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.

Evolutionary Dynamics of Avian Influenza Viruses Isolated from Wild Birds in Moscow.

Forty-five strains of AIVs were isolated from wild aquatic birds during their autumn migration through Moscow (Russia). The aim of this work is to study the dynamics of AIV genomes in their natural habitat. Viruses were isolated from fecal sample in embryonated chicken eggs; their complete genomes were sequenced, and a phylogenetic analysis was performed. The gene segments of the same lineage persisted over the years in the absence of persistence of complete viral genomes. The genes for internal proteins of the same lineage were often maintained by the viruses over few years; however, they were typically associated with the genes of novel HA and NA subtypes. Although frequent reassortment events were observed for any pair of internal genes, there was no reassortment between HA and NA segments. The differences in the persistence of phylogenetic lineages of surface and internal proteins and the different evolutionary strategy for these two types of genes of AIVs in primary hosts are discussed.

Phenotypic effects of mutations observed in the neuraminidase of human origin H5N1 influenza A viruses.

Global spread and regional endemicity of H5Nx Goose/Guangdong avian influenza viruses (AIV) pose a continuous threat for poultry production and zoonotic, potentially pre-pandemic, transmission to humans. Little is known about the role of mutations in the viral neuraminidase (NA) that accompanied bird-to-human transmission to support AIV infection of mammals. Here, after detailed analysis of the NA sequence of human H5N1 viruses, we studied the role of A46D, L204M, S319F and S430G mutations in virus fitness in vitro and in vivo. Although H5N1 AIV carrying avian- or human-like NAs had similar replication efficiency in avian cells, human-like NA enhanced virus replication in human airway epithelia. The L204M substitution consistently reduced NA activity of H5N1 and nine other influenza viruses carrying NA of groups 1 and 2, indicating a universal effect. Compared to the avian ancestor, human-like H5N1 virus has less NA incorporated in the virion, reduced levels of viral NA RNA replication and NA expression. We also demonstrate increased accumulation of NA at the plasma membrane, reduced virus release and enhanced cell-to-cell spread. Furthermore, NA mutations increased virus binding to human-type receptors. While not affecting high virulence of H5N1 in chickens, the studied NA mutations modulated virulence and replication of H5N1 AIV in mice and to a lesser extent in ferrets. Together, mutations in the NA of human H5N1 viruses play different roles in infection of mammals without affecting virulence or transmission in chickens. These results are important to understand the genetic determinants for replication of AIV in mammals and should assist in the prediction of AIV with zoonotic potential.

Molecular modeling and phylogenetic analyses highlight the role of amino acid 347 of the N1 subtype neuraminidase in influenza virus host range and interspecies adaptation.

The N1 neuraminidases (NAs) of avian and pandemic human influenza viruses contain tyrosine and asparagine, respectively, at position 347 on the rim of the catalytic site; the biological significance of this difference is not clear. Here, we used molecular dynamics simulation to model the effects of amino acid 347 on N1 NA interactions with sialyllacto-N-tetraoses 6'SLN-LC and 3'SLN-LC, which represent NA substrates in humans and birds, respectively. Our analysis predicted that Y347 plays an important role in the NA preference for the avian-type substrates. The Y347N substitution facilitates hydrolysis of human-type substrates by resolving steric conflicts of the Neu5Ac2-6Gal moiety with the bulky side chain of Y347, decreasing the free energy of substrate binding, and increasing the solvation of the Neu5Ac2-6Gal bond. Y347 was conserved in all N1 NA sequences of avian influenza viruses in the GISAID EpiFlu database with two exceptions. First, the Y347F substitution was present in the NA of a specific H6N1 poultry virus lineage and was associated with the substitutions G228S and/or E190V/L in the receptor-binding site (RBS) of the hemagglutinin (HA). Second, the highly pathogenic avian H5N1 viruses of the Gs/Gd lineage contained sporadic variants with the NA substitutions Y347H/D, which were frequently associated with substitutions in the HA RBS. The Y347N substitution occurred following the introductions of avian precursors into humans and pigs with N/D347 conserved during virus circulation in these hosts. Comparative evolutionary analysis of site 347 revealed episodic positive selection across the entire tree and negative selection within most host-specific groups of viruses, suggesting that substitutions at NA position 347 occurred during host switches and remained under pervasive purifying selection thereafter. Our results elucidate the role of amino acid 347 in NA recognition of sialoglycan substrates and emphasize the significance of substitutions at position 347 as a marker of host range and adaptive evolution of influenza viruses.

