CN116096363A - Coronavirus proliferation method - Google Patents

Abstract

The present invention provides a method for efficiently propagating coronaviruses, which are materials for vaccines, in a host. A method of propagating a coronavirus in a host, wherein the method comprises the step of inhibiting Bax transfer of a host cell to the inner mitochondrial membrane.

Description

Coronavirus proliferation method

Technical Field

The present invention relates to a method for propagating coronaviruses in a host.

Background

The common cold and acute respiratory syndrome (covd-19, middle Eastern Respiratory Syndrome (MERS), severe Acute Respiratory Syndrome (SARS)) are infections caused by coronaviruses (SARS coronavirus, SARS coronavirus-2, MERS coronavirus, human coronavirus OC43, human coronavirus 229E, human coronavirus HKU1, human coronavirus NL 63) and are respiratory tract infections accompanied by strong systemic symptoms such as hyperpyrexia, cough, headache, myalgia, arthralgia, pneumonia, shortness of breath, lassitude, fatigue, sore throat, etc., due to droplet infection, contact infection, aerosol infection, air infection, etc. Coronavirus vaccines have been developed worldwide for the purpose of preventing infections caused by coronaviruses, and vaccination with coronavirus inactivated coronavirus vaccines is considered as the best means of protection against severe acute respiratory syndrome.

An inactivated coronavirus vaccine is a split vaccine or a subconjugate vaccine in which coronavirus for vaccine production is cultured and propagated by culturing cells, the virus particles are concentrated and purified by centrifugation from the culture solution, the virus particles are treated with a surfactant or the like, and the whole inactivated vaccine or the virus particles are crushed with formalin with diethyl ether or a surfactant and then further purified. The ability of an inactivated SARS coronavirus-2 vaccine under development to induce neutralizing antibodies against coronaviruses was reported (non-patent document 1).

However, in the case of mass production of a vaccine against coronavirus using cultured cells, time, labor and cost are required, and there is a problem in that such supply stability cannot be achieved in a rapid mass production.

In the case of SARS coronavirus, nsp15, which is a viral protein, efficiently proliferates the virus by inhibiting apoptosis of a host, and it is taught that the proliferation of the virus is reduced in a mutant in which the apoptosis inhibition mechanism of Nsp15 is defective (non-patent document 2). However, in other reports, it is reported that SARS coronavirus induces apoptosis due to infection of a host, but replication cannot be inhibited even if an apoptosis signal is inhibited (non-patent document 3). In addition, it is taught that 7A protein of SARS coronavirus inhibits its function by binding to Bcl-XL, bcl-2, mcl-1, bcl-w, A1 proteins as apoptosis inhibitors of the host, and that Bax or Bak as apoptosis-inducing factors does not exhibit binding property, and that they are not controlled (non-patent document 4).

From the results obtained in the above-mentioned non-patent documents, it is possible to infer that apoptosis signals work in a host with coronavirus infection, but it is not clear whether or not a plurality of signal components are necessary for controlling the proliferation of viruses, and whether or not the function of proteins is inhibited or activated by a compound, and the proliferation of viruses is enhanced by the defect of a specific gene. Furthermore, there is no report on inhibition or activation of the function of a protein by a compound and increase of the proliferation of a virus due to a defect of a specific gene.

Prior art literature:

non-patent document 1: science.2020;369:77-81

Non-patent document 2: proc Natl Acad Sci usa.2017;114:E4251-E4260

Non-patent document 3: DNA Cell biol 2005;24:496-502

Non-patent document 4: j Virol.2007;81:6346-5

Disclosure of Invention

The present invention relates to the following 1) to 6).

1) A method of propagating a coronavirus in a host, wherein the method comprises the step of inhibiting Bax transfer of a host cell to the inner mitochondrial membrane.

2) A method for producing a coronavirus particle, wherein the coronavirus is propagated by the method of 1), and the virus particle is recovered from a host.

3) A method for producing a coronavirus vaccine, wherein a vaccine is produced using coronavirus particles produced by the method of 2).

4) A coronavirus proliferation promoter contains Bax inhibitor as effective component.

5) Use of a Bax inhibitor for promoting proliferation of coronaviruses.

6) Use of a Bax inhibitor for the manufacture of a coronavirus proliferation promoter.

Drawings

FIG. 1 shows the effect of promoting the proliferation of coronavirus (HCoV-OC 43) by the addition of Bax inhibitory peptide.

FIG. 2 shows the effect of the Bax inhibitory compound on promoting the proliferation of coronavirus (HCoV-OC 43).

FIG. 3 shows the effect of the Bax inhibitory compound on the proliferation of coronavirus (SARS-CoV-2).

Detailed Description

The present invention relates to a method for more efficiently propagating coronaviruses, which are materials for vaccines, in a host.

