KR0153085B1 - Japanese encephalitis-hemorrhagic fever renal syndrom - Google Patents

Abstract

The present invention is inoculated with Japanese encephalitis virus causing Japanese encephalitis and Hantavirus causing pandemic hemorrhagic fever in 1-7 days old mice or primary hamster kidney epithelial cells, incubated, and the virus antigen is isolated in order by the known method inactivated To provide a Japanese encephalitis- pandemic hemorrhagic fever vaccine consisting of a mixture of the Japanese encephalitis vaccine and the pandemic hemorrhagic fever vaccine prepared through a chemical process. The Japanese encephalitis vaccine of the preparation according to the present invention contains 22 μg / ml of protein and 0.8 mg / ml of myelin-based protein, while the vaccine is reduced to 17 μg / ml of protein and 0.08 mg / ml of myelin-based protein. The occurrence of side effects after inoculation can be prevented. In addition, Hantavirus is cultured in primary hamster kidney epithelial cells to provide a pure Hantan virus tissue culture vaccine free of brain proteins. On the other hand, it is a mixed vaccine that can prevent both Japanese encephalitis and epidemic hemorrhagic fever at the same time, so the convenience of vaccination is increased, and the immunity of the mixed vaccine shows a better effect than the single vaccine.

Description

Japanese encephalitis-pandemic hemorrhagic fever vaccine

1 is an antibody curve measured after inoculating Japanese encephalitis virus and encephalitis vaccine into rat muscle, respectively.

FIG. 2 shows antibody curves measured after inoculation of Hantavirus and Hantavirus vaccine to rat muscle.

FIG. 3 shows the antibody curve of rats injected with Japanese encephalitis-Hantavirus mixed vaccine twice at two week intervals.

4 shows the antibody formation curves after inoculating Japanese encephalitis vaccine and hantavirus vaccine into the hamster muscle, respectively.

Figure 5 shows the antibody formation curve against Japanese encephalitis and Hantavirus after inoculating the hamster twice with Hantan-Japan encephalitis vaccine.

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

FIG. 7 shows the antibody curve after inoculation of hamster muscle with a mixed vaccine made of Japanese encephalitis-hantan virus cultured from primary hamster kidney epithelial cells.

[Purpose of invention]

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a Japanese encephalitis-pandemic hemorrhagic fever mixed vaccine, and more specifically, to completely eliminate the side effects caused by the influx of brain proteins in the existing vaccine, and to provide a preventive effect against two diseases in the city with one preventive vaccine. It relates to a vaccine.

In Korea, Japan, China, and Southeast Asia, the mortality epidemic of Japanese encephalitis and epidemic hemorrhagic fever coexists 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, so prevention measures against these two terrible infectious diseases are urgently needed.

Currently, about 200,000 cases of epidemic hemorrhagic fever occur on earth and the mortality rate is 4 to 60%. Tens of thousands of patients occur in China each year, about 8,000 in Russia, and hundreds-2,000 in Korea, while hundreds of thousands occur in Europe, with severely severe clinical 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, the Japanese encephalitis virus was first isolated from brain tissue of encephalitis patients in Japan in 1935, and in 1954, a human vaccine was developed and widely used in various countries in Asia to contribute to the prevention of Japanese encephalitis. However, currently used Japanese encephalitis vaccines are made from viruses grown in the brains of four-week-old mice, so side effects due to mouse brain cell proteins contained in the vaccines have always been a problem. In the summer of 1994, two Korean high school students who suffered from Japanese encephalitis vaccine died of shock and caused a big bite. The evasion of other vaccines appeared, and the government prepared a compensation system for the vaccine. The problem in Japanese encephalitis vaccine is the content of protein and myelin basic protein of mouse brain tissue, especially the latter is a serious problem because it causes spinal encephalitis, which appears as a nystagmus 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.

Therefore, the present inventors have attempted to develop a mixed vaccine that completely eliminates the side effects of the currently used Japanese encephalitis vaccine and prevents Japanese encephalitis and epidemic hemorrhagic fever at the same time. Therefore, they tried to obtain biologically superior and pure antigen.

[Technical problem to be achieved]

An object of the present invention is to provide a method for preparing safe Japanese encephalitis vaccine and pandemic hemorrhagic fever vaccine which lowered the content of brain tissue protein and myelin-based protein, and also provides a mixed vaccine that can simultaneously prevent Japanese encephalitis and epidemic hemorrhagic fever with a single inoculation. have.

