KR0143925B1 - Japanese encephalitis vaccine production method - Google Patents

Abstract

The present invention relates to a method for preparing Japanese encephalitis vaccine, wherein 1-7 days old mouse or tissue cultured primary hamster kidney epithelium with immature brain cells instead of the 3 to 4 week mice currently used during the manufacturing process of Japanese encephalitis vaccine The vaccine was prepared by culturing the Japanese encephalitis virus in the cells. As a result, the brain protein and Myelin basic protein contents of the newly vaccinated Japanese encephalitis vaccine are significantly reduced to 1/10-1/100 of the previous vaccine, thereby preventing side effects of incorporation of rat brain proteins.

Description

Japanese encephalitis vaccine production method

1 is a harrow curve measured after inoculating Japanese encephalitis virus and encephalitis vaccine into rat muscle

2 is the antibody curve of rats injected with Japanese encephalitis-Hantan virus mixed vaccine twice at intervals of 2 weeks

FIG. 3 shows the formation of harrows after inoculation of the Japanese encephalitis vaccine and Hantan virus vaccine into the muscles of hamsters.

Figure 4 shows the antibody formation curves for Japanese encephalitis and hantavirus after inoculation of hamster-vaccinated vaccine with Hantan-Japan encephalitis twice.

5 is a growth curve of Japanese encephalitis virus and Hantan virus in primary hamster kidney epithelial cells.

FIG. 6 shows the antibody curve after inoculation of hamster muscle with a mixed vaccine made from Japanese encephalitis-Hantan virus cultured from primary hamster kidney epithelial cells.

[Technical Field]

The present invention relates to a method for producing Japanese encephalitis vaccine, and more particularly, to a method for preparing a Japanese encephalitis vaccine that can completely eliminate the side effects of the presence of brain protein in the existing vaccine.

[Cultivation technology]

In Korea, Japan, China, and Southeast Asia, the mortality epidemic of Japanese encephalitis and epidemic hemorrhagic fever coexists with high mortality every year, and many patients die and die. Epidemic hemorrhagic fever occurs not only in Asia, but also in Europe, Africa and the Americas, but Japanese encephalitis occurs only in Asia. Therefore, in South Korea, North Korea, Japan, China, Vietnam, Sri Lanka, India, and Malaysia belonging to Asia, epidemic hemorrhagic fever and Japanese encephalitis patients can occur at the same time.

There are about 200,000 epidemic hemorrhagic fever cases on earth each year with a mortality rate of 4-60%. Tens of thousands of patients occur in China each year, and about 8,000 in Russia and hundreds-2,000 in Korea, while hundreds of thousands of patients occur in Europe, with severe clinical symptoms and mild patients. . In the summer of 1993, there were dozens of new cases of the disease caused by the new Hantan virus and death rate of 70% in the United States. To date, more than 125 patients have occurred in the United States, Canada, and Brazil, with about 86 deaths, with a mortality rate of 69%. Hantan virus, a pathogen of epidemic hemorrhagic fever, was first discovered in Korea in 1976 and a human vaccine was developed in 1989 (Hantavax). On the other hand, Japanese encephalitis virus was first isolated from brain tissue of encephalitis patients in Japan in 1935, and human vaccine was developed in 1954 and widely used in various countries in Asia to contribute to the prevention of Japanese encephalitis. However, currently used Japanese encephalitis vaccines are vaccines made from viruses grown in the brains of four-week-old mice, so side effects have always been a problem with mouse brain cell proteins contained in the vaccine. In the summer of 1994, two Korean high school students who suffered from Japanese encephalitis vaccine died of shock and caused great controversy. The evacuation of other vaccines appeared, and the government prepared a compensation system for the vaccine. The problem with Japanese encephalitis vaccine is the content of protein of mouse brain tissue and Myelin basic protein, especially the latter is a serious problem because it causes spinal encephalitis appearing as a resting period after vaccination. According to research statistics, one out of 100,000 people can develop paralytic spinal encephalitis if they have the current Japanese encephalitis vaccine.

Accordingly, the present inventors have attempted to obtain an antigen of pure Japanese encephalitis that can completely eliminate the side effects of the currently used Japanese encephalitis injection. In addition, the acquisition of pure antigens allows the development of mixed vaccines that can simultaneously prevent Japanese encephalitis and epidemic hemorrhagic fever when mixed with antigens of epidemic hemorrhagic fever.