As a result of intensive studies, the present inventors have found that when Bax, which is known as an apoptosis-promoting protein, is inhibited from migrating to the inner mitochondrial membrane in a host infected with coronavirus to inhibit apoptosis, the proliferation of the virus is improved as compared with that in a steady state (control infected with the virus), and the amount of produced virus is increased.

According to the method of the present invention, coronaviruses can be efficiently propagated, and coronaviruses used for vaccine preparation can be mass-produced.

In the present invention, coronaviruses may be any of the types of genotypes of α, β, γ and δ, but β is preferably exemplified. As the beta-type coronavirus, MERS coronavirus (MERS-CoV), SARS coronavirus (SARS-CoV), SARS coronavirus-2 (SARS-CoV-2), human coronavirus OC43 (HCoV-OC 43), human coronavirus HKU1 (HCoV-HKU 1) can be mentioned. Examples of the α -type coronavirus include human coronavirus 229E (HCoV-229) and human coronavirus NL63 (HCoV-NL 63).

The virus strain to be used is not particularly limited as long as it can infect humans as a host, and other than the virus strain can infect animals raised as primates, domestic animals, poultry, and pets, and it includes mutant virus strains having a subtype to be isolated and identified and a partial mutation of the sequence, in addition to coronaviruses for which separation reports have been made so far.

The coronavirus 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 genetically established from cultured cells.

In the present invention, "Bax" refers to apoptosis-promoting proteins belonging to the Bcl-2 family. Proteins belonging to the Bcl-2 family have more than 1 amino acid sequence called BH (Bcl-2 homology) domains. In addition, since a TM (transmembrane) region having high hydrophobicity is provided on the C-terminal side, it is transferred to the mitochondrial membrane, and apoptosis can be controlled.

The primary function of Bcl-2 family proteins is the control of apoptosis by modulating mitochondrial permeability. The anti-apoptotic proteins Bcl-2 and Bcl-xL are present on the outer wall of mitochondria, inhibiting the release of cytochrome c. Bad, bid, bax and Bim, which are apoptosis-promoting proteins, exist in cytoplasm and migrate to the inner mitochondrial membrane through cell death signals, thereby promoting release of cytochrome c. Bax binds to Ku70 protein and is induced to the vicinity of the mitochondrial outer membrane. Bid is cleaved by Caspase-9 (Caspase-9) to become an activator tBit (truncated Bid (truncated Bid)) which binds to Bax induced toward the outer mitochondrial membrane, thereby inducing penetration of Bax into the inner mitochondrial membrane. It is thought that extracellular outflow of cytochrome c forms a complex with Apaf-1, and activates caspase 9, and thus caspases 3,6, and 7, to cause apoptosis (Annu Rev Genet (2009) 43:95-118).

In the present invention, "inhibiting Bax transfer to the mitochondrial inner membrane" means inhibiting Bax transfer from the cytoplasm to the mitochondrial inner membrane caused by signal transmission generated in the cell during release from viral infection.

The inhibition means is not particularly limited as long as it can inhibit the transfer of Bax existing in the cytoplasm of the host to the mitochondrial inner membrane. Examples thereof include: applying to the cell a molecule that interacts with Bax, inhibits the transfer of Bax to mitochondria, or inhibits a molecule other than Bax that promotes the transfer of Bax to mitochondria (referred to as a "Bax inhibitor"); bax and the like existing in cytoplasm are reduced genetically, but Bax inhibitors are preferably used. That is, in the present invention, the Bax inhibitor is considered as a coronavirus proliferation promoter for proliferating coronaviruses by culturing a host, and can be used for proliferating coronaviruses by culturing a host. In addition, bax inhibitors can also be used in the manufacture of coronavirus proliferation promoters.

The Bax inhibitor is preferably a peptide or a compound that inhibits binding of Ku70 protein to Bax, which is required for transfer of Bax to the mitochondrial inner membrane, and examples thereof include peptides described in Biochem Biophys Res Commun (2004) 321:961-966) or Nat Cell Biol (2003) 5:352-357).

Specifically, val-Pro-Met-Leu-Lys (SEQ ID NO: 1), pro-Met-Leu-Lys-Glu (SEQ ID NO: 2), val-Pro-Thr-Leu-Lys (SEQ ID NO: 3), and Val-Pro-Ala-Leu-Arg (SEQ ID NO: 4), but Val-Pro-Ala-Leu-Lys (SEQ ID NO: 5), pro-Ala-Leu-Lys-Asp (SEQ ID NO: 6), val-Ser-Ala-Leu-Lys (SEQ ID NO: 7), ser-Ala-Leu-Lys-Asp (SEQ ID NO: 8) and the like are exemplified.

