JP2000083657A - Japanese encephalitis virus vaccine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain Japanese encephalitis viruses or their antigen components useful for producing vaccines, etc., in a high yield by infecting established cells with the Japanese encephalitis viruses, culturing the infected cells and purifying the viruses or their antigen components from the culture product. SOLUTION: This method for preparing Japanese encephalitis viruses or their antigen components comprises infecting established cells, such as animal or insect-originated cells, selected from the group consisting of Vero cells, GL37 cells (FERM P-16857), HmLu-1 cells, BHK-21 cells, MDCK cells and C6/36 cells with Japanese encephalitis viruses such as Beijin-1 strain or Nakayama strain, culturing the virus-injected cells by a static culture method, a roller bottle culture method, a suspension culture method, etc., and subsequently purifying the viruses or their antigen components from the culture product by a purification method such as a concentration method using an ultrafiltration membrane or an ion exchange or adsorption chromatography. The obtained Japanese encephalitis viruses or their antigenic components are preferably used to prepare Japanese encephalitis virus vaccines.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a Japanese encephalitis virus (hereinafter sometimes referred to as "JEV") vaccine. More specifically, a method of infecting a cell line derived from African green monkey kidney with JEV, culturing the cell line, preparing a JEV or JEV antigen component from the culture, and a JEV or JEV antigen component obtained by the method The present invention relates to a Japanese encephalitis virus vaccine comprising

[0002]

2. Description of the Related Art Japanese encephalitis is a viral disease caused by a single-stranded RNA virus of 50 nm in diameter belonging to the Flaviviridae family, and is a serious disease with fever, meningeal irritation and encephalitis as main symptoms. . The prognosis is generally poor, with an average fatality rate of about 35%, which is higher for older people.

[0003] There is no other preventive method for Japanese encephalitis than vaccination, and a report by Otani et al. (Acta. Paediatr. Jp.
According to n., 30: 175-184, 1988), a 1: 10-fold neutralizing antibody titer as a minimum effective dose for protecting against viral infection can prevent the development of Japanese encephalitis. The first Japanese encephalitis virus vaccine was manufactured in Japan in 1954,
5% mouse brain emulsion infected with Japanese encephalitis virus Nakayama strain was used as vaccine material. In 1989, the vaccine material was changed to a mouse brain emulsion infected with the Beijing-1 strain, which is more immunogenic and appears to be more antigenic than the field strain.

[0004] Current vaccines are manufactured according to the following method. First, a virus is inoculated into the brain of a mouse 3 to 5 weeks after birth, and the brain immediately before death showing cerebral inflammatory symptoms is collected. This was used as a 20% brain emulsion in buffered saline, and the supernatant was purified by ultrafiltration and concentration, combined with protamine sulfate treatment, and zonal centrifugation to purify the virus, to which 0.08% formalin was added to inactivate the stock solution. And To the final bulk obtained by diluting this stock solution, thimerosal as a preservative and gelatin or Tween 80 as a stabilizer are added to obtain an inactivated vaccine solution.

At present, the Japanese encephalitis virus vaccine used in Japan is a highly purified virus particle obtained by the above-mentioned method, and it has been recognized that it is effective and safe. Exists. That is,
Since the current vaccine uses mouse brain emulsion infected with Japanese encephalitis virus as a starting material, there is a risk of allergic central nervous disorder and contamination with pathogenic microorganisms due to mouse brain contaminants. It has been pointed out that it is becoming difficult to supply mice and that their production costs are high, and that it is not desirable to use animals from the viewpoint of animal welfare.

[0006] To solve the above problems, vaccines using recombinant vaccinia virus are being developed in the United States and Japan. Mason and Koishi et al. Reported that a vaccinia virus incorporating the outer coat glycoprotein (E protein) gene, which is a major protective antigen of the Japanese encephalitis virus, and the PreM gene upstream thereof, was used as a cell line derived from African green monkey kidney (Vero cells). ) And chicken embryo fibroblasts (CE cells) were reported to secrete particle-like structures containing these proteins in the supernatant (Mason PW et al. Vi
rology 180: 294-305, 1991, Koishi E et al. Virolog
y 185: 401-410, 1991). Although these particles are highly immunogenic in mice, many safety and efficacy studies will still be needed before they can be used as vaccines.

