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WO2004009850A1 - Systeme de replication du virus de l'hepatite c et procedes d'utilisation - Google Patents

Systeme de replication du virus de l'hepatite c et procedes d'utilisation Download PDF

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Publication number
WO2004009850A1
WO2004009850A1 PCT/US2003/022590 US0322590W WO2004009850A1 WO 2004009850 A1 WO2004009850 A1 WO 2004009850A1 US 0322590 W US0322590 W US 0322590W WO 2004009850 A1 WO2004009850 A1 WO 2004009850A1
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Prior art keywords
hcv
cells
pcr
infected
vero
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Mohamed Laakel
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Biowest Therapeutics Inc
PEPE JEFFREY C
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Micrologix Biotech Inc
PEPE JEFFREY C
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Priority to AU2003261189A priority Critical patent/AU2003261189A1/en
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    • 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
    • G01N33/502—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 for testing non-proliferative effects
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates generally to methods of replicating hepatitis C virus (HCV) and, more specifically, to cultured human cells lines that can be infected with, and support the expression of, HCV genetic materials and production of HCV particles over many generations.
  • HCV hepatitis C virus
  • the invention further relates to the use of such a cell culture system to identify agents useful in the prevention or treatment of HCV infections.
  • HCV Hepatitis C virus
  • HCV is a major cause of post transfusion hepatitis, which often leads to liver cirrhosis and cancer.
  • HCV is a worldwide health problem. Development of vaccines and other antiviral therapies is awaited. To develop this, cell culture systems for propagating HCV are needed, but the levels of virus replication reported up to now have been too low.
  • Primary human and chimpanzee hepatocytes are susceptible to HCV, and do replicate the virus ⁇ see U.S. Patent No. 6,096,541), but primary hepatocytes are difficult to obtain, and usually survive less than two weeks in culture.
  • HCV RNA Full length HCV RNA has been successfully transferred into a number of cell lines, but replicative levels are not stable and become undetectable within a few weeks. More recently, sub-genomic regions of HCV have been cloned and expressed at high levels in minireplicons, but none of these minireplicons supports virus replication.
  • This invention provides methods for growing cultured human cells under conditions that permit biochemical and genetic manipulations.
  • the lifespan of these cultured human cell lines is prolonged by immortalization.
  • One way in which this is achieved is by using the SV40 large T-antigen but other methods can also be used.
  • the cells lines are transformed so that they carry a chemical marker such as the dominant selection marker neo.
  • This invention also provides methods for infecting human cell lines with HCV under conditions and for a time sufficient to allow propagation of the infected cell lines, stable retention of infection, and production of HVC particles over many generations.
  • an immortalized cell comprising SV40 T Antigen, infected with hepatitis C virus (HCV), wherein said cell replicates HCV.
  • HCV hepatitis C virus
  • the invention provides an immortalized cell selected from ST-E4, ST-E8, ST-D5, Vero-B9, Vero-D7, Vero-E9, Bart-D9, Bart-G3, Bart-E9, and MDBK-B10.
  • the invention also provides methods for using these stably transformed and infected human cell lines for identifying antiviral agents, such as anti-HCV agents (with or without other antiviral compounds), useful for the treatment or prevention of HCV infections.
  • the invention provides a method of screening for an agent that inhibits HCV replication, comprising contacting the immortalized cell according to claim 1 with a candidate agent and determining an inhibition of HCV replication.
  • Figure 1 shows a schematic of exemplary embodiments of the methods of using cell culture to replicate HCV.
  • Figure 2 shows results of RT-PCR of RNA samples isolated from HCV infected cells.
  • Figure 3 shows results of RT-PCR with RNA isolated from HCV infected cells and the effect of the amount of RNA used in the PCR.
  • Figure 4 shows the RT-PCR results using 200 ng RNA isolated from control (non-infected) cells, infected cells, and supernatants.
  • Figure 5 shows primer selection can affect detection of HCV with RT-PCR.
  • Figure 6 shows alignment of candidate HCV genome sequenced from PCR product from infected cells with other known HCV genomes.
  • Figure 7 shows result of plaque assay to examine permissiveness of transformed cell lines to infection by HCV.
  • Figure 8 shows the result of RT-PCR and nested PCR to detect HCV in infected transformed cells.
  • Figure 9 shows alignment of candidate HCV genome sequenced from PCR product from infected cells with other known HCV genomes.
  • Figure 10 shows the result of Southern blot analysis to detect HCV in infected transformed cells.
  • Figure 11 shows the result of a plaque assay to quantify HCV from transformed cell lines infected with HCV.
  • Figure 12 shows the result of a plaque assay to quantify HCV from transformed cell lines infected with HCV (no plaques detected).
  • Figure 13 shows the result of nested PCR on the RT-PCR sample of Vero-B9 infected with S1 supernatant using primers H6 and R3.
  • Figure 14 shows the result of RT-PCR and nested PCR samples from infected ST-E4, ST-E8 and ST-D5 probed with DIG-labeled nested PCR product from Figure 13.
  • Figure 15 shows result of HPLC to quantify HCV-specific PCR products generated from transformed cell lines infected with HCV.
  • Figure 16 shows the detection by immunofluorescense of SV
  • Figure 17 shows the detection by immunofluorescense of SV Large T Ag in ST-E8 cells transformed with pSV3-neo and serially passaged.
