High-titer Japanese encephalitis virus genotype V and its use

Abstract

The present invention relates to a high-titer Japanese encephalitis virus genotype V and its use, and more particularly to a high-titer virus produced by passage culture and mouse cerebral inoculation method and a vaccine composition containing the same.

Description

[Technical Field]

[0001] The present invention relates to a high-titer Japanese encephalitis virus genotype V and uses thereof. [Background technology]

[0002] Japanese encephalitis (JE) is a mosquito-borne infectious disease that causes encephalitis in humans, accompanied by high fever and coma, and ultimately leads to death. In Korea and Japan, JE is known to be transmitted by the Culex tritaeniorhynchus mosquito. In China, India, and other Southeast Asian regions, JE is also known to be transmitted by Culex annulus, Culex pipiens, Culex gelidus, and Anopheles spp. Unlike adults, who show asymptomatic infection, JE infection in children under 14 and the elderly can cause high fever, headache, paresthesia, impaired consciousness, coma, and death. During recovery, symptoms include speech impairment and impaired judgment. Livestock, including pigeons, horses, cattle, sheep, and goats, as well as wild birds, including pigeons and blue herons, and reptiles, are susceptible to this disease. Pigs, in particular, are known to act as an amplification host for JE and are therefore an important target animal for immunization in public health. When Japanese encephalitis virus infects adult and fattening pigs via mosquitoes, it does not show any clinical symptoms even if it causes viremia, but if pregnant pigs are infected with Japanese encephalitis, it induces reproductive disorders such as premature birth, miscarriage, and the birth of weak piglets with neurological symptoms. In boars, the virus invades the reproductive tract and inhibits spermatogenesis, resulting in a decrease in sperm count and an increase in abnormal sperm, lowering the conception rate.

[0003] Japanese encephalitis (JE) is a mosquito-borne zoonotic disease that has been occurring continuously in Korea since the 1950s. It primarily occurs in Southeast Asia, including China, Japan, and Korea. However, it has also been reported outside of Asia, including Australia, Africa, and Russia, and is recognized as an emerging new infectious disease. In humans, more than 67,900 cases are reported annually in Asia alone, resulting in approximately 5,000 deaths. In particular, recent global warming and climate change, along with the development of agricultural irrigation channels and the specialized and large-scale nature of agriculture, are changing the ecological environment of mosquitoes, which are vectors of the disease, potentially significantly affecting the transmission of JE. Furthermore, pigs infected with the JE virus exhibit viremia for two to three days, during which time mosquitoes transmit large amounts of the virus to the mosquito when they feed on blood. After approximately 10 days in the mosquito's body, the JE virus can infect other animals and humans via the mosquito's salivary glands. Therefore, prevention of Japanese encephalitis and the development of immunity in pigs, which are amplification hosts, are extremely important for the control of Japanese encephalitis in humans.

[0004] The Japanese encephalitis virus currently circulating in Korea is genotype I, and the vaccine is being administered against genotype III. In 2015, genotype V was isolated for the first time from a patient infected with Japanese encephalitis virus in Korea (isolate name: K15P38), raising concerns that the existing circulating virus may be replaced. Therefore, preparations for the replacement of new genotypes (V types) are necessary, and the present invention aims to develop a vaccine candidate substance using genotype V viruses isolated from patients.

[0005] Patent Document 1 relates to a vaccine composition for preventing Japanese encephalitis in pigs, containing purified genotype 1 Japanese encephalitis virus, and discloses purified genotype 1 Japanese encephalitis virus KV1899-120P (accession number: KCTC18347P) and a vaccine composition containing the same. Patent Document 2 discloses an inactivated Japanese encephalitis vaccine composition containing a BEI-inactivated Japanese encephalitis virus KV1899 strain antigen and porcine GM-CSF recombinant protein.

[0006] However, the high-titer Japanese encephalitis virus genotype V of the present invention and its use are not disclosed. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Korean Patent Registration No. 1642705 [Patent Document 2] Korean Patent Registration No. 1484605 Summary of the Invention [Problem to be solved by the invention]

[0008] In order to solve the above problems, the present invention aims to prepare for the outbreak of a new Japanese encephalitis virus genotype, type V, by using viruses isolated from Japanese encephalitis patients in Japan to obtain high-titer viruses with improved cell proliferation rates, and to develop these as vaccine candidates. [Means for solving the problem]

[0009] To achieve the objectives of the present invention, the present invention provides a high-titer Japanese encephalitis virus genotype V K15P38-KNIH.

[0010] The Japanese encephalitis virus of the present invention, designated K15P38-KNIH, is derived from the Japanese encephalitis virus that has recently been prevalent in Japan. The K15P38 virus strain was subcultured in Vero cells to obtain the K15P38-V25 virus strain, which was then inoculated into the brain of a mouse to produce the final product. The genome sequence analysis result is the same as sequence number 1.

[0011] The Japanese encephalitis virus may have accession number KCTC15027BP.

[0012] The high titer is 1 x 10 8It may be more than pfu / mL.

[0013] In another example of the present invention, the present invention provides a method for producing high-titer Japanese encephalitis virus genotype V K15P38-KNIH, which comprises the steps of inoculating an isolated Japanese encephalitis virus into a mouse cerebrum and obtaining the Japanese encephalitis virus from the mouse cerebrum.

