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      A novel hemagglutinin protein produced in bacteria protects chickens against H5N1 highly pathogenic avian influenza viruses by inducing H5 subtype-specific neutralizing antibodies

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          Abstract

          The highly pathogenic (HP) H5N1 avian influenza viruses (AIVs) cause a mortality rate of up to 100% in infected chickens and pose a permanent pandemic threat. Attempts to obtain effective vaccines against H5N1 HPAIVs have focused on hemagglutinin (HA), an immunodominant viral antigen capable of eliciting neutralizing antibodies. The vast majority of vaccine projects have been performed using eukaryotic expression systems. In contrast, we used a bacterial expression system to produce vaccine HA protein (bacterial HA) according to our own design. The HA protein with the sequence of the H5N1 HPAIV strain was efficiently expressed in Escherichia coli, recovered in the form of inclusion bodies and refolded by dilution between two chromatographic purification steps. Antigenicity studies showed that the resulting antigen, referred to as rH5- E. coli, preserves conformational epitopes targeted by antibodies specific for H5-subtype HAs, inhibiting hemagglutination and/or neutralizing influenza viruses in vitro. The proper conformation of this protein and its ability to form functional oligomers were confirmed by a hemagglutination test. Consistent with the biochemical characteristics, prime-boost immunizations with adjuvanted rH5- E. coli protected 100% and 70% of specific pathogen-free, layer-type chickens against challenge with homologous and heterologous H5N1 HPAIVs, respectively. The observed protection was related to the positivity in the FluAC H5 test (IDVet) but not to hemagglutination-inhibiting antibody titers. Due to full protection, the effective contact transmission of the homologous challenge virus did not occur. Survivors from both challenges did not or only transiently shed the viruses, as established by viral RNA detection in oropharyngeal and cloacal swabs. Our results demonstrate that vaccination with rH5- E. coli could confer control of H5N1 HPAIV infection and transmission rates in chicken flocks, accompanied by reduced virus shedding. Moreover, the role of H5 subtype-specific neutralizing antibodies in anti-influenza immunity and a novel correlate of protection are indicated.

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          Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution.

          The haemagglutinin glycoprotein of influenza virus is a trimer comprising two structurally distinct regions: a triple-stranded coiled-coil of alpha-helices extends 76 A from the membrane and a globular region of antiparallel beta-sheet, which contains the receptor binding site and the variable antigenic determinants, is positioned on top of this stem. Each subunit has an unusual loop-like topology, starting at the membrane, extending 135 A distally and folding back to enter the membrane.
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            Genetic characterization of the pathogenic influenza A/Goose/Guangdong/1/96 (H5N1) virus: similarity of its hemagglutinin gene to those of H5N1 viruses from the 1997 outbreaks in Hong Kong.

            Analysis of the sequences of all eight RNA segments of the influenza A/G oose/Guangdong/1/96 (H5N1) virus, isolated from a sick goose during an outbreak in Guangdong province, China, in 1996, revealed that the hemagglutinin (HA) gene of the virus was genetically similar to those of the H5N1 viruses isolated in Hong Kong in 1997. However, the remaining genes showed greater similarity to other avian influenza viruses. Notably, the neuraminidase gene did no have the 19-amino-acid deletion in the stalk region seen in the H5N1 Hong Kong viruses and the NS gene belonged to allele B, while that of the H5N1 Hong Kong viruses belonged to allele A. These data suggest that the H5N1 viruses isolated from the Hong Kong outbreaks derived their HA genes from a virus similar to the A/Goose/Guangdong/1/96 virus or shared a progenitor with this goose pathogen.
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              Novel Eurasian Highly Pathogenic Avian Influenza A H5 Viruses in Wild Birds, Washington, USA, 2014

              Novel Eurasian lineage avian influenza A(H5N8) virus has spread rapidly and globally since January 2014. In December 2014, H5N8 and reassortant H5N2 viruses were detected in wild birds in Washington, USA, and subsequently in backyard birds. When they infect commercial poultry, these highly pathogenic viruses pose substantial trade issues.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                17 February 2017
                2017
                : 12
                : 2
                : e0172008
                Affiliations
                [1 ]Institute of Biotechnology and Antibiotics, Warsaw, Poland
                [2 ]Department of Poultry Diseases, National Veterinary Research Institute, Puławy, Poland
                [3 ]Department of Recombinant Vaccines, Intercollegiate Faculty of Biotechnology, University of Gdańsk and Medical University of Gdańsk, Gdańsk, Poland
                University of South Dakota, UNITED STATES
                Author notes

                Competing Interests: All the authors (VS AR KF VC-A MK-B KŚ MO ZM BS GP & AP), except Katarzyna Domańska-Blicharz, have filed the Patent Applications P.408649 (2014-06-24), PCT/PL2015/050025 (2015-06-24): Influenza virus hemagglutinin protein as a vaccine antigen. The Patent Applications contain the findings reported in this paper. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The authors have declared that no competing interests exist.

                • Conceptualization: VS AR KŚ ZM.

                • Formal analysis: VS AR KŚ.

                • Funding acquisition: BS GP.

                • Investigation: AR KF VC-A MK-B MO KD-B.

                • Methodology: VS AR KŚ ZM AP.

                • Resources: KŚ ZM BS GP AP.

                • Visualization: VS.

                • Writing – original draft: VS.

                • Writing – review & editing: BS.

                Article
                PONE-D-16-23239
                10.1371/journal.pone.0172008
                5315377
                28212428
                2bc5c489-ee57-4709-8078-660e3c14ee16
                © 2017 Sączyńska et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 4 July 2016
                : 30 January 2017
                Page count
                Figures: 3, Tables: 1, Pages: 21
                Funding
                This work was supported by the Innovative Economy Operational Program, Grant No. WND-POIG.01.01.02-00-007/08-00, as a part of the project “Centre of medicinal product biotechnology. Package of innovative biopharmaceuticals for human and animal therapy and prophylactics” and Grant No. PBS2/A7/14/2014 (ID: 210068): “Influenza vaccine - innovative obtaining of subunit antigens”. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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                All relevant data are within the paper and its Supporting Information file. The sequence used in the studies is available in EpiFlu Database [ http://platform.gisaid.org]; Accession No. EPI156789.

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