H5N1 influenza virus-like particles produced by

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H5N1 influenza virus-like particles produced by transient expression in mammalian cells induce humoral and cellular immune responses in mice Ling Tao, Jianjun Chen, Zhenhua Zheng, Jin Meng, Zhenfeng Zhang, Yao Chen, Huanle Luo, Hongxia Li, Ze Chen, Qinxue Hu, and Hanzhong Wang

Abstract: Vaccination is an effective way to protect from influenza virus infection. Among the new candidates of influenza vaccines, influenza virus-like particles (VLPs) seem to be promising. Here, we generated 2 types of H5N1 influenza VLPs by co-expressing influenza virus Env (envelope protein) and murine leukemia virus (MLV) Gag–Pol. VLPs generated by cotransfection of pHCMV-wtH5 or pHCMV-mtH5 with pSV-Mo-MLVgagpol and pHCMV-N1 were named as wtH5N1 VLPs or mtH5N1 VLPs. The plasmid of pHCMV-wtH5 encoded the wild-type hemagglutinin (HA) (wtH5) from A/swine/Anhui/ ca/2004 (H5N1) with a multibasic cleavage site, while pHCMV-mtH5 encoded the modified mutant-type (mtH5) with a monobasic cleavage site. Influenza virus HA VLPs were characterized and equal amounts of them were used to immunize mice subcutaneously, intraperitoneally, or intramuscularly. The levels of HA-specific IgG1, IFN-g, and neutralization antibodies were significantly induced in mice immunized with wtH5N1 VLPs or mtH5N1 VLPs via all 3 routes, while HAspecific IgG2a was barely detectable. IL-4 secretion was detected in mice subcutaneously immunized with wtH5N1 VLPs or mtH5N1 VLPs, or intramuscularly immunized with mtH5N1 VLPs. Our results indicated that both H5N1 influenza VLPs could induce specific humoral and cellular immune responses in immunized mice. In conclusion, our study provides helpful information for designing new candidate vaccines against H5N1 influenza viruses. Key words: H5N1, influenza, virus-like particles, mammalian cells, immune response. Résumé : La vaccination est un moyen de protection efficace contre l’infection au virus de l’influenza. Parmi les nouveaux vaccins candidats contre l’influenza, les particules qui ressemblent au virus (VLP) semblent prometteuses. Nous avons généré ici deux types de VLP de l’influenza H5N1 en exprimant conjointement Env et MLV Gag–Pol de l’influenza. Les VLP générées par la transfection conjointe des plasmides pHCMV-wtH5 ou pHCMV-mtH5, pSV-Mo-MLVgagpol et pHCMV-N1 ont été appelées wtH5N1 ou mtH5N1. Le plasmide pHCMV-wtH5 codait l’hémagglutinine sauvage (HA) (wtH5) de la souche A/porcine/Anhui/ca/2004 (H5N1) possédant un site multibase de clivage, alors que pHCMV-mtH5 codait le type mutant modifié (mtH5) possédant un site monobase de clivage. Les VLP de l’HA du virus de l’influenza ont été caractérisées et des quantités égales d’entre elles ont été utilisées pour immuniser des souris par injection sous-cutanée, intra-péritonéale ou intramusculaire. Les niveaux d’IgG1 spécifiques à l’HA, d’IFN-g et d’anticorps neutralisants étaient significativement induits chez les souris immunisées avec les VLP wtH5N1 ou mtH5N1 par les trois routes d’administration, alors que les IgG2a spécifiques à l’HA étaient à peine détectables. Une sécrétion d’IL-4 était détectée chez les souris immunisée par voie sous-cutanée avec les VLP wtH5N1 ou mtH5N1, ou immunisées par voie intramusculaire avec les VLP mtH5N1. Nos résultats ont indiqué que les deux types de VLP de l’influenza pouvaient induire des réponses immunes humorales et cellulaires chez les souris immunisées. En conclusion, notre étude fournit une information utile en vue de concevoir de nouveaux vaccins candidats contre les virus de l’influenza de type H5N1. Mots‐clés : H5N1, influenza, particules ressemblant au virus, cellules de mammifères, réponse immune. [Traduit par la Rédaction]

Introduction Since the highly pathogenic avian influenza virus H5N1 can be transmitted from birds to humans, this raises great concern about its potential to cause pandemics (Guan et al.