In China, attenuated Japanese encephalitis virus (SA-14-14-2 strain) is infected into primary canine kidney cells (PCK cells), and the infected cells are cultured. Attempts have been made to produce this, and many clinical trials have already been conducted using this live attenuated vaccine (Kenn
eth H. Eckels Vaccine Vol. 6, p513, 1988). However, it has been pointed out that this vaccine strain has problems in attenuated toxicity and immunogenicity. From the above background, there is a strong demand for the development of a Japanese encephalitis virus vaccine that is economically inexpensive, effective, safe and can guarantee a certain quality.

In general, it is known that the virus susceptibility to cells, the virus growth in the cells and the immunogenicity of the obtained virus antigen vary greatly depending on the combination of the virus and the cell to be infected. For example, Leucart et al. (PDLukert Am J Vet Res, Vol. 36, No. 4 1975: 539
~ 540) are chicken embryo kidney cells
l) Infectious Fabricius brain disease virus (IB
It has been reported that chickens immunized with the virus antigen of DV) and chickens immunized with the virus antigen of IBDV adapted to Vero cells have different protective abilities against virus attack depending on the administration route. In other words, chickens immunized with chicken embryo kidney cell-derived IBDV antigen were completely protected from virus attack by either the subcutaneous administration or drinking water administration method, whereas the Vero cell-derived IBDV antigen was It is reported that chickens given subcutaneously were protected from virus challenge, but chickens given drinking water were not protected at all.

[0009] The reason is that a virus antigen having a different sugar chain is produced depending on the cell used, and the difference in the sugar chain causes a change in antigenicity. As a result, the ability to induce immunity as a vaccine is affected. It is guessed that it did. As described above, the sensitivity of the virus to the cells and the proliferation of the virus to the cells are recognized, but the antigenicity may vary depending on the combination of the virus and the host cell, and whether or not to produce an antigen suitable as a vaccine is determined. To clarify, it is necessary to examine the immunogenicity of the viral antigen obtained with each virus / cell combination.
Therefore, it is not easy to prepare an effective vaccine material using cultured cells.

[0010]

An object of the present invention is to provide a cell line derived from African green monkey kidney, GL37 or Vero.
It is an object of the present invention to provide a method for preparing a JEV antigen capable of raising a neutralizing antibody against JEV using cells.

Another object of the present invention is to provide a Japanese encephalitis virus vaccine containing JEV or its antigen component as a main component, which is obtained by the above method.

[0012]

Means for Solving the Problems The present inventors have conducted intensive studies in order to achieve the above object, and as a result, JEV
o High sensitivity to and proliferate in GL37 cells and GL37 cells, and intraperitoneal injection of vaccines prepared with viral antigens obtained from JEV-infected Vero cells and GL37 cells As a result, it was found that an antibody having a high neutralizing antibody titer against JEV was induced in the serum of the mouse, thereby completing the present invention.

[0013]

The method of the present invention is characterized by the JEV and the cell line used to propagate the virus. As JEV strains used for manufacturing, for example,
Beijing-1 stock, Zhongshan stock, but preferably Beijing-1 stock
The strain is used. The host cell used for the propagation of JEV can be any cell as long as the cell is a cell line that produces JEV that has good proliferation of JEV and has a highly immunogenic antigen, and that is a cell line that produces JEV. Cell lines derived from animals or insects are used. Specifically, HmLu-1 derived from hamster lung
Cells (Hideo Okumura, Cancer Cells in Culture, 292-2
BHK-21 cells derived from hamster kidney (IAN Mac)
pherson and Michael Stoker Virology 16: 147-151,
1962), African green monkey kidney-derived Vero cells or GL
37 cells, canine kidney-derived MDCK cells, mosquito-derived C6 / 36 cells and the like, and preferably, Vero cells or GL37
Cells are used. The GL37 cells have been deposited by the applicant under the accession number FERM P-16857 on June 19, 1998 at the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology.

As a medium used for cell preparation and cell culture for growing JEV, M199-earle base, Eagle minimum essential medium (E-MEM), Dulbecco m
inimumessential medium (D-MEM), SC-UCM102 (Nissui), VP-SFM (GIBCO BRL), EX-CELL302 (Nichirei), EX-CELL293-S (Nichirei), TFBM-01 (Nichirei), ASF104, etc. Generally, those used for tissue culture are appropriately selected, and those obtained by adding amino acids, salts, antifungal / antibacterial agents, animal serum, and the like are used.