  • Figure 18 shows the detection by immunofluorescense of SV Large T Ag in Vero-B9 cells transformed with pSV3-neo and serially passaged.
  • Figure 19 shows the detection by immunofluorescense of SV
  • Figure 20 shows the result of RT-PCR and nested PCR to detect HCV in transformed cells that had been serially passaged.
  • Figure 21 shows the result of RT-PCR and nested PCR to detect
  • Figure 22 shows the result of RT-PCR to detect HCV negative strand in transformed cells that had been serially passaged 8, 9, and 11 times, and one week post-infection (p.i.).
  • Figure 23 shows the result of a kinetic experiment to detect the negative strand in the cell clones MDBK-B10 and ST-E4.
  • Figure 24 shows the result of nested PCR to detect HCV positive strand in H2.35 cell line infected with HCV.
  • Figure 25 shows the results of the cytopathic effect of HCV supernatant S2 infecting ST-D5, ST-E4 and ST-E8 cell lines.
  • Figure 26 shows the results of immunofluorescence detection of HCV core protein in HCV infected transformed cell lines.
  • Figure 27 shows the results of immunofluorescence detection of HCV protein NS5 in HCV infected transformed cell lines.
  • Figures 28A and 28B show the results of western blot analysis of
  • HCV infected cells to detect the expression of the HCV NS5B (A) and core (B) proteins.
  • the present invention provides compositions and methods for making and using cell lines that stably replicate HCV.
  • the instant invention therefore, relates generally to the surprising discovery that certain immortalized cell lines are useful for the stable propagation of hepatitis C virus (HCV). Accordingly, such cell lines are further useful for screening for and identifying candidate antiviral agents that inhibit HCV replication. Discussed in more detail below are methods of replicating HCV in cell culture and suitable uses thereof, as well as representative compositions and therapeutic uses.
  • any concentration range, percentage range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • the use of the alternative (e.g., "or”) should be understood to mean either one, both or any combination thereof of the alternatives.
  • the individual compounds, or groups of compounds, derived from the various combinations of the sequences, structures, and substituents described herein are disclosed by the present application to the same extent as if each compound or group of compounds was set forth individually. Thus, selection of particular sequences, structures, or substituents is within the scope of the present invention.
  • the instant invention generally provides a method of stably replicating HCV in cells permissive to infection by HCV.
  • cells permissive to infection by HCV and for stable replication of HCV are transformed cells, more preferably the transformed cells are immortalized, and even more preferably the immortalized cells express SV40 large T Ag.
  • the cells suitable for transformation include H2.35, ST, Vero, Bart, and MDBK. Techniques for transformation and immortalization of a variety of cell types (such as primary culture cells, secondary culture cells, cell lines, etc.) is known in art, as described in Salmon et al. ⁇ Mol. Therapy 2:404, 2000, and references cited therein).
  • the immortalized cells are generated by transfection with a nucleic acid molecule that encodes SV40 large T Ag.
  • the T Ag nucleic acid is carried on a vector, such as a plasmid, YAC, shuttle vector, and the like.
  • the vector can further comprise a nucleic acid that encodes a selectable marker, such as neomycin.
  • the vector is a plasmid and more preferably is pSV3-neo.
  • a suitable transformed cell useful in the instant invention can be detected, for example, by indirect immunofluorescence antibody (IFA) analysis.
  • IFA indirect immunofluorescence antibody
  • SV40 large T Ag can be evaluated by IFA as follows: cells are grown on glass coverslips, gently washed with prewarmed phosphate-buffered saline (PBS), fixed with immunofluorescent buffer (IF buffer, Bio-Rad) containing 3% formaldehyde for at least 1h at room temperature, and then treated with 3% Triton X-100 in IF buffer for another 1h.
  • PBS prewarmed phosphate-buffered saline
  • IF buffer immunofluorescent buffer
  • Bio-Rad immunofluorescent buffer
  • mice anti-SV40 Large T Ag monoclonal antibody (alternatively, a human monoclonal antibody against SV40 large T antigen (Ag), clone 101, (Research Diagnostic, Inc.) can be used) for 1h, washed with PBS, and incubated under the same conditions with fluorescein labeled anti-mouse IgG (Sigma) and blue Evans. UV light microscopy can be used to examine the labeled cells.
  • Exemplary transformed cells suitable for use with the instant invention include ST-E4, ST-E8, ST-D5, Vero-B9, Vero-D7, Vero-E9, Bart-D9, Bart-G3, Bart-E9 and MDBK-B10.
  • Preferred transformants should be stable over many passages.
  • the expression of the protein SV40 Large T Ag in transformants H2.35, ST-E8, Vero-B9 and MDBK-B10 remains stable after 40 passages ⁇ see, e.g., Figures 16-19).
  • Infection of transformed cells suitable for use in the instant invention can be accomplished using procedures known in the art ⁇ see, e.g., U.S. Patent No. 6,096,541).
  • the different transformed cells can be infected with HCV and subsequently sequentially passaged, wherein the supernatant of the previous passage is used to infect fresh cells.
  • the presence of HCV will typically be monitored during the sequential infection and passage of the cells to verify the continued permissiveness to HCV infection.
  • the summary of the third and fourth passages is as follows:
  • a cytopathic effect may be observed in some cell clones.
