KR101642705B1 - Attenuated genotype 1 Japanese encephalitis vaccine composition containing the same for preventing Japanese encephalitis in swine - Google Patents

Abstract

The present invention relates to a vaccine composition including an attenuated genotype 1 Japanese encephalitis virus for the prevention of porcine Japanese encephalitis. Specifically, the present invention relates to a novel virus strain obtained by serially injecting various cell strains with a genotype 1 Japanese encephalitis virus KV1899; and to a live vaccine strain which contains the virus strain and thus can secure excellent stability and immunogenicity. In the case of using a live vaccine containing a virus strain KV1899-120P provided according to the present invention, the live vaccine can highly effectively generate an antibody against a genotype 1 Japanese encephalitis virus in the human body, and thus can prevent infection. Especially, the live vaccine can prevent infection in pigs which are amplifying hosts of Japanese encephalitis viruses, and thus can greatly reduce the risk of Japanese encephalitis virus infection in humans.

Description

[0001] The present invention relates to a vaccine composition for preventing swine encephalitis in a Japanese encephalitis vaccine composition,

The present invention relates to a vaccine composition for the prevention of Japanese encephalitis comprising a purified genotype 1 Japanese encephalitis virus, and more particularly, to a vaccine composition for preventing Japanese encephalitis, comprising a novel virus strain obtained by a method of transferring KV1899, The present invention relates to a live vaccine strain capable of providing an immunogen with excellent stability by containing a virus strain.

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. Japanese encephalitis is known to be transmitted by small red house mosquitoes ( Culex. Tritaeniorhynchus ) in Korea and Japan, but 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, which causes high fever, headache, tardive dysfunction, consciousness disorder, coma and death. . Livestock such as pigs, horses, cattle, sheep, goats, wild birds such as pigeons and herons, and reptiles are susceptible to this disease. In particular, pigs are known as amplification hosts for Japanese encephalitis viruses. It is an animal. 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, the infant with the similar acid symptoms such as preterm birth, mummy fetus, Causing reproductive disorder disease. The infestation of spermatozoa inhibits spermatogenesis, decreases the number of spermatozoa and increases the number of spermatozoa.

In Japan, Japanese encephalitis is a common infectious disease mediated by mosquitoes, which has been occurring continuously since 1950, and occurs mainly in Southeast Asia, including China, Japan, and Korea. However, it has been reported in Australia, Africa, Russia, and other non-Asian countries, and is recognized as a new emerging infectious disease. In Asia, only 67,900 cases are reported every year, of which about 5,000 people die. In particular, recent global warming and changes in climate have become more problematic because of the change in the ecological environment of the mosquito, which is the disease mediator, along with the maintenance of agriculture relations channels, the industrialization and scale of agriculture, and the influence of 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, live vaccine has been inoculated into pigs to prevent Japanese encephalitis, but the live vaccine has a problem that the antibody level is not formed high. The Japanese encephalitis virus, which is currently being used, is the genotype 3 type Japanese encephalitis virus vaccine developed in the 1980s, while the Japanese encephalitis virus, which has occurred since 1990, Is only 87% homologous to the gene, so there is a limit to the prevention of the currently prevalent gene 1. Therefore, development of vaccine using genotype 1 is urgent.

Accordingly, the present inventors have made efforts to develop a vaccine against genotype 1 Japanese encephalitis virus, which is superior to conventional live vaccine or inactivated vaccine in terms of safety and immunogenicity. As a result, it was possible to develop a new virus strain from the KV1899 virus strain, and it was confirmed that when the virus strain was used as a live vaccine, it could provide excellent stability and an immunogen, and the present invention was completed.

1. Kwon, Hyeok-jin, Kang, Byung-jik, Lim, Young-moon, Studies on the Vaccine of Japanese Parenteral Encephalitis IV. Immunogenicity, farming test on pigs in tissue culture-purified Japanese encephalitis virus (Anyang, Korea) 19, 7-12, 1978. 2. Fan YC1, Chen JM, Chen YY, Lin JW, Chiou SS. Reduced neutralizing antibody titer against genotype I virus in swine immunized with a live-attenuated genotype III Japanese encephalitis virus vaccine. Vet Microbiol. May 3, 163 (3-4): 248-56.

Therefore, a main object of the present invention is to provide a novel virus strain which can be used as a live vaccine against genotype 1 Japanese encephalitis virus.

Another object of the present invention is to provide a live vaccine against genotype 1 Japanese encephalitis virus which can provide excellent safety and immunogenicity by containing the virus strain.

According to one aspect of the present invention, the present invention provides a purified genotype 1 Japanese encephalitis virus KV1899-120P (Accession No. KCTC18347P).

The Japanese encephalitis virus of the present invention named KV1899-120P is derived from the Japanese encephalitis virus which is recently popular in Korea, and KV1899 virus strain was obtained in relation to Vero cell, TF104 cell, ST cell, A72 cell and PK15 cell . prM, E, and NS5 / 3 'NCR regions, and the results of the genome sequencing analysis are as shown in SEQ ID NO: 1. Since the genome of the Japanese encephalitis virus is RNA, the DNA sequence of SEQ ID NO: 1 is converted from the KV1899-120P genomic RNA sequence of the present invention to DNA.

According to another aspect of the present invention, there is provided a Japanese encephalitis vaccine composition comprising the Japanese encephalitis virus.

The Japanese encephalitis vaccine of the present invention may further include a pharmacologically acceptable carrier or diluent 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, and the like. The carrier may be an aqueous solution or a non-aqueous solution, a suspension or an 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 carriers include water, alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media. 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 (immunosuppressant, immunostimulant) in the form of a diluent.

