KR101755114B1 - A chimeric virus-like particle vaccine confers broadened antibody responses against different clades of highly pathogenic avian influenza H5 viruses - Google Patents

Abstract

The present invention relates to a chimeric virus-like particle vaccine which induces a broad antibody response against the highly pathogenic avian influenza H5 virus of various clades.

Description

A chimeric virus-like particle vaccine that induces a broad antibody response to the highly pathogenic avian influenza H5 virus of various clades.

The present invention relates to a chimeric virus-like particle vaccine which induces a broad antibody response against the highly pathogenic avian influenza H5 virus of various clades.

In 1996, Asian high-pandemic influenza A (H5N1) H5N1 first developed in China, and within the past 100 years, the world's largest pandemic animal disease has begun. Since then, the Asian H5N1 virus has spread to Asia, the Middle East, Europe and Africa, resulting in the death or forcible destruction of more than 400 million poultry.

These HPAI H5 viruses evolved independently into various clades (Donis, RO, and GJ Smith. 2015. Nomenclature updates resulting from the evolution of avian influenza A (H5) virus clades 2.1.3.2a, 2.2.1, and 2.3. 4 during 2013-2014, Influenza and other respiratory viruses doi 10.1111 / irv.12324), which are genetically and antigenically differentiated, making them more difficult to remove. In addition, continuous human infections of HPAI H5N1 cause human epidemics.

A new strategy for HPAI H5 is needed to reduce the economic losses and the risk of human infection in the poultry industry.

Vaccination against HPAI H5 is widely used in various regions. However, current vaccines have limitations in that they do not exhibit a broad protective effect against the HPAI H5 virus of several clades (Grund, C., SM Abdelwabel, AS Arafa, M. Ziller, MK Hassan, MM Aly, HM Hafez, TC Harder, and M. Beer, 2011. Vaccine 29: 5567-5573, doi 10.1016 / J.Vaccine 2011.01.006).

Virus-like particles (hereinafter referred to as "VLPs") have been proposed as novel alternative vaccine candidates. VLPs were produced using the most important immunogenic proteins of the virus, and the VLP vaccine showed a high level of protection against several viral diseases (Lee DH, Park JK, Song CS 2014. Clin Exp Vaccine Res 3: 133- Buonaguro L, Tornesello ML, Tagliamonte M, Gallo RC, Wang LX (1997) J. Virol 71: 7207-7213; Buonaguro L, Tornesello ML, Tagliamonte M, Gallo RC, Wang LX , Kamin-Lewis R, Abdelwahab S, Lewis GK, Buonaguro FM 2006. J Virol 80: 9134-91438-10).

However, there has been no study of VLPs that induce a wide range of immune responses against the HPAI H5 virus of several clades in poultry species.

The present invention has been made in view of the above needs, and it is an object of the present invention to provide a VLP inducing a wide range of immune responses to HPAI H5 virus of various clades.

In order to achieve the above object, the present invention provides a chimeric virus-like particle comprising an HA gene of a clad 1 H5N1 virus, an HA gene of a clad 2 H5N1 virus, and an influenza M1 gene.

In one embodiment of the present invention, the HA gene of the clad 1 H5N1 virus is preferably composed of the nucleotide sequence of SEQ ID NO: 1, and the HA gene of the clade 2 H5N1 virus comprises the nucleotide sequence of SEQ ID NO: 2 And the influenza M1 gene comprises the nucleotide sequence of SEQ ID NO: 3, but is not limited thereto.

The present invention also provides antigenic formulations comprising the chimeric virus-like particles of the invention and an adjuvant or an immune stimulator.

The present invention also provides a vaccine comprising the chimeric virus-like particle of the present invention.

In one embodiment of the present invention, the vaccine preferably has a protective effect against infection by poultry influenza virus, but is not limited thereto.

In another embodiment of the invention, the vaccine is preferably, but not exclusively, formulated with an adjuvant or immunostimulant.