  • ST-transformed cells, MDBK-B10 and Vero-B9 showed some cytopathic effects, and in particular, the ST clones showed subsequent lysis at about seven days post infection (p.i.). The cells are at the
  • Replication of HCV can be monitored directly (e.g., plaque assay) or indirectly (e.g., RT-PCR, nested PCR, HPLC of HCV nucleic acids), and qualitatively (e.g., Southern blots, western blots) or quantitatively (e.g., PCR, plaque assay), and preferably a combination of methods are used to verify the presence of HCV in infected cells.
  • plaque assay e.g., plaque assay
  • RT-PCR e.g., RT-PCR, nested PCR, HPLC of HCV nucleic acids
  • qualitatively e.g., Southern blots, western blots
  • quantitatively e.g., PCR, plaque assay
  • RNA from infected transformed cells can be extracted using Trizol LS reagent (GIBCO BRL).
  • Total RNA isolated from cells can be used in TitaniumTM One-Step RT-PCR (Clontech) to confirm the presence of HCV ⁇ see, e.g., Figure 2).
  • the PCR product is of the expected size (185 base pairs (bp)). Quantification of the concentration of RNA from the three cell lines showed that 2.3 ⁇ g was used per reaction for Vero-B9, 1.7 ⁇ g for MDBK-B10 and 3.6 ⁇ g for Bart-D9.
  • Figure 3 shows that when the RNA concentration varies from 1 to 500 ng, a proportional increase in the RT-PCR product is observed, particularly for MDBK-B10 and Vero-B9 cells. However, for Bart-D10 cells, there is no observable RT-PCR product.
  • lane 4 shows the negative RT control reaction (RT was heat inactivated).
  • Figure 4 shows the RT-PCR results using 200 ng RNA isolated from control (non-infected) cells, infected cells, and supernatants. For Vero-B9, only the infected cells show the PCR product at 185 bp.
  • primers are selected from HCV genome internal sequences, 5' UTR, or 3' UTR.
  • combination primers were designed ⁇ see Figure 5, top). Conditions were used to obtain a PCR product in one step RT- PCR reaction.
  • primers useful in the instant invention include H6 (SEQ ID NO: 1 , sense primer), R3 (SEQ ID NO: 3, anti-sense primer), R7 (SEQ ID NO: 2), H3 (SEQ ID NO: 4), and R8 (SEQ ID NO:5).
  • H6-R3 combination gave a product at the correct size (185 bp).
  • H6-R7 gave a product of the right size, although less product was produced (239 bp).
  • a second round PCR (nested PCR) using different combinations of primers was performed. The results are summarized at the bottom of Figure 5 showing a strong band of the right size with the H6-R7 combination.
  • transformed cells are passaged further (e.g., 7th and 8th passage) and then analyzed again for the presence of HCV.
  • the replication of HCV can be followed by RT-PCR and nested PCR.
  • first round RT-PCR can be performed using primers H3 and R8, wherein the expected product size is 290 bp.
  • the results from the first round PCR can be seen in Figure 8A.
  • Lanes 3, 5, 6, 9 and 10 had faint bands approximately 290 bp in size when compared to the 100 bp DNA ladder in lanes 1 and 8.
  • a second round of PCR was done using internal primers H6 and R3. This primer combination had an expected product size of 185 bp, which was detectable in the samples from the infected cells ( Figure 8B).
  • transformed cells are passaged further (e.g., 9th, 10th and the 11th passage), and the replication of HCV was followed by RT-PCR and nested PCR.
  • Figure 20 shows only some of the cell clones that have been infected with different supernatants of HCV virus. The virus is still able to be detected in these cells clones that have been infected with supernatants from passages 8, 9, and 11.
  • the band at position 185 bp shows that there is the continual presence of the virus in these later passages.
  • the band is from the positive strand of the RNA.
  • Figure 21 shows the detection of the negative strand in one cell clone, MDBK-B10, infected with supernatant Sa/5.
  • the band at position 185 bp. confirms the replication of the virus.
  • the virus must first produce the negative strand in order to make the positive strand.
  • the negative strand is the intermediary step for replication before it passes to the positive strand.
  • the band at position 185 bp. for the negative strand ( Figure 21) was excised, purified and sent for sequencing. The sequence matches the known HCV sequences from the Genebank, confirming that the negative strand is from HCV.
  • the gel in Figure 23 is the result of a kinetic experiment to detect the negative strand in the cell clones MDBK-B10 and ST-E4.
  • lanes 2 and 6 there is the appearance of a band at position 185 bp in Day 1 p.i. for clone MDBK-B10 infected with either S2 or Sa/5.
  • the clone ST-E4 infected with Sa/5 shows a strong band at position 185 bp starting from Day 0 (6 hrs. after the virus was absorbed on the cells) and the intensity slowly decreases by day 3 but the band is always present.
  • the cell clone ST-E4 infected with S2 also shows a band at 185 bp ay Day 0 but it is less intense. The band slowing decreases in intensity by Day 3.
  • the virus is able to replicate early on in these cell clones.
  • FIG 24 shows the replication of HCV in the H2.35 cell line.
  • H2.35 cells are mouse hepatocytes transformed by SV40.
  • a band at position 185 bp is apparent in cells infected with either S1 or S2 supernatant at passage 3, done under two different experimental procedures.
  • the band at 185 bp is the result of a nested PCR using the positive strand of DNA.
