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WO2018101745A1 - Antiviral composition against hepatitis b virus, including interleukin-32 as active ingredient - Google Patents

Antiviral composition against hepatitis b virus, including interleukin-32 as active ingredient Download PDF

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WO2018101745A1
WO2018101745A1 PCT/KR2017/013837 KR2017013837W WO2018101745A1 WO 2018101745 A1 WO2018101745 A1 WO 2018101745A1 KR 2017013837 W KR2017013837 W KR 2017013837W WO 2018101745 A1 WO2018101745 A1 WO 2018101745A1
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expression
hbv
cells
virus
antiviral
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Kyun-Hwan Kim
Doo-Hyun Kim
Yeong-Min Park
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Dandi Bioscience Inc
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5067—Liver cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0337—Animal models for infectious diseases
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    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]

Definitions

  • the present invention relates to an antiviral composition against hepatitis B virus (HBV), and more particularly, to an antiviral composition against hepatitis B virus, including interleukin-32 (IL-32) which is an inhibitor of expression or activation of hepatocyte nuclear factor 4 alpha (HNF4 ⁇ ) or hepatocyte nuclear factor 1 alpha (HNF1 ⁇ ) as an active ingredient, and a method for screening an antiviral substance against the virus.
  • HNF4 ⁇ hepatocyte nuclear factor 4 alpha
  • HNF1 ⁇ hepatocyte nuclear factor 1 alpha
  • HBV chronic hepatitis B
  • liver cirrhosis and hepatocellular carcinoma have emerged globally as public health problems.
  • HBV HBV genome
  • RC DNA relaxed circular DNA
  • cccDNA covalently closed circular DNA
  • HBV expresses 4 types of viral proteins, that is, a polymerase, a surface protein, a core protein, and HBx from four open-reading frames (PreC/C, P, preS1/S2/S and X).
  • PreC/C, P, preS1/S2/S and X open-reading frames
  • HBV replication occurs by reverse transcription from core capsids using the polymerase protein of HBV.
  • 4 types of HBV RNA encoded in cccDNA of HBV are transcripted by an HBV enhancer.
  • HNF hepatocyte nuclear factor
  • C/EBP CCAAT/enhancer binding protein
  • cytokines inhibit gene expression and replication of HBV, and the occurrence of a disease caused thereby by various mechanisms.
  • a host defense system against HBV is known to regulate the life cycle of HBV.
  • TNF- ⁇ and IFN- ⁇ are well known as cytokines inducing an antiviral response in the host defense mechanism.
  • Secretion of TNF- ⁇ and IFN- ⁇ is mediated by a variety of antiviral genes through cytokine-dependent signal transduction pathways such as CIAP2, MxA and PKR pathways.
  • TNF- ⁇ regulates generation of HBV RNA and stability of a capsid
  • IFN- ⁇ removes a capsid including pregenomic RNA from mouse liver cells.
  • TNF- ⁇ and IFN- ⁇ are involved in removal of a non-cytopathic virus.
  • various cytokines also exhibit directly or indirectly an antiviral effect on HBV. Therefore, various cytokines have been known to contribute to the host defense system with respect to HBV infection, but a host immune mechanism with respect to HBV has not been clearly known yet.
  • IL-32 is a type of cytokine, and has been known to induce expression in the pancreas due to the synergistic effect of TNF- ⁇ and IFN- ⁇ .
  • IL-32 is generated by various epithelial cells, and immune cells such as T lymphocytes, NK cells and monocytes.
  • the IL-32 gene may be located on a human chromosome 16p13.3, and IL-32 is known as a pro-inflammatory cytokine that activates signal transduction pathways of a mitogen-activated protein kinase (MAPK) and a nuclear factor kappa-light-chain-enhancer of activated B cells (NF- ⁇ B).
  • MAPK mitogen-activated protein kinase
  • NF- ⁇ B nuclear factor kappa-light-chain-enhancer of activated B cells
  • the IL-32 gene has six selective splicing variants after transcription, and among such variants, IL-32 ⁇ is known as the most activated form.
  • biological functions of isoforms of the IL-32 are not well known.
  • IL-32 is associated with various bacteria and viruses such as Orientia Tsutsugamushi, vesicular stomatitis virus (VSV), human immunodeficiency virus (HIV), hepatitis C virus (HCV), human papilloma virus (HPV), and influenza virus (Immunology 2011;132:410-420).
  • VSV vesicular stomatitis virus
  • HCV hepatitis C virus
  • HPV human papilloma virus
  • influenza virus Immunology 2011;132:410-420
  • the present invention is also directed to providing a method for screening an antiviral substance against HBV.
  • the present invention provides a method for screening an antiviral substance against HBV, which includes the following steps:
  • the cells may be liver cells.
  • the present invention provides a method for treating hepatitis B, which includes administering an antiviral composition including an IL-32 gene or an IL-32 protein, which is an inhibitor of expression or activation of HNF4 ⁇ or HNF1 ⁇ , as an active ingredient.
  • the inventors first identified an IL-32-mediated antiviral effect against HBV and a molecular mechanism thereof by confirming that IL-32 secretion is induced by TNF- ⁇ and IFN- ⁇ cytokines secreted from host cells infected by HBV, the secreted IL-32 reduces the expression of HNF4 ⁇ and HNF1 ⁇ , which are host transcription factors involved in the transcription of a viral gene, by increasing ERK1/2 phosphorylation to exhibit an inhibitory effect on DNA replication and gene expression of the virus, and the IL-32 exhibits the above-mentioned effect only when expressed in the cells. Therefore, an IL-32-mediated antiviral effect on HBV can impart a new understanding for the development of a therapeutic agent for the virus, and the antiviral composition according to the present invention can be effectively used in the development of an antiviral therapeutic agent.
  • FIGS. 2a to 2d show the inhibitory effect of IL-32 on HBV replication, where FIG. 2a shows that all of Huh7 and HepG2 cells do not exhibit an inhibitory effect on HBV replication by treatment of a recombinant IL-32 (rhIL-32 ⁇ ) protein, FIG. 2b shows the ELISA result representing that IL-32 ⁇ is present in most cells, FIG. 2c shows that inhibition of HBV replication is induced by treatment of Huh7 cells with TNF- ⁇ and IFN- ⁇ , and FIG. 2d shows that an inhibitory effect on HBV replication is not produced by treatment of IL-32-specific siRNA.
  • rhIL-32 ⁇ recombinant IL-32
  • FIGS. 3a to 3d show that the inhibitory effect on HBV replication due to ectopic expression of IL-32, where FIG. 3a shows the result of Southern blotting representing that an HBV DNA level is decreased dependently on an IL-32 ⁇ expression concentration after Huh7 cells are transfected with HBV 1.2 and IL-32 ⁇ expression plasmids, FIG. 3b shows that an antiviral effect caused by IL-32 expressed in cells is produced by treating a culture medium with IL-32 antibodies, FIG. 3c shows the result of comparing degrees of inhibiting HBV replication according to expression of IL-32 isoforms (IL-32 ⁇ , IL-32 ⁇ , and IL-32 ⁇ ), and FIG. 3d shows that HBV replication is not inhibited by transfection with IL-32 siRNA after IL-32 ⁇ expression.
  • FIG. 3a shows the result of Southern blotting representing that an HBV DNA level is decreased dependently on an IL-32 ⁇ expression concentration after Huh7 cells are transfected with HBV
  • FIGS. 5a to 5f show IL-32-mediated down-regulation of HNF4 ⁇ and HNF1 ⁇ transcription factors
  • FIG. 5a shows binding sites of host transcription factors involved in HBV RNA transcription on a gene map of an HBV enhancer
  • FIGS. 5b and 5c are the results of quantitative RT-PCR and western blotting showing that a decrease in RNA and protein expression levels of HNF4 ⁇ and HNF1 ⁇ when IL-32 ⁇ is expressed in Huh7 cells, respectively
  • FIGS. 5d and 5e show the result of ChIP analysis indicating that binding efficiency of HNF4 ⁇ binding to RI (Enhancer I) and HNF1 ⁇ binding to RII (Enhancer II) is decreased under a condition for expressing IL-32 ⁇
  • the present invention provides an antiviral composition against HBV, including IL-32 which is an inhibitor of expression or activation of HNF4 ⁇ or HNF1 ⁇ as an active ingredient, and a method for screening an antiviral substance against the virus.
  • antiviral refers to weakening or ending the action of a virus having invading a body by inhibiting viral proliferation in the body, and more specifically, inhibiting viral proliferation by inhibiting nucleic acid synthesis of a virus, gene expression, or viral replication, and in the present invention, HBV is targeted.
  • activation inhibitor refers to a substance causing a reduction in the function of a target protein, and preferably, due to the activation inhibitor, the function of a target protein is undetectable or present at an insignificant level.
  • the expression or activation inhibitor of HNF4 ⁇ and HNF1 ⁇ according to the present invention may consist of a base sequence selected from the group consisting of SEQ ID NOs: 1 to 4, and more specifically, SEQ ID NOs: 1 to 4 are the base sequences of IL-32 isoforms such as IL-32 ⁇ , IL-32 ⁇ , IL-32 ⁇ , and IL-32 ⁇ , respectively, and preferably, the IL-32 gene may consist of the base sequence of SEQ ID NO: 3.
  • HBV replication was inhibited due to IL-32 expression induced by TNF- ⁇ and IFN- ⁇ in a liver cancer cell line, which was not shown when a recombinant IL-32 protein was extracellularly treated (refer to Example 3).
  • an inhibitory effect on HBV replication was shown when intracellular expression of IL-32 is induced by transfecting a liver cancer cell line with plasmids expressing HBV and IL-32, and an antiviral effect was exhibited even when a cell culture medium was treated with an IL-32-specific antibody, and therefore, it was confirmed that HBV replication was inhibited by IL-32 expressed only in cells.
  • all of various isoform proteins of IL-32 exhibited an inhibitory effect on HBV replication, and among the proteins, IFN- ⁇ is most effective (refer to Example 4).
  • HBV gene it was newly identified that the expression and replication of an HBV gene are inhibited by inducing IL-32 expression by TNF- ⁇ and IFN- ⁇ secreted from infected cells when cells are infected with HBV, increasing ERK1/2 phosphorylation, and reducing HBV enhancer activity by inhibiting the expression and activation of HNF4 ⁇ and HNF1 ⁇ involved in HBV gene transcription.