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.

Effects of Receptor Specificity and Conformational Stability of Influenza A Virus Hemagglutinin on Infection and Activation of Different Cell Types in Human PBMCs.

Humans can be infected by zoonotic avian, pandemic and seasonal influenza A viruses (IAVs), which differ by receptor specificity and conformational stability of their envelope glycoprotein hemagglutinin (HA). It was shown that receptor specificity of the HA determines the tropism of IAVs to human airway epithelial cells, the primary target of IAVs in humans. Less is known about potential effects of the HA properties on viral attachment, infection and activation of human immune cells. To address this question, we studied the infection of total human peripheral blood mononuclear cells (PBMCs) and subpopulations of human PBMCs with well characterized recombinant IAVs differing by the HA and the neuraminidase (NA) but sharing all other viral proteins. Monocytes and all subpopulations of lymphocytes were significantly less susceptible to infection by IAVs with avian-like receptor specificity as compared to human-like IAVs, whereas plasmacytoid dendritic cells (pDCs) and myeloid dendritic cells were equally susceptible to IAVs with avian-like and human-like receptor specificity. This tropism correlated with the surface expression of 2-3-linked sialic acids (avian-type receptors) and 2-6-linked sialic acids (human-type receptors). Despite a reduced infectivity of avian-like IAVs for PBMCs, these viruses were not less efficient than human-like IAVs in terms of cell activation as judged by the induction of cellular mRNA of IFN-α, CCL5, RIG-I, and IL-6. Elevated levels of IFN-α mRNA were accompanied by elevated IFN-α protein secretion in primary human pDC. We found that high basal expression in monocytes of antiviral interferon-induced transmembrane protein 3 (IFITM3) limited viral infection in these cells. siRNA-mediated knockdown of IFITM3 in monocytes demonstrated that viral sensitivity to inhibition by IFITM3 correlated with the conformational stability of the HA. Our study provides new insights into the role of host- and strain-specific differences of HA in the interaction of IAVs with human immune cells and advances current understanding of the mechanisms of viral cell tropism, pathogenesis and markers of virulence.

Characterization of changes in the hemagglutinin that accompanied the emergence of H3N2/1968 pandemic influenza viruses.

The hemagglutinin (HA) of A/H3N2 pandemic influenza viruses (IAVs) of 1968 differed from its inferred avian precursor by eight amino acid substitutions. To determine their phenotypic effects, we studied recombinant variants of A/Hong Kong/1/1968 virus containing either human-type or avian-type amino acids in the corresponding positions of HA. The precursor HA displayed receptor binding profile and high conformational stability typical for duck IAVs. Substitutions Q226L and G228S, in addition to their known effects on receptor specificity and replication, marginally decreased HA stability. Substitutions R62I, D63N, D81N and N193S reduced HA binding avidity. Substitutions R62I, D81N and A144G promoted viral replication in human airway epithelial cultures. Analysis of HA sequences revealed that substitutions D63N and D81N accompanied by the addition of N-glycans represent common markers of avian H3 HA adaptation to mammals. Our results advance understanding of genotypic and phenotypic changes in IAV HA required for avian-to-human adaptation and pandemic emergence.

Hemagglutinins of Avian Influenza Viruses Are Proteolytically Activated by TMPRSS2 in Human and Murine Airway Cells.

