KR101484605B1 - Inactivated vaccine composition for Japanese Encephalitis comprising Japanese encephalitis virus KV1899 strain antigen inactivated by binaryethyleneimine and swine GM-CSF recombinant protein - Google Patents

Abstract

The present invention relates to a Japanese encephalitis virus inactivated vaccine composition comprising a Japanese encephalitis virus KV1899 primary antigen and a porcine GM-CSF recombinant protein inactivated with BEI (binary ethyleneimine). The BEI-inactivated Japanese encephalitis virus KV1899 primary antigen and porcine GM-CSF recombinant protein according to the present invention are excellent in immunogenicity, mass production in insect cells and thus suitable for vaccine production, Because the genotype of 1-type Japanese encephalitis virus is identical, it can be used as an effective vaccine against Japanese encephalitis that solves the problems of conventional Japanese encephalitis live vaccine.

Description

Technical Field [0001] The present invention relates to a Japanese encephalitis inactivated vaccine composition comprising a primary antigen and a porcine GM-CSF recombinant protein, which is inactivated by BEI, a Japanese encephalitis virus KV1899 strain, recombinant protein}

The present invention relates to a Japanese encephalitis virus inactivated vaccine composition comprising a BEI-inactivated Japanese encephalitis virus KV1899 primary antigen and a porcine GM-CSF recombinant protein.

Japanese encephalitis is a mosquito-borne infectious disease that causes a person to develop encephalitis accompanied by high fever and coma, eventually leading to death. In Japan and Korea, Japanese encephalitis is a small red house mosquito ( Culex tritaeniorhynchus ), but in China, India, and Southeast Asia, Culex annulus , Culex pipiens , Culex gelidus and Anopheles spp. Mosquitoes are also known to mediate Japanese encephalitis virus. Unlike an adult who presents with an infectious infection, a child or an elderly person under 14 years old is infected with Japanese encephalitis, causing fever, headache, tardy, consciousness, coma, and death. . Wild birds such as pigs, horses, cattle, sheep, goats, and wild birds such as pigeons and herons and reptiles are susceptible to this disease, and pigs are known as amplification hosts for Japanese encephalitis viruses. to be. When the Japanese encephalitis virus is infected with mosquito by the mosquito, it causes clinical symptoms even if it causes viremia. However, when infected with Japanese encephalitis, pregnant piglets are infected with fertile piglets accompanied by similar acid symptoms and neurological symptoms such as premature ejaculation, Causing reproductive disorder disease. The infestation of spermatozoa inhibits spermatogenesis, reduces the number of sperm and increases the number of spermatozoa.

Japanese encephalitis is an infectious disease mediated by mosquitoes that have persistently occurred from the past. It has been reported in Australia, Africa, Russia, and other countries outside Southeast Asia, and is recognized as an emerging common infectious disease. In Asia, 67,900 cases Of which about 5,000 are dead. In particular, recent global warming and climate change are becoming more problematic because of changes in agriculture related channels, agriculture full-scale and scale, and the ecological environment of mosquitoes, which are disease carriers, can greatly affect the spread of Japanese encephalitis.

In addition, when a pig is infected with the Japanese encephalitis virus, it shows vomiting for two or three days, and when the mosquitoes vomit in this period, a large amount of virus is transferred to the mosquito. The Japanese encephalitis virus transferred from pigs can become infected with other animals or people through the salivary glands of mosquitoes after about 10 days in the mosquito's body. Therefore, the prevention of Japanese encephalitis and the formation of immunity against the pig, which is an amplification host, are very important for the management of Japanese encephalitis in humans.

Until now, Japanese encephalitis has been vaccinating pigs with live vaccines, but the live vaccine does not guarantee safety because it can not rule out the possibility of recovering the pathogenicity, and antibody titer is not formed high. In addition, the currently used live vaccine strain (300 strain of Anyang) is the genotype type 3 Japanese encephalitis virus vaccine developed in the 1980s, while the Japanese encephalitis virus that has occurred since 1990 is genotype 1, The homology of the gene to the genotype 3 is only 87%, and it is urgent to develop a vaccine using genotype 1.

Therefore, there is a great demand for development of inactivated vaccine which is superior in safety and immunogenicity to existing inactivated vaccine or purified live vaccine while using genotype 1.