The Bax inhibitory effect of the Bax inhibitor can be evaluated, for example, by measuring the binding inhibitory activity of Bax and Ku70 protein. In addition, the ratio of cells that induce apoptosis in the cells and evaluate the apoptosis can be evaluated by adding a Bax inhibitor.

Further, as another Bax inhibitor, a compound that inhibits the binding of tBit to Bax required for Bax transfer to the mitochondrial inner membrane (Bax-tBit binding inhibitor) is preferable, and examples thereof include a compound described in Biochem j. (2009) 423:381-387, j.med Chem (2003) 46:4365-4368, cell Chem biol (2017) 24:493-506.

Specifically, compounds represented by the following (a) to(s) are given.

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Examples of the method include:

compound (a): (±) -1- (3, 6-Dibromocarbazol-9-yl) -3-piperazin-1-ylpropane-2-ol ((±) -1- (3, 6-Dibromocarbazol-9-yl) -3-piperazin-1-ylpropan-2-ol) (CAS No.335165-68-9;Bax channel blocker,BAI1,iMAC1), < Biochem j. (2009) 423:381-387>

Compound (b): (±) -3,6-Dibromo-9- (2-fluoro-3-piperazin-1-yl-propyl) -carbazole ((±) -3,6-Dibromo-9- (2-fluoro-3-piperazin-1-yl-propyl) -carbazole) (CAS No.335166-36-4; imac2) < Biochem j. (2009) 423:381-387>

Compound (c): 9H-Carbazole-9-ethanol (9H-carbazol-9-ethane), 3, 6-dibromo-alpha- [ [4- (3-phenylpropyl) -1-piperazinyl ] methyl ]

(3,6-dibromo-α-[[4-(3-phenylpropyl)-1-piperazinyl]methyl])(CAS No.335166-34-2；iMAC3)<Biochem J.(2009)423:381-387>

Compound (d): 9H-Carbazole-9-ethanol (9H-carbazol-9-ethane), 3, 6-dibromo-alpha- [ [4- [2- (4-morpholinyl) ethyl ] -1-piperazinyl ] methyl ]

(3,6-dibromo-α-[[4-[2-(4-morpholinyl)ethyl]-1-piperazinyl]methyl])(CAS No.607393-53-3；iMAC4)<Biochem J.(2009)423:381-387>

Compound (e): 9H-Carbazole-9-ethanol (9H-carbazol-9-ethane), 3, 6-dichloro- α - [ [4- [2- (4-morpholinyl) ethyl ] -1-piperazinyl ] methyl ]

(3,6-dichloro-α-[[4-[2-(4-morpholinyl)ethyl]-1-piperazinyl]methyl])(CAS No.1198394-60-3；iMAC5<Biochem J.(2009)423:381-387>

Compound (f): 9H-Carbazole-9-ethanol (9H-carbazol-9-ethane), 3, 6-dibromo-alpha- [ [4- [ (4-methoxyphenyl) methyl ] -1-piperazinyl ] methyl ]

(3,6-dibromo-α-[[4-[(4-methoxyphenyl)methyl]-1-piperazinyl]methyl])(CAS No.758683-33-9)<J.Med Chem.(2003)46:4365-4368>

Compound (g): 1-piperazine carboxamide (1-Piperazinecarboxamide), 4- [3- (3, 6-dibromo-9H-carbazol-9-yl) -2-hydroxypropyl ] -N- (4-fluorophenyl)

(4-[3-(3,6-dibromo-9H-carbazol-9-yl)-2-hydroxypropyl]-N-(4-fluorophen yl))(CAS No.607393-52-2)<J.Med Chem.(2003)46:4365-4368>

Compound (h): 9H-Carbazole-9-ethanol (9H-carbazol-9-ethane), 3, 6-dibromo-alpha- [ [4- [ (4-fluorophenyl) methyl ] -1-piperazinyl ] methyl ]

(3,6-dibromo-α-[[4-[(4-fluorophenyl)methyl]-1-piperazinyl]methyl])(CAS No.335166-30-8)<J.Med Chem.(2003)46:4365-4368>

Compound (i): 9H-Carbazole-9-ethanol (9H-carbazol-9-ethane), 3, 6-dibromo-alpha- [ [4- (4-fluorophenyl) -1-piperazinyl ] methyl ]

(3,6-dibromo-α-[[4-(4-fluorophenyl)-1-piperazinyl]methyl])(CAS No.607393-54-4)<J.Med Chem.(2003)46:4365-4368>

Compound (j): 2-propanone ₍ 2-Propanone), 1- (3, 6-dibromo-9H-carbazol-9-yl) -3- (1-piperazinyl) (1- (3, 6-dibromo-9H-carbazol-9-yl) -3- (1-piperazinyl)) (CAS No. 335166-13-7)<J.Med Chem.(2003)46:4365-4368>