  • the antiviral composition of the present invention may include a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is conventionally used in preparations, and may be, but is not limited to, a saline solution, distilled water, Ringer's solution, buffered saline, a cyclodextrin solution, a dextrose solution, a maltodextrin solution, glycerol, ethanol, or liposomes, etc., and may further include another conventional additive such as an antioxidant or a buffer as needed.
  • the pharmaceutically acceptable carrier may be prepared as injectable preparations such as an aqueous solution, a suspension, and an emulsion, pills, capsules, granules or tablets by further adding diluents, dispersants, surfactants, binders, lubricants, etc.
  • Suitable pharmaceutically acceptable carriers and their preparations may be prepared according to each ingredient using a method disclosed in the Remington’s Pharmaceutical Science.
  • the pharmaceutical composition of the present invention is not limited in formulation, and thus may be prepared as injections, inhalants, external preparations for skin, or oral medications.
  • the antiviral composition of the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, percutaneously, nasally, or intratracheally) according to a desired method, and a dose of the pharmaceutical composition of the present invention may be selected according to a patient’s condition and body weight, severity of a disease, a dosage form, an administration route and time by those of ordinary skill in the art.
  • the effective amount of the composition according to the present invention may vary depending on a patient’s age, sex, and body weight, and may be generally administered at 0.001 to 150 mg and, preferably, 0.01 to 100 mg/kg of body weight daily or every other day, or once to three times a day.
  • the effective amount may vary depending on an administration route, the severity of obesity, sex, body weight or age, and therefore, the scope of the present invention is not limited by the dose in any way.
  • composition of the present invention may be used in various applications such as medications, food and beverages, and in forms of powder, granules, tablets, capsules or drinks.
  • the present invention provides a method for screening an antiviral substance against HBV, which includes the following steps:
  • the cells include liver cells, and any type of liver-derived cells may be used without limitation.
  • the candidate substance may be selected from the group consisting of a compound, a microbial culture medium or extract, a natural substance extract, a nucleic acid, and a peptide
  • the nucleic acid is preferably selected from the group consisting of siRNA, shRNA, microRNA, antisense RNA, an aptamer, a locked nucleic acid (LNA), a peptide nucleic acid (PNA), and a morpholino, but the present invention is not limited thereto.
  • Step (b) measurement of an expression level of IL-32 may be carried out by a polymerase chain reaction (PCR), a microarray, northern blotting, western blotting, an enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunohistochemistry, and immunofluorescence, but the present invention is not limited thereto.
  • PCR polymerase chain reaction
  • ELISA enzyme-linked immunosorbent assay
  • Human liver cancer cell lines such as Huh7 and HepG2 were purchased from American Type Culture Collection (Manassas, VA, USA), and incubated in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco BRL, Oregon, USA) containing 10% fetal bovine serum (FBS, Gibco BRL) inactivated by thermal treatment and 1% penicillin and streptomycin (Gibco BRL) at 37 °C with 5% CO 2 .
  • DMEM Modified Eagle’s Medium
  • FBS fetal bovine serum
  • HBV 1.2 plasmid used in the example of the present invention was obtained from a DNA prep kit (Alphagene, Gyeonggi, Korea), and a pCAGGS mock vector, and IL-32 ⁇ , IL-32 ⁇ and IL-32 ⁇ plasmids were provided by a different team of Konkuk University.
  • An HNF4 ⁇ expression plasmid was amplified by PCR using an HepG2 cDNA library as a template, and subcloned in a pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA).
  • An HBV enhancer luciferase plasmid was amplified by PCR using the HBV 1.2 plasmid.
  • amplification was carried out by PCR using the HBV 1.2 plasmid, and primer sequences used herein are listed in Table 1.
  • Transfection was carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol when cells reached a confluency of 70 to 80% of the area of a culture vessel. 15 hours after transfection, rhIL-32 ⁇ (YbdY, Seoul, Korea), TNF- ⁇ (YbdY), IFN- ⁇ (LG, Jeonbuk, Korea), anti-IL-32 (YbdY) and signal transduction inhibitors were added to new media to treat the cells, and the cells were cultured for 3 days.
  • Cytokine treatment was carried out with rhIL32 (0.1, 0.25, and 0.5 ⁇ g/ml), TNF- ⁇ (0.02 and 0.1 ⁇ g/ml), IFN- ⁇ (500 and 1000 U/ml), and mixed treatment (TNF- ⁇ 0.02 ⁇ g/ml and IFN- ⁇ 500 U/ml).
  • a signal transduction inhibitor, U0126 was treated at a final concentration of 10 ⁇ M, and anti-IL32 was treated at concentrations of 0.2 and 0.5 ⁇ g/ml.
  • the transfected cells were recovered two or three days after the transfection, and then used for a subsequent experiment.
  • IL-32-specific siRNA was synthesized at ST Pharm (Seoul, Korea) to be used in an experiment.
  • the sequence of the siRNA is shown in Table 2 below, and cells were transfected using 10 or 20 nM of Lipofectamine 2000.
  • a cell viability kit was purchased from WELGENE (Seoul, Korea). Huh7 cells were seeded in a 6-well plate, and subjected to transfection or other treatment processes. Afterward, the cell culture medium was replaced with a fresh medium and then the original medium was transferred to a 96-well plate. Then, an XTT reagent and a PMS reagent were added to the 96-well plate, followed by incubating the cells for up to 1 hour, and absorbance was measured at 450 nm using a spectrophotometer.
  • the cells were collected with a scrapper and lysed by adding 100 ⁇ l of a HEPES buffer, and then the core capsids of HBV were precipitated with a polyethylene glycol (PEG) solution. Subsequently, the core capsids were degraded by treatment with a proteinase K-containing SDS solution at 37 °C for 3 hours. Afterward, DNA was isolated from a 1% agarose gel at 100 V for 3 hours, and then transferred to a nitrocellulose membrane (GE Healthcare). To detect the HBV DNA on the membrane, the membrane was hybridized with random hexamers and purified 32P-labeled HBV probes.
  • PEG polyethylene glycol
  • the cells were recovered and lysed with a RIPA buffer [20 mM Tris/HCl, 1% NP-40, 0.5% protease inhibitor cocktail (Sigma, St.Louis, MO), 150 mM NaCl, 2 mM KCl, pH7.4]. After a cell precipitate was filtered, proteins in the cell lysate were divided by size through SDS-PAGE.
  • the proteins in a polyacrylamide gel were transferred onto a PVDF membrane, and then the membrane were treated with a primary antibody, that is, anti-IL-32 ⁇ (YbdY), anti-actin (Sigma), anti-GFP (Sigma), anti-HBsAg (Abcam), anti-HBcAg (DAKO, USA), anti-HNF1 ⁇ (Santa Cruz), anti-HNF3 ⁇ (Santa Cruz), anti-CEBP ⁇ (Santa Cruz), anti-HNF4 ⁇ (Santa Cruz), anti-lamin (Santa Cruz), or anti-tubulin (Santa Cruz), at a 1:2000 dilution.
  • a secondary antibody against the primary antibody an expression level of a target protein was determined, and the membrane was treated with a stripping reagent to remove all of the antibodies and then used to determine the expression level of a different protein.
  • a luciferase reporter assay was carried out by the following method. More specifically, Huh7 cells were seeded in a 12-well plate at a density of 2x10 5 well/cells,andtransfectedwith0.5 ⁇ gofEnhancer-Luc(pEnhI.II,pEnhI.delII,pXp.EnhII,pEnhII/cp),0.5 ⁇ gofIL-32 ⁇ ,or0.25 ⁇ gofa ⁇ -galplasmid.
  • pCAGGS was used as a control vector.
  • Huh7 cells were lysed with a TRIzol reagent (Invitrogen) according to the manufacturer’s protocols to extract total RNA.
  • a reaction solution was prepared by mixing 2 ⁇ g of the extracted RNA and an MMLV reverse transcriptase (Intron Biotechnology) such that the mixed solution had a final volume of 20 ⁇ l, and subjected to reverse transcription PCR (RT-PCR) to synthesize cDNA.
  • RT-PCR reverse transcription PCR
  • the cDNA synthesized as described above was amplified by performing PCR under the following conditions: 40 cycles of first denaturation at 94 °C (5 min), 94 °C (30 sec) and 72 °C (1 min), and final elongation at 72 °C for 5 minutes. Primer sequences used herein are listed in Table 3 below.
  • Chromatin immunoprecipitation assay ChIP
  • ChIP analysis was carried out using a ChIP assay kit (Millipore, Billerica, MA) according to the following method. A supernatant was purified with a protein A-agarose, and incubated by treatment of anti-HNF1 ⁇ (Santa Cruz), anti-HNF4 ⁇ (Santa Cruz), or a normal rabbit IgG antibody as a negative control group. Huh7 cells were cultured in a 6-well plate, and then transfected with or without IL-32 ⁇ before being used in an experiment. Primer sequences used in this experiment are listed in Table 4.
  • Electrophoretic mobility shift assay (EMSA )
  • the cells were recovered, and treated with nuclear and cytoplasmic extraction reagents (Thermo, Rockford, USA) to separate nucleic and cytoplasmic fractions. Subsequently, 2 ⁇ g of the nucleic fraction was precultured, and a [32P]-gamma-labeled dsDNA oligonucleotide was used for HNF4 ⁇ binding.
  • the double-stranded DNA (Enhancer I HNF4 ⁇ ) was purchased from Bioneer (Daejeon, Korea).
  • Huh7 cells were transfected with an IL-32 ⁇ plasmid and treated with TNF- ⁇ and IFN- ⁇ , and after 48 hours, the cells were observed using a confocal microscope (FV-1000 spectral, Olympus). More specifically, the Huh7 cells were cultured to a confluence of 50% of the area of a cover glass, and 48 hours after transfection, the cells were fixed by treatment of acetone.
  • the cells were treated with a 3% BSA solution diluted with PBS at a low temperature of 4 °C for 18 hours to carry out a blocking process, and then washed with PBS, followed by overnight culture at 4 °C by treating the cells with antibodies diluted at a dilution of 1:300.
