JP7072090B2 - Coronavirus propagation method - Google Patents

Description

The present invention relates to a method for growing a coronavirus in a host.

Colds and acute respiratory syndrome (COVID-19, Middle East respiratory syndrome (MERS), severe acute respiratory syndrome (SARS)) are coronaviruses (SARS coronavirus, SARS coronavirus-2, MERS coronavirus, human coronavirus). It is an infectious disease caused by OC43, human coronavirus 229E, human coronavirus HKU1, human coronavirus NL63), and is infected by droplet infection, contact infection, aerosol infection, air infection, etc. It is a respiratory infection with strong systemic symptoms such as joint pain, pneumonia, shortness of breath, malaise, fatigue, and sore throat. Coronavirus vaccines are being developed around the world to prevent infections caused by coronavirus, and coronavirus-inactivated coronavirus vaccines are the best way to prevent the aggravation of acute respiratory syndrome. Considered a means.

In an inactivated coronavirus vaccine, coronavirus for vaccine production is cultured and propagated in cultured cells, concentrated and purified from the culture solution by centrifugation, virus particles are treated with a surfactant or the like, and all particles inactivated with formarin. A split vaccine or a subunit vaccine in which a vaccine or virus particles are crushed with ether or a surfactant and then further purified can be considered. The inactivated SARS coronavirus-2 vaccine under development has been reported to have the ability to induce a neutralizing antibody against coronavirus (Non-Patent Document 1).
However, when a large amount of a vaccine against coronavirus is produced by cultured cells, it takes time, labor and cost, and there is a problem in terms of supply stability that sudden mass production cannot be performed.

In the case of SARS coronavirus, it is suggested that the viral protein Nsp15 efficiently proliferates the virus by suppressing the apoptosis of the host, and that the virus proliferation is reduced in the mutant lacking the apoptosis suppression mechanism of Nsp15. (Non-Patent Document 2). However, another report reports that although SARS coronavirus induces apoptosis by infecting the host, it cannot suppress replication even if the apoptosis signal is inhibited (Non-Patent Document 3). In addition, the 7A protein of SARS coronavirus can suppress its function and induce apoptosis by binding to the host apoptosis suppressors Bcl-XL, Bcl-2, Mcl-1, Bcl-w, and A1 proteins. Although sex is suggested, it does not show binding to or regulates apoptosis-inducing factors Bax and Bak (Non-Patent Document 4).

From the results obtained from the above non-patent literature, it is inferred that the apoptotic signal is activated in the host with the infection of coronavirus, but which protein is regulated among many signal components. It is not clear at this time whether this is essential for virus growth, and whether compound-induced protein function inhibition or activation, or deletion of a specific gene enhances virus growth. In addition, there are no reports that the growth of the virus is enhanced by the functional inhibition or activation of the protein by the compound or the deletion of a specific gene.

Science.2020; 369: 77-81 Proc Natl Acad Sci USA. 2017; 114: E4251-E4260 DNA Cell Biol. 2005; 24: 496-502 J Virol. 200781: 6346-5)

The present invention relates to providing a method for more efficiently propagating a coronavirus, which is a material for a vaccine, in a host.

As a result of diligent research, the present inventors have suppressed the translocation of Bax, which is known as an apoptosis-promoting protein, to the mitochondrial inner membrane in a host infected with coronavirus, thereby suppressing the proliferation of the virus. Was found to be higher than in the steady state (virus-infected control) and increased virus production.

That is, the present invention relates to the following 1) to 4).
1) A method for propagating a coronavirus in a host, comprising the step of suppressing the transfer of Bax to the inner mitochondrial membrane of the host cell.
2) A method for preparing coronavirus particles, in which the coronavirus is propagated by the method of 1) and the virus particles are recovered from the host.
3) A method for producing a coronavirus vaccine, which comprises producing a vaccine using coronavirus particles prepared by the method of 2).
4) A coronavirus growth-promoting agent containing a Bax inhibitor as an active ingredient.

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

Coronavirus (HCoV-OC43) growth promoting effect by adding Bax inhibitory peptide. Coronavirus (HCoV-OC43) growth promoting effect by adding Bax inhibitor compound. Coronavirus (SARS-CoV-2) growth promoting effect by adding Bax inhibitor compound.

