Functional Balance between the Hemagglutinin and Neuraminidase of Influenza A(H1N1)pdm09 HA D222 Variants

D222G/N substitutions in A(H1N1)pdm09 hemagglutinin may be associated with increased binding of viruses causing low respiratory tract infections and human pathogenesis. We assessed the impact of such substitutions on the balance between hemagglutinin binding and neuraminidase cleavage, viral growth and in vivo virulence.Seven viruses with differing polymorphisms at codon 222 (2 with D, 3 G, 1 N and 1 E) were isolated from patients and characterized with regards hemagglutinin binding affinity (Kd) to α-2,6 sialic acid (SAα-2,6) and SAα-2,3 and neuraminidase enzymatic properties (Km, Ki and Vmax). The hemagglutination assay was used to quantitatively assess the balance between hemagglutinin binding and neuraminidase cleavage. Viral growth properties were compared in vitro in MDCK-SIAT1 cells and in vivo in BALB/c mice. Compared with D222 variants, the binding affinity of G222 variants was greater for SAα-2,3 and lower for SAα-2,6, whereas that of both E222 and N222 variants was greater for both SAα-2,3 and SAα-2,6. Mean neuraminidase activity of D222 variants (16.0 nmol/h/10⁶) was higher than that of G222 (1.7 nmol/h/10⁶ viruses) and E/N222 variants (4.4 nmol/h/10⁶ viruses). The hemagglutination assay demonstrated a deviation from functional balance by E222 and N222 variants that displayed strong hemagglutinin binding but weak neuraminidase activity. This deviation impaired viral growth in MDCK-SIAT1 cells but not infectivity in mice. All strains but one exhibited low infectious dose in mice (MID50) and replicated to high titers in the lung; this D222 strain exhibited a ten-fold higher MID50 and replicated to low titers. Hemagglutinin-neuraminidase balance status had a greater impact on viral replication than hemagglutinin affinity strength, at least in vitro, thus emphasizing the importance of an optimal balance for influenza virus fitness. The mouse model is effective in assessing binding to SAα-2,3 but cannot differentiate SAα-2,3- from SAα-2,6- preference, nor estimate the hemagglutinin-neuraminidase balance in A(H1N1)pdm09 strains.     

Amino acid 226 in the hemagglutinin of H4N6 influenza virus determines binding affinity for alpha2,6-linked sialic acid and infectivity levels in primary swine and human respiratory epithelial cells

Avian lineage H4N6 influenza viruses previously isolated from pigs differ at hemagglutinin amino acids 226 and 228 from H4 subtype viruses isolated from birds. Using a parental H4N6 swine isolate and hemagglutinin mutant viruses (at residues 226 and/or 228), we determined that viruses which contain L226 had a higher affinity for sialic acid α2,6 galactose (SAα2,6Gal) and a higher infectivity level for primary swine and human respiratory epithelial cells, whereas viruses which contain Q226 had lower SAα2,6Gal affinity and lower infectivity levels for both types of cells. Using specific neuraminidases, we found that irrespective of their relative binding preferences, all of the influenza viruses examined utilized SAα2,6Gal to infect swine and human cells.     

Glycosylation at 158N of the Hemagglutinin Protein and Receptor Binding Specificity Synergistically Affect the Antigenicity and Immunogenicity of a Live Attenuated H5N1 A/Vietnam/1203/2004 Vaccine Virus in Ferrets