  • the cells were washed with PBS again, treated with Alexa 488 and 568 secondary antibodies (goat and rabbit) to perform a reaction at room temperature for 1 hour, and then washed with PBS.
  • Toppro-3 (1:500) was used to stain nuclear DNA of the cells. Afterward, a cover glass was put on a slide glass for mounting, and then the cells were observed under a confocal microscope.
  • Plasmid DNA (HBV 1.2, IL-32 ⁇ , and ⁇ -gal) was injected into 6-week old male BALB/C mice through hydrodynamic injection. Plasmid DNA-added PBS was injected into a mouse caudal vein at the same amount corresponding to 10% of the body weight of the mouse. DNA flowed into the vein due to a high pressure for 4 to 6 seconds. All experiments were carried out by the approval of the Animal Experiment Ethics Committee of Konkuk University.
  • HBV-infected mouse liver tissue To detect IL-32 ⁇ from HBV-infected liver tissue, immunohistochemistry was carried out using HBV-infected mouse liver tissue through hydrodynamic injection. 4 days after the infection of the mouse with HBV, the mouse was sacrificed to extract the liver, and then the liver tissue was fixed and embedded in a paraffin block. Subsequently, the paraffin block was cut to a thickness of 5 ⁇ m using silane coated glass (MUTO, Japan). Next, the cut tissue sections were washed with an alcohol, pretreated with 0.01M sodium citrate (pH 6.0), and treated with hydrogen peroxide (H 2 O 2 )dilutedwithmethanoltoaconcentrationof3%toinactivateaperoxidaseinthecells.
  • H 2 O 2 hydrogen peroxide
  • tissue section samples were treated with a 3,3’-diaminobenzidine tetrahydrochloride chromogen solution (DAKO), and mounted with a mounting solution after being counterstained with hematoxylin.
  • DAKO 3,3’-diaminobenzidine tetrahydrochloride chromogen solution
  • Virus-specific CD8+ cytotoxic T cells can secrete TNF- ⁇ and IFN- ⁇ .
  • the cytokines secreted from such immune cells allow a host to be protected against viral infections. Therefore, to identify whether IL-32 expression in cells was induced by TNF- ⁇ and IFN- ⁇ , Huh7 cells were treated with each of TNF- ⁇ and IFN- ⁇ , and then subjected to western blotting according to the method described in Example 1-7 to measure the expression level of an IL-32 protein.
  • the IL-32 expression was increased proportionally to the treatment concentrations of TNF- ⁇ and IFN- ⁇ in the cells, and when two types of cytokines were treated together, it was confirmed that the secretion of the IL-32 protein was strongly induced.
  • the cells were treated with each of the cytokines, incubated for 48 hours, and then subjected to an XTT assay, it was confirmed that there was no change in cell viability.
  • FIG. 1b by western blotting and microscopy, it can be seen that the IL-32 protein was mainly expressed in the cytoplasm.
  • Example 2 From the result of Example 2, it was confirmed that IL-32 expression in cells was induced by TNF- ⁇ and IFN- ⁇ , and then it was attempted to verify whether IL-32 exhibited an anti-HBV effect.
  • human liver cancer cell lines such as Huh7 and HepG2 cells, were treated with recombinant human IL-32 ⁇ (rhIL-32 ⁇ ) at 0.1, 0.25, or 0.5 ⁇ g.
  • FIG. 2a it was confirmed that there was no inhibitory effect on HBV replication in both types of the cells above due to rhIL-32 ⁇ .
  • human THP-1 and mouse Raw 264.7 cells were treated with rhIL-32 ⁇ , which is the most activated form of IL-32, at two different concentrations, and subjected to ELISA to measure a secreted amount of IL-32, and therefore, as shown in FIG. 2b, it was confirmed that IL-32 ⁇ was abundantly present in the cells, compared to the culture medium. It also can be seen that, even when either TNF- ⁇ or IFN- ⁇ was treated, IL-32 was expressed in most cells.
  • the cells were treated with each of TNF- ⁇ (20 ng or 100 ng) or IFN- ⁇ (500 U or 1000 U), or both of 20 ng of TNF- ⁇ and 500 U of IFN- ⁇ , and then subjected to southern blotting according to the method described in Example 1-6. Consequently, as shown in FIG. 2c, the antiviral effect on HBV was exhibited due to treatment of TNF- ⁇ and IFN- ⁇ , and when two types of cytokines were treated together, the antiviral effect was strongly induced. In addition, it was confirmed that there was no change in cell viability due to treatment of the substances.
  • Huh7 and HepG2 cell lines were transfected with IL-32-specific siRNA to inhibit the IL-32 expression, and then an antiviral effect on HBV was measured. Consequently, as shown in FIG. 2d, it was confirmed that HBV replication suppressed by the inhibition of the IL-32 expression was restored.
  • IL-32 serves as downstream molecules of TNF- ⁇ and IFN- ⁇ , and intracellular expression thereof mediates an antiviral effect against HBV.
  • Huh7 and HepG2 cells transfected with both HBV 1.2 and IL-32 ⁇ plasmids were introduced, after extraction of HBV DNA, southern blotting was carried out to analyze a degree of HBV replication.
  • Huh7 cells were transfected with IL-32 ⁇ , IL-32 ⁇ and IL-32 ⁇ plasmids or treated with TNF- ⁇ and IFN- ⁇ in the same manner as described above, and then subjected to southern blotting to comparatively analyze a degree of HBV replication. Consequently, as shown in FIG. 3c, it was confirmed that all of the IL-32 ⁇ , IL-32 ⁇ and IL-32 ⁇ reduced HBV replication, and among these, IL-32 ⁇ exhibited the most excellent antiviral effect.
  • FIG. 3d to confirm an HBV replication inhibitory effect due to IL-32 ⁇ expression, when IL-32 expression was inhibited by transfection with IL-32 siRNA, the HBV replication inhibitory effect was not exhibited.
  • Example 4 Based on the results of Examples 3 and 4, to identify at what stage HBV replication was inhibited due to intracellular IL-32 expression, an experiment was carried out in the same manner as described in Example 4, and then an expression level of HBV mRNA was measured through northern blotting. Consequently, as shown in FIG. 4a, it was confirmed that levels of HBV mRNA such as pg/preC RNA and HBV surface RNA (Pre-S/S RNA) were decreased depending on the transfection concentration of IL-32 ⁇ . In addition, as shown in FIG. 4b, it was confirmed that the levels of surface and core proteins of HBV were significantly decreased in proportion to the transfection concentration of IL-32 ⁇ .
  • HBV mRNA such as pg/preC RNA and HBV surface RNA (Pre-S/S RNA) were decreased depending on the transfection concentration of IL-32 ⁇ .
  • FIG. 4b it was confirmed that the levels of surface and core proteins of HBV were significantly decreased in proportion to the transfection concentration of IL-32 ⁇ .
  • HBV RNA production was associated with various transcription factors such as HNF1, HNF3, HNF4, and CEBP, abundantly present in liver cells. This means that HBV utilizes transcription factors present in host liver cells in its replication without producing its own transcription factors. Therefore, the inventors performed an experiment on the assumption that the transcription factors present in the liver cells are associated with an inhibitory effect of IL-32 on transcription of HBV RNA.
  • each transcription factor has its own function by binding to a specific site of the enhancer, and it has been known that, for example, HNF4 promotes viral transcription, and HNF3 inhibits HBV transcription.
  • Example 1-10 ChIP analysis was carried out according to the method described in Example 1-10 to detect whether IL-32 was bound with the transcription factors present in the liver cells in the transfected cells, an immunoprecipitated HBV DNA fragment was detected by semi-quantitative RT-PCR using anti-HNF4 ⁇ and anti-HNF1 ⁇ antibodies. Therefore, as shown in FIGS. 5d and 5e, binding efficiency of HNF4 ⁇ binding to RI (Enhancer I) and HNF1 ⁇ binding to RII (Enhancer II) was reduced when IL-32 was expressed. Such a result shows which method makes binding patterns of the transcription factors changed due to the influence of IL-32 on an HBV enhancer-binding site.
  • EMSA and western blotting were carried out by the method described in Example 1-11, and the western blotting was carried out under the same conditions as those of EMSA. Consequently, as shown in FIG. 5f, it was observed that the HNF4 ⁇ protein was present at the same place as the protein-DNA. However, IL-32 did not bind to an HNF4 ⁇ probe due to the decrease in expression of an HNF4 ⁇ protein, which did not correspond to the ChIP result.
  • MAPKs mitogen-activated protein kinases
  • the inventors measured protein expression levels of several molecules associated with MAPK signal transduction and antiviral signal transduction pathways under the condition in which IL-32 was expressed.
  • a sample treated with only TNF- ⁇ and IFN- ⁇ was used as a positive control. Consequently, as shown in FIG. 6a, it was confirmed that, when IL-32 was expressed in Huh7 cells, the expression of phosphorylated ERK1/2 (p-extracellular signal-regulated kinase 1/2) and p38 protein was increased, but there was no change in the expression of Jun N-terminal kinase (JNK).
  • JNK Jun N-terminal kinase
  • FIG. 7b it was observed that an HBsAg level upon IL-32 ⁇ expression was significantly decreased in a mouse serum, and according to determination of degrees of the expression of IL-32 and a HBV surface protein by immunohistochemistry, as shown in FIG. 7c, the expression of the HBV surface protein was significantly reduced when IL-32 ⁇ was expressed, and therefore it can be seen that the antiviral effect on HBV was mediated by IL-32 ⁇ in a mouse.
  • the inventors first identified an IL-32-mediated antiviral effect on HBV and a molecular mechanism thereof. Therefore, the IL-32-mediated effect on HBV can provide a new understanding for the development of a therapeutic agent for the virus, and an antiviral composition according to the present invention can be effectively used in the development of an antiviral agent.

