JP2007068401A - West nile virus vaccine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a West Nile Virus (WNV) vaccine and to obtain an attenuated WNV vaccine produced by the method. <P>SOLUTION: The method for producing a WNV vaccine comprises culturing an established cell line (Vero cells) originating in the kidney of a WNV-inoculated African green monkey at a high density, attenuating the WNV-infected cells or a liquid culture medium thereof thus obtained and then highly purifying WNV by a sucrose density gradient centrifugation method. The WNV is obtained by the method. The attenuated WNV vaccine comprises the WNV or its antigenic component as a main component and is substantially free from any nucleic acids or proteins originating in a host. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

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

  West Nile virus (WNV) is a single-stranded RNA virus having a diameter of about 50 nm belonging to the Flaviviridae family, which uses birds such as crows, pigeons and sparrows as natural hosts. WNV also infects horses and humans through the transmission of mosquitoes such as the red mosquitoes that breed in paddy fields, the mosquitoes that live in urban areas, and the mosquitoes. In the case of infectious diseases caused by Japanese encephalitis virus (JEV) belonging to the same Flaviviridae family, mosquitoes that carry JEV are almost limited to Culex mosquitoes, so the epidemic range is localized, but WNV infection is Because it is transmitted by various mosquitoes, it spreads over a wide area including urban areas.

  When WNV infects humans, 84% are subclinical or healed after presenting mild symptoms like summer cold, but severe symptoms such as rash, muscle pain, dengue-like symptoms such as hepatitis, and encephalitis. May cause. In the case of such severe symptoms, 93% of the onset patients are forced to be hospitalized, and 31% of them are intensively treated. Of those who develop, 33% have sequelae of the nervous system and 9% die. This tendency is higher for older people.

  WNV was first isolated from a patient with fever in Uganda in 1937. WNV infection has been prevalent in Egypt, Israel, France, South Africa, etc. since the 1950s, and has spread to African countries, the Middle East, Asia and Europe since 1994. When the epidemic occurred in 1999 in four mainland states, 62 infected persons were confirmed. Although the number of infected people once decreased due to mosquito control by spraying insecticides, etc., the virus activity could not be suppressed, and when it became popular in 2002, virus infected people were confirmed from almost the whole mainland USA It was done. The epidemic of the year resulted in a major damage, with 284 deaths among the 4156 infected. Equine vaccines against WNV infection have already been developed and have helped to prevent the infection, but none have been developed for humans. At present, an indirect method using mosquito control is being used to prevent WNV infection, and development of an effective vaccine is desired immediately.

  So far, several different types of NWV vaccines have been developed. One is that WNV is directly used as a vaccine material, and that a live vaccine attenuated by passing WNV with mosquito cells was effective against geese (see Non-Patent Document 1) and It has been reported that after WNV obtained from the brain is inactivated with formalin and administered together with an oil adjuvant, the effectiveness of 52 to 80% was observed when a WNV challenge test was performed (see Non-Patent Document 2). However, for vaccines that use infected mouse brain emulsion as a starting material, there is a risk of contamination with allergic central neuropathy and pathogenic microorganisms caused by contaminants derived from mouse brain. However, the use of animals is required to be avoided as much as possible from the viewpoint of animal welfare. Live attenuated vaccines can be expected to have a long-term vaccine effect, but there is concern over the return of pathogenicity due to mutation.

  In addition, the Flaviviridae virus has an outer glycoprotein called E protein, which is a major protective antigen common to flaviviruses, and the possibility of a WNV vaccine using this is being studied. For example, M. Malkinson et al. Have confirmed a certain WNV protective effect in geese with a commercial vaccine derived from Israeli turkey meningoencephalitis (TME) virus. In the WNV challenge test after administration of TME virus inactivated with formalin together with an oil adjuvant, an effect of 39% to 72% was observed (see Non-Patent Document 2). Tesh et al. Reported that the symptoms after West Nile vaccine infection were reduced by prophylactic administration of Japanese encephalitis vaccine, wild-type St. Louis encephalitis virus and yellow fever vaccine to hamsters (Non-Patent Documents). 3). However, N.I. Kanesa-Thasan et al. Reported that when Japanese encephalitis vaccine and dengue fever vaccine were administered to humans, they could elicit protective antibodies against them but could not induce neutralizing antibodies against West Nile virus (Non-Patent Documents). 4), the effect on WNV infection is not always constant.