The present invention also provides a method for inducing preventive immunity against avian influenza infection in an animal other than a human, comprising administering the above-described antigenic preparation of the present invention to an animal other than a human.

The present invention also provides a method for inducing preventive immunity against avian influenza virus infection in an animal other than a human, which comprises administering the vaccine of the present invention to an animal other than a human.

The present invention also relates to a method for producing a vector containing both the HA gene of the clade 1 H5N1 virus and the HA gene of the clade 2 H5N1 virus and the vector comprising the influenza M1 gene by using a baculovirus expression system, A recombinant baculovirus encoding a clade 2 HA gene and a recombinant baculovirus encoding an influenza M1 gene, and co-infecting the recombinant baculovirus with an insect cell, To provide a method for manufacturing a like particle.

The VLPs of the present invention are used in the preparation of immunogens or pharmaceutical compositions. To do so, the VLP is adjusted to the proper concentration and formulated with any suitable adjuvant, diluent or carrier. A physiologically acceptable medium may be used as carrier and / or diluent. These include, but are not limited to, suitable isotonic medium, glycerol, ethanol and other conventional solvents, phosphate buffered saline, and the like. Suitable adjuvants include synthetic lipid analogs such as aluminum phosphate, aluminum hydroxide, MPL (3-O-deacylated monophosphoryl lipid A; RIBI ImmunoChem Research, Inc., Hamilton, MT, ), Stimulon TM QS-21 (Aquila Biopharmaceuticals, Framingham, MA), IL-12 (Genetics Institute, Cambridge, MA), synthetic polynucleotides such as oligonucleotides containing CpG motifs (US Patent No. 6,207,646 , Heat-labile toxin of E. coli, and cholera toxin (in wild-type or mutant forms, for example, when glutamic acid at amino acid position 29 is replaced by another amino acid, preferably histidine, WO 00/18434 (29)), but are not limited thereto.

In one aspect of the invention, the formulation comprising VLP is for use as an immunogen composition. The virus may be a cold protection additive or stabilizer such as a protein (e.g. albumin, gelatin), a sugar (e.g. sucrose, lactose, sorbitol), an amino acid (e.g. sodium glutamate), saline, ≪ / RTI > The mixture is kept in a liquid state, or is then dried or lyophilized for transport and storage and mixed with water just prior to administration.

Hereinafter, the present invention will be described.

The present inventors have developed chimeric VLPs expressing HA simultaneously from clath 1 and 2 HPAI H5 viruses using the baculovirus expression vector system (BEVS), and demonstrated immunity against the H5 virus of four other clades in two major poultry species And ducks.

Through the present invention it has been shown that the chimeric VLP vaccine of the present invention induces a broad range of antibody responses against multiple clade H5 viruses than conventional VLP vaccines and this provides for better control of the Asian HPAI H5 virus in poultry species And provide strategies for developing improved vaccines.