The above-mentioned medium can be used as a serum-free medium to which no serum is added. The culture temperature and culture period are adjusted by a combination of various cells, virus inoculum amount, culture scale and method, and the like. For example, Vero
JE by static culture or roller bottle culture using cells
When V is grown, the cells are transformed with M199-earle base or gentamicin supplemented with streptomycin (St), kanamycin (Ka) and 5% bovine serum (FBS), non-essential amino acids and 10% bovine serum (FBS). Dulbecco ME with added
In a growth medium consisting of M (D-MEM), a culture temperature of 32 ° C. to 38 ° C.
C., preferably at 37.degree. C., for a culture period of 2 to 7 days, preferably 3 to 4 days. The medium was removed by suction, washed with phosphate buffered saline (PBS (-)), and JEV stam (10% brain emulsion of JEV-infected mice) or JEV passaged with Vero cells was used to obtain MOI = 1 ~
0.0001, preferably inoculated at MOI = 0.01-0.0001,
Incubate at the above culture temperature until CPE reaches 90% to 100%. Preferably, for 3-8 days, more preferably 5 ° C at 37 ° C.
Culture for ~ 7 days. As a maintenance medium after virus inoculation,
E-MEM or D-MEM with L-glutamic acid (L-Glu), heavy sodium, S
t, Ka, inactivated 3-5% FBS added medium or VP-SFM L-
A medium supplemented with glutamic acid (L-Glu) is used.
After completion of the culture, the culture, that is, the cell lysate or the culture supernatant, is collected, and subjected to rough centrifugation or membrane filtration to remove unnecessary substances to obtain a virus stock solution.

When JEV is grown using GL37 cells, the same method as described above is used, except that E-growth is used as a growth medium.
A medium supplemented with L-Glu, L-Glu, gentamicin and 5-10% FBS in MEM is used, and L-Gl in E-MEM is used as a maintenance medium.
A medium supplemented with u, sodium bicarbonate, gentamicin and inactivated 2-8% FBS is used. After virus inoculation, preferably
Incubate at 37 ° C for 3-5 days. In addition, a medium obtained by adding L-Glu to TFBM-01 or VP-SFM is used as a serum-free maintenance medium.

The JEV can also be grown by a suspension culture method in which cells are attached to a carrier such as a microcarrier and the cells are suspended and cultured. The carrier is washed with PBS (-) before attaching the cells, sterilized by high-pressure steam sterilization, and stored in a 2-10% inactivated FBS-containing medium (growth medium). The final concentration of the carrier during culture is used at 3 to 15 g / L. Vero cells or GL37 cells cultured in a static state are treated with trypsin-EDTA, and the cells are detached from the vessel wall. After suspending the suspension in the above-mentioned growth medium, an appropriate amount of a sterilized carrier is added to start the culture. Cells attach to the carrier in 5-6 hours. After attachment, add the same medium at half the final culture volume, and add
Stir at 15 ° C. overnight at 15 ° C. Thereafter, the remaining medium is added, and the cells are cultured at a rotation speed of 30 to 40 rpm for 3 to 5 days. When the cell density exceeds 1 × 10 ⁶ cells / ml, once remove the medium and add 1/5 volume of serum-free medium. The virus solution is inoculated at MOI = 0.1-0.0001 and adsorbed at 34 ° C. for 90 minutes. After virus adsorption, the remaining medium is added and virus growth is initiated. After culturing at 34 ° C. for 3 to 10 days and confirming a sufficient amount of virus, a virus stock solution is recovered.

The JEV antigen is prepared by subjecting the above-mentioned virus stock solution to an ultrafiltration membrane (preferably having a molecular weight cut of 100,000 to 500,000).
And then centrifuged by sucrose density gradient centrifugation to fractionate and pool antigen-positive fractions. Furthermore, a high-purity JEV antigen can be obtained by performing a purification step such as gel filtration, ion exchange, or adsorption chromatography. Preferably, adsorption chromatography by ion exchange is performed. Also,
The above purification step can also be performed prior to sucrose density gradient centrifugation.

The JEV antigen prepared by the method of the present invention can be obtained by inactivating a live attenuated vaccine when using an attenuated strain or by inactivating the virus antigen when using an unattenuated virus strain. It can be used as an inactivated vaccine.

The virus can be inactivated by adding an inactivating agent (preferably, formalin is used) such as formalin, glutaraldehyde or β-propiolactone to the virus stock solution or the purified virus antigen solution. It is achieved by allowing to stand for about 1 to 3 months at about ° C.