  On the other hand, development of a WNV vaccine using genetic recombination technology is also underway. T. T. et al. Wang et al. Revealed that the E protein of WNV expressed in Escherichia coli reacted with the serum of a WNV-infected person, and that a mouse antibody obtained by immunizing this E protein protected WNV infection. The possibility of a component vaccine is reported (refer nonpatent literature 5). In addition, J.H. S. Yang et al. Clarified that immunization of mice with DNA encoding the capsid protein gene of WNV induces strong cellular immunity and humoral immunity (see Non-Patent Document 6). S. Davis et al. Have shown that mice and horses immunized with DNA encoding the preM and E protein genes of WNV are protected from WNV infection (see Non-Patent Document 7). These results suggest the possibility of DNA vaccines that introduce expression vectors directly into the body.

  Furthermore, the possibility of a chimeric vaccine that protects against multiple viral infections is also being investigated. G. Pletnev et al. Have low neuropathogenicity in animal experiments and chimeric mice in which the gene region encoding the premembrane protein (preM) and envelope protein (E) of dengue virus type 4 is replaced with that of West Nile virus. It was clarified that a high protective effect was given to (see Non-Patent Document 8). Further, in the United States, development of a chimeric virus vaccine in which the outer coat glycoprotein (E protein) gene of WNV and the upstream PreM gene are incorporated into the 17D virus genome of yellow fever virus vaccine strain is in progress ( Non-patent document 9). All of the above attempts using genetic recombination technology are currently in the basic examination stage, and there has been no report that they have been highly purified to the extent that they can be used as vaccines. There are many issues that need to be solved, such as optimization methods, safety, effectiveness, and cost.

S. Lustig et al., Viral Immunol, 13,4,401-10 (2000) M. Malkinson et al., N Y Acad Sci, 951, 255-61 (2001) R. B. Tesh et al., Infect Dis, 8, 3, 245-51 (2002) N. Kanesa-Thasan et al., Am J Trop Med Hyg, 66, 2,115-6 (2002) T. Wang et al., J Immunol, 167,9,5273-7 (2001) J. S. Yang et al., J Infect Dis, 184,7,809-16 (2001) B. S. Davis et al., J Virol, 75, 9, 4040-7 (2001) A. G. Pletnev et al., Proc Natl Acad Sci U S A, 99,5,3036-41 (2002)

  As described above, the possibility of various vaccines against NWV infection has been studied. To date, West Nile virus (WNV) infection has been effectively prevented, and safety, low cost, stable supply, etc. have been achieved. There are no satisfactory WNV vaccines and methods for their production.

  Therefore, an object of the present invention is to provide a highly purified WNV vaccine production method and a WNV vaccine produced by the method.

  As a result of intensive studies to achieve the above object, the inventors of the present application inoculated WNV into Vero cells that were attached to microcarriers and cultured at high density, and this was inoculated with serum-free medium VP- for virus culture. It was found that WNV is produced in a very large amount by culturing under appropriate culture conditions in SFM. Furthermore, the WNV is completely inactivated by formalin, the inactivated WNV is highly purified by sucrose density gradient centrifugation, and the inactivated vaccine prepared using this purified antigen is WNV and The inventors have found that an antibody having high neutralizing ability against a related Japanese encephalitis virus is induced, and have completed the present invention.

  Therefore, the present invention was obtained by inoculating WNV on Vero cells that were attached to microcarriers and cultured at high density, and were cultured under appropriate culture conditions in a serum-free medium VP-SFM for virus culture. It includes a method for producing an inactivated WNV vaccine, which comprises a step of inactivating a virus solution with formalin and purifying the solution by a sucrose density gradient centrifugation.