A live attenuated influenza A/Vietnam/1203/2004 (H5N1) vaccine virus (VN04 ca) has receptor binding specificity to α2,3-linked sialosides (α2,3SAL), and a single dose induces a minimal serum antibody response in mice and ferrets. In contrast, A/Hong Kong/213/2003 (H5N1) vaccine virus (HK03 ca) binds to both α2,6SAL and α2,3SAL and generates a stronger serum antibody response in animals. Among the 9 amino acids that differed between the two H5 HA1 proteins, several HK03-specific residues enabled the VN04 ca virus to bind to both α2,3SAL and α2,6SAL receptors, but only the removal of the 158N glycosylation, together with an S227N change, resulted in more-efficient viral replication in the upper respiratory tract of ferrets and an increased serum antibody response. However, the antibody response was HK03 strain specific and did not significantly cross-neutralize VN04 virus. A second approach was taken to adapt the H5N1 VN04 ca virus in MDCK cells to select HA variants with larger plaque morphology. Although a number of large-plaque-size HA variants with amino acid changes in the HA receptor binding region were identified, none of these mutations affected virus receptor binding preference and immunogenicity. In addition, the known receptor binding site changes, Q226L and G228S, were introduced into the HA protein of the VN04 ca virus. Only in conjunction with the removal of the 158N glycosylation did the virus replicate efficiently in the upper respiratory tract of ferrets and became more immunogenic, yet the response was also HK03 specific. Thus, the mask of the antigenic epitopes by 158N glycosylation at the HA globular head and its α2,3SAL binding preference of VN04 ca virus affect virus antigenicity and replication in the host, resulting in a lower antibody response.Influenza A viruses have the potential to cause pandemics of various severities. The emergence of new influenza virus strains to which the general population has low or no immunity, such as the 2009 swine-origin influenza A H1N1 viruses, will continue to challenge public health authorities and the scientific community to develop quick and efficient mitigation responses (18). Highly pathogenic avian influenza A (HPAI) H5N1 viruses pose a serious pandemic threat due to their virulence and high mortality in humans, and their increasingly expanding host reservoir and significant ongoing evolution could enhance their human-to-human transmissibility (8). Currently, the case fatality rate of HPAI H5N1 viruses in humans is estimated to be approximately 60% (30).Although HPAI H5N1 viruses are now endemic in several countries (2), direct transmission of influenza viruses from avian species to humans remains a relatively rare event. The hemagglutinin (HA) protein''s affinity for cell surface sialic acid-containing molecules is one of the determinants of influenza A virus host range restriction. Human and avian influenza virus isolates differ in their recognition of host cell receptors; human strains mainly bind α2,3-linked sialosides (α2,6SAL), whereas the avian strains have a high affinity to α2,3SAL (15, 32). The influenza pandemics of the last century have been suggested to result from switching of HA receptor-binding specificity from α2,3SAL to α2,6SAL receptors (6, 26, 31).The receptor-binding specificity of the HA protein can be influenced by several critical residues. For influenza H3 subtype viruses, substitutions of Q226L and G228S could completely reverse receptor-binding specificity from α2,3SAL to α2,6SAL (4, 21). For the H1 subtype viruses, the E190D and D225G residues switch virus receptor binding specificity from α2,3SAL to α2,6SAL for the 1918 pandemic H1N1 viruses (6, 25). However, based on glycan microarray analysis, the 190E and 225D residues cannot alter the HA binding preference from α2,3SAL to α2,6SAL for H5N1 viruses (26).Vaccination is considered a preferred approach to prevent influenza-related illness in the community. A pandemic influenza vaccine should stimulate protective immunity in the target population using the smallest amount of antigen possible, thus enabling availability of maximal vaccine doses. The inactivated H5N1 VN04 vaccines have been found to be poorly immunogenic in humans, and adjuvants are needed to enhance vaccine immunogenicity (13). Live attenuated influenza vaccines (LAIV) have several desirable attributes: the stimulation of a durable mucosal and systemic immunity, broad efficacy against homologous and drifted strains, and efficient production (17).Several H5N1 LAIV vaccines possessing a modified HA and neuraminidase (NA) of an H5N1 virus and the six internal protein gene segments (PB1, PB2, PA, NP, M, and NS) of the A/Ann Arbor/6/60 (H2N2) cold-adapted (AA ca) master donor virus were previously generated and evaluated for their immunogenicity and efficacy in mice and ferrets (29). A single dose of A/Vietnam/1203/2004 (VN04 ca) LAIV elicited very low levels of serum neutralizing antibodies against homologous and heterologous wild-type (wt) H5N1 viruses 4 weeks after administration to mice and ferrets. In contrast, a single dose of A/Hong Kong/213/2003 (H5N1) (HK03 ca) LAIV was more immunogenic (29). A specific amino acid residue at position 227 in the HK03 HA has been reported to be responsible for the greater immunogenicity of HK03 (9). VN04 and HK03 also differ in their receptor binding specificities. The VN04 HA mainly recognizes α2,3SAL, while the HK03 HA recognizes both α2,3SAL and α2,6SAL (7, 14, 22, 36). Sequence alignment of the two H5 HA proteins revealed nine amino acid differences in their HA1 region (9). The current analysis evaluates the impact of these amino acid differences on H5N1 VN ca vaccine strain replication and immunogenicity. In addition, adaptive mutations selected from MDCK passage of the H5N1 VN04 ca virus and introduction of known receptor binding sites were evaluated for their effect on antigenicity and immunogenicity of the H5N1 VN04 ca virus.     

