Method for Influenza Virus Propagation

Abstract

To provide a method for efficiently growing, in a host, an influenza virus to be the material for a vaccine.SOLUTION: A method for growing an influenza virus in a host is provided, the method including the step of adding, to the host, at least one tankyrase inhibitor selected from [(3aR*,4S*,7R*,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H- isoindole-2-yl]-N-8-quinolinyl benzamide and 4-[4-(4-methoxyphenyl)-5-[[[3-(4-methylphenyl)-1,2,4-oxadiazole-5-yl]methyl]thio]-4H-1,2,4-triazole-3-yl]-pyridine.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for growing influenza virus in a host.

Background Art

[0002] Influenza is an infectious disease caused by influenza virus, which is transmitted through droplet infection or contact infection, etc., and is a respiratory infectious disease accompanied by severe systemic symptoms such as high fever, headache, muscle pain, and joint pain. Vaccination against influenza is the best means of preventing the aggravation of influenza.

[0003] Influenza vaccine is a whole particle vaccine in which influenza virus for vaccine production is inoculated into the chorioallantoic cavity of embryonated chicken eggs for culture and growth, concentrated and purified by centrifugation from allantoic fluid, the virus particles are treated with a surfactant, etc., and inactivated with formalin, or a split vaccine or subunit vaccine in which the virus particles are disrupted with ether or a surfactant and then further purified. However, when producing influenza vaccine using embryonated chicken eggs having embryos as hosts, there are problems in terms of supply stability in that it requires time, labor, and cost and cannot be mass-produced rapidly.

[0004] As an alternative virus production method, a technique of replicating using cultured cells as hosts for influenza virus has been studied, and it has been reported that MDCK cells are appropriate cells for in vitro replication of influenza virus (Non-Patent Document 1). In addition, Patent Document 1 discloses that after removing or reducing trypsin inhibitor secreted in the culture solution of MDCK cells, inoculating the cells with influenza virus and culturing the influenza virus-inoculated cells can increase the virus production amount.

[0005] On the other hand, the Wnt signaling mechanism includes 1) the Wnt / β-catenin pathway, 2) the Wnt-PCP (planar cell polarity) pathway, 3) the Wnt / Ca 2+It is composed of three types of pathways, which form a network within cells and are responsible for controlling functions such as early development / migration / growth of cells. Conventionally, regarding the relationship between the Wnt signaling mechanism and viral infection in a host, it has been reported that in β-catenin knockdown cells, the growth of influenza virus is enhanced (Non-Patent Document 2), and that a compound that inhibits the Wnt / β-catenin signal suppresses the infection rate of influenza virus into cells (Patent Documents 2 and 3). That is, there are reports that inhibiting the Wnt / β-catenin signal promotes the growth of the virus in cells and reports that it is suppressed.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0008] The present invention relates to a method for more efficiently growing influenza virus, which is a material for a vaccine, in a host.

Means for Solving the Problem

[0009] As a result of intensive research, the present inventors have found that when a specific tankyrase inhibitor is added to a host infected with influenza virus, the growth property of the virus is improved and the production amount of the virus increases.

[0010] That is, the present invention relates to the following 1) to 4). 1) A method for growing influenza virus in a host, comprising the step of adding to the host at least one tankyrase inhibitor selected from [(3aR * ,4S * ,7R * ,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolylbenzamide and 4-[4-(4-methoxyphenyl)-5-[[[3-(4-methylphenyl)-1,2,4-oxadiazol-5-yl]methyl]thio]-4H-1,2,4-triazol-3-yl]-pyridine. 2) A method for preparing influenza virus particles, comprising growing influenza virus by the method of 1) and recovering virus particles from the host. 3) A method for producing an influenza vaccine, comprising producing a vaccine using the influenza virus particles prepared by the method of 2). 4) An influenza virus growth promoter comprising as an active ingredient at least one tankyrase inhibitor selected from [(3aR * ,4S * ,7R * ,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolylbenzamide and 4-[4-(4-methoxyphenyl)-5-[[[3-(4-methylphenyl)-1,2,4-oxadiazol-5-yl]methyl]thio]-4H-1,2,4-triazol-3-yl]-pyridine.

Effect of the Invention

[0011] According to the method of the present invention, influenza virus can be efficiently propagated, and influenza virus for vaccine preparation can be mass-produced.