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Abstract

The present invention relates to an antiviral composition against hepatitis B virus, which includes an interleukin-32 (IL-32) gene or an IL-32 protein, which is an inhibitor of expression or activation of hepatocyte nuclear factor 4 alpha (HNF4α) or hepatocyte nuclear factor 1 alpha (HNF1α), as an active ingredient, and a method for screening an antiviral substance against the virus. The inventors experimentally confirmed an inhibitory effect on viral DNA replication and expression by reducing the expression of host transcription factors such as HNF4α and HNF1α, involved in the transcription of the viral gene by increasing ERK1/2 phosphorylation due to IL-32 secretion induced by TNF-α and IFN-γ cytokines secreted from infected cells when host cells were infected with the hepatitis B virus, and therefore an IL-32-mediated antiviral effect on hepatitis B virus and a molecular mechanism thereof were identified. Therefore, the antiviral activity of IL-32 against hepatitis B virus newly identified according to the present invention may provide a new understanding for the development of therapeutic agent against the virus, and may be effectively used in the development of an antiviral agent.

Description

ANTIVIRAL COMPOSITION AGAINST HEPATITIS B VIRUS, INCLUDING INTERLEUKIN-32 AS ACTIVE INGREDIENT
The present invention relates to an antiviral composition against hepatitis B virus (HBV), and more particularly, to an antiviral composition against hepatitis B virus, including interleukin-32 (IL-32) which is an inhibitor of expression or activation of hepatocyte nuclear factor 4 alpha (HNF4α) or hepatocyte nuclear factor 1 alpha (HNF1α) as an active ingredient, and a method for screening an antiviral substance against the virus.
Continuous infection by HBV as well as chronic hepatitis B (CHB), liver cirrhosis and hepatocellular carcinoma have emerged globally as public health problems.
It has been known that the HBV genome (3.2Kb), relaxed circular DNA (RC DNA), is converted into covalently closed circular DNA (cccDNA) in the nucleus. HBV expresses 4 types of viral proteins, that is, a polymerase, a surface protein, a core protein, and HBx from four open-reading frames (PreC/C, P, preS1/S2/S and X). HBV replication occurs by reverse transcription from core capsids using the polymerase protein of HBV. In the nucleus, 4 types of HBV RNA encoded in cccDNA of HBV are transcripted by an HBV enhancer. In addition, various transcription factors such as a hepatocyte nuclear factor (HNF) and a CCAAT/enhancer binding protein (C/EBP) have been known to be involved in producing HBV RNA when binding to enhancers I and II regions of HBV, which means that HBV is able to utilize a host transcription factor for transcription.
Until now, according to a variety of in vitro and in vivo studies, it has been reported that cytokines inhibit gene expression and replication of HBV, and the occurrence of a disease caused thereby by various mechanisms. For example, a host defense system against HBV is known to regulate the life cycle of HBV. Particularly, TNF-α and IFN-γ are well known as cytokines inducing an antiviral response in the host defense mechanism. Secretion of TNF-α and IFN-γ is mediated by a variety of antiviral genes through cytokine-dependent signal transduction pathways such as CIAP2, MxA and PKR pathways. However, according to research of other mechanisms, it has been reported that TNF-α regulates generation of HBV RNA and stability of a capsid, and IFN-γ removes a capsid including pregenomic RNA from mouse liver cells. In addition, it has been known that TNF-α and IFN-γ are involved in removal of a non-cytopathic virus. It has been reported that, other than TNF-α and IFN-γ, various cytokines also exhibit directly or indirectly an antiviral effect on HBV. Therefore, various cytokines have been known to contribute to the host defense system with respect to HBV infection, but a host immune mechanism with respect to HBV has not been clearly known yet.
Meanwhile, IL-32 is a type of cytokine, and has been known to induce expression in the pancreas due to the synergistic effect of TNF-α and IFN-γ. IL-32 is generated by various epithelial cells, and immune cells such as T lymphocytes, NK cells and monocytes. The IL-32 gene may be located on a human chromosome 16p13.3, and IL-32 is known as a pro-inflammatory cytokine that activates signal transduction pathways of a mitogen-activated protein kinase (MAPK) and a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). The IL-32 gene has six selective splicing variants after transcription, and among such variants, IL-32γ is known as the most activated form. However, biological functions of isoforms of the IL-32 are not well known. According to conventional studies, it has been reported that IL-32 is associated with various bacteria and viruses such as Orientia Tsutsugamushi, vesicular stomatitis virus (VSV), human immunodeficiency virus (HIV), hepatitis C virus (HCV), human papilloma virus (HPV), and influenza virus (Immunology 2011;132:410-420). However, an antiviral effect of IL-32 on HBV and a direct mechanism thereof have not been defined.
As a result of research conducted to define factors inducing antiviral effects by TNF-α and IFN-γ and molecular action mechanisms thereof in an immune defense system of a host against HBV, the inventors first identified that replication of the virus is inhibited by reducing the enhancer activity of hepatitis B virus by inhibiting expression or activation of HNF4α or HNF1α, which is a transcription factor present in liver cells of a host, by IL-32 expression induced by TNF-α and IFN-γ. Based on this finding, the present invention was completed.
Therefore, the present invention is directed to providing an antiviral composition against HBV, including an IL-32 gene or IL-32 protein which is an inhibitor of expression or activation of HNF4α or HNF1α as an active ingredient.
The present invention is also directed to providing a method for screening an antiviral substance against HBV.
However, technical problems to be solved in the present invention are not limited to the above-described problems, and other problems which are not described herein will be fully understood by those of ordinary skill in the art from the following descriptions.
To achieve the above-mentioned objects of the present invention, the present invention provides an antiviral composition against HBV, including an IL-32 gene or IL-32 protein which is an inhibitor of expression or activation of HNF4α or HNF1α as an active ingredient.
In one exemplary embodiment of the present invention, the IL-32 gene may consist of a base sequence selected from the group consisting of SEQ ID NOs: 1 to 4.
In another exemplary embodiment of the present invention, the IL-32 protein may consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 8.
In still another exemplary embodiment of the present invention, the IL-32 may inhibit the expression or activation of HNF4α or HNF1α by increasing ERK1/2 protein phosphorylation in cells.
In yet another exemplary embodiment of the present invention, the composition may inhibit virus replication by reducing the enhancer activity of HBV by inhibiting expression or activation of HNF4α or HNF1α.
In addition, the present invention provides a method for screening an antiviral substance against HBV, which includes the following steps:
(a) in vitro treating cells with a candidate substance;
(b) measuring an expression level of IL-32 in the cells; and
(c) selecting a substance increasing the expression of IL-32 compared to a candidate substance-untreated group as an antiviral substance against HBV.
In an exemplary embodiment of the present invention, the cells may be liver cells.
In another exemplary embodiment of the present invention, the candidate substance may be selected from the group consisting of a compound, a microbial culture medium or extract, a natural substance extract, a nucleic acid and a peptide.
In still another exemplary embodiment of the present invention, the nucleic acid may be selected from the group consisting of siRNA, shRNA, microRNA, antisense RNA, an aptamer, a locked nucleic acid (LNA), a peptide nucleic acid (PNA), and a morpholino.
In yet another exemplary embodiment of the present invention, Step (b) may be carried out by performing measurement using a method selected from the group consisting of a polymerase chain reaction (PCR), a microarray, northern blotting, western blotting, an enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunohistochemistry, and immunofluorescence.
In addition, the present invention provides a method for treating hepatitis B, which includes administering an antiviral composition including an IL-32 gene or an IL-32 protein, which is an inhibitor of expression or activation of HNF4α or HNF1α, as an active ingredient.
Moreover, the present invention provides a use of an antiviral composition including an IL-32 gene or IL-32 protein, which is an inhibitor of expression or activation of HNF4α or HNF1α, as an active ingredient.
The inventors first identified an IL-32-mediated antiviral effect against HBV and a molecular mechanism thereof by confirming that IL-32 secretion is induced by TNF-α and IFN-γ cytokines secreted from host cells infected by HBV, the secreted IL-32 reduces the expression of HNF4α and HNF1α, which are host transcription factors involved in the transcription of a viral gene, by increasing ERK1/2 phosphorylation to exhibit an inhibitory effect on DNA replication and gene expression of the virus, and the IL-32 exhibits the above-mentioned effect only when expressed in the cells. Therefore, an IL-32-mediated antiviral effect on HBV can impart a new understanding for the development of a therapeutic agent for the virus, and the antiviral composition according to the present invention can be effectively used in the development of an antiviral therapeutic agent.
FIGS. 1a and 1b show IL-32 expression induced by TNF-α and IFN-γ, where FIG. 1a shows the result of western blotting for measuring expression levels of an IL-32 protein after a Huh7 liver cancer cell line is treated with TNF-α and IFN-γ at different concentrations, and the change in cell viability by treatment of the two types of cytokines, and FIG. 1b shows the result of microscopy representing that an IL-32 protein is mainly expressed in the cytoplasm.
FIGS. 2a to 2d show the inhibitory effect of IL-32 on HBV replication, where FIG. 2a shows that all of Huh7 and HepG2 cells do not exhibit an inhibitory effect on HBV replication by treatment of a recombinant IL-32 (rhIL-32γ) protein, FIG. 2b shows the ELISA result representing that IL-32γ is present in most cells, FIG. 2c shows that inhibition of HBV replication is induced by treatment of Huh7 cells with TNF-α and IFN-γ, and FIG. 2d shows that an inhibitory effect on HBV replication is not produced by treatment of IL-32-specific siRNA.
FIGS. 3a to 3d show that the inhibitory effect on HBV replication due to ectopic expression of IL-32, where FIG. 3a shows the result of Southern blotting representing that an HBV DNA level is decreased dependently on an IL-32γ expression concentration after Huh7 cells are transfected with HBV 1.2 and IL-32γ expression plasmids, FIG. 3b shows that an antiviral effect caused by IL-32 expressed in cells is produced by treating a culture medium with IL-32 antibodies, FIG. 3c shows the result of comparing degrees of inhibiting HBV replication according to expression of IL-32 isoforms (IL-32α, IL-32β, and IL-32γ), and FIG. 3d shows that HBV replication is not inhibited by transfection with IL-32 siRNA after IL-32γ expression.