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 dosage of the vaccine composition for the purpose of the present invention is not limited as it can be adjusted depending on the condition of the subject to be administered, the route of administration and the administration form. As a matter of course, Usually 0.01 to 1 ml (10 ^5.0 TCID ₅₀ / ml or more) per 1 kg body weight of the subject to be treated, preferably 0.01 to 0.03 ml per 1 kg of body weight, Intermittent administration is possible.

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.

In the present invention, the stability and efficacy of the virus of the present invention as a vaccine were evaluated in mice, guinea pigs and pigs. Since animals have been proven to be stable in both mouse, guinea pig and pigs, animals such as livestock including pigs, horses, cows, sheep and goats, wild birds such as pigeons, herons and herons, and reptiles It can be used to prevent encephalitis. In addition, a separate stability experiment has been conducted, and if it is judged to be safe for the human body, it can be used for human.

When the present invention is used as a live vaccine containing KV1899-120P virus strain newly provided by the present invention, an antibody against genotype 1 Japanese encephalitis virus can be generated very effectively in the body to prevent infection, As a result, the risk of infection with Japanese encephalitis virus is expected to be greatly reduced.

1 is a microscopic photograph of a plaque formed by infection with the Japanese encephalitis virus strain (KV1899-120P).
FIG. 2 is a microscopic photograph of the specific cytopathic effect of Vero cells, which appeared as an infection of the Japanese encephalitis virus strain (KV1899-120P).
Figure 3 shows the result of a fluorescent antibody test performed using a specific monoclonal antibody (3A9 clone; manufactured by Agriculture, Forestry and Livestock Quarantine Division) that appeared as an infection of Japanese encephalitis virus strain (KV1899-120P).
4 is a photograph of a Japanese encephalitis test vaccine sample lyophilized with 10 ^7.0 TCID ₅₀ / ml of the Japanese encephalitis virus strain (KV1899-120P).
Fig. 5 shows the result of checking for contamination with the Japanese encephalitis virus strain (KV1899-120P) sample using primers specific for mycoplasma, parainfluenza, rabies, Akabane, and epidemic virus. The sample contained no viruses other than Japanese encephalitis virus.
6 shows the survival rates of the Japanese encephalitis virus strain (KV1899-10P) (A) in the 10th generation and the Japanese encephalitis virus strain (KV1899-120P) (B) And 40% of the mice inoculated with the undiluted solution of KV1899-10P and the 10-fold diluted solution died from the mice that were killed and 1,000 times inoculated. However, inoculation into the brain by diluting 1,000 times from the stock solution of KV1899-120P survived in all mice.
FIG. 7 is a graph showing the results of hemagglutination inhibition antibody titer after inoculation of Japanese encephalitis virus strain (KV1899-120P) into guinea pig.
8 is a graph showing the results of hemagglutination inhibition antibody titers after inoculation of Japanese encephalitis virus strain (KV1899-120P) into SPF piglets (A) and pregnant sows (B).
FIG. 9 is a graph showing the results of inoculation of mice with the Japanese encephalitis virus strain (KV1899-120P) and inactivated Japanese encephalitis virus vaccine strain (KV1899-10P) twice in the mouse and inoculation with K87 strain (genotype 3 Japanese encephalitis virus) to be. Mice inoculated with the Japanese encephalitis virus strain (KV1899-120P) survived 100% while those inoculated with the inactivated vaccine survived 60%.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example 1. Purification of Japanese encephalitis virus

In order to purify the genotype 1 Japanese encephalitis virus host KV1899, the following experiment was carried out. The KV1899 virus strain was isolated from the pig blood of the pig farm in Gyeonggi-do in 1999.

1-1. Transmission of KV1899 in various cells

KV1899 was used to obtain Japanese encephalitis virus purified from 5 different cells. As shown in Table 1, in the case of 1 to 5 animals, TF104 (MA104, cells derived from monkey kidney cells), 6 to 10 animals were observed in Vero (monkey kidney derived) (Puer kidney cells) cells in 61 to 80 cells, Vero cells in 81 to 120 cells in 51 to 60 cells in A72 (dog tumor cell) cells, 21 to 45 cells in TF104 cells, 46 to 50 cells in Vero cells, . The content of total virus was low at 10 ^4.5 TCID ₅₀ / ㎖ from the initial separation stage to the 5th generation. However, the content of virus increased during the period of 6 generations, and the virus content increased from 10 ^8.0 while raised to TCID ₅₀ / ㎖ showed a virus content of 10 ^7.0 TCID ₅₀ / ㎖ at 120.

The Japanese encephalitis virus (KV1899) was transfected with cells and virus content Number of passages cell Virus content Remarks 1-5 Vero cell 10 ^4.5 TCID ₅₀ / ml 6 to 10 TF104 cell 10 ^7.0 TCID ₅₀ / ml Passage by Limit Dilution Method 11-20 ST cell 10 ^7.0 TCID ₅₀ / ml Passage by Limit Dilution Method 21 ~ 45 TF104 cell 10 ^8.0 TCID ₅₀ / ml Passage by Limit Dilution Method 46 to 50 Vero cell 10 ^7.3 TCID ₅₀ / ml Passage by Limit Dilution Method 51 ~ 60 A72 cell 10 ^7.5 TCID ₅₀ / ml Passage by Limit Dilution Method 61 to 80 PK15 cell 10 ^7.0 TCID ₅₀ / ml Passage by Limit Dilution Method 81-120 Vero cell 10 ^7.0 TCID ₅₀ / ml Passage by Limit Dilution Method

After the 5th passage, the virus was cloned into the TF104 cells to maximize the dilution. In other words, Japanese encephalitis virus was diluted decisively and continued to pass the virus identified in the last dilution. The cloning method by the final dilution method is as follows.