  The present invention also includes an inactivated WNV vaccine highly purified by the above production method and having neutralizing activity against Japanese encephalitis virus.

  According to the method of the present invention, a highly purified inactivated WNV vaccine and a method for producing the same are provided. It is possible to obtain a virus solution having a high concentration by infecting Vero cells attached to a microcarrier with WNV and culturing the cells at a high density. In the present invention, a serum-free, low-concentration protein medium is used in order to reduce the amount of impure protein to be removed in the culture solution during virus growth. Thereby, since a refinement | purification step can be simplified, shortening of manufacturing time, the yield improvement of WNV, and acquisition of high purity WNV can be achieved. This enables low cost, mass production and stable supply of WNV vaccines. Moreover, since a serum-free medium is used, the possibility of contamination with unknown pathogens derived from serum can be reduced.

  Moreover, since the inactivated WNV vaccine of this invention has the neutralization activity with respect to both WNV and Japanese encephalitis virus, it has the effect which protects not only the infection by WNV but the infection by Japanese encephalitis virus.

  The method of the present invention comprises (1) a step of inoculating WNV on Vero cells attached to a microcarrier and culturing at a high density, (2) a step of inactivating WNV with formalin, and (3) a sucrose density gradient centrifugation. It is characterized by the manufacturing method of the inactivated WNV vaccine including the process of refine | purifying WNV, and the inactivated WNV vaccine obtained by the manufacturing method.

  As WNV used in the present invention, clinical isolates can be used, but the strain type is not particularly limited. In the present invention, Eg101 (hereinafter also referred to as EG strain) distributed by Mr. Koichi Morita of Nagasaki University Institute of Tropical Medicine and Mr. Koichi Morita of the US CDC (Centers for Disease Control and Prevention) NY99-35262-11 (hereinafter also referred to as NY strain) distributed by him is used.

  Any host cell can be used as a host cell for WNV propagation, as long as it is an animal cell in which WNV is proliferated. It is preferred to use cells. Examples of such cell lines include Vero cells, CHO cells, MDCK cells, and the like, preferably Vero cells.

  As a medium used for the growth of Vero cells, a commercially available medium generally used for tissue culture such as M199 medium and Eagle MEM medium may be prepared according to the attached method of use. Preferably, Dulbecco's MEM medium is added with amino acids, salts, antifungal / antibacterial agents, animal serum and the like. 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 (GIBUCO), EX-CELL series (Nichirei), SFM-101 (Nissui), and the like are commercially available. Preferably, VP-SFM is used. The pH of the medium is adjusted to 7-8, preferably 7.4, suitable for animal cell growth.

  As microcarriers for attaching Vero cells, various types of microbeads of different types such as size, shape, density, surface charge, and surface coat material are commercially available, and may be appropriately selected from these. Examples thereof include microbeads such as Cytodex, Biosilon (Narugenunk International), and CELLYARD (Pentax), and Cytodex I (Amersham Bioscience) is preferable. The amount of cytodex used is 1 to 10 g, preferably 3 to 5 g, per liter of the culture solution.

High-density culture is achieved by culturing cells attached to a microcarrier with a fermenter by a fed-batch method. To attach Vero cells to Cytodex, place Cytodex and the aforementioned culture solution in a fermenter so that the concentration is 3 g / L, add 1 to 5 × 10 ⁸ cells thereto, and add the culture solution at a rate of 20 to 40 rpm. Incubate with rotary agitation to allow cells to attach to Cytodex and grow. Although culture | cultivation temperature and culture | cultivation period are adjusted with the combination of the number of cells when making it adhere, a culture | cultivation scale, etc., they are culture | cultivation temperature 32 degreeC-38 degreeC, and culture | cultivation period 2-7 days.

