Abstract
BackgroundA novel influenza virus, subtype H7N9, circulated through China in 2013–2014. Its higher rates of human infection in a wide range of locations within China and the associated increased likelihood of human-to-human transmission have caused global concern. Recombinant subunit vaccines provide safe and targeted protection against viral infections. However, the protective efficacy of recombinant subunit vaccines tends to be less potent than vaccines made from whole viruses. Studies have shown that bacterial flagellin has strong adjuvant activity and induces protective immune responses.ResultsIn this study, we used overlap-PCR to generate an H7N9 influenza recombinant subunit vaccine that fused the globular head domain (HA1-2, aa 62–284) of the protective hemagglutinin (HA) antigen with the potent TLR5 ligand, Salmonella typhimurium flagellin (fliC). The resulting fusion protein, HA1-2-fliC, was efficiently expressed in an Escherichia coli prokaryotic expression system, and Western blotting and TLR5-stimulating activity analysis confirmed that the HA1-2-fliC moiety could be faithfully refolded to take on the native HA and fliC conformations. In a C3H/HeJ mouse model of intraperitoneal vaccination, the fusion protein elicited significant and robust HA1-2-specific serum IgG titers, maintaining high levels for at least 3 months in the vaccinated animals, and induced similar levels of HA1-2-specific IgG1 and IgG2a that were detectable 12 days after the third immunization. HA1-2-fliC was also found to be capable of triggering the production of neutralizing antibodies, as assessed by measuring hemagglutination inhibition titers.ConclusionsWe conclude that immunization with HA1-2-fliC induces potent HA1-2-specific responses, offering significant promise for the development of a successful recombinant subunit vaccine for avian influenza A (H7N9).
Highlights
A novel influenza virus, subtype H7N9, circulated through China in 2013–2014
We demonstrated that the mice immunized intraperitoneally with the HA1-2-fliC fusion protein developed significantly higher HA1-2-specific serum IgG and hemagglutination inhibition (HAI) titers compared with the mice injected with HA1-2 alone
Horseradish peroxidase (HRP)-conjugated goat antimouse IgG, IgG1, or IgG2a (Invitrogen, USA) were incubated for 1 h at 37 °C as the secondary antibody. 3, 3′, 5, 5′-tetramethybenzidine was used as a substrate to Expression and characterization of the recombinant proteins To create a fusion protein of HA1-2 and fliC, a fragment containing the HA1-2 gene fused to the N-terminus of fliC gene was designed (Fig. 2)
Summary
A novel influenza virus, subtype H7N9, circulated through China in 2013–2014. Its higher rates of human infection in a wide range of locations within China and the associated increased likelihood of human-to-human transmission have caused global concern. Avian-origin influenza A (H7N9) virus emerged as a human pathogen in China in spring 2013 and, as of October 2014, it caused 453 human cases and 175 deaths [1]. At this rate, it will soon match or surpass the burden of avian influenza A (H5N1) (676 cases, as of December 2014) [2]. Live-attenuated, inactivated whole virus or split vaccines produced in embryonated hens’ eggs are used to control influenza. Production of these types of H7N9 influenza vaccines often has several hurdles [4]. Some laboratories have studied the liveattenuated H7N9 virus vaccine candidate [5] and other H7 subtype virus vaccines [6, 7] for their ability to protect from H7N9 virus infection
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