses in birds and mammals each year. The viral surface protein hemagglutinin initiates infection by binding to host cell terminal sialic acids. Hemagglutinin adaptations affect the binding affinity to these sialic acids and the potential host species targeted. While avian and human IAVs tend to bind to N-acetylneuraminic acid (sialic acid), equine H7 viruses prefer binding to N-glycolylneuraminic acid (NeuGc). To better understand the function of NeuGc-specific adaptations in hemagglutinin and to elucidate interspecies transmission potential NeuGc-adapted viruses, we evaluated the effects of NeuGc-specific mutations in avian H7 viruses in chickens and ducks, important economic hosts and reservoir birds, respectively. We also examined the impact on viral replication and found a binding affinity to tri-antennary N-glycans containing different terminal epitopes. These findings are significant as they contribute to the understanding of the role of receptor binding in avian influenza infection.

Assessment of the antigenic evolution of a clade 6B.1 human H1N1pdm influenza virus revealed differences between ferret and human convalescent sera.

BACKGROUND: Influenza viruses continually acquire mutations in the antigenic epitopes of their major viral antigen, the surface glycoprotein haemagglutinin (HA), allowing evasion from immunity in humans induced upon prior influenza virus infections or vaccinations. Consequently, the influenza strains used for vaccine production must be updated frequently. METHODS: To better understand the antigenic evolution of influenza viruses, we introduced random mutations into the HA head region (where the immunodominant epitopes are located) of a pandemic H1N1 (H1N1pdm) virus from 2015 and incubated it with various human sera collected in 2015-2016. Mutants not neutralized by the human sera were sequenced and further characterized for their haemagglutination inhibition (HI) titers with human sera and with ferret sera raised to H1N1pdm viruses from 2009 to 2015. FINDINGS: The largest antigenic changes were conferred by mutations at HA amino acid position 187; interestingly, these antigenic changes were recognized by human, but not by ferret serum. H1N1pdm viruses with amino acid changes at position 187 were very rare until the end of 2018, but have become more frequent since; in fact, the D187A amino acid change is one of the defining changes of clade 6B.1A.5a.1 viruses, which emerged in 2019. INTERPRETATION: Our findings indicate that amino acid substitutions in H1N1pdm epitopes may be recognized by human sera, but not by homologous ferret sera. FUNDING: This project was supported by funding from the NIAID-funded Center for Research on Influenza Pathogenesis (CRIP, HHSN272201400008C).

Differential Protection of Chickens against Highly Pathogenic H5 Avian Influenza Virus Using Polybasic Amino Acids with H5 Cleavage Peptide.

BACKGROUND: Highly pathogenic H5Nx viruses cause avian influenza, a zoonotic disease that can infect humans. The vaccine can facilitate the prevention of human infections from infected poultry. Our previous study showed that an H5 cleavage-site peptide vaccine containing the polybasic amino acid RRRK could protect chickens from lethal infections of the highly pathogenic H5N6 avian influenza virus. METHODS: Chickens immunized with the various polybasic amino combinations (RRRK, RRR, RR, R, RK, and K) of H5 cleavage-site peptides were challenged with highly pathogenic H5N6 avian influenza viruses. The challenged chickens were monitored for survival rate, and viral titers in swabs and tissue samples were measured in Madin-Darby canine kidney (MDCK) cells using the median tissue culture infectious dose 50 (log10 TCID50/mL). RESULTS: Most H5 cleavage-site vaccines containing various combinations of polybasic amino acids protected chickens from lethal infection. Chickens immunized with the RK-containing peptide combination of the H5 cleavage site were not protected. CONCLUSIONS: The polybasic amino acids (RRRK) of H5 cleavage cleavage-site peptide vaccines are important for protecting chickens against HP H5N6 avian influenza virus. The H5 cleavage cleavage-site peptide containing RK did not protect chickens against the virus.

Evolution and biological characterization of H5N1 influenza viruses bearing the clade 2.3.2.1 hemagglutinin gene.