Compound (k): 9H-Carbazole-9-ethylamine (9H-carbazol-9-ethane), 3, 6-dibromo-alpha- (1-piperazinylmethyl) (3, 6-dibromo-alpha- (1-piperazinylethyl)) (CAS No. 607393-56-6) < J.Med chem. (2003) 46:4365-4368>

Compound (l): 9H-Carbazole (9H-Carbazole), 3,6-dibromo-9- [2,2-difluoro-3- (1-piperazinyl) propyl ] (3, 6-dibromo-9- [2,2-difluoro-3- (1-piperazinyl) propyl ]) (CAS No. 607393-55-5) < J.Med chem. (2003) 46:4365-4368>

Compound (m): 2-Piperidinecarboxamide (2-Piperidinecarboxamide), N- (6-aminohexyl) -1- (1-oxotridecyl) (N- (6-aminoxyl) -1- (1-oxotricyl)) (CAS No.355138-94-2,355138-95-3) Bci1< Biochem J. (2009) 423:381-387>

Compound (n): 2-Pyrrolidinecarboxamide (2-Pyrrolidinecarboxamide), N- (5-aminopentyl) -1- (1-oxotridecyl) (N- (5-aminopentyl) -1- (1-oxotricyl)) (CAS No. 355139-25-2) Bci < Biochem J. (2009) 423:381-387>

Compound (o): 2-benzofurancarboxylic acid (2-Benzofurancarboxylic acid), 3- [ [ (4-bromophenyl) methyl ] amino ] -2, 3-dihydro-6-hydroxy-, ethyl ester, (2S, 3S)

(3-[[(4-bromophenyl)methyl]amino]-2,3-dihydro-6-hydroxy-,ethyl ester,(2S,3S))(CAS No.2251048-54-9；BJ-1)<Cell Chem Biol.

(2017)24:493-506>

Compound (p): 2-benzofurancarboxylic acid (2-Benzofurancarboxylic acid), 6-hydroxy-, ethyl ester (6-hydroxy-, ethyl ester) (CAS No.906448-92-8; BJ-1-BP) < Cell Chem biol (2017) 24:493-506>

Compound (q): (S) -N- {1- [ (3-Amino-propyl) - (4-bromo-benzyl) -carbamoyl ] -2-phenyl-ethyl } -benzamide ((S) -N- {1- [ (3-Amino-propyl) - (4-bromoo-benzyl) -carbamoyl ] -2-phenyl-eth yl } -benzamide) (DAN 004) < Cell Chem biol (2017) 24:493-506>

Compound (r): n- {1 (S) - [ [3 (S) -Amino-6- (2-methoxy-ethoxymethoxy) -2, 3-dihydro-benzofuran-2 (R) -ylmethyl ] - (4-bromo-benzyl) -carbamoyl ] -2-phenyl-ethyl } -benzamide (N- {1 (S) - [ [3 (S) -Amino-6- (2-method) -2,3-dihydro-benz ofan-2 (R) -ylmethyl ] - (4-bromoo-benzyl) -carbamoyl ] -2-phenyl-ethyl } -benz amide) (CAS No.1592908-75-2; MSN-50) < Cell Chem Biol.

(2017)24:493-506>

Compound(s): n- {1 (S) - [ [2 (R) -Aminomethyl-6- (2-methoxy-ethoxymethoxy) -2, 3-dihydro-benzofuran-3 (S) -yl ] - (4-bromo-benzyl) -carbamoyl ] -2-phenyl-ethyl } -benzamide (N- {1 (S) - [ [2 (R) -amino methyl-6- (2-method) -2, 3-dihydro-benzofuran-3 (S) -yl ] - (4-bromoo-benzyl) -carbamoyl ] -2-phenyl-ethyl } -benz amide) (CAS No.1592908-16-1; MSN-125) < Cell Chem Biol.

(2017) 24:493-506>, etc.

The Bax inhibitory effect in the Bax inhibitor can be evaluated, for example, by measuring the binding inhibitory activity of Bax to tBit protein. In addition, the Bax inhibitor can be evaluated by inducing apoptosis in the cells, evaluating the proportion of the cells that undergo apoptosis, and measuring the membrane potential of mitochondria and the amount of cytochrome c released.

In the present invention, the Bax inhibitor may be used in combination with 1 or 2 or more selected from the group consisting of the above peptides and the compounds represented by the above (a) to(s) as appropriate.