In studying Japanese encephalitis virus vaccine, the inventors of the present invention found that a recently introduced genotype type 1 Japanese encephalitis virus, KV1899, is an antigen immobilized with BEI (binary ethyleneimine), a granulocyte macrophage colony-stimulating factor ) Is excellent in immunogenicity of recombinant protein, is produced in insect cells, is excellent in safety and can be mass-produced, and is suitable for vaccine production. Thus, the present invention has been completed.

Accordingly, the present invention provides a Japanese encephalitis-inactivated vaccine composition comprising a BEI-inactivated Japanese encephalitis virus KV1899 primary antigen and a porcine GM-CSF recombinant protein.

The present invention provides a Japanese encephalitis inactivated vaccine composition comprising a BEI-inactivated Japanese encephalitis virus KV1899 primary antigen and a porcine GM-CSF recombinant protein.

The present invention also provides a method for preventing or treating Japanese encephalitis by administering the vaccine composition to an animal other than a human.

The BEI-inactivated Japanese encephalitis virus KV1899 primary antigen and porcine GM-CSF recombinant protein according to the present invention are excellent in immunogenicity, mass production in insect cells and thus suitable for vaccine production, Because the genotype of 1-type Japanese encephalitis virus is identical, it can be used as an effective vaccine against Japanese encephalitis that solves the problems of conventional Japanese encephalitis live vaccine.

1 is a graph showing a result of a fluorescent antibody test performed using a monoclonal antibody (3A9 clone; manufactured by Agriculture, Forestry and Fisheries Quarantine Inspection Headquarters) specific to Japanese encephalitis virus (KV1899 strain).
Fig. 2 is a diagram showing electrophoresis results of the prME gene of amplified Japanese encephalitis virus.
Fig. 3 is a graph showing changes in weight (3A) and survival rate (3B) of intraperitoneally administered group mice of Japanese encephalitis virus (KV1899 strain).
Fig. 4 shows the results of the HI test (hemagglutinin inhibition test) of the Japanese encephalitis virus (4A) inactivated by BEI and the Japanese encephalitis virus (4B) inactivated by formalin.
FIG. 5 is a graph showing the results of immunization with BEI-inactivated viruses of the Japanese encephalitis virus (KV1899) and the viruses cultured in the 47th passage in the Vero cells in the 9th passage, and the ISS1313, cabopol, or rehydragel adjuvant Lt; RTI ID = 0.0 > HI < / RTI > test results.
6 is a diagram showing GM-CSF gene recombinant baculovirus of a purely isolated pig.
Fig. 7 shows HI test results of a GM-CSF recombinant protein-containing or non-containing Japanese encephalitis inactivating vaccine.

Hereinafter, the present invention will be described in detail.

The present invention provides a Japanese encephalitis inactivated vaccine composition comprising a BEI-inactivated Japanese encephalitis virus KV1899 primary antigen and a porcine GM-CSF recombinant protein.

The Japanese encephalitis virus KV1899 strain is one of the two Japanese encephalitis virus strains currently isolated in Korea. It is one of the four genotypes classified by the nucleotide sequence analysis of prM, E, and NS5 / 3 'NCR regions, Of viruses.

In addition, BEI (binary ethyleneimine) is effective as a strong alkali-resistant virus and is inactivated by alkylation of a lactic acid. Therefore, it is an inactivating agent that does not affect virus antigen and has a function of enhancing stability of antigen protein. BEI has no bactericidal effect on bacteria, but greatly weakens the virulence of virus, maintains immunogenicity by protecting viral protein, neutralizes by using sodium thiosulfate, Which is more safe than formalin.

In the present invention, the swine granulocyte macrophage colony-stimulating factor (GM-CSF) protein of the present invention is a kind of cytokine that functions as a growth factor of leukocyte. The stem cell is transformed into granulocyte (neutrophils, eosinophils, and basophils) (monocyte). It is a protein that increases the number of immune cells as well as the activation of macrophages in the body so that it can overcome the infection of external antigen quickly.

The BEI-inactivated Japanese encephalitis virus KV1899 primary antigen and porcine GM-CSF recombinant protein according to the present invention are excellent in immunogenicity, suitable for mass production, and superior in safety compared to conventional vaccines, It can be useful as an aviation vaccine.

