WO2022119560A1 - Cell strain having increased virus production ability and production method thereof - Google Patents
Cell strain having increased virus production ability and production method thereof Download PDFInfo
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- WO2022119560A1 WO2022119560A1 PCT/US2020/062749 US2020062749W WO2022119560A1 WO 2022119560 A1 WO2022119560 A1 WO 2022119560A1 US 2020062749 W US2020062749 W US 2020062749W WO 2022119560 A1 WO2022119560 A1 WO 2022119560A1
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C07K2319/00—Fusion polypeptide
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- C07K2319/41—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a Myc-tag
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- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16151—Methods of production or purification of viral material
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- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
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- C12N2770/24011—Flaviviridae
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- C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
- C12N2770/24151—Methods of production or purification of viral material
- C12N2770/24152—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
Definitions
- the present invention relates to a cell line having an increased ability to produce virus and a method for preparing thereof, and more particularly, to a cell line that has an increased ability to produce virus, due to the overexpression of a CCCH-type zinc finger protein and to a production method thereof.
- Cell-culture based vaccine production may offer distinct advantages over the conventional methods of vaccine production (ie, egg-based or mouse brain-based vaccine production) including the following: (1) the source of the cell lines for vaccine production are more easily and conveniently, which can rapidly produce vaccine supplies in the event of a pandemic; (2) offer a higher similarity with the circulating strains and potentially improved protection; (3) a more controlled production process and faster start-up of the vaccine manufacturing process; (4) avoid the risk of severe allergic reaction from egg-based and mouse brain-based vaccines.
- the cell-culture production process might cause lower virus yield and cost-effectiveness. Thus, it will be a key issue in improving cell-culture based vaccine production with higher virus yield.
- CCCH-type zinc finger proteins are RNA-binding molecules involved in RNA metabolism pathways such as RNA splicing, polyadenylation, and messenger RNA (mRNA) decay.
- mRNA messenger RNA
- CCCH-type zinc finger family such as ZC3H12A (MCP-1 induced protein 1), ZFP36 (Tristetraproline, TTP) and ZC3H2 (Zinc finger CCCH- type antiviral protein 1, also known as Zinc finger antiviral protein (ZAP)) were suggesting that they can directly bind to specific viral RNA sequences through its CCCH-type zinc finger motifs and inhibits the replication of some virus.
- ZAP Zinc finger antiviral protein
- the present invention demonstrates that cells overexpressing ZC3HAV1L (Zinc finger CCCH-type, antiviral 1-like) protein resulted in higher levels of virus production.
- one aspect of the invention relates to a virus-producing cell which introduced ZC3HAV1L gene in a cell having an ability to produce virus, wherein the cell having an increased ability to produce virus by overexpressing the product of said ZC3HAV1L gene.
- the cell is infected with a virus.
- the virus is Flavivirus or Alphainfluenzavirus.
- the virus is selected from the group consisting of Japanese encephalitis virus (JEV), Dengue virus (DENV), and Influenza A virus (IAV).
- JEV Japanese encephalitis virus
- DEV Dengue virus
- IAV Influenza A virus
- the mutant cell is a Vero cell.
- the present invention is related to a method for preparing a virus-producing cell, the method comprising introducing a ZC3HAV1L gene to a virus-producing cell.
- the method further comprising infecting the mutant cell with a virus.
- the virus is Flavivirus or Alphainfluenzavirus.
- the virus is selected from the group consisting of Japanese encephalitis virus (JEV), Dengue virus (DENV), and Influenza A virus (IAV).
- JEV Japanese encephalitis virus
- DEV Dengue virus
- IAV Influenza A virus
- the mutant cell is an animal cell.
- the animal cell is a Vero cell.
- Another aspect of the present invention relates to a method for producing a desired virus, the method comprising the steps of:
- the mutant cell is infected with 0.01-0.1 MOI of the desired virus.
- a further aspect of the present invention relates to a method for producing a desired virus, the method comprising the steps of: (a) infecting the mutant cell of claim 1 with the desired virus; (b) culturing the mutant cell infected with the desired virus, and then harvesting the supernatant of the culture to recover the desired virus; and (c) attenuating or inactivating the recovered virus.
- Fig. 4 shows the immunofluorescence of the stable cell line with ZC3HAVlL-flag overexpression in Vero cells.