2004; Peiris et al. 2007). In 1997, avian influenza virus H5N1 was first transmitted to humans in Hong Kong (Claas et al. 1998; Subbarao et al. 1998). Since 2003, 15 countries in Asia, Europe, and Africa have reported 332 deaths among

Received 19 October 2011. Accepted 16 December 2011. Published at www.nrcresearchpress.com/cjm on XX March 2012. L. Tao, Z. Zheng, J. Meng, Z. Zhang, Y. Chen, and H. Luo. State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, No. 44, Xiaohongshan, Wuhan 430071, People’s Republic of China; Graduate School, Chinese Academy of Sciences, No. 19, Yuquan Road, Shijingshan, Beijing 100049, People’s Republic of China. J. Chen, H. Li, Z. Chen, Q. Hu, and H. Wang. State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, No. 44, Xiaohongshan, Wuhan 430071, People’s Republic of China. Corresponding author: Hanzhong Wang (e-mail: [email protected]). Can. J. Microbiol. 58: 391–401 (2012)

doi:10.1139/W2012-006

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566 confirmed H5N1-infected human cases, about a 59% mortality rate (WHO 2011). Avian influenza virus subtype H5N1 belongs to the species Influenza A virus in the genus Influenzavirus A within the Orthomyxoviridae family (Palese and Shaw 2007). Influenza A viruses have 8 RNA segments and encode 11 proteins. Hemagglutinin (HA) is the main surface glycoprotein and is involved in receptor-binding, membrane fusion, and induction of neutralization antibodies in hosts (Skehel and Wiley 2000). HA of most avian and mammalian influenza viruses has a monobasic amino acid at the cleavage site and can be activated by secreted trypsin-like proteases; thus these viruses can only initiate infection in the respiratory or intestinal tracts of the host (Klenk and Garten 1994; Klenk et al. 1975; Steinhauer 1999). In contrast, HA of highly pathogenic influenza viruses, such as H5N1 influenza viruses, has multibasic amino acids at the cleavage site and can be cleaved by ubiquitously expressed endogenous proteases in various types of cells in the host; therefore, viruses with biologically active HA can spread through the host and cause systemic infection (Horimoto et al. 1994; Okumura et al. 2010; Stieneke-Gröber et al. 1992). In the production of traditional vaccines against influenza virus H5N1 from chick embryos, the multibasic cleavage site of H5-HA is modified to be monobasic to weaken the virulence of recombinant viruses (Bresson et al. 2006; Lin et al. 2006; Subbarao et al. 2003; Webby et al. 2004). It is reported that the antigenicity of HA is retained, but its cleavability is changed and viral virulence is attenuated (Li et al. 1999; Subbarao et al. 2003; Suguitan et al. 2009). However, in the development of new candidate vaccines such modification of HA may not be necessary, especially in the production of influenza virus-like particles (VLPs) (Kang et al. 2009b; Szécsi et al. 2006; Wu et al. 2010). Influenza VLPs, which are produced by the baculovirus– insect cell system, closely resemble the authentic influenza virus (Latham and Galarza 2001). They can induce broad immune responses in mice and provide cross-protective immunity against influenza viruses (Bright et al. 2007; Kang et al. 2009b; Quan et al. 2007). Plasmid-based systems have also been used to generate influenza VLPs from mammalian cells (Chen et al. 2007; Neumann et al. 2000; Szécsi et al. 2006; Wu et al. 2010). Szécsi et al. (2006) reported that influenza VLPs packaging envelope proteins of the highly pathogenic influenza viruses H5N1 or H7N1 around the empty “core” particles of murine leukemia virus (MLV) can induce hightiter neutralization antibodies in BALB/c mice. In the current study, we used MLV Gag–Pol as the budding engine to generate influenza VLPs by transient expression in mammalian 293T cells. We packaged 4 types of VLPs: (i) control VLPs only contained the core of MLV Gag–Pol, (ii) N1 VLPs contained the core of MLV Gag–Pol and neuraminidase (NA) (N1) of H1N1 (A/Puerto Rico/8/34) in the envelope, (iii) wtH5N1 VLPs contained the core of MLV Gag–Pol and the wild-type HA (wtH5) of H5N1 (A/ swine/Anhui/ca/2004) and N1 in the envelope, (iv) mtH5N1 VLPs contained the core of MLV Gag–Pol and the mutanttype of H5-HA (mtH5) and N1 in the envelope. By vaccinating mice with these VLPs via 3 different routes, we investigated the specific humoral and cellular immune responses in