FIGS. 4a to 4d show inhibition of viral gene expression by down-regulation of an IL-32 HBV enhancer, where FIG. 4a shows the result of northern blotting representing that HBV mRNA expression is decreased after Huh7 cells are transfected with HBV 1.2 and IL-32γ expression plasmids, FIG. 4b shows that a decrease in the expression of surface and core proteins of HBV is dependent on an IL-32γ transfection concentration, FIG. 4c is a diagram illustrating HBV enhancer luciferase reporter plasmid variants manufactured to identify a mechanism for reducing HBV mRNA expression by IL-32γ, and FIG. 4d shows that HBV enhancer activity is decreased by IL-32γ according to the result of analyzing a luciferase reporter.
FIGS. 5a to 5f show IL-32-mediated down-regulation of HNF4α and HNF1α transcription factors, where FIG. 5a shows binding sites of host transcription factors involved in HBV RNA transcription on a gene map of an HBV enhancer, FIGS. 5b and 5c are the results of quantitative RT-PCR and western blotting showing that a decrease in RNA and protein expression levels of HNF4α and HNF1α when IL-32γ is expressed in Huh7 cells, respectively, FIGS. 5d and 5e show the result of ChIP analysis indicating that binding efficiency of HNF4α binding to RI (Enhancer I) and HNF1α binding to RII (Enhancer II) is decreased under a condition for expressing IL-32γ, and FIG. 5f shows the result of EMSA, indicating that binding efficiency to a HNF4α-binding viral enhancer is decreased in the presence of IL-32γ, and the result of western blotting using a native gel, indicating that an HNF4α protein is expressed in a corresponding location.
FIGS. 6a to 6e show that the down-regulation of HNF4α and HNF1α due to IL-32 is caused by an ERK1/2-dependent pathway, where FIG. 6a shows that the expression of phosphorylated ERK1/2 and p38 protein is increased when IL-32γ is expressed in Huh7 cells, FIG. 6b shows that the expression of HNF4α and HNF1α proteins decreased by IL-32 is increased again when an ERK1/2 inhibitor, U0126, is treated, FIGS. 6c and 6d show that virus replication is increased again when each of HNF4α and HNF1α is overexpressed, and FIG. 6e shows that an HBV replication level is increased again when an ERK1/2 inhibitor, U0126, is treated.
FIGS. 7a to 7c show IL-32-induced HBV antiviral effects in mouse models, where FIG. 7a shows the result of southern blotting, indicating that HBV replication levels are decreased in mice expressing IL-32γ after HBV 1.2 and IL-32γ expression plasmids are injected into mice, FIG. 7b shows a decrease in an HBsAg level by IL-32γ expression in a mouse serum, and FIG. 7c shows that expression of an HBV surface protein is considerably decreased when IL-32γ is expressed through immunohistochemistry using mouse liver tissue.
FIG. 8 illustrates an antiviral effect against HBV by IL-32 identified in the present invention and a molecular mechanism thereof.
The present invention provides an antiviral composition against HBV, including IL-32 which is an inhibitor of expression or activation of HNF4α or HNF1α as an active ingredient, and a method for screening an antiviral substance against the virus.
Hereinafter, the present invention will be described in detail.
The inventors identified a factor inducing an antiviral effect by TNF-α and IFN-γ and a molecular action mechanism thereof in an immune defense system of a host against HBV, and therefore the present invention was completed.
Therefore, the present invention provides an antiviral composition against HBV, including an IL-32 gene or IL-32 protein, which is an inhibitor of expression or activation of HNF4α or HNF1α as an active ingredient.
The term “antiviral” used herein refers to weakening or ending the action of a virus having invading a body by inhibiting viral proliferation in the body, and more specifically, inhibiting viral proliferation by inhibiting nucleic acid synthesis of a virus, gene expression, or viral replication, and in the present invention, HBV is targeted.
In the present invention, HNF4α and HNF1α are nuclear transcription factors binding to DNA, in which HNF4α is known to regulate expression of various genes including HNF1α, and regulates expression of various genes in liver cells. HNF4α is also known to play an important role in liver, kidney and intestinal development, and to be clinically associated with diabetes called maturity onset diabetes of the young (MODY) and colon cancer. In addition, it has been reported that HNF4α is bound to a viral enhancer to promote transcription of a viral gene.
The term “activation inhibitor” used herein refers to a substance causing a reduction in the function of a target protein, and preferably, due to the activation inhibitor, the function of a target protein is undetectable or present at an insignificant level.
The expression or activation inhibitor of HNF4α and HNF1α according to the present invention, such as the IL-32 gene, may consist of a base sequence selected from the group consisting of SEQ ID NOs: 1 to 4, and more specifically, SEQ ID NOs: 1 to 4 are the base sequences of IL-32 isoforms such as IL-32α, IL-32β, IL-32γ, and IL-32δ, respectively, and preferably, the IL-32 gene may consist of the base sequence of SEQ ID NO: 3.
In the present invention, the IL-32 protein may consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 8, and more specifically, SEQ ID NOs: 5 to 8 are the amino acid sequences of IL-32 isoforms such as IL-32α, IL-32β, IL-32γ, and IL-32δ protein, respectively, and preferably, the IL-32 protein may consist of the amino acid sequence of SEQ ID NO: 7.
Hereinafter, an antiviral effect against HBV by IL-32 and a molecular mechanism thereof will be identified through the exemplary embodiments of the present invention.
In one exemplary embodiment of the present invention, it was confirmed that, to examine induction of IL-32 expression by cytokines secreted from immune cells in viral infections, such as TNF-α and IFN-γ, when a liver cancer cell line was treated with TNF-α and IFN-γ, expression of IL-32 was increased, and when treated with the two types of cytokines, the IL-32 expression was more strongly induced (refer to Example 2).
In another exemplary embodiment of the present invention, it was confirmed that HBV replication was inhibited due to IL-32 expression induced by TNF-α and IFN-γ in a liver cancer cell line, which was not shown when a recombinant IL-32 protein was extracellularly treated (refer to Example 3).
In still another exemplary embodiment of the present invention, an inhibitory effect on HBV replication was shown when intracellular expression of IL-32 is induced by transfecting a liver cancer cell line with plasmids expressing HBV and IL-32, and an antiviral effect was exhibited even when a cell culture medium was treated with an IL-32-specific antibody, and therefore, it was confirmed that HBV replication was inhibited by IL-32 expressed only in cells. In addition, all of various isoform proteins of IL-32 exhibited an inhibitory effect on HBV replication, and among the proteins, IFN-γ is most effective (refer to Example 4).
In yet another exemplary embodiment of the present invention, it was confirmed that the inhibitory effect of IL-32 on HBV replication was exhibited at an HBV RNA transcription level, and this occurs due to the reduction in HBV enhancer activity (refer to Example 5).
In yet another exemplary embodiment of the present invention, it was confirmed that RNA and protein expression of HNF4α and HNF1α was decreased under a condition of IL-32 expression by measuring expression levels of transcription factors present in host liver cells involved in transcription when binding to an HBV enhancer, and degrees of binding HNF4α and HNF1α to the HBV enhancer were decreased. Therefore, it can be seen that transcription and replication of an HBV gene are inhibited by inhibiting expression and activation of HNF4α and HNF1α due to IL-32 (refer to Example 6).
In yet another exemplary embodiment of the present invention, as a result of measuring MAPK signal transduction associated with TNF-α and IFN-γ and protein expression levels of molecules associated with an antiviral signal transduction pathway under a condition of IL-32 expression, it was confirmed that expression of phosphorylated ERK1/2 and p38 was reduced, the expression levels of HNF4α and HNF1α decreased due to IL-32 expression upon the treatment of an ERK1/2 inhibitor were increased again, and an HBV replication level was increased again (refer to Example 7).
In yet another exemplary embodiment of the present invention, an antiviral effect caused by IL-32 expression was confirmed using an HBV-infected mouse model (refer to Example 8).
According to an example of the present invention, it was newly identified that the expression and replication of an HBV gene are inhibited by inducing IL-32 expression by TNF-α and IFN-γ secreted from infected cells when cells are infected with HBV, increasing ERK1/2 phosphorylation, and reducing HBV enhancer activity by inhibiting the expression and activation of HNF4α and HNF1α involved in HBV gene transcription.
The antiviral composition of the present invention may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is conventionally used in preparations, and may be, but is not limited to, a saline solution, distilled water, Ringer's solution, buffered saline, a cyclodextrin solution, a dextrose solution, a maltodextrin solution, glycerol, ethanol, or liposomes, etc., and may further include another conventional additive such as an antioxidant or a buffer as needed. In addition, the pharmaceutically acceptable carrier may be prepared as injectable preparations such as an aqueous solution, a suspension, and an emulsion, pills, capsules, granules or tablets by further adding diluents, dispersants, surfactants, binders, lubricants, etc. Suitable pharmaceutically acceptable carriers and their preparations may be prepared according to each ingredient using a method disclosed in the Remington’s Pharmaceutical Science. The pharmaceutical composition of the present invention is not limited in formulation, and thus may be prepared as injections, inhalants, external preparations for skin, or oral medications.
The antiviral composition of the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, percutaneously, nasally, or intratracheally) according to a desired method, and a dose of the pharmaceutical composition of the present invention may be selected according to a patient’s condition and body weight, severity of a disease, a dosage form, an administration route and time by those of ordinary skill in the art.
The composition of the present invention is administered at a pharmaceutically effective amount. In the present invention, the “pharmaceutically effective amount” refers to an amount sufficient to treat the disease at a reasonable benefit/risk ratio applicable for medical treatment, and an effective dosage may be determined by parameters including a type of a patient’s disease, severity, drug activity, sensitivity to a drug, administration time, an administration route and an excretion rate, the duration of treatment and drugs simultaneously used, and other parameters well known in the medical field. The composition of the present invention may be administered separately or in combination with other therapeutic agents, and may be sequentially or simultaneously administered with a conventional therapeutic agent, or administered in a single dose or multiple doses. In consideration of all of the above-mentioned parameters, it is important to achieve the maximum effect with the minimum dose without a side effect, and such a dose may be easily determined by one of ordinary skill in the art.
Specifically, the effective amount of the composition according to the present invention may vary depending on a patient’s age, sex, and body weight, and may be generally administered at 0.001 to 150 mg and, preferably, 0.01 to 100 mg/kg of body weight daily or every other day, or once to three times a day. However, the effective amount may vary depending on an administration route, the severity of obesity, sex, body weight or age, and therefore, the scope of the present invention is not limited by the dose in any way.