- FIG. 5 shows the production of viral protein NS3 in cells infected with JEV-EGFP using Western blot.
- Fig. 6 illustrates the virus growth curve in JEV-infected cells.
- Fig. 7 illustrates the virus growth curve in DENV-2-infected cells.
- Fig. 8 illustrates the virus growth curve in lAV-infected cells.
- JEV PR-9 strain (GenBank accession: AF014161) (Chen, LK et al. Virology 1996, 223, 79-88.) and DENV-2 PL046 strain (GenBank accession: KJ734727) (Lin, YL et al. J Virol 1998, 72, 9729-9737.) were propagated in mosquito C6/36 cells (ATCC, CRL-1660) maintained in RPMI-1640 medium supplemented with 5% fetal bovine serum (FBS). JEV-EGFP (RP-9 strain) and DENV-2-EGFP (PL046 strain) were a kind gift from Dr. Yi-Ling Lin.
- JEV and DENV titration was determined by plaque-forming assay in baby hamster kidney BHK-21 cells (ATCC, CCL-10) grown in RPMI-1640 medium supplemented with 5% FBS, 2 mM L-glutamine and 1% penicillin-streptomycin (P/S).
- the influenza A/PuertoRico/8/1934 (H1N1) (GenBank accession: CY009444) used in this study was propagated in Madin-Darby canine kidney (MDCK) cells, which were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibico) containing 10% fetal bovine serum (FBS). Viral titration was by plaque-formation assays on MDCK cells.
- Human lung carcinoma A549 cells (ATCC, CCL-185) were cultured in F-12 medium supplemented with 10% FBS, 2 mM L-glutamine and 1% P/S.
- African green monkey kidney Vero cells (ATCC, CCL-81) for ZIKV titration were grown in Minimum Essential Medium (MEM) supplemented with 10% FBS, 2 mM L-glutamine and 1% P/S.
- Human embryonic kidney 293T/17 cells (ATCC, CRL-11268) were cultured in Dulbecco’s Modified Eagle's Medium (DMEM) supplemented with 10% FBS, 2 mM L-glutamine and 1% P/S.
- DMEM Modified Eagle's Medium
- Mouse monoclonal anti-flag antibody from Sigma- Aldrich.
- Mouse monoclonal anti-ZC3HAVlL antibody (F4) (sc-514959) from Santa Cruz Biotechnology.
- Mouse monoclonal anti-JEVNS3 antibody was described previously (Chen, LK et al. Virology 1996, 217, 220-229.).
- ZC3HAV1L Human Tagged ORF Clone (RC203208) was purchased from OriGene Technologies.
- SFFV constitutive spleen focus-forming virus
- lentivirus preparation human embryonic kidney 293 T/17 cells were cotransfected with a lentivirus expression construct (pSIN) and the two helper plasmids, pMD.G and pCMVAR8.91, with Lipofectamine 2000 reagent (Invitrogen). Transfected cells were incubated at 37°C for 4 -5 h and then refed with fresh medium. Cell supernatants containing the viral particles were harvested 24 - 60 h after transfection and stored at -80°C. For ZC3HAVlL-overexpressing Vero cell line, A549 cells were transduced with the lentiviral vector expressing flag-tagged ZC3HAV1L protein or control EGFP protein for 72 h.
- Cells were lysed in sodium dodecyl sulfate (SDS) sample buffer (62.5 mM Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% bromophenol blue) containing a cocktail of protease inhibitors.
- SDS sodium dodecyl sulfate
- Virus titers were calculated as the number of plaque forming units (PFU) per milliliter.
- pSIN vector is used to construct ZC3HAVlL-Myc-Flag/pSIN plasmid.
- A549 cells (ATCC, CCL-185) overexpressing flag-tagged ZC3HAV1L protein are established by lentiviral transduction.
- the expression of flag-tagged ZC3HAV1L protein or control EGFP protein in cells is presented in Western blots ( Figure 1).
- Anti-flag and anti- ZC3HAV1L monoclonal antibodies are used for detection, and the protein size is predicted to be approximately ⁇ 34 kDa
- Example 2 Proviral potential of overexpressing Zc3havlL against Japanese encephalitis virus (JEV) infection
- A549 cells are transduced with the lentiviral vector expressing flag- tagged ZC3HAV1L protein or control mcherry protein, then infected with JEV-EGFP.