The virus stock solution was diluted stepwise from 10 < ^-1 > to 10 <" ⁷ > In other words, 200 μl of virus stock solution and 1.8 ml of dilution were mixed and then 200 μl each was passed to the next dilution step. After appropriate labeling, 0.1 ml of virus was inoculated into each well of Vero cell-grown cells and incubated at 37 ° C for 5 days. Viruses of the last dilution wells showing cytopathic effect were harvested using a pipette. Thus, the Japanese encephalitis virus was transferred to 120 generations, and the resulting virus was named KV1899-120P. The Japanese encephalitis virus was proliferated and then lyophilized to confirm that the virus content was 10 ^7.0 TCID ₅₀ / ml, and the following experiment was conducted.

Example 2. Nucleotide Sequence Analysis of Japanese Encephalitis Virus

2-1. Analysis of the E-protein of the Japanese encephalitis virus group

The amino acid changes of the E protein of Japanese encephalitis virus against the first, ninth, 51st, 96th, 104th, 118th, and 120th generations were observed in several cells. As shown in the following Table 2, amino acids of E73, E132, E138 and E489 were changed to glutamine, proline, lysine and alanine when they were in the ninth generation. When the 96th line was passed, the amino acid of E160 was replaced with glutamic acid, , It was confirmed that the amino acids of E179 and E394 were changed to threonine and glutamine.

Changes in Amino Acids of K Protein E Protein by KV1899 One KV1899 Nine 51 units 96 103 118 120 One E73 R (cga) Q (caa) Q (caa) Q (caa) Q (caa) Q (caa) Q (caa) 2 E132 S (tca) P (cca) P (cca) P (cca) P (cca) P (cca) P (cca) 3 E138 E (gag) K (aag) K (aag) K (aag) K (aag) K (aag) K (aag) 4 E160 G (gga) G (gga) G (gga) E (gaa) E (gaa) E (gaa) E (gaa) 5 E179 K (aag) K (aag) K (aag) K (aag) K (aag) T (acg) T (acg) 6 E394 H (cat) H (cat) H (cat) H (cat) H (cat) Q (caa) Q (caa) 7 E489 G (ggt) A (gct) A (gct) A (gct) A (gct) A (gct) A (gct)

2-2. Analysis of NS1 protein of Japanese encephalitis virus group

1, 9, 11, and 120 years of age. As shown in the following table, the amino acids of NS1-51, 52, 146, 175, 193, 198, 206, 212, 218, 298 and 315 in nine units are alanine, glycine, arginine, asparagine, alanine, leucine, Leucine, glycine, isoleucine, and serine. In the 11th line, NS-1-183 and 339 were changed to valine and lysine, respectively.

2-3. Genetic analysis after the passage of 120 Japanese encephalitis virus

Changes in the genomes of viruses (KV1899-120P) obtained for parental viruses KV1899 and 120 in the subculture were compared. As shown in FIG. 10, KV1899 is composed of 10,963 nucleotides, whereas KV1899-120P of 120 generations has 10,965 nucleotides with two nucleotides. After 120 generations, 223 bases were changed in the genome and 67 amino acids were replaced.

KV1899 Changes in Amino Acids of NS1 Proteins One KV1899 Nine  11  120 One NS1-51 K (aag) A (caa) A (caa) A (caa) 2 NS1-52 E (gaa) G (gga) G (gga) G (gga) 3 NS1-146 H (CaC) R (cGC) R (cGC) R (cGC) 4 NS1-175 T (acc) N (aac) N (aac) N (aac) 5 NS1-183 I (atc) I (atc) V (gtc) V (gtc) 6 NS1-193 T (aca) A (gca) A (gca) A (gca) 7 NS1-198 V (gTG) L (ttg) L (ttg) L (ttg) 8 NS1-206 Y (tac) L (ctc) L (ctc) L (ctc) 9 NS1-212 R (cGT) L (ctt) L (ctt) L (ctt) 10 NS1-218 E (gaa) G (gga) G (gga) G (gga) 11 NS1-298 L (ctc) I (atc) I (atc) I (atc) 12 NS1-315 N (aac) S (agc) S (agc) S (agc) 13 NS1-339 R (agg) R (agg) K (aag) K (aag)

That is, the Japanese encephalitis virus (KV1899-120P) of the present invention was found to have many mutations compared to the gene of the first isolate (KV1899).

Example 3 Evaluation of purified Japanese encephalitis virus (KV1899-120P) as a vaccine

3-1. Identification of Japanese encephalitis virus

The Japanese encephalitis virus was confirmed by various types of cells. As shown in Figs. 1, 2 and 3, the plaque formation of viruses, the cytopathic effect of 10-dilution of the virus with high dilution drainage, and the specific enucleation of cytoplasm by staining with Japanese encephalitis-specific antibody and fluorescent conjugate Using the method of investigation. As shown in FIG. 4, Japanese encephalitis virus was proliferated and then lyophilized to confirm that the virus content was 10 ^7.0 TCID ₅₀ / ml, and the following experiment was conducted.

3-2. Check for contamination

KV1899-120P In order to confirm the contamination of viruses and viruses in Japanese encephalitis virus samples, genetic tests were carried out using primers specific for Mycoplasma, Akabane virus, canine parainfluenza virus, rabies virus, Respectively. As shown in Fig. 5, there was no contamination of mycoplasma, Akabane virus, parainfluenza virus, rabies virus and small-scale heat virus.

3-3. Stability evaluation

In order to confirm the safety of Japanese encephalitis virus in mice, KV1899-10P (virus obtained from 10 generations) and KV1899-120P were respectively proliferated, and the virus content was 10 ^6.5 TCID ₅₀ / ml, 10 ^7.0 TCID ₅₀ / ml The dilution was diluted 10-fold and diluted to 1000-fold. Each dilution was inoculated with 0.03 ml of 4-week-old BALB / c 5 mice in the brain. Clinical symptoms were confirmed. As shown in FIG. 6, 100% of the mice inoculated with the stock solution of KV1899-10P and 10-fold diluted solution died and 40% of the mice inoculated with the 100 and 1000-fold diluted solution died until 9th day after inoculation. On the other hand, mice inoculated with the diluted solution of KV1899-120P, 10, 100, and 1000 times survived until 15 days after inoculation without death. These results indicate that the KV1899-120P virus strain is highly stable.