The vaccine of the present invention may further comprise a pharmacologically acceptable carrier or diluent. Suitable carriers for vaccines are known to those skilled in the art and include, but are not limited to, proteins, sugars, and the like. The carrier may be an aqueous solution, or a non-aqueous solution, suspension or emulsion. Examples of non-aqueous carrier include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable organic ester such as ethyl oleate. Aqueous carrier includes water, alcohol / aqueous solution, emulsion or suspension, including saline and buffered medium. Parenteral carriers include sodium chloride solution, Ringer ' s dextrose, dextrose and sodium chloride, lactic acid treatment linger or fixed oils. The intravenous carrier includes electrolyte supplements, liquids and nutritional supplements, such as those based on Ringer ' s dextrose. Preservatives and other additives may include, for example, antimicrobial agents, antioxidants, chelating agents, inert gases, and the like. Preferred preservatives include formalin, thimerosal, neomycin, polyamicin B and amphotericin B.

In addition, the vaccine composition may further comprise an adjuvant (an immunomodulatory agent, an immunostimulant).

The adjuvant includes any absorption-promoting agent that refers to a compound or mixture that promotes an immune response and / or promotes an absorption rate after inoculation. Acceptable excipients include, but are not limited to, Freund's complete adjuvant, Freund's incomplete adjuvant, saponin, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oils or hydrocarbon emulsions, Non-hemicin, dinitrophenol, and the like. A preferred adjuvant is MONTANIDE IMS1313 adjuvant from SEPPIC.

The dose of the vaccine composition for the purpose of the present invention is preferably 0.01 ml to 1 ml per 1 kg body weight, preferably 0.1 ml to 0.5 ml per kg body weight.

The vaccine composition of the present invention may be administered through the route of administration such as oral, muscular, subcutaneous, intraperitoneal, intravenous, dermal, ocular, intracerebral, etc., but is not limited thereto.

The present invention also provides a method for preventing or treating Japanese encephalitis by administering the above vaccine composition to an animal other than human.

The animal other than human may be at least one animal selected from the group consisting of livestock including pigs, horses, cows, sheep, goats, and wild birds such as doves, herons and herons and reptiles.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example  One. Japanese encephalitis  Identification of genotype of virus

In order to confirm whether the isolated Japanese encephalitis virus strain (KV1899) was genotype 1, the following experiment was conducted.

1-1. Japanese encephalitis Of the virus strain (KV1899)  multiplication

First, the isolated Japanese encephalitis virus strain (KV1899) was proliferated against Vero cells grown in 10% fetal bovine serum (FBS) and a MEM Essential Medium alpha medium supplemented with antibiotics. To confirm the proliferation of Japanese encephalitis virus, a fluorescent antibody test was performed using a monoclonal antibody (3A9 clone; manufactured by Agriculture, Forestry and Fisheries Inspection Headquarters) specific to Japanese encephalitis virus.

The results are shown in Fig.

As shown in Fig. 1, specific fluorescence could be confirmed in the cytoplasm of Vero cells. In addition, when the Japanese encephalitis virus strain (KV1899) was cultured in the Vero cells, it exhibited a high proliferative activity of 10 ^7.0 TCID ₅₀ / ml or more and was suitable for the production of vaccine.

1-2. RT -PCR ( reverse transcriptase - 중합체 chain reaction )

For the detection of Japanese encephalitis virus, genomic RNA was extracted from the virus propagated in 1-1 above using an RNA extraction kit. The extracted RNA was eluted with 50 μl of distilled water.

In order to amplify the extracted RNA gene, a primer specific to Japanese encephalitis virus as shown in Table 1 below was prepared.

≪ Primer for identification of prME gene of Japanese encephalitis virus &

The amplification of the RNA gene was carried out by using primers of prM F / R, E1F / R, E2F / R and E3F / R shown in Table 1 for 30 min at 42 ° C, The cycle of denaturation for 45 seconds, annealing for 45 seconds at 53 DEG C and extension for 45 seconds at 75 DEG C was repeated 35 times and the amplified single stranded cDNA was incubated at 72 DEG C for 15 minutes DNA was double-stranded and electrophoresed on 1.8% agarose gel.