The JEV vaccine is obtained by diluting the thus obtained virus antigen solution by an ultrafiltration method or a dialysis method or diluting it with an appropriate buffer, and then sterile-filtering with a membrane filter. It is prepared by adding an immunoinactivating agent such as aluminum phosphate, potassium phosphate, mineral oil or non-mineral oil, a virus dispersant such as polysorbate 80 and polysorbate 20, and a stabilizer such as amino acid and sugar. Thimerosal or benzoic acid may be used as a preservative. When insoluble matter is generated during dialysis, the mixture is subjected to rough centrifugation or pretreatment with a prefilter, and then sterile-filtered with a membrane filter.

The vaccine of the present invention thus prepared is capable of inducing an antibody having a virus neutralizing activity, and contains other viruses (eg, hepatitis A virus, rabies virus) and bacteria (eg, , Pertussis, diphtheria, tetanus) can be used as a combination vaccine by combining with at least one vaccine selected from the group consisting of vaccines against pertussis, diphtheria and tetanus.

According to the present invention, a JEV inactivated vaccine for preventing JEV infection is provided, and the method of the present invention stably supplies a vaccine of a constant quality at a lower cost than the conventional method. It is possible to do. Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples.

[0024]

[Example] Example 1 In a serum-supplemented medium using cell lines
According JEV the static culture cell line of (Beijing -1 strain), 8 ml per 25 cm ² culture flask (approximately 2x10 ⁵
cell / ml) and grow at 37 ° C in growth medium (0.1% for Vero cells).
M199-earle base medium containing 0.1% sodium bicarbonate, 0.1 mg / ml St, 0.1 mg / ml Ka and 5% FBS, 0.06% L-Glu, 0.
Contains 11% sodium bicarbonate, 0.005% gentamicin and 10% FBS
E-MEM medium, 0.4% L-Glu, 0.5% for HmLu-1 and BHK-21 cells
% Glucose, 0.3% sodium bicarbonate, 0.1 mg / ml St, 0.1 mg / ml
D-MEM medium containing Ka and inactivated 5% FBS, 0.
1% gentamicin, 0.06% L-Glu, 0.3% sodium hydroxide and 5%
E-MEM medium containing FBS, 0.1 mg / ml St, 0.1 for C6 / 36 cells
E-MEMearle base medium containing mg / ml Ka, 0.03% L-Glu, 0.1% sodium bicarbonate, non-essential amino acids and 1% FBS is used)
For 3 to 4 days. Aspirate the medium and remove 2 ml of PBS (-)
Then, 0.2 ml of a virus solution passaged with a virus stamm (10% brain emulsion) or Vero cells was inoculated to a MOI of 0.1 to 0.001, and adsorbed at 37 ° C. for 1 hour. Next, a maintenance medium (for Vero cells, 0.03% L-Glu, 0.3% sodium bicarbonate, 0.
E-ME with 1 mg / ml St, 0.1 mg / ml Ka and inactivated 3% FBS
For M medium and GL37 cells, 0.03% L-Glu, 0.3% sodium bicarbonate, 0.1%
E-MEM containing mg / ml St, 0.1 mg / ml Ka and inactivated 5% FBS
Medium, HmLu-1 and BHK-21 cells contain 0.4% L-Glu, 0.5% glucose, 0.3% sodium bicarbonate, 0.1 mg / ml St, 0.1 mg / ml Ka and D- containing inactivated 1-2% FBS. MEM medium, 0.1 for MDCK cells
E-MEM medium containing gentamicin, 0.06% L-Glu, 0.3% sodium bicarbonate and inactivated 3% FBS, 0.1 mg / ml S for C6 / 36 cells
t, 0.1 mg / ml Ka, 0.03% L-Glu, 0.3% sodium bicarbonate, non-essential amino acids and inactivated 1% FBS) were added at 8 ml / bottle and cultured at 37 ° C for 6-7 days. The culture supernatant was collected, and the infectious titer was measured by a plaque assay. Table 1 shows the results.

