Inclusion of membrane-anchored LTB or flagellin protein in H5N1 virus-like particles enhances protective responses following intramuscular and oral immunization of mice
Introduction
The continued circulation of highly pathogenic avian influenza (HPAI) viruses poses a significant public health threat. The H5N1 subtype of HPAI was first shown to infect humans in 1997 during an outbreak in poultry in China. As of May 3, 2016, 856 confirmed human infections and 452 fatalities have occurred (http://www.fao.org/ag/againfo/programmes/en/empres/news_130516c.html). Since 2003, HPAI H5N1 viruses have become highly enzootic and evolved such that they are now classified into multiple clades based on phylogenetic analyses of the HA gene, and clades 2.3.2.1 and 2.3.4 have become the predominant lineage [1], [2] (http://www.who.int/entity/influenza/vaccines/virus/201402_h5h7h9h10_vaccinevirusupdate.pdf). While the pandemic risk posed by HPAI H5N1 viruses is widely appreciated, predicting the clade most likely to give rise to a virus capable of widespread human infection is difficult; thus, determining which vaccines to stockpile in the event of an influenza pandemic of avian origin is not straightforward [3].
Virus-like particles (VLPs) are reported to be a useful platform for the generation of vaccines against a variety of pathogenic viruses [4], [5], [6]. Recombinant noninfectious VLPs produced using a baculovirus expression system represent a promising system for the production of vaccines against H5N1 viruses, as they circumvent the need for handling replication-competent influenza viruses during the manufacturing process. We previously demonstrated that intramuscular immunization of mice with VLPs produced using a baculovirus expression system containing the HA, NA, and M1 proteins of A/meerkat/Shanghai/SH-1/2012 (clade 2.3.2.1) elicited effective protection against H5 HPAI viruses of different clades [7].
Mucosal administration of vaccines, including via oral and intranasal routes, stimulates both mucosal and systemic immune responses [8]. As intranasal vaccines may have a detrimental effect on people with asthma, chronic pulmonary or cardiovascular disorders and other reactive airway diseases [9], oral vaccines may represent the safest alternative [10]. Additionally, evidence suggests that oral vaccines can result in the transcytosis of molecules across the cells into circulation and prevent infection in the lungs [11], [12]. The Escherichia coli heat-labile enterotoxin B subunit (LTB) protein is widely recognized as a robust immunological adjuvant [13] and can serve as an efficient carrier molecule for target antigens [14]. LTB binds to the monosialotetrahexosylganglioside (GM1) ganglioside receptor on the surface of mammalian cells, and receptor binding is essential for the adjuvant properties of LTB [15]. Similarly, bacterial flagellin (Flic), the structural component of bacterial flagellar filaments, has been shown to possess immunostimulatory properties. The recognition of Flic by Toll-like receptor 5 (TLR5) in human immature dendritic cells (DCs) induces the expression of a variety of chemokines and cytokines. Based on these properties, LTB and Flic have been pursued as potent oral vaccine adjuvants [16], [17], [18].
In this report, we describe the generation of H5N1 VLPs composed of viral HA, NA, and M1 proteins and membrane-anchored forms of the Escherichia coli LTB or Flic protein using a baculovirus expression system. Mice were intramuscularly (IM) or orally immunized with different VLP preparations (with or without LTB or Flic) to assess humoural and cellular responses generated in response to vaccination. We also evaluated the abilities of the different VLP preparations to elicit protective immune responses in the context of lethal challenge with homologous and heterologous H5N1 viruses.
Section snippets
Viruses and cells
The HPAI H5N1 viruses A/meerkat/Shanghai/SH-1/2012 (SH-1; clade 2.3.2.1) and A/duck/Jilin/JL-SIV/2013 (JL-SIV; clade 2.3.4) were isolated and stored at the Changchun Veterinary Research Institute. Spodoptera frugiperda Sf9 insect cells (Invitrogen, USA) were grown in TMN insect medium (Appilchem, Germany) at 27 °C. Madin-Darby canine kidney (MDCK) cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% foetal bovine serum. All experiments using highly pathogenic
Expression and characterization of A/meerkat/Shanghai/SH-1/2012 VLPs with or without LTB or Flic protein
rBVs were constructed to express the HA, NA, and M1 proteins of A/meerkat/Shanghai/SH-1/2012 (H5N1) and membrane-anchored versions of the LTB or Flic protein. Infection of insect cells with rBVs resulted in the expression of influenza proteins as determined by immunofluorescence analysis following incubation with an anti-influenza polyclonal serum, whereas no signal was observed in uninfected cells (data not shown). As expected, culture supernatants from cells infected with rBVs engineered to
Discussion
The emergence of new subclades of H5N1 influenza virus in poultry represents a potential source of future pandemic viruses. Thus, the development of safe and broadly protective vaccines that are effective against a range of different H5 clades is a high priority for pandemic preparedness [2], [22]. However, serological studies on confirmed human cases of influenza A (H5N1) infection suggest that immunological cross-protection between influenza A (H5N1) clades may be limited. The production of
Author contributions
ZR, YG, and XX designed the experiments. ZR, YZ, JL, XJ, LM, TW, WS, KZ, XS, ZY, YL, NF, and HW performed the experiment. ZR, YZ, JL, KZ, XS, ZY, SY, ZY, ZW, YG, and XX analyzed the data. ZR and YG wrote the manuscript. All authors reviewed the manuscript.
Acknowledgments
This work was supported by the National Key R&D Program of China (2017YFD0501705), the National Major Infectious Projects of China (2016ZX10004502-005), the National Science and Technology Major Project of China (2014ZX09102044-007) and the Natural Science Foundation of China (No. U1504823).
Conflicts of interest statement
The authors have no conflicts of interest, and all authors approved the manuscript for publication.
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Flagellin-expressing virus-like particles exhibit adjuvant effects on promoting IgG isotype-switched long-lasting antibody induction and protection of influenza vaccines in CD4-deficient mice
2019, VaccineCitation Excerpt :Flagellin was reported to activate human DCs, resulting in up-regulation of maturation markers and chemokine production via a TLR5 signaling [34], suggesting its potential vaccine adjuvant mechanism. Also, previous studies demonstrated that influenza VLPs co-presenting flagellin and hemagglutinin antigens were more effective in inducing virus-specific Th1-biased humoral immune responses than VLP vaccines without flagellin [24,25,35,36]. Herein, we investigated whether flagellin only expressing VLP (FliC-VLP) would be an independent and effective vaccine adjuvant to promote the immune responses to co-administered vaccine antigens in wild-type and mutant mice in comparison with soluble flagellin.
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These authors contributed equally to this work.