WO2005014803A1

WO2005014803A1 - West nile virus vaccine

Info

Publication number: WO2005014803A1
Application number: PCT/JP2004/011041
Authority: WO
Inventors: Kengo Sonoda; Yuji Ishikawa; Kiyoto Nishiyama; Shoji Kuzuhara; Yoichiro Kino
Original assignee: Juridical Foundation The Chemo-Sero-Therapeutic Research Institute
Priority date: 2003-08-07
Filing date: 2004-08-02
Publication date: 2005-02-17
Also published as: JP2007068401A

Abstract

It is intended to provide process for producing a West Nile Virus (WNV) vaccine and an attenuated WNV vaccine produced by this method. A process which comprises culturing an established cell line (Vero cells) originating in the kidney of a WNV-inoculated vervet monkey at a high density, attenuating the WNV-infected cells or a liquid culture medium thereof thus obtained, and then highly purifying WNV by sucrose density gradient centrifugation; WNV obtained by this process; and an attenuated WNV vaccine comprising the WNV or an antigenic component thereof as the main component and being substantially free from any nucleic acids or proteins originating in a host.

Description

Description Technical field

The present invention relates to a West Nile virus (hereinafter sometimes referred to as “WNV”) vaccine. In detail, WNV-infected cells obtained by culturing cell lines (Vero cells) derived from the kidney of African green lantern seeded with WNV, or a culture solution thereof. A method for highly purifying WNV, which is obtained by the method. The present invention relates to WNV and a vaccine containing the WNV or an antigen component thereof.

^ Scenic technology

[0002] West Nile virus (WNV) is a single-stranded RNA virus having a diameter of about 50 nm and belonging to the Flaviviridae family, which uses birds such as crows, pigeons and sparrows as natural hosts. WNV also infects pests and humans through the transmission of mosquitoes, such as the Koga takae power, which breeds in paddy fields, the mosquito power that lives in urban areas, and the Aedes albopictus power. In the case of infectious diseases caused by the Japanese encephalitis virus (QEV) belonging to the same flaviviridae family, the mosquitoes that carry JEV are almost limited to the mosquito mosquitoes. Because they are transmitted by various mosquitoes, they are widespread, including in urban areas.

[0003] When WNV is transmitted to humans, 84% are subclinical or have mild symptoms like a summer cold and then heal. However, dengue-like symptoms such as rash, myalgia, hepatitis, encephalitis, etc. May cause severe symptoms. In the case of such severe symptoms, 93% of affected individuals are required to be hospitalized, with 31% receiving intensive care. Of the affected individuals, 33% will have sequelae of the nervous system and 9% will die. This tendency is higher for older people.

[0004] WNV was first isolated from a febrile patient in Uganda in 1937. WNV infection has spread in Egypt, Israel, France, South Africa, and elsewhere since the 1950s, and has spread to African countries, the Middle East, Asia, and Europe since 1994. Outbreaks in four states on the mainland in 1999 resulted in 62 cases of infection. Mosquito control by spraying insecticides, etc., has reduced the number of infected people, but it has not been able to control the activity of the virus. confirmed. The epidemic of the year ultimately resulted in 284 deaths out of 4,156 infected individuals. Vaccines for pests against WNV infection have already been developed, and none have been developed for humans, which has greatly contributed to the prevention of infectious diseases. At present, WNV infection is prevented indirectly by mosquito control, and the development of an effective vaccine is urgently needed.

[0005] To date, development of several different types of WNV vaccines has been attempted. One is that WNV is directly used as a vaccine material. A live vaccine attenuated by passage of WNV with mosquito cells was effective against geese (see Non-Patent Document 1). It has been reported that WNV obtained from human brain was inactivated with formalin and administered with oil adjuvant, and then WNV challenge test showed 52-80% efficacy (see Non-Patent Document 2). I have. However, vaccines that use infected mouse brain emulsion as a starting material may cause allergic central nervous system damage or contamination with pathogenic microorganisms due to contaminants derived from mouse brain. It has been pointed out that the use of animals is becoming difficult, and the production cost is high. In addition, the use of animals must be avoided as much as possible from the viewpoint of animal welfare. For live attenuated vaccines, a long-term vaccine effect can be expected, but there is concern that the pathogenicity may be restored by mutation.