Figure 1 shows the preparation and characterization of a chimeric H5 VLP vaccine containing HA protein from several clades of HPAI H5 virus. (A) Recombinant baculoviruses (rBV) encoding clade 2 HA as well as HA gene clade 1 HA, clade 2 HA, or clade 1 HA were used to infect Sf9 cells. Sf9 cells were co-infected with one of the rBVs coding for the HA gene and rBV encoding the influenza M1 gene (B), resulting in the production of clade 1, clade 2, or clade 1 + 2 VLPs. 4 micrograms of clade 1 VLP (lane 1), clade 2 (lane 2), and clade 1 + 2 (lane 3) with (C) anti-clad 1 monoclonal antibody with anti- (D) Western blot using anti-clade 2 monoclonal antibody with anti-influenza M1 antibody. (E) Negative staining transmission electron microscopy of clade 1 + 2 H5 VLPs. Bar represents 100 nm.
Figure 2 is a cross-clad HI test in chicken. Six-week old SPF chickens (n = 10 per group) were immunized with (A) clade 1, (B) clade 2, or (C) chimeric H5 VLP vaccine (40 ug of VLP / 0.5 mL dose). Serum samples were obtained 3 weeks after single immunization and HI titers for HPAI H5N1 viruses of several clades were determined using clades 1, 2.1, 2.3.2.1, and 2.5 H5N1 viruses. The average HI antibody titer of 10 birds (Log 2) was shown.
Figure 3 is a cross-clad HI test in a duck. Five week old SPF chickens (n = 10 per group) were immunized with (A) clade 1, (B) clade 2, or (C) chimeric H5 VLP vaccine (40 ug of VLP / 0.5 mL dose). Serum samples were obtained after 3 weeks of single immunization and treated with RDE to remove nonspecific HI factors. The HI titer for the HPAI H5N1 virus of several clades was determined using clades 1, 2.1, 2.3.2.1, and 2.5 H5N1 viruses. The average HI antibody titer of 10 ducks (Log 2) was shown.
Figure 4 is a statistical analysis of HI titer from chicken immunized with several VLP vaccines. (A) clade 1, (B) clade 2.1, (C) clade 2.3.2, and (3) after 3 weeks of immunization with clay 1, clade 2 or chimeric VLP vaccine (D) Clade 2.5 H5N1 virus. * P <0.05 and *** P <0.001 (ANOVA with Tukey-Kramer post-hoc test compared to other groups).
Figure 5 is a statistical analysis of HI titer from duck immunized with several VLP vaccines. (A) clade 1, (B) clade 2.1, (C) clade 2.3.2, and (3) after 3 weeks of immunization with clay 1, clade 2 or chimeric VLP vaccine (D) Clade 2.5 H5N1 virus. The mean HI antibody titer (Log 2) of 10 ducks was shown. ** P <0.01 and *** P <0.001 (ANOVA with Tukey-Kramer post-hoc test compared to other groups).

Hereinafter, the present invention will be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the present invention and the scope of the present invention is not limited by the following examples.

Example  1: Recombination baculoviruses Manufacturing

For cloning of the full-length HA gene of the clade 1 H5N1 virus, the HA gene of A / Vietnam / 1194/2004 (H5N1) virus was chemically synthesized without MBCS sequence (Bioneer, Korea). To clone the full-length HA gene of the clade 2 H5N1 virus, the viral RNA was extracted from A / mandarine duck / K10-483 / 2010 (H5N1, clade 2.3.2.1) to amplify the HA gene (Hoffmann, E., J. Stech, Y. Guan, RG Webster, and DR Perez, 2001. Universal primer set for the full-length amplification of all influenza viruses. Archives of virology 146: 2275-2289)

MBCS (multibasic cleavage site) is described by Heckman, K. L., and L. R. Pease. 2007. Nature protocols 2: 924-932. doi 10.1038 / nprot.2007.132.

HA genes amplified from clade 1 or clade 2 virus were cloned into vector pFastBac1 (Invitrogen, USA) and the plasmids were designated as pFast_clade 1 and pFast_clade 2, respectively. The vector pFastBac1, which simultaneously contains the clade 1 and clade 2 H5N1 virus-derived HA genes, was constructed by cloning the pFast_clade 2-derived SnaBI- / HpaI-digested fragment into the HpaI-digested region of pFast_clade 1, The resulting plasmid was named pFast_Clade 1 + 2. The vector pFastBac1 containing the influenza matrix1 gene was constructed by cloning the full-length M1 gene of A / Puerto Rico / 8/1934 (H1N1) with the vector pFastBac1 (Park, JK, et al. 2014. Clinical and vaccine immunology: CVI 21 : 360-365, doi 10.1128 / CVI.00636-13), resulting in a plasmid designated pFast_M1.

