Influenza virus‐like particles harboring H9N2 HA and NA proteins induce a protective immune response in chicken

Background Avian influenza viruses represent a growing threat of an influenza pandemic. The co‐circulation of multiple H9N2 genotypes over the past decade has been replaced by one predominant genotype—G57 genotype, which displays a changed antigenicity and improved adaptability in chickens. Effective H9N2 subtype avian influenza virus vaccines for poultry are urgently needed. Objective In this study, we constructed H9N2 subtype avian influenza virus‐like particle (VLP) and evaluated its protective efficacy in specific pathogen‐free (SPF) chickens to lay the foundation for developing an effective vaccine against influenza viruses. Methods Expression of influenza proteins in VLPs was confirmed by Western blot, hemagglutination inhibition (HI), and neuraminidase inhibition (NI). The morphology was observed by electron microscopy. A group of 15 three‐week‐old SPF chickens was divided into three subgroups of five chickens immunized with VLP, commercial vaccine, and PBS. Challenge study was performed to evaluate efficacy of VLP vaccine. Results and Conclusions The hemagglutinin (HA) and neuraminidase (NA) proteins were co‐expressed in the infected cells, self‐assembled, and were released into the culture medium in the form of VLPs of diameter ~80 nm. The VLPs exhibited some functional characteristics of a full influenza virus, including hemagglutination and neuraminidase activity. In SPF chickens, the VLPs elicited serum antibodies specific for H9N2 and induced a higher HI titer (as detected by a homologous antigen) than did a commercial H9N2 vaccine (A/chicken/Shanghai/F/1998). Viral shedding from VLP vaccine subgroup was reduced compared with commercial vaccine subgroup and control subgroup.


| INTRODUCTION
The avian H9N2 influenza virus has been endemic among domestic poultry in Asia since the early 1990s. 1,2 It readily infects both chicken and duck, as well as the minor poultry species quail, pheasant, partridge, pigeon, silky chicken and chukar. 3,4 In 19993,4 In , 20033,4 In , from 20073,4 In to 2009, and 2013 human infection with H9N2 occurred in Hong Kong, Shenzhen, and Hunan, China. [5][6][7][8] Recently, WHO have reported that three human cases of H9N2 infections have been identified in China from September 27, 2016, to February 27, 2017 Its occasional transmission to humans has raised the possibility of a damaging pandemic spreading through an immunologically naive population. 9,10 The conventional production of avian influenza vaccines is complicated by the need for a large number of embryonated chicken eggs and disposal facilities. Studies of virus-like particles (VLPs) in animals and humans indicate that an influenza vaccine platform designed to present both full-length HA and NA antigens in VLP form may better emulate native presentation of these antigens to the immune system, yet at the same time avoid the safety concerns posed by a live, replicating agent. 11 Moreover, it was reported that the heterologous coexpression of the H3N2 genes HA, NA, M1, and M2 in insect cells has been shown to result in the self-assembly of VLPs, which act protectively against H3N2 in mice. 12,13 The development of an H9N2 influenza VLP vaccine effective in BALB/c mice requires the expression of only three viral proteins, namely HA, NA, and M1. 14 Thus, VLPs which resemble infectious virus particles with respect to structure and morphology have been suggested as novel vaccine candidates against various viral infections. 15 Live poultry markets and farms in Shanghai have been under surveillance since 2006; during this time, H9N2 has been detected in both poultry and swine. The co-circulation of multiple H9N2 genotypes over the past decade has led to the emergence of the G57 genotype, which displays a changed antigenicity and improved adaptability in chickens. 16 It has become predominant in vaccinated farm chickens in China, causing widespread influenza outbreaks before the H7N9 viruses emerged in humans; the latter viruses have inherited their internal genes from G57 genotype strains. In a previous study, the immunity efficacy of the currently used vaccine (A/chicken/shanghai/F/1998) against H9N2 has been evaluated and found unable to provide complete vaccine protection. 17 Here, the HA and NA genes from a strain belonging to G57 genotype were used to construct a potential VLP-based vaccine to protect against H9N2.