[0014] The isolated Japanese encephalitis virus may be Japanese encephalitis virus genotype V, the method may further include a step of subculturing the virus in Vero cells, and the inoculation may be performed two or more times.

[0015] In another embodiment of the present invention, the present invention provides a vaccine composition comprising the Japanese encephalitis virus.

[0016] The vaccine composition may further comprise a pharmaceutically acceptable carrier or excipient in addition to the virus. Suitable carriers for vaccines are known to those skilled in the art and include, but are not limited to, proteins, sugars, etc. The carrier may be an aqueous or non-aqueous solution, suspension, or emulsion. Examples of non-aqueous carriers include propylene glycol, polyethylene glycol, edible oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous carriers include electrolyte supplements, such as those based on Ringer's dextrose, fluids, and nutritional supplements. Preservatives and other additives may include, for example, antimicrobial agents, antioxidants, chelating agents, inert gases, etc. Preferred preservatives include formalin, thimerosal, neomycin, polymyxin B, and amphotericin B.

[0017] The vaccine composition may further contain an adjuvant (immunocomposition agent, immunopotentiator) in the form of a diluent.

[0018] The adjuvant refers to a compound or mixture that enhances immune response and / or accelerates absorption after inoculation, including any absorption enhancer. Acceptable adjuvants include, but are not limited to, Freund's complete adjuvant, Freund's incomplete adjuvant, saponin, mineral gel (e.g., aluminum hydroxide), surfactants (e.g., lysolecithin, fluron polyol, polyanion, peptide, oil or hydrocarbon emulsion, keyhole limpet hemocyanin, dinitrophenol, etc.). A preferred adjuvant is Montanide IMS1313 adjuvant manufactured by Seppic.

[0019] The dose of the vaccine composition for the purpose of the present invention can be adjusted depending on the condition of the subject, the route of administration, and the form of administration, and is not limited. Those skilled in the art of the present invention will understand that the dose can be used within various ranges depending on the symptoms. Generally, in the present invention, an experimentally effective dose is 0.01 to 1 mL (10 mL) per kg of the body weight of the subject. 5.0 TCID 50 / mL or more), preferably 0.01 to 0.03 mL per kg of body weight per day, can be administered continuously or intermittently.

[0020] The vaccine composition of the present invention can be administered via administration routes such as oral, intramuscular, subcutaneous, peritoneal, intravenous, dermal, ocular, and intracerebral, but is not limited to these, and is preferably administered via intramuscular administration.

[0021] In yet another embodiment of the present invention, there is provided a method for vaccinating a non-human animal against Japanese encephalitis, comprising the step of administering the vaccine composition to the non-human animal. [Effects of the Invention]

[0022] In order to improve the growth rate of vaccine strains, which is an essential condition for industrial application in vaccine development, the present invention has obtained high-titer viruses by repeatedly inoculating human isolates into animal cells and mouse brains. Although the growth-improved strains have some genetic mutations, they maintain immunogenicity compared to the original virus, making them more useful as vaccine candidate strains. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 shows a comparison of the growth rates of Japanese encephalitis viruses K15P38-V25 and K15P38-KNIH. [Figure 2] Comparison of the immunogenicity of Japanese encephalitis virus K15P38-V25 and K15P38-KNIH viruses. IgG ELISA of serum inoculated with each virus. (A) Original Japanese encephalitis virus attached, (B) K15P38-KNIH virus attached, control group (Ctrl) non-immunized mouse serum. [Figure 3] FIG. 1 shows the results of analyzing the neutralizing antibody titer of serum vaccinated with Japanese encephalitis virus K15P38-KNIH. [Figure 4] This figure shows the results of confirming an increase in immunoglobulin antibody titer (total IgG) in groups vaccinated with Japanese encephalitis virus genotype III and V antigens (8 μg / dose) when the same antigen was administered twice. [Figure 5] FIG. 1 shows the results of determining neutralizing antibody titers (ND50, Karber formula) using mouse sera induced by genotype V immunizing antigens. BEST MODE FOR CARRYING OUT THE INVENTION

[0024] The present invention will be described in more detail with reference to the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

[0025] Example 1: Method for producing Japanese encephalitis virus strains 1) Subcultured strain (K15P38-V25) To produce high-titer virus from the genotype V Japanese encephalitis virus strain K15P38, we performed repeated subculture experiments in Vero cells. The K15P38 virus strain is the original virus isolated from a patient. Vero (derived from monkey kidney epithelial cells) cells were cultured at 5 x 10 6 The cells were seeded at 75 flasks. After 24 hours, the cells were washed twice with DPBS, and 200 μL of the medium was inoculated into 3 mL of serum-free MEM medium and cultured for 1 hour at 5% CO2. 6 mL of 2% serum MEM was added to the culture medium, and the culture was then cultured for 48 hours at 5% CO2. This process was repeated 25 times to generate K15P38-V25.