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mice. Our data demonstrated that influenza VLPs elicited both humoral and cellular immune responses in mice.

Materials and methods Cell culture Cultures of 293T (human kidney epithelium) and Madin– Darby canine kidney (MDCK) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) heat-inactivated (30 min, 56 °C) fetal bovine serum (FBS; GIBCO BRL, Grand Island, New York, USA), 5 mmol/L HEPES, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C and 5% CO2. Antibodies An avian influenza A NA antibody from Abcam (Cambridge, UK); the alkaline phosphatase (AP)-conjugated goat anti-rabbit IgG; the fluorescein isothiocyanate (FITC)conjugated goat anti-rabbit IgG from Pierce (Thermo Scientific, Rockford, Illinois, USA); and the AP-conjugated goat anti-mouse IgG, IgG1, and IgG2a from Southern Biotech (Birmingham, Alabama, USA) were used in this study. Rabbit anti-P30 serum against MLV Gag and rabbit anti-HA1 serum, which were produced in our laboratory, were also used. Plasmids The plasmids pSV-Mo-MLVgagpol and pHCMV-SARSSCD19 were kindly provided by Tsanan Giroglou from the Institute for Biomedical Research, Frankfurt University Medical School (Giroglou et al. 2004). The HA gene from A/ swine/Anhui/ca/2004 (H5N1) was kindly provided by Tianxian Li of the Wuhan Institute of Virology, Chinese Academy of Science. The NA gene (N1) was from A/Puerto Rico/8/34 (H1N1). The entire open reading frame (ORF) of wtH5 and the entire ORF of N1 was amplified by PCR using High Fidelity DNA polymerase KOD -Plus- (Toyobo, Osaka, Japan) by using the following primers: forward primer (5′ACAGGATCCGCCACCATGGAGAAAATAGTG-3′) (BamHI site is underlined) and reverse primer (5′-GGCAAGCTTCTCGAGTTAA-ATGCAAATTCTGC-3′) (XhoI site is underlined) for wtH5; forward primer (5′-GGCCGAATTCGCCACCATGAATCCAAATCAGAAAAT-3′) (EcoRI site is underlined) and reverse primer (5′-GGACTCGAGCTACTTGTCAATGGTGAATGGC-AACTC-3′) (XhoI site is underlined) for N1. The genes were cloned into the pGEM-T easy vector (Promega, Madison, Wisconsin, USA) for sequencing. DNA sequencing of the resulting plasmids pGEM-T easywtH5 and pGEM-T easy-N1 revealed no difference between the actual sequences and the original sequences of wtH5 and N1. The mutant-type of the HA gene (mtH5) was generated from pGEM-T easy-wtH5 by PCR-based site-directed mutagenesis using the following primers: forward primer (5′-CTCAGAAATAGCCCTCAAAGAGAGACAAGAGGACTTTTTGGAGC-3′) and reverse primer (5′-GCTCCAAAAAGTCCTCTTGTCTCTCTTTGAGGGCTATTTCTG-AG-3′). DNA sequencing of the resulting plasmid, pGEM-T easymtH5, revealed the expected changes (nucleotides 1021–1035 AGAAGAAGAAAAAGG→1021–1023 ACA) in mtH5, which led to amino acid changes at position 341–345 (RRRKR→T). The sequenced genes were inserted into pHCMV-SARS-SCD19 to replace the gene of SARS-SCD19: Published by NRC Research Press