[Configuration and Function of Invention]

In order to achieve the above object of the present invention, the present invention is a 1 to 7 day old mouse or primary hamster kidney epithelial cell having immature brain cells in place of the 3 to 4 week mice currently used during the manufacturing process of the Japanese encephalitis vaccine. The vaccine was prepared by culturing the Japanese encephalitis virus. As a result, the contents of brain protein and myelin-based protein of the newly improved Japanese encephalitis vaccine were significantly reduced from 1/10 to 1/100 of the previous vaccine. In addition, Hantan virus causing epidemic hemorrhagic fever was also cultured in primary hamster kidney epithelial cells to prepare a vaccine. On the other hand, the Japanese encephalitis- pandemic hemorrhagic fever mixed vaccine was prepared by mixing the amount of the Japanese encephalitis virus antigen prepared by the above method to 20 to 80 units / 0.1 ml, and the amount of the Hantan virus antigen to 1,024 units / 0.1 ml or more.

Hereinafter, the present invention will be described in more detail with reference to Examples. 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. After inoculating 0.01 ml of Japanese encephalitis virus suspension into the brain of an ICR mouse, about one third of the mice are paralyzed and after 3 to 5 days of death from encephalitis, the brain is aseptically collected to prepare a known Japanese encephalitis vaccine. Vaccines were prepared by adding new filtration methods. The preparation method used alcohol protamine purification method, ultracentrifugation purification method and special filtration method as follows.

Example 2

Antigen titers of Japanese encephalitis vaccine

The Japanese encephalitis virus antigen contained in the vaccine was measured by the ELISA method so that a constant amount of antigen was always contained in the vaccine. The titer of antigen was introduced into the Hantan virus antigen titer and expressed as ESISA unit / 0.1 ml. The antigenic dose of the encephalitis vaccine used in the experiment was 80 u / 0.1 ml and contained 0.02% gelatin.

Example 3

Preparation of Pandemic Hemorrhagic Fever Vaccine

Pandemic hemorrhagic fever vaccine was used with 84/105 strains of Hantan virus (ATCC VR 2250) and / or 76/118 (ATCC VR 938) strains. After inoculation of 0.01 ml into the brain of 1 to 3 days old ICR mice, approximately 25% 4-5 days after rat paralysis and death from encephalitis, aseptic brain tissue was collected and isolated pure viral antigen by alcohol protamine purification, ultra-fast centrifugation and filtration method, and then the virus antigen on alum gel, an immunostimulator After adsorbing, it was inactivated for 2 weeks at 40C with 0.05% formalin and used as a vaccine. The amount of Hantan virus antigen in the vaccine was measured by ELISA method and standardized so that 1,024 units of antigen was contained in 0.1 ml.

Example 4

Japanese encephalitis-pandemic hemorrhagic fever vaccine

Properly mix the vaccine made with the Japanese encephalitis Nakayama strain containing a certain amount of viral antigen with the 84/105 and 76/118 strains of pandemic hemorrhagic fever, respectively, to make a mixed vaccine, and then 0.25 to 0.5 ml of rat, mouse, ham After inoculating the muscles of star and guinea pigs at regular intervals, blood was collected at two-week intervals to measure fluorescent and neutralizing antibodies against Japanese encephalitis and Hantan virus in serum.

Example 5

Comparative experiment with existing vaccine

Antibodies against Japanese encephalitis and Hantan virus were measured by indirect fluorescent antibody method, ELISA method and neutralizing antibody method. The antibody value was expressed as the highest dilution factor of sera that showed positive response. Healthy SPF / ICR mice were used as vaccine animals, and SD rats, mice, guinea pigs, and LVG hamsters were used for virus feeding and antibody analysis. Animals vaccinated with viruses and vaccines were always used for animal breeding. It was raised in a safety cabinet.

1) Improved protein and myelin protein content of Japanese encephalitis vaccine and epidemic hemorrhagic fever vaccine The most problematic problem in Japanese encephalitis vaccine is the content of brain cell protein and myelin basic protein in vaccine. According to the WHO vaccine recommendation criteria, the protein content of Japanese encephalitis vaccine should be less than 80ng / ml and myelin-based protein should be less than 2.0ng / ml.