In addition, 1.0 ml of KV1899-120P sample (10 ^6.0 TCID ₅₀ / ml) was injected intramuscularly with 4 days old Japanese encephalitis virus antibody piglets, and blood was collected on days 1, 2 and 4, and the virus was sacrificed on the 7th day As a result, the virus could be isolated from the blood of the second day. Thus, the Japanese encephalitis virus isolated from the blood was repeatedly inoculated repeatedly to the four-day-old piglets three times, and then the mice were inoculated again to the 4-day-old mammalian mice and the clinical symptoms were observed until day 7 after the inoculation. But it grew normally. This indicates that the piglets are counter-passaged over three generations and are safe even when they are inoculated into the mouse brain again. In addition, the gene of the Japanese encephalitis virus gene was confirmed in the mouse brain. This indicates that KV1899-120P in mammalian mice lacking the immune system is not effectively removed from the mouse body and survives consistently, but does not show any pathogenicity.

3-4. From guinea pig  Immunogenicity evaluation

A vaccine sample was prepared by adding lyophilized Japanese encephalitis virus KV1899-120P and diluent to various kinds of adjuvants and prepared as shown in Table 4 for evaluation of immunogenicity in guinea pig. Immunogenicity of Japanese encephalitis was confirmed by immunizing the vaccine sample two times at intervals of two weeks and collecting blood to confirm hemagglutination inhibition antibody (HI). As shown in FIG. 7, when a vaccine sample was inoculated once to guinea pigs, low HI antibody titer was induced between 40 and 160 times, but when the two shots were inoculated, high boosting effect was obtained and high HI antibody titer was induced from 960 to 1,920 . There was no significant difference in antibody titers obtained from guinea pigs inoculated with various adjuvants in a vaccine sample.

Inoculation to confirm immune response of Japanese encephalitis live vaccine in guinea pig number Group name Diluent (adjuvant) inoculation
Head inoculation
Route Inoculation method One PRME PRME 4 I.M. Every two weeks
2 doses
<Blood collection>
1st - 2nd week
2nd - 2nd week 2 JEV P120 JEV P120 + PBS 4 I.M. 3 IMS1313 JEV P120 + IMS1313 4 I.M. 4 IMS1513 JEV P120 + IMS1513 4 I.M. 5 Come JEV P120 + Montanide gel 4 I.M. 6 ISA206 JEV P120 + ISA206 4 I.M. 7 Control PBS 4 I.M.

3-5. Evaluation of safety and immunogenicity in pigs

To evaluate the safety and immunogenicity of live vaccine (KV1899-120P), six pigs with negative antibody against Japanese encephalitis virus and three sows at 60 days of gestation were selected. Four of six selected piglets were inoculated twice with live vaccine at intervals of 2 weeks. No clinical symptoms or weight loss after the inoculation was observed. As shown in FIG. 8 (A), blood samples were collected at intervals of 2 weeks for piglets, and the HI antibody titer was 60 times or more on average at 2 weeks after the second inoculation. The control group was negative for antibody, and 12 weeks after the vaccination And the HI antibody level was maintained at 20 times. In addition, 2 sows at 60 days of gestation were treated with 1 inoculation group and 1 in control group. Inoculation group was injected twice at 2 - week intervals. Clinical symptoms and weakness of piglets were observed in 3 sows including inoculated group. In total, 30 sows were delivered from 1 to 6 sows, 14 sows from 2 sows, and 10 sows from the control group. There were no stills or mummies. The autopsy of 10 pigs (50%) born in the inoculation group at 2 days showed no specific gross lesions or microscopic lesions. (RT-PCR) to confirm the Japanese encephalitis virus in the liver, spleen, and liver. The Japanese encephalitis virus vaccine was inoculated with the Japanese encephalitis virus vaccine in sows.

As shown in FIG. 8, it was confirmed that the antibody titer was well formed in the sows when the HI antibody value was 200 times or more when the second inoculation was performed.

3-6. Evaluation of efficacy as a vaccine in pigs

The strain K91, a genotype 1 Japanese encephalitis virus, was purchased from the CDC and propagated. The concentration of the virus proliferation was confirmed with 10 ^5.5 TCID ₅₀ / ㎖.

In order to confirm the efficacy of the Japanese encephalitis virus live vaccine strain (KV1899-120P), 1 ml of live vaccinic strain (10 ^6.0 TCID ₅₀ / ml) was inoculated intramuscularly into pigs and 2 weeks after the live vaccination 12 ml of K95 strain inoculation).

As a result, as shown in Table 5, the pigs of the vaccinated group had a mean blood coagulation inhibition value of 215 times at the second week after the K95 attack, whereas the control group showed a high HI antibody level of 4,513 times at the fifth week after the inoculation The antibody titer was 1,447 times. The reason for the high antibody level in the control group is that when the virus is infected with a pathogenic Japanese encephalitis virus in a pig having no antibody, it shows a high antibody titer in a short period of time. Since the vaccine group already inoculated the vaccine, I never do that.

 The vaccination group and the control group after the inoculation showed that the vaccination group was normal as shown in Table 6, but the control group had severe fever symptoms.