Both α2,3- and α2,6-Linked Sialic Acids on O-Linked Glycoproteins Act as Functional Receptors for Porcine Sapovirus

Sapovirus, a member of the Caliciviridae family, is an important cause of acute gastroenteritis in humans and pigs. Currently, the porcine sapovirus (PSaV) Cowden strain remains the only cultivable member of the Sapovirus genus. While some caliciviruses are known to utilize carbohydrate receptors for entry and infection, a functional receptor for sapovirus is unknown. To characterize the functional receptor of the Cowden strain of PSaV, we undertook a comprehensive series of protein-ligand biochemical assays in mock and PSaV-infected cell culture and/or piglet intestinal tissue sections. PSaV revealed neither hemagglutination activity with red blood cells from any species nor binding activity to synthetic histo-blood group antigens, indicating that PSaV does not use histo-blood group antigens as receptors. Attachment and infection of PSaV were markedly blocked by sialic acid and Vibrio cholerae neuraminidase (NA), suggesting a role for α2,3-linked, α2,6-linked or α2,8-linked sialic acid in virus attachment. However, viral attachment and infection were only partially inhibited by treatment of cells with sialidase S (SS) or Maackia amurensis lectin (MAL), both specific for α2,3-linked sialic acid, or Sambucus nigra lectin (SNL), specific for α2,6-linked sialic acid. These results indicated that PSaV recognizes both α2,3- and α2,6-linked sialic acids for viral attachment and infection. Treatment of cells with proteases or with benzyl 4-O-β-D-galactopyranosyl-β-D-glucopyranoside (benzylGalNAc), which inhibits O-linked glycosylation, also reduced virus binding and infection, whereas inhibition of glycolipd synthesis or N-linked glycosylation had no such effect on virus binding or infection. These data suggest PSaV binds to cellular receptors that consist of α2,3- and α2,6-linked sialic acids on glycoproteins attached via O-linked glycosylation.     

Application of deglycosylation and electrophoresis to the quantification of influenza viral hemagglutinins facilitating the production of 2009 pandemic influenza (H1N1) vaccines at multiple manufacturing sites in China

The single radial immunodiffusion (SRID) method currently used to determine the hemagglutinin (HA) content of the inactivated influenza vaccines depends on the availability of reference HA antigen and corresponding anti-serum, updated and provided annually by World Health Organization (WHO) collaborative centers. Particularly early in a pandemic outbreak, reference reagents could be the bottleneck in vaccine development and release. Therefore, other reliable tests capable of quantifying HA content could substantially shorten the time needed for vaccine formulation. Here electrophoretic separation of deglycosylated samples in conjunction with densitometry was used to quantify HA contents of H1N1 vaccine at multiple manufacturing sites. We found the overall consistency between the alternative method and traditional SRID was 88–122% in seven lots of vaccine bulks from four subtypes (types) of influenza vaccine, confirming its suitability to quantify HA content. Moreover, we used the alternative method to prepare a national HA antigen reference in China for quality control of 2009 pandemic influenza A (H1N1) vaccines prior to the arrival of the WHO SRID reference standards, subsequently confirming good agreement between both methods. The alternative method for vaccine quantification enabled the Chinese health authority to approve H1N1 vaccine 1 month earlier than otherwise possible.     

Functional Testing of an Inhalable Nanoparticle Based Influenza Vaccine Using a Human Precision Cut Lung Slice Technique

Annual outbreaks of influenza infections, caused by new influenza virus subtypes and high incidences of zoonosis, make seasonal influenza one of the most unpredictable and serious health threats worldwide. Currently available vaccines, though the main prevention strategy, can neither efficiently be adapted to new circulating virus subtypes nor provide high amounts to meet the global demand fast enough. New influenza vaccines quickly adapted to current virus strains are needed. In the present study we investigated the local toxicity and capacity of a new inhalable influenza vaccine to induce an antigen-specific recall response at the site of virus entry in human precision-cut lung slices (PCLS). This new vaccine combines recombinant H1N1 influenza hemagglutinin (HAC1), produced in tobacco plants, and a silica nanoparticle (NP)-based drug delivery system. We found no local cellular toxicity of the vaccine within applicable concentrations. However higher concentrations of NP (≥10³ µg/ml) dose-dependently decreased viability of human PCLS. Furthermore NP, not the protein, provoked a dose-dependent induction of TNF-α and IL-1β, indicating adjuvant properties of silica. In contrast, we found an antigen-specific induction of the T cell proliferation and differentiation cytokine, IL-2, compared to baseline level (152±49 pg/mg vs. 22±5 pg/mg), which could not be seen for the NP alone. Additionally, treatment with 10 µg/ml HAC1 caused a 6-times higher secretion of IFN-γ compared to baseline (602±307 pg/mg vs. 97±51 pg/mg). This antigen-induced IFN-γ secretion was further boosted by the adjuvant effect of silica NP for the formulated vaccine to a 12-fold increase (97±51 pg/mg vs. 1226±535 pg/mg). Thus we were able to show that the plant-produced vaccine induced an adequate innate immune response and re-activated an established antigen-specific T cell response within a non-toxic range in human PCLS at the site of virus entry.     