[0006] In addition, flaviviridae viruses include a coat glycoprotein called E protein, which is a major infection-protecting antigen common to flaviviruses, and the potential of WNV vaccines using these proteins is also being studied. ing. For example, M. Malkinson and colleagues have confirmed that gooses have some WNV protective effects with a commercial vaccine derived from the Israeli Turkey Meningoencephalitis (TME) virus. In a WNV challenge test after administration of a formalin-inactivated TME virus together with an oil adjuvant, an effect of 39% to 72% was observed (see Non-Patent Document 2).

Tesh et al. Reported that prophylactic administration of Japanese encephalitis vaccine, wild-type St. Louis encephalitis virus, and yellow fever vaccine to hamsters reduced symptoms following West Nile vaccine infection (non-patented). Reference 3). N. Kanesa-Thasan et al. Reported that when humans were administered Japanese encephalitis vaccine and dengue vaccine, protective antibodies against them were elicited, but neutralizing antibodies against West Nile virus could not be induced. (See Non-Patent Document 4), and its effect on WNV infection is not always constant.

[0007] On the other hand, development of WNV vaccines using genetic recombination technology is also in progress. T.

Wang et al. Showed that WNV E protein expressed in Escherichia coli reacted with sera from WNV-infected individuals and that mouse antibodies obtained by immunizing this E protein protected WNV infection. It reports the potential of a recombinant component vaccine (see Non-Patent Document 5). JS Yang et al. Showed that immunization of mice with DNA encoding the WNV force psid protein gene induces strong cellular and humoral immunity (see Non-Patent Document 6). Davis et al. Have shown that mice and horses immunized with DNA encoding the WNV premembrane protein (preM) and E protein gene are protected from WNV infection (see Non-Patent Document 7). These results suggest the possibility of a DNA vaccine that directly introduces an expression vector into a living body.

[0008] Furthermore, the possibility of a chimeric vaccine that protects against multiple virus infections is also being investigated. G.

Pletnev and colleagues reported that a chimeric virus in which the gene regions encoding the dengue virus type 4 premembrane protein (preM) and envelope protein (E) were replaced with that of West Nile virus had low neuropathogenicity in animal experiments in WNV challenge tests. Also conferred a high protective effect on mice (see Non-Patent Document 8). In the United States, the development of a chimeric virus vaccine in which WNV outer coat glycoprotein (E protein) gene and its upstream PreM gene have been integrated into the virus genome of the yellow fever virus bacterium strain 17D strain ( Non-Patent Document 9). All of the above trials using genetic recombination technology are currently in the basic examination stage, and it has not been reported that they have been highly purified enough to be used as vaccines. There are many issues to be resolved, such as methods, safety, effectiveness, and costs.

[0009] Non-patent document 1: S. Lustig et al, Viral Immunology, 13, 4, p.401-10 (2000)

Non-Patent Document 2: M. Malkinson et al., New York Academy of Sciences, 951, p.255-61 (2001)

Non-Patent Document 3: R.B.Tesh et al., Infectious Disease, 8, 3, p.245-51 (2002)

Non-Patent Document 4: N. Kanesa-Thasan et al, American Journal of Tropical Medicine and

Hygiene, 66, 2, p.115-6 (2002) Non-patent document 5: T. Wang et al., Journal of Immunology, 167, 9, p. 5273-7 (2001) Non-patent document 6: JS Yang et al., Journal of Infectious Disease, 184, 7, p. 809-16 (2001)

Non-Patent Document 7: BS Davis et al, Journal of Virology, 75, 9, p. 4040-7 (2001) Non-Noon Document 8: AG Pletnev et al., Proceeding of National Academy of Sciences USA, 99, 5, p. .3036-41 (2002)

Disclosure of the invention

Problems to be solved by the invention

[0010] As described above, the potential of various vaccines against WNV infection is being studied. Until now, West Nile virus (WNV) infection has been effectively prevented, and safety, low cost, and stable supply have been achieved. There is no WNV vaccine that satisfies such conditions and a method for producing the same.

