Influenza Neuraminidase Subtype N1: Immunobiological Properties and Functional Assays for Specific Antibody Response.

Don Changsom,Pirom Noisumdaeng,Witthawat Wiriyarat,Hatairat Lerdsamran,Wanibtisam Masamae,Jarunee Prasertsopon,Bunpote Siridechadilok,Warunya Chakritbudsabong,Pilaipan Puthavathana,Stephen Mark Tompkins

doi:10.1371/journal.pone.0153183

Abstract

Influenza neuraminidase (NA) proteins expressed in TK− cells infected with recombinant vaccinia virus carrying NA gene of highly pathogenic avian influenza H5N1 virus or 2009 pandemic H1N1 (H1N1pdm) virus were characterized for their biological properties, i.e., cell localization, molecular weight (MW), glycosylation and sialidase activity.Immune sera collected from BALB/c mice immunized with these recombinant viruses were assayed for binding and functional activities of anti-NA antibodies. Recombinant NA proteins were found localized in cytoplasm and cytoplasmic membrane of the infected cells. H1N1pdm NA protein had MW at about 75 kDa while it was 55 kDa for H5N1 NA protein. Hyperglycosylation was more pronounced in H1N1pdm NA compared to H5N1 NA according to N-glycosidase F treatment. Three dimensional structures also predicted that H1N1 NA globular head contained 4 and that of H5N1 contained 2 potential glycosylation sites. H5N1 NA protein had higher sialidase activity than H1N1pdm NA protein as measured by both MUNANA-based assay and fetuin-based enzyme-linked lectin assay (ELLA). Plaque reduction assay demonstrated that anti-NA antibody could reduce number of plaques and plaque size through inhibiting virus release, not virus entry. Assay for neuraminidase-inhibition (NI) antibody by ELLA showed specific and cross reactivity between H5N1 NA and H1N1pdm NA protein derived from reverse genetic viruses or wild type viruses. In contrast, replication-inhibition assay in MDCK cells showed that anti-H1N1 NA antibody moderately inhibited viruses with homologous NA gene only, while anti-H5N1 NA antibody modestly inhibited the replication of viruses containing homologous NA gene and NA gene derived from H1N1pdm virus. Anti-H1N1 NA antibody showed higher titers of inhibiting virus replication than anti-H5N1 NA antibody, which are consistent with the results on reduction in plaque numbers and sizes as well as in inhibiting NA enzymatic activity. No assay showed cross reactivity with reassorted PR8 (H1N1) virus and H3N2 wild type viruses.

Highlights

Influenza virus contains two major glycoprotein spikes: neuraminidase (NA) and hemagglutinin (HA) on the virion surface
WB assay was performed to determine the molecular weight (MW) of NA proteins expressed in various virus-cell systems, i.e., TK− cells infected with recombinant vaccinia viruses and Madin-Darby canine kidney (MDCK) cells infected with parental wild-type viruses
TK− cells infected with rVac-H5N1 NA virus or MDCK cells infected with KAN-1 H5N1 wild type virus produced NA proteins with MWs of about 55 kDa

Summary

Introduction

Influenza virus contains two major glycoprotein spikes: neuraminidase (NA) and hemagglutinin (HA) on the virion surface. NA may contribute to the initial step of viral infection by promoting virus entry [3] or removing decoy receptors from human airway epithelial cells [4, 5], facilitating virus invasion. Epidemiological data in a human population suggested that pre-existing antibody to N2 NA induced by prior infections with the pandemic Asian influenza A (H2N2) virus in 1957 might have contributed to the partial protection against the pandemic caused by Hong Kong A (H3N2) virus in 1968 [7, 8]. Several studies employed animal models to explore the role of NA immunity against influenza virus. The NA proteins produced in those studies were not well characterized; and the methods to determine functional activities of NA antibody against homologous and heterologous NA strains and subtype are quite limited

Methods

Results

Conclusion