In the present invention, the coronavirus may be any genotype of alpha type, beta type, gamma type and delta type, but the beta type can be preferably exemplified. Examples of beta-type coronaviruses include MERS coronavirus (MERS-CoV), SARS coronavirus (SARS-CoV), SARS coronavirus-2 (SARS-CoV-2), human coronavirus OC43 (HCoV-OC43), and human coronavirus. The virus HKU1 (HCoV-HKU1) can be mentioned. Examples of the alpha coronavirus include human coronavirus 229E (HCoV-229) and human coronavirus NL63 (HCoV-NL63).

In addition, the target virus strain may be any virus strain that can infect humans as a host, and is not particularly limited as long as it is a virus strain that can infect primates, livestock, poultry, and animals raised as pets. In addition to the coronaviruses that have been reported to be isolated, subtypes that will be isolated and identified in the future are also included.

Further, 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 engineered in cultured cells.

In the present invention, "Bax" refers to an apoptosis-promoting protein belonging to the Bcl-2 family. Proteins belonging to the Bcl-2 family have one or more amino acid sequences called BH (Bcl-2 homology) domains. In addition, since it has a highly hydrophobic TM (transmembrane) region on the C-terminal side, it can migrate to the mitochondrial membrane and control apoptosis.

The main function of the Bcl-2 family proteins is the regulation of apoptosis by regulating mitochondrial permeability. The anti-apoptotic proteins Bcl-2 and Bcl-xL are present on the outer wall of mitochondria and inhibit the release of cytochrome c. The apoptotic proteins Bad, Bid, Bax and Bim are present in the cytoplasm and migrate to the inner mitochondrial membrane by the signal of cell death, where they promote the release of cytochrome c. Bax binds to the Ku70 protein and is induced near the outer mitochondrial membrane. The Bid is cleaved by Caspase-9 to become an activator tBit (truncated Bid), which binds to the Bax induced near the outer mitochondrial membrane to induce the penetration of the Bax into the inner mitochondrial membrane. Cytochrome c that has flowed out of the cell forms a complex with Apaf-1, activates caspase-9, and further activates caspases 3, 6, and 7 to cause apoptosis (Annu Rev Genet (Annu Rev Genet). 2009) 43: 95-118).

In the present invention, "suppression of the translocation of Bax to the inner mitochondrial membrane" means suppressing the translocation of Bax from the cytoplasm to the inner mitochondrial membrane due to signal transduction that occurs inside the cell in the process of release from viral infection. Means.

The suppressing means is not particularly limited as long as it can suppress the transfer of Bax present in the cytoplasm of the host to the inner mitochondrial membrane. For example, applying a molecule that interacts with Bax to suppress the mitochondrial translocation of Bax or a molecule other than Bax that promotes mitochondrial translocation of Bax (referred to as "Bax inhibitor") to the cell, intracytoplasmic reticulum. The use of a Bax inhibitor is preferable, although it may be mentioned that the Bax present in the above is genetically engineered. That is, in the present invention, the Bax inhibitor can be said to be a coronavirus growth promoter for growing coronavirus by culturing the host, and can be used to grow coronavirus by culturing the host. It can also be said that the Bax inhibitor can be used to produce a coronavirus growth promoter.

Bax inhibitors preferably include peptides and compounds that inhibit the binding of Bax to the Ku70 protein required for Bax translocation to the mitochondrial inner membrane, eg, Biochem Biophys Res Commun (2004) 321: 961-. The peptides described in 966) and Nat Cell Biol (2003) 5: 352-357) can be mentioned.
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 also Val-Pro-Ala-Leu-Lys (SEQ ID NO: 5) and Pro-Ala-Leu-Lys-Asp (SEQ ID NO: 5). No. 6), Val-Ser-Ala-Leu-Lys (SEQ ID NO: 7), Ser-Ala-Leu-Lys-Asp (SEQ ID NO: 8) and the like. Here, the amino acid sequences shown in SEQ ID NOs: 1 and 2 are the sequences of the Bax binding region in human Ku70, SEQ ID NO: 3 is the sequence of the Bax binding region in mouse Ku70, and SEQ ID NO: 4 is the sequence of the Bax binding region in rat Ku70. Is an array of Bax binding regions in. Peptides consisting of these amino acid sequences inhibit the binding of Ku70 to Bax and suppress the function of Bax (Biochem Biophys Res Commun (2004) 321: 961-966), Nat Cell Biol (2003) 5: 352- 357). The amino acid sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 are the amino acid sequences of the Bax-binding region of Ku70 derived from water birds, and the amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8 are the Bax binding of Ku70 derived from chickens. It is specified as the amino acid sequence of the region, and it can be said that it is highly probable that it binds to Bax and suppresses the function of Bax, similarly to the peptides of SEQ ID NOs: 1 to 4.