The clade 1 HA gene, the clade 2 HA gene, the clade 1 clade 2 HA gene, or the influenza 1 gene were cloned using pFast_clade 1, pFast_clade 2, pFast_clade 1 + 2 and pFast_M1 Recombinant baculovirus (rBV) encoding the M1 gene was constructed using the Bac-to-Bac baculovirus expression system (Invitrogen). As a result, rBVs were cloned into rBV clade 1, rBV clade 2, rBV clade 1 +2, or rBV_M1. The titer of rBVs was measured by Spodoptera frugiperda (Sf9) insect cells. The rBV clade 1 + 2 was deposited with KCTC 12845BP on June 12, 2015 at Korea Gene Bank, Yuseong-gu, Daejeon, Korea.

Example  2: H5 VLPs And VLP  Manufacture of vaccines

For clade 1 H5 VLP production, Sf9 cells were co-infected with rBV_clade 1 and rBV_M1 in a multiplicity of infection (MOI) of 5. For clade 2 H5 VLP production, Sf9 cells were co-infected with rBV_clade 2 and rBV_M1 at 5 MOI (multiplicity of infection).

For chimeric H5 VLP production, Sf9 cells were co-infected with rBV_clade 1 + 2 and rBV_M1 in a multiplicity of infection (MOI) of 5. After 72 hours of infection, the culture medium containing VLPs was collected and cleaned with low speed centrifugation (2,000 xg, 30 min, 4 캜) to remove large cell debris and pelleted with purified supernatant derived VLPs (30,000 xg, 1.5 h , 4 [deg.] C). The pellet was resuspended in phosphate-buffered saline (PBS) (pH 7.2) and loaded on a 20-50% (w / v) discontinuous sucrose density gradient to give ultra-high speed centrifugation (150,000 xg, 1.5 h, ). The presence of VLPs was observed with a transmission electron microscope (Tecnai G2 Spirit, FEI, Netherlands, Korea Basic Science Institute) using negative staining method.

Expression of HA and M1 proteins in each VLP was detected using mouse anti-clade 1 H5 monoclonal antibody (Median diagnostics, Korea), anti-clade 2 H5 monoclonal antibody (Bionote, Korea) and rabbit anti-M1 polyclonal antibody , USA) and detected by Western blot with detection with HRP (horseradish peroxidase) -conjugated goat anti-mouse or anti-rabbit IgG (AbD Serotec, UK). The final protein concentration of each H5 VLPs was quantified by Bradford protein assay (Pierce, USA) according to the manufacturer's instructions.

For the production of the H5 VLP vaccine, each H5 VLPs was emulsified in the oil adjuvant Montanide ISA70 (SEPPIC, France) at a ratio of 30:70 (v / v) to obtain a final concentration of 40 ug of VLP / 0.5 mL · dose.

Example  3: Determination of animal immunity and serological immune response

A total of 30 6-week-old SPF white leghorn chickens (Namduck Sanitec, Korea) were divided into three groups (10 chickens / group). A total of 30 5-week-old Peckin ducks (provided by Moran Food & Breeding Company, Korea) were divided into three groups (10 ducks / group). Each group of chickens or ducks was intramuscularly immunized (0.5 mL / animal) with a clade 1, clade 2, or chimeric H5 VLP vaccine.

To determine the serologic immune response after immunization, sera were collected from VLP vaccinated chickens and ducks three weeks after immunization for cross-clad HI (hemagglutination inhibition) testing with multiple clade-derived H5N1 viruses.

A / Vietnam / 1194/2004 (Clade 1), A / Indonesia / 5/2005 (Claude 2.1), A / mandarin duck / K10-483 / 2010 / ES / 2003 (Clade 2.5) H5N1 virus containing the HA gene without the MBCS sequence of Lee, DH et al. 2013. PloS one 8: e58186. was prepared using a reverse genetics (RG) system as described in doi 10.1371 / journal.pone.0058186. The HI test was performed according to the OIE standard method using the H5N1 virus of 4 HA units. To remove nonspecific HI factors, one volume of duck serum was treated with 3 volumes of receptor-destroying enzyme (Denka Seiken Co., JAPAN) for 16 hours at 37 ° C.

