title: A novel hemagglutinin protein produced in bacteria protects chickens against H5N1 highly pathogenic avian influenza viruses by inducing H5 subtype-specific neutralizing antibodies.
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.
authors.affiliation: Institute of Biotechnology and Antibiotics, Warsaw, Poland.
authors.forename: Violetta
authors.initials: V
authors.lastname: Sączyńska
authors.affiliation: Institute of Biotechnology and Antibiotics, Warsaw, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Romanik
authors.affiliation: Institute of Biotechnology and Antibiotics, Warsaw, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Florys
authors.affiliation: Institute of Biotechnology and Antibiotics, Warsaw, Poland.
authors.forename: Violetta
authors.initials: V
authors.lastname: Cecuda-Adamczewska
authors.affiliation: Institute of Biotechnology and Antibiotics, Warsaw, Poland.
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Kęsik-Brodacka
authors.affiliation: Department of Poultry Diseases, National Veterinary Research Institute, Puławy, Poland.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Śmietanka
authors.affiliation: Department of Poultry Diseases, National Veterinary Research Institute, Puławy, Poland.
authors.forename: Monika
authors.initials: M
authors.lastname: Olszewska
authors.affiliation: Department of Poultry Diseases, National Veterinary Research Institute, Puławy, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Domańska-Blicharz
authors.affiliation: Department of Poultry Diseases, National Veterinary Research Institute, Puławy, Poland.
authors.forename: Zenon
authors.initials: Z
authors.lastname: Minta
authors.affiliation: Department of Recombinant Vaccines, Intercollegiate Faculty of Biotechnology, University of Gdańsk and Medical University of Gdańsk, Gdańsk, Poland.
authors.forename: Bogusław
authors.initials: B
authors.lastname: Szewczyk
authors.affiliation: Institute of Biotechnology and Antibiotics, Warsaw, Poland.
authors.forename: Grażyna
authors.initials: G
authors.lastname: Płucienniczak
authors.affiliation: Institute of Biotechnology and Antibiotics, Warsaw, Poland.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Płucienniczak
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology*; D000918:Antibody Specificity; D002645:Chickens; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology*; D006388:Hemagglutinins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology*; D056724:Immunity, Humoral; D053124:Influenza A Virus, H5N1 Subtype / Q000276:immunology*; D013045:Species Specificity
chemical_list: D057134:Antibodies, Neutralizing; D029968:Escherichia coli Proteins; D006388:Hemagglutinins