Patent Application
20110189684
The present invention relates to a method for determining drug resistance mutations in any of the non-structural protein regions NS3 to NS5B of Hepatitis C Virus (HCV) for genotypes 1 to 6, more in particular for subtype specific genotypes 1a, 1b, 2a, 2b, 3a, 4a and 4d.
HCV is a single stranded, positive-sense RNA virus, with a genome of around 9,600 bases belonging to the Flaviviridae family of viruses in the hepacivirus genus. The NS5B region of the RNA polygene encodes a 65 kDa RNA dependent RNA polymerase (RdRp), which is essential to viral replication. Following the initial acute infection, a majority of infected individuals develop chronic hepatitis because HCV replicates preferentially in hepatocytes but is not directly cytopathic. In particular, the lack of a vigorous T-lymphocyte response and the high propensity of the virus to mutate appear to promote a high rate of chronic infection. Chronic hepatitis can progress to liver fibrosis, leading to cirrhosis, end-stage liver disease, and HCC (hepatocellular carcinoma), making it the leading cause of liver transplantations.
Transmission of HCV can occur through contact with contaminated blood or blood products, for example following blood transfusion or intravenous drug use. The introduction of diagnostic tests used in blood screening has led to a downward trend in post-transfusion HCV incidence. However, given the slow progression to the end-stage liver disease, the existing infections will continue to present a serious medical and economic burden for decades.
There are six major HCV genotypes and more than 50 subtypes, which are differently distributed geographically. HCV genotype 1 is the predominant genotype in Europe and the USA. The extensive genetic heterogeneity of HCV has important diagnostic and clinical implications, perhaps explaining difficulties in vaccine development and the lack of response to current therapy.
The genetic variability of HCV complicates amplification, sequencing and genotyping processes. These processes rely on the use of so-called primers complementary to and capable of hybridizing to corresponding nucleic acid sequences of the HCV genome. Due to the high degree of variability of the HCV genome, primers complementary to one HCV species may not be complementary to another species.
To determine the subtype of an HCV clinical isolate an accurate and direct method is sequencing the viral genome in a region that is sufficiently divergent among various species in order to distinguish between HCV genotypes and subtypes accordingly. Phylogenetic analysis of the sequences generated from these regions is used to determine the subtype of clinical isolates.
Several selective and potent antiviral drugs against chronic hepatitis C virus (HCV) infection are currently evaluated in clinical trials. The emergence of drug resistance mutations was proven in previous trials, creating a need for patients to be monitored for the development of such drug-resistance mutations.
In order to improve the identification of HCV types and subtypes for purposes of clinical analysis and therapeutic decision making by a treating physician, there is a continuing need to improve sequencing-based HCV assays.
The hepatitis C virus is, as mentioned above, currently classified into at least 6 major genotypes (
The present invention relates to the development of subtype-specific assays for HCV genotype resistance analysis suitable for clinical trial support and regulatory filings.
In more detail the invention relates to genotyping assays covering the complete coding region from NS3 to NS5B as developed on a large panel of clinical samples including protocols for subtypes 1a, 1b, 2a, 2b, 3a, 4a and 4d.
The current invention relates to a NS5B sequence-based subtyping assay detecting all six HCV genotypes and discriminating between the different subtypes.
One aspect of the invention concerns a method for determining drug resistance mutations in any of the non-structural protein regions NS3 to NS5B of Hepatitis C Virus (HCV) for genotypes 1 to 6, more in particular for subtype specific genotypes 1a, 1b, 2a, 2b, 3a, 4a and 4d, present in a sample comprising:
Another embodiment of the current invention is that above method further comprises the steps for performing a NS3 phenotyping assay by
In another embodiment the invention relates to the above mentioned method further comprising the steps for performing a NS5B phenotyping assay by
Part of the invention is also a vector comprising the HCV genome and a deletion spanning the HCV NS3 N-terminal 181 amino acid region, in particular vector pFK I341 PI luc ΔNS3 7-192_ET (SEQ ID NO.10) and a vector comprising the HCV genome and a deletion spanning the HCV NS5B region, in particular vector pFK_I1341_PI_NS3-3_ET_dNS5a/b—5a440-5b591-Scla (SEQ ID NO 21) and the vector comprising the HCV genome and a deletion spanning the HCV NS5B region, in particular vector pFK_I341_PI_NS3-3_ET_dNS5a/b—5a440-5b591-XbaI (SEQ ID NO 27).
Besides the use of any of the above vectors in any of the methods mentioned, also the primers with SEQ ID NO 1-5 for the amplification of the HCV NS5B region, as obtained from a sample of an HCV-infected patient, belong to the invention.