Conservation and Diversity of Influenza A H1N1 HLA-Restricted T Cell Epitope Candidates for Epitope-Based Vaccines

Background

The immune-related evolution of influenza viruses is exceedingly complex and current vaccines against influenza must be reformulated for each influenza season because of the high degree of antigenic drift among circulating influenza strains. Delay in vaccine production is a serious problem in responding to a pandemic situation, such as that of the current H1N1 strain. Immune escape is generally attributed to reduced antibody recognition of the viral hemagglutinin and neuraminidase proteins whose rate of mutation is much greater than that of the internal non-structural proteins. As a possible alternative, vaccines directed at T cell epitope domains of internal influenza proteins, that are less susceptible to antigenic variation, have been investigated.

Methodology/Principal Findings

HLA transgenic mouse strains expressing HLA class I A*0201, A*2402, and B*0702, and class II DRB1*1501, DRB1*0301 and DRB1*0401 were immunized with 196 influenza H1N1 peptides that contained residues of highly conserved proteome sequences of the human H1N1, H3N2, H1N2, H5N1, and avian influenza A strains. Fifty-four (54) peptides that elicited 63 HLA-restricted peptide-specific T cell epitope responses were identified by IFN-γ ELISpot assay. The 54 peptides were compared to the 2007–2009 human H1N1 sequences for selection of sequences in the design of a new candidate H1N1 vaccine, specifically targeted to highly-conserved HLA-restricted T cell epitopes.

Conclusions/Significance

Seventeen (17) T cell epitopes in PB1, PB2, and M1 were selected as vaccine targets based on sequence conservation over the past 30 years, high functional avidity, non-identity to human peptides, clustered localization, and promiscuity to multiple HLA alleles. These candidate vaccine antigen sequences may be applicable to any avian or human influenza A virus.     

Evaluation of Live Attenuated H7N3 and H7N7 Vaccine Viruses for Their Receptor Binding Preferences,Immunogenicity in Ferrets and Cross Reactivity to the Novel H7N9 Virus

Live attenuated influenza vaccine (LAIV) candidates of the H7 subtype, A/Netherlands/219/03 (H7N7, NL03 ca) and A/chicken/British Columbia/CN-6/2004 (H7N3, BC04 ca), were evaluated for their receptor binding specificity and immunogenicity in ferrets. The BC04 ca virus exhibited α2,3-SA and α2,6-SA dual receptor binding preference while the NL03 ca virus preferentially bound to α2,3-SA. Substitution of the Q226 and G228 (Q-G) by the L226 and S228 (L-S) residues in the HA improved binding to α2,6-SA for NL03 ca. The vaccine viruses with L-S retained the attenuation phenotype. NL03 L-S ca replicated more efficiently than the original NL03 ca virus in the upper respiratory tract of ferrets, and induced higher levels of humoral and cellular immune responses. Prior vaccination with seasonal LAIV reduced H7-specific antibody responses, but did not reduce the H7N7 vaccine mediated protection against a heterologous H7N3 BC04 wt virus infection in ferrets. In addition, the H7N3 and H7N7 vaccine immunized ferret sera cross reacted with the newly emerged H7N9 virus. These data, in combination with the safety data from previously conducted Phase 1 studies, suggest that these vaccines may have a role in responding to the threat posed by the H7N9 virus.     

Vaccination against Influenza with Recombinant Hemagglutinin Expressed by Schizochytrium sp. Confers Protective Immunity

For the rapid production of influenza vaccine antigens in unlimited quantities, a transition from conventional egg-based production to cell-based and recombinant systems is required. The need for higher-yield, lower-cost, and faster production processes is critical to provide adequate supplies of influenza vaccine to counter global pandemic threats. In this study, recombinant hemagglutinin proteins of influenza virus were expressed in the microalga Schizochytrium sp., an established, fermentable organism grown in large scale for the manufacture of polyunsaturated fatty acids for animal and human health applications. Schizochytrium was capable of exporting the full-length membrane-bound proteins in a secreted form suitable for vaccine formulation. One recombinant hemagglutinin (rHA) protein derived from A/Puerto Rico/8/34 (H1N1) influenza virus was evaluated as a vaccine in a murine challenge model. Protective immunity from lethal challenge with homologous virus was elicited by a single dose of 1.7, 5 or 15 µg rHA with or without adjuvant at survival rates between 80–100%. Full protection (100%) was established at all dose levels with or without adjuvant when mice were given a second vaccination. These data demonstrate the potential of Schizochytrium sp. as a platform for the production of recombinant antigens useful for vaccination against influenza.     

Enhanced susceptibility of nasal polyp tissues to avian and human influenza viruses

Background

Influenza viruses bind and infect respiratory epithelial cells through sialic acid on cell surface. Differential preference to sialic acid types contributes to host- and tissue-tropism of avian and seasonal influenza viruses. Although the highly pathogenic avian influenza virus H5N1 can infect and cause severe diseases in humans, it is not efficient in infecting human upper respiratory tract. This is because of the scarcity of its receptor, α2,3-linked sialic acid, in human upper airway. Expression of sialic acid can be influenced by various factors including inflammatory process. Allergic rhinitis and nasal polyp are common inflammatory conditions of nasal mucosa and may affect expression of the sialic acid and susceptibility to influenza infection.