[0026] 2) Mouse cerebral inoculation strain (K15P38-KNIH) This experiment was conducted to generate high-titer virus by repeated intracerebral administration of K15P38-V25 virus into BALB / c mice. Five female mice per set were anesthetized intramuscularly using a mixture of anesthetics (Zoletil: 50 μL / kg, Rompun: 10 mg / kg). A 1 cm incision was made in the scalp, and the mouse's head was fixed in a stereotaxic device for deep brain imaging. After confirming the bregma, a hole was drilled in the right skull over the hippocampus, and 5 μL of K15P38-V25 was administered over 2 minutes (2.5 μL / min). The skin was sutured, and recovery was induced. Disease status was monitored from day 0 of K15P38-V25 administration until days 5 and 6. If abnormal symptoms (piloerection, abnormal gait, or extension) occurred, brain tissue was removed. 1 mL of PBS was added to the brain tissue, and the tissue was crushed and centrifuged (12,000 rpm, 20 minutes). The separated supernatant was then inoculated into mice again, and this process was repeated.

[0027] <Example 2> Confirmation of virus titer by cell passage culture and repeated inoculation into mouse cerebral cortex 1) Confirmation of virus titer using plaque assay The titers of the virus obtained by passage culture using Vero cells and repeated inoculation into the brain after passage culture on Vero cells were compared. The JE genotype V isolate was passage cultured only on cells (black blocks in Figure 1). After passage culture on cells was repeated 1, 20, and 25 times, the maximum titer was 6.5 × 10 6 pfu / mL, but after repeated inoculation into mouse brains, the growth rate improved with 1, 3, and 5 subcultures on Vero cells (white blocks in Figure 1), and the titer was 1.15 × 10 8 It was confirmed that the pfu / mL

[0028] When the clinical specimen isolated virus (K15P38) was passaged in Vero cells (name: K15P38-V25), it was difficult to improve the growth rate. However, after subsequent inoculation into mouse brains (name: K15P38-KNIH, accession number: KCTC15027BP), the virus showed improved growth rate and a high titer. Even after repeated passages in mouse brains, a higher titer of virus was confirmed, even with a short passage in cells, compared to the titer obtained through cell passage (Figure 1).

[0029] Example 3: Confirmation of genetic mutations in K15P38-V25 and K15P38-KNIH viruses 1) Analysis of full-length gene mutations of Japanese encephalitis virus For the base sequence analysis of Japanese encephalitis virus that was subcultured in Vero cells (passages 1 and 25) and repeatedly inoculated into the brain to obtain high-titer virus, full-length gene mutations were analyzed using Blast 2 sequencing. As a result of the analysis, the identity of the Japanese encephalitis virus DNA was 98% (10925 / 10945), and it was confirmed that there were some gene mutations (Table 1). The sequence of the final virus strain obtained (K15P38-KNIH) is the same as SEQ ID NO: 1 below. JPEG0007763347000001.jpg154170