Tao et al.

for wtH5 or mtH5 the genes were inserted between the BamHI and XhoI sites, and for N1 (1 EcoRI site from the forward primer and the other from the pGEM-T easy vector) the gene was inserted between the EcoRI sites with the ORF in the right direction; thus influenza virus Envexpressing vectors were generated and named as pHCMVwtH5, pHCMV-N1, and pHCMV-mtH5, respectively. The nature of these constructs were confirmed by restriction enzyme digestion, PCR identification, and sequencing. Plasmids were purified by using a Qiagen EndoFree Plasmid Maxi kit (Qiagen, Hilden, Germany) and dissolved in Tris–EDTA buffer. Protein expression Recombinant plasmids pHCMV-wtH5, pHCMV-mtH5, pHCMV-N1, or empty vector (2 µg each plasmid per well) were transfected into 293T cells in a 6-well plate (Greiner Bio-One, Frickenhausen, Germany) using a calcium phosphate transfection kit (Promega) following the manufacturer’s instructions. These cells were harvested at 54 h posttransfection for Western blotting analysis. Recombinant plasmids pHCMV-wtH5, pHCMV-mtH5, or empty vector (1 µg each plasmid per well) were transfected into 293T cells on sterile glass coverslips in a 12-well plate (Greiner Bio-One) and were harvested at 30 h post-transfection for immunofluorescence staining. Production of VLPs VLPs were produced by transient transfection of 293T cells as previously described with slight modifications (Giroglou et al. 2004). Briefly, pSV-Mo-MLVgagpol and influenza virus Env-expressing plasmids were transfected into cells using the calcium phosphate transfection kit with dosages of plasmids as follows: 20 µg pSV-Mo-MLVgagpol for control VLPs; 20 µg pSV-Mo-MLVgagpol and 2 µg pHCMV-N1 for N1 VLPs; 20 µg pSV-Mo-MLVgagpol, 2 µg pHCMV-N1, and 1 µg pHCMV-wtH5 for wtH5N1 VLPs; 20 µg pSV-MoMLVgagpol, 2 µg pHCMV-N1, and 1 µg pHCMV-mtH5 for mtH5N1 VLPs. The medium was replaced with 7 mL of DMEM–2% FBS at 6 h post-transfection. Supernatants containing VLPs were harvested after an additional 24 or 48 h and then cleared, filtered, and stored at 4 °C before ultracentrifugation. Sedimentation of VLPs was obtained by ultracentrifugation of 26 mL supernatants per tube in a Type 70 Ti Beckman rotor (Beckman Coulter, Fullerton, California, USA) at 222 000g for 1 h at 4 °C. The sediment was dissolved in 100 µL of phosphate-buffered saline (PBS, pH 7.2–7.4) per tube. The protein concentration of VLPs was measured by a bicinchoninic acid protein assay. VLPs were aliquoted and stored at –80 °C until needed. Western blotting analysis To detect wtH5, mtH5, and N1 protein expression, 293T cells in the 6-well plate transfected with the plasmid DNA as indicated above were harvested, treated, and detected as previously described (Ren et al. 2008). Samples were separated by 10% SDS–PAGE. The rabbit anti-HA1 serum (1:1000) or the avian influenza A NA antibody (1:1000) was used as the first antibody. Concentrated VLPs of 3.5 µg (10 µL per lane) were measured as described above, and the expression of P30, a protein of the MLV capsid, was also detected by the rabbit anti-P30 serum (1:1000).

