A protective measles virus-derived vaccine inducing long-lasting immune responses against influenza A virus H7N9.

A novel Influenza A virus (subtype H7N9) emerged in spring 2013 and caused considerable mortality in zoonotically infected patients. To be prepared for potential pandemics, broadly effective and safe vaccines are crucial. Recombinant measles virus (MeV) encoding antigens of foreign pathogens constitutes a promising vector platform to generate novel vaccines. To characterize the efficacy of H7N9 antigens in a prototypic vaccine platform technology, we generated MeVs encoding either neuraminidase (N9) or hemagglutinin (H7). Moraten vaccine strain-derived vaccine candidates were rescued; they replicated with efficiency comparable to that of the measles vaccine, robustly expressed H7 and N9, and were genetically stable over 10 passages. Immunization of MeV-susceptible mice triggered the production of antibodies against H7 and N9, including hemagglutination-inhibiting and neutralizing antibodies induced by MVvac2-H7(P) and neuraminidase-inhibiting antibodies by MVvac2-N9(P). Vaccinated mice also developed long-lasting H7- and N9-specific T cells. Both MVvac2-H7(P) and MVvac2-N9(P)-vaccinated mice were protected from lethal H7N9 challenge.

Characterization of Influenza Virus Binding to Receptors on Isolated Cell Membranes.

An interplay between receptor-binding properties of influenza viruses (IVs) and spectrum of sialic acid-containing receptors on target cells in birds and mammals determine viral host range, tissue tropism, and pathogenicity. Here, we describe method that allows to characterize binding of IVs to biologically relevant cellular receptors using a conventional solid-phase enzyme-linked assay. In this method, we isolate plasma membranes from respiratory and intestinal epithelial cells of animal origin (Subheading 3.2). We adsorb the membranes in the wells of 96-well ELISA plates, incubate the membrane-coated wells with serially diluted IVs, and determine amounts of IVs attached to the membranes using viral ability to bind peroxidase-labeled sialoglycoprotein fetuin. Based on the concentration dependence of IV binding to the membrane, we estimate binding avidity and number of binding sites. We describe two variants of the assay in Subheadings 3.6 and 3.7 and provide examples.

Receptor-binding properties of influenza viruses isolated from gulls.

Ducks, gulls and shorebirds represent the major hosts of influenza A viruses (IAVs) in nature, but distinctions of IAVs in different birds are not well defined. Here we characterized the receptor specificity of gull IAVs with HA subtypes H4, H6, H14, H13 and H16 using synthetic sialylglycopolymers. In contrast to duck IAVs, gull IAVs efficiently bound to fucosylated receptors and often preferred sulfated and non-sulfated receptors with Galß1-4GlcNAc cores over the counterparts with Galß1-3GlcNAc cores. Unlike all other IAVs of aquatic birds, H16 IAVs showed efficient binding to Neu5Acα2-6Gal-containing receptors and bound poorly to Neu5Acα2-3Galß1-3-terminated (duck-type) receptors. Analysis of HA crystal structures and amino acid sequences suggested that the amino acid at position 222 is an important determinant of the receptor specificity of IAVs and that transmission of duck viruses to gulls and shorebirds is commonly accompanied by substitutions at this position.

Solid-phase assays of receptor-binding specificity.

Influenza virus attachment to sialic acid-containing molecules on the cell surface initiates the infection. The spectrum of functional receptors on target cells and decoy receptors on cells and epithelial mucus varies substantially between animal species leading to variations in the receptor-binding specificity of viruses circulating in these species. Analysis of the receptor specificity of different animal and human influenza viruses can give insight into factors and mechanisms that determine viral host range, tissue and cell tropism, replication efficiency, and pathogenesis. Knowledge of viral receptor specificity may also be useful for the development of more efficient influenza vaccines and anti-influenza drugs.A majority of known receptor specificity assays measure influenza virus binding to sialic acid-containing natural and synthetic compounds (receptor analogues). Here, we describe protocols of two solid-phase enzyme-linked receptor-binding assays which are technically similar to standard ELISA. Each assay determines binding of the virus immobilized in the wells of 96-well plate to receptor analogues in solution. In the direct binding assay, the virus binds to either synthetic biotinylated sialylglycopolymers or to peroxidase-labeled sialylglycoprotein fetuin (Fet-HRP); the apparent association constants of the virus-receptor complexes are calculated from the Scatchard plots of the binding data. In the fetuin-binding inhibition assay, the virus is incubated with a mixture of unlabeled receptor analogue and standard preparation of Fet-HRP; the association constant for analogue is calculated based on the level of its competition with Fet-HRP.

