CN1695736A - Japanese encephalitis virus vaccine and preparation method thereof - Google Patents

Abstract

A vaccine of the encephalitis B virus for preventing viral encephalitis B is prepared through inoculating said virus into human diploid cells, culturing, naturalizing, adapting, amplifying, concentrating and purifying.

Description

Japanese encephalitis virus vaccine and preparation method thereof

technical field

The invention relates to a Japanese encephalitis virus vaccine prepared by human diploid cell culture. Specifically, the present invention relates to a method of inoculating Japanese encephalitis virus in human diploid cells for cultivation, and after domestication, adaptation and amplification processes, the Japanese encephalitis virus reproduces in large numbers in human diploid cells, It is then concentrated and purified to prepare a Japanese encephalitis virus vaccine. The virus vaccine can prevent Japanese encephalitis disease caused by Japanese encephalitis virus infection in infants, children, teenagers and adults. The present invention also relates to the preparation method of this virus vaccine.

Background technique

Japanese encephalitis is an infectious disease caused by acute viral infection of the central nervous system. The annual incidence rate in endemic areas is as high as 100-100/100,000. Nearly 3 billion people live in Japanese encephalitis endemic areas. In this area, more than 100,000 newborns are born every year. 70 million. Among viral encephalitis in Asia, Japanese encephalitis is the most serious disease. It causes at least 50,000 clinical cases and 10,000 deaths each year, the vast majority occurring in children. In recent decades, several outbreaks of Japanese encephalitis have increasingly spread to areas that were once non-endemic, accompanied by high case fatality rates and severe persistent neurological sequelae. , Japanese encephalitis has become a serious public health problem.

Epidemic encephalitis virus (Japanese Encephalitis Virus) is referred to as JE virus for short. Virus particles have an icosahedral symmetrical structure with a diameter of 45-50nm. Virus particles are composed of core, envelope and spikes. It is a kind of flavivirus. The gene is a positive-strand single-stranded RNA, and the RNA is coated in the capsid C of the polypeptide, which constitutes the core of the virus particle. There are glycosylated protein E and non-glycosylated protein prM/M in its envelope.

Mosquitoes are the main carrier of JE virus in the route of JE transmission, pigs are the main intermediate host, and humans are infected by the bites of infected mosquitoes. At present, there is no specific drug for the treatment of Japanese encephalitis, and the effect of controlling its spread from the environment is not satisfactory. The immunization of Japanese encephalitis vaccine is the most economical and effective measure to control Japanese encephalitis disease.

There are three types of Japanese encephalitis vaccines used on a large scale, inactivated vaccines prepared by cultured rat brain tissue, inactivated vaccines prepared by cultured primary hamster kidney cells or Vero cells, and attenuated vaccines prepared by cultured primary hamster kidney cells. Virus live vaccine.

Since the basic immunization of Japanese encephalitis vaccine begins with healthy infants aged 6 months, this requires that the vaccine must first ensure sufficient safety as a prerequisite. No matter what kind of culture system is used for the Japanese encephalitis vaccine mentioned above, it is prepared by culturing animal-derived tissues or animal cells. The disadvantages of these vaccines are: firstly, the contamination of animal-derived exogenous factors cannot be completely removed. Secondly, it is impossible to further increase the antigen content to generate a higher immune response in the human body and provide higher and longer immune protection. The reason is that if the virus antigen content is further increased, the accompanying animal-derived substances such as miscellaneous proteins, nucleic acids, and lipids will also increase, causing severe hypersensitivity reactions, so the side effects of the vaccine will increase. For example, local side effects of pain, redness, and swelling of inactivated vaccines cultured from rat brain tissue are about 20%, and systemic reactions including headache, fever, myalgia, malaise, and gastrointestinal symptoms are 10-30% . In Denmark and Australia in 1989 and 1990, 31 cases and 7 cases of severe allergic reactions of urticaria and angioedema were found respectively. The fever of inactivated vaccines cultured by primary hamster kidney cells exceeding 38°C was as high as 12%, and after reducing the residual amount of bovine serum, the fever exceeding 38°C was reduced to 6% (Tsai, Theodore F, Japanese encephalitis vaccines, http : //www.cdc.gov . publication date: 01/01/1990). The live attenuated vaccine uses non-clean grade hamster kidney primary cells as the vaccine culture substrate, which is difficult to avoid the contamination of exogenous factors of mouse origin, and the possible contamination is difficult to detect by current biological methods. Therefore, it cannot be accepted by the World Health Organization's vaccine manufacturing verification regulations (Zui Wenyuan, Planned Immunology, Shanghai Science and Technology Literature Publishing House, 2001, page 484). Although the safety of the inactivated vaccine prepared by Vero cell culture has been significantly improved, there are tumorigenic phenomena in nude mice after Vero cells exceed 232 passages (Levenbook IS, et al, Tumorigenicity of vero cells.J Biol Stand 1984 Oct ; 12(4):391-8). Therefore, the U.S. FDA requires that vaccines produced with Vero cells must have a cell removal process during the production process, and must ensure that there are no residual cells in the finished product (FDA letter to vaccine manufacturers concerning the use of Vero cells.http://www.fda .gov/cber/letters.htm).