The electrophoresis results are shown in Fig.

As shown in Fig. 2, specific amplification of the prME gene of Japanese encephalitis virus was confirmed.

1-3. Japanese encephalitis  virus( KV1899 )

In order to confirm the genotype of the Japanese encephalitis virus (KV1899), the amplified prME gene was purified using a gel extraction kit, and then cloned into a pGEMT vector and sequenced to obtain 300 enantiomers of Japanese encephalitis genotype 3 And K94P05 strain of genotype 1.

The nucleotide sequence analysis result of Japanese encephalitis virus (KV1899) is shown in SEQ ID NO: 1.

As shown in SEQ ID NO: 1, the nucleotide sequence of the prME gene was analyzed. As a result, the Japanese encephalitis virus KV1899 strain showed 87.1% homology with the present 300 strain of Japanese encephalitis live bean, And 96.4% homology with the Japanese encephalitis genotype 1, K94P05 strain, was confirmed, confirming the Japanese encephalitis type 1 strain.

Example  2. Japanese encephalitis  Pathogenicity of virus

In order to confirm the pathogenicity of Japanese encephalitis virus (KV1899), two groups of 4 week old Balb / c mice (intraperitoneal administration group and intracerebral administration group) were used. 10 ^6.0 TCID ₅₀ / ml of the virus was decanted to 10, 100, and 1000 times, and 0.2 ml was injected into the peritoneal cavity of peritoneal-administered mice, 0.03 ml was injected into the brain of the intrabuccine mice, Respectively.

The results are shown in FIG.

As shown in Fig. 3, all of the intraperitoneally administered group mice continuously increased for 15 days without decreasing body weight, and did not die (3A). However, the mice in the intracerebral group were all killed on the 10th day in the undiluted solution and in the 10-fold diluted group, and the survival rate of the virus diluted 1000 times was 60%, indicating that 4-week old mice were pathogenic (3B).

Example  3. Japanese encephalitis  Virus inactivated vaccine Inactivating agent  Selection

In order to select the inactivating agent for the Japanese encephalitis virus inactivating vaccine, 10 ^7.3 TCID ₅₀ / ml of the virus propagated in Vero cells was inactivated with BEI (binary ethyleneimine) and formalin, respectively, Respectively.

In the inactivation method using BEI, 0.1 M BEI was diluted to 1% of the viral antigen, stirred at 37 캜 with a magnetic bar and left for 18 hours. After cooling to 4 ° C, 20% sodium thiosulfate solution was diluted 20-fold to neutralize the action of BEI.

The formalin-inactivated method was added to the virus to a concentration of 0.2%, and the mixture was stirred at 37 ° C for 18 hours.

The inactivated Japanese encephalitis virus antigens were inoculated twice in each of the above two methods. Blood samples were collected two weeks after the second inoculation and the antibody titers were measured by HI test (hemagglutinin inhibition test).

The results are shown in Fig.

As shown in Fig. 4, in the mouse group inoculated with the BEI-inactivated antigen, the HI antibody level was higher than that in the mouse group inoculated with the formalin-inactivated antigen, and thus the BEI was inactivated It was confirmed that it is preferable to use it as a topic.

Example  4. Japanese encephalitis  Virus of virus inactivation vaccine Number of passages  And Adjunct Burnt  Selection

In order to select the optimal virus strain number and ejuvant for the Japanese encephalitis virus inactivated vaccine, the Japanese encephalitis virus (KV1899) and the 47th passage cultivated virus (KV1899) cultured in Vero cells in the ninth passage were inactivated with BEI Each vaccine was prepared by adding 10% of IMS1313, cabopol, or rehydragel adjuvant to the antigen.

The prepared vaccine was inoculated twice with 0.2 ml of each mouse in each group of 2 mice at intervals of 2 weeks. In guinea pigs, 1 ml of each 4 mice per group was inoculated with 2 intramuscular injections every two weeks. Blood samples were collected two weeks after the second inoculation and HI test was performed to measure antibody titer.

The results are shown in Fig.

As shown in Fig. 5, the Japanese encephalitis virus inactivated vaccine, which was cultured in the ninth passage in both mouse and guinea pig, showed a higher level of HI antibody than the Japanese encephalitis virus inactivated vaccine cultured in the 47th passage, It was confirmed that it is preferable to use as inactivated vaccine.