  • the band in lane 5 was excised, purified and sent for sequencing.
  • the band containing the candidate nucleic acid was excised from the gel, purified, quantified by spectrophotometry (Beckman DU 520 General Purpose Spectrophotometer), and then sequenced using methods known in the art. Sequence analysis and alignment was done using Lasergene ® software (DNASTAR' Inc., Madison, Wl). This alignment was compared to other HCV genotypes (e.g., 1a and 1b in Figure 6, and see Figure 9). The nucleic acid sequence of the purified PCR product shows almost perfect identity with other known HCV genome sequences.
  • HCV genome specific labeled probe Another way to confirm the presence of HCV is to perform the Southern blot hybridization using an HCV genome specific labeled probe.
  • the anti-sense primer R3 labeled with DIG was used ⁇ see, e.g., Example 12). Briefly, the RT-PCR and nested PCR products were transferred to a nylon membrane and subsequently detected by DIG-labeled R3 probe ( Figure 10). The three nested PCR sample bands were detected by autoradiography. The specificity of the probe is evident since only the amplified HCV products were detected and not the ⁇ -actin despite the fact that ⁇ -actin had a very intense band on the gel. No bands were detected in the control lane showing there was no contamination during amplification by PCR or during subsequent steps.
  • IFA Indirect immunofluorescence antibody analysis was carried out with an anti -Human SV40 large T ag, clone 101 , monoclonal antibody (Research Diagnostic, Inc.). This expression of SV40 large T ag was evaluated by IFA as follows: cell grown on glass coverslips were gently washed with prewarmed phosphate buffered saline (PBS) and fixed with immunofluorescent buffer (IF buffer, Bio-Rad) containing 3% formaldehyde for at least 1h at room temperature, then treated with 3% Triton X-100 in IF buffer for another 1h.
  • PBS prewarmed phosphate buffered saline
  • IF buffer immunofluorescent buffer
  • Bio-Rad immunofluorescent buffer
  • the cells were incubated with the mouse anti-SV40 Large ag monoclonal antibody for 1h, washed with PBS and incubated under the same conditions the fluorescein-labeled anti-mouse IgG (Sigma) and blue Evans. The cells were then examined under UV light microscopy.
  • the slides in Figure 26 confirm the presence of the HCV virus at the protein level.
  • the anti-core antibody to HCV was used in this experiment.
  • the fluorescence is evident in all the infected clones, ST-E4, ST-D5, ST-E8, H2.35, Vero-B9 and MDBK-B10. There is no evidence of fluorescence in the non-infected cells.
  • the slides in Figure 27 confirm the presence of the HCV virus at the protein level.
  • the anti-NS5 antibody to HCV was used in this experiment.
  • the fluorescence is evident in all the infected clones, ST-E4, ST-D5, ST-E8, H2.35, Vero-B9 and MDBK-B10. There is no evidence of fluorescence in the non-infected cells.
  • Figure 28A and 28B show the results of Western blotting ( Figure 28A and 28B) using protein extracted from different cell clones infected with different supernatants.
  • Figure 28A shows the results of probing with the monoclonal mouse-anti NS5B antibody. The control is purified NS5B protein (Fract. II). There is a band at position 68 kD in all the infected cell clones. There is no band at this position in the uninfected sample (ST-D5 NI).
  • Figure 28B shows the results of probing with the monoclonal antibody mouse anti-core antibody. There is a band at position 21 kD in all the infected samples. There is no band at this position in the uninfected samples (Vero NI and MDBK NI). These blots confirm that at the there is expression of the HCV NS5B and core proteins in the infected cell clones. Quantification of the HCV
  • ST clones ST-D5, ST-E8, ST-E4
  • Vero-D10 Vero clone
  • plaques were quantified by manual counting after the cells were fixed with 7% formaldehyde and stained with 0.5% crystal violet.
  • both the ST-T and the clone ST-D5 produced high levels of HCV replication ⁇ see, e.g., Figure 7).
  • the results of the plaque assay are from the supernatants of cells of the invention in the 4th passage.
  • Figure 11 shows that the infectivity of HCV varied from clone to clone and from transformed cell line to another. Both ST-T and the clone ST-E4 presented high level of HCV replication.
  • Figure 12 also shows that in the MDBK-B10 and Vero-B9 tested there is no production of the plaques, which may or may not mean that there is no replication of HCV in these transformed cell lines.
  • replication of HCV is directly detected in a transformed cell line.
  • the method of direct detection is the plaque assay.
  • HCV infection of transformed cells is detected by dot blot hybridization.
  • the PCR Digoxigenin (DIG) Probe Synthesis kit (Roche) can be used to put a DIG label on PCR products. Briefly, a nested PCR on the RT-PCR sample of Vero-B9 infected with S1 supernatant using H6 and R3 as the primers was used and the PCR DIG labeling mix. The controls are an unlabeled Vero-B9 S1 PCR product and the control template supplied with the kit. The cycling conditions are described previously in the materials and methods for the nested PCR. The samples were run on a 2% agarose gel at 100V for 1 hour.
  • the kit control gave a band at the expected 500 bp size (data not shown).
  • the nested PCR product for the unlabeled sample gave the expected band at 185 bp.
  • the DIG labeled nested PCR product gave a band at a higher position than 185 bp.
  • the increase in the band size is due to the presence of the DIG label. This confirms that the nested PCR has a DIG label on it.