The Bax inhibitor is used at a concentration of 0.001 μm or more, preferably 0.01 μm or more, more preferably 0.1 μm or more, and 200 μm or less, preferably 100 μm or less, more preferably 50 μm or less, and further 0.001 to 200 μm, preferably 0.01 to 100 μm, more preferably 0.1 to 50 μm, relative to a host for propagating coronaviruses (α, β, γ, and δ). In addition, by using 1Bax inhibitor or 2 or more kinds in combination, the coronavirus proliferation ability can be further improved as compared with the use of the peptide or compound alone.

Specifically, the propagation of coronavirus is performed by a step of infecting coronavirus in a host and a step of culturing the infected host under conditions capable of replicating the virus, but in the present invention, the step of inhibiting Bax migration to the mitochondrial inner membrane is performed, for example, before virus infection, after virus infection, or simultaneously with virus infection, but it is preferable to inhibit Bax migration to the mitochondrial inner membrane within 3 hours after virus infection. As a preferred mode, the Bax inhibitor is added to the host before, after or simultaneously with the viral infection, preferably within 3 hours after the viral infection.

As a host for propagation of coronavirus, cultured cells are preferred.

Any cell can be used as long as it is sensitive to coronavirus. Examples of such cells include Vero cells (strain cells derived from African mouse kidney), vero E6 rhesus monkey kidney cells (ATCC CRL-1586), vero FM rhesus monkey kidney cells (ATCC CCL-81), HCT-8 cells (ATCC CCL-244), MRC-5 cells (ATCC CCL-171), caCo-2 human colon cancer (ATCC HTB-37), calu 1 human lung cancer (ICLC HTL 95002), calu 6 human lung cancer (ICLC HTL 97003), POEK porcine fetal kidney (cell culture collection, the Robert Koch-Institute (RKI), berlin, germany), PK13 porcine kidney (cell culture collection, the Bernhard-Nocht-Institute (BNI), hamburg, germany), 293 human embryo kidney (ATCC CRL-1573), FEA embryo fibroblasts (established by Dr. Marcel, newInc), human Swine, human RK, and UK 7 Ml, and Kidney cell (Lab-Ml 7). These cells may be genetically modified for the purpose of changing the sensitivity to coronaviruses or for the purpose of enhancing the viral proliferation, and the cells are not limited to the above-described strain cells, but primary culture cells may be used, but are not limited thereto, as long as they are cells showing sensitivity to coronaviruses.

When cultured cells are used as a host, the Medium used for culturing the cells may be any Medium commonly used for cell culture, such as MEM Medium (manufactured by Wako Co., ltd.) containing Fetal Bovine Serum (FBS), serum-Free Medium (manufactured by ThermoFisher Co., ltd.), RPMI Medium (manufactured by ThermoFisher Co., ltd.), DMEM Medium (manufactured by ThermoFisher Co., ltd.), or the like.

To this medium, a non-essential amino acid or L-glutamine may be added in order to increase the proliferation efficiency of cells. In addition, in order to achieve efficient virus propagation in the culture of coronaviruses, proteases such as trypsin and acetyltrypsin may be added. In order to avoid contamination with microorganisms, 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 to 8, preferably 6.8 to 7.3, suitable for proliferation of animal cells by means of a suitable buffer (e.g. sodium bicarbonate, HEPES).

The method of cell culture includes stationary culture in which cells are attached to the bottom of a culture vessel and suspension culture in which cells are suspended in a medium, but in the case of industrial production, suspension culture is preferable. The suspension culture method includes a method of suspending cells on a carrier such as a microcarrier and culturing the cells in suspension, and a method of suspending cells without using a carrier, but any method may be used.

The cell culture (mixture of cultured cells and medium) may be used directly for the inoculation of coronavirus, but in the case of coronavirus inoculation, it is preferable to wash the cells with fresh medium or a suitable buffer, e.g. PBS, tris buffer.

Specifically, cells grown in a spin Flask (spin flash) or the like are subjected to low-speed centrifugation or membrane filtration, separated into cells and culture supernatant, and fresh medium is added to the cells in the centrifugal sediment or membrane filtration concentrate to suspend the cells, whereby medium exchange is performed.

The cell culture thus obtained is subjected to culture under a certain condition by adding a coronavirus solution thereto. The initial cell density at the beginning of virus culture may be 0.001 to 100X 10 ⁶ cells/mL, preferably 0.01 to 10X 10 ⁶ cells/mL, more preferably 0.1 to 10X 10 ⁶ cells/mL. The measurement of the cell density may be performed by a general method such as a blood cell counting plate. The coronavirus solution to be added to the cell culture may be added so that the infection number MOI (Multiplicity of infection) becomes 0.00001 to 10, but is preferably added so that the infection number becomes 0.0001 to 0.1, more preferably 0.0001 to 0.01.