The use of the primers with SEQ ID NO 1-5 for the preparation of a sequence-based subtyping HCV assay to detect HCV genotypes 1, 2, 3 and 4 and to discriminate between the subtypes 1a, 1b, 2a, 2b, 3a, 4a and 4d, belongs in particular to the current invention.
A panel of 603 clinical samples covering all six genotypes (G) was collected. Two test systems were developed: a NS5B sequence-based subtyping assay and a set of subtype-specific genotyping assays to determine drug-resistance mutations in the following target regions: (1) protease inhibitors (complete NS3/4A and the N-terminal 181 as of NS3), (2) polymerase inhibitors (complete NS5B), and (3) others (complete NS4B/5A region). All primer sets have been optimized for subtype specificity and to allow the same PCR protocol to be used for a target region independent of the subtype (
The NS5B sequence-based subtyping assay was tested on a set of 603 clinical samples containing all six genotypes with a clinical sensitivity (amplification success rate of high viral load samples) of 91%.
For the subtype-specific genotyping assays, sets of clinical samples of, on average, n=94 for G1a/b, n=16 for G2a/b, n=76 for G3a, and n=83 for G4a/d were tested in the different assays to evaluate the clinical sensitivity. Amplification success rates between 90% and 100% and sequencing success rates between 95% and 100% were achieved (
General Outline:
The general process flow is visualized in
1. Subtyping
An amplicon was generated from patient-derived viral plasma RNA by One-step RT-PCR followed by nested PCR. This amplicon, further referred to as the NS5B subtyping amplicon, contains a 329 by sequence of the NS5B polymerase domain, which is used for phylogenetic tree analysis in order to obtain subtype information of the clinical isolate. The assay is called the NS5B sequence-based subtyping assay. The subtype information of the clinical isolate will be used in a next step to select the appropriate subtype-specific amplification and sequencing primers in order to obtain sequence information of the region of interest in the genotyping assay.
2. Genotyping
Using subtype-specific primers, an amplicon of the NS3 protease domain (containing the catalytic domain) is generated from patient-derived viral plasma RNA by One-Step RT-PCR followed by nested PCR. This amplicon, further referred to as the NS3 amplicon, contains the catalytic domain of the NS3 protease. This amplicon is going to be sequenced with subtype-specific sequencing primers in the HCV NS3 protease genotypic assay.
Using subtype-specific primers, an amplicon of the complete NS5B polymerase can also be generated from patient-derived viral plasma RNA by One-Step RT-PCR followed by nested PCR. This amplicon, further referred to as the NS5B amplicon, contains the complete NS5B gene. This amplicon is going to be sequenced with subtype-specific sequencing primers in the HCV NS5B polymerase genotypic assay.
The same can be achieved using subtype-specific primers for other dedicated HCV regions like NS3/NS4A or NS4B/NS5A and the like.
3. Phenotyping
A NS3-deleted replication incompetent shuttle vector, further referred to as the delta[NS3] backbone, has been generated based on the subgenomic replicon con1b sequence. The NS3 amplicon is generated, from patient-derived material, replicon plasmid DNA, synthetic genes or PCR products of replicon RNA, by PCR using the One-Step RT-PCR product of the HCV NS3 protease genotypic assay. In-Fusion™ cloning (Clontech) of the PCR-generated NS3 amplicon and the delta[NS3] backbone resulted in a replication-competent recombinant HCV replicon that was used in experiments to evaluate HCV NS3 phenotypic drug resistance.
A NS5B-deleted replication incompetent shuttle vector, further referred to as the delta[NS5B] backbone has been generated based on the subgenomic replicon con1b sequence. The NS5B amplicon is generated, from patient-derived material, replicon plasmid DNA, synthetic genes or PCR products of replicon RNA, by PCR using the One-Step RT-PCR product of the HCV NS5B polymerase genotypic assay. In vitro cloning (using BD In-Fusion™, Clontech Laboratories Inc.) of the PCR-generated NS5B amplicon and the delta[NS5B] backbone resulted in a replication-competent recombinant HCV replicon that was used in experiments to evaluate HCV NS5B phenotypic drug resistance.
A. RNA-Extraction
From a total 500 μl of plasma, total RNA was extracted using the EasyMAG™
RNA extraction platform (BioMerieux). After elution in 60 μl elution buffer, RNA was stored at −80° C. until use for amplicon generation.
