Virus production method

Abstract

The present invention relates to: a virus production method comprising a step for mixing a virus-infected cell and a non-infected cell and co-culturing the same to proliferate a virus; a virus strain for vaccination characterized by having 0.6 times or more of the expression level of the membrane surface glycoprotein gp51 as compared with the expression level of the capsid protein p24; and a vaccine composition characterized by comprising the vaccine virus strain that has been inactivated.

Description

Virus production method

[0001] The present invention relates to a technology for producing virus-related vaccines, and more specifically to a method for producing viruses belonging to the Retroviridae, Flaviviridae, Picornaviridae, Reoviridae, Coronaviridae, Paramyxoviridae, Rhabdoviridae, Flaviviridae, Circoviridae, and Herpesviridae families, as well as virus strains and vaccine compositions for vaccine production.

[0002] Viruses are particles with a basic structure consisting of viral nucleic acid and a protein shell called a capsid that encases it. Viral nucleic acid is usually either DNA or RNA, and viruses are broadly classified as DNA viruses or RNA viruses depending on the type of nucleic acid they possess.

[0003] Viruses cannot replicate on their own; they can only replicate using mechanisms inherent in prokaryotic or eukaryotic cells. Therefore, in order for viruses to replicate, they must "infect" a cell. The first step in viral infection is adsorption via receptors expressed on the cell surface. Once adsorbed to the cell surface receptor, virus particles then enter the cell, where actual replication occurs. Once inside the cell, the virus undergoes initial disassembly of the capsid, releasing viral nucleic acid from its interior (uncoating). Protein synthesis from the viral nucleic acid and the genome replication process then begin. For example, in retroviruses, viral nucleic acid is enveloped in a complex of viral and cellular proteins, such as reverse transcriptase and integrase, and is "reverse transcribed" from RNA to DNA by the action of reverse transcriptase. The reverse-transcribed viral nucleic acid then migrates to the nucleus and is integrated into the cellular DNA by the action of viral integrase. Similar to cellular genes, new viral particles are then formed through the processes of transcription, translation, and post-translational modification, and these viral particles are released outside the cell, thereby replicating the virus.

[0004] Vaccines are pharmaceuticals that contain attenuated or inactivated pathogens such as viruses and their toxins, and are administered to hosts to induce specific immunity against the pathogens and prevent infectious diseases. Commonly used vaccines are broadly divided into live vaccines and inactivated vaccines. Live vaccines are used for immunization by repeatedly passaged pathogens such as viruses in other hosts (animals) or tissue culture (cell) / medium, resulting in attenuated strains (attenuated strains) that are less pathogenic to the original host. On the other hand, inactivated vaccines are used for immunization by culturing the pathogen in a host (animal) or tissue culture / medium, inactivating it, purifying it, and using it as an antigen.

[0005] The amount of virus used as the vaccine raw material is important in the development of these vaccines. In the production of viral antigens, it is ideal to establish cells that divide and proliferate while persistently infected with the virus and continue to produce high titers of viral particles. However, cells that produce large amounts of viral particles have their original functions restricted by viral replication, resulting in a decrease in cell proliferation capacity. On the other hand, cells with high proliferation capacity often have suppressed virus production, and cell proliferation and viral particle production are incompatible. For example, in the production of bovine leukemia virus (BLV) antigens, methods for producing the virus from virus-infected host cells are known (Patent Documents 1 to 3). However, even when using these host cells, it is difficult to ensure sufficient BLV production, which is one of the challenges in BLV vaccine production.

[0006] JP-A No. 63-44531 JP-A No. 1-125329 JP-A No. 1-123152

[0007] Therefore, an object of the present invention is to provide a method for efficiently producing viruses that have been difficult to produce industrially, and to provide a virus strain for vaccine use that is easier to use as a vaccine than naturally occurring viruses, and a vaccine composition using the virus strain for vaccine use.

[0008] In order to solve the above problems, the present inventors conducted extensive research and found that viruses that have been difficult to produce industrially can be efficiently produced by mixing virus-infected cells with non-infected cells and co-culturing them to grow the virus, and that the virus strains produced by this method have an expression level of the membrane surface glycoprotein gp51, which is useful as a vaccine antigen, at a ratio of 0.6 times or more to the expression level of the capsid protein p24, thereby completing the present invention.

[0009] That is, the gist of the present invention is: [1] a method for producing a virus, comprising a step of mixing virus-infected cells and non-infected cells and co-culturing them to grow the virus; [2] the method for producing a virus according to [1] above, comprising a step of mixing and co-culturing the virus-infected cells obtained after a passage period by co-cultivation with non-infected cells; [3] the method for producing a virus according to [2] above, which repeats the step of mixing and co-culturing the virus-infected cells obtained after a passage period by co-cultivation with non-infected cells; [4] the method for producing a virus according to any of [1] to [3] above, wherein the serum concentration in the medium used for the co-cultivation is less than 10% by volume; [5] a method for producing a virus, comprising a step of growing the virus by continuing to passage the virus-infected cells obtained by the method for producing a virus according to any of [1] to [4] above, without adding new non-infected cells; [6] the method for producing a virus according to [5] above, wherein the number of passages continued without adding new non-infected cells is two or more; [7] The method for producing a virus according to any one of [1] to [6] above, wherein the uninfected cells are one or more types of cells selected from an established cell line derived from sheep kidney, an established cell line derived from pig kidney, an established cell line derived from bovine kidney, an established cell line derived from bovine testis, and an established cell line derived from pig oviduct epithelium; [8] The method for producing a virus according to any one of [1] to [7] above, wherein the virus is one or more types of viruses selected from the Retroviridae family, Flaviviridae family, Picornaviridae family, Reoviridae family, Coronaviridae family, Paramyxoviridae family, Rhabdoviridae family, Flaviviridae family, Circoviridae family, and Herpesviridae family; [9] A virus strain for use in a vaccine, characterized in that the expression level of membrane surface glycoprotein gp51 is 0.6 times or more compared to the expression level of capsid protein p24; and

[10] A vaccine composition, characterized in that it contains the inactivated virus strain for use in a vaccine according to [9] above.