[0011] Accordingly, an object of the present invention is to provide a highly purified WNV, a method for producing a WNV actin using the purified WNV, and a WNV vaccine produced by the method.

Means for solving the problem

[0012] The inventors of the present application have conducted intensive studies in order to achieve the above object, and as a result, WNV was inoculated into Vero cells adhered to microcarriers and cultured at a high density, and this was inoculated with serum-free serum for virus culture. Medium We found that WNV was produced in extremely large amounts by culturing under appropriate culture conditions in VP-SFM. In addition, this WNV was completely inactivated by formalin, and the inactivated WNV was highly purified by sucrose density gradient centrifugation and, optionally, further column chromatography. The inactivated vaccine prepared using purified WNV was found to induce antibodies having high neutralizing ability against WNV and related Japanese encephalitis virus, and completed the present invention.

[0013] Accordingly, the present invention provides a method of inoculating WNV into Vero italocytes adhered to microcarriers and cultured at high density, and culturing them in a serum-free medium for virus culture VP-SFM under appropriate culture conditions. And a method for producing a purified and inactivated WNV, which comprises a step of inactivating the obtained virus solution with formalin, and purifying the virus solution by sucrose density gradient centrifugation, and optionally column chromatography. [0014] The present invention also includes purified and inactivated WNV highly purified by the above-mentioned production method and having a neutralizing activity against WNV and Japanese encephalitis virus.

The invention's effect

According to the present invention, a highly purified inactivated WNV and a method for producing the same are provided. By infecting Vero cells attached to microcarriers with WNV and culturing them at high density, it is possible to obtain a high-concentration virus solution. In the present invention, a serum-free, low-concentration protein medium is used to reduce the amount of impurity protein to be removed from the culture solution during virus growth. As a result, the purification step can be simplified, so that the production time can be reduced, the yield of WNV can be improved, and high-purity WNV can be obtained. This will enable low cost, mass production and stable supply of WNV vaccines. In addition, since a serum-free medium is used, the possibility of contamination with unknown pathogens derived from serum can be reduced.

[0016] Further, since the purified and inactivated WNV of the present invention has an ability to induce a neutralizing antibody against both WNV and Japanese encephalitis virus, it protects not only infections caused by WNV but also infections caused by Japanese encephalitis virus. It can be used as a vaccine with the efficacy of BEST MODE FOR CARRYING OUT THE INVENTION

[0017] The present invention provides (1) a step of inoculating WNV into Vero cells attached to a microcarrier and culturing it at a high density, (2) a step of inactivating WNV with formalin, and (3) a sucrose gradient. The present invention relates to a method for producing purified inactivated WNV, which further comprises a step of optionally purifying WNV by column chromatography, and a purified inactivated WNV obtained by the production method.

[0018] As the WNV used in the present invention, a clinical isolate can be used, but the type of the strain is not particularly limited. In a preferred embodiment of the present invention, the CDC (Centers for Disease Control) in the United States is provided through EglOl (hereinafter, also referred to as “EG strain”) and Koichi Morita donated by Mr. Koichi Morita of the Institute of Tropical Medicine, Nagasaki University. and Prevention), NY99-35262-ll (hereinafter sometimes referred to as “NY stock”) distributed by Gubler.

[0019] As the host cell used for WNV growth, any animal cell can be used as long as it is an animal cell in which WNV is grown. However, in the present invention, a host cell for obtaining a high-concentration virus solution is used. It is preferable to use a cell line that allows density culture. Such cell lines include Vero cells, CHO cells, MDCK cells and the like. Preferably, Vero cells are used.