| Ethics statement
The conduct of this study followed animal welfare guidelines set out by the World Organization for Animal Health and was approved by the Shanghai Municipal Commission of Agriculture (Permit number: 2013 18 ).

| Cloning of HA and NA genes
Viral RNA was extracted from a 200 μL volume of a centrifuged suspension of H9N2 virus (A/chicken/Shanghai/06/2015) using a QIAamp ® MinElute ® Virus Spin kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. Reverse transcription PCR was performed using a One-Step RT-PCR system (Takara Bio Inc., Dalian, China) based on gene-specific oligonucleotide primers. Following the RT-PCR, the resulting cDNA sequences (containing the H9N2 HA and NA genes) were cloned into the pFastBac™ dual vector (Invitrogen, Waltham, MA, USA). The HA and NA sequences were found to be identical to those deposited in GenBank (respectively, KU720440 and KU720446).

| Generation of recombinant baculoviruses
The HA gene was represented by a 1.  When HI titers to homologous virus reached 2 8 -2 10 , blood from the four chickens was mixed. H9N2-specific serum (A/chicken/ Shanghai/06/2015) was inactivated at 60°C for 10 minutes and stored at −30°C until use.

| Production of H9N2 VLP and preparation of VLP vaccine
As the VLP antigen used was not sucrose-purified (to reduce production costs), its antigen content was quantified in terms of hemagglutination units (HAU) as described in the OIE manual, 18 rather than by measuring the total protein concentration. 19

| Electron microscopy
The culture medium containing VLPs was treated for 24 hour at 4°C with 2% glutaraldehyde in phosphate-buffered saline (PBS) (pH 7.2), adsorbed on a freshly discharged plastic/carbon-coated grid, which was then rinsed in deionized water (East China Normal University, Shanghai, China). The grids were finally stained with 2% sodium phosphotungstate (pH 6.5), and the VLPs were observed by transmission electron microscopy at a magnification of 6000-100 000×.

| Neuraminidase assay
Neuraminidase activity was determined using a Neuraminidase Assay Kit (Sigma), according to the supplier's protocol.

| Statistical analysis
Statistical comparisons between vaccinated and nonvaccinated chickens were performed using Fisher's exact test. An analysis of variance using the Tukey-Kramer post-test was used to compare virus shedding titers. Statistical significance was assigned to differences applying a P-value threshold of .05.

| Expression of VLP vaccine for influenza A/ chicken/Shanghai/06/2015 (H9N2) virus
The VLPs generated by the recombinant baculoviruses harboring the genes encoding H9N2 HA and NA proteins were tested for the presence of the influenza proteins using SDS-PAGE and Western blotting, and also a hemagglutination and a neuraminidase assay. The    viruses have developed the ability to escape from immunization with changed antigenicity and improved fitness. Furthermore, our study also showed that antigenic groups typically correspond with the phylogenetic relationships between viruses and that currently used commercial vaccine provides a reduced level of protection against more recent H9N2 strains. 17 Thus, this underlines the need for updating the vaccine used to give protection against current strains.

| The immune response to the H9N2 VLPs
Although the first cell-based and recombinant subunit influenza vaccines have been introduced and approved by the regulatory agencies, the majority of influenza vaccines are still made in eggs, and if there is an outbreak of avian influenza or other disease that affects chicken flocks, the supply of vaccine could be threatened. 23,24 Moreover, cost is also one of the major impediments to the more widespread use of vaccination in poultry production. response at the site of inoculation, which may be responsible for the enhanced immunogenicity of VLP vaccination in poultry. 33 According to this character, our baculovirus-expressed influenza VLP antigen was elaborated without extensive purification: all that was required was to include a low-speed centrifugation step in order to remove large cell debris. To note, our results showed that no inflammation or other adverse events were observed at the sites of injection in our research.
In summary, these results indicate that nonreplicating influenza VLPs represent a promising strategy for the development of a safe and effective vaccine to prevent and control H9N2 influenza viruses.