Influenza A virus carries hundreds of trimeric hemagglutinin (HA) proteins on its viral envelope that interact with various sialylated glycans on a host cell. This interaction represents a multivalent binding event that is present in all the current receptor binding assays, including those employing viruses or precomplexed HA trimers. To study the nature of such multivalent binding events, we fused a superfolder green fluorescent protein (sfGFP) to the C-terminus of trimeric HA to allow for direct visualization of HA-receptor interactions without the need for additional fluorescent antibodies. The multivalent binding of the HA-sfGFP proteins was studied using glycan arrays and tissue staining. The HA-sfGFP with human-type receptor specificity was able to bind to a glycan array as the free trimer. In contrast, the HA-sfGFP with avian-type receptor specificity required multimerization by antibodies before binding to glycans on the glycan array could be observed. Interestingly, multimerization was not required for binding to tissues. The array data may be explained by the possible bivalent binding mode of a single human-specific HA trimer to complex branched N-glycans, which is not possible for the avian-specific HA due to geometrical constrains of the binding sites. The fact that this specificity pattern changes upon interaction with a cell surface probably represents the enhanced amount of glycan orientations and variable densities versus those on the glycan array.

The present vaccine against influenza virus has the inevitable risk of antigenic discordance between the vaccine and the circulating strains, which diminishes vaccine efficacy. This necessitates new approaches that provide broader protection against influenza. Here we designed a vaccine using the hypervariable receptor-binding domain (RBD) of viral hemagglutinin displayed on a nanoparticle (np) able to elicit antibody responses that neutralize H1N1 influenza viruses spanning over 90 years. Co-display of RBDs from multiple strains across time, so that the adjacent RBDs are heterotypic, provides an avidity advantage to cross-reactive B cells. Immunization with the mosaic RBD-np elicited broader antibody responses than those induced by an admixture of nanoparticles encompassing the same set of RBDs as separate homotypic arrays. Furthermore, we identified a broadly neutralizing monoclonal antibody in a mouse immunized with mosaic RBD-np. The mosaic antigen array signifies a unique approach that subverts monotypic immunodominance and allows otherwise subdominant cross-reactive B cell responses to emerge.

Conserved epitopes shared between virus subtypes are often subdominant, making it difficult to induce broadly reactive antibodies by immunization. Here, we generate a plasmid DNA mix vaccine that encodes protein heterodimers with sixteen different influenza A virus hemagglutinins (HA) representing all HA subtypes except H1 (group 1) and H7 (group 2). Each single heterodimer expresses two different HA subtypes and is targeted to MHC class II on antigen presenting cells (APC). Female mice immunized with the plasmid mix produce antibodies not only against the 16 HA subtypes, but also against non-included H1 and H7. We demonstrate that individual antibody molecules cross-react between different HAs. Furthermore, the mix vaccine induces T cell responses to conserved HA epitopes. Immunized mice are partially protected against H1 viruses. The results show that application of valency-based immuno-selection to diversified antigens can be used to direct antibody responses towards conserved (subdominant) epitopes on viral antigens.

As targets of adaptive immunity, influenza viruses are characterized by the fluidity with which they respond to the selective pressure applied by neutralizing antibodies. This mutability of structural determinants of protective immunity is the obstacle in developing universal influenza vaccines. Towards the development of such vaccines and other immune therapies, our studies are designed to identify regions of influenza viruses that are conserved and that mediate virus neutralization. We have specifically focused on viruses of the H3N2 subtype, which have persisted as a principal source of influenza-related morbidity and mortality in humans since the pandemic of 1968. Three monoclonal antibodies have been identified that are broadly-neutralizing against H3 influenza viruses spanning 40 years. The antibodies react with the hemagglutinin glycoprotein and appear to bind in regions that are refractory to the structural variation required for viral escape from neutralization. The antibodies demonstrate therapeutic efficacy in mice against H3N2 virus infection and have potential for use in the treatment of human influenza disease. By mapping the binding region of one antibody, 12D1, we have identified a continuous region of the hemagglutinin that may act as an immunogen to elicit broadly protective immunity to H3 viruses. The anti-H3 monoclonal antibodies were identified after immunization of mice with the hemagglutinin of four different viruses (A/Hong Kong/1/1968, A/Alabama/1/1981, A/Beijing/47/1992, A/Wyoming/3/2003). This immunization schedule was designed to boost B cells specific for conserved regions of the hemagglutinin from distinct antigenic clusters. Importantly, our antibodies are of naturally occurring specificity rather than selected from cloned libraries, demonstrating that broad-spectrum humoral immunity to influenza viruses can be elicited in vivo.