Brief Description of the Drawings

[0012]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0013] In the present invention, the influenza virus may be any of type A, type B, type C, and type D, and type A and type B can be preferably exemplified. In addition, the type of haemagglutinin (HA: haemagglutinin) and the type of neuraminidase (NA type) of the influenza virus are not particularly limited. For example, in addition to currently known subtypes such as H1N1 strain, H2N2 strain, H3N2 strain, H4N2 strain, H4N6 strain, H5N1 strain, H5N2 strain, H7N7 strain, H7N9 strain, H9N2 strain, etc., subtypes to be isolated and identified in the future are also included.

[0014] In addition, the target virus may be any virus that can infect humans, and may also be a virus that has the ability to infect pigs, chickens, horses, cows, etc.

[0015] In addition, the influenza virus of the present invention may be a strain isolated from an infected individual such as an infected animal or a patient, or may be a recombinant virus established in cultured cells by genetic engineering.

[0016] In the present invention, a tankyrase inhibitor is a compound that inhibits the activity of tankyrase, which is one of the axin-degrading enzymes. A tankyrase inhibitor is known as a compound that promotes the degradation of β-catenin by stabilizing axin and inhibits the activation of receptors downstream of the Wnt signaling pathway (Palazzo et al. Biochem Soc Trans. 2018;46(6):1681-1695.).

[0017] "β-Catenin" refers to a protein involved in cell adhesion and transcriptional regulation and regulation of genes, and functions as an intracellular signaling factor in the Wnt / β-catenin pathway, which is one of the Wnt signaling pathways responsible for functional control such as cell early development / migration / growth. The regulation and degradation of β-catenin are carried out by the β-catenin destruction complex (see Figure 1). The "β-catenin destruction complex" is a complex that efficiently degrades β-catenin, and consists of axin as a scaffold protein, APC (Adenoma polyposis coli), GSK-3β (Glycogen synthase kinase-3β), CK-1 (casein kinase 1α) and β-catenin. β-catenin is phosphorylated in sequence by CK-1 and GSK-3β, and the phosphorylated β-catenin undergoes ubiquitination and is finally degraded by the proteasome.

[0018] In the Wnt signaling pathway, it is considered that the Wnt protein transmits signals into cells and controls various cell functions by binding directly to the Frizzled (Fz) or LRP (low-density lipoprotein receptor-related protein) 5 / 6 receptors on the cell membrane or to the cytoplasmic protein Dishevelled (Dvl) (see Figure 1). In the Wnt / β-catenin pathway, in the absence of Wnt, the protein amount of β-catenin in the cytoplasm is kept low. This is because β-catenin is phosphorylated in the above-mentioned β-catenin destruction complex and is ultimately degraded by the proteasome. On the other hand, when Wnt is secreted extracellularly and binds to Frizzled and LRP5 / 6 on the target cell membrane, the Wnt signal is transmitted into the cell, Dvl suppresses the phosphorylation of GSK-3β-dependent β-catenin, and β-catenin accumulates in the cytoplasm because it is spared from degradation by the proteasome. Subsequently, β-catenin that has translocated into the nucleus forms a complex with the transcription factor Tcf / Lef and promotes the expression of target genes, thereby controlling various cell functions (see Figure 1). Thus, in the Wnt / β-catenin pathway, gene expression via Tcf / Lef is controlled by regulating the protein amount of β-catenin in the cytoplasm.

[0019] The tankyrase inhibitor of the present invention is a β-catenin destruction complex-stabilizing compound that increases the protein amount of axin (axin1, axin2), which is a scaffold protein that contributes to the stability of the β-catenin destruction complex. Tankyrase inhibitors are classified into three types: compounds that bind to the Adenosine subsite (type A), compounds that bind to the Nicotinamide subsite (type B), and compounds that bind to the Dual-binding subsite (type C) depending on the binding site to tankyrase. Among these, the compound that exhibits an influenza virus growth effect is a compound that binds to the Adenosine subsite and has the following structure: [(3aR * ,4S * ,7R *,7aS)-1,3,3a,4,7,7a-Hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolinylbenzamide (Compound 1; Endo-IWR1) and 4-[4-(4-methoxyphenyl)-5-[[[3-(4-methylphenyl)-1,2,4-oxadiazol-5-yl]methyl]thio]-4H-1,2,4-triazol-3-yl]-pyridine (Compound 2; JW74).