In addition, as a result of comparing the antibody titers of the adjuvant, carbopole showed the highest antibody value in the mouse, and IMS1313 showed the highest antibody value in the guinea pig, but in the mouse, IMS1313 showed a difference between the carbopol and the large antibody , It was confirmed that IMS1313 should be used as an adjuvant in the production of Japanese encephalitis inactivated vaccine.

Example  5. Pigs GM - CSF  Preparation of recombinant proteins

GM-CSF recombinant protein was prepared by cloning pig GM-CSF gene to use porcine GM-CSF recombinant protein as an adjuvant for enhancing immunogenicity of Japanese encephalitis virus inactivating vaccine.

First, the GM-CSF gene of the porcine of SEQ ID NO: 2 was cloned into pGEMT vector and cloned into pBlueBac4.5His vector, a baculovirus transfer vector, and transfected into insect cell sf-9 cells. . Transfected sf-9 cells were purified using blue-containing agar overlay using X-gal containing medium to purify genetically engineered baculoviruses. GM-CSF recombinant protein of porcine was prepared by inoculating again insect cells with baculovirus which was genetically recombined to express GM-CSF gene.

The purified recombinant baculovirus is shown in Fig.

Example  6. Japanese encephalitis  Preparation of virus inactivated vaccine and evaluation of immunogenicity

In order to prepare a Japanese encephalitis virus inactivating vaccine, Japanese encephalitis virus KV1899 strain was cultured in Vero cells in 9 passages and confirmed to be 10 ^7.5 TCID ₅₀ / ml. In the same manner as in Example 3, the virus was inactivated with BEI and then treated with sodium thiosulfate Neutralized. In addition, GM-CSF recombinant protein produced in Example 5 was also inactivated with BEI by the method described above, confirming that the virus content was 10 ^6.5 TCID ₅₀ / ml.

Japanese encephalitis virus inactivated vaccine was prepared using 60% of Japanese encephalitis virus, 10% of GM-CSF recombinant protein and 30% of IMS1313. The control group was composed of 70% of Japanese encephalitis virus and 30% of IMS1313 One Japanese encephalitis inactivating vaccine was used. 2 ml of the two kinds of vaccines were inoculated into 8 pigs which did not have Japanese encephalitis virus antibody twice at intervals of 2 weeks. Then, blood samples were collected 2 weeks after the second inoculation and HI test was performed. And the immunogenicity of each vaccine was compared. The results are shown in Fig.

As shown in Fig. 7, in the pig group inoculated with the Japanese encephalitis-inactivating vaccine containing the GM-CSF recombinant protein, the antibody level higher than that of the porcine group inoculated with the Japanese encephalitis-inactivating vaccine containing no GM-CSF recombinant protein Respectively. Therefore, it was confirmed that the Japanese encephalitis-inactivated vaccine containing the GM-CSF gene recombinant protein of pigs exhibited better immunogenicity.