Inoculation with WNV is performed after the growth of Vero cells has reached a stable phase. Cells are removed by aspiration and cells washed several times with medium without addition of serum or phosphate buffered saline are inoculated with a viral load of 0.01 to 0.0001 infection efficiency (MOI). The The culture of the virus-infected cells is performed at 32 ° C. to 38 ° C. for 1 to 5 days using a medium obtained by adding L-glutamic acid to a VP-SFM medium. Preferably, it is cultured at 37 ° C. for 2 to 3 days. 10% to 20%, preferably 15% sodium carbonate solution containing 2% to 10%, preferably 6% glucose by the fed-batch method is added as needed to reduce pH and nutrient deficiencies that occur during the culture period. PH maintenance and nutrient replenishment are performed. The culture solution after completion of the culture is subjected to coarse centrifugation or membrane filtration in order to remove insoluble matters. The virus-containing solution thus obtained (hereinafter sometimes referred to as virus stock solution) contains an extremely high amount of virus.
The virus inactivation treatment is performed by concentrating the virus stock solution with an ultrafiltration membrane having an exclusion limit molecular weight of 100,000 to 500,000, preferably 300 to 500,000, and then having a final concentration of 0.02 to 0.00. Formalin is added so as to be 10%, and the mixture is allowed to stand at around 4 ° C. for 1 to 3 months. The inactivation may be performed on the virus solution during or after purification. Completion of inactivation is confirmed by inoculating a part of the inactivated virus stock solution into Vero cells and culturing the cells to check for the presence of virus growth.

  The virus stock solution after the inactivation treatment is purified by sucrose density gradient centrifugation at 20000 to 50000 rpm for 2 to 24 hours. Preferably, sucrose density gradient centrifugation is performed at 30,000 rpm for 16 hours. The virus-containing fraction obtained in this way is highly purified, desugared by ultrafiltration or dialysis, etc., or diluted with an appropriate buffer, and then sterile filtered with a membrane filter. used. This vaccine material includes immunostimulators such as aluminum hydroxide, aluminum phosphate, mineral oil and non-mineral oil, polysorbate 80, stabilizers such as amino acids and sugars such as lactose and sucrose, and formalin, thimerosal, 2-phenoxyethanol, benzyl Formulation is carried out by appropriately selecting and adding preservatives such as alcohol, benzethonium chloride and benzalkonium chloride. Further, when a sugar such as lactose or sucrose having an effect as an excipient is added, it can be formulated as a lyophilized preparation.

The virus-containing fraction after the 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 commonly used for purification of proteins and polypeptides. Preferably, cellulose sulfate gel capable of purifying WNV particles with high purity is used.
Efficacy as a vaccine can be examined by vaccinating the animal and conducting an challenge test or measuring the virus neutralizing ability of the sera of the vaccinated animal. More specifically, a mouse is immunized with an appropriately diluted WNV vaccine solution to produce an antibody against WNV. By measuring the neutralizing activity of this antibody by the 50% plaque reduction method, the effectiveness as a vaccine can be examined. The immunization method for mice may be in accordance with the usual immunization method. For example, immune sera can be obtained by boosting 1 to 3 weeks after the initial vaccination with the WNV vaccine, voting one week after the boosting, and separating the serum.

The vaccine obtained by the present invention is a specific vaccine that not only induces neutralizing antibodies against WNV but also induces neutralizing antibodies against Japanese encephalitis virus, as is apparent from the results of Example 6 described later. is there. According to a report by Ben-Nathan D et al. (J Infect Dis 2003 Jul 1; 188 (1) 5-12), it has been clarified that antibodies play an important role in the treatment and prevention of WNV infection. It is suggested that the inventive WNV vaccine is very effective in preventing WNV infection. In addition, the vaccine of the present invention is expected to be a more effective vaccine by mixing with vaccines derived from other WNV strains, for example, EG strains. Furthermore, combining with 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, diphtheria, tetanus) Can be used as a combination vaccine.
Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples.