Receptor-binding profiles of H7 subtype influenza viruses in different host species.

Influenza viruses of gallinaceous poultry and wild aquatic birds usually have distinguishable receptor-binding properties. Here we used a panel of synthetic sialylglycopolymers and solid-phase receptor-binding assays to characterize receptor-binding profiles of about 70 H7 influenza viruses isolated from aquatic birds, land-based poultry, and horses in Eurasia and America. Unlike typical duck influenza viruses with non-H7 hemagglutinin (HA), all avian H7 influenza viruses, irrespective of the host species, displayed a poultry-virus-like binding specificity, i.e., preferential binding to sulfated oligosaccharides Neu5Acα2-3Galß1-4(6-O-HSO(3))GlcNAc and Neu5Acα2-3Galß1-4(Fucα1-3)(6-O-HSO(3))GlcNAc. This phenotype correlated with the unique amino acid sequence of the amino acid 185 to 189 loop of H7 HA and seemed to be dependent on ionic interactions between the sulfate group of the receptor and Lys193 and on the lack of sterical clashes between the fucose residue and Gln222. Many North American and Eurasian H7 influenza viruses displayed weak but detectable binding to the human-type receptor moiety Neu5Acα2-6Galß1-4GlcNAc, highlighting the potential of H7 influenza viruses for avian-to-human transmission. Equine H7 influenza viruses differed from other viruses by preferential binding to the N-glycolyl form of sialic acid. Our data suggest that the receptor-binding site of contemporary H7 influenza viruses in aquatic and terrestrial birds was formed after the introduction of their common precursor from ducks to a new host, presumably, gallinaceous poultry. The uniformity of the receptor-binding profile of H7 influenza viruses in various wild and domestic birds indicates that there is no strong receptor-mediated host range restriction in birds on viruses with this HA subtype. This notion agrees with repeated interspecies transmission of H7 influenza viruses from aquatic birds to poultry.

Altered receptor specificity and fusion activity of the haemagglutinin contribute to high virulence of a mouse-adapted influenza A virus.

The viral haemagglutinin (HA) and the viral polymerase complex determine the replication fitness of a highly virulent variant of influenza A virus strain A/PR/8/34 (designated hvPR8) and its high pathogenicity in mice. We report here that the HA of the hvPR8 differs from the HA of a low virulent strain (lvPR8) by the efficiency of receptor binding and membrane fusion. hvPR8 bound to 2,6-linked as well as 2,3-linked sialic acid-containing receptors, whereas lvPR8 bound exclusively to 2,3-linked sialic acids with high avidity. Remarkably, hvPR8 infected its target cells faster than lvPR8 and tolerated an elevated pH for efficient membrane fusion. In spite of these differences, both viruses targeted type II but not type I pneumocytes in the lung of infected mice. The HA of hvPR8 differs from that of lvPR8 by 16 aa substitutions and one insertion. Mutational analyses revealed that amino acid at HA position 190 (H3 numbering) primarily determined the specificity of receptor binding, while the insertion at position 133 influenced the avidity of receptor binding. Both amino acid positions also strongly influenced viral virulence. Furthermore, leucine at position 78 and glutamine at position 354 were critical determinants of increased fusion activity and virulence of hvPR8. Our data suggest that the HA of hvPR8 enhances virulence by mediating optimal receptor binding and membrane fusion thereby promoting rapid and efficient viral entry into host cells.

Aprotinin, a protease inhibitor, suppresses proteolytic activation of pandemic H1N1v influenza virus.