[Problems to Solve Invention]

An object of the present invention is to provide a method for preparing a safe Japanese encephalitis vaccine lowering the content of brain tissue protein and Myelin basic protein.

[Means for solving the problem]

The present invention provides a 1-7 day old mouse or tissue cultured primary hamster kidney with immature brain cells instead of the 3 to 4 week old mice currently used during the manufacturing process of the Japanese encephalitis vaccine to achieve the object of the present invention described above. A vaccine was prepared by culturing Japanese encephalitis virus in epithelial cells. As a result, the contents of brain protein and Myelin basic protein of the newly improved Japanese encephalitis vaccine were significantly reduced to 1/10-1/100 of the previous vaccine.

Hereinafter, the present invention will be described in more detail with reference to Examples. Vaccines mixed with the Japanese encephalitis vaccine alone and the epidemic hemorrhagic fever vaccine were prepared, and antibody titers were measured in each animal. The culture medium of each virus was also diversified into mice and tissue culture cells. The examples are only illustrative of the present invention and the scope of the present invention is not limited only to the matters described in the following examples.

Example 1

Preparation of Japanese Encephalitis Vaccine

The Japanese encephalitis virus used for the Japanese encephalitis vaccine was Nakayama, an international standard strain. Two vaccines were used for the production of vaccines: four-week-old mice and seven-day-old mice. Preparation of a known Japanese encephalitis vaccine by inoculating 0.01 ml of Japanese encephalitis virus suspension into the brain of 1CR mouse and aseptically extracting the brain after 3-5 days of paralysis and death of encephalitis. The vaccine was prepared by adding a new filtration method to the method. As the preparation method, alcoholol protamine purification method, ultracentrifugation precipitation purification method and special filtration method were used as follows.

Example 2

Flavor Potency of Japanese Encephalitis Vaccine

The Japanese encephalitis virus source contained in the vaccine was measured by the ELISA method so that a certain amount of the source was always contained in the vaccine. The titer of fragrance was introduced by Hantan virus fragrance titer, ELISA unit / 0.1ml and contained 0.02% gelatin.

Example 3

Comparative experiment with existing vaccine

Antibodies against Japanese encephalitis and Hantan virus were measured by indirect fluorescent method, ELISA method and neutralizing antibody method, and antibody titer was expressed as the highest dilution factor of seropositive serum. Healthy SPE / ICR mice were used as vaccine animals, and SD rats, mice, guinea pigs, and LVG hamsters were used for virus propagation and antibody analysis. Virus and vaccine animals were always used for animal breeding. Animals vaccinated and vaccinated in safety cabinets were always housed in animal safety cabinets. 1) Contents of improved Japanese encephalitis vaccine and epidemic hemorrhagic fever vaccine and Myelin basic protein

The most problematic in Japanese encephalitis vaccines is the amount of brain cell protein and Myelin basic protin in the vaccine. According to the WHO vaccine recommendation criteria, the protein content of Japanese encephalitis vaccine should be less than 80µg / ml, and Myelin basic protin should be less than 2.0ng / ml. In this experiment, we compared the titer and protein amount of the Japanese encephalitis virus aroma extracts from brain tissues of 4 and 7 days old mice, and the results are shown in Table 1 below.

*-World Health Organization recommends 80ug / ml.

**-World Health Organization recommendation standard is 2.0g / ml.

The protein contained in the 4-week-old vaccine was 22 µg / ml and Myelin basic protin content was 0.8 ng / ml. However, the protein content of Japanese encephalitis vaccine in 7-day-old mice was 17 µg / ml and Myelin basic protin was 0.08 ng / ml. The Japanese encephalitis vaccine produced in 7-day-old mice contained 5 µg / ml less than the vaccine produced in 4-week-old mice. In particular, the amount of Myelin basic protin was only one tenth of that produced in four-week-old mice.

The protein contained in the epidemic hemorrhagic fever vaccine produced in 1 week old mice was 10-12 ㎍ / ml and Myelin basic protin was less than 0.01 ng / ml.