(Vero cell culture)
Vero cells (CCL-81 strain) purchased from ATCC were used as cell lines for propagating viruses. This was suspended in 10% bovine serum-containing M199 medium and cultured in a 5% carbon dioxide incubator at 37 ° C. for 5 to 7 days. The obtained cells were stored as a cell bank. Cell growth up to the tank culture was performed using Dulbecco's MEM medium containing 5% bovine serum under the same conditions as described above. A 5 liter fermentation tank was used for tank culture, 5 × 10 ⁸ Vero cells and 15 g cytodex were added to 5 L of Dulbecco's MEM medium containing 5% bovine serum, and cultured at 37 ° C. and 40 rpm for 3 to 7 days. This culture yields 1 × 10 ⁶ cells or more per mL.

(Virus culture using VP-SFM)
In order to remove cell metabolites generated during cell culture, bovine serum, etc., the cells adhering to Cytodex were washed with Dulbecco's MEM without serum, and then infected with 0.01% VP-SFM medium. 1 L of WNV solution adjusted so as to be added 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 VP-SFM4L, the number of revolutions was increased to 40 rpm, and the culture was continued while maintaining the dissolved oxygen amount of 2 ppm. The pH during the culture was maintained at 7.4 by adding 15% sodium carbonate solution containing 6% glucose (Glu) as needed. After 3 days, the culture broth was collected and the virus content was measured by 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. A numerical value shows a virus production amount (nLog10).

(Inactivation of virus suspension and sucrose density gradient centrifugation)
The harvested virus solution was subjected to rough centrifugation at 3000 rpm for 5 minutes to remove cytodex and cells, and then concentrated to 10 to 30 times with an ultrafiltration membrane (exclusion limit molecular weight of 300,000). For ultrafiltration concentration, SARTOCON SLICE DISPORABLE (Fuji Filter) was used. Formalin was added to the concentrate to 0.08%, and the virus was inactivated by leaving it at 4 ° C. for 70 to 120 days. Viral particles were purified by sucrose density gradient centrifugation after surviving virus could not be confirmed in the viral load measurement or cell inoculation test. Using a rotor PR42 (Hitachi), 10-30 mL of a 50% sucrose solution, 10-30 mL of a 30% sucrose solution, and 10-30 mL of a virus inactivated sample solution are sequentially layered on a centrifuge tube, and 30,000 rpm, 4 ° C., 16 Centrifugation was performed for a time. After completion of the centrifugation, 100 mL of fractions having a sucrose concentration of 40% or more where OD280 absorbance was observed were pooled. Subsequently, after dialyzing in 10 L of phosphate buffer at 4 ° C. for 1 day, the solution was aseptically filtered to obtain a purified inactivated antigen. This was diluted with phosphate buffered saline so as to contain 30 μg of protein per mL, and a solution obtained by adding polysorbate 80 to 0.01% was used as a prototype vaccine.

(Quantification of nucleic acid from prototype vaccine host)
All nucleic acid quantification was performed according to the method attached to the kit. A DNA extractor kit (Wako) was used for nucleic acid extraction from the specimen. The obtained nucleic acid was blotted onto a nylon membrane using Biodot SF (Bio-Rad), then hybridized using Vero cell-derived DNA labeled with Gene Image Labeling System (Amersham Pharmacia Biotech), and Gene Image Detection was performed using Detection Kit (Amersham Pharmacia Biotech). The chemiluminescence signal was digitized, and the amount of nucleic acid in the sample was determined using the standard sample as the standard. A known amount of nucleic acid derived from Vero cells was used as a standard sample. As a result, as shown in Table 2, the host-derived DNA content was 1 ng or less per 1 inoculum (protein amount 15 μg). This is a value less than 10 ng per inoculum recommended by WHO.

(Quantification of host-derived protein in prototype vaccine)
Rabbits and guinea pigs were immunized with proteins obtained from Vero cell culture supernatant to produce anti-Vero protein antibodies. Anti-Vero protein guinea pig antibody was adsorbed to a 96-well plate (Nunc) at 500 ng per well, and an appropriately diluted prototype vaccine solution was added thereto, followed by incubation at 37 ° C. for 2 hours. Next, 100 ng of anti-Vero protein rabbit antibody was added per well and incubated at 37 ° C. for 2 hours. Then, peroxidase-labeled anti-rabbit IgG (ZYMED) and a chromogenic substrate (orthophenylenediamine; OPD) were added to cause color development. . The amount of protein was determined from a calibration curve prepared using a known amount of Vero protein. As a result, as shown in Table 3, it was shown that it was 100 ng or less per inoculum (protein amount 15 μg).