B. One-Step RT-PCR
Five μl RNA were mixed with 2× reaction buffer, 120 ng/ml yeast tRNA (Ambion Inc., Woodward, USA), 0.2 μM primer NS5Bsubtype_A (TGGGGTTCGCGTATGATACCCGCTGCTTTGA) (SEQ ID NO: 1), 0.2 μM primer NS5Bsubtype_B (TGGGGTTTTCTTACGACACCAGGTGCTTTGA) (SEQ ID No: 2), 0.2 μM primer Pr2 (published in Sandres-Saunes et al. 2003) and 0.5 μl of the Superscript™ III RT/Platinum Taq High Fidelity enzyme mix from the SuperScript™ III One-Step RT-PCR System (Invitrogen) in a total volume of 25 μl. The cDNA synthesis is performed for 30 min at 52° C. followed by a denaturation step at 94° C. for 2 min. Thermal cycling consisted of 50 cycles of denaturation at 94° C. for 15 s, annealing at 63° C. for 30 s and elongation at 72° C. for 30 s. Final extension took place at 72° C. for 5 min. An aliquot of the resulting amplification product was used for a nested PCR step.
C. Inner PCR
For the nested PCR, 2.5 μl from the One-Step RT-PCR product were mixed with 10× buffer 2 from the Expand™ High Fidelity kit (Roche), 0.35 mM dNTPs (Promega), 0.4 μM primer NS5Bsubtype_C (CCGTATGATACCCGCTGCTTTGACTCAAC) (SEQ ID NO: 3), 0.3 μM primer NS5Bsubtype_D (TCCTACGACACCAGGTGCTTTGATTCAAC) (SEQ ID NO: 4), 0.4 μM primer NS5Bsubtype_E (AATTCCTGGTCATAGCCTCCGTGAAGACTC) (SEQ ID NO: 5) and 0.075 U/μl of DNA polymerase (Roche, Basel, Switzerland) to give a total volume of 50 μl. Initial denaturation was 94° C. for 2 min and thermal cycling consisted of 30 cycles of denaturation at 94° C. for 15 s, annealing at 56° C. for 30 s and elongation at 72° C. for 30 s. Final extension took place at 72° C. for 5 min. The amplicons were purified using the QIAQuick 96 PCR purification kit (Qiagen,). Final volume of purified amplicons was 100 μl.
D. Raw Sequence Analysis
Sequencing reaction was performed according to standard procedures by using the primers from the nested PCR for sequencing of both directions, forward and reverse. Electropherograms were retrieved from the ABI3730 capillary sequencer and imported into Seqscape v2.5 (Applied Biosystems). Sequence ends were trimmed based on quality values and the 329 by length of the subtyping reference sequence; the latter spanned the regions between the amplification primers. No insertions, deletions or STOP codons were allowed to occur in the sequences.
E. Phylogenetic Tree Analysis
The sample sequences with a length of 329 by were merged with subtype reference sequences in BioEdit ((Ibis Therapeutics; public source internet: www.mbio.ncsu.edu/BioEdit/bioedit.html) and subsequently analysed in MEGA v3.1 (public source, internet: http://www.megasoftware.net/) using Neighbour-Joining tree and Jukes-Cantor distance model.
Results:
NS5B Subtyping Sequences from HCV-1b Infected Patients:
These sequences were subtyped using phylogenetic analysis. Table 1 shows the result.
Based on the NS5B sequence-based subtype information, the appropriate subtype-specific primers were selected for the amplification of the NS3 protease domain.
HCV NS3 Genotyping Assay
A. One-Step RT-PCR
Five μl RNA were mixed with 2× reaction buffer, 120 ng/ml yeast tRNA (Ambion), 0.2 μM forward primer 1b-NS3_out_F (GCGTGTGGGGACATCATCTTAGG) (SEQ ID NO: 6), 0.2 μM primer 1b_NS3_out_R (GCTGCCAGTGGGAGCGTG) (SEQ ID NO: 7) and 0.5 μl of the Superscript™ III RT/Platinum Taq High Fidelity enzyme mix from the SuperScript™ III One-Step RT-PCR System (Invitrogen) in a total volume of 25 μl. The cDNA synthesis is performed for 30 min at 52° C. followed by a denaturation step at 94° C. for 2 min. Thermal cycling consisted of 50 cycles of denaturation at 94° C. for 15 s, annealing at 58° C. for 30 s and elongation at 72° C. for 1 min. Final extension took place at 72° C. for 5 min. An aliquot of the resulting amplification product was used for a nested PCR step.
B. Inner PCR
For the nested PCR, 2.5 μl from the One-Step RT-PCR product was mixed with 10× buffer 2 from the Expand™ High Fidelity kit (Roche), 0.35 mM dNTPs (Promega), 0.4 μM primer 1b_NS3_in_F (TCATCTTAGGCCTGCCCGTCTC) (SEQ ID NO: 8), 0.4 μM primer 1b_NS3_in_R (GGGAGCGTGTAGATGGGCCAC) (SEQ ID NO: 9) and 0.075 U/μl of Expand™ High Fidelity DNA polymerase (Roche) to give a total volume of 50 μl. Initial denaturation was 94° C. for 2 min and thermal cycling consisted of 30 cycles of denaturation at 94° C. for 15 s, annealing at 58° C. for 30 s and elongation at 72° C. for 1 min. Final extension took place at 72° C. for 5 min. The amplicons were purified using the QIAQuick 96 PCR purification kit (Qiagen). Final volume of purified amplicons was 100 μl.