Antibody changes by attack inoculation after Japanese encephalitis live vaccination division The mean hemagglutination inhibition (HI titer) 0 weeks 2 weeks 4 weeks 8 weeks 12 weeks Two weeks after the attack 5 weeks after the attack KV1899-120P
Inoculation group 0 40 67 34 28 215 452 Control group 0 0 0 0 0 4513 1447

Clinical Significance of Inoculation after Japanese Encephalitis Live Vaccination division Pig number Clinical symptoms after inoculation 0 One 2 3 4 5 6 7 8 9 10 11 12 Vaccinated group One N N N N N N N N N N N N N 2 N N N N N N N N N N N N N 3 N N N N N N N N N N N N N 4 N N N N N N N N N N N N N Control group 5 N N F F F F F F N N N N N 6 N N F F F F F F N N N N N

N: normal, F: fever

3-7. Evaluation of efficacy as a vaccine in mice

First, the genotype 3 K87 strain expressing the virulence of the mouse in the mouse was distributed from the CDC to proliferate the virus. The virus content of K87 was confirmed to be 10 ^5.0 TCID ₅₀ / ml. In order to determine the pathogenicity of the K87 careful virus propagated in Vero cells resulting juice was inoculated into the abdominal cavity to 5-week-old mice was 100%, a 10-fold dilution is 60%, and 100-fold dilution is indicated mortality 40% of median lethal dose is 31.6 LD ₅₀ / 0.5 ml.

In order to confirm the efficacy of Japanese encephalitis virus live vaccine strain (KV1899-120P), live vaccine strain and unstimulated vaccine strain were diluted 10-fold and infused twice at 4-day intervals. On day 8, 10 LD ₅₀ / Japanese encephalitis virus was inoculated 0.5ml into peritoneal cavity and the efficacy test was carried out. As shown in FIG. 9, mice vaccinated with the live vaccine showed 100% survival and 100% protective efficacy. The mice inoculated with the inactivated vaccine had a survival rate of 60%, whereas the control survived only 30%. As shown in FIG. 9, the change of the average weight of the mice after the inoculation with the vaccine was confirmed, and it was confirmed that the mice vaccinated with the live vaccine continuously increased without decreasing the body weight. However, inactivated vaccine group and control mice lost weight.