The Bax inhibitory effect of such a Bax inhibitor can be evaluated, for example, by measuring the binding inhibitory activity between Bax and the Ku70 protein. Further, it can be evaluated by inducing apoptosis in the cells under the condition to which the Bax inhibitor is added and evaluating the ratio of the apoptotic cells.

Further, as another Bax inhibitor, preferably, a compound (Bax-tBit binding inhibitor) that inhibits the binding between tBit and Bax, which is necessary for the translocation of Bax to the inner mitochondrial membrane, can be mentioned, for example, Biochem J. (2009) 423: 381-387, J. Med Chem. (2003) 46: 4365-4368, Cell Chem Biol. (2017) 24: 493-506.
Specific examples thereof include the compounds shown in the following (a) to (s).

Compound (a): (±) -1- (3,6-Dibromocarbazol-9-yl) -3-piperazin-1-ylpropan-2-ol (CAS No. 335165-68-9; Bax channel blocker, BAI1, BAI1, iMAC1), <Biochem J. (2009) 423: 381-387>
Compound (b): (±) -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, 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, 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, 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, 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-Piperazinecarboxamide, 4- [3- (3,6-dibromo-9H-carbazol-9-yl) -2-hydroxypropyl] -N- (4-fluorophenyl) (CAS No. 607393-52- 2) <J. Med Chem. (2003) 46: 4365-4368>
Compound (h): 9H-Carbazole-9-ethanol, 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, 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, 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-ethanamine, 3,6-dibromo-α-(1-piperazinylmethyl) (CAS No. 607393-56-6) <J. Med Chem. (2003) 46: 4365-4368 ＞
Compound (l): 9H-Carbazole, 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, N- (6-aminohexyl) -1- (1-oxotridecyl) (CAS)
No. 355138-94-2, 355138-95-3) Bci1 <Biochem J. (2009) 423: 381-387>
Compound (n): 2-Pyrrolidinecarboxamide, N- (5-aminopentyl) -1- (1-oxotridecyl) (CAS No. 355139-25-2) Bci2 <Biochem J. (2009) 423: 381-387>
Compound (o): 2-Benzofurancarboxylic acid, 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, 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 (DAN004) <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 (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 (CAS No. 1592908-16-1; MSN-125) <Cell Chem Biol. (2017) 24: 493-506> and the like.

The Bax inhibitory effect of such a Bax inhibitor can be evaluated, for example, by measuring the binding inhibitory activity between Bax and the tBit protein. Further, it can be evaluated by inducing apoptosis in the cells under the condition of adding a Bax inhibitor and evaluating the ratio of the apoptotic cells, or by measuring the membrane potential of mitochondria and the amount of cytochrome c released.

In the present invention, such a Bax inhibitor may be used in an appropriate combination of one or two or more selected from the group consisting of the above peptides and the group consisting of the compounds represented by the above (a) to (s). can.

The Bax inhibitor has a concentration of 0.001 μM or more, preferably 0.01 μM or more, and more preferably 0.1 μM with respect to the host for growing the coronavirus (alpha type, beta type, gamma type, and delta type). As described above, it is used at 200 μM or less, preferably 100 μM or less, more preferably 50 μM or less, and 0.001 to 200 μM, preferably 0.010.1 to 100 μM, more preferably 0.1 to 50 μM. Further, the coronavirus growth ability may be further improved as compared with the use of the peptide or the compound alone by using one kind of Bax inhibitor or using two or more kinds of them in combination.