  • This band was excised from the gel and purified using the Qiagen gel purification kit. The purified DIG labeled PCR product was used as a probe in DOT blot and Northern blot experiments.
  • Samples were prepared and deposited in a dot blot apparatus for subsequent probing with the DIG-labeled nested PCR product.
  • the samples that were deposited on the nylon membrane are as follow.
  • tenfold serial dilutions of unlabeled Vero-B9 infected with S1 supernatant were deposited in duplicata in 10-fold decreasing concentrations from 25 ng/ ⁇ L, 2.5 ng/ ⁇ L, etc. to 2.5 fg/ ⁇ L.
  • a DIG labeled DNA marker was deposited in a single lane at tenfold serial dilutions from 25 ng/ ⁇ L to 2.5 fg/ ⁇ L.
  • the RT- PCR and the nested PCR samples from infected ST-E4, ST-E8 and ST-D5 were deposited in duplicata, each undiluted and diluted 1/10.
  • a scheme of the deposit layout is below.
  • the nylon membrane was UV-cross-linked for 8 min. to fix the samples on the membrane and then prehybridized in DIG hybridization solution for 2 hours at 55°C.
  • the membrane was sealed in a pouch with a DIG-labeled PCR probe and further incubated overnight at 55°C. The membrane was then washed in the same manner as for a Southern blot ⁇ see Example 12).
  • the hybridized membrane was exposed for a minimum of 3 hours to Kodak BioMax film.
  • the unlabeled PCR product gave a signal that was proportional to the concentration deposited on the membrane.
  • the three samples showed an intense signal, and even the RT-PCR samples showed a good signal.
  • the aforementioned method is useful for the detection and the quantification of HCV genome according to the instant invention.
  • HPLC A new method for the detection and the quantification oh HCV genome using the HPLC was established as follows. The first step is to set up the optimal conditions for the separation of the different reagents of RT-PCR reactions, ⁇ see Figure 15).
  • the instant invention provides methods for screening agents that inhibit the replication of HCV by contacting candidate agents with transformed cells infected with HCV and detecting a decrease or abolishment of HCV replication.
  • One such agent tested was suramin.
  • the cytopathic effect of HCV supernatant S2 upon infection of ST-D5, ST-E4 and ST-E8 cell lines is shown in Figure 25.
  • the top row shows the effect of different dilutions of the virus 10 "2 , 10 "4 , 10 "6 ten days post-infection. The cytopathic effect is evident because the virus has killed of all the cells.
  • the control (NI, untreated cells) cells were not affected.
  • the present invention also pertains to methods for treating or preventing HCV infection, comprising administering to a subject in need thereof a composition comprising at least one agent that inhibits HCV replication as identified in the methods of the instant invention, and a pharmaceutically acceptable carrier, diluent, or excipient, at a dose sufficient to inhibit HCV replication.
  • an infection is due known group or subgroup of HCV.
  • a subject suitable for treatment with a HCV replication inhibitor formulation may be identified by well-established indicators of risk for developing a disease or well-established hallmarks of an existing disease. For example, indicators of an infection include fever, pus, microorganism positive cultures, inflammation, and the like.
  • Infections that may be treated with a HCV replication inhibitor of the subject invention include those caused by or due to HCV, whether the infection is primary, secondary, opportunistic, and the like. Examples of HCV include any antigenic variant of these viruses.
  • compositions that contain one or more HCV replication inhibitor of the invention may be in any form that allows for the composition to be administered to a subject, such as a human or animal.
  • compositions of the present invention may be prepared and administered as a liquid solution or prepared as a solid form ⁇ e.g., lyophilized), which may be administered in solid form, or resuspended in a solution in conjunction with administration.
  • An HCV replication inhibitor composition is formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a subject or patient or bioavailable via slow release.
  • compositions that will be administered to a subject or patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of one or more compounds of the invention in aerosol form may hold a plurality of dosage units.
  • any of the aforementioned pharmaceutical compositions comprising a HCV replication inhibitor or cocktail of inhibitors of the invention are in a container, preferably in a sterile container.
  • the HCV replication inhibitor pharmaceutical composition is preferably sterile.
  • the cells used are MDBK (ATCC), Vero (ATCC), and ST (ATCC).
  • the two first cell lines were maintained in EMEM and ST were maintained in
  • DMEM media supplemented with glutamine 2 mM, 1% of
  • EXAMPLE 2 PLASMID PREPARATION pSV3 neo was obtained from ATCC. It was propagated in E. coli and supercoiled DNA was isolated according to QIAGEN plasmid purification handbook (QIAfilter plasmid Maxi Protocol). The yield and purity of the DNA were very good. The DNA concentration was about 1.8 ⁇ g/ ⁇ L for about 600 ⁇ L of total volume.
  • Supercoiled DNA was introduced into tissue culture cells ( ⁇ 10 g for approximately 1-5x10 6 cells in six well plates). About 72 h after exposure to DNA, the cells were trypsinized, replated and G418 was added to the medium at the concentration as indicated in Table 1. At this step, the transformed cells were passedged four time in the selection medium and with Vero, ST and MDBK transformed cells the fourth passage was successful. The next step in our planning is to do the sub-cloning of the already transformed cells by limiting dilution method. Cultures were diluted and plated at 60, 50, 40, 30, 20, 10, 5, and I cells per 200 1 in 96 well plates (12 wells per each dilution). The cultures were observed microscopically for identification of wells containing single cells, and subsequently for their proliferation.