- Example 3 The Western blot analysis of JEV proteins NS3
- JEV-EGFP 0.01, 0.1, 1
- NS3 a non- structural protein produced by JEV, is determined by western blot to demonstrate the replication of JEV can be perform effectively in cells overexpressing ZC3HAV1L protein ( Figure 4).
- African green monkey kidney Vero cells are transduced with the lentiviral vector expressing flag-tagged ZC3HAV1L protein for 72 h.
- the transduced cells and parental cells are prepared for indirect immunofluorescence using a mouse anti -FL AG antibody and an anti-mouse IgG antibody (Alexa-488) as the first and secondary antibodies, respectively ( Figure 5).
- Figure 5 Compared with parental cells, almost all cells overexpressing flag- tagged ZC3HAV1L can be stained.
- Example 5 The virus growth in JE V-infected cells with ZC3HAV IL overexpression
- Example 7 The virus growth in lAV-infected cells with ZC3HAV1L overexpression
- ZC3HAV1L is identified as a novel host factor and significantly increases virus production.
- cells overexpressing ZC3HAV1L protein result in approximately more than 100-folds increases in viral yield of JEV and DENV, and more than 10-folds increases in viral yield of IAV
- the present invention provides higher viral yield and lower cost-effectiveness in the production of live-attenuated and inactivated virus vaccines.
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Abstract
The present invention relates to a cell line having an increased ability to produce virus and a method for preparing thereof, and more particularly, to a cell line that has an increased ability to produce virus, due to the overexpression of a CCCH-type zinc finger protein and to a production method thereof. According to the present invention, when the engineered cell line is used as a virus-producing cell line, the production yield of virus can be increased. In addition, the engineered cell line can be used for the production and research of vaccines for preventing and treating viral diseases.
Description
Cell strain having increased virus production ability and production method thereof
BACKGROUND OF THE INVENTION
Technical Field of the Invention
[ 0001 ] The present invention relates to a cell line having an increased ability to produce virus and a method for preparing thereof, and more particularly, to a cell line that has an increased ability to produce virus, due to the overexpression of a CCCH-type zinc finger protein and to a production method thereof.
Background
[0002] The vaccination of conventional vaccines including live-attenuated and inactivated vaccines has proven to be the most effective strategy for the prevention and control of viral diseases. Cell-culture based vaccine production may offer distinct advantages over the conventional methods of vaccine production (ie, egg-based or mouse brain-based vaccine production) including the following: (1) the source of the cell lines for vaccine production are more easily and conveniently, which can rapidly produce vaccine supplies in the event of a pandemic; (2) offer a higher similarity with the circulating strains and potentially improved protection; (3) a more controlled production process and faster start-up of the vaccine manufacturing process; (4) avoid the risk of severe allergic reaction from egg-based and mouse brain-based vaccines. However, the cell-culture production process might cause lower virus yield and cost-effectiveness. Thus, it will be a key issue in improving cell-culture based vaccine production with higher virus yield.
[0003 ] CCCH-type zinc finger proteins are RNA-binding molecules involved in RNA metabolism pathways such as RNA splicing, polyadenylation, and messenger RNA
(mRNA) decay. As a consequence of whole genome searching, totally 57 CCCH genes were identified in human and can be divided into 6 groups based on the amino acid sequence alignment (Liang, J et al. PLoS one 2008, 3(8), e2880.).
[0004 Some members of CCCH-type zinc finger family, such as ZC3H12A (MCP-1 induced protein 1), ZFP36 (Tristetraproline, TTP) and ZC3H2 (Zinc finger CCCH- type antiviral protein 1, also known as Zinc finger antiviral protein (ZAP)) were suggesting that they can directly bind to specific viral RNA sequences through its CCCH-type zinc finger motifs and inhibits the replication of some virus. (Zhou, L et al. Circ Res 2006, 98, 1-10; Taylor, GA et al. Immunity 1996 4, 445-454; Gao, G et al. Science 2002, 297, 1703- 1706.).