The pandemic threat posed by emerging zoonotic influenza A viruses necessitates development of antiviral agents effective against various antigenic subtypes. Human monoclonal antibody (hMAb) targeting the hemagglutinin (HA) stalk offers a promising approach to control influenza virus infections. Here, we investigated the ability of the hMAb 81.39a to inhibit in vitro replication of human and zoonotic viruses, representing 16 HA subtypes. The majority of viruses were effectively neutralized by 81.39a at a 50% effective concentration (EC50) of <0.01 to 4.9 μg/ml. Among group 2 HA viruses tested, a single A(H7N9) virus was not neutralized at 50 μg/ml; it contained HA2-Asp19Gly, an amino acid position previously associated with resistance to neutralization by the group 2 HA-neutralizing MAb CR8020. Notably, among group 1 HA viruses, H11-H13 and H16 subtypes were not neutralized at 50 μg/ml; they shared the substitution HA2-Asp19Asn/Ala. Conversely, H9 viruses harboring HA2-Asp19Ala were fully susceptible to neutralization. Therefore, amino acid variance at HA2-Asp19 has subtype-specific adverse effects on in vitro neutralization. Mice given a single injection (15 or 45 mg/kg of body weight) at 24 or 48 h after infection with recently emerged A(H5N2), A(H5N8), A(H6N1), or A(H7N9) viruses were protected from mortality and showed drastically reduced lung viral titers. Furthermore, 81.39a protected mice infected with A(H7N9) harboring HA2-Asp19Gly, although the antiviral effect was lessened. A(H1N1)pdm09-infected ferrets receiving a single dose (25 mg/kg) had reduced viral titers and showed less lung tissue injury, despite 24- to 72-h-delayed treatment. Taken together, this study provides experimental evidence for the therapeutic potential of 81.39a against diverse influenza A viruses.

Glycan structures on hemagglutinin (HA) of influenza A viruses have been analyzed previously to understand their significance. However, the formerly established methods using mass spectrometry present disadvantages such as procedure complexity, sensitivity, and throughput. Our study has established a novel method for analyzing glycan profiles of HA using lectin microarray techniques. We successfully obtained glycan profiles of HA starting from 1 ml of the 106 TCID50 samples through simple antigen enrichment using optimized immunoprecipitation. The profiles were reasonably consistent with known glycan structures of HA. Next, we compared glycan profiles of the HAs prepared from chicken embryos, MDCK, Vero, and A549 cells, and demonstrated the host cell-specific HA glycan profiles. Notably, the HA from MDCK cells was α1-3 galactosylated. Our method provides a highly sensitive and simple procedure for glycan profiling of the viral glycoproteins, thereby paving way for direct glycan analyses of human- and animal-derived virions.

Influenza B viruses (IBV) cause respiratory disease epidemics in humans and are therefore components of seasonal influenza vaccines. Serological methods are employed to evaluate vaccine immunogenicity prior to licensure. However, classical methods to assess influenza vaccine immunogenicity such as the hemagglutination inhibition assay (HI) and the serial radial hemolysis assay (SRH), have been proven to have many limitations. As such, there is a need to develop innovative methods that can improve on these traditional assays and provide advantages such as ease of production and access, safety, reproducibility, and specificity. It has been previously demonstrated that the use of replication-defective viruses, such as lentiviral vectors pseudotyped with influenza A hemagglutinins in microneutralization assays (pMN) is a safe and sensitive alternative to study antibody responses elicited by natural influenza infection or vaccination. Consequently, we have produced Influenza B hemagglutinin-pseudotypes (IBV PV) using plasmid-directed transfection. To activate influenza B hemagglutinin, we have explored the use of proteases in increasing PV titers via their co-transfection during pseudotype virus production. When tested for their ability to transduce target cells, the influenza B pseudotypes produced exhibit tropism for different cell lines. The pseudotypes were evaluated as alternatives to live virus in microneutralization assays using reference sera standards, mouse and human sera collected during vaccine immunogenicity studies, surveillance sera from seals, and monoclonal antibodies (mAbs) against IBV. The influenza B pseudotype pMN was found to effectively detect neutralizing and cross-reactive responses in all assays and shows promise as an effective and versatile tool in influenza research.