[0025]

[Table 1]

Example 2 In a serum-free medium using cell lines
According JEV the static culture cell line of (Beijing -1 strain), 8 ml per 25 cm ² culture flask (approximately 2x10 ⁵
cell / ml) and grow at 37 ° C in growth medium (0.1% for Vero cells).
M199-earle base medium containing 0.1% sodium bicarbonate, 0.1 mg / ml St, 0.1 mg / ml Ka and 5% FBS, 0.06% L-Glu, 0.
Contains 11% sodium bicarbonate, 0.005% gentamicin and 10% FBS
(E-MEM medium) for 3 to 4 days. The medium was aspirated off, washed with 2 ml of PBS (-), and the virus solution passaged with virus stams (10% brain emulsion) or Vero cells was removed at MOI = 0.01.
0.2 ml, and adsorbed at 37 ° C. for 1 hour. Next, the cells were cultured for 7 days in a serum-free medium (VP-SFM containing 2 mM L-glutamin for Vero cells, and TFBM-01 medium for GL37 cells). Table 2 shows the results.

[0027]

[Table 2] * Unit: PFU / ml

Example 3 Using Vero cells and GL37 cells
JEV (Beijing-1 strain) bead-cultured Vero cells and GL37 cells were used as medium (0.03% LG for Vero cells).
lu, 0.3% sodium bicarbonate, 0.1 mg / ml St, 0.1 mg / ml Ka and 3%
E-MEM medium with inactivated FBS, 0.06% L-Gl for GL37 cells
u, 0.11% sodium bicarbonate, 0.005% gentamicin and 5% inactivated FBS are used in E-MEM medium), and the passaged virus solution is MOI = 0.01 for Vero cells and 0.01 for GL37 cells.
Inoculation was performed so that MOI = 0.01 and 0.001, and the cells were adsorbed at 37 ° C. for 1 hour. After the completion of the adsorption, 0.2 g of a carrier (calcifer: TOYOBO) previously coated with FBS at 4 ° C. overnight and 50 ml of the above medium were added to make 50 ml.
Spin at a speed of 30-40 rpm for 1 minute. This operation was repeated six times at intervals of once every 30 minutes.
Thereafter, another 50 ml of the above medium was added, and the cells were cultured at 37 ° C. for 7 days at a rotation speed of 30 to 40 rpm. The culture supernatant was collected over time and the infectious titer was measured by plaque assay.
Table 3 shows the results. Viral load was determined by Vero cells and GL
The highest values were reached on day 5 using any of the 37 cells.

Example 4 JEV using Vero cells (Beijing-1)
Take out the Vero cells cryopreserved in a large volume of liquid nitrogen at 37 ° C.
Immediately after melting with 2 ml of 10% FBS-M199 earle base
/ well in 6-well plate filled at 37 ℃, 5% CO
The cells were cultured in the incubator of No. ² for 5 to 7 days. Cells grown in a monolayer were recovered by trypsin-EDTA treatment. 5% FBS-DMEM (Dulbecco's modified MEM)
Medium) and seeded in a T-flask (175-225 cm ² )
The cells were cultured in an incubator at 37 ° C. and 5% CO ² for 5 to 7 days. When the cell number reaches 1 × 10 ⁸ cells, trypsin-EDTA
After treatment, the cells were collected and subcultured into a 1 L spinner flask (Cell Master, Wakaku Co., Ltd.) or 5 to 10 disposable roller bottles (Falcon, 850 cm ² ).
In the case of a spinner-flask, while controlling the temperature (37 ° C.) and the dissolved oxygen (DO = 2 to 3 ppm) at a stirring speed of 30 to 40 rpm,
Cultured for 4-6 days. The medium used was D-MEM containing 2 to 5% FBS, and the carrier employed was Cytodex1 (3 g / L). On the other hand, in the case of roller bottles, the cells are
And the cells were sealed and cultured at a rotation speed of 0.5 rpm at 37 ° C.