BACKGROUND: The recent emergence of pandemic influenza virus H1N1v stresses the need for the development of new anti-influenza drugs. METHODS: Host proteases responsible for viral haemagglutinin (HA) cleavage are attractive targets for such drugs. Aprotinin, a natural 58-amino-acid polypeptide from bovine lungs, was chosen for this purpose because it is a drug already approved for human use as an antiprotease compound to treat pancreatitis and bleeding, and because it inhibits a wide spectrum of serine proteases, some of which are involved in influenza virus activation. RESULTS: First, we show that HA of pandemic H1N1v was intensively cleaved and activated in different host systems (human tracheo-bronchial epithelium, human intestinal Caco-2 cells and chicken embryonated eggs). Second, aprotinin inhibited HA cleavage and replication of pandemic influenza virus H1N1v in all host systems, including human tracheo-bronchial epithelium. Third, aprotinin did not induce any apparent toxic side effects in these hosts. CONCLUSIONS: Aprotinin can be considered a promising drug against the novel H1N1v pandemic influenza virus.

6-sulfo sialyl Lewis X is the common receptor determinant recognized by H5, H6, H7 and H9 influenza viruses of terrestrial poultry.

BACKGROUND: Influenza A viruses of domestic birds originate from the natural reservoir in aquatic birds as a result of interspecies transmission and adaptation to new host species. We previously noticed that influenza viruses isolated from distinct orders of aquatic and terrestrial birds may differ in their fine receptor-binding specificity by recognizing the structure of the inner parts of Neu5Ac alpha 2-3Gal-terminated sialyloligosaccharide receptors. To further characterize these differences, we studied receptor-binding properties of a large panel of influenza A viruses from wild aquatic birds, poultry, pigs and horses. RESULTS: Using a competitive solid-phase binding assay, we determined viral binding to polymeric conjugates of sialyloligosaccharides differing by the type of Neu5Ac alpha-Gal linkage and by the structure of the more distant parts of the oligosaccharide chain. Influenza viruses isolated from terrestrial poultry differed from duck viruses by an enhanced binding to sulfated and/or fucosylated Neu5Ac alpha 2-3Gal-containing sialyloligosaccharides. Most of the poultry viruses tested shared a high binding affinity for the 6-sulfo sialyl Lewis X (Su-SLex). Efficient binding of poultry viruses to Su-SLex was often accompanied by their ability to bind to Neu5Ac alpha 2-6Gal-terminated (human-type) receptors. Such a dual receptor-binding specificity was demonstrated for the North American and Eurasian H7 viruses, H9N2 Eurasian poultry viruses, and H1, H3 and H9 avian-like virus isolates from pigs. CONCLUSION: Influenza viruses of terrestrial poultry differ from ancestral duck viruses by enhanced binding to sulfated and/or fucosylated Neu5Ac alpha 2-3Gal-terminated receptors and, occasionally, by the ability to bind to Neu5Ac alpha 2-6Gal-terminated (human-type) receptors. These findings suggest that the adaptation to receptors in poultry can enhance the potential of an avian virus for avian-to-human transmission and pandemic spread.

Neuraminidase inhibitor-resistant influenza viruses may differ substantially in fitness and transmissibility.

Mutations of the conserved residues of influenza virus neuraminidase (NA) that are associated with NA inhibitor (NAI) resistance decrease the sialidase activity and/or stability of the NA, thus compromising viral fitness. In fact, clinically derived NAI-resistant variants with different NA mutations have shown different transmissibilities in ferrets (M. L. Herlocher, R. Truscon, S. Elias, H. Yen, N. A. Roberts, S. E. Ohmit, and A. S. Monto, J. Infect. Dis. 190:1627-1630, 2004). Molecular characterization of mutant viruses that have a homogeneous genetic background is required to determine the effect of single mutations at conserved NA residues. We generated recombinant viruses containing either the wild-type NA (RG WT virus) or a single amino acid change at NA residue 119 (RG E119V-NA virus) or 292 (RG R292K-NA virus) in the A/Wuhan/359/95 (H3N2) influenza virus background by reverse genetics. Both mutants showed decreased sensitivity to oseltamivir carboxylate, and the RG R292K-NA virus showed cross-resistance to zanamivir. We also observed differences between the two mutants in NA enzymatic activity and thermostability. The R292K mutation caused greater reduction of sialidase activity and thermostability than the E119V mutation. The NA defect caused by the R292K mutation was associated with compromised growth and transmissibility, whereas the growth and transmissibility of the RG E119V-NA virus were comparable to those of RG WT virus. Our results suggest that NAI-resistant influenza virus variants may differ substantially in fitness and transmissibility, depending on different levels of NA functional loss.