Methodology/Principal Finding

To test this hypothesis, we detected α2,3- and α2,6-linked sialic acid in human nasal polyp and normal nasal mucosal tissues by lectin staining and infected explants of those tissues with avian influenza viruses H5N1 and seasonal influenza viruses. We show here that mucosal surface of nasal polyp expressed higher level of α2,3- and α2,6-linked sialic acid than normal nasal mucosa. Accordingly, both H5N1 avian influenza viruses and seasonal influenza viruses replicated more efficiently in nasal polyp tissues explants.

Conclusions/Significance

Our data suggest a role of nasal inflammatory conditions in susceptibility to influenza infection, especially by avian influenza viruses, which is generally inefficient in infecting human upper airway. The increased receptor expression may contribute to increased susceptibility in some individuals. This may contribute to the gradual adaptation of the virus to human population.     

Single-amino-acid substitution in an antigenic site of influenza virus hemagglutinin can alter the specificity of binding to cell membrane-associated gangliosides.

Antigenic variants of influenza virus A/Mem/1/71-Bel/42 (H3N1) selected with monoclonal antibodies and having single substitutions in their hemagglutinins were examined for their ability to hemagglutinate and hemolyse erythrocytes coated with different gangliosides. The majority of variants, including one with a substitution near the receptor-binding site (Asn-133----Lys), did not differ from the parent in specificity for receptor molecules. However, a substitution in HA1 at residue 205 (Ser----Tyr), which is distant from the receptor-binding site in antigenic site D, affected hemagglutination and hemolysis of erythrocytes coated with sialyl-paraglobosides. The variant preferentially recognized N-acetylneuraminic acid-alpha 2,6-galactose linkages to sialylparaglobosides, whereas the parent and other variants preferentially recognized N-acetylneuraminic acid-alpha 2,3-galactose linkages. In the trimeric hemagglutinin molecule, residue 205 is located across the subunit interface from the receptor-binding site. The bulky hydrophobic tyrosine in the variant may cause a conformational change in the receptor-binding pocket on the neighboring subunit and influence receptor binding.     

Expression of N-Acetylglucosaminyltransferase III Suppresses α2,3-Sialylation,and Its Distinctive Functions in Cell Migration Are Attributed to α2,6-Sialylation Levels

N-Acetylglucosaminyltransferase III (GnT-III), which catalyzes the addition of the bisecting GlcNAc branch on N-glycans, is usually described as a metastasis suppressor. Overexpression of GnT-III inhibited migration in multiple types of tumor cells. However, these results seem controversial to the clinical observations for the increased expression of GnT-III in human hepatomas, glioma, and ovarian cancers. Here, we present evidence that these inconsistencies are mainly attributed to the different expression pattern of cell sialylation. In detail, we show that overexpression of GnT-III significantly inhibits α2,3-sialylation but not α2,6-sialylation. The migratory ability of cells without or with a low level of α2,6-sialylation is consistently suppressed after GnT-III overexpression. In contrast, the effects of GnT-III overexpression are variable in tumor cells that are highly α2,6-sialylated. Overexpression of GnT-III promotes the cell migration in glioma cells U-251 and hepatoma cells HepG2, although it has little influence in human breast cancer cell MDA-MB-231 and gastric cancer cell MKN-45. Interestingly, up-regulation of α2,6-sialylation by overexpressing β-galactoside α2,6-sialyltranferase 1 in the α2,6-hyposialylated HeLa-S3 cells abolishes the anti-migratory effects of GnT-III. Conversely, depletion of α2,6-sialylation by knock-out of β-galactoside α2,6-sialyltranferase 1 in α2,6-hypersialylated HepG2 cells endows GnT-III with the anti-migratory ability. Taken together, our data clearly demonstrate that high expression of α2,6-sialylation on the cell surface could affect the anti-migratory role of GnT-III, which provides an insight into the mechanistic roles of GnT-III in tumor metastasis.     

Haemagglutinin substitutions N125D,D127E,D222G and R223Q improve replicative fitness and vaccine effectiveness of an A/H1N1pdm09 live attenuated influenza vaccine virus by enhancing α-2,6 receptor binding