[ 0005 ] The functions of most CCCH-type zinc finger proteins are still unclear, and the interaction with viruses and the role they play are rarely discussed. Not only the antiviral activities, other members may have proviral activities that can enhance viral replication, which can be utilized to provide higher viral yield and lower cost-effectiveness in the production of vaccines using cell-culture based platform.
SUMMARY OF INVENTION
[0006] Based on the above objects, the present invention demonstrates that cells overexpressing ZC3HAV1L (Zinc finger CCCH-type, antiviral 1-like) protein resulted in higher levels of virus production.
[0007] Accordingly, one aspect of the invention relates to a virus-producing cell which introduced ZC3HAV1L gene in a cell having an ability to produce virus, wherein the cell having an increased ability to produce virus by overexpressing the product of said ZC3HAV1L gene.
[0008] In some examples of the present invention, the cell is infected with a virus.
[0009 In some examples of the present invention, the virus is Flavivirus or Alphainfluenzavirus.
[0010] In some examples of the present invention, the virus is selected from the group consisting of Japanese encephalitis virus (JEV), Dengue virus (DENV), and Influenza A virus (IAV).
[0011 ] In other examples of the present invention, the mutant cell is a Vero cell.
[0012] In another aspect of the present invention, it is related to a method for preparing a virus-producing cell, the method comprising introducing a ZC3HAV1L gene to a virus-producing cell.
[0013 ] In some examples of the present invention, the method further comprising infecting the mutant cell with a virus.
[0014] In some examples of the present invention, the virus is Flavivirus or Alphainfluenzavirus.
[0015] In some examples of the present invention, the virus is selected from the group consisting of Japanese encephalitis virus (JEV), Dengue virus (DENV), and Influenza A virus (IAV).
[0016] In other examples of the present invention, the mutant cell is an animal cell. Preferably, the animal cell is a Vero cell.
[0017] Another aspect of the present invention relates to a method for producing a desired virus, the method comprising the steps of:
(a) infecting the mutant cell of claim 1 with the desired virus; and (b) culturing the mutant cell infected with the desired virus, and then harvesting the supernatant of the culture to recover the desired virus.
[0018] In certain examples of the present invention, the mutant cell is infected with 0.01-0.1 MOI of the desired virus.
[0019 ] A further aspect of the present invention relates to a method for producing a desired virus, the method comprising the steps of: (a) infecting the mutant cell of claim 1 with the desired virus; (b) culturing the mutant cell infected with the desired virus, and then harvesting the supernatant of the culture to recover the desired virus; and (c) attenuating or inactivating the recovered virus.
BREIF DESCRIPTION OF THE DRAWINGS
[0020] Fig. 1 demonstrates Western blot analysis for the expression of flag- tagged ZC3HAV1L protein in A549 cells transduced with lentiviruses.
[0021 ] Fig. 2 demonstrates JEV replication in A549 cells overexpressing ZC3HAV1L protein by using JEV-GFP (MOI = 0.01).
[0022] Fig. 3 demonstrates JEV replication in A549 cells overexpressing ZC3HAV1L protein by using JEV-GFP (MOI = 0.1).
[0023 ] Fig. 4 shows the immunofluorescence of the stable cell line with ZC3HAVlL-flag overexpression in Vero cells.
[0024] Fig. 5 shows the production of viral protein NS3 in cells infected with JEV-EGFP using Western blot.
[0025] Fig. 6 illustrates the virus growth curve in JEV-infected cells.
[0026] Fig. 7 illustrates the virus growth curve in DENV-2-infected cells.
[0027 ] Fig. 8 illustrates the virus growth curve in lAV-infected cells.
DETAILED DESCRIPTION OF THE INVENTION
[0028] Other features and advantages of the present invention will be further exemplified and described in the following examples, which are intended to be illustrative only and not to limit the scope of the invention.
[0029] Virus, cell lines, chemicals and antibodies
[0030] JEV PR-9 strain (GenBank accession: AF014161) (Chen, LK et al.