The above results are expected to have no side effects because the new vaccine is 10 times biochemically pure in mice 7 days older than the Japanese encephalitis vaccine. Therefore, in case of mixed vaccine mixed with epidemic hemorrhagic fever vaccine made in 7-day-old mouse, the content of Myelin basic protin is less than 0.08ng / ml, so there will be no problem. Therefore, the inventive encephalitis- pandemic hemorrhagic fever vaccine can eliminate the side effects that sometimes occur in the Japanese encephalitis vaccine, which is used alone, and inoculate the mixed vaccine of encephalitis and hemorrhagic fever. .

2) The encephalitis vaccine and pandemic hemorrhagic fever vaccines made from old tissues of 4 weeks old mouse and 7 days old mouse with fluorescent antibody of the newly inoculated Japanese encephalitis vaccine and epidemic hemorrhagic fever vaccine Investigations on the anti-diarrheal agents for hantan virus are shown in Table 2 below.

There was no difference in immunity between the Japanese encephalitis vaccines made in 4 week-old mature mice and those of 7-day-old suckling mice. Hantan virus vaccines were compared to those with and without adjuvant. Antibodies were produced only with adjuvant. In a mixed vaccine made of a combination of the Japanese encephalitis vaccine and an adjuvant-free epidemic hemorrhagic fever vaccine, only the form for the Japanese encephalitis virus was formed. The combined vaccine, which was made by mixing the Japanese encephalitis vaccine and the epidemic hemorrhagic fever vaccine with adjuvant, simultaneously demonstrated high titers of antibodies against the Japanese encephalitis virus and Hantan virus. Particularly in such mixed vaccines, the antibody against the Japanese encephalitis virus was much higher than when the encephalitis vaccine was injected alone. It is interpreted that the synergy was shown by stimulating.

In the Japanese encephalitis vaccine, gelatin solution was not used as the adjuvant such as alumgel, which uses 0.02% gelatin solution used as stabilizer for viral sensitization in the Japanese encephalitis vaccine, as the adjuvant in the hantan virus vaccine. It was found that he played the role of Juvant.

It can be seen that the mixed vaccine made by mixing the Hantan virus vaccine containing alum gel and the Japanese encephalitis vaccine containing gelatin solution produces more antibodies to the Japanese encephalitis virus than the hantan virus alone. FIG. 1 shows the antibody curve after inoculation of live Japanese encephalitis virus and killed Japanese encephalitis vaccine into the muscles of rats. The neutralizing antibody titer was 1: 512 at live virus injection and 1: 100 at vaccine injection. FIG. 2 shows the antibody curve of Japanese encephalitis-Hantan virus mixed vaccine injected into rats' muscles twice every two weeks. High fluorescence antibodies (2,048-8192) against Japanese encephalitis and Hantan virus were demonstrated, respectively, and the titers of neutralizing antibodies were 1: 512 and 1: 128, respectively.

3) Neutralizing antibodies of animals inoculated with pandemic hemorrhagic fever vaccine, Japanese encephalitis vaccine and mixed vaccine

According to the National Institutes of Health Biologic Standard Evaluation Criteria, anti-neutral antibody formation against these viruses was inoculated into the muscles of guinea pigs and mice by inoculating Hantan virus and Hantan virus vaccine, Japanese encephalitis virus and Japanese encephalitis vaccine, and Hantan-Japan encephalitis vaccine It is shown in Table 4 below.

As shown in Table 4, the neutralizing antibody titer of the guinea pigs inoculated with 2,560 antigen units of live hantan virus was 1: 113, and the neutralizing antibody titer of the guinea pigs inoculated with the same amount of pandemic hemorrhagic fever vaccine was 1: 105. In addition, the antibody value of guinea pigs inoculated with 5,120 units, which doubled the amount of aroma, was 1: 300. Antibody titers of vaccines made with the Hantan virus 84/105 and 76/118 strains were similar at 310 and 300, respectively. The guinea pigs and mice inoculated with 200 units of live Japanese encephalitis virus had a neutralizing antibody titer of 1: 4,581 or more, and the mice with the Japanese encephalitis vaccine containing 12.5 units of antigen showed a 1: 4,266 neutralizing antibody titer. However, in guinea pigs inoculated with a vaccine containing a small amount of Hantan virus (512u / 0.1ml), the neutralizing antibody titer was 1:30, and a mixed vaccine containing a large amount of Hantanvirus antigen (2,048u / 0.1ml) was used. Inoculated animals were able to demonstrate a high neutralizing antibody of 1: 289 against Hantan virus and a neutralizing antibody against Japanese encephalitis virus of 1: 4,291. Neutralizing antibodies against hantan virus were well formed in guinea pigs but not good in mice. Table 5 shows the results of measuring neutralizing antibodies in tissue culture cells by inoculation of guinea pigs and mice with a combination vaccine containing a certain amount of Japanese encephalitis virus antigen and various amounts of Hantan virus antigen. Similar to the grades shown in Table 4.