During 2013–14 and 2015–16, A/H1N1pdm09 live attenuated influenza vaccine (LAIV) viruses replicated inefficiently in primary human nasal epithelial cells (hNEC). This led to reduced vaccine effectiveness (VE) in quadrivalent formulations, mediated by inter-strain competition. By mutating the haemagglutinin (HA) protein, we aimed to enhance hNEC replication of a novel A/H1N1pdm09 vaccine strain to overcome competition and improve VE. Combinations of N125D, D127E, D222G and R223Q substitutions were introduced to the HA protein of A/Slovenia/2903/2015 (A/SLOV15). A/SLOV15 S13, containing all four HA substitutions, produced approximately 1000-fold more virus than parental V1 during hNEC infection. Immunogenicity in ferrets was increased by approximately 10-fold, without compromising yield in eggs or antigenic match to wild-type (wt) reference strains. Despite S13 and V1 being antigenically similar, only S13 protected ferrets from wt virus shedding and fever post-challenge. Crucially, these data suggested that enhanced fitness allowed S13 to overcome inter-strain competition in quadrivalent LAIV (QLAIV). This improved efficacy was later validated by real-world VE data. S13 displayed increased binding avidity to a mammalian-like α-2,6 receptor analogue (6-SLN), relative to V1, while maintaining avian-like 3-SLN avidity. In silico modelling of the HA receptor binding site revealed additional interactions in the S13:6-SLN binding network and a mild increase in 6-SLN binding energy, indicating a possible mechanism for increased α-2,6 receptor-binding avidity. These data confirm that rational HA mutagenesis can be used to optimise hNEC replication and VE for A/H1N1pdm09 LAIV viruses.     

Biophysical Measurement of the Balance of Influenza A Hemagglutinin and Neuraminidase Activities

The interaction of influenza A viruses with the cell surface is controlled by the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). These two glycoproteins have opposing activities: HA is responsible for binding the host receptor (sialic acid) to allow infection, and NA is responsible for cleaving the receptor to facilitate virus release. Several studies have demonstrated that compatible levels of HA and NA activity are required for a virus to replicate efficiently. This is consequently of great interest for determining virus transmissibility. The concurrent role of these two proteins in receptor binding has never been directly measured. We demonstrate a novel biophysical approach based on bio-layer interferometry to measure the balance of the activities of these two proteins in real time. This technique measures virus binding to and release from a surface coated with either the human-like receptor analog α2,6-linked sialic acid or the avian-like receptor analog α2,3-linked sialic acid in both the presence and absence of NA inhibitors. Bio-layer interferometry measurements were also carried out to determine the effect of altering HA receptor affinity and NA stalk length on receptor binding.     

Amino Acid Substitutions That Affect Receptor Binding and Stability of the Hemagglutinin of Influenza A/H7N9 Virus

Receptor-binding preference and stability of hemagglutinin have been implicated as crucial determinants of airborne transmission of influenza viruses. Here, amino acid substitutions previously identified to affect these traits were tested in the context of an A/H7N9 virus. Some combinations of substitutions, most notably G219S and K58I, resulted in relatively high affinity for α2,6-linked sialic acid receptor and acid and temperature stability. Thus, the hemagglutinin of the A/H7N9 virus may adopt traits associated with airborne transmission.     

NKp46 O-Glycan Sequences That Are Involved in the Interaction with Hemagglutinin Type 1 of Influenza Virus