Virology 1996, 223, 79-88.) and DENV-2 PL046 strain (GenBank accession: KJ734727) (Lin, YL et al. J Virol 1998, 72, 9729-9737.) were propagated in mosquito C6/36 cells (ATCC, CRL-1660) maintained in RPMI-1640 medium supplemented with 5% fetal bovine serum (FBS). JEV-EGFP (RP-9 strain) and DENV-2-EGFP (PL046 strain) were a kind gift from Dr. Yi-Ling Lin. JEV and DENV titration was determined by plaque-forming assay in baby hamster kidney BHK-21 cells (ATCC, CCL-10) grown in RPMI-1640 medium supplemented with 5% FBS, 2 mM L-glutamine and 1% penicillin-streptomycin (P/S). The influenza A/PuertoRico/8/1934 (H1N1) (GenBank accession: CY009444) used in this study was propagated in Madin-Darby canine kidney (MDCK) cells, which were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibico) containing 10% fetal bovine serum (FBS). Viral titration was by plaque-formation assays on MDCK cells. Human lung carcinoma A549 cells (ATCC, CCL-185) were cultured in F-12 medium supplemented with 10% FBS, 2 mM L-glutamine and 1% P/S. African green monkey kidney Vero cells (ATCC, CCL-81) for ZIKV titration were grown in Minimum Essential Medium (MEM) supplemented with 10% FBS, 2 mM L-glutamine and 1% P/S. Human embryonic kidney 293T/17 cells (ATCC, CRL-11268) were cultured in Dulbecco’s Modified Eagle's Medium (DMEM) supplemented with 10% FBS, 2 mM L-glutamine and 1% P/S. Lipofectamine 2000 reagent was from Invitrogen. Mouse monoclonal anti-flag antibody from Sigma- Aldrich. Mouse monoclonal anti-ZC3HAVlL antibody (F4) (sc-514959) from Santa Cruz Biotechnology. Mouse monoclonal anti-JEVNS3 antibody was described previously (Chen, LK et al. Virology 1996, 217, 220-229.).
[0031 ] Plasmid constructs, lentivirus generation and establishment of ZC3HAVlL-overexpressing stable cell lines
[0032] ZC3HAV1L (NM_080660) Human Tagged ORF Clone (RC203208) was purchased from OriGene Technologies. The cDNA of human ZC3HAV1L fused with
Flag-tag ZC3HAV1L Human Tagged ORF Clone was subcloned to the self-inactivating lentiviral vector (pSIN), in which the expression of an inserted gene is under the control of a constitutive spleen focus-forming virus (SFFV) promoter (Godfrey, A et al. Blood 2005, 105, 2510-2518.). For lentivirus preparation, human embryonic kidney 293 T/17 cells were cotransfected with a lentivirus expression construct (pSIN) and the two helper plasmids, pMD.G and pCMVAR8.91, with Lipofectamine 2000 reagent (Invitrogen). Transfected cells were incubated at 37°C for 4 -5 h and then refed with fresh medium. Cell supernatants containing the viral particles were harvested 24 - 60 h after transfection and stored at -80°C. For ZC3HAVlL-overexpressing Vero cell line, A549 cells were transduced with the lentiviral vector expressing flag-tagged ZC3HAV1L protein or control EGFP protein for 72 h.
[0033 ] Western immunoblot analysis
[0034] A549 cells were transduced with the lentiviral vector expressing flag- tagged ZC3HAV1L protein or control mcherry protein for 72 h, then infected with JEV- EGFP (MOI= 0.01 or 0.1 or 1) for 16h. Cells were lysed in sodium dodecyl sulfate (SDS) sample buffer (62.5 mM Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% bromophenol blue) containing a cocktail of protease inhibitors. Similar amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Hybond-C Super; Amersham). The nonspecific antibody-binding sites were blocked with 5% skim milk in TBS-T (25 mM Tris, 0.8% NaCl, and 2.68 mM KC1 [pH 7.4], with 0.1% Tween 20) prior to the addition of the primary antibody. The blots were then treated with a horseradish peroxidase-conjugated secondary antibody (Amersham) and developed with an ECL system (Amersham).
[0035] Immunofluorescence assay
[0036] A549 cells were transduced with the lentiviral vector expressing flag-
tagged ZC3HAV1L protein or control mcherry protein for 72 h, then infected with JEV- EGFP (MOI= 0.01 or 0.1) for indicated hours. EGFP protein expression was were observed under a fluorescence microscope.