[0020]

Table 1

[0021] As shown in the examples described below, by adding Compound 1 (endo-IWR1) or Compound 2 (JW74), which are tankyrase inhibitors, to the host, influenza virus can be efficiently propagated. On the other hand, it was also confirmed that there are compounds that do not show an influenza virus growth-promoting effect even though they are tankyrase inhibitors. Therefore, by adding one or more tankyrase inhibitors selected from [(3aR * ,4S * ,7R * ,7aS)-1,3,3a,4,7,7a-Hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolinylbenzamide (Compound 1) and 4-[4-(4-methoxyphenyl)-5-[[[3-(4-methylphenyl)-1,2,4-oxadiazol-5-yl]methyl]thio]-4H-1,2,4-triazol-3-yl]-pyridine (Compound 2) to the host cell, it is possible to grow influenza virus in the host, and it can be said that the tankyrase inhibitor is an influenza virus growth promoter for growing influenza virus by culturing the host, and it can be used for growing influenza virus by culturing the host. Also, [(3aR * ,4S * ,7R *,7aS)-1,3,3a,4,7,7a-Hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolinylbenzamide (Compound 1) and 4-[4-(4-methoxyphenyl)-5-[[[3-(4-methylphenyl)-1,2,4-oxadiazol-5-yl]methyl]thio]-4H-1,2,4-triazol-3-yl]-pyridine (Compound 2), one or more tankyrase inhibitors selected therefrom, can be said to be used for producing an influenza virus growth promoter.

[0022] The tankyrase inhibitor of the present invention is used for a host for growing influenza virus (type A, B, C, D) at a concentration of 0.01 μM or more, preferably 0.1 μM or more, more preferably 1 μM or more, and 100 μM or less, preferably 50 μM or less, more preferably 20 μM or less, and also 0.01 to 100 μM, preferably 0.1 to 50 μM, more preferably 1 to 20 μM.

[0023] The growth of influenza virus is specifically carried out by a step of infecting a host with influenza virus and a step of culturing the infected host under conditions where the virus can replicate. In the present invention, the step of adding a tankyrase inhibitor to the host is carried out, for example, before virus infection, after virus infection, or simultaneously with virus infection. Preferably, the tankyrase inhibitor is added to the host within 3 hours after virus infection.

[0024] As the host used for the growth of influenza virus, either cultured cells or developing chicken eggs may be used, but it is preferable to use cultured cells from the viewpoint of supply stability. As the cultured cells, any cells can be used as long as they are sensitive to influenza virus. Examples of such cells include MDCK cells (established cells derived from canine kidney), Vero cells (established cells derived from African green monkey kidney), PER.C6 (established cells derived from human retinal cells), SK-NEP-1 cells (established cells derived from human kidney), A549 (human alveolar basal epithelial adenocarcinoma cells), and Duck embryo cells (duck embryo cells). These cells are registered with ATCC (American Type Culture Collection) as CCL-34, CCL-81, CCL-107, HTB-48, CCL-185, CCL-141, etc., respectively, and can also be purchased commercially. In addition, as chicken-derived cells sensitive to influenza virus, CEF cells (Chicken embryonic fibroblast cell: fibroblast cells derived from chicken embryo) can be used. Note that CEF cells include not only isolated cells but also cells present in developing chicken eggs. In addition, for the growth of influenza virus, cell lines developed for efficiently growing influenza virus can also be used. Examples of such cell lines include, but are not limited to, EB66 (registered trademark), DuckCelt-T17 (registered trademark), EBx (registered trademark), etc.

[0025] When using cultured cells as the host, as the medium for culturing the cells, media usually used for cell culture, for example, MEM medium containing fetal bovine serum (FBS) (manufactured by Wako), serum-free medium (Serum-Free Medium) (manufactured by ThermoFisher), etc. can be used, and any of them can be used.

[0026] To this medium, non-essential amino acids and L-glutamine can be added to increase the cell growth efficiency. Also, in the culture of influenza virus, proteases such as trypsin and acetylated trypsin can be added for the purpose of promoting the cleavage of hemagglutinin. Further, to avoid microbial contamination, antibiotics commonly used in cell culture such as penicillin, streptomycin, and gentamicin may be added. The pH of the medium is adjusted to 6.5 - 8, preferably 6.8 - 7.3, suitable for the growth of animal cells with an appropriate buffer (e.g., sodium bicarbonate, HEPES).