[0030] [SEQ ID NO: 1] K15P38-KNIH full-length genome sequence TAGAAGTTTATCTGTGTGAACTTCTTGGCTTAGTATTGTTGAGAGGAATCGAGAGATTAGTGCAGTTTAAACAGTTTTTTAGAACGGAAGAAAACCATGA 100 CTAAAAAACCAGGAGGGCCCGGTAAAAACCGGGCTATCAATATGCTGAAACGCGGCATACCCCGCGTATCCCCACTTGTGGGGGTGAAGAGGGTAATTAT 200 GAACTTGCTCGACGGCAGAGGGCCAATACGATTCGTTTTGGCTCTCTTGGCGTTTTTCAATTTCCACATCACTAGCCCCGACCAAGGCACTCGTTAGCCGA 300 TGGAAGGCAGTATAGAAAACGTTGCCAATGAAACATCTCACCAGTTTCAAAAAGGAACTGGGAACGCTCATCAACGCTGTAAATAAGAGGGGCAAAAAC 400 AAAACAAAAGAGGAGGAAGTAATGGGACAGTCATCTGGATAATGGGCTTGGCAGTTGTGGTTGCCAGTGTGAGTGCAATCAAGCTGTCAAATTTTCAAGG 500 CAAGGTGCTGATGACAATCAACAACACCGATGTGGCTGATGTGATCACCATTCCCACCTCAAAAGGGAGTAATAGATGTTGGGTCCGAGCTATAGATGTG 600 GGACACATGTGCGAGACACAATCACTTATGAATGCCCAAAGCTTGACGCTGGCAACGACCCGGAGGACATTGACTGCTGGTGCGACAAACAAGCCGTGT 700 ATGTCCAGTATGGACGTTGCACTAGGACCAGGCACTCCAGGAGAAGTAGAAGATCTGTGTCAGTGCAAACCCACGGAGAAAGTTCTTTTAGTGAACAAAA 800 AGAAGCCTGGATGGATTCGACGAAAGCCACTCGGTATCTCATGAAAACAGAGAATTGGATCATACGGAATCCAGGCTATGCTCTTGTGGCAGTGGCACTT 900 GGATGGATGCTTGGTAGCAACAACGGCCAGCGTGTGGTATTCACAATTCTCTTGCTGTTGGTCGCACCCGCATATAGCTTCAACTGCTTAGGTATGGGAA 1000 ATCGTGACTTCATCGAAGGAGTCAGTGGAGCGACGTGGGTGGACCTGGTGCTGGAAGGAGACAGTTGCCTCACCATCATGGCGAACGACAAACCAACATT 1100 GGACGTGCGCATGATAAACATCGAGGCCACGGAACTGGCTGAGGTGCGAACCTACTGCTACCACGCCACAGTGGCTGACATCTCAACAGTGGCAAGATGC 1200 CCCACGACTGGAGAAGCCCATAACACGAAGCGAGCTGACAGCAGCTATGTCTGCAAGCAAGGCTACACTGATCGTGGATGGGGAAACGGATGTGGGTTGT 1300 TTGGGAAAGGCAGCATTGACACATGCGCTAAATTTGTTTGCAGCCACAAGGCCATTGGAAAGATAATCCAGCCAGAAAACATCAAATATGAAGTTGGAGT 1400 GTTTGTTCATGGAACCACAACAGCAGAGAACCACGGAAATTACACAGCCCAGATTGGGGCTTCCCAGGCTGCCAAGTTCACCATCACGCCCAATGCTCCT 1500 TCCATCACTCTGAAGCTTGGAGATTACGGAGAAGTCACGATGGATTGCGAGCCGCGTAGTGGATTTAATACTGAGGCATTTTACGTGCTGACCGTAGGGT 1600 CTAAGTCGTTCCTAGTCCACCGCGAATGGTTCAATGATCTTGCGCTTCCGTGGCTATCTCCATCTAGCACAAATTGGAGAAATAGAGAGATCCTGATGGA 1700 GTTCGAAGAGGCTCACGCGACAAAACAGTCTGTCGTCGCGCTTGGATCACAAGAGGGAGCCTTGCATCAGGCATTGGCTGGTGCCATAGTGGTGGAGTAC 1800 TCTAGCTCAGTGAAGTTGACATCTGGTCACCTCAAATGCAGACTAAGAATGGAGAAGTTGGCGTTGAAAGGGACCACCTATGGTATGTGCACAGAGAAGT 1900 TCTCTTTTTCCAAGAATCCAGCTGACACTGGTCATGGTACGGTTGTCATAGAATTGCAGTACACCGGCACTGACGGACCTTGCAAGATACCCATTTCTTC 2000 GGTGGCCAGTCTGAATGATTTAACTCCAGTTGGTAGATTGGTGACAGTCAATCCTTTTGTTGCCACATCCACCGCCAATTCGAAGGTTTTGGTAGAATTG 2100 GAACCACCATTTGGAGATTCATTCATTGTTGTCGGAAGAGGAGATAAGCAGATCAATCACCATTGGCACAAGGCTGGCAGTTCACTGGGAAAGGCTTTCA 2200 CGACTACTTTGAAAGGAGCTCAGAGGCTGGCAGCCCTTGGTGACACCGCTTGGGACTTTGGATCCATTGGAGGGGTTTTCAATTCCATTGGTAAGGCCGT 2300 GCACCAAGTGTTTGGAGGAGCTTTCAGAACCCTTTTTGGTGGCATGTCCTGGATAACACAAGGATTGATGGGAGCGCTGCTGCTGTGGATGGGTATCAAC 2400 GCACGAGATCGGTCGATTGCACTAGCTTTTCTTGCTACAGGAGGTGTGCTCTTGTTTCTGGCCACCAATGTCCACGCCGATACCGGCTGCGCCATCGATA 2500 TAACCAGAAAAGAAATGAGGTGTGGTAGTGGCATATTTGTGCACAATGACGTGGAGGCTTGGGTTGATAGATACAAGTATCTACCTGAGACTCCCAAGTC 2600 TTTAGCCAAAATAGTCCACAAAGCACACAAGGAAGGCATTTGTGGAGTGAGATCAGTCACCAGACTGGAACACCAAATGTGGGAGGCCGTCAGGGACGAG 2700 TTAAATGTCTTGTTGAAGGAGAATGCGGTAGATCTTAGTGTGGTGGTGGACAAACCATCAGGAAGATACCGACCAGCTCCATTGCGGTTGGCCATGACTC 2800 AGGAAAAGTTTGAGATGGGTTGGAAAGCATGGGGGAAGAGCATTCTCTTTGCGCCGGAACTGGCCAATTCCACGTTTGTGATCGACGGACCTGAAACCAA 2900 AGAGTGTCCAGATGAGCGTAGAGCATGGAACAGCATGCAGATTGAGGATTTTGGGTTTGGCATTACGTCGACTCGAGTGTGGTTGAAGATCAGGGAGGAG 3000 CGCACGAATGAATGTGATGGAGCCATCATCGGCACGGCTGTTAAAGGGAACATGGCAGTGCACAGTGACTTGTCATACTGGATTGAAAGCCATCTCAACG 3100 ACACCTGGAAGCTCGAGAGAGCTGTGTTTGGAGAGATAAAGTCTTGCACTTGGCCAGAAACACACACGCTCTGGGGAGATGGTGTTGAGGAAAGTGAGTT 3200 GATAATTCCACACACACTCGCTGGACCCAAAAGCAAGCATAACAGAAGAGAGGGTTACAAGACACAGAATCAGGGACCATGGGACGAGAGTGAGATCACT 3300 CTTGATTTTGACTACTGTCCAGGGACCACAGTCACCATTGCTGAGGGATGTGGGAAAAGGGGGCCCTCAATCAGGACCACCACTGATAGTGGAAAATTAA 3400 TCACTGATTGGTGTTGCAGGAGCTGCACTTTGCCGCCACTGAGGTTCAGGACAGCCAGTGGGTGCTGGTATGGAATGGAAATACGGCCCATGAAgCATGA 3500 TGAATCCACGCTCGTGAAATCACAAGTCAACGCGTTTAATGGGGAGATGATTGATCCTTTTCAGTTGGGCCTTCTGGTGATGTTTCTGGCCACCCAGGAG 3600 