[0037] Virus growth curve assay
[0038] For JEV and DENV-2 infection, monolayers of the cells grown in 12- well plates were initially adsorbed with virus at the indicated multiplicity of infection (MOI) for 1 h at 37°C. After 1 h of adsorption, the unbound virus was removed from cells by gentle washing with RPMI 1640 medium. At indicated time point post-infection (p.i.), culture media were harvested for the plaque-forming assay in baby hamster kidney BHK-21 cells.
[0039] For influenza A virus infection, monolayers of the cells grown in 12- well plates were initially adsorbed with virus at the indicated multiplicity of infection (MOI = 0.01) for 1 h at 37°C. After 1 h of adsorption, the unbound virus was removed from cells by gentle washing with PBS medium. At indicated time point post-infection (p.i.), culture supernatants containing viral particles were harvested. Viral titer was determined by plaque assay. Briefly, MDCK cells were seeded at 6.0 x 105 cells/well in 6-well plates before virus infection for 24 h at 37 °C. Virus titers were evaluated by serial 10-fold dilutions in 6-well plates at 37 °C. At 1 h post-infection, cells were washed twice with 1 mL PBS, and the cells overlaid with 2 mL of DMEM/ HG medium supplemented with 0.3% agarose. After incubation for 48 h at 37 °C, the cells were fixed in 10% formalin for at least 1 h before crystal violet staining. Virus titers were calculated as the number of plaque forming units (PFU) per milliliter.
[0040] Example 1. Overexpression of Zc3havlL in cell lines
[0041 ] pSIN vector is used to construct ZC3HAVlL-Myc-Flag/pSIN plasmid. A549 cells (ATCC, CCL-185) overexpressing flag-tagged ZC3HAV1L protein are established by lentiviral transduction. The expression of flag-tagged ZC3HAV1L protein or
control EGFP protein in cells is presented in Western blots (Figure 1). Anti-flag and anti- ZC3HAV1L monoclonal antibodies are used for detection, and the protein size is predicted to be approximately ~34 kDa
[0042] Example 2. Proviral potential of overexpressing Zc3havlL against Japanese encephalitis virus (JEV) infection
[0043 ] A549 cells are transduced with the lentiviral vector expressing flag- tagged ZC3HAV1L protein or control mcherry protein, then infected with JEV-EGFP. As shown in Figure 2, GFP expression is observed after 6 h of JEV-EGFP infection (MOI = 0.01) in cells transduced with lentiviruses expressing ZC3HAV1L and 24 h in mcherry counterpart thereof. No significant GFP fluorescence was observed in mcherry control group until 16 h in a higher MOI (0.1) of JEV-EGFP infection (Figure 3).
[0044] Example 3. The Western blot analysis of JEV proteins NS3
[0045] A549 cells are transduced with the lentiviral vector expressing flag- tagged ZC3HAV1L protein or control mcherry protein, then infected with JEV-EGFP (MOI = 0.01, 0.1, 1) for 16 h. NS3, a non- structural protein produced by JEV, is determined by western blot to demonstrate the replication of JEV can be perform effectively in cells overexpressing ZC3HAV1L protein (Figure 4).
[0046] Example 4. Establishment of stable cell lines overexpressing ZC3HAV1L
[0047] African green monkey kidney Vero cells are transduced with the lentiviral vector expressing flag-tagged ZC3HAV1L protein for 72 h. The transduced cells and parental cells are prepared for indirect immunofluorescence using a mouse anti -FL AG antibody and an anti-mouse IgG antibody (Alexa-488) as the first and secondary antibodies, respectively (Figure 5). Compared with parental cells, almost all cells overexpressing flag- tagged ZC3HAV1L can be stained.
[ 0048 ] Example 5. The virus growth in JE V-infected cells with ZC3HAV IL overexpression
[ 0049 ] Vero cells with or without ZC3HAV1L overexpression are infected with JEV (MOI = 0.01) and analyzed for viral replication. Viral titers at 24, 48, 60, 72 hours post infection are determined using plaque assay (Figure 6). Compared with the parental cells and GFP-overexpressing cells, the virus production in cells overexpressing ZC3HAV1L protein increase nearly 100 folds.
[0050 Example 6. The virus growth in DENV-2-infected cells with ZC3HAV1L overexpression
[ 0051 ] Vero cells with or without ZC3HAV1L overexpression are infected with DENV-2 (MOI = 0.01) and analyzed for viral replication. Viral titers at 24, 48, 72, 96 hours post infection are determined using plaque assay (Figure 7). Compared with the parental cells and GFP-overexpressing cells, the virus production in cells overexpressing ZC3HAV1L protein increase nearly 100 folds.