The 1 × 10 ⁹ cells grown in the above step were
After recovering by trypsin-EDTA treatment and neutralizing trypsin with 2% FBS-DMEM, immediately inoculate in 5 L jar fermenter (Able; BCP-07) and then 5 L of 2% FBS
-Inject DMEM and Cytodex 1 (15g / 5L) and rotate at 15 rpm
For 4 to 6 hours. After confirming that the Vero cells adhere to the carrier by 90% or more, the culture conditions were changed to 40 rpm,
The temperature was set at 37 ° C., pH 7.3, and dissolved oxygen (DO) 2 ppm, and stirring culture was continued. 2 × 10 ⁶ cells / m on the 5th or 6th day of culture
l, digesting the cells by trypsin-EDTA treatment,
After neutralizing trypsin with 1 L of 2% FBS-DMEM, the carrier (15
(0 g; 3 g / L). The 8 L spinner flask was aseptically connected to a 50 L culture tank, and the carriers and cells were pumped into a 50 L culture tank filled with 50 L of medium (2% FBS-DMEM). Control of 50L culture tank is pH 7.3, D
The mixture was stirred at O = 2.0 ppm and a stirring speed of 20 rpm for 4 to 6 hours. pH
7.3, DO = 2.0 ppm, stirring speed 40 rpm, temperature 37 ° C, exhaust pressure 0.
Culture was continued at 3 kgf / cm ² . After reaching a cell density of 1 × 10 ⁶ cells / ml or more, Japanese encephalitis virus (Beijing strain) was
Inoculated at OI = 0.01-0.0001. If MOI = 0.001,
The infectious titre reached 10 ^8.9 PFU / ml 5 days after inoculation (Table 3).
Two days later, the culture was subjected to a pre-filter (Sartorius; Sarto Pure PP2, 10 inches 0.65μ + 1.2μ) to remove cell components and unprecipitated beads, and then subjected to an ultrafiltration concentrator (estimated molecular weight cut-off 300K Dalton, 0.45 m ² × 2)
Was concentrated 10 to 25 times.

[0031]

[Table 3]

Example 5 Purified and inactivated JE derived from GL37 cell culture
V immunogenicity -80 ° C frozen GL37 cells were grown in growth medium (0.06% L-Glu, 0.11%
EM with heavy sodium, 0.005% gentamicin and 10% FBS
After reconstitution with an EM medium), the cells were cultured at 37 ° C. for 1 week with 4-fold expansion.
The subculture was repeated, and when the cells were subcultured to 300 roller bottles, the medium was removed, the cells were washed with PBS (-), the subculture virus was inoculated at MOI = 0.01, and adsorbed at 37 ° C for 1 hour. After completion of the adsorption, 150 ml of a maintenance medium containing 5% FBS (E-MEM medium containing 0.03% L-Glu, 0.3% sodium bicarbonate, 0.1 mg / ml St, 0.1 mg / ml Ka and inactivated 5% FBS) The cells were added and cultured at 37 ° C. for 5 days. On the 5th day after inoculation, 45 L of the culture supernatant was harvested and concentrated to about 5 L by ultrafiltration concentration (molecular weight cut off: 300,000). Concentrate
The cells were inactivated with 0.08 w / v% formalin at 4 ° C. for 18 days, and purified by sucrose density gradient centrifugation (30,000 rpm, 1 hr). After centrifugation,
The antigen-positive fractions were pooled and placed in 100 L of M / 100 PBS (-) at 4 ° C,
After dialysis for one day, sterile filtration is performed and purified inactivated antigen (ELISA
(Unit: 104 u / ml).

The inactivated JEV antigen derived from the GL37 cell culture was added to thimerosal (final concentration; 0.001%) and Tween 80 (final concentration; 0.01).
%) To give a stock solution of 35 u / ml in ELISA. Also, an antigen solution was prepared by adding aluminum hydroxide gel to this to a final concentration of 200 μg / ml. These antigen solutions were diluted with stock solution and diluted 32 ×, inoculated intraperitoneally to 10 DDY mice (female, 4.5 weeks old) into 10 mice at 0.5 ml each, and one week later, the same amount was boosted, followed by blood sampling and serum separation. . As a reference, the current Kaketsuken vaccine derived from mouse brain (Lot No. P-
26A) was immunized in the same manner by diluting the current vaccine for national assay (Lot. 184-P) by 16 × according to the standard as a reference. Neutralizing antibody titers of each of 10 pooled sera were measured by a plaque reduction method using Vero cells. Hemagglutination (HI) antibody titers were also measured. As a result, it was shown that the inactivated JEV antigen derived from the GL37 cell culture had immunogenicity equal to or higher than that of the current vaccine and the reference product. Table 4 shows the results.