C. Raw Sequence Analysis
Sequencing reaction was performed according to standard procedures by using the primers from the nested PCR for sequencing of both directions, forward and reverse (SEQ ID No's 8-9). Electropherograms were retrieved from the ABI3730 capillary sequencer and imported into Seqscape v2.5 (Applied Biosystems). Sequence ends were trimmed based on quality values and the 543 by (coding sequence for the N-terminal 181 aa of NS3) length of the subtyping reference sequence; the latter spanned the regions between the amplification primers. No insertions, deletions or STOP codons were allowed to occur in the sequences.
Result:
NS3 Protease Sequences from Five (5)HCV-1b Patient Isolates
D. Alignment of Sequences with Reference Sequence
The alignment shows the nucleotide sequence of the NS3 protease domain of an HCV-1b isolate from an untreated patient. The sequences were aligned against a reference sequence. Homologies between the two sequences are plotted as dots.
The following shows the amino acid sequence of the NS3 protease domain of an HCV-1b isolate from an untreated patient. The sequences were aligned against a reference sequence. Homologies between the two sequences are plotted as dots.
NS3 amplicons from these five HCV-1b isolates were further used in the NS3 replicon phenotyping assay.
HCV NS5B Polymerase Genotyping Assay
One-Step RT-PCR:
Five μl RNA were mixed with 2× reaction buffer, 120 ng/ml yeast tRNA (Ambion Inc.), 0.2 μM primer 1b_NS5B_out_F (TAGAGTCCTGGAAGGACCCGG) (Sequence ID NO:13), 0.2 μM primer 1b_NS5B_out_R (GGCCTGGAGTGGTTAGCTCCCC) (Sequence ID NO:14) and 0.5 μl of the Superscript™ III RT/Platinum Taq High Fidelity enzyme mix from the SuperScript™ III One-Step RT-PCR System (Invitrogen) in a total volume of 25 μl. The cDNA synthesis is performed for 30 min at 47° C. followed by a denaturation step at 94° C. for 2 min. Thermal cycling consisted of 50 cycles of denaturation at 94° C. for 15 s, annealing at 59° C. for 30 s and elongation at 68-° C. for 2 min 30 s. Final extension took place at 68° C. for 5 min. An aliquot of the resulting amplification product was used for a nested PCR step.
Inner PCR:
For the nested PCR, 2.5 μl from the One-Step RT-PCR product was mixed with 10× buffer 1 from the Expand™ Long Template High Fidelity kit (Roche, Basel, Switzerland), 0.35 mM dNTPs (Promega), 0.4 μM primer 1b_NS5B_in_F (TGGAAGGACCCGGACTACG) (Sequence ID NO:15), 0.4 μM primer 1b_NS5B_in_R (GAGTGGTTAGCTCCCCGTTCA) (Sequence ID NO:16) and 0.075 U/μl of Expand™ High Fidelity DNA polymerase (Roche,) to give a total volume of 50 μl. Initial denaturation was 94° C. for 2 min and thermal cycling consisted of 30 cycles of denaturation at 94° C. for 15 s, annealing at 59° C. for 30 s and elongation at 68° C. for 2 min 30 s. Final extension took place at 68° C. for 5 min. The amplicons were purified using the QIAQuick 96 PCR purification kit (Qiagen). Final volume of purified amplicons was 100 μl.
Raw Sequence Analysis
Sequencing reaction was performed according to standard procedures by using 8 sequencing primers (SEQ ID No's 15-16 and 87-92) to cover both directions, forward and reverse. Electropherograms were retrieved from the ABI3730 capillary sequencer and imported into Seqscape v2.5 (Applied Biosystems). Sequence ends were trimmed based on quality values and the 1776 by (coding sequence of the NS5B polymerase) length of the subtype-specific reference sequence; the latter spanned the regions between the amplification primers. No insertions, deletions or STOP codons were allowed to occur in within the sequences.
Result:
NS5B Polymerase Sequences from Five HCV-1 b Clinical Isolates
D. Alignment of Sequences with Reference Sequence
The alignment shows the nucleotide sequence of the NS5B polymerase domain of an HCV-1b isolate from an untreated patient. The sequence was aligned against a reference sequence. Homologies between the two sequences are plotted as dots.