GTCCTTCGCAAGAAGGTGGACGGCCAGACTGACGATTCCTGCGGTTTTGGGGGCCCTACTTGTTCTGATGCTTGGGGGCATCACCTACACTGATCTAGTG 3700 AGATATGTGGTGTTGGTGGCTGCTGCTTTCGCTGAAGCTAACAATGGAGGCGATGTAGTTCATTTGGCCCTGATTGCCGTATTTAAAATCCAACCGGCAT 3800 TCCTAGTCATGAGCATAGCAAGAACAAATTGGACAAACCAGGAAAACATTGCCCTAGTGCTAGGAGCTGCTTTCTTTCAGATGGCCTCAACAGACCTGGA 3900 GTTTGGTATCCATGGGCTGCTAAATGCAGCGGCGACGGCCTGGATGGTAGTGCGGGCGATCACGTTTCCCACGACCTCCACCATCACAATGCCCATACTA 4000 GCTTTGCTGGCGCCAGGAATGAGAGCTCTCCATCTTGACACCTACAGAATCTTTTTGCTTATCATCGGGGTCTGTGCTCTGCTGCATGAAAGGAGGAAAa 4100 aCCATGGCGAAGAAGAAAGGTGCTGTCCTTTTGGGTCTAGCCCTAAGTTCCACTGGGTGGTTTTCACCAGCCATTATGGCAGCTGGGCTCATGGCGTGCA 4200 ATCCAAACAAAAAGAGAGGATGGCCAGCGACAGAATTTCTGTCTGCGATTGGGTTGATGTTTGCTATTGTTGGGGGCCTGGCCGAGTTGGACATCGACTC 4300 TATGGCGATACCTTTCATGTTAGCTGGGCTTATGGCAGTGTCATATGTGGTATCAGGAAAAGCAACGGACATGTGGCTAGAACGTGCGGCCGACATTAGT 4400 TGGGAAGTGGATGCAGCGATCACAGGCAGCAGCCGAAGGTTGGACGTCAAACTAGATGATGATGGAGATTTCCACCTTATTGATGATCCAGGCGTCCCAT 4500 GGAAAATTTGGGTACTGCGCATGTCTTGCATAGGATTGGCGGCCTTCACACCATGGGCCATCATCCCAGCGGCTTTTGGATATTGGCTGACTTTGAAAAC 4600 CACAAAGAGAGGAGGCGTTTTTTGGGACACACCATCACCCAAAGTCTACGCAAAAGGGGACACAACCACAGGAGTGTACAGGATAATGGCTCGAGGGATC 4700 TTTGGCGTCTACCAAGCAGGTGTTGGAGTGATGTATGAGAACGTGTTCCACACTCTGTGGCACACGACCAGAGGAGCTGCCATAATGAGTGGAGAAGGAA 4800 AGTTGACGCCGTACTGGGGAAGCGTTAAGGAGGACCGCATAACTTATGGGGGCCCATGGAGATTCGATCGAAAATGGAATGGAGTGGATGACGTGCAAAT 4900 GATTGTAGTTGAACCAGGAAAAGCAGCTGTAAATGTCCAAACAAAGCCGGGAGTGTTTCGGACTCCACACGGAGAAATTGGAGCTGTTAGCCTGGATTAC 5000 CCTAGTGGGACATCGGGCTCACCCATCCTGGACATCAACGGTGATATCATTGGATTGTATGGGAACGGAGTTGAACTTGGAGATGGCTCATATGTGAGCG 5100 CCATTGTGCAGGGTGAACGGCAAGAAGAGCCCATCCCCGATGCATACAATCCAAACATGCTCAAGAAAAGGCAGCTGACCGTGTTGGACTTGCATCCAGG 5200 ATCTGGGAAAACAAGGAAAATCTTACCCCAAATCATCAAGGATGCCATTCAACAGCGTCTCAGAACAGCTGTTCTGGCGCCCACTCGGGTTGTTGCAGCT 5300 GAGATGGCAGAAGCTCTTAGAGGACTTCCCGTTAGATACCAAACTTCAGCGGTTCCGCGAGAACATCAGGGGAATGAGATAGTTGATGTCATGTGTCACG 5400 CCACCTTAACGCATAAGCTGATGTCACCAAATCGCGTCCCCAATTACAACTTGTTTGTCATGGATGAGGCCCATTTCACAGACCCAGCCAGCATCGCCGC 5500 TAGAGGATACATATCCACCAGAGTGGAATTGGGAGAAGCCGCGGCTATCTTCATGACTGCCACTCCACCAGGAACGACTGACCCCTTCCCTGACTCCAAC 5600 GCTCCCATTCATGATTTGCAGGACGAGATCCCTGACAGAGCATGGAGCAGTGGGTATGAATGGATAACTGAGTACTCTGGGAAGACAGTATGGTTTGTGG 5700 CGAGTGTGAAAATGGGCAACGAAATCGCAGTGTGTCTACAGAGAGCCGGAAAGAGAGTCATCCAGTTAAATCGGAAATCTTATGACACCGAGTACCCCAA 5800 ATGTAAGAATGGGGATTGGGATTTTGTCATCACCACGGACATTTCTGAAATGGGGGCCAACTTTGGAGCGAGCAGAGTGATTGATTGTAGGAAGAGTGTG 5900 AAACCCACCATTCTGGAAGAAGGAGAAGGAAGAGTCATTCTCAGCAACCCATCGCCTATCACCAGTGCGAGTGCAGCCCAGCGGAGAGGCAGAGTGGGCA 6000 GAAATCCAAATCAGGTTGGAGATGAGTACCATTACGGAGGGGTCACAAGTGAAGATGACACCAACCTAGCACACTGGACAGAAGCCAAGATCATGTTGGA 6100 CAACATCCACCTGCCAAATGGGTTGGTAGCTCAGCTCTATGGACCTGAAAGGGAAAAGGCCTTCACAATGGACGGTGAGTATCGATTGAGGGGTGAAGAA 6200 AAGAAGAACTTTCTGGAGTTAATCAGAACAGCCGACCTCCCCGTATGGCTAGCTTACAAAGTGGCTTCAAATGGAATACAGTACACCGATAGGAGATGGT 6300 GTTTTGATGGACCTCGGACGAATGCTATCTTAGAAGACAGCACTGAAGTAGAGATAATCACCAGAACGGGAGAAAGGAAAATTCTAAAACCAAGATGGCT 6400 GGACGCACGGGTGTACGCAGATCACCAGGCTTTGAAGTGGTTTAAGGACTTCGCAGCAGGAAAGAGATCAGCTGTCAGTTTTCTAGAGGTGCTTGGGCGC 6500 ATGCCAGAGCACTTCATGGGGAAAACGCGTGAAGCTCTTGATACAATGTACCTGGTTGCCACGGCAGAAAAAGGGGGAAAAGCTCACCGAATGGCCCTGG 6600 AGGAACTGCCAGATGCACTGGAGACGGTGACACTCATCGCAGCGCTTGCCGTGATGACAGGTGGGTTCTTTCTACTCATGATGCAACGAAAGGGCATAGG 6700 AAAAATGGGCCTCGGGGCTCTCGTGCTCACCCTGGCCACTTTCTTCTTGTGGATGGCAGAGGTCTCAGGGACGAAAATAGCCGGGACCTTACTCATAGCG 6800 TTGCTGCTTATGGTGGTACTCATCCCGGAGCCGGAGAAGCAAAGATCCCAAACGGACAACCAGTTGGCCGTGTTTCTGATCTGCGTCCTCACCGTAGTGG 6900 GAATTGTGGCTGCTAATGAGTATGGCATGCTCGAAAAGACCAAGGAAGACATAAGGAATGTGTTTGGTAACAAGGTTCAGACATCCAATGCACCTGGAAG 7000 CCTATCAAGTCTGGCGCTCGATCTACGACCAGCAACGGCTTGGGCCTTATATGGAGGTAGTACAGTGATTTTAACTCCACTGCTGAAACATTTGATCACC 7100 TCTGAGTATGTGACAACATCACTAGCTTCAATCAACTCACAGGCCGGCTCACTTTTTGTTTTGCCAAAAGGCATGCCTTTCACGGATTTGGATCTGACGG 7200 TTGGACTCGTCTTTCTGGGTTGTTGGGGGCAAATCACTCTCACCACTTTTCTGACAGCTGGAGTGTTAGTAGTTCTGCATTACGGCTATATGCTCCCTGG 7300 