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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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          Most cited references 44

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          Antibody recognition of a highly conserved influenza virus epitope.

          Influenza virus presents an important and persistent threat to public health worldwide, and current vaccines provide immunity to viral isolates similar to the vaccine strain. High-affinity antibodies against a conserved epitope could provide immunity to the diverse influenza subtypes and protection against future pandemic viruses. Cocrystal structures were determined at 2.2 and 2.7 angstrom resolutions for broadly neutralizing human antibody CR6261 Fab in complexes with the major surface antigen (hemagglutinin, HA) from viruses responsible for the 1918 H1N1 influenza pandemic and a recent lethal case of H5N1 avian influenza. In contrast to other structurally characterized influenza antibodies, CR6261 recognizes a highly conserved helical region in the membrane-proximal stem of HA1 and HA2. The antibody neutralizes the virus by blocking conformational rearrangements associated with membrane fusion. The CR6261 epitope identified here should accelerate the design and implementation of improved vaccines that can elicit CR6261-like antibodies, as well as antibody-based therapies for the treatment of influenza.
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            Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes.

            A real-time reverse transcriptase PCR (RRT-PCR) assay based on the avian influenza virus matrix gene was developed for the rapid detection of type A influenza virus. Additionally, H5 and H7 hemagglutinin subtype-specific probe sets were developed based on North American avian influenza virus sequences. The RRT-PCR assay utilizes a one-step RT-PCR protocol and fluorogenic hydrolysis type probes. The matrix gene RRT-PCR assay has a detection limit of 10 fg or approximately 1,000 copies of target RNA and can detect 0.1 50% egg infective dose of virus. The H5- and H7-specific probe sets each have a detection limit of 100 fg of target RNA or approximately 10(3) to 10(4) gene copies. The sensitivity and specificity of the real-time PCR assay were directly compared with those of the current standard for detection of influenza virus: virus isolation (VI) in embryonated chicken eggs and hemagglutinin subtyping by hemagglutination inhibition (HI) assay. The comparison was performed with 1,550 tracheal and cloacal swabs from various avian species and environmental swabs obtained from live-bird markets in New York and New Jersey. Influenza virus-specific RRT-PCR results correlated with VI results for 89% of the samples. The remaining samples were positive with only one detection method. Overall the sensitivity and specificity of the H7- and H5-specific RRT-PCR were similar to those of VI and HI.
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              A neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins.

              The isolation of broadly neutralizing antibodies against influenza A viruses has been a long-sought goal for therapeutic approaches and vaccine design. Using a single-cell culture method for screening large numbers of human plasma cells, we isolated a neutralizing monoclonal antibody that recognized the hemagglutinin (HA) glycoprotein of all 16 subtypes and neutralized both group 1 and group 2 influenza A viruses. Passive transfer of this antibody conferred protection to mice and ferrets. Complexes with HAs from the group 1 H1 and the group 2 H3 subtypes analyzed by x-ray crystallography showed that the antibody bound to a conserved epitope in the F subdomain. This antibody may be used for passive protection and to inform vaccine design because of its broad specificity and neutralization potency.
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                Author and article information

                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.

                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
                PONE-D-16-23239
                10.1371/journal.pone.0172008
                5315377
                28212428
                © 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.

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                Figures: 3, Tables: 1, Pages: 21
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                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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