H5N1 avian influenza viruses bearing the clade 2.3.2.1 hemagglutinin (HA) gene have been widely detected in birds and poultry in several countries. During our routine surveillance, we isolated 28 H5N1 viruses between January 2017 and October 2020. To investigate the genetic relationship of the globally circulating H5N1 viruses and the biological properties of those detected in China, we performed a detailed phylogenic analysis of 274 representative H5N1 strains and analyzed the antigenic properties, receptor-binding preference, and virulence in mice of the H5N1 viruses isolated in China. The phylogenic analysis indicated that the HA genes of the 274 viruses belonged to six subclades, namely clades 2.3.2.1a to 2.3.2.1f; these viruses acquired gene mutations and underwent complicated reassortment to form 58 genotypes, with G43 being the dominant genotype detected in eight Asian and African countries. The 28 H5N1 viruses detected in this study carried the HA of clade 2.3.2.1c (two strains), 2.3.2.1d (three strains), or 2.3.2.1f (23 strains), and formed eight genotypes. These viruses were antigenically well-matched with the H5-Re12 vaccine strain used in China. Animal studies showed that the pathogenicity of the H5N1 viruses ranged from non-lethal to highly lethal in mice. Moreover, the viruses exclusively bound to avian-type receptors and have not acquired the ability to bind to human-type receptors. Our study reveals the overall picture of the evolution of clade 2.3.2.1 H5N1 viruses and provides insights into the control of these viruses.

A new class of antibodies that overcomes a steric barrier to cross-group neutralization of influenza viruses.

Antibody titers that inhibit the influenza virus hemagglutinin (HA) from engaging its receptor are the accepted correlate of protection from infection. Many potent antibodies with broad, intra-subtype specificity bind HA at the receptor binding site (RBS). One barrier to broad H1-H3 cross-subtype neutralization is an insertion (133a) between positions 133 and 134 on the rim of the H1 HA RBS. We describe here a class of antibodies that overcomes this barrier. These genetically unrestricted antibodies are abundant in the human B cell memory compartment. Analysis of the affinities of selected members of this class for historical H1 and H3 isolates suggest that they were elicited by H3 exposure and broadened or diverted by later exposure(s) to H1 HA. RBS mutations in egg-adapted vaccine strains cause the new H1 specificity of these antibodies to depend on the egg adaptation. The results suggest that suitable immunogens might elicit 133a-independent, H1-H3 cross neutralization by RBS-directed antibodies.

Molecular Mechanisms behind Conformational Transitions of the Influenza Virus Hemagglutinin Membrane Anchor.

Membrane fusion is a fundamental process that is exploited by enveloped viruses to enter host cells. In the case of the influenza virus, fusion is facilitated by the trimeric viral hemagglutinin protein (HA). So far, major focus has been put on its N-terminal fusion peptides, which are directly responsible for fusion initiation. A growing body of evidence points also to a significant functional role of the HA C-terminal domain, which however remains incompletely understood. Our computational study aimed to elucidate the structural and functional interdependencies within the HA C-terminal region encompassing the transmembrane domain (TMD) and the cytoplasmic tail (CT). In particular, we were interested in the conformational shift of the TMD in response to varying cholesterol concentration in the viral membrane and in its modulation by the presence of CT. Using free-energy calculations based on atomistic molecular dynamics simulations, we characterized transitions between straight and tilted metastable TMD configurations under varying conditions. We found that the presence of CT is essential for achieving a stable, highly tilted TMD configuration. As we demonstrate, such a configuration of HA membrane anchor likely supports the tilting motion of its ectodomain, which needs to be executed during membrane fusion. This finding highlights the functional role of, so far, the relatively overlooked CT region.

Palmitoylation of the hemagglutinin of influenza B virus by ER-localized DHHC enzymes 1, 2, 4, and 6 is required for efficient virus replication.

IMPORTANCE: Influenza viruses are a public health concern since they cause seasonal outbreaks and occasionally pandemics. Our study investigates the importance of a protein modification called "palmitoylation" in the replication of influenza B virus. Palmitoylation involves attaching fatty acids to the viral protein hemagglutinin and has previously been studied for influenza A virus. We found that this modification is important for the influenza B virus to replicate, as mutating the sites where palmitate is attached prevented the virus from generating viable particles. Our experiments also showed that this modification occurs in the endoplasmic reticulum. We identified the specific enzymes responsible for this modification, which are different from those involved in palmitoylation of HA of influenza A virus. Overall, our research illuminates the similarities and differences in fatty acid attachment to HA of influenza A and B viruses and identifies the responsible enzymes, which might be promising targets for anti-viral therapy.

Structural basis for cross-group recognition of an influenza virus hemagglutinin antibody that targets postfusion stabilized epitope.