In view of the above reasons, it is necessary to revolutionize the existing Japanese encephalitis virus vaccine cell lines, find new virus culture cell lines, and avoid the safety hazards of the above-mentioned rat brain tissue, hamster kidney primary cells and Vero cell lines. , to prepare a Japanese encephalitis vaccine that can meet the needs of inoculation from infants.

It has been confirmed that the origin of diploid cells derived from humans is quite clear, including the age, sex, race, region, physical fitness and health status of the donor, the tissue or organ from which the cells are directly derived, and the number of cell generations. After decades of research, human diploid cell lines have been fully verified for their growth characteristics, genetically stable characteristics, contamination checks for exogenous factors (bacteria, fungi, mycoplasma and viruses), and negative tumorigenicity. More importantly, human diploid cells themselves are cells derived from the human body. When used in vaccine production, the residual components of human diploid cells will not become hypersensitivity allergens, and basically will not cause hypersensitivity reactions in vaccinators. Therefore, from the perspective of quality control, compared with the above-mentioned mouse brain tissue, hamster kidney primary cells and Vero cell lines, human diploid cells have no potential safety hazards and are ideal cell lines for preparing vaccines.

It has been further confirmed that vaccines prepared with human diploid cell lines have been used more and more, such as rubella virus vaccines prepared with MRC-5 and/or WI-38 cell lines, hepatitis A virus vaccines prepared with MRC-5 cell lines , the rotavirus vaccine prepared with the FrhL-2 cell line, the varicella virus vaccine prepared with the MRC-5 cell line, and the internationally recognized gold standard rabies vaccine are also human diploid cell culture vaccines. Therefore, using human diploid cells to prepare Japanese encephalitis virus vaccines can significantly improve the quality of current Japanese encephalitis virus vaccines.

The core content of the present invention is that the human diploid cell line is used as the cell matrix for Japanese encephalitis virus cultivation and propagation, and the Japanese encephalitis virus is domesticated and adapted in the human diploid cells at the same time, and both the virus and the cells have Good stability, and then prepared into Japanese encephalitis virus vaccine.

In summary, the Japanese encephalitis virus vaccine of the present invention has solved the key points. Human diploid cells are used to replace rat brain tissue, hamster kidney primary cells and Vero cells to carry out Japanese encephalitis virus culture, and then further make the vaccine . Vaccines prepared with human diploid cells can significantly reduce the side effects of vaccines, especially the hazards of allergic reactions and animal-derived viruses, remove potential safety hazards, and ensure the use of vaccines in children and adolescents and adults in non-epidemic areas security.

In order to overcome the deficiencies of the prior art, the object of the present invention is to provide a novel Japanese encephalitis virus vaccine and a preparation method thereof.

Contents of the invention

In order to accomplish the purpose of the present invention, the present invention provides a Japanese encephalitis virus vaccine, which is characterized in that the Japanese encephalitis virus antigen contained in the vaccine is Japanese encephalitis virus cultured by human diploid cells, propagated, Purified and extracted. The Japanese encephalitis virus vaccine is an inactivated vaccine, and also includes live attenuated vaccines, split vaccines and/or subunit vaccines and combined vaccines with other vaccines.

The Japanese encephalitis virus vaccine is prepared from any strain of Japanese encephalitis virus SA ₁₄ strain, SA _14-14-2 strain, P ₃ strain, Nakayama strain and other isolated strains of Japanese encephalitis virus.

The human diploid cell is any one of 2BS, MRC-5, KMB-17, WI-38 or HL cell lines.