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Immunofluorescence staining and confocal microscopy As indicated previously, transfected 293T cells in the 12well plate were fixed by 4% p-formaldehyde and permeabilized by 0.2% Triton X-100. After blocking with PBS supplemented with 2% BSA and 5% normal goat serum, cells were incubated with the rabbit anti-HA1 serum (1:100) as the primary antibody and then with the FITC-conjugated goat antirabbit IgG (1:200) as the secondary antibody. Hoechst 33258 staining buffer (Beyotime, Haimen, Jiangsu, China) was used for staining nuclei of 293T cells. Cells were observed with a Leica TCS SP2 laser scanning confocal microscope with a 40× plan apochromat objective, numerical aperture 1.40 (Leica Camera AG, Solms, Germany). Electron microscopy Concentrated VLPs were stained as previously described (Ren et al. 2008) and examined at 200 kV in a Tecnai G2 Spirit Transmission Electron Microscope (Financial Executives International, Morristown, New Jersey, USA). Hemagglutination assay Two-fold serial dilutions of VLPs (beginning concentration 25 ng/µL) were used for the hemagglutination assay following the WHO Manual on Animal Influenza Diagnosis and Surveillance (WHO 2002). The HA titer was calculated as the highest dilution of VLPs agglutinating chicken red blood cells and presented as HA units. NA activity assay NA activity was detected by a Neuraminidase Assay kit (Beyotime), according to the manufacturer’s instruction. Briefly, VLPs (500 ng/µL) were added to the reaction buffer and then MilliQ H2O was added with controls set in parallel. The fluorescence substrate was added and the fluorescence intensity was measured by excitation at 360 nm and recording the emission at 440 nm in a Synergy HT Multi-Mode Microplate Reader (Bio-TEK, Winooski, Vermont, USA). Immunization protocols Female BALB/c mice, 6 to 8 weeks old, were purchased from the Centers for Disease Control and Prevention (Changsha, Hunan, China) and maintained in a special pathogen-free environment. All animal experiments were carried out according to the regulations by the Experimental Animal Society of Hubei Province. Mice were randomly divided into 12 groups, 6 mice per group, and immunized 3 times at 2-week intervals with 20 µg VLPs (dissolved in 100 µL of PBS) subcutaneously, intraperitoneally, or intramuscularly. ELISA analysis At 1 week after the final immunization, mice were bled and sacrificed by cervical dislocation, and splenocytes were isolated. Serum samples were collected and stored at –20 °C in aliquots before use. The levels of HA-specific antibodies (IgG, IgG1, and IgG2a) in serum were assessed by an indirect ELISA using recombinant HA1 protein (amino acids 1– 328 of wtH5 HA0) as the detection antigen. ELISA was done as previously described with slight modifications (Hu et al. 2009). Briefly, 5 µg/mL concentration of antigen was coated onto 96-well ELISA plates (Greiner Bio-One) overnight at 4 °C. After overnight blocking with PBS – 1% BSA at 4 °C Published by NRC Research Press