NS3 Phenotyping Assay
Construction of Delta [NS3] Shuttle Vector
The plasmid 11pFK I341 PI luc NS3-3′_ET is based on the construct described in Krieger et al. 2001 and was kindly provided by Prof. Bartenschlager (Heidelberg, Germany). In order to generate a shuttle vector for NS3 phenotyping, it was modified by site-directed mutagenesis to introduce two new SacII restriction sites at position 3338 and 3899. In a next step, the modified plasmid was digested with SacII and subsequently religated to give the delta[NS3] shuttle vector pFK I341 PI luc ΔNS3 7-192_ET (SEQ ID No 10).
For InFusion cloning, the delta[NS3] backbone pFK I341 PI luc ΔNS3 7-192_ET (SEQ ID NO: 10) was linearized by SacII digestion.
Cloning of the NS3 PCR Amplicons from Infected Patients into the Delta[NS3] Shuttle Vector
A. NS3 Amplicon Generation from Isolates of HCV-Infected Patients
For the PCR, 1 μl from the One-Step RT-PCR product of the NS3 genotyping assay was mixed with 0.2 μM primer 1b_InFu_NS3_F (SEQ ID NO: 11), 0.2 μM primer 1b_InFu_NS3_R (SEQ ID NO: 12) and 2× Herculase™ Hotstart master mix (Stratagene) to give a total volume of 50 μl. Initial denaturation was 95° C. for 2 min and thermal cycling consisted of 10 cycles followed by another 20 cycles consisting of denaturation at 95° C. for 30 s, annealing at 60° C. for 30 s and elongation at 72° C. for 1 min (plus 10 s per cycle). Final extension took place at 72° C. for 10 min. The amplicons were purified using the QIAQuick gel purification kit (Qiagen).
B. Delta [NS3] Shuttle Vector Preparation
The NS3 subgenomic shuttle backbone was digested with an excess of the restriction endonuclease SacII (NEB) and 1× restriction enzyme buffer 4 (NEB) at 37° C. overnight. In a next step, calf intestine phosphatase was added and the mixture incubated for 40 min at 37° C. in order to dephosphorylate the linearized shuttle backbone. The dephosphorylated vector was purified via agarose gel electrophoresis (crystal violet) followed by gel extraction using the kit from QIAGEN. The linearized vector was stored at −20° C. until further use.
C. Cloning of the NS3 Derived from Patient Isolates into the Linearized Delta [NS3] Shuttle Vector
The PCR products and the linearized vector were thawed and the PCR product was stored on ice until cloning. Immediately before the In-Fusion™ cloning, the linearized vector was denatured for 5 min at 60° C. and subsequently put on ice. For the cloning reaction, 1 μl of the PCR product and 1 μl of the vector preparation were added to 8 μl Dnase/Rnase-free water. The complete mix (10 μl) was added into one tube containing the Dry-Down In-Fusion™ reaction mix (Clontech) and carefully pipetted up and down. The pipetting steps were performed on ice. The PCR tubes containing the In-Fusion™ cloning mix were subsequently transferred to a thermocycler and incubated for 30 min at 42° C. After incubation the tubes were immediately transferred to ice.
D. Transformation of Recombinant Replicon DNA
The transformation of Escherichia coli cell was performed immediately after the In-Fusion™ cloning step. The XL10-Gold® Ultracompetent Cells (Stratagene) were used for the transformation. 50 μl of the cells were transformed with 5 μl of the In-Fusion™ cloning mix according to the protocol from Stratagene. The complete transformation mix was plated onto ampicillin-containing LB Petri dishes and incubated overnight at 37° C. Colonies were pooled by applying 1 ml of ampicillin-containing LB medium onto the Petri dishes and removing the colonies by scraping. The bacterial suspension was transferred into a 15 ml-Falcon tube. The Petri dishes were washed for a second time with 1 ml of the ampicillin-containing LB medium and the solution was again transferred into the Falcon tube. 2 ml of ampicillin-containing LB medium were added and cells were grown at 37° C. until they reached the logarithmic phase (approximately 4-5 hours). 1.5 ml of the cell culture was used for inoculation of 200 ml ampicillin-containing LB medium. Cells were grown overnight at 37° C. for the DNA preparation. The DNA was prepared using the Maxiprep DNA purification kit from QIAGEN.
Replicon NS3 Phenotypic Assay
A. Recombinant Replicon Plasmid DNA Linearization
The replicon plasmid DNA (10 μg per sample) was linearized using 1.5 μl of Asel (NEB) and 3 μl ScaI (NEB) together with 4 μl NEB buffer 3 to give a total volume of 40 μl. The reaction mix was incubated for 4 hours at 37° C. The linearized vector was separated from the resulting fragments via agarose gel electrophoresis and purified using the gel extraction kit from QIAGEN. DNA concentration was measured using the Nanodrop® spectrophotometer (ratio OD260 nm/OD280 nm). The purified DNA was stored at −20° C. until further use.