Korea Biotechnology Research Institute KCTC18347P 20150116

<110> REPUBLIC OF KOREA (Ministry of Agriculture, Food and Rural Affairs, Animal and Plant Quarantine Agency) <120> Attenuated genotype 1 Japanese encephalitis vaccine composition          containing the same for preventing Japanese encephalitis in swine <130> PA140827-C01 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 10965 <212> DNA <213> Japanese encephalitis virus KV1899-120P full genome sequence <400> 1 agaagtttat ctgtgtgaac ttcttggctt agtatcgttg agaagaatcg agagattagt 60 gcagtttaaa cagtttttta gaacggaaga acaaccatga ctaaaaaacc aggagggccc 120 gggaaaaacc gggccatcaa tatgctgaaa cgcggattac cccgcgtatt cccactagtg 180 ggagtgaaga gggtagtgat gagcttgttg gacggcagag ggccagtacg atttgtgctg 240 gctcttatca cgttcttcaa gtttacagca ttagccccga ccaaggcgct tttgggccgc 300 tggagagcag tggaaaaaag tgtggcaatg aaacatctta ccagtttcaa acgagaactt 360 ggaacactca tcgacgccgt gaacaagcgg ggcaaaaaac aaaccaaaag aggagggaat 420 gaaagctcga ttatgtggct tgccagcttg gcaatcgtaa cagcctgtgc cggagccatg 480 aagctatcaa actttcaagg aaagcttctg atgaccatca acaacacgga cattgcggac 540 gtcatcgtga tccccacctc aaaaggtgaa aacagatgtt gggtccgagc aatcgacgtt 600 ggttacatgt gtgaagacac catcacgtac gaatgtccga agcttgccgt gggcaacgat 660 ccggaagacg tggactgctg gtgcgacaat caagaagtct acgtgcagta tggtcgctgc 720 acacggacca ggcattccaa acgaagcaga agatccgttt cggtccaaac gcatggggaa 780 agctcactag tgaacaaaaa agaggcttgg ctggattcaa cgaaggccac gcgatacctc 840 atgaaaacgg agaactggat tataaggaac cctggttatg ctttcctggc ggcggcactt 900 ggatggatgc ttggcagcaa cagtggccaa cgtgtggtgt ttactattct cttgctgttg 960 gtcgccgg cttacagttt caactgtctg ggaatgggaa atcgggattt catagaagga 1020 gccagtggag ccacttgggt ggatctggtg ttagaaggag atagctgttt gacaatcatg 1080 gcaaacgaca aaccaacact agatgtccgc atgatcaaca ttgaagctag ccaacttgct 1140 gaagtcagga gttactgcta ccacgcttca gtcacggaca tttcaacggt ggctcaatgc 1200 cccacgactg gagaagccca caacgaaaaa cgtgctgaca gcagctacgt gtgcaaacaa 1260 ggctttactg accgcggatg gggaaatgga tgtggacttt tcgggaaagg aagcattgac 1320 acatgcgcaa aattttcttg taccagtaag gccattggaa gaatgatcca accagagaac 1380 atcaagtaca aggttggcat attcgtgcac ggaaccacca cctcggaaaa ccatgggaat 1440 tactcagcgc aagtagaagc gtctcaagca gcaaagttta ctgtgactcc aaatgctccc 1500 tcaataaccc tcacgcttgg tgattatgga gaagtcacac tggattgtga accaaggagt 1560 ggactgaaca ctgaagcgtt ctatgtcatg accgtgggtt cgaagtcatt cttagtccat 1620 agggaatggt tccatgacct ttctcttccc tggacgtccc cctcaagcac ggcatggaga 1680 aacagagaac tcctcatgga atttgaagag gcacatgcca caaaacaatc tgtcgtagct 1740 cttgggtcac aggagggagg cctccatcaa gcgttggcag gagccatcgt ggtggagtac 1800 tcgagctcag tgaagttgac atcaggtcac ctgaaatgca ggctaaaaat ggacaaactg 1860 gctctgaagg gcacgactta tggcatgtgt acagaaaaat tctcgttcgc gaaaaatcca 1920 gcggacacag gccatggaac agttgtcatt gagctcacat actctggaag tgatggtccc 1980 tgtaaaattc cgattgtctc agtcgcgagt ttaaacgaca tgacccctgt ggggaggctg 2040 gtaacagtaa accccttcgt cgcgacatct agctccaact caaaggtgct ggttgagatg 2100 gaacctccct tcggagactc ttatatcgtg gttggaagag gggacaagca gattaaccaa 2160 cactggcaca aagctggaag cacgctgggt aaagccttct caacaacttt gaaaggggct 2220 cagagactag cagcgctagg tgacacagcc tgggacttcg gctccattgg aggggtattc 2280 aactccatag ggaaagctgt tcaccaagta tttggcggtg cattcagaac gctctttggg 2340 ggaatgtctt ggatcacaca aggactaatg ggggccttac ttctttggat gggtgtcaac 2400 gcacgagatc ggtcaattgc cctggctttt ctggccacgg gagctgtgct cgtgttttta 2460 gcgaccaatg tgcatgccga cactggctgt gccattgaca tcacaagaaa agagatgagg 2520 tgtggaagcg gcatcttcgt gcacaacgac gtagaggctt gggtagatag atacaaatat 2580 ctgccagaga cgcccagatc cttagcgaag atcgtccaca aagcacatca agaaggagtg 2640 tgcggggtca gatccgtcac tagactggaa caccagatgt gggaatctgt gcgggacgaa 2700 ctgaatgtct tgctcaaaga gaacgcggtg gatctcagtg tggtggtgaa caagcccgtg 2760 gggagatatc gctcagcccc caaacgccta tccatgactc aagaaaagtt tgagatgggc 2820 tggaaagcat ggggaaaaag cattcttttc gcccccgaat tggccaactc cacgttcgtc 2880 gtggatggac ccgagacaaa ggaatgccct gatgagcgca gagcctggaa cagcatgcag 2940 atcgaagact tcggcttcgg catcacatca acccgagtgt ggctgaaaat tagagaggag 3000 aacactgatg agtgtgatgg agcagtcata gggacagctg ttaaagggca tgtggcagtt 3060 cacagtgact tgtcgtactg gattgagagc cgtctcaatg acacctggaa acttgagagg 3120 gctgtcttcg gagaggtgaa atcttgcact tggcccgaga cacacactct ttggggtgac 3180 ggtgttgagg agagcgagct tatcatccca cataccatag ctggaccgag aagcaagcac 3240 aaccggagag aagggtataa aacacaaaac cagggaccct gggatgagaa cggcatcgtc 3300 cttgactttg actattgtcc aggaacaaaa gtcaccatca cagaggactg tggcaagagg 3360 ggtccctcaa tcagaaccac tactgacagt ggaaagctga tcaccgattg gtgttgccgc 3420 agctgttctc taccgccttt acggttccgg acagaaaatg gttgctggta tgggatggaa 3480 atcagacctg ttaagcatga