Virus-like Particles Vaccine For Avian Influenza Of H5N1 Subtype

H5N1 avian influenza initially garnered widespread attention following the first human cases of infection recorded in Hong Kong in 1997[1]. Avian influenza viruses represent a growing threat for an influenza pandemic. Vaccination is an effective way to control influenza virus infection[2]. Today, the vast majority of influenza vaccines are produced by growing the target influenza virus in fertile chicken eggs. However, manufacturing difficulty made this way of production fragile in obtaining an timely and adequate supply of influenza viruses vaccine. More importantly, the egg-based technology may not be suitable to respond to a pandemic crisis. Since the H5N1 avian influenza virus strains are often lethal to chicken eggs[3], it will take several months to get a high-yield strain grown in chicken eggs following the identification of new potential strains. In addition, due to the high pathogenicity of avian influenza virus strains, the conventional production of avian influenza vaccines would require biosafety level 3 containment facilities. Therefore, a strategy that can rapidly produce new influenza vaccines is needed as a priority for pandemic preparedness.Virus-like particles (VLPs) are multiple protein structures that mimic the organization and conformation of a authentic native virus, but without DNA/RNA inside. Compared with individual protein or peptide, VLPs present conformational epitopes more similar to the native virus, and hence possessing higher immunogenicity. Due to their highly repetitive surface, VLPs are able to induce strong B-cell responses in the absence of adjuvants by efficiently cross-linking specific receptors on B cells[4,5].The baculovirus expression vector system (BEVS) is a useful tool for the expression of recombinant proteins in insect cells. This system has several unique features which account for its popularity. First, expression in eukaryotic milieu makes folding, post-transcriptional modications and assembly of target protein more close to its native ways. Then, this system is safe and could achieve high expression of target proteins. In addition, it also offers the possibility to express two or more proteins simultaneously in one cell by coinfection, making this system especially suitable for the expression of multisubunit protein complexes such as VLPs[6]. Here, we produced the influenza VLPs in insect cells co-infected with two rBVs, one expressing HA and NA proteins of A/goose/Jilin/hb/2003(H5N1), and the other expressing M1 and M2 proteins of A/PR/8/34(H1N1).First, the mutated HA gene (ΔHA) was inserted downstream of promoter of the Autographa californica multiple nuclear polyhedrosis virus(AcMNPV)polyhedrin gene and the NA gene was inserted downstream of the AcMNPV p10 promoter into pFastBac Dual bacmid transfer vector[7], the resultant plasmid was named pFastBac Dual-ΔHA-NA. Similarly, the M2 gene was inserted downstream of the AcMNPV polyhedrin promoter and the M1 gene was inserted downstream of the AcMNPV p10 promoter into pFastBac Dual bacmid transfer vector, and the resultant plasmid named pFastBac Dual-M1-M2.Recombinant bacmids were produced by site-specific homologous recombination following transformation of the transfer plasmids(pFastBac Dual-ΔHA-NA,pFastBac Dual-M1-M2)into E. coli DH10Bac competent cells, which contains the AcMNPV baculovirus genome and the helper plasmid which encodes the transposase and so posseses the Tn7 transposition function [8]. Blue/white colony selection was applied to identify colonies containing the recombinant bacmid and the presence of the influenza genes was verified in these colonies by PCR analysis. Bacmid DNA were purified from DH10Bac? transformants, and named rBacmid-ΔHA-NA and rBacmid-M1-M2.Then, the recombinant bacmid DNA was transfected into the Spodoptera frugiperda Sf9 insect cells. The recombinant baculoviruses(rBVs) was harvested in the culture medium 5 days post transfection. The titer of baculoviral stock was determined by the Viral Plaque Assay. The viral stock was amplified by infecting cells at a multiplicity of infection (MOI) 0.1, to generate a higher titer P2 stock. To produce high titer P3 stock, we scaled up the amount of cells and volume of virus used correspondantly, and finally achieved production of the P3 viral stock with a titer of 1×108pfu/ml . The baculoviral stock rBV-ΔHA-NA-P3 or rBV-M1-M2-P3 were used to infect insect cells and expression of the recombinant proteins ( HA,NA or M1,M2) was determined by SDS-PAGE and Western Blot.VLPs were produced in HighFive insect cells co-infected with two rBVs at MOI of 5 for each. The cells were growed in suspension in serum-free medium. The culture supernatants were harvested at 72 h post infection, VLPs in the supernatants were pelleted by ultracentrifugation at 100,000×g for 4h. The pellets were resuspended and loaded into Cellufine Sulfate affinity chromatography colum for purification. Preparations of VLPs were examined by transmission electron microscopy and revealed particles of about 80nm in diameter with spikes on the surface which resemble shape of influenza virions. VLPs formation were analyzed by Western-Blot to determine the distribution of HA, NA, M1 and M2 proteins. In addition, VLPs preparations could hemagglutinate chicken erythrocytes with titers about 128. Therefore, influenza VLPs produced by cells infected with rBVs were similar to influenza virions in structure, size, and morphology despite lacking the viral ribonucleoprotein components.The last, BALB/c mice were intramuscularly immunized with VLP(s10ug)and adjuvant, and a boost was given 21 days after the priming dose. All mice remained healthy and showed no signs of abnormal behavior after vaccination. Serum samples were taken before the primary inoculations and at day 7, 14, 21, 28, 35, 42, 49 post priming immunization. The first (priming) immunization of mice with VLPs induced low or undetectable levels of antibodies as measured by ELISA with H5N1 HA as antigen expressed from 293cells or by inactivated H5N1viruses from eggs.The levels of virus-specific antibodies were gradually increased after boost immunization. At four weeks after boost immunization, End-point ELISA HA specific titers reached to 1:40,000, Virus specific titers reached to 1:200,000, HAI titers to1:1600, and neutralizing antibodies to 1:160。VLPs comprised of the HA and NA protein of A/goose/Jilin/hb/2003(H5N1), the M1 and M2 of A/PR/8/34(H1N1)exhibited functional characteristics of ifluenza virus including hemagglutination activities.In BALB/c mice,VLPs elicited serum antibodies specific for influenza A/goose/Jilin/hb/2003(H5N1) virus Thus,VLPs represent a potential strategy for the development of human vaccines against avian influenza H5N1 viruses.