The culture conditions may be any conditions as long as they allow propagation of coronaviruses in the host. The cell type, virus inoculation amount, culture scale, method, and the like can be appropriately adjusted. For example, when cultured cells are used as a host, the culture temperature is 32 to 39 ℃, preferably 33 to 38 ℃, and the culture period is 1 to 10 days, preferably 2 to 7 days, the carbon dioxide concentration is 3 to 8%, preferably 4 to 5%, the oxygen concentration is 17 to 25%, preferably 20 to 22%.

According to the method of the present invention, coronaviruses can be efficiently proliferated. The virus content in the host can be determined by measuring the virus titer of the plaque, TCID ₅₀ Real-time PCR, indirect immunoperoxidase staining (Indirect Immunoperoxidase Assay) (IPA method) and the like, which can measure the amount of viral RNA.

Coronaviruses are contained in the cells of infected cultured cells and in the culture supernatant. After the completion of the culture, virus particles are recovered from the virus suspension or cell lysate in the host, and concentrated, purified and inactivated, whereby virus particles for inactivated whole-particle vaccines or inactivated split vaccines can be produced. In the case of use as a live vaccine, an attenuated live vaccine, it can be prepared as a virus particle for coronavirus vaccine after concentration and purification.

The recovery of the virus particles is performed by clarifying the virus suspension, specifically by centrifugation or filtration, followed by ultrafiltration for concentration. The virus may be purified by means of ultracentrifugation such as size exclusion chromatography or sucrose density gradient centrifugation, or liquid chromatography. The purified virus solution is inactivated by formalin treatment, ultraviolet irradiation, beta-propiolactone, binary ethyleneimine, etc. under the condition of inactivating whole-grain vaccine or inactivated split vaccine. When used as a live vaccine or an attenuated live vaccine, the purified virus solution is prepared as a virus particle for coronavirus vaccine. The steps of the inactivation treatment and purification are not particularly limited, and may be performed first.

The coronavirus particles can be appropriately added with pharmaceutically acceptable carriers (buffers, emulsifiers, preservatives (e.g., thimerosal), isotonic agents, pH adjusters, adjuvants (e.g., aluminum hydroxide gel), etc.), to produce vaccines in various forms.

With respect to the above-described embodiments, the following modes are also disclosed in the present invention.

<1> a method for propagating coronaviruses in a host, wherein the method comprises a step of inhibiting the transfer of Bax of a host cell to the inner mitochondrial membrane.

<2> the method according to <1>, wherein the step of inhibiting the transfer of Bax to the inner mitochondrial membrane is a step of adding a Bax inhibitor to the host.

<3> the method according to <2>, wherein the Bax inhibitor is 1 or more selected from the group consisting of Val-Pro-Met-Leu-Lys (SEQ ID NO: 1), pro-Met-Leu-Lys-Glu (SEQ ID NO: 2), val-Pro-Thr-Leu-Lys (SEQ ID NO: 3), val-Pro-Ala-Leu-Arg (SEQ ID NO: 4), val-Pro-Ala-Leu-Lys (SEQ ID NO: 5), pro-Ala-Leu-Lys-Asp (SEQ ID NO: 6), val-Ser-Ala-Leu-Lys (SEQ ID NO: 7) and Ser-Ala-Leu-Lys-Asp (SEQ ID NO: 8), and the group of compounds represented by the following (a) to(s).

<4> the method of any one of <1> to <3>, wherein the host is selected from Vero E6 rhesus monkey kidney cells (ATCC CRL-1586), vero FM rhesus monkey kidney cells (ATCC CCL-81), HCT-8 cells (ATCC CCL-244), MRC-5 cells (ATCC CCL-171), caCo-2 human colon cancer (ATCC HTB-37), calu 1 human lung cancer (ICLC HTL 95002), calu 6 human lung cancer (ICLC HTL 97003), poe k porcine fetal kidney (cell culture collection, the Robert Koch-Institute (RKI), berlin, germany), PK13 porcine kidney (cell culture collection, the Bernhard-Nocht-Institute (BNI), hamburg, germany), human embryo kidney (ATCC CRL-1573), FEA embryo fibroblasts (established by dr. Cel, new inc), new, colne), human rbc, and rpc (rkus cells in laboratory, rkus, and rfs cell culture in the laboratory, rat kidney (rks).

The method of any one of <2> to <4>, wherein the Bax inhibitor is used at a concentration of 0.001. Mu.M or more, preferably 0.01. Mu.M or more, more preferably 0.1. Mu.M or more, and 200. Mu.M or less, preferably 100. Mu.M or less, more preferably 50. Mu.M or less, and further 0.001 to 200. Mu.M, preferably 0.01 to 100. Mu.M, more preferably 0.1 to 50. Mu.M, with respect to the host for propagation of coronavirus.

<6> a method for producing coronavirus particles, wherein coronavirus is propagated by the method of any one of <1> to <5>, and virus particles are recovered from a host.