<110> REPUBLIC OF KOREA (Management: Ministry for Food, Agriculture, Forestry and Fisheries. National Veterinary Research and Quarantine Service (NVRQS)) <120> Inactivated vaccine composition for Japanese Encephalitis          Japanese encephalitis virus KV1899 strain antigen          inactivated by binary ethyleneimine and swine GM CSF recombinant          protein <130> ydk1-28p <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 3731 <212> DNA <213> Japanese encephalitis virus <220> <221> gene &Lt; 222 > (1) .. (3731) <223> JEV KV1899 strain prME gene <400> 1 agaagtttat ctgtgtgaac ttcttggctt agtatcgttg agaagaatcg agagattagt 60 gcagtttaaa cagtttttta gaacggaaga aaaccatgac taaaaaacca ggagggcccg 120 gaaaaaaccg ggccatcaat atgctgaaac gcggattacc ccgcgtattc ccactagtgg 180 gagtgaagaa ggtagtaatg agcttgttgg acggcagagg gccagtacga tttgtgctgg 240 ctcttatcac gttcttcaag tttacagcat tagccccgac caaggcgctt ttgggccgct 300 ggagagcagt ggaaaaaagt gtggcaatga aacatcttac cagtttcaaa cgagaacttg 360 gaacactcat cgacgtcgtg aacaagcggg gcaaaaaaca aaacaaaaga ggagggaatg 420 aaagtacgat catgtggctc gcaagcttgg caatcgtaac agcctgtgcc ggagccatga 480 agctatcaaa ctttcaagga aagcttttga tgaccatcaa caacacggac attgcggacg 540 tcatcgtgat ccccacctca aaaggtgaaa acagatgttg ggtccgagca atcgacgttg 600 gttacatgtg tgaagacacc atcacgtacg aatgtccgaa gcttgccgtg ggcaacgatc 660 cggaagacgt ggattgctgg tgcgacaatc aagaagtcta cgtgcagtat ggtcgctgca 720 cacggaccag gcattccaaa cgaagcagaa gatccgtttc ggtccagacg catggggaaa 780 gctcactagt gaacaaaaaa aaggcttggc tggattcaac gagggccacg cgatacctca 840 tgaaaacgga gaactggatc atacggaacc ctggttacgc tttcctggcg gcggcacttg 900 gatggatgct tggcagcaac agtggccaac gtgtggtgtt cactattctc ttgctgctgg 960 tcgctccggc ttacagtttt aactgtctgg gaatggggaa ccgggatttc atagaaggag 1020 ccagtggagc tacttgggtg gatctggtgt tagaaggaga tagctgtttg acaatcatgg 1080 caaacgacaa accaacacta gatgtccgca tgatcaacat tgaagctagc caacttgctg 1140 aagtcaggag ttactgctat cacgcttcag tcactgacat ttcaacggtg gctcgatgcc 1200 ccacgactgg agaagcccac aacgaaaaac gtgctgacag cagctacgtg tgcaaacaag 1260 gctttactga ccgcggatgg ggaaatggat gtggactttt cgggaaagga agcattgaca 1320 catgcgcaaa attttcttgt accagtaagg ccattggaag aatgatccaa tcagagaaca 1380 tcaagtacga ggttggcata ttcgtgcacg gaaccaccac ctcggaaaac catgggaatt 1440 actcagcgca agtaggagcg tctcaagcag caaagtttac tgtaactcca aatgctccct 1500 caataccct caagcttggt gattatggag aggtcacact ggattgtgaa ccaaggagtg 1560 gactgaacac tgaagcgttc tatgtcatga ccgtgggttc gaagtcattc ttagtccata 1620 gggaatggtt ccatgacctt tctcttccct ggacgtcccc ctcaagcacg gcatggagaa 1680 acagagaact cctcatggaa tttgaagagg cacatgccac aaaacaatct gtcgtagctc 1740 ttgggtcaca ggagggaggc ctccatcaag cgttggcagg agccatcgtg gtggagtact 1800 cgagctcagt gaagttgaca tcaggtcacc tgaaatgcag gctaaaaatg gacaaactgg 1860 ctctgaaggg cacgacttat ggcatgtgta cagaaaaatt ctcgttcgcg aaaaatccag 1920 cggacacagg ccatggaaca gttgtcattg agctcacata ctctggaagt gatggtccct 1980 gtaaaattcc gattgtctca gtcgcgagtt taaacgacat gacccctgtg gggaggctgg 2040 taacagtaaa ccccttcgtc gcgacatcta gctctaactc aaaggtgctg gttgagatgg 2100 aacctccctt cggagactct tacatcgtgg ttggaagagg ggacaagcag attaaccatc 2160 actggcacaa agctggaagc acgctgggta aagccttctc aacaactttg aaaggggctc 2220 agagactagc agcgctaggt gacacagcct gggacttcgg ctccattgga ggggtattca 2280 actccatagg aaaagctgtc caccaagtgt ttggcggtgc attcaggacg ctctttgggg 2340 gaatgtcctg gatcacacaa ggactaatgg gggccttact tctttggatg ggtgtcaacg 2400 ccgagaccg