Binding of influenza viruses to target cells is mediated by the viral surface protein hemagglutinin. To determine the presence of binding sites for influenza A viruses on cells and tissues, soluble hemagglutinins of the H7 and H9 subtype were generated by connecting the hemagglutinin ectodomain to the Fc portion of human immunoglobulin G (H7Fc and H9Fc). Both chimeric proteins bound to different cells and tissues in a sialic acid-dependent manner. Pronounced differences were observed between H7Fc and H9Fc, in the binding both to different mammalian and avian cultured cells and to cryosections of the respiratory epithelium of different virus host species (turkey, chicken and pig). Binding of the soluble hemagglutinins was similar to the binding of virus particles, but showed differences in the binding pattern when compared to two sialic acid-specific plant lectins. These findings were substantiated by a comparative glycan array analysis revealing a very narrow recognition of sialoglycoconjugates by the plant lectins that does not reflect the glycan structures preferentially recognized by H7Fc and H9Fc. Thus, soluble hemagglutinins may serve as sialic acid-specific lectins and are a more reliable indicator of the presence of binding sites for influenza virus HA than the commonly used plant lectins.

Vaccines that elicit broadly cross-neutralizing antibodies, including antibodies that target the conserved stem of hemagglutinin (HA), are being developed as a strategy for next-generation influenza vaccines that protect against influenza across multiple years. However, efficient induction of cross-neutralizing antibodies remains a challenge, and potential escape mutations have not been well characterized. Here we elicited cross-neutralizing antibodies by immunizing animals with the hemagglutinins from H5 and H9 subtype influenza A viruses that are sensitive to neutralization by stem antibodies. We further isolated and characterized an HA stem monoclonal antibody 4C2 that broadly neutralizes group 1 influenza viruses and identified HA mutations that reduced sensitivity to stem antibodies. Our results offer insights for next-generation influenza vaccine strategies for inducing cross-neutralizing antibodies.

Highly pathogenic avian influenza viruses (HPAIVs) typically emerge from low-pathogenic avian influenza viruses (LPAIVs) of the H5 and H7 subtypes upon spillover from wild aquatic birds into poultry. The conversion from LPAIV to HPAIV is characterized by the acquisition of a multibasic cleavage site (MBCS) at the proteolytic cleavage site in the viral binding and fusion protein, hemagglutinin (HA), resulting in cleavage and activation of HA by ubiquitously expressed furin-like proteases. The ensuing HPAIVs disseminate systemically in gallinaceous poultry, are endotheliotropic, and cause hemorrhagic disease with high mortality. HPAIV infections in wild aquatic birds are generally milder, often asymptomatic, and generally not associated with systemic dissemination nor endotheliotropic. As MBCS cleavage by host proteases is the main virulence determinant of HPAIVs in poultry, we set out to determine whether cleavage of HPAIV HA by host proteases might influence the observed species-specific pathogenesis and tropism. Here, we sequenced, cloned, and characterized the expression and functionality of duck furin. The furin sequence was strongly conserved between chickens and ducks, and duck furin cleaved HPAIV and tetrabasic HA in an overexpression system, confirming its functionality. Furin was expressed ubiquitously and to similar extents in duck and chicken tissues, including in primary duck endothelial cells, which sustained multicycle replication of H5N1 HPAIV but not LPAIVs. In conclusion, differences in furin-like protease biology between wild aquatic birds and gallinaceous poultry are unlikely to largely determine the stark differences observed in species-specific pathogenesis of HPAIVs. IMPORTANCE HPAIV outbreaks are a global concern due to the health risks for poultry, wildlife, and humans and their major economic impact. The number of LPAIV-to-HPAIV conversions, which is associated with spillover from wild birds to poultry, has been increasing over recent decades. Furthermore, H5 HPAIVs from the A/goose/Guangdong/1/96 lineage have been circulating in migratory birds, causing increasingly frequent epizootics in poultry and wild birds. Milder symptoms in migratory birds allow for dispersion of HPAIVs over long distances, justifying the importance of understanding the pathogenesis of HPAIVs in wild birds. Here, we examined whether host proteases are a likely candidate to explain some differences in the degree of HPAIV systemic dissemination between avian species. This is the first report to show that furin function and expression is comparable between chickens and ducks, which renders the hypothesis unlikely that furin-like protease differences influence the HPAIV species-specific pathogenesis and tropism.