[0027] Examples of cell culture methods include static culture in which cells are attached to the bottom of the incubator and suspension culture in which cells are suspended in the medium and cultured. When carried out at the industrial production level, suspension culture is preferred. Examples of suspension culture methods include a method in which cells are attached to a carrier such as a microcarrier and suspended for culture or a method in which cells are suspended and cultured without using a carrier. Any method can be used.

[0028] The cell culture (a mixture of cultured cells and medium) can be directly used for inoculation with influenza virus. However, when inoculating with influenza virus, it is preferably washed with fresh medium or an appropriate buffer, such as PBS or Tris buffer. Specifically, cells cultured and grown in a spinner-flask or the like are centrifuged at low speed or filtered through a membrane, separated into cells and culture supernatant, fresh medium is added to the cells in the centrifugal sediment or membrane filtration concentrate, and the medium is exchanged by suspending the cells.

[0029] To the cell culture thus obtained, an influenza virus solution is added and cultured under certain conditions. The initial cell density at the start of virus culture can be 0.001 - 100×10 6 cells / mL, preferably 0.01 - 10×10 6 cells / mL, more preferably 0.1 - 10×10 6It is cells / mL. The cell density can be measured according to a general method using a hemocytometer or the like. The influenza virus solution added to the cell culture can be added so that the infection titer MOI (Multiplicity of infection) is 0.00001 to 10, preferably 0.0001 to 0.1, and more preferably 0.0001 to 0.01.

[0030] When using a developing chicken egg as a host, it can be incubated under conditions of 33°C to 38°C, preferably 35 to 37°C, and humidity conditions of 40 to 60%, preferably 45 to 55%, and rotated 1 to 24 times a day, preferably 4 to 12 times, to obtain a developed chicken egg for use. Influenza virus can be infected using a chicken egg on the 8th to 13th day of development, but preferably it can be infected with a chicken egg on the 10th to 12th day. The amount of virus to be infected can be 1 to 1×10 50 EID 6 EID 50 / Egg, but preferably 1×10 2 ~1×10 5 EID 50 / Egg, more preferably 1×10 3 ~1×10 4 EID 50 / Egg can be infected. The infection site is preferably within the chorioallantoic membrane (in the allantoic cavity fluid) of the chicken egg, but it can also be within the amnion (in the amniotic fluid), and is not limited as long as it is a site where influenza virus grows in the chicken egg. It is desirable that the addition site of the tankyrase inhibitor of the present invention coincides with the virus infection site, but is not limited as long as it is a site where influenza virus grows within the chorioallantoic membrane (in the allantoic cavity fluid), within the amnion (in the amniotic fluid), and in the chicken egg. As described above, the addition of the tankyrase inhibitor of the present invention can be added to the host before virus infection, after virus infection, or simultaneously with virus infection, but preferably it is desirable to use it in a state of being mixed with the virus simultaneously with virus infection.

[0031] The culture conditions can be any conditions as long as the influenza virus can grow in the host. They are appropriately adjusted according to combinations such as cell type, virus inoculum amount, and culture scale and method. For example, when using cultured cells as the host, the culture temperature is 33°C to 39°C, preferably 34 to 38°C, the culture period is 1 to 10 days, preferably 3 to 7 days, the carbon dioxide concentration is 3 to 8%, preferably 4 to 5%, and the oxygen concentration is 17 to 25%, preferably 20 to 22%. Also, when using a developing chicken egg as the host, after infection, it is cultured under the conditions of 33°C to 38°C, preferably 34 to 36°C, the culture period is 1 to 5 days, preferably 2 to 4 days, and the humidity condition is 40 to 60%, preferably 45 to 55%. Since the conditions for maximizing growth vary depending on the virus strain, the culture period, culture temperature, humidity, etc. can be appropriately combined.

[0032] According to the method of the present invention, influenza virus can be efficiently grown. The virus content in the host can be measured by hemagglutination method (dilution factor) using red blood cells such as guinea pigs, ELISA method (μg / mL) using antibodies against hemagglutinin, plaque assay for measuring virus infectivity titer, TCID 50 , and real-time PCR capable of measuring the amount of viral RNA, etc.

[0033] When the host is a developing chicken egg, the influenza virus is contained in the allantoic cavity fluid (urine-like fluid) or amniotic fluid. When the host is cultured cells, it is contained in the culture supernatant. After the culture is completed, virus particles are recovered from the virus suspension in the host, and by concentrating, purifying, and inactivating, virus particles for inactivated whole particle vaccine or inactivated split vaccine can be prepared. When used as a live vaccine or attenuated live vaccine, it can be prepared as virus particles for influenza vaccine after concentration and purification.