[0010] The virus production method of the present invention allows efficient production of viruses belonging to the Retroviridae, Flaviviridae, Picornaviridae, Reoviridae, Coronaviridae, Paramyxoviridae, Rhabdoviridae, Flaviviridae, Circoviridae, and Herpesviridae families. Furthermore, the virus strains obtained by the virus production method of the present invention express a useful membrane surface glycoprotein gp51 at a significantly higher level than virus strains obtained from infected primary cells that have not been co-cultured and virus strains produced by conventional methods. Therefore, these virus strains are suitable for vaccines, and vaccine compositions with excellent vaccine efficacy can be produced by using these virus strains.

[0011] 1 is a diagram showing immunoblotting analysis images of virus particle-forming proteins produced in the culture supernatant of various BLV-infected cells, as performed in Example 1.

[0033] FIG. 2 is a table showing the doubling time of BLV-infected cells with various culture medium compositions, as performed in Example 2.

[0034] FIG. 3 is a diagram showing immunoblotting analysis images of virus particle-forming proteins produced in the culture supernatant of BLV-infected cells with various culture medium compositions, as performed in Example 2.

[0035] FIG. 4 is a graph showing the results of measuring the intensity of the protein bands shown in FIG. 3 using a densitometer.

[0036] FIG. 5 is a schematic diagram showing an overview of cell culture performed in Example 3.

[0037] FIG. 6 is a graph showing the results of examining the Env concentration in the supernatant of cultures with various combinations of culture medium compositions and mixing ratios of BLV-infected cells and non-infected cells, as performed in Example 3.

[0038] FIG. 7 is a graph showing the results of examining the Env production amount contained in the culture supernatant prepared in the test described in Example 4, as performed by antigen capture ELISA.

[0039] FIG. 8 is a diagram showing immunoblotting analysis images of virus particle-forming proteins produced in the culture supernatant of various BLV-infected cells, as performed in Example 5.

[0012] In the present invention, the term "virus" refers to an RNA virus or a DNA virus, preferably a virus of the Retroviridae, Flaviviridae, Picornaviridae, Reoviridae, Coronaviridae, Paramyxoviridae, Rhabdoviridae, Flaviviridae, Circoviridae, or Herpesviridae families. Examples of the virus include, but are not limited to, bovine infectious lymphoma virus, bovine viral diarrhea virus, foot-and-mouth disease virus, bovine rotavirus, bovine coronavirus, rinderpest virus, vesicular stomatitis virus, bovine ephemeral fever virus, feline leukemia virus, swine fever virus, porcine circovirus, and porcine cytomegalovirus. In the present invention, the virus may be one or more types.

[0013] In the present invention, the term "virus-infected cells" refers to cells infected with the virus, and examples of the cells include one or more types of cells selected from a mutton kidney-derived cell line, a porcine kidney-derived cell line, a bovine kidney-derived cell line, a bovine testis-derived cell line, and a porcine oviduct epithelium-derived cell line.

[0014] Methods for infecting the cells with the virus include, but are not limited to, "adsorption," in which a virus solution is directly exposed to the surface of a monolayer cell for a certain period of time, and "centrifugal inoculation," in which a virus solution is layered on a monolayer cell, or cells are suspended in the virus solution and then centrifuged for a certain period of time. Whether the cells are virus-infected can be confirmed by known methods such as syncytium formation by co-culture with feline-derived cells (CC81 cells) or the like, immunocytochemical detection of viral proteins expressed in the cells, or PCR, which amplifies proviral DNA using cellular DNA as a template.

[0015] In the present invention, the term "uninfected cells" refers to cells that are not infected with the virus, and examples of the cells include one or more types of cells selected from a mutton kidney-derived cell line, a porcine kidney-derived cell line, a bovine kidney-derived cell line, a bovine testis-derived cell line, and a porcine oviduct epithelium-derived cell line.

[0016] (Virus Production Method) A first embodiment of the virus production method according to the present invention comprises the step of mixing virus-infected cells with uninfected cells and co-culturing them to grow the virus.

[0017] In the present invention, "co-culture" refers to culturing the virus-infected cells in a mixed state with non-infected cells. When virus-infected cells and non-infected cells are mixed and co-cultured in this manner, the virus produced by the virus-infected cells is transferred to the non-infected cells, increasing the number of cells capable of viral replication and thereby increasing the amount of virus produced. In particular, even viruses that have traditionally been difficult to produce industrially can be efficiently mass-produced by using the virus production method of the present invention. Furthermore, the virus-infected cells obtained by co-culture of virus-infected cells and non-infected cells in the present invention have a good balance between virus production ability and cell proliferation ability and are cells that can maintain virus production ability even through subculture (persistently infected cells), providing superior effects compared to conventional methods.