While single-cell sequencing technologies have revealed tissue heterogeneity, resolving mixed cellular libraries into cellular clones is essential for many pooled screens and clonal lineage tracing. Fluorescent proteins are limited in number, while DNA barcodes can only be read after cell lysis. To overcome these limitations, we used influenza virus hemagglutinins to engineer a genetically encoded cell-surface protein barcoding system. Using antibodies paired to hemagglutinins carrying combinations of escape mutations, we developed an exponential protein barcoding system which can label 128 clones using seven antibodies. This study provides a proof of principle for a strategy to create protein-level cell barcodes that can be used in vivo in mice to track clonal populations.

CL-385319, an N-substituted piperidine, is effective in inhibiting infection of H1-, H2-, and to a lesser extent, H3-typed influenza A viruses by interfering with the fusogenic function of the viral hemagglutinin. Here we show that CL-385319 is effective in inhibiting infection of highly pathogenic H5N1 influenza A virus in Madin-Darby Canine Kidney (MDCK) cells with an IC50 of 27.03±2.54 μM. This compound with low cytotoxicity (CC50=1.48±0.01 mM) could also inhibit entry of pseudoviruses carrying hemagglutinins from H5N1 strains that were isolated from different places at different times, while it had no inhibitory activity on the entry of VSV-G pseudotyped particles. CL385319 could not inhibit N1-typed neuraminidase activity and the adsorption of H5-typed HA to chicken erythrocytes at the concentration as high as 1 mg/ml (2.8 mM). Computer-aid molecular docking analysis suggested that CL-385319 might bind to the cavity of HA2 stem region which was known to undergo significant rearrangement during membrane fusion. Pseudoviruses with M24A mutation in HA1 or F110S mutation in HA2 were resistant to CL-385319, indicating that these two residues in the cavity region may be critical for CL-385319 bindings. These findings suggest that CL-385319 can serve as a lead for development of novel virus entry inhibitors for preventing and treating H5N1 influenza A virus infection.

Seasonal influenza virus infections cause significant morbidity and mortality every year. Annual influenza virus vaccines are effective but only when well matched with circulating strains. Therefore, there is an urgent need for better vaccines that induce broad protection against drifted seasonal and emerging pandemic influenza viruses. One approach to design such vaccines is based on targeting conserved regions of the influenza virus hemagglutinin. Sequential vaccination with chimeric hemagglutinin constructs can refocus antibody responses towards the conserved immunosubdominant stalk domain of the hemagglutinin, rather than the variable immunodominant head. A complementary approach for a universal influenza A virus vaccine is to induce T-cell responses to conserved internal influenza virus antigens. For this purpose, replication deficient recombinant viral vectors based on Chimpanzee Adenovirus Oxford 1 and Modified Vaccinia Ankara virus are used to express the viral nucleoprotein and the matrix protein 1. In this study, we combined these two strategies and evaluated the efficacy of viral vectors expressing both chimeric hemagglutinin and nucleoprotein plus matrix protein 1 in a mouse model against challenge with group 2 influenza viruses including H3N2, H7N9 and H10N8. We found that vectored vaccines expressing both sets of antigens provided enhanced protection against H3N2 virus challenge when compared to vaccination with viral vectors expressing only one set of antigens. Vaccine induced antibody responses against divergent group 2 hemagglutinins, nucleoprotein and matrix protein 1 as well as robust T-cell responses to the nucleoprotein and matrix protein 1 were detected. Of note, it was observed that while antibodies to the H3 stalk were already boosted to high levels after two vaccinations with chimeric hemagglutinins (cHAs), three exposures were required to induce strong reactivity across subtypes. Overall, these results show that a combinations of different universal influenza virus vaccine strategies can induce broad antibody and T-cell responses and can provide increased protection against influenza.