It was found that high titers of neutralizing antibodies can only be produced in the anti-hanthan vaccine, but at least 1,024 u / 0.1 ml of the anti-tantan virus must be present in the mixed vaccine to obtain sufficient neutralizing antibodies against the anti-hantan virus. Has been proven.

[Antibody Formation in Hamsters Inoculated with Pandemic Hemorrhagic Fever and Japanese Encephalitis Vaccine]

Figure 3 shows the antibody formation curves after inoculation of hamster muscles with pandemic hemorrhagic vaccines made with Japanese encephalitis vaccine and 84/105 and 76/118 strains, respectively. Two doses of the Japanese encephalitis vaccine and the epidemic hemorrhagic fever vaccine at about 1 month intervals demonstrated a high titer of fluorescent antibodies of 1: 2,048 for the epidemic hemorrhagic fever vaccine made with 1: 256 and 84/105 strains. Figure 4 shows the antibody formation curve after inoculating the hamster twice with the Japanese encephalitis-hantan mixed vaccine. The antibody against the Japanese encephalitis virus was higher than when the encephalitis vaccine was injected alone, but the antibody against the hantan virus was injected alone. It proved weaker when done.

This result is almost similar to the results obtained when the Japanese encephalitis-hantan combination vaccine was injected into rats or guinea pigs.

[Mixed vaccine made with Japanese encephalitis virus and Hantan virus grown in tissue culture cells]

Japanese encephalitis virus and Hantan virus were cultured in primary hamster kidney epithelial cells in tissue culture and used as a vaccine virus virus house. 5 is a growth curve of Japanese encephalitis virus and Hantan virus in primary hamster kidney cells. Japanese encephalitis virus when inoculated with the virus in tissue culture cells 100 ID ₅₀ when you could get a virus antigen of approximately 10 ^7.0 / 1㎖ inoculated with the virus to 100 ID ₅₀ 76/118 Hantaan virus after 7 days after 9 days about 10 ^6.0 / 1 ml of viral antigen was obtained, and 84/105 strains obtained 10 ^5.0 / 1 ml of viral antigen after 7 days of inoculation. Pure virus fragrance was isolated by ultra-fast centrifugation and treated with 0.05% formarin for 2 weeks at 4 ° C. Encephalitis virus was adsorbed in 0.02% gelatin solution and Hantan virus was used as vaccine. Tissue cultured Japanese encephalitis- pandemic hemorrhagic fever vaccines were prepared by mixing a certain amount of viral antigens, such as vaccines made in mouse brain tissues, and then injected into the hamster and guinea pig muscles and examined for antibody responses. As shown in FIG. 6, 3 weeks after the inoculation of the tissue cultured Japanese encephalitis-Hantan virus vaccine, the fluorescent antibody titer of the Japanese encephalitis virus was measured at 1: 512, and the fluorescent antibody titer of the hantan virus was 1: 1,024. .

[Benefit]

The newly made Japanese encephalitis vaccine contains 22µg / ml protein and 0.8ng / ml Myelin basic protein, thus preventing side effects after encephalitis vaccine. In addition, biologically pure antigens are required in the case of mixed vaccines. By developing a Japanese encephalitis- pandemic hemorrhagic fever vaccine combined with the Japanese encephalitis antigen and the Hantan virus antigen according to the preparation method, it is possible to simultaneously prevent two diseases with a single inoculation. Can be harvested.

Claims (2)

Preparation of encephalitis vaccine prepared by inoculation of Japanese encephalitis virus causing Japanese encephalitis in 1-7 days old mouse or tissue cultured primary hamster kidney epithelial cells, purely isolated viral antigen by a known method, and then inactivated Way. The method for producing a Japanese encephalitis vaccine according to claim 1, wherein the adjuvant of the Japanese encephalitis vaccine is gelatin.