  • Vero transformed cells For Vero transformed cells, they were at the 33th passages and obtained 8 potential clones which were growing in 12.5 cm 2 flasks.
  • MDBK transformed cells For MDBK transformed cells, they were at the 33th passages and obtained 3 potential clones which were growing in 12.5 cm 2 flasks.
  • RNA extraction were done using Trizol LS from GibcoBRL. RNA was extracted from cells and supernatants. 0.75 ml of Trizol LS was added for every 0.25 ml of supernatant and mixed. After 5 minutes of incubation at room temperature, 0.2 ml of chloroform was added to each sample followed by a further 15 minutes of incubation at room temperature. Samples were centrifuged for 15 minutes at 4°C at 12 000 rpm to separate the phases. The upper aqueous phase containing the RNA was removed and placed in a clean Eppendorf tube to which was added 0.5 ml of isopropanol.
  • RNA samples were quantified by spectrophotometry.
  • RT-PCR is a very sensitive and versatile technique that is used to measure gene expression in cultured cells.
  • the RT-PCR was made using the
  • Titanium One-Step RT-PCR kit (Clontech Cat# K1403-1). It is a unique method since the RT-PCR can be performed in a single step, in a single tube. There is an advantage using this Titanium kit since traditional RT-PCR reactions are usually performed in two consecutive steps, in two different reactions , the first a first-strand cDNA synthsis step using reverse transcriptase followed by a PCR step using a thermostable DNA polymerase, Taq Polymerase. The two-step procedures require either multiple tubes or the sequential addition of enzymes and reagents. Another advantage of the Titanium kit is that since no additional reagents are needed after the reaction is initiated, the possibility of cross- contamination is reduced.
  • RNA sample 200 ng/ ⁇ L
  • PCR Primer mix 50 pmol/ ⁇ L
  • DEPC H 2 O DEPC H 2 O
  • the primers (Invitrogen Life Technologies) that we used for the RT-PCR at the 3' UTR region are:
  • H6 GTG CAG CCT CCA GGA CC (SEQ ID NO: 1)
  • R3 GTA CCA CAA GGC CTT TC (SEQ ID NO: 3)
  • RT-PCR reactions were run in hot-lid Biometra thermocycling machines using the following program: 50°C for 1 hr., 94°C for 2 min. and 35 cycles of 94°C for 30 sees, 60°C for 30 secsl and 72 °C for 1 min. the 72° C for 8 min.
  • Nested PCR consists of using the RT-PCR product (1 st round) in a second PCR reaction.
  • the first consists of preparing a 1x reaction mix using 1 ⁇ L dNTP mix, 3.75 ⁇ L 20 mM MgCI 2 , 5 ⁇ L 10x Buffer, 1 ⁇ L of forward primer, 2 ⁇ L of reverse primer, 0.36 ⁇ L of Taq polymerase enzyme, 2 ⁇ L of the RNA sample and H 2 O to complete the volume to 50 ⁇ L.
  • the second method consists of using the Platinum Super Mix (Canadian Life Technologies).
  • RNA sample For one reaction prepare a reaction mix with 45 ⁇ L of the Platinum Super Mix, 1 ⁇ L of forward primer (50 pmol/ ⁇ L), 2 ⁇ L of reverse primer (50 pmol/ ⁇ L), and 2 ⁇ L of the RNA sample (200ng/ ⁇ L).
  • the primers (Invitrogen Life Technologies) that were used at the
  • H6 GTG CAG CCT CCA GGA CC (SEQ ID NO: 1 )
  • R7 ATG GTG CAC GGT CTA CGA GAC (SEQ ID NO: 2)
  • R3 GTA CCA CAA GGC CTT TC (SEQ ID NO: 3)
  • H3 GAA AGC GTC TAG CCA TGG CGT (SEQ ID NO: 4)
  • R8 GGT TTA GGA TTC GTG CTC ATG G (SEQ ID NO:5)
  • PCR reactions were run in hot-lid Biometra thermocycling machines using the following program: 94°C for 2 min., and 35 cycles of 94°C for 30 sees, 60 °C for 30 sees, and 72°C for 1 min. the 72° C for 8 min.
  • An agarose was gel was prepared using 1.5% agarose and 4% ethidium bromide diluted in 1x TAE (0.04M Tris -acetate , 0.001 M EDTA Buffer). The samples were mixed with 6x loading buffer (0.25% bromophenol blue and 30% glycerol in water) and loaded onto the gel. The gel was run at 100V for 1hr. in 1x TAE as the running buffer. The gel was observed under UV light (Herolab) and photographed using a Biometra software.
  • RT-PCR and nested PCR samples from cells and / or supernatants were deposited as well as RT-PCR samples of ⁇ -actin and H 2 O as controls. 10 ⁇ l of each sample was loaded with 2 ⁇ l of 6x loading buffer and 2 ⁇ l of ethidium bromide. 5 ⁇ l of DIG DNA marker (Roche) was also deposited. A 1.5% agarose gel was prepared and run at 60V for 30 minutes then at 80V for 3 hours. Southern hybridization was carried out according to Sambrook et al. using DIG detection as opposed to radioactivity. After gel electrophoresis, the gel was denatured for 45 minutes in 1.5 M NaCI, 0.5 N NaOH with gentle agitation.
