Characterization of the neuraminidase of the H1N1/09 pandemic influenza virus

The envelope of influenza A viruses contains two large antigens, hemagglutinin (HA) and neuraminidase (NA). Conventional influenza virus vaccines induce neutralizing antibodies that are predominantly directed to the HA globular head, a domain that is subject to extensive antigenic drift. Antibodies directed to NA are induced at much lower levels, probably as a consequence of the immunodominance of the HA antigen. Although antibodies to NA may affect virus release by inhibiting the sialidase function of the glycoprotein, the antigen has been largely neglected in past vaccine design. In this study, we characterized the protective properties of monospecific immune sera that were generated by vaccination with recombinant RNA replicon particles encoding NA. These immune sera inhibited hemagglutination in an NA subtype-specific and HA subtype-independent manner and interfered with infection of MDCK cells. In addition, they inhibited the sialidase activities of various influenza viruses of the same and even different NA subtypes. With this, the anti-NA immune sera inhibited the spread of H5N1 highly pathogenic avian influenza virus and HA/NA-pseudotyped viruses in MDCK cells in a concentration-dependent manner. When chickens were immunized with NA recombinant replicon particles and subsequently infected with low-pathogenic avian influenza virus, inflammatory serum markers were significantly reduced and virus shedding was limited or eliminated. These findings suggest that NA antibodies can inhibit virus dissemination by interfering with both virus attachment and egress. Our results underline the potential of high-quality NA antibodies for controlling influenza virus replication and place emphasis on NA as a vaccine antigen. IMPORTANCEThe neuraminidase of influenza A viruses is a sialidase that acts as a receptor-destroying enzyme facilitating the release of progeny virus from infected cells. Here, we demonstrate that monospecific anti-NA immune sera inhibited not only sialidase activity, but also influenza virus hemagglutination and infection of MDCK cells, suggesting that NA antibodies can interfere with virus attachment. Inhibition of both processes, virus release and virus binding, may explain why NA antibodies efficiently blocked virus dissemination in vitro and in vivo. Anti-NA immune sera showed broader reactivity than anti-HA sera in hemagglutination inhibition tests and demonstrated cross-subtype activity in sialidase inhibition tests. These remarkable features of NA antibodies highlight the importance of the NA antigen for the development of next-generation influenza virus vaccines.

Section: Discussionmentioning

confidence: 99%

Biological and Protective Properties of Immune Sera Directed to the Influenza Virus Neuraminidase

Halbherr¹,

Ludersdorfer²,

Ricklin³

et al. 2015

“…pHW2000 plasmid was kindly provided by Erich Hoffmann and Robert Webster (St. Jude Children's Research Hospital, Memphis, TN). pHW2000 plasmids containing HA genes of A/Hong Kong/1/ 1968 (H3N2) and A/Hamburg/5/2009 (H1N1) were described previously (31,34). pHW2000 plasmid containing HA gene of A/swine/Marseille/ 2260/1980 (H1N1) was prepared as described previously (32).…”

Section: Cells Mdck and Hela Cells Were Propagated In Dulbecco=s Modmentioning

confidence: 99%

H1N1 Swine Influenza Viruses Differ from Avian Precursors by a Higher pH Optimum of Membrane Fusion

Baumann

Kouassi

Foni

et al. 2016

The H1N1 Eurasian avian-like swine (EAsw) influenza viruses originated from an avian H1N1 virus. To characterize potential changes in the membrane fusion activity of the hemagglutinin (HA) during avian-to-swine adaptation of the virus, we studied EAsw viruses isolated in the first years of their circulation in pigs and closely related contemporary H1N1 viruses of wild aquatic birds. Compared to the avian viruses, the swine viruses were less sensitive to neutralization by lysosomotropic agent NH 4 Cl in MDCK cells, had a higher pH optimum of hemolytic activity, and were less stable at acidic pH. Eight amino acid substitutions in the HA were found to separate the EAsw viruses from their putative avian precursor; four substitutions-T49 2 S, N72 2 D, R75 2 K, and S113 2 F-were located in the structural regions of the HA2 subunit known to play a role in acid-induced conformational transition of the HA. We also studied low-pH-induced syncytium formation by cell-expressed HA proteins and found that the HAs of the 1918, 1957, 1968, and 2009 pandemic viruses required a lower pH for fusion induction than did the HA of a representative EAsw virus. Our data show that transmission of an avian H1N1 virus to pigs was accompanied by changes in conformational stability and fusion promotion activity of the HA. We conclude that distinctive host-determined fusion characteristics of the HA may represent a barrier for avian-to-swine and swine-to-human transmission of influenza viruses. IMPORTANCEContinuing cases of human infections with zoonotic influenza viruses highlight the necessity to understand which viral properties contribute to interspecies transmission. Efficient binding of the HA to cellular receptors in a new host species is known to be essential for the transmission. Less is known about required adaptive changes in the membrane fusion activity of the HA. Here we show that adaptation of an avian influenza virus to pigs in Europe in 1980s was accompanied by mutations in the HA, which decreased its conformational stability and increased pH optimum of membrane fusion activity. This finding represents the first formal evidence of alteration of the HA fusion activity/stability during interspecies transmission of influenza viruses under natural settings.