CTGGCAAGCTGAAGCCCTGAGGGCAGCTCAGAGACGAACAGCTGCGGGTATCATGAAGAACGCCGTTGTGGATGGGATGGTTGCCACTGATGTGCCTGAA 7400 TTGGAAAGAACAACACCCCTAATGCAGAAGAAGGTGGGGCAAGTGTTGCTAATAGGAGTCAGTATAGCAGCTTTTCTTGTCAATCCCAATGTCACCACCG 7500 TGCGAGAAGCCGGTGTGCTGGTGACCGCTGCTACGCTCACCCTATGGGATAATGGAGCAAGTGCCGTCTGGAATTCAACTACAGCCACAGGACTCTGCCA 7600 CGTCATGCGAGGCAGTTACTTGGCTGGTGGTTCAATAGCTTGGACTCTCATCAAGAATGTTGATAAACCATCCTTGAAAAGAGGAAGGCCTGGAGGAAGA 7700 ACGCTGGGTGAGCAATGGAAAGAAAAATTGAACGCCATGAACAAAGAAGAGTTTTTCAGGTACAGGAAAGAAGCCATAGTTGAGGTGGACCGCACAGAGG 7800 CACGCAGGGCTAGACGAGAGAATAACAAAGTGGGAGGTCATCCCGTGTCACGAGGTTCAGCAAAGCTCCGATGGATGGTGGAGAAAGGGTTTGTCTCCCC 7900 TGTTGGAAAGGTTGTGGACCTTGGCTGTGGGCGGGGAGGATGGTGCTATTATGCTGCCACTCTGAAAAAAGTGCAAGAAGTTAAAGGTTATACAAAAGGA 8000 GGGGCCGGACATGAGGAACCGATGCTGATGCAAAGCTATGGTTGGAATCTGGTCACGATGAAGAGTGGAGTGGATGTGTTCTACAGACCTTCAGAGCCTA 8100 GTGACACCCTGCTCTGTGACATAGGGGAGTCTTCTCCAAGTCCAGACGTCGAGGAGCAACGCACTCTGCGGGTTCTGGAAATGGCATCAGAGTGGCTACA 8200 CCGAGGACCCAGAGAATTCTGCATTAAAGTTTTGTGTCCATACATGCCAAAGGTGATAGAAAAGATGGAAACGCTACAACGTCGCTTTGGAGGCGGACTG 8300 GTGCGCGTTCCCCTGTCACGCAACTCAAATCACGAAATGTACTGGGTTAGTGGGGCTGCTGGGAACGTGGTACATGCTGTAAACATGACCAGTCAAGTTT 8400 TGCTGGGGCGAATGGACCGACCAGTCTGGAGAGGACCCAAATATGAAGAAGATGTCAACTTGGGAAGCGGGACCAGAGCTGTAGGGAAAGGTGAGGTTCA 8500 CAGTGACCAAGGAAAAATCAAGAAGCGGATAGAGAAGCTGAAAGAAGAGTATGCAGCGACGTGGCACGAGGACCCTGAACATCCATACCGCACTTGGACA 8600 TACCATGGAAGCTATGAAGTGAAGGCCACCGGTTCAGCCAGCTCCCTTGTCAACGGAGTGGTTAAGCTCATGAGTAAACCTTGGGATGCCATCACTAGTG 8700 TCACCACCATGGCCATGACTGACACTACTCCCTTTGGTCAGCAGAGAGTCTTCAAAGAAAAAGTTGACACCAAGGCGCCTGAGCCACCTGCAGGAGTTCG 8800 GGAAGTGCTGGACGAAACTACCAACTGGCTGTGGGCCTACTTGTCAAGAGAGAAAAGACCTCGCTTGTGTACGAGAGAGGAGTTCATCCGGAAAGTCAAC 8900 AGCAACGCAGCTCTTGGAGCCATGTTTGCCGAGCAAAATCAGTGGAGCTCAGCCAGGGAGGCTGTTAGTGACCCGGCCTTCTGGAACATGGTCGACATTG 9000 AAAGAGAGAACCACCTACGAGGGGAGTGCCACACCTGCATCTACAACATGATGGGAAAAAGAGAAAAGAAACCTGGTGAGTTTGGGAAGGCTAAAGGAAG 9100 CAGAGCTATCTGGTTCATGTGGCTCGGAGCCCGCTACCTGGAATTCGAGGCACTCGGGTTCCTGAACGAGGACCATTGGCTGAGCAGGGAGAACTCTGGA 9200 GGAGGAGTGGAAGGCTCAGGCATACAGAAGCTAGGGTACATTTTGCGAGACATCTCAACGAAATCTGGAGGGAAAATGTATGCTGATGACACCGCAGGCT 9300 GGGACACTAGGATCACAAGGGTTGATCTGGACAATGAGGCAAAGGTGCTGGAACTTCTGGATGGGGAGCACAGGATGTTGGCCCGTGCTATTATAGAATT 9400 GACTTACAAACACAAAGTTGTCAAAGTAATGAGGCCAGCAGTAGGTGGAAAGACCGTGATGGATGTGATCTCTAGAGAAGATCAAAGAGGGAGTGGGCAA 9500 GTGGTCACATATGCTCTCAACACCTTCACAAACATAGCTGTCCAGCTAGTGAGGTTGATGGAGGCTGAAGGGGTTGTCGGTCCACAGGATGTGGAGCAGC 9600 TCCCCAGGAAAACCAAATTCGCAGTCAGGACATGGCTTTTTGAAAATGGAGAAGAGAGAGTCACCAGAATGGCAGTGAGTGGGGATGACTGCGTTGTCAA 9700 ACCTTTGGATGACAGATTCGCGCATGCTTTACACTTCTTGAATGCGATGTCAAAGGTGAGGAAAGACATACAAGAATGGAAGCCATCTCAAGGCTGGCAC 9800 GACTGGCAGCAAGTCCCTTTCTGCTCGAACCACTTTCAGGAGATTGTGATGAAGGATGGTAGAAGCCTTGTTGTGCCCtGCCGGGGACAGGATGAGTTAA 9900 TAGGCAGGGCCCGGATTTCACCAGGAGCAGGATGGAATGTGAGAGACACAGCCTGCCTAGCTAAGGCCTACGCCCAAATGTGGCTCCTCCTCTACTTTCA 10000 CCGGAGAGACTTGCGCCTTATGGCCAACGCGATCTGCTCAGCCGTTCCGGTAGACTGGGTACCCACAGGTAGGACATCATGGTCGATACACTCAAAAGGA 10100 GAGTGGATGACAACAGAAGACATGCTGCAGGTGTGGAACAGGGTATGGATTGAGGAGAATGAATGGATGAGAGACAAAACCCCCGTCGCCAGTTGGACCG 10200 ACGTCCCATACGTCGGAAAGAGGGAAGACATCTGGTGCGGCAGCTTGATCGGAACGCGGACAAGGGCCACCTGGGCAGAAAACATCTATGCAGCAATAAA 10300 CCAAGTGAGAGCAATAATTGGAAATGAAAAGTATGTGGACTACATGACATCACTTAGGAGGTATGAAGACACTTTGGTCCAGGAAGATAGAGTAATTTAA 10400 AAAACTTTTGGTAATGAGTGTAAATAGTAGTATTTATTGTAAATAGTGTAAATAAACAAATTTAAATAGGAAGTCAGGCCGACGTAAGTCGCCACCGGAT 10500 GCTGAGTAGACGGTGCTGCCTGCGCCTCAGCCCCAGGAGGACTGGGTTAACAAATCTGACAACCGAAGGTAGGAAAGCCCTCAAAACCGTCTCGGAAGAA 10600 GGTCCCTGCTTACTGGAGGTTGGAAGACCGTGTCAGGCCACGCAAGTGCCACTTCGCTGAGGAGTGCAGCCTGTACAGCCCCGGGAGGACCGGGTAAACA 10700 AAGCCGATGAGGCCCCCACGGCCCAAACCTCATCCAGGATGCAATGGATGAGGCGTAAGGACTAGAGGTTAGAGGAGACCCCGTGGAAAAGAAAATGCGG 10800 CCCAAACTCTTTcGAAGCTGTAGAAGGAGTGGAAGGACTAGAGGTTAGAGGAGACCCCGCATTTGCATCAAAACAGCATATTGACACCTGGGATTAGACT 10900 AGGAGATCTTCTGATCTATCTCAACATCAGCTACAAGGCACAGAGCGCCGAAATATGTAGCTGGTGGTGGGGAAGAACACAGGATCT 10987