The growth of coronavirus is specifically carried out by a step of infecting the host with the coronavirus and a step of culturing the infected host under conditions in which the virus can replicate. In the present invention, the mitochondria of Bax are carried out. The step of suppressing the transfer to the intima is performed, for example, before the virus infection, after the virus infection, or at the same time as the virus infection, but within 3 hours after the virus infection, the transfer of Bax to the mitochondrial inner membrane is suppressed. preferable. A preferred embodiment is to add the Bax inhibitor to the host before, after the virus infection, or at the same time as the virus infection, preferably to add the Bax inhibitor to the host within 3 hours after the virus infection. Can be mentioned.

As a host used for the growth of coronavirus, cultured cells are preferably preferable.
As the cultured cells, any cells that are sensitive to coronavirus can be used. Such cells include Vero cells (strained cells derived from African green monkey kidney), such as Vero E6 rhesus kidney cells (ATCC CRL-1586), Vero FM rhesus kidney cells (ATCC CCL-81), HCT-8 cells (ATCC). CCL-244), MRC-5 cells (ATCC CCL-171), CaCo-2 human kidney carcinoma (ATCC HTB-37), Calu 1 human lung carcinoma (ICLC HTL95002), Calu 6 humanKlCl porcine focal kidney (cell culture collection of the)
Ｒｏｂｅｒｔ Ｋｏｃｈ－Ｉｎｓｔｉｔｕｔｅ（ＲＫＩ），Ｂｅｒｌｉｎ，Ｇｅｒｍａｎｙ）、ＰＫ１３ ｐｏｒｃｉｎｅ ｋｉｄｎｅｙ（ｃｅｌｌ ｃｕｌｔｕｒｅ ｃｏｌｌｅｃｔｉｏｎ ｏｆ ｔｈｅ Ｂｅｒｎｈａｒｄ－Ｎｏｃｈｔ－Ｉｎｓｔｉｔｕｔｅ（ＢＮＩ），Ｈａｍｂｕｒｇ，Ｇｅｒｍａｎｙ）、２９３ ｈｕｍａｎ ｅｍｂｒｙｏｎｉｃ ｋｉｄｎｅｙ（ＡＴＣＣ ＣＲＬ－１５７３）、ＦＥＡ ｆｅｌｉｎｅ ｅｍｂｒｙｏｎｉｃ fibroblast (estavlyshed by Dr. Marcel Asper, NewLab Inc., Koch Institute), RD human rhabdomyosarcoma cells (RKI), PS porcine Kidney Cell .. These cells may be genetically modified for the purpose of further changing the susceptibility to coronavirus or enhancing the viral proliferation, and if the cells show susceptibility to coronavirus, the above-mentioned strains may be obtained. Not limited to cells, primary cultured cells may be used, and the present invention is not limited thereto.

When cultured cells are used as the host, the medium for culturing the cells is a medium usually used for cell culture, for example, a MEM medium containing fetal bovine serum (FBS) (manufactured by Wako), a serum-free medium (Serum-Free). Medium) (manufactured by Thermo Fisher), RPMI medium (manufactured by Thermo Fisher), DMEM medium (manufactured by Thermo Fisher) and the like may be used.

Non-essential amino acids and L-glutamine can be added to the medium in order to increase the growth efficiency of cells. Further, in the culture of coronavirus, proteases such as trypsin and acetylated trypsin can be added in order to realize efficient viral growth. Further, in order to avoid contamination of microorganisms, antibiotics generally used for 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 a suitable buffer (eg, sodium hydrogen carbonate, HEPES).

Examples of the cell culture method include static culture in which cells are attached to the bottom of an incubator and suspension culture in which cells are suspended and cultured in a medium, but suspension culture is preferable when performing at an industrial production level. Examples of the method of suspension culture include a method of attaching cells to a carrier such as a microcarrier and suspending and culturing the cells, a method of suspending and culturing cells without using a carrier, and the like. You may use it.