[0018] In the first embodiment, the mixing ratio of the virus-infected cells to the non-infected cells is, for example, from the viewpoint of efficiently transmitting the virus from the virus-infected cells to the non-infected cells, in terms of the cell number ratio (virus-infected cells:non-infected cells), which is 99:1 to 1:99, preferably 1:1 to 1:99, and more preferably 1:9 to 3:7.

[0019] The culture conditions for the co-culture may be any known culture conditions appropriate for the type of cells used. Examples of culture media include, but are not limited to, Eagle's minimum essential medium (E-MEM), Dulbecco modified Eagle's medium (Dulbecco's MEM), and Roswell Park Memorial Institute 1640 medium. The medium may contain serum, bactopeptone, lactalbumin hydrolysate, glucose, sodium pyruvate, L-glutamine, gentamicin, HEPES buffer, sodium bicarbonate, and the like. Examples of serum include fetal bovine serum (FBS), newborn calf serum, bovine serum, human serum, horse serum, goat serum, rabbit serum, pig serum, chicken serum, sheep serum, guinea pig serum, mouse serum, rat serum, goat serum, and dog serum. The culture temperature may be adjusted to a range of 35 to 40°C, preferably 36 to 38°C from the viewpoint of optimal cell growth. The pH of the medium is not particularly limited, but may be adjusted to a range of 6.5 to 8.0, and from the viewpoint of better cell growth, may be adjusted to a range of 6.9 to 7.6. For the co-culture, a known culture vessel or culture device may be used, but is not particularly limited.

[0020] Furthermore, since the virus production method of the present invention allows efficient virus production without weakening virus-infected cells, it can be carried out even when the serum concentration in the medium used for co-culture is less than 10% by volume. A serum concentration of less than 10% by volume has the advantage of reducing production costs and being economical. The serum concentration may be less than 5% by volume or may be 2-3% by volume. When the serum concentration is less than 10% by volume, other nutrients such as bactopeptone, lactalbumin hydrolysate, and glucose may be added to the medium to ensure efficient cell growth. The content of the nutrients in the medium used for co-culture may be adjusted appropriately depending on the type of cells to be cultured; for example, the total amount of nutrients may be 0.5-5% by weight.

[0021] In the first embodiment, the method may include a step of co-culturing the virus-infected cells obtained through the co-culture passage period with uninfected cells. By including such a step, a culture containing many cells in which virus production has reached its peak can be achieved, thereby improving the amount of virus produced.

[0022] In the first embodiment, the term "passaging by co-culture" refers to collecting a portion of the virus-infected cells obtained by the co-culture, transferring them to another culture vessel or culture device, and co-culturing them in a new medium containing non-infected cells. The term "passaging period" refers to the period during which the passaging is carried out.

[0023] In the first embodiment, the step of co-culturing the virus-infected cells obtained through the co-culture passage period with uninfected cells may be repeated. By repeating the co-culturing step in this manner, the amount of virus produced can be improved.

[0024] In the first embodiment, the number of passages is not particularly limited, but is preferably less than 30 times, and more preferably 1 to 10 times.

[0025] The completion of one passage can be confirmed by the presence of numerous nodular cell masses that have uniformly filled the bottom of the culture dish, or by the presence of four or more nodules per field of view observed under an inverted microscope with a 4x objective and a 10x eyepiece.

[0026] Furthermore, a second embodiment of the present invention includes a step of propagating the virus by continuing to passage the virus-infected cells obtained by the virus production method of the first embodiment without adding any new non-infected cells. The second embodiment was developed based on the discovery that, for unknown reasons, the virus-infected cells obtained by mixing and co-culturing virus-infected cells with non-infected cells in the first embodiment can be continuously cultured in a state of high virus production even when passaging is continued without adding any new non-infected cells. By continuing to passage the cells without adding non-infected cells in this way, production costs can be reduced with a simpler method without requiring much effort, and this has the advantage of being highly economical.

[0027] In the second embodiment, the passaging without adding non-infected cells refers to collecting a portion of the virus-infected cells obtained by the co-culture, transferring them to another culture vessel or culture device, and culturing them in a new medium that does not contain non-infected cells.

[0028] As in the first embodiment, the culture conditions in the second embodiment may be any known culture conditions suitable for the type of cells used.

[0029] In the second embodiment, the number of passages may be two or more, and may be three to ten, from the viewpoint of efficient virus production.

[0030] The completion of one passage can be confirmed by the presence of numerous nodular cell masses that have uniformly filled the bottom of the culture dish, or by the presence of four or more nodules per field of view observed under an inverted microscope with a 4x objective and a 10x eyepiece.

[0031] In conventional methods, a cell line that continues to stably produce virus particles at a low level is established, and virus particles are collected from the culture supernatant. In contrast, in the first and second embodiments of the present invention, although the amount of virus produced in virus-infected cells tends to decrease with continued passage, by collecting virus particles before the amount of virus production decreases, more virus particles can be collected than in the conventional method of collecting virus particles from the culture supernatant of virus-infected cells, thereby enabling efficient production of virus particles.

[0032] Methods for recovering a virus from cultured virus-infected cells or the like include, but are not limited to, concentrating virus particles released into the culture supernatant based on their physical or immunological properties after recovering the supernatant, disrupting the cells using a relatively slow method such as freezing and thawing, and concentrating the particles accumulated within the cells using a method similar to that described above, or using a combination of these methods.