[0031] <Example 4>Confirmation of Immunogenicity of K15P38 and K15P38-KNIH 1) Immunogenicity Analysis Using ELISA Mice were immunized with K15P38 and K15P38-KNIH viruses, and serum was collected. The serum obtained after immunization with each virus was used to perform ELISA to confirm serum immunoglobulin G (IgG) production and cross-reactivity immunogenicity against each virus. The original Japanese encephalitis virus (K15P38) or K15P38-KNIH virus before cell passage was coated onto an ELISA plate, and the virus-specific immunoglobulin G (IgG) production of serum immunized with each cell-passaged virus (K15P38-V25) or mouse brain-passaged virus (K15P38-KNIH) was compared. Results confirmed that there was no difference in immunogenicity between the cell-passaged and brain-passaged viruses (Figure 2).

[0032] - We confirmed that there was no difference in antibody responses to the original isolate between the two subculture methods (Figure 2A), and that the immunogenicity to the brain-passaged virus was similar with no significant difference (Figure 2B).

[0033] 2) Neutralizing antibody formation analysis using the PRNT (Plaque Reduction Neutralization Test) assay Using mouse serum immunized with Japanese encephalitis virus K15P38-KNIH, neutralizing antibody titers (ND) against the original virus (K15P38) and high-titer virus (K15P38-KNIH) were measured. 50 , Karber formula) were compared.