B. Preparation of In Vitro Transcribed Replicon RNA
The in vitro transcription was performed using the MEGAscript High Yield Transcription kit (Ambion) according to protocol HCV_SP—038.vs2 in the Laboratory Operation Unit at Tibotec. Briefly, 1 μg of the linearized and purified replicon DNA was used per reaction for in vitro transcription and were added to a mix containing 44 μl nuclease-free water, 4 μl ATP solution, 4 μl CTP solution, 4 μl GTP solution, 4 μl UTP solution and 10× reaction buffer. Four μl of the enzyme mix were subsequently added. The pipetting was performed at room temperature. The reaction mix was incubated for 4 hours at 37° C. Two μl of TURBO DNase (Ambion) were subsequently added and the mixture was incubated for 15 min at 37° C. in order two destroy the DNA template. The RNA was purified using the MEGAclear™ kit (Ambion). RNA was quantified using the Nanodrop® spectrophotometer (ratio OD260 nm/OD280 nm). The purified RNA was stored in 10 μg aliquots at −80° C. until further use.
C. Hepatoma Cell Line
Cured hepatoma cell line Huh7 were cultured at 37° C. in a humidified atmosphere with 5% CO2 in Dulbecco's Modified Eagle medium (DMEM, Biowhittaker, Cat no BE12-917F) supplemented with L-Glutamine and 10% FCS.
D. Determination of Transient Replicon Replication
4×106 cells were transfected with 10 μg of in vitro transcribed replicon RNA via electroporation. For EC50 determination 4,000 cells/well were seeded in a volume of 30 μl medium in white 384-well compound plates. Compound plates contained 10 μl/well of the respective compound dilution in medium (containing 2% DMSO), leading to a total volume of 40 μl per well with a final concentration of 0.5% DMSO. Compound dilutions were prepared in quadruplates. Cell culture plates were incubated for 48 h at 37° C. and 5% CO2. Experiments were performed in triplicates. The firefly luciferase chemiluminescence read-out was performed using the Steady-Lite reagent (PerkinElmer). The EC50 values were assessed as the inhibitor concentration at which a 50% reduction in the level of firefly luciferase reporter was observed as compared to the level of firefly luciferase signal without the addition of compounds. Results of studies testing the inhibitory effect of an example protease inhibitor, SCH 503034, on replication of WT replicon and replicons with patient-derived NS3 sequences are shown in Table 2.
Table 2 shows that the NS3-restored shuttle vector is replicating. GND serves as a non-replicating replicon control.
Table 3 shows EC50 values of an HCV protease inhibitor tested in the NS3 replicon shuttle system with 5 patient isolates.
Results:
1RLU represents level of firefly luciferase signal observed after 48 hours post-transfection.
2Replication level is compared to wild type (WT) vector.
NS5B Phenotyping Assay
Construction of Delta [NS5B] Backbone
The plasmid 11 pFK I341 PI luc NS3-3′_ET is based on the construct described in Krieger et al. 2001 and was kindly provided by Prof. Bartenschlager (Heidelberg, Germany). In order to generate a shuttle vector, it was modified by site-directed mutagenesis to introduce two AfIII restriction sites at position 7481 and 9287. First, an AfIII restriction site was introduced by site directed mutagenesis into the 3′ NCR directly after the stop codon of NS5B at Medigenomix (Munich, Germany) resulting in plasmid pFKi341Luc_NS3-3′-ET-AfIII (Sequence ID NO:17). Next, a second AfIII restriction site 8aa upstream of the NS5A/NS5B cleavage site was introduced using the Quick Change Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif., USA) according to the manufacturers recommendations with SDM primer pair AfIII-5A-fwd (5′-accgtaagcgaggagcttaaggctagtgaggacgtc-3′) (Sequence ID NO:18) and AfIII-5A-rev (5′-gacgtcctcactagccttaagctcctcgcttacggt-3′) (Sequence ID NO:19) resulting in plasmid pFKi341Luc_NS3-3′-ET-2×AfIII (Sequence ID NO:20). In a next step, the modified plasmid was digested with AfIII and subsequently re-ligated resulting in the delta[NS5B] backbone pFK_I341_PI_NS3-3_ET_dNS5a/b—5a440-5b591-ScaI (Sequence ID NO:21).