(Immune effect of prototype vaccine)
Prototype vaccine solution as it is or diluted 4-fold with phosphate buffered saline, inoculate 20 ddY mice (female, 4 weeks old) into the abdominal cavity of 20 mice, and boost the same amount one week later. After one week, blood was collected and serum was separated. About the obtained serum, the neutralizing antibody titer by the 50% plaque reduction method using Vero cells was measured. As a control, Japanese encephalitis vaccine immune serum was used. The results are shown in Table 4.

Inactivated West Nile virus (WNV) obtained by the method of the present invention is highly purified and has neutralizing activity against WNV and Japanese encephalitis virus, either alone or with various stabilizers, By using together with additives such as protective agents and preservatives, it can be used as an antigen material for vaccines against WNV and Japanese encephalitis virus infections.
In addition, the WNV of the present invention can be used as an antigen in producing a monoclonal antibody / polyclonal antibody, or as a research material related to the binding of an anti-WNV antibody to WNV, for example, a detection system material such as EISA or WB.

Claims (8)

An isolated West Nile virus having a host-derived nucleic acid amount of 10 pg to 10 ng and a host-derived protein amount of 10 to 100 ng per 10 μg of total protein, and neutralizing antibodies against West Nile virus and Japanese encephalitis virus The West Nile virus characterized by having an inducible antigenicity. The West Nile virus according to claim 1, which is obtained by a production method including the following steps (1) to (3).
(1) A step of inoculating West Nile virus on a cell line attached to a microcarrier and culturing the cell 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) Step of purifying West Nile virus by sucrose density gradient centrifugation The West Nile virus according to claim 1, which is obtained by a production method including the following steps (1) to (3).
(1) Inoculating West Nile virus on Vero cells attached to Cytodex I and culturing at high density in VP-SFM while adding 2 to 10% glucose by the fed-batch method,
(2) a step of inactivating West Nile virus with a final concentration of 0.02 to 0.10% formalin after filtration through an ultrafiltration membrane with a molecular weight cut off of 300 to 500,000,
(3) A step of purifying West Nile virus by sucrose density gradient centrifugation at 20000 to 50000 rpm for 2 to 24 hours. The West Nile virus according to any one of claims 1 to 3 is contained as a main component, and is obtained by adding at least one of an immunostimulant, a dispersant, a stabilizer and a preservative. Nile virus inactivated vaccine. The immunostimulant is selected from the group consisting of aluminum hydroxide, aluminum phosphate, mineral oil and non-mineral oil, the stabilizer is selected from polysorbate 80, amino acid or sugar, and the preservative is formalin, thimerosal, 2-phenoxyethanol, benzyl alcohol. The West Nile virus inactivated vaccine according to claim 4, wherein the vaccine is selected from the group consisting of benzethonium chloride and benzalkonium chloride. The West Nile virus inactivated vaccine according to claim 5, which is a freeze-dried preparation. The manufacturing method of the West Nile virus inactivation vaccine characterized by including the process of following (1)-(3).
(1) A step of inoculating West Nile virus on a cell line attached to a microcarrier and culturing the cell 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) Step of purifying West Nile virus by sucrose density gradient centrifugation The manufacturing method of the West Nile virus inactivation vaccine characterized by including the process of following (1)-(3).
(1) A step of inoculating West Nile virus on Vero cells attached to Cytodex I and culturing at a high density in VP-SFM while adding 1 to 10% glucose by a fed-batch method,
(2) a step of inactivating West Nile virus with a final concentration of 0.02 to 0.10% formalin after filtration through an ultrafiltration membrane with a molecular weight cut off of 300 to 500,000,
(3) A step of purifying West Nile virus by sucrose density gradient centrifugation at 20000 to 50000 rpm for 2 to 24 hours.