In this experiment, we compared the titer and protein content of the Japanese encephalitis virus antigens extracted from brain tissues of 4 and 7 days old mice. The results are shown in Table 1 below.

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

The protein contained in the epidemic hemorrhagic fever vaccine produced in a week-old mouse was 10-12 ng / ml, and the myelin-based protein was 0.01 ng / ml or less, which could not be detected by the current measuring method.

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 the mixed vaccine mixed with Japanese encephalitis vaccine made in 7 days old mouse and epidemic hemorrhagic fever vaccine made in 7 days old mouse, myelin-based protein content is less than 0.08ng / ml, so there will be no problem. Therefore, the inventive encephalitis- pandemic hemorrhagic fever vaccine eliminates the side effects that sometimes occur in the Japanese encephalitis vaccine, which is currently used alone, and is expected to have a single benefit of preventing both diseases by inoculating a mixed vaccine of encephalitis and hemorrhagic fever. Can be.

2) Fluorescent antibodies of animals vaccinated with the newly improved Japanese encephalitis vaccine and pandemic bleeding vaccine

Japanese encephalitis vaccine and epidemic hemorrhagic fever vaccine made from 4 weeks old mouse and 7 days old mouse brain tissue were inoculated into hamster, and the fluorescent antibodies against Japanese encephalitis virus and Hantan virus were examined and shown in Table 2 below.

There was no difference in immunity between Japanese encephalitis vaccines made in 4 week-old mature mice and Japanese encephalitis vaccines produced in 7-day-old suckling mice. Hantan virus vaccines were compared with and without adjuvant, but antibodies were only produced with adjuvant. In a mixed vaccine made of a mixture of the Japanese encephalitis vaccine and an adjuvant-free epidemic hemorrhagic fever vaccine, only antibodies to the Japanese encephalitis virus were 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.

In particular, in the mixed vaccine, the antibody against the Japanese encephalitis virus was much higher than when the encephalitis vaccine was injected alone. The result shows that the Japanese encephalitis- pandemic hemorrhagic fever vaccine produces the antibody more in the process of antibody formation than the Japanese encephalitis vaccine alone. It is interpreted that the cells are stimulated by stimulation.

We investigated whether 0.02% gelatin solution used as stabilizer of viral antigen in Japanese encephalitis vaccine could be used in place of alumgel used as adjuvant in Hantan virus vaccine. As shown in Table 3, the gelatin solution did not show the role of adjuvant such as alumgel in the Hantan vaccine, whereas in the Japanese encephalitis vaccine, the gelatin solution acted as the adjuvant.

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. Figure 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. Figure 2 shows the antibody curve after inoculation of the live vaccines of the live hantan virus at 84/105, 76/118 and 84/105 and 76/118, respectively, to the rat muscles. Fluorescent antibody titer was shown, which was not significantly different from that of live Hantan virus. However, the titer of neutralizing antibody was 1: 316 at the time of live virus injection, much higher than the antibody titer of 1: 100 after vaccination.

FIG. 3 shows the antibody curve in which the Japanese encephalitis-Hantan virus mixed vaccine was injected twice into the rat muscle at 2 week intervals. 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) Neutralized antibodies in animals vaccinated with pandemic hemorrhagic fever vaccines, Japanese encephalitis vaccines and mixed vaccines; Hantan virus, Hantan virus vaccine, Japanese encephalitis virus, Japanese encephalitis vaccine and Hantan-Japan encephalitis vaccine according to the National Institute of Health Biologic Standard Neutralizing antibody formation for these viruses after inoculation into the muscles of guinea pigs and mice is shown in Table 4 below.

As shown in Table 4, the neutralizing antibody titer of guinea pigs inoculated with live Hantan virus of 2,560 antigen units was 1: 113, and the neutralizing antibody titer of 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 antigen was 1: 310. Antibody titers of vaccines made with the Hantan virus 84/105 and 76/118 strains were similar at 310 and 300, respectively. In guinea pigs and mice inoculated with 200 units of live Japanese encephalitis virus, neutralizing antibody titers of 1: 4,581 or more were observed, and in mice vaccinated with Japanese encephalitis vaccine containing 12.5 units of antigen, they showed 1: 4,266 neutralizing antibody titers. However, in guinea pigs inoculated with a mixed vaccine containing a small amount of Hantan virus antigen (512u / 0.1ml), the neutralizing antibody value was 1: 30, and a mixed vaccine containing a large amount of Hantanvirus antigen (2,048u / 0.1ml) Animals vaccinated 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 obtained by inoculating guinea pigs and mice with a combination vaccine containing a certain amount of Japanese encephalitis virus antigen and various amounts of Hantan virus antigen, and measuring neutralizing antibodies in tissue culture cells according to the National Institutes of Health test. The results were similar to the results shown in Table 4.