[0034]

[Table 4]

Example 6 Purified and inactivated JE derived from Vero cell culture
V immunogenicity -80 ° C frozen Vero cells were grown in growth medium (0.1%
M199-earle ba containing g / ml St, 0.1 mg / ml Ka and 5% FBS
After reconstitution in a medium (se medium), the cells were cultured at 37 ° C. for 1 week with 6-fold expansion.
The passage was repeated, and when passage was performed up to 120 roller bottles, the medium was removed, the cells were washed with PBS (-), the passage virus was inoculated at MOI = 0.01, and adsorbed at 37 ° C for 1.5 hours. After the adsorption, 150 ml of VP-SFM medium containing 2.mM L-glutamin
The cells were added and cultured at 37 ° C. for 5 days. Culture supernatant on day 5 of inoculation 16.
7 L was harvested and concentrated to about 3 L by ultrafiltration concentration (molecular weight cut off 300,000). Concentrate at 0.08 w / v% formalin at 4 ° C
After inactivation for 4 weeks, sucrose density gradient centrifugation (30,000rpm, 1h
Purified in r). After centrifugation, the antigen-positive fractions were pooled, dialyzed against 100 L of M / 100 PBS (−) at 4 ° C. for 3 days, sterile-filtered, and a concentrated solution of purified inactivated antigen (ELISA unit: about 20 u /
ml). A part after sucrose density gradient centrifugation was analyzed by HPLC (Tosoh G3000SW). Comparison of the elution pattern with the control (elution buffer) revealed no contaminating proteins (FIG. 1). In addition, EL
ISA antigen protein and Lowry protein were measured and their ratio (ELISA / Lo
When wry) was calculated, a value of 100 to 120% was obtained, indicating that the sample was a high-purity sample containing substantially no contaminating proteins derived from cells.

The obtained inactivated JEV antigen derived from Vero cell culture, the present Kaketsuken Labs vaccine (Lot. 29A) whose ELISA unit was adjusted to about 20 u / ml, and a reference product for national assay (Lot. 184-P) were used. 4X
Diluted to ~ 32X, intraperitoneally into DDY mice (female, 4.5 weeks old)
Ten mice were inoculated in 0.5 ml portions, and one week later, the same amount was boosted, followed by blood sampling and serum separation. Neutralizing antibody titers of each of 10 pooled sera were measured by a plaque reduction method using Vero cells. Hemagglutination (HI) antibody titers were also measured. Based on the neutralizing antibody titer obtained by immunizing each antigen,
As a result of selecting three dilution folds showing linearity and performing a parallel line assay, the inactivated JEV antigen derived from Vero cell culture and the current vaccine when the relative titer of the reference product was 1.00 were 4.5 and 1.2, respectively. Met. From the above results, it was shown that the inactivated JEV antigen derived from Vero cell culture has immunogenicity equal to or higher than that of the current vaccine and the reference product. Tables 5 and 6 show the results.

[0037]

[Table 5]

[0038]

[Table 6]

[0039]

[Brief description of the drawings]

FIG. 1 is a view showing a fractionation pattern of JEV-inactivated purified antigen by analytical HPLC. (a) is an elution buffer containing a JEV antigen,
(b) shows the result of analyzing only the elution buffer. The vertical axis is 280
Absorbance in nm, horizontal axis shows retention time (min).

Claims (9)

[Claims] A method for preparing Japanese encephalitis virus or an antigen component thereof, comprising infecting a cell line with the Japanese encephalitis virus, culturing the virus-infected cell, and removing the virus or the virus antigen component from the culture. The above method, wherein the method is purified. 2. The method according to claim 1, wherein the cell line is a cell derived from an animal or an insect. 3. The method according to claim 1, wherein the cell derived from an animal or insect is Vero cell, GL.
37 cells, HmLu-1 cells, BHK-21 cells, MDCK cells and C6 / 36
3. The method according to claim 2, wherein the cells are selected from the group consisting of cells. 4. The method according to claim 1, wherein the Japanese encephalitis virus is a Beijing-1 strain or a Zhongshan strain. 5. The method according to claim 1, wherein the culture step is a culture method selected from the group consisting of a stationary culture method, a roller bottle culture method, and a suspension culture method. . 6. The purification step comprises: (1) concentration by an ultrafiltration membrane, (2) inactivation by a chemical substance, (3) purification by sucrose density gradient centrifugation, (4) purification by ion exchange or adsorption chromatography. The method according to any one of claims 1 to 5, further comprising (5) a step consisting of desalting by dialysis or dilution. 7. A Japanese encephalitis virus or an antigenic component thereof substantially free of cell components, obtained by the method according to any one of claims 1 to 6. 8. The Japanese encephalitis virus or an antigenic component thereof according to claim 7, which is capable of inducing an antibody having a neutralizing activity against the Japanese encephalitis virus. 9. A Japanese encephalitis virus vaccine comprising the Japanese encephalitis virus according to claim 7 or 8 or an antigen component thereof.