All animal experiments performed in the present invention were conducted under the review, approval and supervision of Konkuk University Animal Experimental Ethics Committee.

Analysis of variance (ANOVA) following Tukey-Kramer post-hoc testing was performed for comparison of serum antibody titer between groups. A P value, <0.05 was considered statistically significant.

The results of the above embodiment are as follows.

Chimeric H5 VLP Expression and properties

H5 VLPs containing clade 1 HA, clade 2 HA, or clade 1 and clade 2 HA were produced in Sf9 cells using BEVS (Fig. 1). After concentration by ultrafast centrifugation, purification was performed using sucrose density gradient centrifugation. The band located at the top of the 50% sucrose density was found to contain both HA and M1 proteins by Western blot (Fig. 1B), and the bands identified were about 68 kDa and 28 kDa, . As expected, chimeric H5 VLP-derived HA could be detected in both clade 1-specific and clade 2-specific mAb.

Investigation of the negatively stained formulation by TEM showed the presence of chimeric H5 VLPs at a diameter of about 100 nm (Fig. 1D), which is a typical size of influenza VLPs.

Chimeric H5 VLP Vaccination  After extensive antibody reaction

Three weeks after immunization, a cross-clad HI test was performed on the H5N1 virus of four different clades. As shown in Figures 2 and 4, chickens immunized with the chimeric H5 VLP vaccine exhibited a significantly broader immune response to several clade 1 and 2 H5N1 viruses. The results of HI test of duck serum were more remarkable. As shown in Figures 3 and 5, ducks immunized with a standard VLP vaccine containing either clade 1 HA or clade 2 HA were able to induce the lowest level of antibody response to the H5N1 virus of several clades. Immunization of ducks with the chimeric VLP vaccine, however, was able to induce a broad antibody response against several other clade-derived H5N1 viruses.