[0033] (Virus strain for vaccine) The virus strain obtained by the vaccine production method according to the present invention is characterized by being rich in the membrane surface glycoprotein gp51 compared to virus strains obtained from infected primary cells that have not been co-cultured and virus strains produced by conventional methods.

[0034] In viruses, the envelope (Env) is a membranous structure found in some virus particles, such as retroviruses. In these viruses, the envelope is located at the outermost part of the virus particle and covers the viral genome and the protein shell / layer called the capsid, which are the basic structure of the virus. For example, BLV Env is synthesized in the rough endoplasmic reticulum as a type I single-pass transmembrane protein. After synthesis in the host cell, it is transported to the Golgi apparatus and glycosylated. During this process, it is degraded by host proteases into a surface glycoprotein (gp51), and the degraded gp51 is incorporated into the virus particle and functions.

[0035] gp51 is a protein with a molecular weight of approximately 51,000 daltons that plays a crucial role in viral infection and is known to contain many epitopes that are targets of host immunity. An epitope, also known as an antigenic determinant, is a part of an antigen that is recognized by the immune system, particularly antibodies, B cells, and T cells. When antibodies bind to pathogenic microorganisms or viruses, they do not recognize the entire antigen, but rather recognize and bind to only a relatively small portion of the antigen's surface. This antibody-binding site is called the antigen's epitope. An epitope is the smallest unit of antigenicity. Therefore, the virus strains rich in gp51 obtained by the present invention are expected to be highly effective as vaccine antigens.

[0036] Specifically, the virus strain for vaccine use according to the present invention is characterized in that the expression level of the membrane surface glycoprotein gp51 is 0.6 times or more higher than the expression level of the capsid protein p24.

[0037] Capsid protein p24 is a known protein with a molecular weight of approximately 24,000 daltons, which includes the viral genome, enzymes, etc. and exhibits antigenicity recognized by the host immune system. In the present invention, when the expression level of gp51 relative to the expression level of p24 is 0.6-fold or more, it can be said that gp51 is abundant compared to the expression levels of gp51 relative to the expression level of p24 (0.46-0.55-fold) in virus strains obtained from infected primary cells that have not been co-cultured and virus strains produced by conventional methods. The expression level of gp51 relative to the expression level of p24 is preferably 1-fold or more. There is no particular upper limit to the expression level of gp51 relative to the expression level of p24, as long as it does not adversely affect the vaccine's applicability.

[0038] The expression levels of p24 and gp51 can be determined by measuring the density of each band confirmed by electrophoresis using a densitometer or the like.

[0039] (Vaccine Composition) The vaccine composition according to the present invention contains the inactivated virus strain for vaccine use.

[0040] The inactivation method may be any known method for inactivating viruses, and examples thereof include treatment with chemicals such as formalin and β-propiolactone, heat treatment, and ultraviolet treatment.

[0041] The vaccine composition of the present invention may contain pharmacologically acceptable components in addition to the inactivated virus strain, such as, but not limited to, sterile water, physiological saline, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, fragrance, excipient, vehicle, preservative, binder, diluent, isotonicity agent, soothing agent, bulking agent, disintegrant, buffer, coating agent, lubricant, coloring agent, sweetener, thickener, flavoring agent, solubilizing agent, or other additive.

[0042] Furthermore, to enhance the immune response, an adjuvant can be further added to the vaccine composition of the present invention. Examples of adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, oil adjuvant, aluminum hydroxide, aluminum phosphate, saponin, vitamin E, etc., but there is no particular limitation as long as the desired vaccine effect is exerted.

[0043] The animals to which the vaccine composition of the present invention is administered are not particularly limited as long as they are animals (non-human animals) that can be infected with viruses, and may be livestock, companion animals (pets), laboratory animals, or animals for other purposes. Examples of animals include, but are not limited to, cows, pigs, horses, sheep, goats, chickens, ducks, geese, wild goats, quails, pheasants, pigeons, turkeys, guinea fowls, dogs, and cats.

[0044] The vaccine composition of the present invention can be administered to a living body by a known administration route, including subcutaneous, intradermal, intravenous, intramuscular, oral, and intranasal, to confer immunity to the living body. When the vaccine composition of the present invention is a combination drug, each formulation may be administered simultaneously or at a time lag within a range that does not diminish the effect of the combination.

[0045] The dose of the vaccine composition of the present invention may be any amount that is capable of inducing an immune response in the body, and may vary depending on the age or weight of the animal, the type of animal, the method and route of administration, etc. The single dose of the inactivated virus strain, which is the active ingredient, is typically 0.1 μg to 500 μg, preferably 1 μg to 100 μg, and more preferably 10 μg to 50 μg. Administration may be carried out multiple times, in which case the interval between doses is typically 1 to 2 weeks.

[0046] (Cells Used) - Uninfected Cells: A fetal lamb kidney-derived cell line (FLK cells) was transformed with SV40 T antigen using a commercially available recombinant lentiviral vector according to standard procedures. (Hereinafter, also referred to as FLKBL.LS40 cells.) - BLV-Infected Primary Cells: The uninfected cells were infected with bovine infectious lymphoma virus (BLV), a member of the Retroviridae family, according to standard procedures. (Hereinafter, also referred to as virus-primary-infected FLKBL.LS40 cells.) - BLV-Infected Cells: Primary virus-infected FLKBL.LS40 cells were cultured alone to grow, and cells capable of stably producing BLV were used as BLV-infected cells. (Hereinafter, also referred to as 9054#2 cells) FLK-BLV cells: Virus-infected cells established by American researchers in the 1970s, used as a control (Van der Maaten MJ & Miller JM (1976). Replication of bovine leukemia virus in monolayer cell cultures. Bibl. Haematol. 43 360-362.) (Culture media used): E-MEM containing 10% by volume FBS (E-MEM-10) (Anti-BLV Env gp51 monoclonal antibody): An anti-BLV Env gp51 monoclonal antibody was produced based on the method described by Koehler et al., "Continuous cultures of fused cells secreting antibody of predefined specificity," Nature. 1975 Aug 7;256(5517):495-7. In this example, hybridomas were produced using polyethylene glycol instead of Sendai virus.