Natural killer (NK) cells serve as a crucial first-line defense against tumors and virus-infected cells. We previously showed that lysis of influenza virus (IV)-infected cells is mediated by the interaction between the NK receptor, NKp46, and the IV hemagglutinin (HA) type 1 expressed by the infected cells. This interaction requires the presence of sialyl groups on the NKp46-T225 O-glycoforms. In the current study, we analyzed the O-glycan sequences that are imperative for the interaction between recombinant NKp46 (rNKp46) and IV H1N1 strains. We first showed that rNKp46 binding to IV H1N1 is not mediated by a glycoform unique to the Thr225 site. We then characterized the O-glycan sequences that mediate the interaction of rNKp46 and IV H1N1; we employed rNKp46s with dissimilar glycosylation patterns and IV H1N1 strains with different sialic acid α2,3 and α2,6 linkage preferences. The branched α2,3-sialylated O-glycoform Neu5NAcα2,3-Galβ1,4-GlcNAcβ1,6[Neu5NAcα2,3-Galβ1,3]GalNAc competently mediated the interaction of rNKp46 with IV H1N1, manifesting a preference for α2,3 linkage. In contrast, the linear α2,3-sialylated O-glycoform Neu5NAcα2,3-Galβ1,3-GalNAc was not correlated with enhanced interaction between rNKp46 and IV H1N1 or a preference for α2,3 linkage. The branched α2,3- and α2,6-sialylated O-glycoform Neu5NAcα2,3-Galβ1,3[Neu5NAcα2,6]GalNAc competently mediated the interaction of rNKp46 with IV H1N1, manifesting a preference for α2,6 linkage. Previous viral HA-binding-specificity studies were performed with glycopolymer conjugates, free synthetic sialyl oligosaccharides, and sialidase-treated cells. This study shed light on the O-glycan sequences involved in the interaction of glycoprotein and viral hemagglutinins and may help in the design of agents inhibitory to hemagglutinin for influenza treatment.Hemagglutinin (HA) is the receptor-binding and membrane fusion protein of influenza virus (IV), as well as the target for infectivity-neutralizing antibodies (27). Terminal sialic acids of glycoproteins and glycolipids are the cellular receptors for the IV HA (27). Two major linkages between sialic acid and the penultimate galactose residues of carbohydrate side chains are found in nature, Neu5NAcα(2,3)-Gal and Neu5NAcα(2,6)-Gal (27); different HAs have different recognition specificities for these linkages and the sugar backbone beneath (23, 26, 30). However, all of the HA-binding specificity studies were performed with glycopolymer conjugates, free synthetic sialyl oligosaccharides, and sialidase-treated cells (8, 10, 20, 25). This could be sufficient for the design of IV-inhibitory agents, and yet, it contributes only partially to the understanding of the interaction of IV HAs with glycoproteins and glycolipids. We aimed to further explore the exact glycoform sequences conjugated to a specific glycoprotein''s glycosylation site that is recognized by different IV strains.For this purpose, we took advantage of our findings on the interaction of natural cytotoxicity receptors (NCRs) and IV HAs (2, 3, 13, 18, 19, 22, 34). We showed that the NKp44 and NKp46 NCRs but not the NKp30 NCR interact with IV HAs. This interaction requires the sialylation of NKp44 and NKp46 oligosaccharides, and the binding of these NCRs to viral HA is required for the lysis of virus-infected cells by NK cells (3, 13, 18). NKp46 displays two putative O-linked glycosylation sites at Thr125 and Thr225 and one N-linked glycosylation site at Asn216. In order to determine the specific sugar-carrying residue that is important for the HA1 recognition, site-directed mutagenesis of the three residues was performed to carry the glycan modifications. Only when Thr225 was replaced was a sharp decrease in the enhanced binding to IV HA1 and IV H1N1-infected cells observed (2). Therefore, for the NKp46 receptor, the interaction with IV HA1 is restricted to Thr225, one of its three glycosylation sites (2).We already showed that producing recombinant NKp46 (rNKp46) in different cell lines resulted in dissimilar glycosylation patterns and had a strong effect on the binding to its ligands (11). Therefore, we analyzed the O-glycan patterns of rNKp46 produced from various cell lines and utilized the dissimilar glycosylation patterns to elucidate the NKp46 O-glycan sequences that mediate the interaction with IV H1N1 strains. To associate the results with the IV preference for sialic acid α2,3 and/or α2,6 linkages, we employed A/PR/8/34 (H1N1), A/NC/20/99 (H1N1), and A/Brisbane/59/2007 (H1N1) grown in either hen egg amnion or Madin-Darby canine kidney (MDCK) cells. Our results pointed to two branched O-glycan sequences that mediated the interaction of the NKp46 glycoprotein with IV H1N1 in correlation with the sialic acid linkage preference of the IV strain.     

In Vitro Assessment of Attachment Pattern and Replication Efficiency of H5N1 Influenza A Viruses with Altered Receptor Specificity