The composition of the present invention may be used in various applications such as medications, food and beverages, and in forms of powder, granules, tablets, capsules or drinks.
As another aspect of the present invention, the present invention provides a method for screening an antiviral substance against HBV, which includes the following steps:
(a) in vitro treating cells with a candidate substance;
(b) measuring an expression level of IL-32 in the cells; and
(c) selecting a substance increasing the expression of IL-32 compared to a candidate substance-untreated group as an antiviral substance against HBV.
In the present invention, the cells include liver cells, and any type of liver-derived cells may be used without limitation.
The candidate substance may be selected from the group consisting of a compound, a microbial culture medium or extract, a natural substance extract, a nucleic acid, and a peptide, and the nucleic acid is preferably selected from the group consisting of siRNA, shRNA, microRNA, antisense RNA, an aptamer, a locked nucleic acid (LNA), a peptide nucleic acid (PNA), and a morpholino, but the present invention is not limited thereto.
In Step (b), measurement of an expression level of IL-32 may be carried out by a polymerase chain reaction (PCR), a microarray, northern blotting, western blotting, an enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunohistochemistry, and immunofluorescence, but the present invention is not limited thereto.
Hereinafter, exemplary examples will be provided to help in understanding of the present invention. However, the following examples are merely provided to more easily understand the present invention, and the scope of the present invention is not limited to the following examples.
[Examples]
Example 1. Experimental preparations and methods
1-1. Cell culture
Human liver cancer cell lines, such as Huh7 and HepG2, were purchased from American Type Culture Collection (Manassas, VA, USA), and incubated in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco BRL, Oregon, USA) containing 10% fetal bovine serum (FBS, Gibco BRL) inactivated by thermal treatment and 1% penicillin and streptomycin (Gibco BRL) at 37 °C with 5% CO2.
1-2. Preparation of plasmid
An HBV 1.2 plasmid used in the example of the present invention was obtained from a DNA prep kit (Alphagene, Gyeonggi, Korea), and a pCAGGS mock vector, and IL-32α, IL-32β and IL-32γ plasmids were provided by a different team of Konkuk University. An HNF4α expression plasmid was amplified by PCR using an HepG2 cDNA library as a template, and subcloned in a pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA). An HBV enhancer luciferase plasmid was amplified by PCR using the HBV 1.2 plasmid. In addition, to manufacture an HBV enhancer luciferase reporter, amplification was carried out by PCR using the HBV 1.2 plasmid, and primer sequences used herein are listed in Table 1.
Primer Sequence SEQ ID NO:
pEnhI.II-Luc_Forward 5'-GGG GTA CCT AAA TAG ACC TAT TGA T-3' 9
pXp.EnhII-Luc_Forward 5'-GGG GTA CCG CGC ATG C-3' 10
pNRE.EnhII-Luc_Forward 5'-GGG GTA CCG GAA ATA CAC CTC-3' 11
pEnhII/cp-Luc_Forward 5'-GGG GTA CCT CAC CTC TGC-3' 12
pEnhI.ΔII-Luc_Reverse 5'-AGA GAT CTA GCG AAG TCA CAC-3' 13
pEnh-Luc_Reverse 5'-TGA GAT CTA CAG ACC AAT TTA TGC-3' 14
1-3. Transfection and treatment conditions
Transfection was carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol when cells reached a confluency of 70 to 80% of the area of a culture vessel. 15 hours after transfection, rhIL-32γ (YbdY, Seoul, Korea), TNF-α (YbdY), IFN-γ (LG, Jeonbuk, Korea), anti-IL-32 (YbdY) and signal transduction inhibitors were added to new media to treat the cells, and the cells were cultured for 3 days. Cytokine treatment was carried out with rhIL32 (0.1, 0.25, and 0.5 μg/ml), TNF-α (0.02 and 0.1 μg/ml), IFN-γ (500 and 1000 U/ml), and mixed treatment (TNF-α 0.02 μg/ml and IFN-γ 500 U/ml). A signal transduction inhibitor, U0126, was treated at a final concentration of 10 μM, and anti-IL32 was treated at concentrations of 0.2 and 0.5 μg/ml. The transfected cells were recovered two or three days after the transfection, and then used for a subsequent experiment.
1-4. RNA interference of IL-32
To specifically inhibit the expression of IL-32 mRNA, IL-32-specific siRNA was synthesized at ST Pharm (Seoul, Korea) to be used in an experiment. The sequence of the siRNA is shown in Table 2 below, and cells were transfected using 10 or 20 nM of Lipofectamine 2000.
siRNA Sequence SEQ. ID NO:
IL-32 siRNA sense 5'-GGC UUA UUA UGA GGA GCA GTT-3' 15
antisense 5'-CUG CUC CUC AUA AUA AGC CTT -3' 16
1-5. Cell viability analysis
To analyze cell viability, a cell viability kit was purchased from WELGENE (Seoul, Korea). Huh7 cells were seeded in a 6-well plate, and subjected to transfection or other treatment processes. Afterward, the cell culture medium was replaced with a fresh medium and then the original medium was transferred to a 96-well plate. Then, an XTT reagent and a PMS reagent were added to the 96-well plate, followed by incubating the cells for up to 1 hour, and absorbance was measured at 450 nm using a spectrophotometer.
1-6. Southern blotting
Three days after cell transfection, the cells were collected with a scrapper and lysed by adding 100 μl of a HEPES buffer, and then the core capsids of HBV were precipitated with a polyethylene glycol (PEG) solution. Subsequently, the core capsids were degraded by treatment with a proteinase K-containing SDS solution at 37 °C for 3 hours. Afterward, DNA was isolated from a 1% agarose gel at 100 V for 3 hours, and then transferred to a nitrocellulose membrane (GE Healthcare). To detect the HBV DNA on the membrane, the membrane was hybridized with random hexamers and purified 32P-labeled HBV probes.
1-7. Western blotting
Two or three days after cell transfection, the cells were recovered and lysed with a RIPA buffer [20 mM Tris/HCl, 1% NP-40, 0.5% protease inhibitor cocktail (Sigma, St.Louis, MO), 150 mM NaCl, 2 mM KCl, pH7.4]. After a cell precipitate was filtered, proteins in the cell lysate were divided by size through SDS-PAGE. Subsequently, the proteins in a polyacrylamide gel were transferred onto a PVDF membrane, and then the membrane were treated with a primary antibody, that is, anti-IL-32γ (YbdY), anti-actin (Sigma), anti-GFP (Sigma), anti-HBsAg (Abcam), anti-HBcAg (DAKO, USA), anti-HNF1α (Santa Cruz), anti-HNF3β (Santa Cruz), anti-CEBPα (Santa Cruz), anti-HNF4α (Santa Cruz), anti-lamin (Santa Cruz), or anti-tubulin (Santa Cruz), at a 1:2000 dilution. Afterward, by treatment of a secondary antibody against the primary antibody, an expression level of a target protein was determined, and the membrane was treated with a stripping reagent to remove all of the antibodies and then used to determine the expression level of a different protein.
1-8. Luciferase reporter assay
To measure HBV enhancer activity, a luciferase reporter assay was carried out by the following method. More specifically, Huh7 cells were seeded in a 12-well plate at a density of 2x105well/cells,andtransfectedwith0.5μgofEnhancer-Luc(pEnhI.II,pEnhI.delII,pXp.EnhII,pEnhII/cp),0.5μgofIL-32γ,or0.25μgofaβ-galplasmid. Here, to normalize a total amount of the transfected plasmid DNA, pCAGGS was used as a control vector. 48 hours after transfection, the cells were recovered and lysed, followed by measuring luciferase activity using a reagent Steady Glo-Luciferase system (Promega, Madison, WI). Experimental data was derived from values obtained by experiments repeated at least three times.
1-9. Real-time quantitative PCR
Huh7 cells were lysed with a TRIzol reagent (Invitrogen) according to the manufacturer’s protocols to extract total RNA. A reaction solution was prepared by mixing 2 μg of the extracted RNA and an MMLV reverse transcriptase (Intron Biotechnology) such that the mixed solution had a final volume of 20 μl, and subjected to reverse transcription PCR (RT-PCR) to synthesize cDNA. The cDNA synthesized as described above was amplified by performing PCR under the following conditions: 40 cycles of first denaturation at 94 °C (5 min), 94 °C (30 sec) and 72 °C (1 min), and final elongation at 72 °C for 5 minutes. Primer sequences used herein are listed in Table 3 below. A real time PCR amplification process was carried out using an ABI PRISM 7500 sequence detection machine with a SYBR Green PCR master mix (Applied Biosystems), and relative mRNA quantitative analysis was carried out using a ΔΔCt method. The results are expressed as a relative n-fold difference with respect to a calibrator (RQ=2- ΔΔCt).
Primer Sequence SEQ ID NO:
HNF1α Forward 5'-TGTGCGCTATGGACAGCCTGC-3' 17
Reverse 5'-CTGTGTTGGTGAACGTAGGA-3 18
HNF4α Forward 5'-GAGTGGGCCAAGTACATCCCAG-3' 19
Reverse 5'-GCTTTGAGGTAGGCATACT-3' 20
HNF3b Forward 5'-AAGATGGAAGGGCACGAGC-3' 21
Reverse 5'-TGTACGTGTTCATGCCGTTCA-3' 22
C/EBPa Forward 5'-CCTTGTGCAATGTGAATGTGC-3' 23
Reverse 5'-CGGAGAGTCTCATTTTGGCAA-3' 24
GAPDH Forward 5'-ATCATCCCTGCCTCTACTGG-3' 25
Reverse 5'-TGGGTGTCGCTGTTGAAGTC-3' 26
1-10. Chromatin immunoprecipitation assay ( ChIP )
ChIP analysis was carried out using a ChIP assay kit (Millipore, Billerica, MA) according to the following method. A supernatant was purified with a protein A-agarose, and incubated by treatment of anti-HNF1α (Santa Cruz), anti-HNF4α (Santa Cruz), or a normal rabbit IgG antibody as a negative control group. Huh7 cells were cultured in a 6-well plate, and then transfected with or without IL-32γ before being used in an experiment. Primer sequences used in this experiment are listed in Table 4.