[0047] Example 1: Production of Env-rich viral particles by co-culturing BLV-infected and non-infected cells When BLV-infected primary cells are cultured alone to become BLV-infected cells, the amount of viral particles produced gradually increases, but at the same time, the apparent proliferation ability of the cells decreases, and the cost of producing viral particles increases significantly.

[0048] Therefore, in order to renew virus-infected cells, 3 × 10５ BLV-infected cells and 7 × 10 ５ The uninfected cells were mixed, suspended in 10 mL of culture medium, and seeded in a 100 mm diameter Petri dish and co-cultured for 1 week at 37°C in the presence of 5% by volume carbon dioxide. The culture supernatant was then recovered and clarified by low-speed centrifugation (310 × g, 8 minutes), and then filtered through a membrane filter with a pore size of 0.45 mm to obtain a virus-containing solution.

[0049] Ten milliliters of the virus-containing solution was layered on 2 milliliters of 20% sucrose / Tris-buffered saline in a centrifuge tube, loaded into a swinging bucket (SW 40 Ti, Beckman-Coulter), and centrifuged at 151,000 × g and 4°C for 45 minutes using a preparative ultracentrifuge (XPN-80, Beckman-Coulter) (ultracentrifugal concentration). After removing the supernatant, the sediment was dissolved in 10 μl of Tricine Sample Buffer (Bio-Rad), and 10 μl of Tris-buffered saline was added to prepare the sample.

[0050] Culture supernatants from BLV-infected cells and FLK-BLV cells alone were also treated in the same manner.

[0051] Proteins contained in these samples were detected by immunoblotting. Specifically, 10 μl of the sample was subjected to 4-15% wt. gradient polyacrylamide gel electrophoresis. The sample was transferred to a polyvinylidene fluoride (PVDF) membrane and reacted with anti-BLV Gag p24 and Env gp51 monoclonal antibodies (BLV2 and BLV3, VMRD). Antibodies bound to the p24 antigen or gp51 antigen were reacted with horseradish peroxidase (HRP)-conjugated anti-mouse antibody (Cytiva), followed by a chemiluminescent substrate (Immunostazeta, Fujifilm Wako Pure Chemical Industries, Ltd.). The luminescence was recorded using an image analyzer (Amersham Imager 600, Cytiva). Figure 1 shows the immunoblotting analysis results for the viral particle-constituting proteins produced in the culture supernatant.

[0052] As shown in Figure 1, electrophoresis of virus particles prepared in the same manner from the culture supernatant of FLK-BLV cells (positive control) reveals that the p24 antigen, Gag p24 protein (hereafter referred to as p24), appears as a band approximately halfway between the 20 kD and 25 kD molecular weight markers, and the gp51 antigen, Env gp51 protein (hereafter referred to as gp51), appears as a band with a lower mobility than the 50 kD marker. p24 and gp51 were also detected in virus particles derived from the culture supernatant of BLV-infected cells (9054#2). Although virus production was enhanced in the BLV-infected cells, the apparent proliferation rate of the cells themselves decreased, and they no longer became as confluent as before, even when the same number of cells were seeded and cultured for a certain period of time.

[0053] On the other hand, when the supernatant (9054#2 + FLKBL.LS40) was examined after one week of co-culture of BLV-infected and uninfected cells, the bands for both p24 and gp51 were observed to be thicker than those for other culture supernatants (9054#2, FLK-BLV). Analysis of the intensities of these bands revealed that p24 was 1.17-fold stronger after co-culture than before co-culture, and gp51 was 1.44-fold stronger after co-culture than before co-culture.

[0054] The ultracentrifugation conditions employed sediment virus particles but do not sediment free proteins not associated with the particles. Furthermore, while the number of Gag molecules constituting one particle is strictly determined, there is no principle that dictates the number of Env protein molecules. Therefore, while an increase in Gag p24 indicates enhanced virus particle production, if the increase in Env gp51 is not the same, it is possible that the number of Env gp51 molecules incorporated into one particle increased. That is, it is possible that the virus replicated in the uninfected cells upon mixing with uninfected cells, resulting in the production of virus particles, which increased the number of virus particles and the number of Env gp51 protein molecules per particle. Although the mechanism is unclear, viral replication is unnecessary for cellular metabolism and division. However, it is thought that in virus-producing cells (also referred to as persistently infected cells in this invention) obtained by co-culture of BLV-infected and uninfected cells, a balance between viral replication and self-proliferation is achieved. In these persistently infected cells, viral replication is somewhat suppressed, whereas in the majority of newly infected cells, the balance between viral replication and proliferation is lost, leading to cell death, but resulting in the production of more viral particles.