[0020] As a medium used for growing Vero cells, a commercially available medium generally used for tissue culture, such as an Ml99 medium or an Eagle MEM medium, may be prepared according to the attached use method. Preferably, Dulbecco's MEM medium supplemented with amino acids, salts, antifungal agents, animal serum and the like is used. When culturing infected cells infected with WNV, it is preferable to use a serum-free, low-concentration protein medium that does not contain animal serum. As such a medium, VP-SFM (GIBC II), EX-CELL series (Nichirei), SFM-101 (Nissi), etc. are commercially available. Preferably, VP-SFM is used. The pH of the medium is adjusted to 78, preferably 7.4, suitable for growing animal cells.

[0021] As microcarriers for attaching Vero cells, various types of microbeads having different types such as size, shape, density, surface charge, and surface coat material are commercially available. And use it. For example, microbeads such as Cytodex I (Cytodex I, Amersham Bioscience), Biosilon (Nargennuk International), and CELLYARD (Pentax) are preferred. The amount of the cytodettas used is 1 to 10 g, preferably 3 to 5 g, per liter of the culture solution.

[0022] High-density culturing is achieved by culturing cells adhered to the microcarrier in a fermenter by the Fedo batch method. To attach Vero cells to cytodettas, put the cytodettas and the above-mentioned culture solution into a fermenter so that the concentration becomes 3 g / L, add 15 × 10 ⁸ cells to this, and add the culture solution at a speed of 20 40 rpm. Is cultured while rotating and agitating, and the Itoda cells are allowed to adhere to cytodettas and proliferate. The cultivation temperature and cultivation period are controlled by a combination of the number of cells to be attached, the culture scale, etc. The cultivation temperature is 32 to 38 ° C, and the cultivation period is 2 to 7 days.

[0023] WNV inoculation is performed after the growth of Vero cells reaches a stable period. The medium is aspirated off, and cells without serum or cells washed several times with phosphate buffered saline are inoculated with a viral load of 0.01 0.0001 (MOI). Culture of virus-infected cells Cultivation is performed at 32-38 ° C for 115 days using a VP-SFM medium supplemented with L-glutamic acid. Preferably, the cells are cultured at 37 ° C for 2-3 days. For the pH drop and nutrient deficiency that occur during the culture period, add 2 to 10%, preferably 6% glucose, 10 to 20%, preferably 15% sodium carbonate solution as needed by the Fued-batch method. PH is maintained and nutrients are replenished. After completion of the culture, the culture solution is subjected to rough centrifugation or membrane filtration to remove insolubles. The virus-containing solution obtained in this manner (hereinafter sometimes referred to as "no-nores") contains an extremely high concentration of virus.

[0024] In the virus inactivation treatment, the above-mentioned virus stock solution is concentrated with an ultrafiltration membrane having an exclusion limit molecular weight of 100,000 to 500,000, preferably 30.500,000, and a final concentration of 0.02 0.1% This is done by adding formalin to the soy sauce and leaving it at about 4 ° C for 11 months. The inactivation may be performed on the virus solution during or after purification. Completion of the inactivation is confirmed by inoculating a part of the inactivated virus stock solution into Vero cells, culturing the cells, and observing the presence or absence of virus growth.

[0025] The virus stock solution after the inactivation treatment is purified by sucrose density gradient centrifugation at 20,000-50,000 rpm for 2-24 hours. Preferably, sucrose density gradient centrifugation at 30,000 rpm for 16 hours is performed. The virus-containing fraction thus obtained is highly purified, and is desalted by ultrafiltration or dialysis, or diluted with an appropriate buffer, and then sterile-filtered with a membrane filter to obtain the vaccine starting material. Used as The vaccine raw materials include immunostimulants such as aluminum hydroxide, aluminum phosphate, mineral oil and non-mineral oil, stabilizers such as polysorbate 80, sugars such as amino acids and ratatose sucrose, and formalin, thimerosal, and 2-phenoki. Formulation is carried out by appropriately selecting and adding a preservative such as ciethanol, benzyl alcohol, benzethonium chloride, and salt of benzenikonium. When a sugar such as ratatose or sucrose having an effect as an excipient is added, it can be formulated as a lyophilized preparation.