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and a 1 h incubation with 1:100 diluted mouse sera at 37 °C, the plates were washed extensively, and bound antibodies were detected by the AP-conjugated goat anti-mouse IgG, IgG1, or IgG2a (1:3000). The reaction was visualized by adding p-nitrophenyl phosphate substrate, and the absorbance at 405 nm was measured in a Synergy HT Multi-Mode Microplate Reader (Bio-TEK). Neutralization assay HA pseudotypes have been used as safe, quantitative, and high-throughput tools for sensitive and specific detection of neutralizing antibodies against influenza viruses (Szécsi et al. 2006; Wang et al. 2008, 2010). Here, we used wtH5N1 pseudotypes to test serum neutralization antibodies. The pseudotypes were produced by transient transfection of 293T cells with 7.5 µg of pMP71-eGFP-pre, 12.5 µg of pSV-Mo-MLVgagpol, 2 µg of pHCMV-wtH5, and 1 µg of pHCMV-N1 per 100 mm plate, using the calcium phosphate transfection methods as described previously (Giroglou et al. 2004). Cells were examined under an IX51-A22PH inverted fluorescence microscope (Olympus, Tokyo, Japan) to inspect the expression of eGFP (enhanced green fluorescent protein) and to monitor the efficacy of transfection at about 20 h post-transfection. Supernatants were harvested at 24 and 48 h post-transfection and then cleared, filtered through 0.45 µm filters (Millipore, Co., Cork, Ireland), and stored at –80 °C in aliquots until used. The titer of wtH5N1 pseudotypes was measured on transduced MDCK cells and expressed as transducing units (TU), representing clusters of eGFP-positive cells. Neutralization assays were performed as previously described with some modifications (Lu et al. 2010). In brief, the mixture of wtH5N1 pseudotypes (400 TU) and serum (2-fold diluted in DMEM – 2% FBS) were incubated at 37 °C for 1 h and then added to MDCK cells. After incubation at 37 °C for 1 h, mixtures were replaced with DMEM – 2% FBS. Then, the plates were incubated at 37 °C for 48 h. Finally, the clusters of fluorescent cells (TU) per well were examined, and the titers of neutralizing antibodies were defined as the highest dilution of tested samples that reduced the TU by 75% in comparison with the negative serum control. ELISpot assays Preparation of spleen single-cell suspensions and ELISpot assays were carried out as previously described (Lu et al. 2009). A Mouse IFN-g ELISpotPLUS kit (HRP) and a Mouse IL-4 ELISpotPLUS PLUS kit (HRP) (Mabtech AB, Nacka Strand, Sweden) were used. Splenocytes (2.5 × 105 per well) were stimulated with 20 µg/mL purified recombinant HA1 protein, and all stimulation conditions were tested in quadruplicate, with controls in parallel. The spots were scanned and counted in an ELISpot reader (BioReader 4000 Pro-X; BioSys GmBH, Hamburg, Germany). Statistical analysis All data are presented as the mean ± standard deviation (SD). Statistical analysis was done with SPSS version 13.0 for Windows (SPSS Inc., Chicago, Illinois. USA) and assessed by the Student’s t test. A P value of 0.05) between groups immunized with wtH5N1 or mtH5N1 VLPs. The HA-specific IgG2a was hardly detected in groups immunized with influenza VLPs via all 3 routes (Fig. 4C). Therefore, both influenza VLPs were immunogenic and could induce HA-specific humoral immune responses with a predominantly IgG1 subclass in mice. Serum neutralization activity HA is the major viral antigen responsible for eliciting neutralization antibodies to protect from the infection of influenza virus. The serum neutralization activity against wtH5N1 pseudotypes was detected on MDCK cells as described in Materials and methods. From all 3 routes, groups immu-

nized with influenza VLPs induced specific serum neutralization antibodies in comparison with groups immunized with control or N1 VLPs (P < 0.05, Table 1). The titer of the group immunized with mtH5N1 VLPs intramuscularly was significantly higher than that immunized with wtH5N1 VLPs (P < 0.05), whereas the titer of the group immunized with mtH5N1 VLPs intraperitoneally was significantly lower than that immunized with wtH5N1 VLPs (P < 0.05). However, there was no significant difference (P > 0.05) between the subcutaneously immunized groups. Our results demonstrated that influenza VLPs induced serum neutralization activity. Cellular immune responses to influenza VLPs The cellular immune responses of immunized mice were evaluated by ELISpot assays at 1 week after the final boost, by detecting HA-specific IFN-g and IL-4 secretion. In all 3 administration routes, groups immunized with influenza VLPs secreted higher levels of specific IFN-g compared with groups immunized with control or N1 VLPs (P < 0.05, Fig. 5A). The levels of IFN-g secretion in groups immunized with mtH5N1 VLPs were significantly higher than those immunized with wtH5N1 VLPs via all 3 routes (P < 0.05). HA-specific IL-4 secretion was not induced in all groups of mice immunized intraperitoneally with influenza VLPs (Fig. 5B). However, IL-4 secretion was observed in the groups immunized subcutaneously with influenza VLPs and Published by NRC Research Press