[0055] (Example 2: Determination of the composition of a culture medium for virus production with the aim of reducing FBS) BLV-infected cells have been cultured in a culture medium containing 10% by volume FBS, but the production costs are high due to the expense of FBS, the high protein concentration makes it difficult to handle, and there is a possibility of side effects when inoculated into animals. Therefore, the composition of a culture medium with a reduced concentration of FBS was investigated.

[0056] (Preliminary Study) To reduce FBS, FLK cells were infected with a different BLV strain of the same lineage as the BLV-infected primary cells by standard methods to produce 3-B11 cells (another BLV-infected primary cell line). Studies were performed using these 3-B11 cells.

[0057] The serum concentration in the culture medium was reduced to 1, 2, and 3% by volume, and 0.5% by weight of Bacto-peptone (manufactured by Becton Dickinson and Company, hereinafter abbreviated as "P") and 0.3% by weight of lactalbumin hydrolysate (manufactured by the same company, hereinafter abbreviated as "L") were added as substitute nutrients to form culture mediums (E-MEM-1.L.P, E-MEM-2.L.P, E-MEM-3.L.P). The effects of these culture mediums, excluding L and P (E-MEM-1, E-MEM-2, E-MEM-3), and those supplemented with 0.5% by weight of glucose (hereinafter abbreviated as "G") (E-MEM-1.L.P.+G, E-MEM-2.L.P.+G, E-MEM-3.L.P.+G), were investigated using cell proliferation and virus production as indicators. The same number of cells (2 × 10 ５ or 4 x 10 ５ The cells (800 cells) were suspended in 10 mL of each culture medium and seeded in a 100 mm diameter Petri dish. The cells were passaged every 3 to 4 days, and this procedure of seeding and culturing the same number of cells was repeated 7 times. The number of cells recovered at each passage was calculated, and the doubling time (unit: hours) was calculated from the time elapsed after passage. After a further 7 passages, the culture supernatant was subjected to ultracentrifugal concentration and immunoblotting in the same manner as in Example 1 to evaluate virus production. The results are shown in Figure 2.

[0058] Regarding cell proliferation, cells cultured in additive-free culture media (E-MEM-1, E-MEM-2, E-MEM-3) at each FBS concentration showed the shortest average doubling time (E-MEM-1: 17.0 hours, E-MEM-2: 16.5 hours, E-MEM-3: 16.5 hours, Figure 2). Furthermore, cells cultured at higher FBS concentrations (2% and 3% by volume) showed higher proliferation than those cultured at 1% by volume, but there was no difference between 2% and 3% by volume. The addition of alternative nutrients prolonged doubling time at each FBS concentration, but the presence or absence of glucose did not have any significant effect.

[0059] For each culture medium, BLV capsid protein p24 was detected by immunoblotting analysis. The immunoblotting analysis images of the viral particle-constituting proteins obtained are shown in Figure 3. The band intensities shown in Figure 3 indicate that cells cultured in nutrient-supplemented culture medium at each FBS concentration produced more viral particles.

[0060] The band intensity shown in Figure 3 was measured using a densitometer (Amersham™ Imager AI600, Cytiva; the same applies hereinafter). The results are shown in Figure 4. The results in Figure 4 indicate that virus production increased when L or P was added at each FBS concentration, but when G was further added, virus production actually decreased slightly at FBS concentrations of 1% by volume and 2% by volume. E-MEM-3.L.P.+G produced the highest virus production under the conditions examined. However, because the addition of G decreased the hydrogen ion concentration of the culture medium, we concluded that adding G was unnecessary, considering the balance between the stability of the culture system and the virus production resulting from its addition.

[0061] Because the replication process of retroviruses, including gene expression from the provirus and protein synthesis, is completely dependent on cellular machinery, we expected that higher overall cell proliferation activity would result in higher virus production. However, in reality, cells that slowed proliferation due to nutrient addition resulted in higher virus production. It was also suggested that added nutrients may preferentially express specific transcription factors that activate provirus transcription. This may slow cell proliferation, as cellular machinery is used for virus production. Furthermore, increasing the FBS concentration to 2-3% by volume (1.5x) also increased virus production by 1.5 times. While the FBS cost per unit of viral protein production remains unchanged, the increased protein concentration due to increased FBS content could adversely affect the amount of contaminating proteins present during production as an immunizing antigen. Therefore, we selected E-MEM-2.L.P. as the culture medium for virus production, considering the balance between FBS addition and viral particle production.

[0062] (Example 3: Application of preliminary study results to BLV-infected cells (main study)) The composition of the culture medium for virus production determined for 3-B11 cells was applied to BLV-infected cells, and the culture conditions for most efficient virus production when mixed with non-infected cells were investigated. BLV-infected and non-infected cells had previously been cultured only in E-MEM containing 10% by volume FBS (E-MEM-10), but in this study, they were cultured in E-MEM-10 or E-MEM-2.L.P. from the first passage onwards. Figure 5 shows an overview of the cell culture performed in Example 3.

[0063] In addition, infected cells and non-infected cells were mixed at a ratio of 3:7 or 1:9 (cell number ratio, the same applies below). Furthermore, cultures were set up in which infected cells were mixed with non-infected cells at passage 1, and then continued to be passaged without mixing with non-infected cells from passage 2 onwards. These cultures were passaged five times every seven days, and the culture supernatants at passage 5 were quantified by antigen capture ELISA.