In parallel, a NS5B phenotyping construct with an XbaI restriction site at the 3′ end was generated using plasmid pFKi341Luc_NS3-3′-ET-2×AfIII (Sequence ID NO:20) as template. First, an XbaI site in the gene of the firefly luciferase was mutated by a site directed mutagenesis approach, resulting in a silent mutation, using primer pair XbaI-mut-fwd (5′-ggcgccattctatccactagaggatggaacc-3′) (Sequence ID NO:22) and XbaI-mut-rev (5′-ggttccatcctctagtggatagaatggcgcc-3′) (Sequence ID NO:23). In a second SDM reaction, an XbaI restriction site was introduced at the 3′ end of the HCV 3′ NCR instead of the ScaI site using primer pair XbaI-add-fwd (5′-gagtgctgatactggcctctctgcagatcaagtctagaaagtccctttagtgagggttaattc-3′) (Sequence ID NO:24) and XbaI-add-rev (5′-gaattaaccctcactaaagggactttctagacttgatctgcagagaggccagtatcagcactc-3′) (Sequence ID NO:25) resulting in plasmid pFKi341Luc_NS3-3′-ET-2×AfIII-XbaI (Sequence ID NO:26). In a next step, the modified plasmid was digested with AfIII and subsequently re-ligated resulting in the delta[NS5B] backbone pFK_I341_PI_NS3-3_ET_dNS5a/b—5a440-5b591-XbaI (Sequence ID NO:27). Linearization with XbaI results in an authentic HCV 3′ end and offered the possibility to shuttle amplicons of clinical isolates which harbor a ScaI site in the NS5B coding sequence.
For InFusion cloning, the delta[NS5B] backbone pFK_I341_PI_NS3-3_ET_dNS5a/b—5a440-5b591-ScaI (SEQ ID NO:21) or pFK_I341_PI_NS3-3_ET_dNS5a/b—5a440-5b591-XbaI (SEQ ID NO:27) was linearized by AfIII digestion.
Cloning of the NS5B PCR Amplicons from HCV-Infected Patients into the Delta [NS5B] Shuttle Vector
A. NS5B Amplicon Generation from Isolates of HCV-Infected Patients
For the InFusion™ PCR, 1 μl from the One-Step RT-PCR product of the NS5B genotyping assay was mixed with 0.2 μM primer 1b_NS5B_F_AfIII-Infusion (5′-AAGCGAGGAGCTTAAGGCYRGTGAGGACGT-3′) (SEQ ID NO:28), 0.2 μM primer 1b_NS5B_R_AfIII-Infusion (5′-AGCTCCCCGTCTTAAGTCAYCGGT TGGGG-3′) (SEQ ID NO:29) and 2× Herculase™ Hotstart master mix (Stratagene, La Jolla, Calif., U.S.) to give a total volume of 50 μl. Initial denaturation was 95° C. for 2 min and thermal cycling consisted of 10 cycles followed by another 20 cycles consisting of denaturation at 95° C. for 30 s, annealing at 60° C. for 30 s and elongation at 72° C. for 1 min 30 s (plus 10 s per cycle). Final extension took place at 72° C. for 10 min. The amplicons were purified using the QIAQuick gel purification kit (Qiagen, Hilden, Germany). Final volume of purified amplicons was 30 μl.
B. Delta [NS5B] Shuttle Vector Preparation
The NS5B subgenomic shuttle backbone was digested with an excess of the restriction endonuclease AfIII (NEB) and 1× restriction enzyme buffer 4 (NEB) at 37° C. overnight. In a next step, calf intestine phosphatase was added and the mixture incubated for 1 h at 37° C. in order to dephosphorylate the linearized shuttle backbone. The dephosphorylated vector was purified via agarose gel electrophoresis (crystal violet) followed by gel extraction using the kit from QIAGEN. The linearized vector was stored at −20° C. until further use.