“…The BAL fluid antiviral activity shown in this study is consistent with these inhibitors and may be the result of the combined effect of these inhibitors. The NA activity of the 2009 H1N1 influenza virus was previously shown to be stronger than that of seasonal influenza viruses for both ␣2,3-and ␣2,6-linked sialic acid (30). In general, avian influenza viruses have high NA activity against ␣2,3-linked sialic acid but low activity against ␣2,6-linked sialic acid, whereas swine influenza viruses have high NA activity against ␣2,6-linked sialic acid but low activity against ␣2,3-linked sialic acid.…”

Section: Discussionmentioning

confidence: 96%

“…Seasonal influenza viruses, on the other hand, have relatively lower activity against both ␣2,3-and ␣2,6-linked sialic acid (30). In contrast, the 2009 H1N1 virus was shown to have high NA activity against ␣2,3-and ␣2,6-linked sialic acid at a level comparable to those of avian and swine influenza viruses, respectively (30). This is in agreement with our data showing the role of NA in the resistance of pandemic H1N1 virus to human BAL fluid.…”

Section: Discussionmentioning

confidence: 99%

“…In general, avian influenza viruses have high NA activity against ␣2,3-linked sialic acid but low activity against ␣2,6-linked sialic acid, whereas swine influenza viruses have high NA activity against ␣2,6-linked sialic acid but low activity against ␣2,3-linked sialic acid. Seasonal influenza viruses, on the other hand, have relatively lower activity against both ␣2,3-and ␣2,6-linked sialic acid (30). In contrast, the 2009 H1N1 virus was shown to have high NA activity against ␣2,3-and ␣2,6-linked sialic acid at a level comparable to those of avian and swine influenza viruses, respectively (30).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Neuraminidase Activity and Resistance of 2009 Pandemic H1N1 Influenza Virus to Antiviral Activity in Bronchoalveolar Fluid

Ruangrung

Suptawiwat

Maneechotesuwan

et al. 2016

Human bronchoalveolar fluid is known to have anti-influenza activity. It is believed to be a frontline innate defense against the virus. Several antiviral factors, including surfactant protein D, are believed to contribute to the activity. The 2009 pandemic H1N1 influenza virus was previously shown to be less sensitive to surfactant protein D. Nevertheless, whether different influenza virus strains have different sensitivities to the overall anti-influenza activity of human bronchoalveolar fluid was not known. We compared the sensitivities of 2009 pandemic H1N1, seasonal H1N1, and seasonal H3N2 influenza virus strains to inhibition by human bronchoalveolar lavage (BAL) fluid. The pandemic and seasonal H1N1 strains showed lower sensitivity to human BAL fluid than the H3N2 strains. The BAL fluid anti-influenza activity could be enhanced by oseltamivir, indicating that the viral neuraminidase (NA) activity could provide resistance to the antiviral defense. In accordance with this finding, the BAL fluid antiinfluenza activity was found to be sensitive to sialidase. The oseltamivir resistance mutation H275Y rendered the pandemic H1N1 virus but not the seasonal H1N1 virus more sensitive to BAL fluid. Since only the seasonal H1N1 but not the pandemic H1N1 had compensatory mutations that allowed oseltamivir-resistant strains to maintain NA enzymatic activity and transmission fitness, the resistance to BAL fluid of the drug-resistant seasonal H1N1 virus might play a role in viral fitness. IMPORTANCEHuman airway secretion contains anti-influenza activity. Different influenza strains may vary in their susceptibilities to this antiviral activity. Here we show that the 2009 pandemic and seasonal H1N1 influenza viruses were less sensitive to human bronchoalveolar lavage (BAL) fluid than H3N2 seasonal influenza virus. The resistance to the pulmonary innate antiviral activity of the pandemic virus was determined by its neuraminidase (NA) gene, and it was shown that the NA inhibitor resistance mutation H275Y abolished this resistance of the pandemic H1N1 but not the seasonal H1N1 virus, which had compensatory mutations that maintained the fitness of drug-resistant strains. Therefore, the innate respiratory tract defense may be a barrier against NA inhibitor-resistant mutants, and evasion of this defense may play a role in the emergence and spread of drug-resistant strains.