The cell culture (mixture of cultured cells and medium) can be used as it is for coronavirus inoculation, but when inoculating coronavirus, fresh medium or a suitable buffer, for example, PBS, Tris buffer, etc. It is preferable that the cells are washed with the virus.
Specifically, cells cultured and proliferated in a spinner flask or the like are subjected to slow centrifugation or membrane filtration, separated into cells and culture supernatant, and fresh medium is added to the cells of the centrifugal sediment or membrane filtration concentrate to suspend the cells. By doing so, the medium is exchanged.

A coronavirus solution is added to the cell culture thus obtained, and the culture is carried out under certain conditions. The initial cell density at the start of virus culture can be 0.001 to 100 × 10 ⁶ cells / mL, preferably 0.01 to 10 × 10 ⁶ cells / mL, more preferably 0.1 to 10 ×. It is 10 ⁶ cells / mL. The cell density may be measured according to a general method using a blood cell calculator or the like. The coronavirus solution added to the cell culture can be added so that the infectious titer MOI (Multiplicity of infection) is 0.00001 to 10, but is preferably 0.0001 to 0.1, more preferably 0.0001. It can be added at ~ 0.01.

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

According to the method of the present invention, the coronavirus can be efficiently propagated. The virus content in the host can be measured by a plaque assay for measuring the virus infectivity titer, a TCID ₅₀ , a real-time PCR capable of measuring the amount of virus RNA, an Indirect Immunoperoxidase Assay (IPA method), or the like.

Coronavirus is contained in the cells of infected cultured cells and in the culture supernatant. After the culture is completed, virus particles can be recovered from the virus suspension or cell lysate in the host and concentrated, purified and inactivated to prepare virus particles for inactivated whole-grain vaccine or inactivated split vaccine. can. When used as a live vaccine or an attenuated live vaccine, it can be prepared as virus particles for a coronavirus vaccine after concentration and purification.

Recovery of virus particles is performed by clarification of the virus suspension, specifically by centrifugation or filtration, followed by extrafiltration for concentration. Purification of the virus can be performed by means such as ultracentrifugation such as size exclusion chromatography and sucrose density gradient centrifugation, and liquid chromatography. In the case of an inactivated whole-grain vaccine or an inactivated split vaccine, the purified virus solution is inactivated by formalin treatment, ultraviolet irradiation, beta-propiolactone, binary ethyleneimine, or the like. When used as a live vaccine or an attenuated live vaccine, the purified virus solution is prepared as virus particles for a coronavirus vaccine. Either the inactivation treatment or the purification step may be performed first, and is not particularly limited.

A carrier (buffer, emulsifier, preservative (eg, Timerosar), tonicity agent, pH adjuster, adjuvant (eg, aluminum hydroxide gel), etc., which is appropriately acceptable as a medicine, is added to such coronavirus particles. , Various types of vaccines can be produced.