<7> the method of <6>, wherein the virus particle is used in the manufacture of a vaccine for preventing diseases caused by coronaviruses.

<8> the method of <6>, wherein the coronavirus is MERS coronavirus, SARS coronavirus-2, human coronavirus OC43, human coronavirus HKU1, human coronavirus 229E or human coronavirus NL63.

<9> a method for producing a coronavirus vaccine, wherein a vaccine is produced using coronavirus particles produced by the method of any one of <6> to <8 >.

<10> a coronavirus proliferation promoter comprising a Bax inhibitor as an active ingredient.

<11> use of bax inhibitors for promoting proliferation of coronaviruses.

<12> use of bax inhibitor for the manufacture of a coronavirus proliferation promoter.

<13> the preparation according to <10> or the use according to <11> or <12>, wherein the Bax inhibitor is 1 or more selected from the group consisting of Val-Pro-Met-Leu-Lys (SEQ ID NO: 1), pro-Met-Leu-Lys-Glu (SEQ ID NO: 2), val-Pro-Thr-Leu-Lys (SEQ ID NO: 3), val-Pro-Ala-Leu-Arg (SEQ ID NO: 4), val-Pro-Ala-Leu-Lys (SEQ ID NO: 5), pro-Ala-Leu-Lys-Asp (SEQ ID NO: 6), val-Ser-Ala-Leu-Lys (SEQ ID NO: 7) and Ser-Ala-Leu-Lys-Asp (SEQ ID NO: 8), and the group of compounds represented by the following (a) to(s).

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Examples (example)

Example 1 coronavirus proliferation promoting effect by addition of Bax inhibitor.

(1) HCT-8 cells (cell lines from human colon cancer cells, obtained from DS-pharma Biochemical Co.) were cultured in RPMI medium (manufactured by Wako Co.) containing 10% Horse serum (Horse serum; HS) at 37℃and 5% CO ₂ Culturing in the presence of a culture medium. The above HCT-8 cells were inoculated into 24-well plates and used in experiments in the state of cell confluence (Confluent). HCT-8 cells inoculated into the 24-well plate were washed with PBS, and then, RPMI medium (containing high glucose; wako Co.) containing 2% HS was added at 400. Mu.L/well to acclimate the cells for 1 hour.

(2) Coronavirus B1 (Betacoronavirus 1) (HCoV-OC 43 strain: ATCC VR-1558) as a beta-type coronavirus was made to become a Multiplicity of infection (MOI)The cells were infected and incubated for 1 hour with the method of injection) =0.0005. At the time of infection, the virus used RPMI medium (with high glucose) containing 2% HS, 300. Mu.L per 1 well. Next, bax inhibitor (VPMLK (SEQ ID NO: 1) or PMLKE (SEQ ID NO: 2) or iMAC2 (Compound (b)) or BAI1 (Compound (a)); manufactured by TOCRIS bioscience Co.) was diluted to a concentration of 0 to 200. Mu.M in virus infection medium, acetylated trypsin (manufactured by Sigma Co.) was added to a concentration of 2.0. Mu.g/mL, 300. Mu.L/Kong Liang was added thereto, and the mixture was cooled to 33℃and 5% CO ₂ Culturing for 3-5 days under the concentration condition (the final concentration of the acetylated trypsin is 1.0 mug/mL). 3-5 days after infection, the cells were repeatedly thawed by freezing 3 times for the purpose of eluting the viruses in the cells, and the culture supernatant and the cell lysate were collected, centrifuged at 1,200Xg for 10 minutes at 4℃to remove impurities, and the cells were purified by the TCID described below ₅₀ The number of coronaviruses infected was measured (FIG. 1). In the subsequent experiments, RPMI medium (containing high glucose) containing 1.0. Mu.g/mL-acetylated trypsin and 2% -HS as final concentrations was used for the culture of coronaviruses in experiments for evaluating the proliferation of coronaviruses. The test was performed independently 3 times.