Primer Sequence SEQ ID NO:
ChIP R1 Forward 5′ -TAAATAGACCTATTGATTGGAAAGTATGT-3′ 27
Reverse 5′ -GAGAGAGGACAACAGAGTTGTCAG-3′ 28
ChIP R2 Forward 5′ -TCACCTCTGCACGTCGCATG-3′ 29
Reverse 5′ -ACAGACCAATTTATGCCTACAGCC-3′ 30
ChIP R3 Forward 5′ -CTACTGTACCTGTCTTTAATCCTGAGTGG-3′ 31
Reverse 5′ -CTGTGTGTAGTTTCTCTCTTATATAGAATG-3' 32
1-11. Electrophoretic mobility shift assay ( EMSA )
18 hours after transfection of the Huh7 cells, the cells were recovered, and treated with nuclear and cytoplasmic extraction reagents (Thermo, Rockford, USA) to separate nucleic and cytoplasmic fractions. Subsequently, 2 μg of the nucleic fraction was precultured, and a [32P]-gamma-labeled dsDNA oligonucleotide was used for HNF4α binding. The double-stranded DNA (Enhancer I HNF4α) was purchased from Bioneer (Daejeon, Korea). After the binding reaction was performed on ice, a DNA-protein complex was subjected to electrophoresis at a low temperature using a 6% polyacrylamide gel, and then the gel was dried at 70 °C for 30 minutes. Unlabeled HNF4α DNA was added to the gel, and incubated for 10 minutes before treatment of [32P]-gamma-labeled probes.
1-12. Immunofluorescence analysis
To observe expression of IL-32γ, HNF4α, and HNF1α, immunofluorescence analysis was performed. Huh7 cells were transfected with an IL-32γ plasmid and treated with TNF-α and IFN-γ, and after 48 hours, the cells were observed using a confocal microscope (FV-1000 spectral, Olympus). More specifically, the Huh7 cells were cultured to a confluence of 50% of the area of a cover glass, and 48 hours after transfection, the cells were fixed by treatment of acetone. Afterward, the cells were treated with a 3% BSA solution diluted with PBS at a low temperature of 4 °C for 18 hours to carry out a blocking process, and then washed with PBS, followed by overnight culture at 4 °C by treating the cells with antibodies diluted at a dilution of 1:300. The cells were washed with PBS again, treated with Alexa 488 and 568 secondary antibodies (goat and rabbit) to perform a reaction at room temperature for 1 hour, and then washed with PBS. Toppro-3 (1:500) was used to stain nuclear DNA of the cells. Afterward, a cover glass was put on a slide glass for mounting, and then the cells were observed under a confocal microscope.
1-13. Hydrodynamic injection
Plasmid DNA ((HBV 1.2, IL-32γ, and β-gal) was injected into 6-week old male BALB/C mice through hydrodynamic injection. Plasmid DNA-added PBS was injected into a mouse caudal vein at the same amount corresponding to 10% of the body weight of the mouse. DNA flowed into the vein due to a high pressure for 4 to 6 seconds. All experiments were carried out by the approval of the Animal Experiment Ethics Committee of Konkuk University.
1-14. Immunohistochemistry for mouse liver tissue
To detect IL-32γ from HBV-infected liver tissue, immunohistochemistry was carried out using HBV-infected mouse liver tissue through hydrodynamic injection. 4 days after the infection of the mouse with HBV, the mouse was sacrificed to extract the liver, and then the liver tissue was fixed and embedded in a paraffin block. Subsequently, the paraffin block was cut to a thickness of 5 μm using silane coated glass (MUTO, Japan). Next, the cut tissue sections were washed with an alcohol, pretreated with 0.01M sodium citrate (pH 6.0), and treated with hydrogen peroxide (H2O2)dilutedwithmethanoltoaconcentrationof3%toinactivateaperoxidaseinthecells. Subsequently, to prevent non-specific binding, a blocking process was performed on the tissue section samples, followed by overnight culture at room temperature by treatment of the samples with an IL-32 antibody. After the reaction, a tissue section slide was treated with a 3,3’-diaminobenzidine tetrahydrochloride chromogen solution (DAKO), and mounted with a mounting solution after being counterstained with hematoxylin.
Example 2. Confirmation of IL-32 induction in TNF -α and IFN -γ-mediated cells
Virus-specific CD8+ cytotoxic T cells can secrete TNF-α and IFN-γ. The cytokines secreted from such immune cells allow a host to be protected against viral infections. Therefore, to identify whether IL-32 expression in cells was induced by TNF-α and IFN-γ, Huh7 cells were treated with each of TNF-α and IFN-γ, and then subjected to western blotting according to the method described in Example 1-7 to measure the expression level of an IL-32 protein.
Consequently, as shown in FIG. 1a, the IL-32 expression was increased proportionally to the treatment concentrations of TNF-α and IFN-γ in the cells, and when two types of cytokines were treated together, it was confirmed that the secretion of the IL-32 protein was strongly induced. Here, to verify whether the treatment of TNF-α and IFN-γ affects cell viability, the cells were treated with each of the cytokines, incubated for 48 hours, and then subjected to an XTT assay, it was confirmed that there was no change in cell viability. In addition, as shown in FIG. 1b, by western blotting and microscopy, it can be seen that the IL-32 protein was mainly expressed in the cytoplasm.
Example 3. Confirmation of inhibitory effect on HBV replication due to IL-32 expression induced by TNF-α and IFN-γ
From the result of Example 2, it was confirmed that IL-32 expression in cells was induced by TNF-α and IFN-γ, and then it was attempted to verify whether IL-32 exhibited an anti-HBV effect. To this end, human liver cancer cell lines, such as Huh7 and HepG2 cells, were treated with recombinant human IL-32γ (rhIL-32γ) at 0.1, 0.25, or 0.5 μg.
Consequently, as shown in FIG. 2a, it was confirmed that there was no inhibitory effect on HBV replication in both types of the cells above due to rhIL-32γ. Based on the result, to identify the biological activity of rhIL-32γ, human THP-1 and mouse Raw 264.7 cells were treated with rhIL-32γ, which is the most activated form of IL-32, at two different concentrations, and subjected to ELISA to measure a secreted amount of IL-32, and therefore, as shown in FIG. 2b, it was confirmed that IL-32γ was abundantly present in the cells, compared to the culture medium. It also can be seen that, even when either TNF-α or IFN-γ was treated, IL-32 was expressed in most cells.
Based on the result, it was attempted to evaluate an anti-HBV effect caused by TNF-α and IFN-γ treatment in the Huh7 cell line. To this end, the cells were treated with each of TNF-α (20 ng or 100 ng) or IFN-γ (500 U or 1000 U), or both of 20 ng of TNF-α and 500 U of IFN-γ, and then subjected to southern blotting according to the method described in Example 1-6. Consequently, as shown in FIG. 2c, the antiviral effect on HBV was exhibited due to treatment of TNF-α and IFN-γ, and when two types of cytokines were treated together, the antiviral effect was strongly induced. In addition, it was confirmed that there was no change in cell viability due to treatment of the substances.
To verify the antiviral effect once again, Huh7 and HepG2 cell lines were transfected with IL-32-specific siRNA to inhibit the IL-32 expression, and then an antiviral effect on HBV was measured. Consequently, as shown in FIG. 2d, it was confirmed that HBV replication suppressed by the inhibition of the IL-32 expression was restored.
The results refer that IL-32 serves as downstream molecules of TNF-α and IFN-γ, and intracellular expression thereof mediates an antiviral effect against HBV.
Example 4. Confirmation of inhibition of HBV replication due to ectopic expression of IL-32
To examine a possible regulatory effect of IL-32γ in HBV replication, Huh7 and HepG2 cells transfected with both HBV 1.2 and IL-32γ plasmids were introduced, after extraction of HBV DNA, southern blotting was carried out to analyze a degree of HBV replication.
Consequently, as shown in FIG. 3a, it was confirmed that an HBV DNA level was considerably decreased dependent on the IL-32γ expression concentration, and from a phosphoimager result, it was confirmed that the replication of HBV DNA decreased approximately 70 to 80%. Here, it was also confirmed that there was no change in cell viability according to the expression level of IL-32 in cells.
In addition to the above results, to verify whether the IL-32 secreted out of the cells affects HBV replication inhibiting ability in the cells, an IL-32 antibody was added to a culture medium for Huh7 cells to induce a neutralizing action on IL-32 in the culture medium, and then a titer of HBV was measured. Consequently, as shown in FIG. 3b, it was confirmed that, there was no change in the degree of inhibiting HBV replication due to IL-32 even when an antibody against IL-32 was treated. Such a result refers that HBV replication is inhibited by only IL-32γ expressed in cells.
Further, to verify whether other isoforms of IL-32 also exhibit the same antiviral effect as IL-32γ, Huh7 cells were transfected with IL-32α, IL-32β and IL-32γ plasmids or treated with TNF-α and IFN-γ in the same manner as described above, and then subjected to southern blotting to comparatively analyze a degree of HBV replication. Consequently, as shown in FIG. 3c, it was confirmed that all of the IL-32α, IL-32β and IL-32γ reduced HBV replication, and among these, IL-32γ exhibited the most excellent antiviral effect. In addition, as shown in FIG. 3d, to confirm an HBV replication inhibitory effect due to IL-32γ expression, when IL-32 expression was inhibited by transfection with IL-32 siRNA, the HBV replication inhibitory effect was not exhibited.
Example 5. Confirmation of inhibition of viral gene transcription and protein expression through IL-32-mediated down-regulation of enhancer activity
Based on the results of Examples 3 and 4, to identify at what stage HBV replication was inhibited due to intracellular IL-32 expression, an experiment was carried out in the same manner as described in Example 4, and then an expression level of HBV mRNA was measured through northern blotting. Consequently, as shown in FIG. 4a, it was confirmed that levels of HBV mRNA such as pg/preC RNA and HBV surface RNA (Pre-S/S RNA) were decreased depending on the transfection concentration of IL-32γ. In addition, as shown in FIG. 4b, it was confirmed that the levels of surface and core proteins of HBV were significantly decreased in proportion to the transfection concentration of IL-32γ.