The continuous circulation of the highly pathogenic avian influenza (HPAI) H5N1 virus has been a cause of great concern. The possibility of this virus acquiring specificity for the human influenza A virus receptor, α2,6-linked sialic acids (SA), and being able to transmit efficiently among humans is a constant threat to human health. Different studies have described amino acid substitutions in hemagglutinin (HA) of clinical HPAI H5N1 isolates or that were introduced experimentally that resulted in an increased, but not exclusive, binding of these virus strains to α2,6-linked SA. We introduced all previously described amino acid substitutions and combinations thereof into a single genetic background, influenza virus A/Indonesia/5/05 HA, and tested the receptor specificity of these 27 mutant viruses. The attachment pattern to ferret and human tissues of the upper and lower respiratory tract of viruses with α2,6-linked SA receptor preference was then determined and compared to the attachment pattern of a human influenza A virus (H3N2). At least three mutant viruses showed an attachment pattern to the human respiratory tract similar to that of the human H3N2 virus. Next, the replication efficiencies of these mutant viruses and the effects of three different neuraminidases on virus replication were determined. These data show that influenza virus A/Indonesia/5/05 potentially requires only a single amino acid substitution to acquire human receptor specificity, while at the same time remaining replication competent, thus suggesting that the pandemic threat posed by HPAI H5N1 is far from diminished.Influenza A virus is a negative-strand RNA virus with a segmented genome within the family of Orthomyxoviridae. Influenza A viruses are divided into subtypes based on the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Currently, 16 subtypes of HA and 9 subtypes of NA have been identified in the natural reservoir of all influenza A viruses, wild aquatic birds (24). Occasionally, viruses from this reservoir cross the species barrier into mammals, including humans. When animal influenza viruses are introduced in humans, the spread of the virus is generally limited but may on occasion result in sustained human-to-human transmission. Three influenza A virus subtypes originating from the wild bird reservoir—H1, H2, and H3—have formed stable lineages in humans, starting off with a pandemic and subsequently causing yearly influenza epidemics. In the 20th century, three such pandemics have occurred, in 1918 (H1N1), 1957 (H2N2), and 1968 (H3N2). In 2009, the swine-origin H1N1 virus caused the first influenza pandemic of the 21st century (23).Efficient human-to-human transmission is a prerequisite for any influenza A virus to become pandemic. Currently, the determinants of efficient human-to-human transmission are not completely understood. However, it is believed that a switch of receptor specificity from α2,3-linked sialic acids (SA), used by avian influenza A viruses, to α2,6-linked SA, used by human influenza viruses, is essential (6, 17, 31). It has been shown that the difference in receptor use between avian and human influenza A viruses combined with the distribution of the avian and human virus receptors in the human respiratory tract results in a different localization of virus attachment (26, 33-35). Human viruses attach more abundantly to the upper respiratory tract and trachea, whereas avian viruses predominantly attach to the lower respiratory tract (5, 33-35). Theoretically, the increased presence of virus in the upper respiratory tract, due to the specificity of human influenza A viruses for α2,6-linked SA, could facilitate efficient transmission.Since 1997, highly pathogenic avian influenza (HPAI) H5N1 virus has been circulating in Southeast Asia and has spread westward to Europe, the Middle East, and Africa, resulting in outbreaks of HPAI H5N1 virus in poultry and wild birds and sporadic human cases of infection in 15 different countries (38). The widespread, continuous circulation of the HPAI H5N1 strain has spiked fears that it may acquire specificity for α2,6-linked SA, potentially resulting in a pandemic. Given the currently high case fatality rate of HPAI H5N1 virus infection in humans of ca. 60%, the effect of such a pandemic on the human population could be devastating. In recent years, several amino acid substitutions in HA of HPAI H5N1 viruses have been described, either in virus isolates from patients or introduced experimentally, that increased the binding of the HPAI H5N1 HA to α2,6-linked SA (1, 2, 10, 14, 16, 29, 39, 40). However, none of the described substitutions conferred a full switch of receptor specificity from α2,3-linked SA to α2,6-linked SA and the substitutions were described in virus strains of different geographical origins. Furthermore, it is unknown whether these substitutions led to increased attachment of the virus to cells of the upper respiratory tract, the primary site of replication of human influenza A viruses.Here, we have introduced all of the 21 previously described amino acid substitutions or combinations thereof that changed the receptor specificity of HPAI H5N1 virus strains and six additional combinations not previously described, into HA of influenza virus A/Indonesia/5/05 (IND05). Indonesia is the country that has the highest cumulative number of human cases of HPAI H5N1 virus infection (38). The receptor specificity of 27 mutant H5N1 viruses was determined and the attachment pattern of a subset of these viruses to tissues of the respiratory tract of ferret and human was determined and compared to the attachment pattern of human influenza A virus (H3N2). Subsequently, the role of NA in efficient replication of these mutant viruses was investigated. The data presented here show that receptor specificity of HA of the IND05 virus can be changed by introducing a single amino acid substitution in the receptor-binding domain, resulting in replication competent viruses that attach abundantly to the human upper respiratory tract.     

A Trivalent Virus-Like Particle Vaccine Elicits Protective Immune Responses against Seasonal Influenza Strains in Mice and Ferrets

There is need for improved human influenza vaccines, particularly for older adults who are at greatest risk for severe disease, as well as to address the continuous antigenic drift within circulating human subtypes of influenza virus. We have engineered an influenza virus-like particle (VLP) as a new generation vaccine candidate purified from the supernatants of Sf9 insect cells following infection by recombinant baculoviruses to express three influenza virus proteins, hemagglutinin (HA), neuraminidase (NA), and matrix 1 (M1). In this study, a seasonal trivalent VLP vaccine (TVV) formulation, composed of influenza A H1N1 and H3N2 and influenza B VLPs, was evaluated in mice and ferrets for the ability to elicit antigen-specific immune responses. Animals vaccinated with the TVV formulation had hemagglutination-inhibition (HAI) antibody titers against all three homologous influenza virus strains, as well as HAI antibodies against a panel of heterologous influenza viruses. HAI titers elicited by the TVV were statistically similar to HAI titers elicited in animals vaccinated with the corresponding monovalent VLP. Mice vaccinated with the TVV had higher level of influenza specific CD8+ T cell responses than a commercial trivalent inactivated vaccine (TIV). Ferrets vaccinated with the highest dose of the VLP vaccine and then challenged with the homologous H3N2 virus had the lowest titers of replicating virus in nasal washes and showed no signs of disease. Overall, a trivalent VLP vaccine elicits a broad array of immunity and can protect against influenza virus challenge.