[0034] We confirmed that mouse sera obtained by immunization with the K15P38-KNIH virus strain had an effective neutralizing ability against the original virus that was comparable to that of the high-titer virus (Figure 3).

[0035] - The neutralizing antibodies produced after immunization with a high-titer virus (K15P38-KNIH) were confirmed to have effective neutralizing ability against the original isolate and the high-titer virus, and it was confirmed that some gene mutations did not have a significant impact on immunogenicity.

[0036] 3) Confirmation of immunogenicity of vaccine candidate substances using an animal model (BALB / C) We secured inactivated antigens of the vaccine strain genotype III (Beijing) and vaccine candidate substance type V (K15P38-KNIH) used in Japanese encephalitis virus immunogenicity experiments.

[0037] In the Japanese encephalitis virus genotype III and V antigen (8 μg / dose) vaccination groups, when the same antigen was administered twice, an increase in immunoglobulin and antibody titers (total IgG) was confirmed, and there was no significant difference in the immunogenicity of the inactivated antigen (Figure 4).

[0038] Neutralizing antibody titers (ND 50, Karber formula) were determined using mouse serum induced by genotype V immunization antigens, and it was confirmed that the neutralizing ability against genotype III was significantly lower than that against genotype V virus, which suggests that cross-protection between genotypes is low.

[0039] Although certain aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents.

[0040] [Accession number] Depository institution name: Biological Resource Center Deposit number: KCTC15027BP Entrustment date and time: July 8, 2022

Claims

1. A high-titer Japanese encephalitis virus genotype V K15P38-KNIH with deposit number KCTC15027BP.

2. The high-titer Japanese encephalitis virus genotype V K15P38-KNIH, having the deposit number KCTC15027BP, according to claim 1, characterized in that the Japanese encephalitis virus consists of the gene of SEQ ID NO:

1.

3. A vaccine composition comprising the Japanese encephalitis virus of claim 1 or claim 2.

4. 4. The vaccine composition of claim 3, further comprising a pharmaceutically acceptable carrier or excipient.

5. A method for vaccinating against Japanese encephalitis, comprising the step of administering the vaccine composition according to claim 3 to a non-human animal.

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