Based on this result, to examine by which mechanism the mRNA level of HBV was decreased by IL-32γ, the activity of an HBV enhancer playing a critical role in transcription of HBV mRNA was measured through luciferase reporter analysis. To this end, as illustrated in FIG. 4c, as an HBV enhancer luciferase reporter plasmid, an HBV wild-type enhancer and its variants from which a part is deleted were used. Consequently, as shown in FIG. 4d, it was confirmed that the activity of the HBV enhancer was reduced due to IL-32γ. Accordingly, it was seen that IL-32γ-induced suppression of HBV replication in Huh7 cells was caused by inhibition of the activity of the HBV enhancer.
Example 6. Confirmation of down-regulation of HNF4α and HNF1α binding to HBV enhancer due to IL-32
According to recent studies, it was reported that HBV RNA production was associated with various transcription factors such as HNF1, HNF3, HNF4, and CEBP, abundantly present in liver cells. This means that HBV utilizes transcription factors present in host liver cells in its replication without producing its own transcription factors. Therefore, the inventors performed an experiment on the assumption that the transcription factors present in the liver cells are associated with an inhibitory effect of IL-32 on transcription of HBV RNA.
First, on the gene map of an HBV enhancer, binding sites of various transcription factors associated with the transcription of HBV RNA were analyzed. Consequently, as shown in FIG. 5a, four types, CEBP, HNF1, HNF3 and HNF4 transcription factors, were able to bind different sites of an enhancer I/II sequence, and the binding sites of these factors had very close or overlapping regions. In addition, each transcription factor has its own function by binding to a specific site of the enhancer, and it has been known that, for example, HNF4 promotes viral transcription, and HNF3 inhibits HBV transcription.
Therefore, to find transcription factors reducing the enhancer activity of HBV, the change in expression level of the transcription factors present in liver cells in Huh7 cells was measured by quantitative RT-PCR and western blotting analyses according to the methods described in Examples 1-9 and 1-7, respectively. Therefore, as shown in FIGS. 5b and 5c, when IL-32γ was expressed, both RNA and protein expression of HNF4α and HNF1α were reduced, and it was confirmed that there was no difference in expression levels of HNF3 and CEBP.
Moreover, to investigate the correlation between IL-32 and the transcription factors, ChIP analysis was carried out according to the method described in Example 1-10 to detect whether IL-32 was bound with the transcription factors present in the liver cells in the transfected cells, an immunoprecipitated HBV DNA fragment was detected by semi-quantitative RT-PCR using anti-HNF4α and anti-HNF1α antibodies. Therefore, as shown in FIGS. 5d and 5e, binding efficiency of HNF4α binding to RI (Enhancer I) and HNF1α binding to RII (Enhancer II) was reduced when IL-32 was expressed. Such a result shows which method makes binding patterns of the transcription factors changed due to the influence of IL-32 on an HBV enhancer-binding site.
To more clearly verify the result, EMSA and western blotting were carried out by the method described in Example 1-11, and the western blotting was carried out under the same conditions as those of EMSA. Consequently, as shown in FIG. 5f, it was observed that the HNF4α protein was present at the same place as the protein-DNA. However, IL-32 did not bind to an HNF4α probe due to the decrease in expression of an HNF4α protein, which did not correspond to the ChIP result.
The above results show that intracellular IL-32 inhibits HBV replication by changing the binding pattern of the transcription factors.
Example 7. Confirmation of IL-32-mediated inhibition of HBV replication through ERK1 /2-dependent down-regulation of HNF4α
It has been known that TNF-α and IFN-γ induce activation of mitogen-activated protein kinases (MAPKs), and MAPK signal transduction in host cells plays an important role in an antiviral signal transduction network.
Therefore, the inventors measured protein expression levels of several molecules associated with MAPK signal transduction and antiviral signal transduction pathways under the condition in which IL-32 was expressed. Here, a sample treated with only TNF-α and IFN-γ was used as a positive control. Consequently, as shown in FIG. 6a, it was confirmed that, when IL-32 was expressed in Huh7 cells, the expression of phosphorylated ERK1/2 (p-extracellular signal-regulated kinase 1/2) and p38 protein was increased, but there was no change in the expression of Jun N-terminal kinase (JNK).
Based on the result, to investigate regulatory effects of HNF4α and HNF1α, cells were treated with an ERK1/2 inhibitor such as U0126 at a concentration of 10 μM to inhibit the production of p-ERK1/2. Consequently, as shown in FIG. 6b, it was confirmed that, when the amount of p-ERK1/2 protein was reduced, the expression of the HNF4α and HNF1α proteins reduced by IL-32 was increased again. In addition, since it has been known that the expression of transcription factors present in the liver is regulated by the MAPK signal transduction pathways, it was confirmed from the results that the expression of HNF4α and HNF1α was reduced due to IL-32, and such result was mediated by the MAPK signal transduction pathways.
In addition, as a result of the measurement of a replication level of HBV when HNF4α and HNF1α were overexpressed, as shown in FIGS. 6c and 6d, it was confirmed that viral replication reduced by IL-32 was increased again. Further, as shown in FIG. 6e, it was confirmed that, even when an ERK1/2 phosphorylation inhibitor was treated, an HBV replication level decreased by IL-32 was increased again.
The above results show that the HNF4α and HNF1α expression was reduced by IL-32-mediated induction of ERK1/2 phosphorylation, thereby inhibiting HBV replication.
Example 8. Confirmation of HBV inhibition by IL-32 in mouse model
To verify the results deduced from the examples, an antiviral effect of IL-32 was to be verified in vivo using an HBV-infected mouse model.
To this end, an HBV 1.2 expression plasmid and an IL-32γ expression plasmid were injected into mice using a hydrodynamic injection technique. Subsequently, HBV DNA was extracted from 50 mg of mouse liver tissue and subjected to southern blotting to measure a replication level of HBV DNA. Consequently, as shown in FIG. 7a, in a mouse into which both of the HBV and IL-32γ expression plasmids were injected, compared to a mouse into which only the HBV expression plasmid was injected, it was confirmed that the replication level of HBV DNA was reduced. In addition, it was confirmed that a relative replication level of HBV DNA was the same as those obtained using a phosphoimager.
Further, as shown in FIG. 7b, it was observed that an HBsAg level upon IL-32γ expression was significantly decreased in a mouse serum, and according to determination of degrees of the expression of IL-32 and a HBV surface protein by immunohistochemistry, as shown in FIG. 7c, the expression of the HBV surface protein was significantly reduced when IL-32γ was expressed, and therefore it can be seen that the antiviral effect on HBV was mediated by IL-32γ in a mouse.
Therefore, the above results are summarized and illustrated in FIG. 8. More specifically, when cells were infected with HBV, HBV produces its own mRNA using host transcription factors such as HNF4α and HNF1α, immune cells around the infected cells secrete TNF-α and IFN-γ to kill the virus in the infected cells. As the cytokines induced secretion of IL-32, and the secreted IL-32 increases ERK1/2 phosphorylation, the expression of the transcription factors such as HNF4α and HNF1α were reduced. Consequently, in the present invention, it was identified that HBV DNA replication and viral RNA production are reduced by the above-mentioned mechanism of IL-32.
It should be understood by those of ordinary skill in the art that the above description of the present invention is exemplary, and the exemplary embodiments disclosed herein can be easily modified into other specific forms without departing from the technical spirit or essential features of the present invention. Therefore, the exemplary embodiments described above should be interpreted as illustrative and not limited in any aspect.
The inventors first identified an IL-32-mediated antiviral effect on HBV and a molecular mechanism thereof. Therefore, the IL-32-mediated effect on HBV can provide a new understanding for the development of a therapeutic agent for the virus, and an antiviral composition according to the present invention can be effectively used in the development of an antiviral agent.

Claims (12)

  1. An antiviral composition against hepatitis B virus, comprising:
    an interleukin-32 (IL-32) gene or an IL-32 protein, which is an inhibitor of expression or activation of hepatocyte nuclear factor 4 alpha (HNF4α) or hepatocyte nuclear factor 1 alpha (HNF1α), as an active ingredient.
  2. The composition of claim 1, wherein the IL-32 gene consists of a base sequence selected from the group consisting of SEQ ID NOs: 1 to 4.
  3. The composition of claim 1, wherein the IL-32 protein consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 8.
  4. The composition of claim 1, wherein the IL-32 inhibits the expression or activation of HNF4α or HNF1α by increasing ERK1/2 protein phosphorylation in cells.
  5. The composition of claim 1, wherein the composition inhibits viral replication by reducing the enhancer activity of hepatitis B virus by inhibiting the expression or activation of HNF4α or HNF1α.
  6. A method for screening an antiviral substance against hepatitis B virus, comprising the following steps:
    (a) in vitro treating cells with a candidate substance;
    (b) measuring an expression level of interleukin-32 (IL-32) in the cells; and
    (c) selecting a substance increasing the IL-32 expression compared to a candidate substance-untreated group as an antiviral substance against hepatitis B virus.
  7. The method of claim 6, wherein the cells are liver cells.
  8. The method of claim 6, wherein the candidate substance is selected from the group consisting of a compound, a microbial culture medium or extract, a natural substance extract, a nucleic acid, and a peptide.
  9. The method of claim 8, wherein the nucleic acid is selected from the group consisting of siRNA, shRNA, microRNA, antisense RNA, an aptamer, a locked nucleic acid (LNA), a peptide nucleic acid (PNA), and a morpholino.
  10. The method of claim 6, wherein Step (b) is carried out using a method selected from the group consisting of a polymerase chain reaction (PCR), a microarray, northern blotting, western blotting, an enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunohistochemistry, and immunofluorescence.
  11. A method for treating hepatitis B, comprising:
    administering an antiviral composition comprising an interleukin-32 (IL-32) gene or an IL-32 protein, which is an inhibitor of expression or activation of hepatocyte nuclear factor 4 alpha (HNF4α) or hepatocyte nuclear factor 1 alpha (HNF1α), as an active ingredient.
  12. A use of an antiviral composition for treating hepatitis B, wherein the antiviral composition comprises an interleukin-32 (IL-32) gene or an IL-32 protein, which is an inhibitor of expression or activation of hepatocyte nuclear factor 4 alpha (HNF4α) or hepatocyte nuclear factor 1 alpha (HNF1α), as an active ingredient.
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