[0064] Antigen capture ELISA was performed as follows. 20 μg of anti-BLV Env gp51 monoclonal antibody was dispensed into a 96-well flat-bottom plate and immobilized at 4°C for at least 16 hours. After washing each well twice with phosphate-buffered saline (PBS), the wells were incubated with blocking buffer at 37°C for 1 hour and then washed with PBS containing 0.05% Tween 20 (PBS-T). 100 μl of sample was added to each well and incubated at 37°C for 90 minutes, followed by three washes with PBS-T. Subsequently, 0.05 μg of a detection antibody, a biotin-labeled anti-BLV Env gp51 monoclonal antibody recognizing a different epitope from the capture antibody, was added per well and incubated at 37°C for 1 hour. After washing each well three times with PBS-T, 100 μl of HRP-labeled avidin (1:5,000 dilution, Proteintech) was added to each well and incubated at room temperature for 1 hour. Then, each well was washed four times with PBS-T, and a substrate (TMB, Thermo) was added. The incubation was continued for 15-30 minutes at room temperature. The reaction was stopped with 1N sulfuric acid (Fujifilm Wako Pure Chemical Industries, Ltd.). The absorbance at 450 nm was measured using an ELISA reader ("Muiltiskan FC," Thermo). A dilution series of a standard containing known Env was simultaneously measured, and a calibration curve was created from the absorbance to determine the Env concentration (mg / mL) of the sample.

[0065] Figure 6 shows the results of Env concentration analysis in the supernatant of cultures with various combinations of culture medium compositions and mixing ratios of BLV-infected and non-infected cells. A 10% MIX [10%:10%=7:3] represents virus production when BLV-infected cells and non-infected cells are mixed at a ratio of 7:3. This condition was used as the benchmark for this study. When the mixing ratio was changed to 9:1, the amount of virus production decreased slightly (benchmark: 0.57 mg / mL to 10% MIX [10%:10%=9:1]: 0.51 mg / mL). E-MEM-2.L.P. When uninfected cells were cultured in E-MEM-10 (hereafter, 2% FBS) and mixed at a ratio of 7:3 with BLV-infected cells cultured in E-MEM-10 (hereafter, 10% FBS), and then co-cultured with 10% FBS, the Env level in the culture supernatant increased by 1.4-fold (MIX 10% [2%:10% = 7:3]: 0.8 mg / mL). However, when the mixing ratio was increased to 9:1, the level was 0.5 mg / mL, which was below the benchmark (MIX 10% [2%:10% = 9:1]). When uninfected cells cultured in 2% FBS and BLV-infected cells cultured in 10% FBS were mixed and then co-cultured with 2% FBS, the concentration increased to 1.1 mg / mL at a mixing ratio of 7:3 (2% MIX [2%:10% = 7:3]), but fell to 0.5 mg / mL below the benchmark at a mixing ratio of 9:1 (10% MIX [2%:10% = 9:1]). When both BLV-infected and uninfected cells were cultured at 2% FBS and co-cultured with 2% FBS after mixing, the concentration remained at 1.1 mg / mL, almost the same as the previous conditions, at a mixing ratio of 7:3, but improved to 0.8 mg / mL at a mixing ratio of 9:1, although inferior to the 7:3 ratio (2% MIX [2%:2% = 7:3], 2% MIX [2%:2% = 9:1]).

[0066] In contrast, when cells initially cultured in 10% FBS were mixed and then passaged at 10% FBS without mixing with uninfected cells from the second passage onward, Env production fell below the benchmark (NO MIX 10% [10%:10%], 0.45 mg / mL). When uninfected cells were cultured in 2% FBS and mixed with BLV-infected cells cultured in 10% FBS, and passaged at 10% FBS, Env production further decreased (NO MIX 10% [2%:10%], 0.36 mg / mL). When cells were mixed under these conditions and then co-cultured with 2% FBS instead of 10% FBS, Env production dramatically increased (NO MIX 2% [2%:10%], 1.40 mg / mL). When BLV-infected cells were cultured with 2% FBS by volume and then mixed and co-cultured under the above conditions, Env production increased slightly, reaching 1.45 mg / mL (NO MIX 2% [2%:2%]), a 2.5-fold increase in Env production compared to the benchmark.

[0067] These results led to the discovery of a novel finding that bactopeptone and lactalbumin hydrolysate, added as FBS substitutes, actually enhance virus production. The mechanism by which these additives increase virus production is unknown; however, possible mechanisms include preferential proviral transcription, enhanced viral protein trafficking, suppression of cellular viral inhibitory factors, or sequestration of expressed factors. Furthermore, when comparing the 7:3 and 9:1 mixture ratios, higher Env production was observed at the 7:3 mixture ratio. It is believed that the mixed culture results in virus transmission from infected to uninfected cells, leading to new virus production. However, this transmission is thought to occur not via the virus particles produced but via the viral synapse formed at the contact between infected and uninfected cells. Therefore, the rate of transmission in vitro was slower at the 9:1 mixture ratio, and over a one-week culture period, the ratio of infected cells was lower than at the 7:3 mixture ratio, which likely resulted in lower virus production.

[0068] (Example 4: Confirmation of continuity of enhanced virus production) As shown in the results of Examples 1 and 3, virus production was enhanced by co-culturing virus-infected cells with uninfected cells, and the continuity of this effect was examined. From the cultures prepared in the test described in Example 3, 10% MIX [10%:10%=7:3], 2% MIX [2%:2%=7:3], 10% NO MIX [2%:10%], and 2% NO MIX [2%:2%] were selected, and the amount of Env produced in the supernatant after 10 passages was examined by antigen capture ELISA. The results are shown in Figure 7.