[0034] The recovery of virus particles is carried out by clarifying the virus suspension, specifically by centrifugation or filtration, and then ultrafiltration is carried out for concentration. The purification of the virus can be carried out using means such as ultracentrifugation such as sucrose density gradient centrifugation, size exclusion chromatography, and liquid chromatography. The purified virus solution is inactivated by formalin treatment, ultraviolet irradiation, beta-propiolactone, binary ethyleneimine, etc. in the case of inactivated whole particle vaccines and inactivated split vaccines. When used as a live vaccine or a live attenuated vaccine, the above purified virus solution is prepared as virus particles for influenza vaccines.

[0035] To such influenza virus particles, carriers (buffering agents, emulsifying agents, preservatives (e.g., thimerosal), isotonic agents, pH adjusters, adjuvants (e.g., aluminum hydroxide gel), etc.) that can be appropriately acceptable as pharmaceuticals are added, and vaccines of various dosage forms can be produced.

[0036] Regarding the above-described embodiments, the present invention further discloses the following aspects. <1>A method for growing influenza virus in a host, comprising the step of adding one or more tankyrase inhibitors selected from [(3aR * ,4S * ,7R * ,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolinylbenzamide and 4-[4-(4-methoxyphenyl)-5-[[[3-(4-methylphenyl)-1,2,4-oxadiazol-5-yl]methyl]thio]-4H-1,2,4-triazol-3-yl]-pyridine to the host. <2>The method of <1>, wherein the host is a cultured cell or a developing chicken egg. <3>The method according to <2>, wherein the cell is an MDCK cell (a cell line derived from canine kidney), a Vero cell (a cell line derived from African green monkey kidney), PER.C6 (a cell line derived from human retinal cells), an SK-NEP-1 cell (a cell line derived from human kidney), A549 (human alveolar basal epithelial adenocarcinoma cells), Duck embryo cells (duck embryo cells), fibroblast cells derived from chicken embryo, EB66 (registered trademark), DuckCelt-T17 (registered trademark), or EBx (registered trademark). <4>The tankyrase inhibitor is used at a concentration of 0.01 μM or more, preferably 0.1 μM or more, more preferably 1 μM or more, and 100 μM or less, preferably 50 μM or less, more preferably 20 μM or less, and also 0.01 to 100 μM, preferably 0.1 to 50 μM, more preferably 1 to 20 μM, for a host for growing influenza virus (type A, B, C, D) in the method according to any one of <1> to <3>. <5>A method for preparing influenza virus particles, comprising growing influenza virus by any one of the methods according to <1> to <4> and recovering virus particles from the host. <6>The method according to <5>, wherein the virus particles are used for manufacturing an influenza vaccine. <7>A method for manufacturing an influenza vaccine, comprising manufacturing a vaccine using the influenza virus particles prepared by the method according to <5> or <6>. <8>[(3aR * ,4S * ,7R * An influenza virus growth promoter comprising, as an active ingredient, at least one tankyrase inhibitor selected from [(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolinylbenzamide and 4-[4-(4-methoxyphenyl)-5-[[[3-(4-methylphenyl)-1,2,4-oxadiazol-5-yl]methyl]thio]-4H-1,2,4-triazol-3-yl]-pyridine.

Examples

[0037] Example 1: Viral growth evaluation test using a cell line derived from canine kidney tubular epithelial cells (MDCK cells) (1) MDCK cells (a cell line derived from canine kidney tubular epithelial cells, obtained from DS Pharma Biomedical Co., Ltd.) were cultured in MEM medium (manufactured by Wako Pure Chemical Industries, Ltd.) containing 5% fetal bovine serum (FBS) at 37°C in the presence of 5% CO2. The above MDCK cells were seeded in a 24-well plate and used for the test in a confluent state. After washing the MDCK cells seeded in the above 24-well plate with PBS, Serum free medium (SFM; manufactured by Gibco) was added at 400 μL / well and the cells were acclimated for 1 hour.