C. Cloning of the NS5B Derived from Patient Isolates into the Linearized Delta [NS5B] Shuttle Vector
The PCR products and the linearized vector were thawed and the PCR product was stored on ice until cloning. Immediately before the In-Fusion™ cloning, the linearized vector was denatured for 5 min at 60° C. and subsequently put on ice. For the cloning reaction, 2 μl of the PCR product and 1-3 μl of the vector preparation were added to 5-8 μl Dnase/Rnase-free water. The complete mix (10 μl) was added into one tube containing the Dry-Down In-Fusion™ reaction mix (Clontech) and carefully pipetted up and down. The pipetting steps were performed on ice. The PCR tubes containing the In-Fusion™ cloning mix were subsequently transferred to a thermocycler and incubated for 30 min at 42° C. After incubation the tubes were immediately transferred to ice
D. Transformation of Recombinant Replicon DNA
The transformation of Escherichia coli cell was performed immediately after the In-Fusion™ cloning step. The XL10-Gold® Ultracompetent Cells (Stratagene) were used for the transformation. 50 μl of the cells were transformed with 5 μl of the In-Fusion™ cloning mix according to the protocol from Stratagene. The complete transformation mix was plated onto ampicillin-containing LB Petri dishes and incubated overnight at 37° C. Colonies were pooled by applying 1 ml of ampicillin-containing LB medium onto the Petri dishes and removing the colonies by scraping. The bacterial suspension was transferred into a 15 ml-Falcon tube. The Petri dishes were washed for a second time with 1 ml of the ampicillin-containing LB medium and the solution was again transferred into the Falcon tube. 2 ml of ampicillin-containing LB medium were added and cells were grown at 37° C. until they reached the logarithmic phase (approximately 4-5 hours). 1.5 ml of the cell culture was used for inoculation of 200 ml ampicillin-containing LB medium. Cells were grown overnight at 37° C. for the DNA preparation. The DNA was prepared using the Maxiprep DNA purification kit from QIAGEN.
Replicon NS5B Phenotypic Assay
A. Recombinant Replicon Plasmid DNA Linearization
The replicon plasmid DNA (10 μg per sample) was linearized using 3 μl of XbaI (NEB) together with 10 μl NEB buffer 4 and 1 μl of a 100× concentrated BSA stock solution (NEB) to give a total volume of 100 μl. The reaction mix was incubated for 4 hours at 37° C. The linearized vector was separated from the resulting fragments via agarose gel electrophoresis and purified using the gel extraction kit from QIAGEN. DNA concentration was measured using the Nanodrop® spectrophotometer (ratio OD260 nm/OD280 nm). The purified DNA was stored at −20° C. until further use.
B. Preparation of In Vitro Transcribed Replicon RNA
The in vitro transcription was performed using the MEGAscript High Yield Transcription kit (Ambion) according to protocol HCV_SP—038.vs2 in the Laboratory Operation Unit at Tibotec. Briefly, 1 μg of the linearized and purified replicon DNA was used per reaction for in vitro transcription and were added to a mix containing 44 μl nuclease-free water, 4 μl ATP solution, 4 μl CTP solution, 4 μl GTP solution, 4 μl UTP solution and 10× reaction buffer. Four μl of the enzyme mix were subsequently added. The pipetting was performed at room temperature. The reaction mix was incubated for 4 hours at 37° C. Two μl of TURBO DNase (Ambion) were subsequently added and the mixture was incubated for 15 min at 37° C. in order two destroy the DNA template. The RNA was purified using the MEGAclear™ kit (Ambion). RNA was quantified using the Nanodrop® spectrophotometer (ratio OD260 nm/OD280 nm). The purified RNA was stored in 10 μg aliquots at −80° C. until further use.
C. Hepatoma Cell Line
Cured hepatoma cell line Huh7 were cultured at 37° C. in a humidified atmosphere with 5% CO2 in Dulbecco's Modified Eagle medium (DMEM, Biowhittaker, Cat no BE12-917F) supplemented with L-Glutamine and 10% FCS.
D. Determination of Transient Replicon Replication
4×106 cells were transfected with 10 μg of in vitro transcribed replicon RNA via electroporation. For EC50 determination 4,000 cells/well were seeded in a volume of 30 μl medium in white 384-well compound plates. Compound plates contained 10 μl /well of the respective compound dilution in medium (containing 2% DMSO), leading to a total volume of 40 μl per well with a final concentration of 0.5% DMSO. Compound dilutions were prepared in quadruplates. Cell culture plates were incubated for 48 h at 37° C. and 5% CO2. Experiments were performed in at least duplicates. The firefly luciferase chemiluminescence read-out was performed using the Steady-Lite reagent (PerkinElmer). The EC50 values were assessed as the inhibitor concentration at which a 50% reduction in the level of firefly luciferase reporter was observed as compared to the level of firefly luciferase signal without the addition of compounds. Results of studies testing the inhibitory effect of an example polymerase inhibitor, Thiophene-2-carboxylic acid, on replication of WT replicon and replicons with patient-derived NS5B sequences are shown in Table 4.
Table 4 shows EC50 values of an HCV polymerase inhibitor tested in the NS5B replicon shuttle system with 5 patient isolates.
Results:
| Number | Date | Country | Kind |
|---|---|---|---|
| 08165949.2 | Oct 2008 | EP | regional |
This application is the national stage of PCT Application No. PCT/EP2009/062986 filed Oct. 6, 2009, which claims priority from European Patent Application No. 08165949.2, filed Oct. 6, 2008, the entire disclosures of which are hereby incorporated in their entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP09/62986 | 10/6/2009 | WO | 00 | 4/5/2011 |