Regarding the above-described embodiments, the following aspects are further disclosed in the present invention.
<1> A method for propagating a coronavirus in a host, which comprises a step of suppressing the transfer of Bax to the inner mitochondrial membrane of a host cell.
<2> The method of <1>, wherein the step of suppressing the transfer of Bax to the inner mitochondrial membrane is a step of adding a Bax inhibitor to the cells.
<3> The Bax inhibitor is 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: 1). 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- Selected from the peptide group consisting of Ser-Ala-Leu-Lys (SEQ ID NO: 7) and Ser-Ala-Leu-Lys-Asp (SEQ ID NO: 8), and the compound group represented by the following (a) to (s). The method of <2>, which is 1 or more.
<4> The host is Vero E6 rhesus kidney cells (ATCC).
CRL-1586), Vero FM kidney cells (ATCC CCL-81), HCT-8 cells (ATCC CCL-244), MRC-5 cells (ATCC CCL-171), CaCo-2 human lung cellinoma (ATCC CCL-37C) ）、Ｃａｌｕ １ ｈｕｍａｎ ｌｕｎｇ ｃａｒｃｉｎｏｍａ（ＩＣＬＣ ＨＴＬ９５００２）、Ｃａｌｕ ６ ｈｕｍａｎ ｌｕｎｇ ｃａｒｃｉｎｏｍａ（ＩＣＬＣ ＨＴＬ９７００３）、ＰＯＥＫ ｐｏｒｃｉｎｅ ｆｏｅｔａｌ ｋｉｄｎｅｙ（ｃｅｌｌ ｃｕｌｔｕｒｅ ｃｏｌｌｅｃｔｉｏｎ ｏｆ ｔｈｅ Ｒｏｂｅｒｔ Ｋｏｃｈ－Ｉｎｓｔｉｔｕｔｅ（ＲＫＩ），Ｂｅｒｌｉｎ，Ｇｅｒｍａｎｙ）、ＰＫ１３ ｐｏｒｃｉｎｅ ｋｉｄｎｅｙ（ｃｅｌｌ culture collection of the Bernhard-Nocht-Institute (BNI), Hamburg, Germany), 293 human
ｅｍｂｒｙｏｎｉｃ ｋｉｄｎｅｙ（ＡＴＣＣ ＣＲＬ－１５７３）、ＦＥＡ ｆｅｌｉｎｅ ｅｍｂｒｙｏｎｉｃ ｆｉｂｒｏｂｌａｓｔ（ｅｓｔａｂｌｉｓｈｅｄ ｂｙ Ｄｒ．Ｍａｒｃｅｌ Ａｓｐｅｒ，ＮｅｗＬａｂ Ｉｎｃ．，Ｃｏｌｏｇｎｅ）、ＲＤ ｈｕｍａｎ ｒｈａｂｄｏｍｙｏｓａｒｃｏｍａ ｃｅｌｌｓ（ＲＫＩ）、ＰＳ ｐｏｒｃｉｎｅ ｋｉｄｎｅｙ ｃｅｌｌｓ（ＲＫＩ）、及びＬＬＣ－ＭＫ２ Ａｆｒｉｃａｎ
The method according to any one of <1> to <3>, which is a cultured cell selected from green monkey kidney cells (ATTC CCL-7).
<5> The Bax inhibitor has 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, with respect to the host for growing the coronavirus. The method according to any one of <2> to <4>, which is more preferably 50 μM or less, and more preferably 0.001 to 200 μM, preferably 0.01 to 100 μM, and more preferably 0.1 to 50 μM.
<6> A method for preparing coronavirus particles, wherein the coronavirus is propagated by any of the methods <1> to <5>, and the virus particles are recovered from the host.
<7> The method of <6>, wherein the virus particles are used in the production of a vaccine for preventing a disease caused by a coronavirus.
<8> The method of <6>, wherein the coronavirus is MERS coronavirus, SARS 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, which comprises producing a vaccine using coronavirus particles prepared by any of the methods <9> and <8>.
<10> A coronavirus growth-promoting agent containing a Bax inhibitor as an active ingredient.
<11> Use of a Bax inhibitor to promote the growth of coronavirus.
<12> Use of a Bax inhibitor for producing a coronavirus growth promoter.
<13> Bax inhibitors are 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: 1). 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- Selected from the peptide group consisting of Ser-Ala-Leu-Lys (SEQ ID NO: 7) and Ser-Ala-Leu-Lys-Asp (SEQ ID NO: 8), and the compound group represented by the following (a) to (s). Use of <10> agent or <11> or <12>, which is 1 or more.

Example 1 Coronavirus growth promoting effect by addition of Bax inhibitor (1) HCT-8 cells (cell line derived from human colon cancer cells, obtained from DS Pharma Biochemical) 10% horse serum (HS) The cells were cultured in a containing RPMI medium (manufactured by Wako) at 37 ° C. in the presence of 5% CO ₂ . The above HCT-8 cells were seeded on a 24-well plate and used in the test in a confluent state. The HCT-8 cells seeded on the above-mentioned 24-well plate were washed with PBS, and then 2% HS-containing RPMI medium (high glucose content; manufactured by Wako) was added at 400 μL / well and acclimatized for 1 hour.