Pre-pandemic development of an inactivated, split-virion avian influenza vaccine is challenged by the lack of pre-existing immunity and the reduced immunogenicity of some H5 hemagglutinins compared to that of seasonal influenza vaccines. Identification of an acceptable effective adjuvant is needed to improve immunogenicity of a split-virion avian influenza vaccine.

Zoonotic avian influenza virus infections may lead to epidemics or pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its H1 hemagglutinin was identified as a key mammalian virulence factor. A chimeric 1918 virus expressing a contemporary avian H1 hemagglutinin, however, displayed murine pathogenicity indistinguishable from that of the 1918 virus. Here, isogenic chimeric avian influenza viruses were constructed on an avian influenza virus backbone, differing only by hemagglutinin subtype expressed. Viruses expressing the avian H1, H6, H7, H10, and H15 subtypes were pathogenic in mice and cytopathic in normal human bronchial epithelial cells, in contrast to H2-, H3-, H5-, H9-, H11-, H13-, H14-, and H16-expressing viruses. Mouse pathogenicity was associated with pulmonary macrophage and neutrophil recruitment. These data suggest that avian influenza virus hemagglutinins H1, H6, H7, H10, and H15 contain inherent mammalian virulence factors and likely share a key virulence property of the 1918 virus. Consequently, zoonotic infections with avian influenza viruses bearing one of these hemagglutinins may cause enhanced disease in mammals.

Influenza serology has traditionally relied on techniques such as hemagglutination inhibition, microneutralization, and ELISA. These assays are complex, challenging to implement in a format allowing detection of several types of antibody-analyte interactions at once (multiplex), and troublesome to implement in the field. As an alternative, we have developed a hemagglutinin microarray on the Arrayed Imaging Reflectometry (AIR) platform. AIR provides sensitive, rapid, and label-free multiplex detection of targets in complex analyte samples such as serum. In preliminary work, we demonstrated the application of this array to the testing of human samples from a vaccine trial. Here, we report the application of an expanded label-free hemagglutinin microarray to the analysis of avian serum samples. Samples from influenza virus challenge experiments in mallards yielded strong, selective detection of antibodies to the challenge antigen in most cases. Samples acquired in the field from mallards were also analyzed, and compared with viral hemagglutinin inhibition and microneutralization assays. We find that the AIR hemagglutinin microarray can provide a simple and robust alternative to standard methods, offering substantially greater information density from a simple workflow.

We examined serum samples from adults ages 48-64 who received multiple seasonal influenza vaccines from 2004 to 2009 for cross-neutralizing antibodies to potential pandemic strains. Using pseudoviruses bearing various hemagglutinins (HA-pseudoviruses), we found serum neutralization titers (≥160) in 100% against A/Japan/305/1957 (H2N2), 53% against A/Hong Kong/1073/99 (H9N2), 56% against the H3N2 variant A/Indiana/08/11 (H3N2v), 11% against A/Hong Kong/G9/97 (H9N2), and 36% A/chicken/Hong Kong/SF4/01 (H6N1). None had titers >160 to A/Shanghai/2/13 (H7N9) or A/Netherlands/219/03 (H7N7). Thirty-six percent to 0% had neutralization titers to various H5N1 strains. Titers to H9, H6, and H5 HA-pseudoviruses correlated with each other, but not with H3N2v, suggesting group-specific cross-neutralization.