Plasticity of influenza virus hemagglutinin (HA) conformation increases an opportunity to generate conserved non-native epitopes with unknown functionality. Here, we have performed an in-depth analysis of human monoclonal antibodies against a stem-helix region that is occluded in native prefusion yet exposed in postfusion HA. A stem-helix antibody, LAH31, provided IgG Fc-dependent cross-group protection by targeting a stem-helix kinked loop epitope, with a unique structure emerging in the postfusion state. The structural analysis and molecular modeling revealed key contact sites responsible for the epitope specificity and cross-group breadth that relies on somatically mutated light chain. LAH31 was inaccessible to the native prefusion HA expressed on cell surface; however, it bound to the HA structure present on infected cells with functional linkage to the Fc-mediated clearance. Our study uncovers a novel non-native epitope that emerges in the postfusion HA state, highlighting the utility of this epitope for a broadly protective antigen design.

H3N2 influenza A virus gradually adapts to human-type receptor binding and entry specificity after the start of the 1968 pandemic.

To become established upon zoonotic transfer, influenza A viruses (IAV) need to switch binding from "avian-type" α2-3-linked sialic acid receptors (2-3Sia) to "human-type" Siaα2-6-linked sialic acid receptors (2-6Sia). For the 1968 H3N2 pandemic virus, this was accomplished by two canonical amino acid substitutions in its hemagglutinin (HA) although a full specificity shift had not occurred. The receptor repertoire on epithelial cells is highly diverse and simultaneous interaction of a virus particle with a range of low- to very low-affinity receptors results in tight heteromultivalent binding. How this range of affinities determines binding selectivity and virus motility remains largely unknown as the analysis of low-affinity monovalent HA-receptor interactions is technically challenging. Here, a biolayer interferometry assay enabled a comprehensive analysis of receptor-binding kinetics evolution upon host-switching. Virus-binding kinetics of H3N2 virus isolates slowly evolved from 1968 to 1979 from mixed 2-3/2-6Sia specificity to high 2-6Sia specificity, surprisingly followed by a decline in selectivity after 1992. By using genetically tuned HEK293 cells, presenting either a simplified 2-3Sia- or 2-6Sia-specific receptor repertoire, receptor-specific binding was shown to correlate strongly with receptor-specific entry. In conclusion, the slow and continuous evolution of entry and receptor-binding specificity of seasonal H3N2 viruses contrasts with the paradigm that human IAVs need to rapidly acquire and maintain a high specificity for 2-6Sia. Analysis of the kinetic parameters of receptor binding provides a basis for understanding virus-binding specificity, motility, and HA/neuraminidase balance at the molecular level.

Bioinformatics and Structural Analysis of Antigenic Variation in the Hemagglutinin Gene of the Influenza A(H1N1)pdm09 Virus Circulating in Shiraz (2013 to 2015).

Circulating influenza A virus provided an excellent opportunity to study the adaptation of the influenza A(H1N1)pdm09 virus to the human host. Particularly, due to the availability of sequences taken from isolates, we could monitor amino acid changes and the stability of mutations that occurred in hemagglutinin (HA). HA is crucial to viral infection because it binds to ciliated cell receptors and mediates the fusion of cells and viral membranes; because antibodies that bind to HA may block virus entry to the cell, this protein is subjected to high selective pressure. In this study, the locations of mutations in the structures of mutant HA were analyzed and the three-dimensional (3D) structures of these mutations were modeled in I-TASSER. Also, the location of these mutations was visualized and studied using Swiss PDB Viewer software and the PyMOL Molecular Graphics System. The crystal structure of the HA from A/California/07/2009 (3LZG) was used for further analysis. The new noncovalent bond formations in mutant luciferases were analyzed via WHAT IF and PIC, and protein stability was evaluated in the iStable server. We identified 33 and 23 mutations in A/Shiraz/106/2015 and A/California/07/2009 isolates, respectively; some mutations are located on the antigenic sites of Sa, Sb, Ca1, Ca2, and Cb HA1 and the fusion peptide of HA2. The results show that with the mutation some interactions are lost and new interactions are formed with other amino acids. The results of the free-energy analysis suggested that these new interactions have a destabilizing effect, which needs confirmation experimentally. IMPORTANCE Due to the fact that the mutations that occurred in the influenza virus HA cause the instability of the protein produced by the virus and antigenic changes and the escape of the virus from the immune system, the mutations that occurred in A/Shiraz/1/2013 were investigated in terms of energy level and stability. The mutations located in a globular portion of the HA are S188T, Q191H, S270P, K285Q, and P299L. On the other hand, the E374K, E46K-B, S124N-B, and I321V mutations are located in the stem portion of the HA (HA2). The change V252L mutation eliminates interactions with Ala181, Phe147, Leu151, and Trp153 and forms new interactions with Gly195, Asn264, Phe161, Met244, Tyr246, Leu165, and Trp167 which can change the stability of the HA structure. The K166Q mutation, which is located within the antigenic site Sa, causes the virus to escape from the immune response.