(2) The cells were infected with Betacoronavirus 1 (HCoV-OC43 strain: ATCC VR-1558), which is a betacoronavirus, so that the infection value was MOI (Multiplicity of infection) = 0.0005, and the cells were incubated for 1 hour. At the time of infection, 300 μL of virus was added per well using RPMI medium containing 2% HS (containing high glucose). Then, a 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) was added to a virus infection medium at a concentration of 0 to 200 μM. Dilute with, add acetylated trypsin (manufactured by Sigma) to a concentration of 2.0 μg / mL, add 300 μL / well, and incubate under the conditions of 33 ° C. and 5% CO ₂ concentration for 3 to 5 days (). The final concentration of acetylated trypsin is 1.0 μg / mL). After 3 to 5 days of infection, freeze-thaw was repeated 3 times for the purpose of elution of the virus in the cells, and the culture supernatant and cell lysate were collected and centrifuged at 1,200 × g for 10 minutes under the condition of 4 ° C. , The impurities were removed, and the infectious titer of coronavirus was measured by TCID50 measurement described later (FIG. 1). In the subsequent experiments to evaluate the growth of coronavirus, RPMI medium (containing high glucose) containing 1.0 μg / mL-acetylated trypsin and 2% -HS at the final concentration was used for coronavirus culture. Using. The test was performed independently three times.

(3) TCID50 measurement HCT-8 cells were cultured on a flat 96-well plate and used in the test in a confluent state. After washing the cells with PBS and virus culture medium, the coronavirus culture supernatant was diluted 1000-fold, and the virus solution prepared with 2-fold dilution series was infected with 6 to 8 wells / dilution series. The liquid volume was 100 μL / well, and the cells were infected for 1 hour under 33 ° C. conditions, and then 100 μL / L of RPMI medium (high glucose content) containing 1.0 μg / mL-acetylated trypsin and 2% -HS at the final concentration. Wells were added and the cells were cultured at 33 ° C. and 5% CO ₂ concentration for 3 to 5 days. After culturing, the cells were immobilized with methanol cooled to −20 ° C., and the minimum dilution concentration at which coronavirus infection was observed was detected. For detection of infected cells, 0.5% bovine serum albumin (BSA, manufactured by Wako) was added at 50 μL / well and reacted at room temperature for 2 hours, and then Anti-Coronavirus Group Antibodies (nucleoprotein) were used as the primary antibody against coronavirus. A solution of of OC-43, clone 542-7D; manufactured by Merck, MAB9013) diluted 2000-fold with PBS (containing 0.05% BSA) was added at 50 μL / well and reacted overnight at 4 ° C. Then, it was washed 5 times with PBS, and a solution obtained by diluting goat anti-mouse IgG + IgM HRP antibody (manufactured by Jackson) 2000 times with PBS (containing 0.05% BSA) as a secondary antibody was added at 50 μL / well. The reaction was carried out at room temperature for 2 hours, washed 5 times with PBS, and HRP was developed using the DEPDA reaction. Then, TCID50 was calculated according to the Reed and Muench method (Am J Hyg 1938; 27: 493-497) based on the wells in which color development was observed. The test detected virus-infected cells by 6-fold measurement. The detection results are shown as the average value ± standard error of the results for the three tests, with the TCID50 value detected by six-fold measurement each time after three independent growth evaluations (procedure described in (2) above). 1 and 2).

(4) As a result of this study, it was clarified that the addition of the Bax inhibitor increases the amount of infectious virus of coronavirus (FIGS. 1 and 2). Among the Bax-tBit binding inhibitors, the virus growth-promoting effect of BAI1 was particularly remarkable.

Example 2 SARS-CoV-2 proliferation promoting effect by addition of Bax inhibitor (1) VeroE6 / TMPRSS2 cells (JCRB1819, distributed by National Institute of Infectious Diseases) in DMEM medium containing 10% FBS (manufactured by Wako). The cells were cultured at 37 ° C. in the presence of 5% CO ₂ . The VeroE6 cells were seeded on a flat 6-well plate and used in the test in a confluent state. VeroE6 cells seeded on the above-mentioned flat 6-well plate were washed with PBS and then cultured in serum-free DMEM medium (manufactured by Wako) until virus infection.