[0026] The virus-containing fraction after the above sucrose density gradient centrifugation is further purified by column chromatography depending on the purpose of use. Examples of such column chromatography include cation chromatography, anion chromatography, and adsorption chromatography, which are generally used for purification of proteins and polypeptides. Preferably, WNV granules Cellulose sulfate gel which can purify the particles with high purity is used.

[0027] Efficacy as a vaccine can be determined by inoculating an animal with the vaccine and conducting an challenge test or by measuring the virus neutralizing ability of the serum of the vaccinated animal. More specifically, mice are immunized with a suitably diluted WNV vaccine solution to produce antibodies against WNV. The effectiveness of the antibody as a vaccine can be examined by measuring the neutralizing activity of this antibody by the 50% plaque reduction method. The method of immunization of a mouse may be a commonly used immunization method. For example, an immune serum is obtained by booster injection 13 to 13 weeks after the first vaccination with WNV vaccine, blood sampling 1 week after the booster vaccination, and serum separation.

[0028] As is clear from the results of Example 6 described below, the vaccine obtained according to the present invention not only induces a neutralizing antibody against W NV but also can induce a neutralizing antibody against Japanese encephalitis virus. Vaccine. According to a report by Ben-Nathan D et al. (J. Infect. Dis., 2003 Jul. 1; 188 (1), 5_12), it is clear that antibodies play an important role in the treatment and prevention of WNV infection. It has been suggested that the WNV vaccine of the present invention is extremely effective in preventing WNV infection. In addition, the vaccine of the present invention is expected to be a more effective vaccine when mixed with a vaccine derived from another W NV strain, for example, an EG strain. In addition, at least one vaccine selected from the group consisting of vaccines against other viruses (eg, Japanese encephalitis virus, hepatitis A virus, rabies virus) and bacteria (eg, pertussis and diphtheria 'tetanus bacteria). By combining them, they can be used as a combination vaccine.

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.

Example 1

[0029] (Culture of Vero cells)

Vero cells (CCL-81 strain) purchased from ATCC were used as cell lines for virus propagation. This was suspended in M199 medium containing 10% serum and cultured in a 5% CO 2 incubator at 37 ° C. for 5-7 days. The obtained cells were stored as a cell bank. Cells were grown up to the tank culture using Dulbecco's MEM medium containing 5% Under the same conditions. For tank cultivation, use a 5 liter fermentation tank. Add 5 x 10 ⁸ Vero cells and 15 g of cytodetase to 5 L of Dulbecco's MEM medium containing 5% serum, and add 3 to 7 at 37 ° C and 40 rpm. Cultured for days. This culture yields 1 × 10 ⁶ or more cells per mL.

Example 2

[0030] (Virus culture using VP-SFM)

The cells attached to the cytodettas were washed with serum-free Dulbecco's MEM to remove serum and other cell metabolites generated during cell culture, and then infected with VP-S FM medium to an infection efficiency of 0.01%. One liter of a WNV solution adjusted to be 1 was added, and the culture was continued. As an initial culture, the cells were cultured at 37 ° C. and a rotation speed of 20 rpm for 90 minutes. Then, after adding 4 L of VP-SFM, the rotation speed was increased to 40 rpm, and the culture was continued while maintaining the dissolved oxygen content of 2 ppm. The pH during the culture was maintained at 7.4 by adding a 15% sodium carbonate solution containing 6% gnorecose (Glu) as needed. Three days later, the culture solution was collected, and the virus content was measured by the plaque assay. Table 1 shows the relationship between the above pH maintenance and the presence or absence of glucose addition and the virus content in the medium. Numerical values indicate virus production (nLoglO).