[0069] As shown in Figure 7, Env production decreased after 10 passages in all cultures tested. The greatest decrease was observed in the 2% MIX (2%:2% = 7:3), where Env production decreased from 1.12 mg / mL to 0.11 mg / mL (10%, 19% of the benchmark). The smallest decrease was observed in the 10% NO MIX (10%:10%), where Env production decreased from 0.45 mg / mL to 0.13 mg / mL (29%, 23% of the benchmark). Overall, serum concentration did not appear to be related to the decrease in Env production with passaging. Furthermore, no significant decrease in Env production after passaging was observed when Env production levels were high or low initially. The Env production level of the 2% NO MIX [2%:2%], which showed the highest value after 10 passages, was 0.33 mg / mL. This corresponds to 58% of the 0.57 mg / mL produced by the benchmark 10% MIX [10%:10% = 7:3] at the initial stage of mixing, which can be considered a high value. The reason for the decrease in Env production with passaging is unclear, but microscopic observation of the mixed culture revealed floating cells with balloon-like degeneration and nuclear condensation, suggesting cell damage. It is thought that the populations susceptible to infection by these viruses and causing cell death upon infection are killed by passaging, leaving behind a less susceptible population, resulting in a decrease in production.

[0070] Example 5: We investigated whether virus production could be restored by co-culturing virus-infected cells with newly added uninfected cells, which had reduced Env production as shown in the results of Example 4. Specifically, cells (9054#2.P7 post-cc) whose virus production had gradually decreased after seven passages after mixing with uninfected cells and cells (9054#2 P7 post-cc + uninfected cells) prepared by mixing these cells with uninfected FLKBL.LS40 cells at a cell number ratio of 3:7 were cultured for one week, and the virus particles produced in the culture supernatant were examined by immunoblotting using the same method as in Example 1. The supernatant of FLK-BLV cells cultured in the same way was also used as a control. Furthermore, the amount of Env protein was examined by antigen capture ELISA as described above using the supernatant before ultracentrifugal concentration. The immunoblotting analysis results of the obtained virus particle-constituting proteins are shown in Figure 8.

[0071] The results shown in Figure 8 indicate that BLV-infected cells co-cultured with uninfected cells once and then passaged for seven generations without further mixing with uninfected cells showed a gradual decrease in virus production compared to immediately after co-culture (9054#2.P7.post-cc, Env content in the supernatant: 0.51 mg / mL). When uninfected cells were added to these (9054#2.P7.post-cc) cells as described above and cultured for one week (9054#2.P7 post-cc + uninfected cells), the Env content in the supernatant increased 1.57-fold (0.80 mg / mL). These supernatants and the positive control (FLK-BLV culture supernatant) were concentrated by ultracentrifugation, and the precipitated virus particles were examined by immunoblotting. The p24 band was nearly identical before and after mixing with uninfected cells. This suggests that there was no significant difference in virus production before and after mixing. In contrast, the gp51 band was significantly stronger after mixing. Densitometry of the bands showed that the density after mixing was 2.4 times that before mixing.

[0072] These results demonstrate that even if BLV-infected cells, which showed increased Env production upon co-culture with uninfected cells, experienced a decrease in virus production after passage, production could be restored by co-culturing with new uninfected cells. Furthermore, the gp51 to p24 ratio was 0.55 for FLK-BLV, whereas it increased from 0.46 before mixing to 1.13 after mixing. This indicates that co-culturing BLV-infected cells with uninfected cells produces virus particles richer in Env than FLK-BLV-derived virus particles. Because Env is believed to be the primary target of protective immunity against BLV infection, the virus particles produced by these cells are highly effective as vaccine antigens.

Claims

1. A method for producing a virus, comprising the step of mixing virus-infected cells and non-infected cells and co-culturing them to grow the virus.

2. A method for producing the virus according to claim 1, comprising a step of mixing and co-culturing the virus-infected cells obtained through the co-culture passage period with non-infected cells.

3. A method for producing a virus as described in claim 2, which comprises repeating the step of mixing and co-culturing the virus-infected cells obtained after the co-culture passage period with non-infected cells.

4. The method for producing a virus according to claim 1, wherein the serum concentration in the medium used for the co-culture is less than 10% by volume.

5. A method for producing a virus, comprising the step of propagating the virus by continuing to passage the virus-infected cells obtained by the method for producing a virus described in claim 1 without adding new non-infected cells.

6. The method for producing a virus according to claim 5, wherein the number of passages continued without the addition of new uninfected cells is two or more.

7. The method for producing a virus described in claim 1, wherein the uninfected cells are one or more types of cells selected from an established cell line derived from ovine kidney, an established cell line derived from porcine kidney, an established cell line derived from bovine kidney, an established cell line derived from bovine testis, and an established cell line derived from porcine oviduct epithelium.

8. The method for producing a virus according to claim 1, wherein the virus is one or more viruses selected from the family Retroviridae, Flaviviridae, Picornaviridae, Reoviridae, Coronaviridae, Paramyxoviridae, Rhabdoviridae, Flaviviridae, Circoviridae, and Herpesviridae.

9. A virus strain for use as a vaccine, characterized in that the expression level of the membrane surface glycoprotein gp51 is 0.6-fold or more higher than the expression level of the capsid protein p24.

10. A vaccine composition comprising the inactivated virus strain for a vaccine according to claim 9.

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