[0052] Example 7. The virus growth in lAV-infected cells with ZC3HAV1L overexpression
[ 0053 ] Vero cells with or without ZC3HAV1L overexpression are infected with IAV (MOI = 0.01) and analyzed for viral replication. Viral titers at 24, 48, 64, 80 hours post infection are determined using plaque assay (Figure 8). Compared with the parental cells, the virus production in cells overexpressing ZC3HAV1L protein increase more than 10 folds.
[0054] As shown in the examples, ZC3HAV1L is identified as a novel host factor and significantly increases virus production. As compared to parental cells, cells overexpressing ZC3HAV1L protein result in approximately more than 100-folds increases in viral yield of JEV and DENV, and more than 10-folds increases in viral yield of IAV As compared with the cell-culture based vaccine platforms in the art, the present invention
provides higher viral yield and lower cost-effectiveness in the production of live-attenuated and inactivated virus vaccines.
Claims
1 . A virus-producing mutant cell introduced ZC3HAV1L gene in a cell having an ability to produce virus, wherein the mutant cell having an increased ability to produce virus by overexpressing the product of said ZC3HAV1L gene.
2. The virus-producing mutant cells of claim 1, wherein the mutant cell is infected with a virus.
3. The virus-producing mutant cell of claim 2, wherein the virus is Flavivirus or Alphainfluenzavirus.
4. The virus-producing mutant cell of claim 3, wherein the virus is selected from the group consisting of Japanese encephalitis virus (JEV), Dengue virus (DENV), and Influenza A virus (IAV).
5. The virus-producing mutant cell of claim 1, the mutant cell is an animal cell.
6. The virus-producing mutant cell of claim 5, the animal cell is a Vero cell.
7. A method for preparing a virus-producing mutant cell, the method comprising introducing a ZC3HAV1L gene to a virus-producing cell.
8. The method of claim 7, wherein the method further comprising infecting the mutant cell with a virus.
9. The method of claim 8, wherein the virus is Flavivirus or Alphainfluenzavirus.
10. The method of claim 9, wherein the virus is selected from the group consisting of Japanese encephalitis virus (JEV), Dengue virus (DENV), and Influenza A virus (IAV)
11. The method of claim 7, wherein the mutant cell is an animal cell.
12. The method of claim 11, wherein the animal cell is a Vero cell.
13. A method for producing a desired virus, the method comprising the steps of:
(a) infecting the mutant cell of claim 1 with the desired virus; and
(b) culturing the mutant cell infected with the desired virus, and then harvesting the supernatant of the culture to recover the desired virus.
14. The method of claim 13, wherein the mutant cell is infected with 0.01-0.1 MOI of the desired virus.
15. A method for producing a desired virus, the method comprising the steps of:
(a) infecting the mutant cell of claim 1 with the desired virus;
(b) culturing the mutant cell infected with the desired virus, and then harvesting the supernatant of the culture to recover the desired virus; and
(c) attenuating or inactivating the recovered virus.
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WO2013166264A2 (en) * | 2012-05-02 | 2013-11-07 | University Of Georgia Research Foundation, Inc. | Methods for altering virus replication |
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WO1999047645A1 (en) * | 1998-03-18 | 1999-09-23 | Fundação Oswaldo Cruz - FIOCRUZ | Process for the production of virus in cell cultures |
WO2013166264A2 (en) * | 2012-05-02 | 2013-11-07 | University Of Georgia Research Foundation, Inc. | Methods for altering virus replication |
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LIN REN-JYE, HUANG CHIH-HENG, LIU PING-CHENG, LIN I-CHIEH, HUANG YU-LING, CHEN AN-YU, CHIU HSIN-PING, SHIH SHIN-RU, LIN LI-HSIUNG,: "Zinc finger protein ZFP36L1 inhibits influenza A virus through translational repression by targeting HA, M and NS RNA transcripts", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, GB, vol. 48, no. 13, 1 January 2020 (2020-01-01), GB , XP055942128, ISSN: 0305-1048, DOI: 10.1093/nar/gkaa458 * |
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