Expression and Characterization of Recombinant, Tetrameric and Enzymatically Active Influenza Neuraminidase for the Setup of an Enzyme-Linked Lectin-Based Assay.

Marua Prevato,Ilaria Ferlenghi,Roberta Cozzi,Domenico Maione,John Laird Telford,Sylvie Bertholet,Francois Legay,Giulia Anselmi,Fabiola Giusti,Ethan C Settembre,Alessandra Bonci,Yasushi Uematsu,Zhiping Ye

doi:10.1371/journal.pone.0135474

Marua Prevato, Ilaria Ferlenghi + Show 11 more

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https://doi.org/10.1371/journal.pone.0135474

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Journal: PloS one	Publication Date: Aug 17, 2015
Citations: 19	License type: CC BY 4.0

Affiliation: Novartis (Italy), University of Siena

Abstract

Developing a universal influenza vaccine that induces broad spectrum and longer-term immunity has become an important potentially achievable target in influenza vaccine research and development. Hemagglutinin (HA) and neuraminidase (NA) are the two major influenza virus antigens. Although antibody responses against influenza virus are mainly directed toward HA, NA is reported to be more genetically stable; hence NA-based vaccines have the potential to be effective for longer time periods. NA-specific immunity has been shown to limit the spread of influenza virus, thus reducing disease symptoms and providing cross-protection against heterosubtypic viruses in mouse challenge experiments.The production of large quantities of highly pure and stable NA could be beneficial for the development of new antivirals, subunit-based vaccines, and novel diagnostic tools. In this study, recombinant NA (rNA) was produced in mammalian cells at high levels from both swine A/California/07/2009 (H1N1) and avian A/turkey/Turkey/01/2005 (H5N1) influenza viruses. Biochemical, structural, and immunological characterizations revealed that the soluble rNAs produced are tetrameric, enzymatically active and immunogenic, and finally they represent good alternatives to conventionally used sources of NA in the Enzyme-Linked Lectin Assay (ELLA).

Highlights

Influenza virus infections affect mainly the upper respiratory tract and occasionally lung, and are responsible for high fever, cough, headache, muscle and joint pain
The globular head domains of both swine A/California/07/2009 (H1N1) and avian A/turkey/ Turkey/1/2005 (H5N1) NAs plus extra amino acids at the C-terminus of the stalk regions were fused to an artificial N-terminal stem composed of a secretion murine Ig-κ chain leader sequence, a 6xHis-tag, and a tetrabrachion tetramerization domain (Fig 1A)
Based on the comparisons of the elution volumes (Ev) of both swine and avian recombinant NA (rNA) with the Ev of molecular weight (MW) standards run in the same conditions, the calculated MW of these species consistent with a NA tetramer (%240 kDa)

Summary

Introduction

Influenza virus infections affect mainly the upper respiratory tract and occasionally lung, and are responsible for high fever, cough, headache, muscle and joint pain. Functional and biochemical characterization of rNAs, in addition to 3D reconstruction of NA’s structure corroborated the successful production of correctly folded NA tetramers in the mammalian expression system, as alternative to NA purification from live virus or by baculovirus expression. These rNAs have been exploited as the NA source in an rNA-based functional ELLA assay

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