(2) SARS-CoV-2 NIID isolate (JPN / TY / WK-521, National Institute of Infectious Diseases isolate), which is a new type of coronavirus, was applied to the cells with an infection titer MOI (Multiplicity of infection) = 0.001. Infected to become and incubated for 1 hour. At the time of infection, the virus was diluted with EMEM medium (low glucose content, manufactured by Sigma), and as described in (1) above, the culture medium of VeroE6 cells cultured in serum-free DMEM medium was removed, and then per well. 100 μL of virus solution was added to infect cells. After infection with the virus, the added virus solution is removed by suction, and then a medium obtained by diluting the Bax inhibitor (VPMLK (SEQ ID NO: 1) or PMLKE (SEQ ID NO: 2) with a serum-free EMEM medium to a concentration of 0 to 100 μM) is used. The cells were added at 2 mL / well and cultured under the conditions of 37 ° C. and 5% CO ₂ concentration. 48 hours after infection, the culture supernatant was frozen at -80 ° C for the purpose of elution of the virus in the cells, and thawed. The cell lysate was collected and centrifuged at 1,000 × g for 15 minutes under the condition of 4 ° C. to remove impurities, and the infectious titer of coronavirus was measured by the plaque measurement described later of the obtained coronavirus eluent. (Fig. 3). The test was independently performed 3 times.

(3) Plaque measurement VeroE6 cells were cultured on a flat 6-well plate and used for the test in a confluent state. After washing the cells with PBS and serum-free EMEM medium, the coronavirus eluate was appropriately diluted with serum-free EMEM medium, and the prepared virus solution was infected with a 3-well / dilution series (triple measurement). The liquid volume at the time of infection was 100 μL / well, infected under 37 ° C. conditions for 1 hour, washed with PBS, and then 2 mL / well of EMEM medium containing 0.8% agarose was added to 37 ° C. and 5% CO ₂ concentration. Under the conditions, the cells were cultured for 42 hours until plaques were formed. After culturing, the number of plaques was visually counted and virus-infected cells were detected in PFU / mL units. As for the detection results, the amount of virus detected by triple measurement was shown as an average value ± standard error after three independent growth evaluations (procedure described in (2) above) (FIG. 3). The significance level was a risk rate of 5% (student's t-test).

(4) As a result of this study, it was clarified that the addition of the Bax inhibitor increases the amount of infectious virus of SARS coronavirus-2 (SARS-CoV-2) (Fig. 3). Regarding the evaluation of the proliferation of SARS-CoV-2 using VeroE6 cells, VPMLK (SEQ ID NO: 1) was found to have an effect of enhancing the proliferation of the virus at a concentration of 100 μM, and PMLKE (SEQ ID NO: 2) had a low concentration of 10-50 μM. Showed a virus growth promoting effect.

Claims (7)

A method for propagating coronavirus in a host other than human, including a step of suppressing the transfer of Bax to the mitochondrial inner membrane of a host cell, and a step of suppressing the transfer of Bax to the mitochondrial inner membrane is a Ku70 protein. A method of adding a Bax inhibitor that inhibits binding to Bax or inhibits binding of tBit to Bax to the host . Bax inhibitors are 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 -One or more selected from the peptide group consisting of Leu-Lys (SEQ ID NO: 7) and Ser-Ala-Leu-Lys-Asp (SEQ ID NO: 8), and the compound group represented by the following (a) to (s). , The method according to claim 1 .

A method for preparing coronavirus particles, wherein the coronavirus is propagated by the method according to claim 1 or 2 , and the virus particles are recovered from the culture supernatant. The method of claim 3 , wherein the virus particles are used in the production of a vaccine for preventing a disease caused by a coronavirus. The method according to claim 4 , wherein the coronavirus is MERS coronavirus, SARS coronavirus, SARS coronavirus-2, human coronavirus OC43, human coronavirus HKU1, human coronavirus 229E or human coronavirus NL63. A method for producing a coronavirus vaccine, which comprises producing a vaccine using coronavirus particles prepared by the method according to any one of claims 3 to 5 . A coronavirus growth-promoting agent containing a Bax inhibitor as an active ingredient .
Bax inhibitors are 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 -One or more selected from the peptide group consisting of Leu-Lys (SEQ ID NO: 7) and Ser-Ala-Leu-Lys-Asp (SEQ ID NO: 8), and the compound group represented by the following (a) to (s). , Corona virus growth promoter .

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