tgaaacaaca ctcgttagat cacaggttga cgctttcaac 3540 ggcgaaatga ttgatccttt tcagctgggc cttctggtga tgtttctggc cacccaggag 3600 gtccttcgca agaggtggac ggccagattg acgattcctg cggttttggg ggctctactt 3660 gtgctgatgc ttgggggcat cacttacact gacctggcaa gatatgtggt gctagttgct 3720 gcggctttcg cggaggccaa cagtggggga gatgttctgc acctcgcttt gatagccgtc 3780 ttcaagatcc aaccagcttt tctggtcatg aacatgctta gcgcgagatg gacgaaccaa 3840 gagaacgtgg ttctggtcct gggggcggct tttttccaac tagcttcagt ggagttacag 3900 atcggagtcc acggaatcct gaacgccgca gccatagcat ggatgatcgt ccgagcgatc 3960 acatttccct caacttctac cgttgccatg ccagtcttag cgctcctaac tccgggaatg 4020 agggccttgt atctggacac ctacagaatc attcttcttg tcataggaat ttgttccctg 4080 ctgcaagaga ggagaaagac catggcgaag aagaaaggag ccgtgctctt gggcttagcg 4140 ctcacatcta ccggatggtt ttcgcccacc actatcgcag ccggactaat ggtctgcaac 4200 ccaaacaaga agagagggtg gccagctacc gagttccttt cagcggttgg gttgatgttt 4260 gccattgtgg gaggtctagc cgagttggac atcgaatcta tgtcaatacc cttcatgctg 4320 gcagggctc tggcagtgtc ctacgtggtg tcaggaaagg caaccgacat gtggctggac 4380 cgggctgccg atatcagctg ggagatggaa gctgcaatca ccggaagtag ccggaggctg 4440 gatgttaaat tggatgacga cggcgacttt cacttgattg atgatcccgg tgttccatgg 4500 aaagtctggc tcttgcgtat gtcttgtatc ggtttagccg ctctgacacc ctgggctatc 4560 gttcctgccg cttttggcta ttggctcact ctgaaaacaa caaaaagagg gggcgtgttc 4620 tgggacacgc catctccaaa gccttgccta aagggggaca ccaccacagg agtttaccga 4680 attatggcta gggggattct cggcacttac caggccggcg ttggagtcat gtacgagaat 4740 gttttccaca cattgtggca cacaactaga ggagcagcca tcatgagtgg agaaggaaag 4800 ctaacgccat actggggtag tgtgaaggaa gaccgcataa gctacggagg cccgtggagg 4860 ttcgaccgga agtggaatgg aacagatgat gtgcaagtga tcgtggtgga accagggaaa 4920 cctgcagtga acatccagac aaaaccggga gtgtttcgca cccctttcgg ggaggtcgga 4980 gcagtcagct tggactaccc acggggaaca tccggctcac ccatcctaga ttccaatgga 5040 gatatcatag gcttgtatgg caatggagtt gaactcggcg atggatcgta cgtcagcgcc 5100 attgtgcagg gcgaccgtca agaggaatca gttccagatg cctacactcc aagcatgctg 5160 aaaaagagac agatgactgt gctggacctg cacccaggtt cgggaaaaac caggaagatc 5220 ctaccccaga taatcaagga tgccatccag cagcgcttga gaacagccgt gttggcaccc 5280 acacgagtgg tagcagcaga aatggcggaa gctttgagag gactcccagt acgatatcaa 5340 acctcagcag tgcagaggga gcatcaggga aacgaaatag tagacgtaat gtgccatgcc 5400 actctgaccc atagactaat gtcaccaaac agggtgccca actacaactt attcgtgatg 5460 gcgaggctc acttcactga cccagctagc atcgccgctc ggggttatat cgcaaccaag 5520 gtggaactgg gggaggcagc agccattttt atgacggcga ccccgcccgg gaccactgat 5580 ccctttcccg actcaaatgc cccagttcat gacctgcagg atgagatccc agacagggca 5640 tggagcagtg gatacgaatg gatcacggac tatgtgggga aaactgtgtg gttcgtggca 5700 agtgtgaaaa tggggaatga gatcgcaatg tgccttcaaa gagcggggaa aaaggtcatc 5760 cagctcaatc gtaagtctta tgacacagaa tacccaaaat gcaaaaatgg agattgggac 5820 tttgttatca ccactgacat ctctgagatg ggggccaatt ttggtgcgag cagggtcatt 5880 gactgcagaa agagtgtgaa acccaccatc ctagaggagg gagaaggtag agtcattctt 5940 ggaaacccat cccccataac cagtgcaaat gcagcccaac ggagaggtag agtgggcagg 6000 aatcccaacc aagttggaga tgaataccat tacggggggg ccaccagtga agatgacagc 6060 aacttagccc actggacaga ggcaaaaatt atgctagaca acatacacat gcctaatgga 6120 ttagtggctc agctgtacgg gccagagagg gaaaaggctt tcacaatgga tggagagtac 6180 cgtctcagag gtgaggagaa gaagaacttc ttggagctgc ttagaacggc tgacctccca 6240 gtatggctag cctacaaggt ggcgtccaat ggcattcagt acactgacag aaaatggtgc 6300 tttgatggac cacgcacaaa tgccatacta gaagataaca ctgaggtgga gatagtcacc 6360 cgaatgggtg agagaaagat cctcaagccg agatggctcg acgcgagggt ttatgcagac 6420 caccaggccc tcaagtggtt taaggatttt gcagcgggca agagatcagc cgtcagtttc 6480 atagaggtgc tcggtcgcat gcctgagcat ttcatgggaa agacacggga agccttagac 6540 acaatgtatc tggtggctac agctgagaaa ggtggaaagg cacaccgcat ggctcttgag 6600 gaactgcccg acgcattgga gaccattaca ctcatcgttg ccatcactgt gatgacagga 6660 ggattcttcc tgctcatgat gcagagaaag ggtataggaa aaatgggtct aggggctcta 6720 gtgctcacgc tggccacctt tttcctatgg gcggcggagg ttcctggaac caaaatagcg 6780 ggtaccttac tagtcgccct gttgctgatg gtggtcctca ttccggaacc agaaaaacag 6840 cgtggtcgga 6900 gtggtggcag caaatgagta cggaatgctg gaaaaaacca aagcagacct taagagcatg 6960 tttggcggaa ggacgcaagc atcaggactg accggattgc caagcatggc actggacttg 7020 cgcccagcca cagcttgggc gctgtatggg gggagcacag ttgtgttaac ccctctcctg 7080 aagcacctaa tcacctcaga atacgtcacc acatcgttag cttcaatcag ttcacaagcg 7140 ggttcgctgt ttgtcttgcc gcgaggcgtg cctttcactg acttggatct aaccgttggc 7200 cttgtctttc ttggctgttg gggccaaatc accctcacca cgttcttaac agctatggtg 7260 ctagtgacac tccactatgg atacatgctc cctgggtggc aagcagaggc actcagagct 7320 gctcagagaa gaacagcggc tggcataatg aagaatgccg tcgtggacgg aatggtcgcc 7380 actgatgtgc ccgaactgga aagaaccact cctttgatgc aaaagaaagt cggccaagtg 