(3)TCID ₅₀ Measurement

HCT-8 cells were cultured in planar 96-well plates and used for assays in the state of cell confluence (conflux). After washing the cells with PBS and virus culture medium, the coronavirus culture supernatant was diluted 1000-fold, from which 2-fold dilution series each were prepared, and 6-well/dilution series were used to infect the virus solution. The liquid amount was 100. Mu.L/well, and the cells were infected at 33℃for 1 hour, then 100. Mu.L/well of RPMI medium (containing high glucose) containing 1.0. Mu.g/mL-acetylated trypsin and 2% -HS as final concentrations was added, and the cells were incubated at 33℃with 5% CO ₂ Culturing for 3-5 days under the concentration condition. After incubation, the minimal dilution at which coronavirus infection was observed was detected by immobilization with methanol cooled to-20 ℃. The detection of infected cells was carried out by adding 0.5% bovine serum albumin (BSA, manufactured by Wako Co.) to 50. Mu.L/well, reacting at room temperature for 2 hours, and then reacting with an Anti-coronavirus antigen antibody (Anti-Coronavirus Group Antigen A)ntibody) (nucleoprotein of OC-43, clone542-7D; the solution obtained by diluting MAB9013 with PBS 2000 times (containing 0.05% BSA) was added at 50. Mu.L/well, and reacted overnight at 4 ℃. Then, the secondary antibody, goat anti-mouse IgG+IgM HRP antibody (manufactured by Jackson corporation) was washed 5 times with PBS, and a 2000-fold solution (containing 0.05% BSA) diluted with PBS was added thereto at 50. Mu.L/well, and the mixture was reacted at room temperature for 2 hours, washed 5 times with PBS, and the HRP was developed by DEPDA reaction. Then, based on the well in which color development was confirmed, TCID was calculated according to the half-maximal cumulative (Reed and Muench) method (Am J Hyg 1938; 27:493-497) ₅₀ . The assay detects virus-infected cells by six-way assays. The detection results were subjected to independent 3-time proliferation evaluation (step (2) above), and then TCID detected by each six-way test was performed ₅₀ Values are expressed as mean ± standard error of the results of 3 trials (figures 1 and 2).

(4) The results of this study demonstrate that the addition of Bax inhibitors increases the infectious viral load of coronaviruses (fig. 1 and 2). Especially, the effect of the BAI1 on the increase in virus proliferation is remarkable.

Sequence listing

<110> Kagaku Kogyo Co Ltd

<120> coronavirus proliferation method

<130> KS1761

<150> JP 2020-135920

<151> 2020-08-11

<150> JP 2021-002367

<151> 2021-01-08

<160> 8

<170> PatentIn version 3.5

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Claims (11)

1. A method for propagating coronavirus in a host, wherein,

the method includes a step of inhibiting the transfer of Bax of the host cell to the mitochondrial inner membrane.

2. The method of claim 1, wherein,

the step of inhibiting the transfer of Bax to the mitochondrial inner membrane is a step of adding a Bax inhibitor to the host.

3. The method of claim 2, wherein,

bax inhibitors are at least 1 peptide selected from the group consisting of Val-Pro-Met-Leu-Lys (SEQ ID NO: 1), pro-Met-Leu-Lys-Glu (SEQ ID NO: 2), val-Pro-Thr-Leu-Lys (SEQ ID NO: 3), val-Pro-Ala-Leu-Arg (SEQ ID NO: 4), val-Pro-Ala-Leu-Lys (SEQ ID NO: 5), pro-Ala-Leu-Lys-Asp (SEQ ID NO: 6), val-Ser-Ala-Leu-Lys (SEQ ID NO: 7) and Ser-Ala-Leu-Lys-Asp (SEQ ID NO: 8), and the compounds represented by the following (a) to(s),

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4. a method for producing coronavirus particles, wherein,

the method according to any one of claims 1 to 3, wherein the coronavirus is propagated and virus particles are recovered from the culture supernatant.

5. The method of claim 4, wherein,

the virus particles are used in the manufacture of vaccines for preventing diseases caused by coronaviruses.

6. The method of claim 5, wherein,

the coronavirus is MERS coronavirus, SARS coronavirus-2, human coronavirus OC43, human coronavirus HKU1, human coronavirus 229E or human coronavirus NL63.

7. A method for producing a coronavirus vaccine, wherein,

vaccine manufactured using coronavirus particles prepared by the method of any one of claims 4 to 6.

8. A coronavirus proliferation promoter, wherein,

bax inhibitor is used as effective component.

Use of a bax inhibitor for promoting the proliferation of coronaviruses.

Use of a bax inhibitor for the manufacture of a coronavirus proliferation promoter.

11. The formulation according to claim 8 or the use according to claim 9 or 10, wherein,

bax inhibitors are at least 1 peptide selected from the group consisting of Val-Pro-Met-Leu-Lys (SEQ ID NO: 1), pro-Met-Leu-Lys-Glu (SEQ ID NO: 2), val-Pro-Thr-Leu-Lys (SEQ ID NO: 3), val-Pro-Ala-Leu-Arg (SEQ ID NO: 4), val-Pro-Ala-Leu-Lys (SEQ ID NO: 5), pro-Ala-Leu-Lys-Asp (SEQ ID NO: 6), val-Ser-Ala-Leu-Lys (SEQ ID NO: 7) and Ser-Ala-Leu-Lys-Asp (SEQ ID NO: 8), and the compounds represented by the following (a) to(s),

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