[0038] (2) The above cells were infected with H1N1 influenza virus strain (A / Puerto Rico / 8 / 34), which is an influenza A virus, at a multiplicity of infection (MOI) of 0.001, H1N1pdm (A / California / 07 / 2009), H3N2 (A / Wisconsin / 15 / 2009), and influenza B virus (B / Lee / 1940) at an MOI of 0.05, and incubated for 1 hour. Thereafter, a washing operation was performed with SFM, and compound 1 (at a concentration of 0 - 12.5 μM), compound 2 (at a concentration of 12.5 μM), and compounds 3 - 7 (each at a concentration of 12.5 μM) shown in Table 2-1 and Table 2-2 were added to the SFM culture medium containing 2.0 μg / mL - acetylated trypsin (manufactured by Sigma), and the volume was adjusted to 500 μL / well. H1N1 influenza virus was cultured for 23 hours, and H1N1pdm, H3N2, and influenza B virus were cultured for 45 hours. The culture supernatant was collected 24 hours after infection, and influenza virus was quantified by the following focus assay and HA assay.

[0039]

Table 2-1

[0040]

Table 2-2

[0041] (3) HA assay Using a U-bottom 96-well plate, 50 μL of the influenza virus culture supernatant was serially diluted 2-fold from 2 to 1024 times. Then, 50 μL of PBS containing 0.7% guinea pig red blood cells was added, and the mixture was allowed to stand at 4°C for 2 hours. Subsequently, hemagglutination was confirmed, and the dilution concentration at which no agglutination was observed was defined as the HA titer (Table 3).

[0042] (4) As shown in Table 3, it was revealed that the addition of Compound 1 increased the HA titers of H1N1, H1N1pdm, H3N2, and influenza B viruses, and the addition of Compound 2 increased the HA titer of H1N1 influenza virus.

[0043]

Table 3

[0044] (5) Focus assay MDCK cells were cultured to confluence in a 12-well plate, washed with PBS, and then acclimated in SFM for 1 hour. The influenza virus culture supernatant collected 24 hours after infection was diluted 10 5 -10 8 times, added to the MDCK cells cultured in the above 12-well plate at 1 mL / well, and incubated for 1 hour to infect the influenza virus. This test was performed in triplicate. After infection, a washing operation with SFM was carried out, and SFM containing 1.2% Theolas (Asahi Kasei Chemicals, RC591) and 2.0 μg / mL acetylated trypsin (manufactured by Sigma) was added at 2.0 mL / well, and the cells were cultured for 21 hours. After culturing, the wells were washed three times with PBS cooled to 4°C, and then 100% methanol (manufactured by FUJIFILM Wako Pure Chemical Corporation) cooled to -20°C was added to immobilize the cells. The immobilized cells were reacted with the primary antibody: Mouse monoclonal anti-influenza NP antibody (manufactured by Invitrogen) and the secondary antibody: HRP linked goat Anti-mouse IgG antibody (manufactured by FUJIFILM Wako Pure Chemical Corporation), reacted with HRP using the DEPDA reaction, and the number of stained foci was counted. The focus assay was performed as independent triplicate measurements, and statistical analysis was performed using one-way analysis of variance and Dunnett's test, with a significance level of 5% risk rate.

[0045] (6) As shown in Figure 2, the addition of Compound 1 at 3.13 μM or 12.5 μM resulted in a statistically significantly higher viral growth of H1N1 influenza virus (***; P < 0.001 (vs. control group)).

Claims

A method for growing influenza virus in a host other than human, comprising the step of adding to the host one or more tankyrase inhibitors selected from [(3aR*,4S*,7R*,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolinylbenzamide and 4-[4-(4-methoxyphenyl)-5-[[[3-(4-methylphenyl)-1,2,4-oxadiazol-5-yl]methyl]thio]-4H-1,2,4-triazol-3-yl]-pyridine. Claim 2 The method according to claim 1, wherein the host is a cultured cell or a developing chicken egg. Claim 3 A method for preparing influenza virus particles, comprising growing influenza virus by the method according to claim 1 or 2 and recovering virus particles from the host. Claim 4 The method according to claim 3, wherein the virus particles are used for manufacturing an influenza vaccine. Claim 5 A method for manufacturing an influenza vaccine, comprising manufacturing a vaccine using the influenza virus particles prepared by the method according to claim 3 or 4. Claim 6 An influenza virus growth promoter comprising, as an active ingredient, one or more tankyrase inhibitors selected from [(3aR*,4S*,7R*,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolinylbenzamide and 4-[4-(4-methoxyphenyl)-5-[[[3-(4-methylphenyl)-1,2,4-oxadiazol-5-yl]methyl]thio]-4H-1,2,4-triazol-3-yl]-pyridine.

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