An aptamer is a synthetic oligonucleotide with a unique spatial structure that provides specific binding to a target. To date, several aptamers to hemagglutinin of the influenza A virus have been described, which vary in affinity and strain specificity. Among them, the DNA aptamer RHA0385 is able to recognize influenza hemagglutinins with highly variable sequences. In this paper, the structure of RHA0385 was studied by circular dichroism spectroscopy, nuclear magnetic resonance, and size-exclusion chromatography, demonstrating the formation of a parallel G-quadruplex structure. Three derivatives of RHA0385 were designed in order to determine the contribution of the major loop to affinity. Shortening of the major loop from seven to three nucleotides led to stabilization of the scaffold. The affinities of the derivatives were studied by surface plasmon resonance and an enzyme-linked aptamer assay on recombinant hemagglutinins and viral particles, respectively. The alterations in the loop affected the binding to influenza hemagglutinin, but did not abolish it. Contrary to aptamer RHA0385, two of the designed aptamers were shown to be conformationally homogeneous, retaining high affinities and broad binding abilities for both recombinant hemagglutinins and whole influenza A viruses.

Human antibody-based immunity to influenza A virus is limited by antigenic drift resulting from amino acid substitutions in the hemagglutinin (HA) head domain. Glycan addition can cause large antigenic changes but is limited by fitness costs to viral replication. Here, we report that glycans are added to H1 and H3 HAs at discrete 5-to-7-year intervals, until they reach a functional glycan limit, after which glycans are swapped at approximately 2-fold-longer intervals. Consistent with this pattern, 2009 pandemic H1N1 added a glycan at residue N162 over the 2015-2016 season, an addition that required two epistatic HA head mutations for complete glycosylation. These strains rapidly replaced H1N1 strains globally, by 2017 dominating H3N2 and influenza B virus strains for the season. The pattern of glycan modulation that we outline should aid efforts for tracing the epidemic potential of evolving human IAV strains.IMPORTANCE Frequent mutation of its major antibody target, the glycoprotein hemagglutinin, ensures that the influenza virus is perennially both a rapidly emerging virus and a major threat to public health. One type of mutation escapes immunity by adding a glycan onto an area of hemagglutinin that many antibodies recognize. This study revealed that these glycan changes follow a simple temporal pattern. Every 5 to 7 years, hemagglutinin adds a new glycan, up to a limit. After this limit is reached, no net additions of glycans occur. Instead, glycans are swapped or lost at longer intervals. Eventually, a pandemic replaces the terminally glycosylated hemagglutinin with a minimally glycosylated one from the animal reservoir, restarting the cycle. This pattern suggests the following: (i) some hemagglutinins are evolved for this decades-long process, which is both defined by and limited by successive glycan addition; and (ii) hemagglutinin's antibody dominance and its capacity for mutations are highly adapted features that allow influenza to outpace our antibody-based immunity.

The viral hemagglutinins of conventional influenza A viruses (IAVs) bind to sialylated glycans on host cell surfaces for attachment and subsequent infection. In contrast, hemagglutinins of bat-derived IAVs target major histocompatibility complex class II (MHC-II) for cell entry. MHC-II proteins from various vertebrate species can facilitate infection with the bat IAV H18N11. Yet, it has been difficult to biochemically determine the H18:MHC-II binding. Here, we followed a different approach and generated MHC-II chimeras from the human leukocyte antigen DR (HLA-DR), which supports H18-mediated entry, and the nonclassical MHC-II molecule HLA-DM, which does not. In this context, viral entry was supported only by a chimera containing the HLA-DR α1, α2, and β1 domains. Subsequent modeling of the H18:HLA-DR interaction identified the α2 domain as central for this interaction. Further mutational analyses revealed highly conserved amino acids within loop 4 (N149) and β-sheet 6 (V190) of the α2 domain as critical for virus entry. This suggests that conserved residues in the α1, α2, and β1 domains of MHC-II mediate H18-binding and virus propagation. The conservation of MHC-II amino acids, which are critical for H18N11 binding, may explain the broad species specificity of this virus.