gtcaatcgcc ctggcttttc tggccacggg aggtgtgctc gtgtttttag 2460 cgaccaatgt gcatgccgac actggatgtg ccattgacat cacaagaaaa gagatgaggt 2520 gtggaagtgg catcttcgtg cacaacgacg tagaggcctg ggtagatagg tacaaatatc 2580 tgccagagac gcccagatcc ttagcgaaga tcgtccacaa agcacacaag gaaggagtgt 2640 gcggggtcag atccgtcact agactggaac accagatgtg ggaatctgtg cgggacgaac 2700 tgaatgtctt gctcaaagag aacgcggtgg atctcagtgt ggtggtgaac aagcccgtgg 2760 gtagatatcg ctcagccccc aaacgcctat ccatgactca agaaaagttt gagatgggct 2820 ggaaagcatg gggaaaaagc attctcttcg cccccgaatt ggccaactcc acgttcgtcg 2880 tggatggacc cgagacaaag gaatgccctg atgagcacag agcttggaac agcatgcaaa 2940 tcgaagactt cggcttcggc atcacatcaa cccgagtgtg gctgaaaatt agagaggaga 3000 ccactgatga gtgtgatgga gcaatcatag gcacggctgt caaagggcat gtgacagttc 3060 atagtgacgt gtcgtactgg attgagagtc gctacaatga cacgtggaaa cgtgagaggg 3120 ctgtcttcga agaggtgaaa tcttgcactt ggcccgagac acacactctt tggggtgatg 3180 gtgttgagga gagcgagctt attatcccac acaccatagc tggaccgaga agcaagcaca 3240 accggagaga agggtataaa acacaaaacc agggaccctg ggatgagaac ggcatcgtcc 3300 ttgactttga ctattgtcca ggaacgaaag tcaccatcac agaggactgt ggcaagaggg 3360 gtccctcact cagaaccact actgacagtg gaaagctgat caccgattgg tgctgccgca 3420 actgttctct accgccttta cggttccgga cagaaaatgg ttgctggtat gggatggaaa 3480 tcagacctgt taggcatgat gaaacaacac tcgtcagatc acaggttgat gctttcaacg 3540 gcgaaatggt tgaccctttt cagctgggcc ttctggtgat gtttctggcc acccaggagg 3600 tccttcgcaa gaggtggacg gccagattga cgattcctgc ggttttgggg gctctacttg 3660 tgctgatgct tgggggcatc acttacactg acctggcaag atatgtggtg ctagttgctg 3720 cggctttcgc g 3731 <210> 2 <211> 435 <212> DNA <213> Sus scrofa <220> <221> gene &Lt; 222 > (1) .. (435) <223> Sus scrofa GM-CSF gene <400> 2 atgtggctgc agaacctgct tctcctgggc actgtggtct gcagcatctc cgctcccacc 60 cgcccaccca gccctgtcac ccggccctgg cagcatgtgg atgccatcaa agaagccctg 120 agccttctaa acaacagtaa tgacacagcg gctgtgatga atgaaaccgt agacgtcgtc 180 tgtgaaatgt ttgaccccca ggagccgaca tgcgtgcaga ctcgcctgaa cctgtacaag 240 cagggcctgc ggggcagcct cactaggctc aagagcccct tgactctgtt ggccaagcac 300 tatgagcagc actgccccct caccgaggaa acttcctgtg aaacccagtc tatcaccttc 360 aaaagtttca aagacagtct gaacaaattt ctttttacca tcccctttga ctgctggggg 420 ccagtcaaaa agtaa 435

Claims (7)

For the entire vaccine composition;
58 to 62% by volume of Japanese encephalitis virus KV1899 primary antigen inactivated with BEI (binary ethyleneimine), 8 to 12% by volume of porcine GM-CSF recombinant protein encoded by the nucleotide sequence of SEQ ID NO: 1, 28 to 32% by volume of IMS1313 (Montanide IMS1313).
The vaccine composition according to claim 1, wherein the vaccine composition further comprises at least one selected from the group consisting of a pharmaceutically acceptable carrier, diluent, and adjuvant.
delete delete The vaccine composition according to claim 1, wherein the vaccine composition is administered through one or more routes selected from the group consisting of oral, muscular, subcutaneous, intraperitoneal, intravenous, dermal, .
A method for preventing or treating Japanese encephalitis by administering the vaccine composition of claim 1 to an animal other than a human.
The method for preventing or treating Japanese encephalitis according to claim 6, wherein said animal is at least one selected from the group consisting of pigs, horses, cows, sheep, goats, pigeons, herons and herons.

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