The invention also relates to a method for domesticating, adapting and amplifying Japanese encephalitis virus in human diploid cells.

In other words, the present invention provides that the Japanese encephalitis virus is inoculated in human diploid cells for cultivation, and after domestication, adaptation and amplification processes, the Japanese encephalitis virus reproduces in large quantities in the human diploid cells, and then sterilized. live, concentrated and purified to prepare a Japanese encephalitis virus vaccine. The invention also provides a method for domesticating, adapting and amplifying Japanese encephalitis virus in human diploid cells. The Japanese encephalitis virus vaccine prepared with human diploid cells can respectively prevent Japanese encephalitis diseases caused by Japanese encephalitis virus infection in infants, children, teenagers and adults.

The concept and method mainly introduced in the present invention are to transform and upgrade the current Japanese encephalitis virus vaccine to achieve better safety and immune effect. The present invention uses human diploid cells to replace rat brain tissue, hamster kidney primary cells and Vero cells for culturing Japanese encephalitis virus, and the harvested virus liquid is prepared into Japanese encephalitis virus liquid after being inactivated, concentrated and purified. Inflammatory virus vaccine. This Japanese encephalitis virus vaccine prepared with human diploid cells does not contain any residual components derived from animal tissues or animal cells except for the presence of immunogenic virus antigens, fundamentally eliminating the possibility of causing hypersensitivity. Reactive animal protein, nucleic acid and lipid, etc. Due to the significant improvement in safety, the vaccine can further increase the immune dose and increase the protective efficacy of the vaccine, thereby overcoming the immune limitations and adverse reactions after vaccination of the current Japanese encephalitis virus commercial vaccine.

The Japanese Encephalitis Virus seed used in the present invention was purchased from China Institute for the Control of Pharmaceutical and Biological Products. The names of the poisonous species are: SA ₁₄ , SA _14-14-2 , P ₃ , Nakayama. The China Institute for the Control of Pharmaceutical and Biological Products is responsible for the preparation and distribution of the above-mentioned virus seeds.

In one embodiment of the present invention, any one of the above virus species SA ₁₄ , SA _14-14-2 , P ₃ , and Nakayama can be used for culturing Japanese encephalitis virus. Other suitable virus species can also be used if desired, and the virus species given here are only for illustrative purposes of the present invention.

The Japanese encephalitis virus culture system of the present invention is any strain in human diploid cell line 2BS, MRC-5, KMB-17, WI-38 or HL cell line. Other suitable human diploid cells may also be used if desired, and the cells presented here are merely illustrative of the invention.

The invention provides a method for domesticating, adapting and amplifying Japanese encephalitis virus in human diploid cells. Specifically, the virus titer of JE virus in human diploid cells gradually increased, ≥7.5 lgPFU/ml, and tended to be stable.

Each dose of the Japanese encephalitis virus vaccine of the present invention contains 5-90 μg of viral protein. As for the upper limit of the dosage, those skilled in the art can finally determine it according to the data of vaccine human clinical trials.

The Japanese encephalitis virus vaccine prepared by human diploid cells has good immunogenicity, and the neutralizing antibody titer is greater than or equal to the Japanese encephalitis virus vaccine prepared by hamster kidney cells and Vero cells.

The Japanese encephalitis virus vaccine prepared by the human diploid cell of the present invention has good protective effect, and the efficacy neutralization index is higher than that of the Japanese encephalitis virus vaccine prepared by hamster kidney cells and Vero cells.

The Japanese encephalitis virus vaccine of the present invention can be combined with a combination vaccine formed by coupling meningococcal polysaccharides of groups A and C with a protein carrier to form a combined Japanese encephalitis-meningococcal vaccine.

The Japanese encephalitis virus vaccine of the present invention can further form a combined vaccine with other vaccines, such as: acellular diphtheria vaccine—inactivated poliovirus vaccine—Hib vaccine—hepatitis B vaccine—meningococcal conjugate vaccine— Japanese encephalitis virus vaccine, measles-JE combined vaccine, measles-rubella-mumps-JE combined vaccine, yellow fever-JE combined vaccine, etc.