De novo design of protein structure and function with RFdiffusion.

There has been considerable recent progress in designing new proteins using deep-learning methods1-9. Despite this progress, a general deep-learning framework for protein design that enables solution of a wide range of design challenges, including de novo binder design and design of higher-order symmetric architectures, has yet to be described. Diffusion models10,11 have had considerable success in image and language generative modelling but limited success when applied to protein modelling, probably due to the complexity of protein backbone geometry and sequence-structure relationships. Here we show that by fine-tuning the RoseTTAFold structure prediction network on protein structure denoising tasks, we obtain a generative model of protein backbones that achieves outstanding performance on unconditional and topology-constrained protein monomer design, protein binder design, symmetric oligomer design, enzyme active site scaffolding and symmetric motif scaffolding for therapeutic and metal-binding protein design. We demonstrate the power and generality of the method, called RoseTTAFold diffusion (RFdiffusion), by experimentally characterizing the structures and functions of hundreds of designed symmetric assemblies, metal-binding proteins and protein binders. The accuracy of RFdiffusion is confirmed by the cryogenic electron microscopy structure of a designed binder in complex with influenza haemagglutinin that is nearly identical to the design model. In a manner analogous to networks that produce images from user-specified inputs, RFdiffusion enables the design of diverse functional proteins from simple molecular specifications.

Relevance of Host Cell Surface Glycan Structure for Cell Specificity of Influenza A Viruses.

Influenza A viruses (IAVs) initiate infection via binding of the viral hemagglutinin (HA) to sialylated glycans on host cells. HA's receptor specificity towards individual glycans is well studied and clearly critical for virus infection, but the contribution of the highly heterogeneous and complex glycocalyx to virus-cell adhesion remains elusive. Here, we use two complementary methods, glycan arrays and single-virus force spectroscopy (SVFS), to compare influenza virus receptor specificity with virus binding to live cells. Unexpectedly, we found that HA's receptor binding preference does not necessarily reflect virus-cell specificity. We propose SVFS as a tool to elucidate the cell binding preference of IAVs, thereby including the complex environment of sialylated receptors within the plasma membrane of living cells.

Genetic characterization of a new candidate hemagglutinin subtype of influenza A viruses.

ABSTRACTAvian influenza viruses (AIV) have been classified on the basis of 16 subtypes of hemagglutinin (HA) and 9 subtypes of neuraminidase. Here we describe genomic evidence for a new candidate HA subtype, nominally H19, with a large genetic distance to all previously described AIV subtypes, derived from a cloacal swab sample of a Common Pochard (Aythya ferina) in Kazakhstan, in 2008. Avian influenza monitoring in wild birds especially in migratory hotspots such as central Asia is an important approach to gain information about the circulation of known and novel influenza viruses. Genetically, the novel HA coding sequence exhibits only 68.2% nucleotide and 68.5% amino acid identity with its nearest relation in the H9 (N2) subtype. The new HA sequence should be considered in current genomic diagnostic AI assays to facilitate its detection and eventual isolation enabling further study and antigenic classification.

Structural insights into the broad protection against H1 influenza viruses by a computationally optimized hemagglutinin vaccine.

Influenza virus poses an ongoing human health threat with pandemic potential. Due to mutations in circulating strains, formulating effective vaccines remains a challenge. The use of computationally optimized broadly reactive antigen (COBRA) hemagglutinin (HA) proteins is a promising vaccine strategy to protect against a wide range of current and future influenza viruses. Though effective in preclinical studies, the mechanistic basis driving the broad reactivity of COBRA proteins remains to be elucidated. Here, we report the crystal structure of the COBRA HA termed P1 and identify antigenic and glycosylation properties that contribute to its immunogenicity. We further report the cryo-EM structure of the P1-elicited broadly neutralizing antibody 1F8 bound to COBRA P1, revealing 1F8 to recognize an atypical receptor binding site epitope via an unexpected mode of binding.