7440 ctcctcatag gggttagcgt ggcggcgttt ctcgtcaacc ccaatgttac caccgtgaga 7500 gggcaggtg tgttggtgac ggcagccaca ctcaccttgt gggacaatgg agccagtgca 7560 gtctggaatt ccaccaccgc tacggggctc tgccatgtca tgcgaggcag ctacctagct 7620 ggcggctcca ttgcctggac tctcatcaag aacgctgaca agccctcctt aaaaaggggg 7680 aggcctggag gcaggacgtt aggggagcag tggaaggaaa agttgaatgc tatgagtagg 7740 gatgagttct tcaaatacag aagagaggcc ataattgagg tggaccgcac tgaagcacgc 7800 agggctaggc gcgagaacaa catagtggga ggacatccag tctcgcgagg gtcagcaaag 7860 ctccgctggc ttgtggaaaa aggatttgtc tcaccaatag gaaaagtcat agatctggga 7920 tgcgggcgcg gaggctggag ctactacgca gcaactctga aaaaagttca ggaagtcaaa 7980 gggtacacga aaggtggggc gggacacgaa gaaccgatgc tcatgcagag ttacggttgg 8040 aacctggtct cgctaaaaag tggggtggac gtattctaca aaccctcgga gcctagtgac 8100 accctgttct gtgacatagg tgaatcttcc ccaagtccgg aggtggagga acaacgcact 8160 ctgcgcgtct tagaaatgac atctgactgg ttacatcggg gacctagaga gttctgcata 8220 aaagtgctct gcccttacat gcctaaagtt atagaaaaga tggaagtcct gcaacgccgt 8280 ttcggtggcg ggctggtgcg ccttcccctg tctcgaaact ccaaccatga gatgtactgg 8340 gtgagtggag ctgctggcaa tgtggtgcat gcggtcaaca tgaccagcca agtgctgcta 8400 gggcgaatgg atcgcacagt gtggagaggg ccaaagtatg aagaggatgt caacctgggc 8460 agcgggacga gagctgtggg gaagggagag gtccatagca accaggaaaa aattaggaag 8520 agaattcaga aactcagaga ggaattcgct acaacctggc acaaagaccc cgagcatcca 8580 taccgaactt ggacctatca cggaagttac gaagtgaagg ccactggctc agcaagctct 8640 ctcgtcaatg gggtagtaaa gcttatgagc aaaccctggg acgccatcgc caatgtcacc 8700 acaatggcca tgactgacac cacccccttt ggccagcaga gggtctttaa agagaaggtt 8760 gacacgaagg ctccagagcc accagcagga gtcaaggaag tgctcaacga gactaccaac 8820 tggctgtggg cccacttgtc acgggagaaa cgacctcgct tgtgcactaa ggaagaattc 8880 ataaagaaag tcaacagcaa cgcagctctc ggagcagtgt tcgctgaaca gaatcaatgg 8940 agcacggcgc gggaagccgt gggtgaccct ctgttctggg agatggtcaa tgaagaaagg 9000 gaaaaccatt tgcgagggga gtgccacacg tgcatttaca acatgatggg aaaaagagag 9060 aaaaaacccg gagagttcgg aaaggctaaa gggagtaggg ctatctggtt catgtggctc 9120 ggagctcggt acctagagtt cgaagcccta ggatttctaa atgaagatca ttggctgagc 9180 cgagagaatt caggaggtgg ggtggaaggt tcaggcgtcc aaaagctggg atacattctc 9240 cgtgacatag caggtaagca aggaggtaaa atgtatgccg atgacactgc cgggtgggac 9300 accagaatca ccagaaccga cttggaaaat gaagccaaag tgctggagct tttggatggt 9360 gaacaccgca tgctcgcccg agccataatt gaactaacgt ataggcacaa agtggttaag 9420 gttatgagac ctgcagcagg aggaaagaca gtgatggacg tgatatcacg agaagaccaa 9480 agggggagcg ggcaggtggt gacctacgct ctcaacacat tcacgaacat tgccgtccag 9540 cttgtccgct tgatggaggc tgagggggtc attggaccac aacacttgga acagctgccc 9600 aggaaaaaca aaatagctgt taggacctgg ctctttgaga atggagagga gagagtgacc 9660 aggatggcga tcagtggaga cgactgtgtc gtcaagccgc tggatgacag attcgccacg 9720 gctctccatt tcctcaacgc aatgtcgaag gtcagaaaag atatccaaga atggaagcct 9780 tcgcatggtt ggcaggactg gcagcaggtt cctttttgct ccaatcattt tcaggagatt 9840 gtgatgaaag atggaaggag catagtcgtc ccgtgcagag ggcaggatga gctgattggc 9900 agggcgcgca tctccccagg agctggatgg aatgtgaagg acacagcttg cctggctaaa 9960 gcgtatgcac agatgtggct gcttctatac ttccatcgga gggacctacg cctcatggca 10020 aatgcaatct gctcagcagt tccagtggac tgggtaccca caggcagaac atcctggtca 10080 ctacactcaa aaggagagtg gatgaccact gaagacatgc tgcaagtctg gaacagggta 10140 tggattgaag aaaatgaatg gatgatggac aagaccccaa tcacaagctg gacagacgtt 10200 ccgtacgtgg gaaagcgtga ggacatctgg tgtggcagtc tcatcggaac gcgatccagg 10260 gcaacatggg ctgagaacat ctacgcggca ataaaccaag tgagggccat cattggaaaa 10320 gaaaattatg ttgattacat gacttccctc agaagatatg aggatgtatt gattcaggag 10380 gacagggtca tttagacagg attaagtcat gtgtgtaatg tgagataaga aaatgtgcat 10440 gtggagtcag gccagcaaaa gctgccaccg aatactgagt agacggtgct gcctgcgtct 10500 cagttccagg aggactgggt taacaaatct gacaacggaa ggtgggaaag ccctcagaac 10560 cgtctcggaa gcaggtccct gctcaccgga agttgaaaga ccaacgtcag gccacaattt 10620 tgtgccactc cgctggggag tgcggcctgc gcagccccag gaggactggg ttaacaaagc 10680 cgttgaggcc cccacggccc aagcctcgtc taagatgcaa tagactaggt gtaaggacta 10740 gaggttagag gagaccccgt ggaaacaaca tcatgcggcc caagccccct cgaagctgta 10800 gaggaggtgg aaggactaga ggttagagga gaccccgcat ttgcatcaaa acagcatatt 10860 gacacctggg aatagactgg gagatcttct gctctatctc aacatcagct acttggcaca 10920 gagcgccgaa gtatgtagct ggtggtgagg aagaacacag gatct 10965

Claims (3)

Purified gene 1 Japanese encephalitis virus KV1899-120P (Accession No. KCTC18347P). A Japanese encephalitis vaccine composition comprising the Japanese encephalitis virus of claim 1. 3. The vaccine composition for the prevention of Japanese encephalitis according to claim 2, wherein the vaccine composition is a vaccine composition for pigs.

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