The invention provides a preparation method of Japanese encephalitis virus vaccine. The cell line used for virus cultivation in the preparation process of the vaccine is human diploid cell. In the method of the present invention, the human diploid cell line is selected from any one of 2BS, MRC-5, KMB-17, WI-38 or HL cell lines. And described Japanese encephalitis virus vaccine can be inactivated vaccine, and inactivated vaccine is by Japanese encephalitis virus SA ₁₄ strain, SA _14-14-2 strain, P ₃ strain or Nakayama strain or other isolated B prepared from any of the encephalitis virus strains. The Japanese encephalitis virus vaccine can also be an attenuated live vaccine, a purified vaccine, a split vaccine/or a subunit vaccine.

In addition, the Japanese encephalitis virus vaccine can also be a combined vaccine with other vaccines, including: measles-JE combined vaccine, JE-menorrhea combined vaccine, measles-rubella-mumps-JE combined vaccine, yellow encephalitis combined vaccine, Fever-JE combined vaccine.

Through the method of the invention, a Japanese encephalitis virus vaccine with higher immunogenicity and efficacy, safer and easier acceptance by vaccinators can be prepared.

Detailed ways

The present invention is described below in conjunction with the examples, Japanese encephalitis virus is domesticated, adapted and amplified in human diploid cells, the harvested virus liquid is concentrated and purified, and then suitably prepared into Japanese encephalitis virus vaccine . The Japanese encephalitis virus vaccine prepared with diploid cells has been proved by animal experiments and laboratory tests to have reliable safety, excellent immunogenicity and excellent protective effect.

Example 1:

Preparation of human diploid cells

The preparation of human diploid cells takes MRC-5 as an example: the cell culture medium is DMEM (other suitable cell nutrient solutions such as MEM, 199 medium, 1640 medium, etc. can also be used), and 4-10% fetal bovine serum is added (No exogenous factor contamination) and kanamycin (or gentamicin) with a concentration of 100-200U/ml, adjust the pH to between 7.0-8.0. Cells are cultured at 35-37°C with Koshik flasks or roller bottles. After 4-8 days, they are cultured into dense monolayer cells, and then expanded at a ratio of 1:2 until the production batch is expanded. After 3-5 days After culturing, the cells grow into dense monolayer cells, and the cell culture medium is discarded. The surface of the cells was rinsed with phosphate buffer solution of pH 7.0-8.0 to wash away the residual bovine serum. Prepare for virus inoculation.

Example 2:

Domestication, adaptation and expansion of Japanese encephalitis virus in human diploid cells

Take human diploid cells and MRC-5 as an example: because the virus reproduction titer of JE virus in human diploid cells is low, it is necessary to gradually domesticate and adapt JE virus in human diploid cells. Inoculate the Japanese encephalitis virus P3 strain (or other virus strains such as SA14) into the human diploid cells that have grown to a monolayer, and continue to culture at 30-37°C. When the cytopathy reaches 50-70%, the cell supernatant is harvested, which is the Japanese encephalitis virus liquid that grows and reproduces in the human diploid cells. Successive generation-to-generation adaptations are carried out. When the Japanese encephalitis virus titer in the harvested Japanese encephalitis virus liquid reaches more than 7.5lgPFU/ml, it is used as the seed virus for the production of Japanese encephalitis vaccine. According to the requirements of "China Biological Products Regulations" (Chemical Industry Press, 2000), it can be used for the production and preparation of vaccines after passing various tests and tests.

Example 3:

Passage Stability of Japanese Encephalitis Virus in Human Diploid Cells

Carry out successively by the test procedure of embodiment 2, can observe that the virus titer of Japanese encephalitis virus in human diploid cell improves gradually, more than or equal to 7.5lgPFU/ml, and tends towards stability, the virus titer of each generation is shown in the table 1.

Table 1. Passage stability of Japanese encephalitis virus in human diploid cells Virus Adaptation Generation Virus titer (lgpfu/ml) 1 4.58 2 4.95 3 5.68 4 5.80 5 6.39 6 7.56 7 7.80 8 7.87

Example 4:

Preparation of Japanese encephalitis virus inactivated vaccine

Prepare human diploid cells according to Example 1, and prepare virus seeds for vaccine production according to Example 3. According to Example 3, the generation whose virus titer reaches the vaccine production requirement is selected as the working seed for vaccine production. The cells rinsed with phosphate buffer saline were inoculated with Japanese encephalitis virus into human diploid cells at a ratio of MOI of 0.1-0.00001. The cell maintenance solution is DMEM cell nutrient solution containing 0.1-0.5% human serum albumin, and the pH is between 7.0-8.0. Cultivate at 30-37°C, and harvest the virus supernatant after 3 days. (Can harvest continuously as culturing with microcarriers) The viral titer of the harvested virus liquid reaches more than 7.5 lgpfu/ml, and the sterility test is negative, and the virus liquid after the harvest can be properly merged. The combined liquid is the vaccine stock solution. Add a suitable inactivator 1:2000 formaldehyde solution (or 1:4000 β-propiolactone, or other suitable inactivator, this is just an example). Crude vaccine was obtained after virus inactivation. Then use an ultrafilter with a cut-off of 100,000 to 300,000 molecular weight to concentrate the vaccine 10 to 50 times, and perform two-step column chromatography on an ion exchange column (DEAE-Sepharose FF) and a gel filtration column (Sepharose 4FF) to obtain a purified vaccine. Add human white protective agent and make vaccine after dilution.

Example 5:

Safety Test of Japanese Encephalitis Virus Vaccine

The safety of the Japanese encephalitis virus vaccine prepared by human diploid cells was evaluated, and its safety was evaluated by abnormal toxicity tests in mice and guinea pigs. Choose clean-grade guinea pigs with a body weight of 250-350 g, use 2 guinea pigs for each vaccine, and inject 5.0 ml of Japanese encephalitis virus vaccine into the abdominal cavity of each guinea pig. Weigh the body weight before and 7 days after the injection, and observe the vaccine at any time. reaction. Choose clean-grade mice with a body weight of 18-20 g, and inject 0.5 ml of Japanese encephalitis virus vaccine intraperitoneally into each of 5 mice. Weigh their body weight before and 7 days after injection, and observe the reaction after vaccination at any time. . The test results are listed in Table 2.

Table 2. Japanese encephalitis virus vaccine safety test Vaccine Type Animal species and average body weight before injection (g) observation period response Average body weight 7 days after injection (g) in conclusion Diploid inactivated vaccine (90μg) guinea pig, 278 All survive without abnormal reaction 309 Safety Diploid inactivated vaccine (90μg) mouse, 20 All survive without abnormal reaction twenty three Safety Diploid inactivated vaccine (5μg) guinea pig, 295 All survive without abnormal reaction 326 Safety Diploid inactivated vaccine (5μg) mouse, 19 All survive without abnormal reaction twenty two Safety

Animal safety tests have shown that the Japanese encephalitis virus vaccine prepared with human diploid cells has no unsafe acute toxicity factors, which can ensure the safety of human use.

Embodiment 6:

Comparison of Immunogenicity Tests of Japanese Encephalitis Virus Vaccines

Select 20 mice with a weight of 12-14 g for each vaccine, and each mouse is immunized with 0.5 ml intraperitoneally, and immunized twice with an interval of 7 days. Blood was collected 15 days after primary immunization, and left overnight at 2-8°C. The serum was separated the next day and inactivated at 56°C for 30 minutes. Neutralizing antibody titers were determined using a plaque reduction neutralization assay. The JE neutralizing virus strains were SA ₁₄ and P ₃ strains. The test results are shown in Table 3.

Table 3. Japanese encephalitis virus vaccine neutralizing antibody titer test Vaccine Type Neutralizing antibody titer Neutralize virus SA ₁₄ Neutralizing virus P ₃ Diploid inactivated vaccine (30μg) 1:10 1:10 Diploid inactivated vaccine (50μg) 1:20 1:20 Diploid inactivated vaccine (70μg) 1:40 1:40 Hamster Kidney Inactivated Vaccine 1:2.5 1:5 Vero cell inactivated vaccine 1:10 1:10 Hamster kidney attenuated live vaccine 1:10 1:10

The neutralizing antibody titer test showed that the Japanese encephalitis virus vaccine prepared by human diploid cells had good immunogenicity, and the neutralizing antibody titer was higher than the Japanese encephalitis virus vaccine prepared by hamster kidney cells and Vero cells.