  • the gel was then rinsed briefly with ddH20 followed by 30 minutes of soaking in neutralizing solution (0.5 M Tris, 1.5 M NaCI) with gentle agitation. The neutralizing solution was changed and the gel left to soak for a further 15 minutes.
  • the wick (long piece of 3MM Whatman paper) was wet in 10x SSC and placed over the glass support with the ends of the wick in the glass dish filled with 10x SSC (liquid should reach almost to the level of the glass support).
  • the nylon membrane (Nytran Supercharge, Schleicher & Schuell) was soaked briefly in ddH2O until it was completely wet then soaked for 5 minutes in 10x SSC. Three pieces of 3MM Whatman paper were cut to the size of the gel and soaked in 2x SSC until wet.
  • 10x SSC was used as the transfer buffer.
  • the apparatus was assembled and left to transfer for 2 days. Once the transfer was complete the membrane was fixed with UV light for 7 minutes. The gel was stained with ethidium bromide for twenty minutes to ensure that the transfer was successful.
  • the DIG Luminescence Detection Kit for Nucleic Acids (Roche) was used for detection. The membrane was prehybridized at 55°C for 2 hours. Hybridization with the DIG-labeled oligo probe complimentary to the DNA samples was carried out overnight at 55°C. Following hybridization, the membrane was washed twice in 2x SSC, 0.1% SDS for 20 minutes followed by two 30 minute washes in 0.1x SSC, 0.1% SDS at 55°C and a third at room temperature.
  • the membrane was then blocked with 1x blocking solution (Roche blocking reagent in maleic acid) for 2 hours with gentle agitation.
  • the membrane was incubated with the DIG-alkaline phosphatase antibody for 4 hours at room temperature, and then was equilibrated for 5 minutes in detection buffer.
  • the detection substrate (CSPD, an alkaline phosphatase substrate) was added and left on the membrane for 10 minutes in the dark then removed and the membrane incubated at 37°C for 10 minutes to enhance luminescence.
  • the membrane was exposed to Biomax film (Kodak) overnight.
  • the membrane was washed twice for 20 minutes in 2x SSC, 0.1% SDS at room temperature, twice for 30 minutes at 55°C in 0.1x SSC, 0.1% SDS, and once for 30 minutes in 0.1 x SSC, 0.1% SDS with the oven off.
  • the membrane was blocked in 1x blocking solution for 2 hours at room temperature.
  • the membrane was placed in a plastic Kapak pouch with DIG-alkaline phosphatase antibody for 4 hours at room temperature.
  • the membrane was rinsed briefly in washing buffer then washed overnight in washing buffer at room temperature. The following morning, the membrane was washed twice for 30 minutes at room temperature.
  • IFA Indirect immunofluorescence antibody analysis was carried out with an anti - Human SV40 large T ag, clone 101 , monoclonal antibody (Research Diagnostic, Inc.). This expression of SV40 large T ag was evaluated by IFA as follows: cell grown on glass coverslips were gently washed with prewarmed phosphate-buffered saline (PBS) and fixed with immunofluorescent buffer (IF buffer, Bio-Rad) containing 3% formaldehyde for at least 1h at room temperature, then treated with 3% Triton X-100 in IF buffer for another 1h.
  • PBS prewarmed phosphate-buffered saline
  • IF buffer immunofluorescent buffer
  • Bio-Rad immunofluorescent buffer
  • the cells were incubated with the mouse anti-SV40 Large T ag monoclonal antibody for 1 h, washed with PBS and incubated under the same conditions the fluorescein-labeled anti-mouse IgG (Sigma), and blue Evans. The cells were then examined under UV light microscopy.
  • the cell clones ST-D5, ST-E4 and ST-E8 were plated at a concentration of 1x 10 5 cells/ well in 12-well plates. Each cell clone was infected with S2 supernatant from P12 of the respective clone in the following dilutions 10 "2 , 10 "4 , 10 "6 . The cell clones were infected first and then treated with Suramin sodium salt (Sigma S-2671) at two concentrations (0.5 g/mL and 0.25 g/mL). The controls for each cell clone include non-infected cells treated with the same concentrations of Suramin, infected cells not treated with Suramin and non-infected cells not treated with Suramin. The virus was incubated on the cells overnight. EXAMPLE 16 NEGATIVE STRAND DETECTION
  • the first step is to prepare the cDNA from RNA extracted from the infected cell clones. Only the forward primer H6 (5 1 - GTG CAG CCT CCA GGA CC- 3') (SEQ ID NO: 1) was used here.
  • a 1x reaction mix consisted of 400 ng of RNA, 2 ⁇ L of H6 primer (50 pmol/ ⁇ L), I ⁇ L RNAsin RNAase Inhibitor (40 U/ ⁇ L)(Promega), 1 ⁇ L of 0.1 M DTT, 1 ⁇ L of 2.5 mM dNTP mix (Invitrogen), 4 ⁇ L of 5X RT Buffer and DEPC ddH 2 O to complete the volume to 20 ⁇ L. Aliquot the samples. Heat them for 15 minutes at 65°C. Put the samples on ice and add MMLV-RT enzyme (1 U/rxn) (Pharmacia). Incubate at 42°C for 45 minutes. Add more MMLV-RT enzyme (0.5U/rxn) and incubate 30-45 min longer at 42°C.