Cleavage of the influenza A virus (IAV) hemagglutinin (HA) by host proteases is indispensable for virus replication. Most IAVs possess a monobasic HA cleavage site cleaved by trypsin-like proteases. Previously, the transmembrane protease TMPRSS2 was shown to be essential for proteolytic activation of IAV HA subtypes H1, H2, H7, and H10 in mice. In contrast, additional proteases are involved in activation of certain H3 IAVs, indicating that HAs with monobasic cleavage sites can differ in their sensitivity to host proteases. Here, we investigated the role of TMPRSS2 in proteolytic activation of avian HA subtypes H1 to H11 and H14 to H16 in human and mouse airway cell cultures. Using reassortant viruses carrying representative HAs, we analyzed HA cleavage and multicycle replication in (i) lung cells of TMPRSS2-deficient mice and (ii) Calu-3 cells and primary human bronchial cells subjected to morpholino oligomer-mediated knockdown of TMPRSS2 activity. TMPRSS2 was found to be crucial for activation of H1 to H11, H14, and H15 in airway cells of human and mouse. Only H9 with an R-S-S-R cleavage site and H16 were proteolytically activated in the absence of TMPRSS2 activity, albeit with reduced efficiency. Moreover, a TMPRSS2-orthologous protease from duck supported activation of H1 to H11, H15, and H16 in MDCK cells. Together, our data demonstrate that in human and murine respiratory cells, TMPRSS2 is the major activating protease of almost all IAV HA subtypes with monobasic cleavage sites. Furthermore, our results suggest that TMPRSS2 supports activation of IAV with a monobasic cleavage site in ducks. IMPORTANCE Human infections with avian influenza A viruses upon exposure to infected birds are frequently reported and have received attention as a potential pandemic threat. Cleavage of the envelope glycoprotein hemagglutinin (HA) by host proteases is a prerequisite for membrane fusion and essential for virus infectivity. In this study, we identify the transmembrane protease TMPRSS2 as the major activating protease of avian influenza virus HAs of subtypes H1 to H11, H14 and H15 in human and murine airway cells. Our data demonstrate that inhibition of TMPRSS2 activity may provide a useful approach for the treatment of human infections with avian influenza viruses that should be considered for pandemic preparedness as well. Additionally, we show that a TMPRSS2-orthologous protease from duck can activate avian influenza virus HAs with a monobasic cleavage site and, thus, represents a potential virus-activating protease in waterfowl, the primary reservoir for influenza A viruses.

Diversity and Reassortment Rate of Influenza A Viruses in Wild Ducks and Gulls.

Influenza A viruses (IAVs) evolve via point mutations and reassortment of viral gene segments. The patterns of reassortment in different host species differ considerably. We investigated the genetic diversity of IAVs in wild ducks and compared it with the viral diversity in gulls. The complete genomes of 38 IAVs of H1N1, H1N2, H3N1, H3N2, H3N6, H3N8, H4N6, H5N3, H6N2, H11N6, and H11N9 subtypes isolated from wild mallard ducks and gulls resting in a city pond in Moscow, Russia were sequenced. The analysis of phylogenetic trees showed that stable viral genotypes do not persist from year to year in ducks owing to frequent gene reassortment. For comparison, similar analyses were carried out using sequences of IAVs isolated in the same period from ducks and gulls in The Netherlands. Our results revealed a significant difference in diversity and rates of reassortment of IAVs in ducks and gulls.

MD simulation of the interaction between sialoglycans and the second sialic acid binding site of influenza A virus N1 neuraminidase.

The neuraminidases (NAs) of avian influenza viruses (IAVs) contain a second sialic acid-binding site (2SBS), historically known as the hemadsorption site, which is separated from the sialyl-hydrolase catalytic site and serves to facilitate NA catalytic activity towards multivalent sialyl-capped glycoconjugates. Transmission and adaptation of avian IAVs to humans decreases hemadsorption and catalytic activities of the NA. Here, we report the molecular recognition features of the NA 2SBS of two pandemic H1N1 IAVs, A/Brevig Mission /1/1918 (BM18) and A/California/04/2009 (CA09), differing by their 2SBS activity. Using explicit solvent MD simulation, molecular mechanics, and glycosidic conformation analysis we initially analyzed the interactions of BM18 2SBS with two sialyllacto-N-tetraose pentasaccharides, 3'SLN-LC and 6'SLN-LC, which are models for the glycan receptors of IAVs in birds and humans, respectively. These studies characterize the binding specificity of BM18 2SBS towards human-type and avian-type receptors and identifies the key amino acids that affects binding. We next compared the interactions of the 2SBSs of BM18 and CA09 with 6'SLN-LC, revealing the critical effect of amino acid 372 on binding. Our results expand the current knowledge of the molecular features of NA 2SBSs and its alteration during the adaptation of avian IAVs to humans.