[0031] [Table 1]

Example 3

[0032] (Inactivation of virus suspension and sucrose density gradient centrifugation)

The harvested virus solution was subjected to coarse centrifugation at 3,000 rpm for 5 minutes to remove cytodotes and cells, and then concentrated to 10 to 30 times using an ultrafiltration membrane (exclusion limit molecular weight: 300,000). SARTOCON SLICE DISPORSABLE (Fuji filter) was used for ultrafiltration and concentration. Formalin was added to the concentrated solution to a concentration of 0.08% and left at 4 ° C for 70-120 days to inactivate the virus. For surviving viruses in viral load measurement and cell inoculation tests After no longer being able to be confirmed, virus particles were purified by sucrose density gradient centrifugation. Rotor PR42 (Hitachi) was used, and 10-30 mL of 50% sucrose solution, 10-30 mL of 30% sucrose solution, and 10-30 mL of virus-inactivated sample solution were sequentially layered on a centrifuge tube, and then 30,000 rpm, 4 Centrifugation was performed at ° C for 16 hours. After centrifugation, sucrose concentration 40 at which OD280 absorbance is observed. Fractions lOOmL above / o were pooled. Then, after dialysis in 10 L of phosphate buffer at 4 ° C. for 1 day, the solution was sterile filtered to obtain a purified inactivated antigen. This was diluted with phosphate buffered saline so as to contain a protein amount of 30 zg / mL, and polysonolate 80 was added to this at a concentration of 0.01% to prepare a prototype vaccine.

[0033] A part of the purified inactivated antigen obtained by the sucrose density gradient ultracentrifugation was dialyzed against a buffer having a pH shown in Table 2 below. The same buffer was applied to a column filled with sulfated cell mouth fines. After washing with the same buffer, elution was carried out with the same buffer containing 0-3 M sodium chloride. The recovery was calculated from the absorbance of the eluted fraction and the liquid volume.

[0034] [Table 2]

[0035] As shown in Table 2, at pH 6.7, a white precipitate was generated in the sample, and purification by chromatography was not possible. Recovery was observed between pH 7.0 and 9.0, with 61% at pH 7.5 and 93% at pH 7.0. Analysis of the collected fraction at pH 7.0 showed a decrease in host-derived proteins and host-derived DNA as impurities, as shown in Tables 3 and 4 below, indicating that it was effective as an additional purification method. .

[0036] The purified inactivated antigen obtained by additional purification by column chromatography as described above was diluted with phosphate-buffered saline to contain a protein amount of 30 zg / mL, and polysorbate 80 was added thereto. The prototype vaccine was added to make it 01%.

Example 4

(Quantification of Nucleic Acid Derived from Host of Prototype Vaccine Obtained in Example 3)

All nucleic acid quantifications were performed according to the method attached to the kit. DNA extractor kit (Wako) was used for nucleic acid extraction from the specimen. The obtained nucleic acid is converted to Biodot SF ( Bio-Rad), followed by hybridization with Vero cell-derived DNA labeled with the Gene Image Labeling System (Amersham Pharmacia Biotech), followed by Gene Image Detection Kit (Amersham). (Pharmacia Biotech). The signal from chemiluminescence was quantified, and the amount of nucleic acid in the sample was determined using the standard sample as a standard. A known amount of Vero cell-derived nucleic acid was used as a standard sample. The detection limit was 6.72 pg / mL, and the relative reproducibility was 37.04%. As a result, as shown in Table 3, the content of DNA derived from the host was less than lng per inoculum (15 μg of protein) before purification by chromatography, and less than lOOpg after purification by chromatography. These are the direct recommendations of the WHO, below 10 ng per dose.