Korea Biotechnology Research Institute KCTC12845 20150612

<110> Konkuk University Industrial Cooperation Corp <120> A chimeric virus-like particle vaccine confers broadened antibody          responses against different clades of highly pathogenic avian          influenza H5 viruses <130> HY151012 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 1695 <212> DNA <213> A / Vietnam / 1194/2004 <400> 1 atggagaaaa tagtgcttct ttttgcaata gtcagtcttg ttaaaagtga tcagatttgc 60 attggttacc atgcaaacaa ctcgacagag caggttgaca caataatgga aaagaacgtt 120 actgttacac atgcccaaga catactggaa aagacacaca atgggaagct ctgcgatcta 180 gatggagtga agcctctaat tttgagagat tgtagtgtag ctggatggct cctcggaaac 240 ccaatgtgtg acgaattcat caatgtgccg gaatggtctt acatagtgga gaaggccaat 300 ccagtcaatg acctctgtta cccaggggat ttcaatgact atgaagaatt gaaacaccta 360 ttgagcagaa taaaccattt tgagaaaatt cagatcatcc ccaaaagttc ttggtccagt 420 catgaagcct cattgggggt gagctcagca tgtccatacc agggaaagtc ctcctttttc 480 agaaatgtgg tatggcttat caaaaagaac agtacatacc caacaataaa gaggagctac 540 aataatacca accaagaaga tcttttggta ctgtggggga ttcaccatcc taatgatgcg 600 gcagagcaga caaagctcta tcaaaaccca accacctata tttccgttgg gacatctaca 660 ctaaaccaga gattggtacc aagaatagct actagatcca aagtaaacgg gcaaagtgga 720 cgagagtaat 780 ggaaatttca ttgctccaga atatgcatac aaaattgtca agaaagggga ctcaacaatt 840 atgaaaagtg aattggaata tggtaactgc aacaccaagt gtcaaactcc aatgggggcg 900 ataaactcta gcatgccatt ccacaatata caccctctca ccatcgggga atgccccaaa 960 tatgtgaaat caaacagatt agtccttgcg actgggctca gaaatagccc tcaaagagag 1020 aagagaggat tatttggagc tatagcaggt tttatagagg gaggatggca gggaatggta 1080 gatggttggt atgggtacca ccatagcaac gagcagggga gtgggtacgc tgcagacaaa 1140 gaatccactc aaaaggcaat agatggagtc accaataagg tcaactcgat tattgacaaa 1200 atgaacactc agtttgaggc cgttggaagg gaatttaaca acttagaaag gagaatagag 1260 aatttaaaca agaagatgga agacgggttc ctagatgtct ggacttataa tgctgaactt 1320 ctagttctca tggaaaacga gagaactcta gactttcatg actcaaatgt caagaacctt 1380 tacgacaagg tccgactaca gcttagggat aatgcaaagg agctgggtaa cggttgtttc 1440 gagttctatc ataaatgtga taatgaatgt atggaaagtg taagaaacgg aacgtatgac 1500 tacccgcagt attcagaaga agcaagacta aaaagagagg aaataagtgg agtaaaattg 1560 gaatcaatag gaatttacca aatattgtca atttattcta cagtggcgag ctccctagca 1620 ctggcaatca tggtagctgg tctatcctta tggatgtgct ccaatgggtc gttacaatgc 1680 agaatttgca tttaa 1695 <210> 2 <211> 1695 <212> DNA <213> A / mandarine duck / K10-483 / 2010 <400> 2 atggagaaaa tagtgcttct ctttacaaca atcagccttg ttaaaagcga tcatatttgc 60 attggttatc atgcaaataa ctcgacagag caggttgaca caataatgga aaagaacgtt 120 actgttacac atgcccaaga catactggaa aagacacaca acgggaagct ctgcgatcta 180 aatggagtga agcctctgat tttaaaagat tgtagtgtag cgggatggct cctcggaaac 240 ccattgtgtg acgaattcat caatgtgcca gaatggtctt acatagtaga gaaggccaag 300 ccagccaatg acctctgtta cccagggaat ttcaacgatt atgaagaatt gaaacaccta 360 ttgagcagga taaaccattt tgagaaaata cagatcatcc ccaaagactc ttggtcagaa 420 catgaagcct cattgggggt gagcgcagca tgttcatacc agggaaattc ctccttcttc 480 agaaatgtgg tatggcttat caaaaaggac aatgcatacc caacaataaa gaaaggctac 540 aataatacca accaagaaga tctcttggta ctgtggggga ttcaccatcc taatgatgag 600 gcagagcaaa caaggctcta tcaaaaccca accacctata tttccattgg gacatcaaca 660 ctaaaccaga gattggtacc aaaaatagcc actagatcca aaataaacgg gcaaagtggc 720 aggatagatt tcttctggac aattttaaaa ccgaatgatg caatccactt cgagagtaat 780 ggaaatttca ttgctccaga atatgcatac aaaattgtca agaaaggaga ctccacaatt 840 atgaaaagtg aagtggaata tggtaactgc aacaccaggt gtcagactcc gataggggcg 900 ataaactcta gtatgccatt ccacaacata caccctctca ccatcggaga atgtcccaaa 960 tatgtgaaat caaacaaatt agtccttgcg actgggctca gaaatagtcc tcaaagagag 1020 aaaagaggac tgtttggagc tatagcaggt tttatagagg gaggatggca gggaatggta 1080 gatggttggt atgggtacca ccacagcaat gagcagggga gtgggtacgc tgcagacaaa 1140 gaatctactc aaaaggcaat agacggagtc accaataagg tcaactcgat cattgacaaa 1200 atgaacactc agtttgaggc cgtaggaagg gaatttaata acttagagag gagaatagag 1260 aatttaaaca agaagatgga agacggattc ctagatgttt ggacttataa cgctgaactt 1320 ctggttctca tggaaaatga gagaactcta gatttccatg actcaaatgt caagaacctt 1380 tacgataagg tcagactaca gcttaaggat aatgcaaaag agttgggtaa cggttgtttc 1440 gagttctatc acaaatgtaa taatgaatgt atggaaagtg taagaaacgg aacgtatgac 1500 tacccgcagt attcagaaga agcaagacta aaaagagagg aaataagtgg agtaaaattg 1560 gaatcaatag gaatctacca aatactgtca atttattcaa cagtggcgag ttccctagtg 1620 ctggcaatca tgatggctgg tctgtcttta tggatgtgtt ccaacggatc gttacagtgc 1680 agaatttgca tttaa 1695 <210> 3 <211> 759 <212> DNA <213> A / Puerto Rico / 8/1934 <400> 3 atgagtcttc taaccgaggt cgaaacgtac gttctctcta tcatcccgtc aggccccctc 60 aaagccgaga tcgcacagag acttgaagat gtctttgcag ggaagaacac cgatcttgag 120 gttctcatgg aatggctaaa gacaagacca atcctgtcac ctctgactaa ggggatttta 180 ggatttgtgt tcacgctcac cgtgcccagt gagcgaggac tgcagcgtag acgctttgtc 240 caaaatgccc ttaatgggaa cggggatcca aataacatgg acaaagcagt taaactgtat 300 aggaagctca agagggagat aacattccat ggggccaaag aaatctcact cagttattct 360 gctggtgcac ttgccagttg tatgggcctc atatacaaca ggatgggggc tgtgaccact 420 gaagtggcat ttggcctggt atgtgcaacc tgtgaacaga ttgctgactc ccagcatcgg 480 tctcataggc aaatggtgac aacaaccaac ccactaatca gacatgagaa cagaatggtt 540 ttagccagca ctacagctaa ggctatggag caaatggctg gatcgagtga gcaagcagca 600 gaggccatgg aggttgctag tcaggctagg caaatggtgc aagcgatgag aaccattggg 660 actcatccta gctccagtgc tggtctgaaa aatgatcttc ttgaaaattt gcaggcctat 720 cagaaacgaa tgggggtgca gatgcaacgg ttcaagtga 759