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Fig. 3. Characterization of influenza virus-like particles (VLPs). (A) Western blotting of concentrated VLPs. VLPs were lysed and subjected to Western blotting with the rabbit anti-P30 serum (1:1000, bottom panel), the avian influenza A neuraminidase antibody (1:1000, middle panel), or the rabbit anti-HA1 serum (1:1000, top panel). Lanes: C, control VLPs; N1, N1 VLPs; wt, wtH5N1 VLPs; mt, mtH5N1 VLPs. Lane M, molecular size standards of the prestained protein marker. (B) Electron micrograph of negatively stained VLPs. (C) Hemagglutination assay. Hemagglutination was carried out with 1% chicken erythrocytes with VLPs (beginning concentration 25 ng/µL) by a 2-fold serial dilution method. Three stocks of concentrated VLPs were used in the assay, and results (HAU, hemagglutinin units) are expressed as means ± SD (n = 3). (D) Neuraminidase (NA) activity assay. The NA activity of concentrated VLPs (500 ng/µL) was determined by a kit as described in Materials and methods. Three stocks of concentrated VLPs were used in the assay and results are displayed as means ± SD (n = 3).

in the group immunized intramuscularly with mtH5N1 VLPs compared with groups immunized with control or N1 VLPs (P < 0.05, Fig. 5B). There was no significant difference in IL-4 secretion between the groups immunized with influenza VLPs subcutaneously. Therefore, immunization with influenza VLPs resulted in predominantly HA-specific IFN-g secretion, and the cellular immune responses were enhanced in groups immunized with mtH5N1 VLPs, with more robust IFN-g secretion observed in all 3 routes. IL-4 secretion was also promoted in the intramuscularly immunized group.

Discussion Since H5N1 influenza viruses cause case fatality in human beings and have spread across the world, they have raised great concern about their potential for causing future human influenza pandemics. Vaccination is the most efficient strategy in the prevention of H5N1 influenza virus infection, and

many vaccines against H5N1 influenza viruses have been developed, such as traditional vaccines, including inactivated vaccines (Bresson et al. 2006; Lin et al. 2006) and live attenuated vaccines (Karron et al. 2009); and a variety of new candidate vaccines, including adenovirus-based vaccines (Gao et al. 2006), baculovirus-based vaccines (Prabakaran et al. 2010), DNA vaccines (Kodihalli et al. 1999; Smith et al. 2010), and VLPs-based vaccines (Kang et al. 2009b; Szécsi et al. 2006; Wu et al. 2010). Influenza VLPs represent a promising candidate vaccine because they are nonreplicating and safe, contain viral structural proteins, and show robust immunogenicity (Kang et al. 2009a). By expressing 3 structural proteins, HA, NA, and M1 (Bright et al. 2008; Bright et al. 2007; Crevar and Ross 2008; Kang et al. 2009b; Mahmood et al. 2008), or HA, NA, and the Gag protein of MLV (Haynes et al. 2009), a variety of H5N1 influenza VLPs are produced from baculovirus–insect cell systems.. These VLPs are effective for inducing broad, Published by NRC Research Press


398 Fig. 4. Hemagglutinin (HA)-specific humoral immune responses. Mice (n = 6 per group) were immunized 3 times with 20 µg of influenza virus-like particles, via 3 administration routes, including subcutaneous injection (s.c.), intraperitoneal injection (i.p.), and intramuscular injection (i.m.) at 2-week intervals. The sera of immunized mice were collected 1 week after the final immunization, and HA-specific IgG (A), IgG1 (B), and IgG2a (C) in serum were detected by an indirect ELISA. Absorbance (at 405 nm) is expressed as means ± SD (n = 6).

Can. J. Microbiol. Vol. 58, 2012 Table 1. Serum neutralization antibody titers against wtH5N1 pseudotypes. Routea s.c.

i.p.

i.m.

Immunogen Control VLPs N1 VLPs wtH5N1 VLPs mtH5N1 VLPs Control VLPs N1 VLPs wtH5N1 VLPs mtH5N1 VLPs Control VLPs N1 VLPs wtH5N1 VLPs mtH5N1 VLPs

Titer (2n)b