Embodiment 6:

Experimental Comparison of Protective Efficacy of Japanese Encephalitis Virus Vaccines

Neutralizing antibody assays were performed using immunized mice. Neutralizing antibody was determined by plaque reduction test. Reference vaccines ( _RA and _RB ) and neutralizing test positive serum were purchased from China Institute for the Control of Pharmaceutical and Biological Products. Both the vaccine to be tested and the reference vaccine (R) were diluted 1:32, respectively, and 10 mice weighing 12-14 g were immunized intraperitoneally, each with 0.5 ml, immunized twice with an interval of 7 days. Blood was collected 7 days after the second immunization, the serum was separated, mixed with equal amounts of serum from mice in the same group, inactivated at 56°C for 30 minutes, diluted serum, and mixed with diluted virus (about 200PFU/0.4ml) in equal amounts. At the same time, dilute the diluted virus again 1:2, as a virus control, place it in a water bath at 37°C for 90 minutes, inoculate BHK21 cells in a 6-well plate, 0.4ml per well, incubate at 37°C for 90 minutes, add methylcellulose medium The overlay was cultured in a CO ₂ incubator for 5 days, stained, plaques were counted, and the plaque reduction rate was calculated. The average number of plaques in the virus control group should be between 50 and 150, and the qualified criterion is T≥( _RA + _RB )/2-0.33. The test results are shown in Table 4.

Table 4. Japanese encephalitis virus vaccine protective efficacy test Vaccine Type neutralization index Diploid inactivated vaccine (30μg) 1.63 Diploid inactivated vaccine (50μg) 1.75 Diploid inactivated vaccine (70μg) 1.96 Hamster Kidney Inactivated Vaccine 1.43 Vero cell inactivated vaccine 1.59

Efficacy tests showed that the Japanese encephalitis virus vaccine prepared by human diploid cells had good protective efficacy, and the efficacy neutralization index was higher than that of the Japanese encephalitis virus vaccine prepared by hamster kidney cells and Vero cells.

Claims (12)

1, a kind of encephalitis b virus vaccine through human diploid cell cultivation preparation is characterized in that the used cell line of this vaccine Virus culture in preparation process is human diploid cell.

2, encephalitis b virus vaccine according to claim 1, it is characterized in that the used human diploid cell system of described encephalitis b virus vaccine Virus culture in preparation process is the arbitrary strain in 2BS, MRC-5, KMB-17, WI-38 or the HL cell line.

3, encephalitis b virus vaccine according to claim 1 is characterized in that, described encephalitis b virus vaccine is an inactivated vaccine.

4, encephalitis b virus vaccine according to claim 1 is characterized in that, described encephalitis b virus vaccine is attenuated live vaccine, purified vaccine, split vaccine/or subunit vaccine also.

5, encephalitis b virus vaccine according to claim 1, it is characterized in that, described encephalitis b virus vaccine also can and with the combined vaccine of other vaccine, for example: measles-encephalitis B combined vaccine, encephalitis b-epidemic encephalitis combined vaccine, measles-rubella-parotitis-encephalitis B combined vaccine, yellow heat-encephalitis B combined vaccine etc.

6, encephalitis b virus vaccine according to claim 3 is characterized in that, described inactivated vaccine is by arbitrary strain preparation in encephalitis b virus SA14 strain, SA14-14-2 strain, P3 strain or Nakayama strain or other the isolating encephalitis b virus strain.

7, a kind of preparation method of encephalitis b virus vaccine is characterized in that, the used cell line of this vaccine Virus culture in preparation process is human diploid cell.

8, the preparation method of encephalitis b virus vaccine according to claim 7 is characterized in that, described human diploid cell is the arbitrary strain that is selected from 2BS, MRC-5, KMB-17, WI-38 or the HL cell line.

9, the preparation method of encephalitis b virus vaccine according to claim 7 is characterized in that, described encephalitis b virus vaccine is an inactivated vaccine.

10, the preparation method of encephalitis b virus vaccine according to claim 7 is characterized in that, described encephalitis b virus vaccine is attenuated live vaccine, purified vaccine, split vaccine/or subunit vaccine.

11, the preparation method of encephalitis b virus vaccine according to claim 7, it is characterized in that, described encephalitis b virus vaccine is and the combined vaccine of other vaccine, comprises: measles-encephalitis B combined vaccine, encephalitis b-epidemic encephalitis combined vaccine, measles-rubella-parotitis-encephalitis B combined vaccine, yellow heat-encephalitis B combined vaccine.

12, encephalitis b virus vaccine according to claim 9, it is characterized in that described inactivated vaccine is by arbitrary strain preparation in encephalitis b virus SA14 strain, SA14-14-2 strain, P3 strain or Nakayama strain or other the isolating encephalitis b virus strain.