Receptor-binding properties of swine influenza viruses isolated and propagated in MDCK cells.

To study the receptor specificities of H1 and H3 influenza viruses isolated recently from pigs, we employed the analogues of natural receptors, namely sialyloligosaccharides conjugated with polyacrylamide in biotinylated and label free forms. All Madin-Darby canine kidney (MDCK) cell-propagated viruses with human H3 or classical swine H1 hemagglutinins bound only to Neu5Acalpha2-6Galbeta1-bearing polymers, and not to Neu5Acalpha2-3Galbeta1-bearing polymers. This receptor-binding pattern is typical for human influenza viruses and it differs from the previously described receptor-binding specificity of egg-adapted swine influenza viruses. Swine virus isolates with avian-like H1 and H3 hemagglutinins displayed distinct receptor specificity by binding to both Neu5Acalpha2-6Gal- and Neu5Acalpha2-3Gal-containing receptors. These viruses, as well as egg-adapted swine and turkey viruses with a classical swine HA, differed from the related duck viruses by increased affinity to sulfated sialyloligosaccaride, Su-SiaLe(x). Except for avian-like H3 viruses, none of the studied swine viruses bound to Neu5Gc-containing sialoglycopolymers, suggesting that binding to these sialic acid species abundantly expressed in pigs may not be essential for virus replication in this host.

Neuraminidase is important for the initiation of influenza virus infection in human airway epithelium.

Influenza virus neuraminidase (NA) plays an essential role in release and spread of progeny virions, following the intracellular viral replication cycle. To test whether NA could also facilitate virus entry into cell, we infected cultures of human airway epithelium with human and avian influenza viruses in the presence of the NA inhibitor oseltamivir carboxylate. Twenty- to 500-fold less cells became infected in drug-treated versus nontreated cultures (P < 0.0001) 7 h after virus application, indicating that the drug suppressed the initiation of infection. These data demonstrate that viral NA plays a role early in infection, and they provide further rationale for the prophylactic use of NA inhibitors.

Human and avian influenza viruses target different cell types in cultures of human airway epithelium.

The recent human infections caused by H5N1, H9N2, and H7N7 avian influenza viruses highlighted the continuous threat of new pathogenic influenza viruses emerging from a natural reservoir in birds. It is generally believed that replication of avian influenza viruses in humans is restricted by a poor fit of these viruses to cellular receptors and extracellular inhibitors in the human respiratory tract. However, detailed mechanisms of this restriction remain obscure. Here, using cultures of differentiated human airway epithelial cells, we demonstrated that influenza viruses enter the airway epithelium through specific target cells and that there were striking differences in this respect between human and avian viruses. During the course of a single-cycle infection, human viruses preferentially infected nonciliated cells, whereas avian viruses as well as the egg-adapted human virus variant with an avian virus-like receptor specificity mainly infected ciliated cells. This pattern correlated with the predominant localization of receptors for human viruses (2-6-linked sialic acids) on nonciliated cells and of receptors for avian viruses (2-3-linked sialic acids) on ciliated cells. These findings suggest that although avian influenza viruses can infect human airway epithelium, their replication may be limited by a nonoptimal cellular tropism. Our data throw light on the mechanisms of generation of pandemic viruses from their avian progenitors and open avenues for cell level-oriented studies on the replication and pathogenicity of influenza virus in humans.