[Table 3]

Example 5

(Quantification of Host-Derived Protein in Prototype Vaccine Obtained in Example 3)

The protein obtained from the culture supernatant of Vero cells was immunized to rabbits and guinea pigs to prepare anti-Vero protein antibodies. The anti-Vero protein guinea pig antibody was adsorbed to a 96-well plate (Nunc) at 500 ng / well, and an appropriately diluted prototype vaccine solution was added thereto, followed by incubation at 37 ° C for 2 hours. Next, add anti-Vero protein / sagi antibody in 100 ng / well and incubate at 37 ° C for 2 hours. And developed the color. The protein amount was determined from a calibration curve created using known amounts of Vero protein. The quantitation limit was 2.284 ng / mL, and the relative reproducibility was 8.84%. As a result, as shown in Table 4, it was shown that the amount was less than 100 ng per inoculation amount (protein amount: 15 zg).

[0040] [Table 4] Amount of fe protein from host per inoculation (measured value, 95% confidence interval)

NY strain vaccine EG strain vaccine

Chromatography-Purification Host-derived protein amount 21 Before and after

(Ng per inoculation) (17.3-24.7) 71 27

(58.4-83.6) (22.2-31.8) Example 6

(Immunological Effect of Prototype Vaccine Obtained in Example 3)

The test vaccine solution is used as it is or diluted 4-fold with phosphate-buffered saline, and inoculated in the abdominal cavity of ddY mouse (female, 4-week-old) in 0.5 mL aliquots into 20 mice, and boosted with the same volume one week later After one week, blood was collected and serum separated. The neutralized antibody titer of the obtained serum was measured by a 50% plaque reduction method using Vero cells. As a control, Japanese encephalitis vaccine immune serum was used. Table 5 shows the results.

[Table 5]

Example 7

(Effect of Protecting Onset of Prototype Vaccine Obtained in Example 3)

ddY mice (SPF, 3w female) were purchased from SLC Japan and used. After acclimation for about one week, the vaccines shown in Table 6 below were diluted 4-fold with PBS, and 0.5 mL was intraperitoneally administered twice a week at an interval of one week. One week after the administration, the WNV-NY99 strain was intraperitoneally administered with the dose shown in Table 6 below using the JEV Beijing strain as an aggressive virus, followed by observation for two weeks.

[0044] Table 6 shows the results after two weeks. The number is the number of surviving mice / number of test mice. When WNV was used as the challenge virus, most of the non-immunized mouse groups developed and died. Under these circumstances, the JEV vaccine-administered group partially died of onset, which resulted in death, but there were some individuals who lost their energies, indicating that the protective effect of the JEV vaccine against WNV attack was partial. Indicated. In contrast, the WNV-NY strain vaccine showed perfect protection at all doses, with no transient deprivation. [0045] When JEV was used as the aggressive virus, half of the non-immunized mouse group developed part of the mice and died. Under these circumstances, the JEV vaccine-administered group showed almost perfect protection against onset. W NV—NY strain vaccine showed perfect protection. As described above, the JEV vaccine is effective against JEV, but its efficacy against WNV is partial and insufficient. In contrast, the WNV vaccine did not show any transient desensitization or onset to WNV or JEV, indicating a complete protective effect.

[Table 6]

Industrial applicability

[0047] The inactivated West Nile virus (WNV) obtained by the method of the present invention is highly purified and has a neutralizing activity against WNV and Japanese encephalitis virus. It can be used as an antigen material for vaccines against WNV and Japanese encephalitis virus infection by using it together with immunostimulants, dispersants, stabilizers, preservatives and the like.

[0048] Further, the WNV of the present invention is used as an antigen for producing monoclonal antibodies and polyclonal antibodies, or as a research material relating to the binding of an anti-WNV antibody to WNV, for example, a detection system material such as ELISA and WB. Available.