It was found that the anti-tank vaccine can produce high titer of neutralizing antibody only if there are many fragrant virus antigens.To obtain enough neutralizing antibody against hantan virus, at least 1,024u / 0.1ml of the anti-tantan virus should be in the mixed vaccine. Has been proven.

4) Antibody Formation in Hamsters Inoculated with Pandemic Hemorrhagic Fever and Japanese Encephalitis Vaccine

Figure 4 shows the antibody formation curve 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 approximately one month intervals, the epidemic hemorrhagic fever vaccine made up of 1: 256, 84/105 weeks would be 1: 4,096, 76/118 weeks. A high titer of fluorescent antibody could be demonstrated. Figure 5 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.

5) 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 viral antigen for vaccine. 6 is a growth curve of Japanese encephalitis virus and Hantan virus in primary hamster kidney epithelial cells. Japanese encephalitis virus inoculates tissue culture cells with 100ID virus, approximately 10 days later / Ml of viral antigen was obtained, and Hantan virus 76/118 strains received approximately 10 days after inoculation of 100 ID virus. / Ml viral antigen was obtained, 84/105 weeks 10 after 7 days of inoculation Virus antigen of / ml was obtained. Pure virus antigens were separated by ultra-fast centrifugation and treated with 0.05% formalin at 4 ° C. for 2 weeks, and encephalitis virus was adsorbed in 0.02% gelatin solution and Hantan virus was used as vaccine. Tissue cultured Japanese encephalitis- pandemic hemorrhagic fever vaccine was prepared by mixing a certain amount of viral antigens, such as a vaccine made in mouse brain tissue, and injected into the muscles of hamsters and guinea pigs. . As shown in FIG. 7, the fluorescent antibody titer of Japanese encephalitis virus was measured 1: 512, and the fluorescent antibody titer of hantan virus was 1: 1,024 3 weeks after the inoculation of the tissue culture cultured Japanese encephalitis-Hantan virus vaccine. .

6) Immunization test against Hantan virus of vaccinated dorsal rats

After inoculation twice with the Japanese encephalitis- pandemic hemorrhagic fever vaccine, a large amount of hantan virus was inoculated at 60 days and the results are shown in Table 6. The vaccinated mice were immunized to prevent virus infection and demonstrated high virus proliferation and high antibody titer in normal mice.

[Effects of the Invention]

The newly made Japanese encephalitis vaccine contains 22µg / ml protein and 0.8ng / ml of myelin-based protein, while the anti-tantan vaccine lowers the protein to 17µg / ml and myelin-based protein below 0.08ng / ml. Can be prevented. In addition, Hantan virus was cultured in primary hamster kidney epithelial cells to make a pure Hantan virus tissue culture vaccine without brain proteins. On the other hand, the combination vaccine that can prevent both Japanese encephalitis and epidemic hemorrhagic fever at the same time at the same time, the convenience of vaccination increased and the immunity of the mixed vaccine showed a better effect than the single vaccine.

Claims (3)

Japanese encephalitis virus causing Japanese encephalitis and Hantan virus causing pandemic hemorrhagic fever were inoculated into 1-7 days old mouse or primary hamster kidney epithelial cells, inoculated and cultured. Japanese encephalitis- pandemic hemorrhagic fever vaccine consisting of a mixture of the Japanese encephalitis vaccine and pandemic hemorrhagic fever vaccine obtained by inactivation. 2. The Japanese encephalitis- pandemic hemorrhagic fever combined vaccine according to claim 1, wherein the amount of the Japanese encephalitis virus antigen contained in the mixed vaccine is 20 to 80 units / 0.1 ml of EIA and the amount of the hantan virus antigen is 1,024 unit / 0.1 ml or more. The Japanese encephalitis- pandemic hemorrhagic fever combined vaccine according to claim 1, wherein the inoculated hantan virus is 84/105 strains of ATAN virus (ATCC VR 2250) and / or 76/118 (ATCC VR 938).