  • the next step is the first round PCR using the cDNA and the external primers H3 ( 5' GAA AGC GTC TAG CCA TGG CGT-3') (SEQ ID NO: 4) and R8 (5'- GGT TTA GGA TTC GTG CTC ATG G-3 * ) (SEQ ID NO: 5).
  • a typical reaction mix consisted of 5 ⁇ L of 10x Taq Buffer, 1 ⁇ L of 2.5 mM dNTP mix, 2 ⁇ L of both H3 and R8 (50 pmol/ L). 0.5 ⁇ L of Taq enzyme , 10 ⁇ L of cDNA and ddH 2 O to complete the reaction volume to 50 ⁇ L.
  • the Biometra PCR Thermocycler was used with the cycling conditions for the nested -2 protocol (40 cycles).
  • the last step is a nested PCR using the PCR product from the first round and the inner primers H6 and R3 (5 - GTA CCA CAA GGC CTT TC- 3') (SEQ ID NO: 3).
  • a typical reaction mix consisted of 5 ⁇ L of 10x Taq Buffer, 1 ⁇ L of 2.5 mM dNTP mix, 2 ⁇ l of both H6 and R3 (50 pmol/ ⁇ L) 0.5 ⁇ L of Taq enzyme, 10 ⁇ L of cDNA and ddH 2 O to complete the reaction volume to 50 ⁇ L.
  • the Biometra PCR Thermocycler was used with the cycling conditions for the nested -2 protocol (40 cycles).
  • the proteins were extracted from the infected cell clones using the following procedure. A volume of 750 ⁇ L of Trizol (Invitrogen) was added to disrupt the cells. Then a volume of 200 L of chloroform was added. The aqueous phase was removed after centrifugation at 10000 g for 15 min. at 4°C. The cellular DNA was precipitated with 0.3 mL of 100%) ethanol/ 750 ⁇ L of Trizol. The tubes were vortexed and centrifuged at 2000 g for 5 min. at 4°C. The supernatant is now used for protein precipitation by adding 1.5 mL / 0.75 ml Trizol with isoporopyl alcohol. Store the samples at 15-30°C for 10 min. Centrifuge the protein precipiate at 12 000 g for 10 min. at 4°C.
  • the proteins were quantified by using the DC Protein assay (Bio- Rad). Dilutions of BSA standard were prepared (0.2 mg/mL to 1.5 mg/mL). Add 20 ⁇ L of reagent S to reagent A that will be needed for the experiment. Pipet 5 ⁇ L of standard and protein samples into a clean dry microplate. Add 25 ⁇ L of reagent A into each well. Add 200 ⁇ L of reagent B into each well. Put the plate into a microplate reader (Spectra Max 190), use the mixing function. After 15 min. read the absorbance at 750 nm: SDS-PAGE
  • the membrane in now reading for overnight blocking in 5% milk dissolved in PBS and 0.1 % Tween 20.
  • the primary antibodies (Biodesign) used were monoclonal mouse anti-HCV core antibody (1/50), and monoclonal mouse anti- HCV NS5 antibody (1/100). All the antibodies are dissolved in the blocking buffer.
  • the membranes were incubated for 2 hrs. in the primary antibody. Then the membranes were washed 3x 10 min. each in PBS. Then the membranes were incubated in the secondary antibody for 2 hrs.
  • the secondary antibody was anti-mouse peroxidase (1/2000) for all the primary monoclonal antibodies. The membranes were washed 3x 10 min. each in PBS.
  • the ECL Plus detection system (Amersham Biosciences) was used to reveal the proteins on the membrane. Mix the detection solutions A and B in a ratio of 40:1. A volume of 2mL of solution A and 50 ⁇ L of solution B is used for one membrane. Place the membrane protein side, up on a sheet of Saran wrap. Apply the detection reagent on the membrane and incubate for 5 min. at room temperature. Drain off excess detection reagent and place the membrane protein side down on a clean sheet of Saran wrap. Place the blot protein side up in a X-ray film cassette. Expose it to a sheet of autoradiography film. (Hyperfilm ECL, Amersham-Biosciences) for 15 sees. Develop the film and repeat exposure with a new film based on the results of the first.

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Abstract

La présente invention concerne des procédés de réplication du virus de l'hépatite C (HCV), notamment des lignées cellulaires humaines pouvant être infectées par ledit virus, supportant l'expression du virus de l'hépatite C, des matériaux génétiques du virus de l'hépatite C, et la production de particules du virus de l'hépatite C sur plusieurs générations. L'invention concerne également l'utilisation d'un tel système de culture cellulaire afin d'identifier des agents servant à la prévention ou au traitement d'infections par le virus de l'hépatite C.
PCT/US2003/022590 2002-07-18 2003-07-18 Systeme de replication du virus de l'hepatite c et procedes d'utilisation Ceased WO2004009850A1 (fr)

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WO2015118146A1 (fr) 2014-02-10 2015-08-13 Univercells Nv Système, appareil et procédé de production de biomolécules

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Title
KATO N. ET AL.: "Replication of hepatitis C virus in cultured non-neoplastic human hepatocytes", JAPANESE JOURNAL OF CANCER RESEARCH, vol. 87, August 1996 (1996-08-01), pages 787 - 792, XP001022326 *

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