Claims (11)

A chimeric virus-like particle comprising the hemagglutinin (HA) gene of the clade 1 H5N1 virus, the hemagglutinin (HA) gene of the clade 2 H5N1 virus and the influenza substrate protein M1 gene. 2. The chimeric virus-like particle according to claim 1, wherein the HA gene of the clade 1 H5N1 virus comprises the nucleotide sequence of SEQ ID NO: 1. 2. The chimeric virus-like particle according to claim 1, wherein the HA gene of the clade 2 H5N1 virus comprises the nucleotide sequence of SEQ ID NO: 2. The chimeric virus-like particle according to claim 1, wherein the influenza M1 gene comprises the nucleotide sequence of SEQ ID NO: 3. An antigen formulation comprising the chimeric virus-like particle of claim 1 and an adjuvant or an immune stimulator. A vaccine comprising the chimeric virus-like particle of claim 1. 7. The vaccine according to claim 6, wherein said vaccine has a protective effect against infection with poultry influenza virus. 7. The vaccine of claim 6, wherein the vaccine is formulated with an adjuvant or an immunostimulant. A method for inducing preventive immunity against avian influenza infection in an animal other than a human, comprising administering the antigen formulation of claim 5 to an animal other than a human. A method for inducing preventive immunity against avian influenza virus infection in an animal other than a human, comprising administering the vaccine of claim 6 to an animal other than a human. A vector containing both the HA gene of the clade 1 H5N1 virus and the HA gene of the clade 2 H5N1 virus and the vector containing the influenza M1 gene was cloned 1 and clade 2 HA using a baculovirus expression system A method for producing chimeric virus-like particles according to claim 1, comprising the steps of: preparing a recombinant baculovirus encoding gene and a recombinant baculovirus encoding influenza M1 gene; and co-infecting said recombinant baculovirus with insect cells.

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