Claims

Claims [1] An inactivated West Nile virus isolated and purified after growth in a host cell, wherein the amount of host-derived nucleic acid is 8 pg 10 ng and the amount of host-derived protein is 10-100 ng per 10 zg of total protein. A purified and inactivated West Nile virus, which is purified and has an antigenicity capable of inducing a neutralizing antibody against West Nile virus and Japanese encephalitis virus. [2] Steps (1) and (4) below: (1) Inoculation of West Nile virus into cell lines adhered to microcarriers and high-density culture in serum-free medium; After filtration through an ultrafiltration membrane, a step of inactivating the West Nile virus with formalin, (3) a step of purifying the West Nile virus by sucrose density gradient centrifugation, (4) optionally, a step of purifying the West Nile virus obtained in the step (3). 2. The purified and inactivated West Nile virus according to claim 1, which is obtained by a step of further purifying the Nile virus-containing fraction by column chromatography. [3] Steps (1)-(4) below:

(1) Inoculating West Nile virus into Vero cells attached to cytodettas, and performing high-density culturing in VP-SFM while adding 2-10% gnorecose by the Fued-Batch method;

(2) a step of inactivating West Nile virus with a final concentration of 0.02 0.1% formalin after filtration through an ultrafiltration membrane having a molecular weight cutoff of 300,000 to 500,000;

(3) 20,000-50,000 rpm, 224 days Temple of ノ sucrose density

(4) Optionally, apply the West Nile virus-containing fraction obtained in step (3) to a column packed with cell mouth fine sulfate using a phosphate buffer solution with a pH of about 7.0 8.0, and add 0.3 M sodium chloride. Elution with the same buffer containing

2. The purified and inactivated West Nile virus according to claim 1, which is obtained by:

[4] A West Nile containing the purified inactivated West Nile virus according to any one of claims 1 to 3 as a main component, and at least one of an immunostimulant, a dispersant, a stabilizer and a preservative. Virus inactivated vaccine.

[5] The immunostimulants are aluminum hydroxide, aluminum phosphate, mineral oil and non-mine Ral oil, the stabilizer is selected from polysorbate 80, amino acids or sugars, and the preservative is from the group consisting of formalin, thimerosal, 2-phenoxyethanol, benzyl alcohol, benzethonium chloride and benzalkonium chloride. 5. The West Nile virus inactivated vaccine according to claim 4, wherein the vaccine is selected.

[6] The West Nile virus inactivated vaccine according to claim 5, which is a freeze-dried preparation.

[7] Steps (1)-(5) below:

(1) Inoculating the cell line attached to the microcarrier with West Nile virus and culturing it at high density in a serum-free medium;

(2) a step of inactivating West Nile virus with formalin after filtration with an ultrafiltration membrane,

(3) a step of purifying West Nile virus by sucrose density gradient centrifugation,

(4) optionally further purifying the West Nile virus containing fraction obtained in step (3) by column chromatography,

(5) A step of adding at least one of an immunostimulant, a dispersant, a stabilizer and a preservative to the purified inactivated West Nile virus obtained in step (3) or (4).

A method for producing a West Nile virus inactivated vaccine, comprising:

[8] Steps (1)-(5) below:

(1) Inoculating West Nile virus into Vero cells attached to cytodettas, and performing high-density culturing in VP-SFM while adding 110% gnorecose by the Fued-Batch method.

(2) a step of inactivating West Nile virus with a final concentration of 0.02-0.1% formalin after filtration through an ultrafiltration membrane having a molecular weight cutoff of 300,000 to 500,000;

(3) Purification of West Nile virus by sucrose density gradient centrifugation at 20,000-50,000 rpm for 2-24 hours,

(4) Optionally, apply the West Nile virus-containing fraction obtained in step (3) to a column packed with cell mouth fine sulfate using a phosphate buffer solution with a pH of about 7.0 8.0, and add 0.3 M sodium chloride. Eluting with the same buffer containing

(5) Step of adding at least one of an immunostimulant, a dispersant, a stabilizer and a preservative to the purified inactivated West Nile virus obtained in step (3) or (4)

A method for producing a West Nile virus inactivated vaccine, comprising: