Abstract
With the development of directly acting antivirals, hepatitis C virus (HCV) therapy entered a new era. However, rapid selection of resistance mutations necessitates combination therapy. To study combination therapy in infectious culture systems, we aimed at developing HCV semi-full-length (semi-FL) recombinants relying only on the JFH1 NS3 helicase, NS5B, and the 3′ untranslated region. With identified adaptive mutations, semi-FL recombinants of genotypes(isolates) 1a(TN) and 3a(S52) produced supernatant infectivity titers of ∼4 log10 focus-forming units/ml in Huh7.5 cells. Genotype 1a(TN) adaptive mutations allowed generation of 1a(H77) semi-FL virus. Concentration-response profiles revealed the higher efficacy of the NS3 protease inhibitor asunaprevir (BMS-650032) and the NS5A inhibitor daclatasvir (BMS-790052) against 1a(TN and H77) than 3a(S52) viruses. Asunaprevir had intermediate efficacy against previously developed 2a recombinants J6/JFH1 and J6cc. Daclatasvir had intermediate efficacy against J6/JFH1, while low sensitivity was confirmed against J6cc. Using a cross-titration scheme, infected cultures were treated until viral escape or on-treatment virologic suppression occurred. Compared to single-drug treatment, combination treatment with relatively low concentrations of asunaprevir and daclatasvir suppressed infection with all five recombinants. Escaped viruses primarily had substitutions at amino acids in the NS3 protease and NS5A domain I reported to be genotype 1 resistance mutations. Inhibitors showed synergism at drug concentrations reported in vivo. In summary, semi-FL HCV recombinants, including the most advanced reported genotype 3a infectious culture system, permitted genotype-specific analysis of combination treatment in the context of the complete viral life cycle. Despite differential sensitivity to lead compound NS3 protease and NS5A inhibitors, genotype 1a, 2a, and 3a viruses were suppressed by combination treatment with relatively low concentrations.
INTRODUCTION
Chronic hepatitis C virus (HCV) infection, which causes liver cirrhosis and hepatocellular carcinoma and is the main indication for liver transplantation, poses a significant public health burden (1). HCV is an enveloped virus with a positive-strand RNA genome with 5′ and 3′ untranslated regions (UTRs) and an open reading frame (ORF) encoding a polyprotein processed into structural proteins (core, E1, E2), p7, and nonstructural (NS) proteins NS2, NS3 with protease (NS3P) and helicase (NS3H) domains, NS4A, NS4B, NS5A, and NS5B (1). HCV was classified into seven major genotypes differing in ∼30% of their sequences (2). Genotypes 1, 2, and 3 predominate in Europe, North and South America, Australia, and many Asian countries, such as Japan (1). During the last decade, therapy relied on pegylated alfa interferon and ribavirin, curing ∼50% of treated patients (1). Directly acting antivirals (DAAs) targeting specific HCV proteins have been developed and tested in clinical trials. Lead targets are NS3P, mainly responsible for polyprotein processing; NS5A domain I, of importance for viral replication; and the viral RNA polymerase NS5B (3). In 2011, the first DAAs, targeting NS3P, were licensed for use for treatment of chronic genotype 1 infection in combination with pegylated alfa interferon and ribavirin. However, due to rapid selection of resistant variants, combination of different DAAs will be required to ensure a sustained viral response (3). Combination of NS3P and NS5A inhibitors asunaprevir (4) and daclatasvir (5) recently proved efficient against genotype 1 infection in phase 2 clinical trials (6, 7).
To define new regimens for the treatment of infections caused by the major HCV genotypes, efficient in vitro systems are required. Replicon systems (8, 9) allow the study of viral replication in the host cell and thus recapitulate only a part of the viral life cycle. At the outset of this study, efficient full viral life cycle culture systems relied on genotype 2a isolate JFH1 (10, 11), while genotype 1 full-length culture systems showed low infectivity (12, 13). We previously developed J6/JFH1-based culture-adapted recombinants with genotype-specific NS3P/NS4A (14) or NS5A (15) and used them to study the efficacy of NS3P and NS5A inhibitors, respectively. However, for drug combination studies, recombinants with extended genotype-specific regions are needed. Because the unique replication capacity of JFH1 apparently mainly depended on NS3H, NS5B, and 3′ UTR (16), we constructed genotype 1 to 4 recombinants containing only these JFH1 regions (Fig. 1A). We aimed at adapting these recombinants to Huh7.5 cells and at using them to study the efficacy of combination treatment with NS3P and NS5A inhibitors against different HCV genotypes.
Fig 1.
Development of Huh7.5 cell culture-adapted genotype 1a and 3a semi-FL HCV recombinants. (A) Genome structure indicating genotype- and JFH1-specific genome regions. (B to D) Following transfection of HCV RNA transcripts of the indicated recombinants into Huh7.5 cells, the percentages of HCV core-positive cells in transfection cultures were determined by immunostaining, shown on the left y axis and indicated by lines. Peak infectivity titers in selected culture supernatants are shown on the right y axis as means of 3 replicates with SEMs and are indicated by empty bars. Data from different experiments are shown in the same graph. (B) Of recombinants not spreading during follow-up, 1a(TN) had 5 to 10% infected cells on days 3 to 8 and ≤1% infected cells until day 52 posttransfection. Genotypes 1a(H77) and 1b(J4) did not show infected cells over 29 and 37 days, respectively. Genotype 4a(ED43) showed single infected cells on days 13 and 15 but otherwise no infected cells over 41 days. Except for 2a(J6), one additional transfection experiment was performed and showed similar results (data not shown). NA, not applicable.
MATERIALS AND METHODS
Plasmids.
We replaced the NS3H (nucleotides 3978 to 5312, amino acids 1213 to 1657; nucleotide and amino acid positions are given as absolute H77 [GenBank accession number AF009606] reference numbers, unless otherwise indicated) and NS5B-3′ UTR (nucleotides 7602 to 9646 and amino acids 2421 to 3011 coding for NS5B) sequences of pHC-TN (GenBank accession number EF621489) (17), pCV-H77C (GenBank accession number AF011751) (18), pCV-J4L6S (GenBank accession number AF054247) (19), pJ6CF (GenBank accession number AF177036) (20), pS52 (GenBank accession number GU814264) (21), and pED43 (GenBank accession number GU814266) (21) by the corresponding JFH1 (GenBank accession number AB047639) sequences using fusion PCR- and restriction enzyme-based cloning. After insertion of the JFH1 NS3H and NS5B-3′ UTR in pCV-J4L6S, a KpnI/XbaI fragment was transferred into the J4 5′ UTR-NS2 recombinant (22) to introduce the J4 5′ UTR. Mutations were introduced using fusion PCR- and restriction enzyme-based cloning. The HCV sequences of the final DNA preparations (plasmid maxikit; Qiagen) were confirmed (Macrogen).
Transfection, viral passage, and evaluation of cell cultures.
Transfection of Huh7.5 hepatoma cells with RNA transcripts using Lipofectamine 2000 (Invitrogen) and infection of naïve cells for viral passage with culture supernatant were done as described previously (23). Supernatants collected during experiments were stored at −80°C. The percentage of HCV-infected cells was estimated by immunostaining, using anti-HCV core protein monoclonal antibody (MAb) B2 (Anogen) and Alexa Fluor 594 goat anti-mouse IgG (H+L; Invitrogen) (24) and fluorescence microscopy, assigning values of 0% (no cells infected), 1%, 5%, and 10% to 90% (in steps of 10%). Culture supernatant infectivity titers were determined as the number of focus-forming units (FFU)/ml following immunostaining with hepatitis C core protein MAb C7-50 at a 1:400 dilution (Enzo Life Sciences) (15).
Determination of concentration-response profiles and statistical analysis.
Huh7.5 cultures on poly-d-lysine-coated 96-well plates (Nunc) were infected with the virus stocks specified and treated with antivirals (Acme Bioscience) at 24 h postinfection, immunostained with anti-core MAb C7-50 at a 1:400 dilution at 72 h postinfection, and evaluated by counting of single core-positive cells (14, 15). Cell viability was not affected by the drug concentrations used and was monitored with a CellTiter 96 AQueous One Solution cell proliferation assay (Promega). Concentrations were tested in triplicate. Counts from treated wells were related to the mean of counts from 6 infected, nontreated wells. Following logarithmic transformation of x values, sigmoidal dose-response curves [y = top/(1 + 10[log10 EC50−x] · Hill slope)], where x is the concentration of antiviral, y is the response expressed as the percentage of HCV-infected cells related to nontreated controls, top represents the y value at the top plateau of the fitted curve, and EC50 is the 50% [median] effective concentration, were fitted to the data using GraphPad Prism software. The log10 EC50 and the standard error of the mean (SEM) of the log10 EC50 from replicate experiments were used to calculate inverse variance-weighted means of the log10 EC50 with the SEM and the 95% confidence interval. Mean differences between the log10 EC50s of 2a(JFH1) and other recombinants with SEMs and 95% confidence intervals were calculated from the inverse variance-weighted mean log10 EC50 values. Inverse logarithmic transformation rendered the median EC50 with the 95% confidence interval and the median fold difference with the 95% confidence interval. P values were determined by the Z test; P values of <0.0001 were considered significant.
Long-term treatment experiments.
Huh7.5 cells (3.6 × 105) were plated per well of 6-well plates and on the following day were infected at a multiplicity of infection of 0.05 with the virus stocks specified. After 24 h, cells were transferred to T25 flasks and treatment with asunaprevir and/or daclatasvir was initiated using a cross-titration scheme with concentrations of 0-, 0.8-, 4-, 20-, and 100-fold the EC50. Three times weekly, when cells were split, treatment was administered and cultures were immunostained with anti-core B2, until the occurrence of viral escape, defined as viral spread to ≥80% of cells, or on-treatment virologic suppression, defined as the absence of HCV core-positive cells for at least 14 days under continued treatment.
Drug combination studies and synergy quantification.
The synergy of asunaprevir and daclatasvir for virus treatment was evaluated by the method of Chou and Talalay with CompuSyn freeware (ComboSyn Inc.) (25, 26). The experimental setup was similar to that described above for determination of concentration-response profiles. In brief, 5,000 Huh7.5 cells per well that had been plated on the previous day on poly-d-lysine-coated 96-well plates were infected with the virus stocks specified and treated with antivirals at 24 h postinfection. For treatments with single drugs, 2-fold dilutions were used, applying concentrations determined by the EC50 values previously determined. In the same experiment, viruses were treated with 2-fold dilutions of a combination of both drugs, using a constant concentration ratio, determined by the EC50 values of the drugs for the respective virus. Concentrations were tested in 3 to 4 replicates. Following 48 h of incubation, cultures were immunostained with a mix of primary antibodies, anti-core MAb C7-50 at a 1:400 dilution and anti-NS5A MAb 9E10 at a 1:100 dilution (11), and were evaluated by counting of single HCV-positive cells. Cell viability was not affected by the drug concentrations used and was monitored with the CellTiter 96 AQueous One Solution cell proliferation assay. Counts from treated wells were related to the mean of the counts from at least 6 infected, nontreated wells to calculate the percent inhibition, and these values were entered into CompuSyn as fractional effect (Fa) values over the range of 0.01 to 0.99. For each treatment experiment, 5 to 7 data points were entered. The software was used to determine dose-effect curves for single and combination treatments. Further, combination index (CI) values were calculated in relation to Fa values. In the following, we use verbal descriptions for synergism or antagonism, as recommended by CompuSyn. The CI values reported were in the following categories: 0.3 to 0.7, synergism; 0.7 to 0.85, moderate synergism; 0.85 to 0.9, slight synergism; 0.9 to 1.1, nearly additive; 1.1 to 1.2, slight antagonism; 1.2 to 1.45, moderate antagonism; 1.45 to 3.3, antagonism; 3.3 to 10, strong antagonism.
Sequencing of cell culture-produced HCV.
Extraction of RNA from culture supernatants, reverse transcription-nested PCR, and direct sequencing were described elsewhere (24). Primers and conditions for amplifications are specified in Tables S1 to S5 in the supplemental material.
RESULTS
Culture viability of HCV genotype 1 to 4 semi-full-length (semi-FL) recombinants.
We constructed HCV genomes with 5′ UTR-NS3P and NS4A-NS5A of reference genotypes(isolates) 1a(H77 and TN) (17, 18), 1b(J4) (19), 2a(J6) (20), 3a(S52) (21), and 4a(ED43) (21) and with NS3H, NS5B, and the 3′ UTR of JFH1 (10) (Fig. 1A). Recombinants are designated according to the genotype(isolate) of the genotype-specific sequence; reference recombinant J6/JFH1 (11) is designated 2a(J6/JFH1). Huh7.5 cells were transfected with RNA transcripts, and the percentage of HCV core-expressing cells was evaluated by immunostaining. The 2a(J6/JFH1), 2a(J6), and 3a(S52) viruses infected the majority of culture cells on days 4, 10, and 48, respectively, with supernatant infectivity titers reaching 4.5, 3.4, and 3.3 log10 FFU/ml, respectively (Fig. 1B). In contrast, transfection of genotype 1a, 1b, and 4a transcripts did not result in viral spread (for details, see the Fig. 1B legend). We did not pursue further development of 2a(J6), because we succeeded in developing full-length J6cc (27), designated 2a(J6cc). To further promote culture adaptation of 3a(S52), we inoculated naïve Huh7.5 cells with supernatant from the peak of infection of the transfection culture (Fig. 1B); the 1st-passage virus peak titer was 3.1 log10 FFU/ml (Tables 1 and 2).
Table 1.
Coding nucleotide changes of 3a(S52) semi-FL recombinant recovered from Huh7.5 cellsa
| 3a(S52) virus | Passage (day) | Infectivity titer (log10 FFU/ml) | Nucleotide identityb at the indicated nucleotide positionsc in the following HCV gene: |
||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Core (366/368) | Core (668/670) | E1 (1372/1374) | E2 (1552/1554) | E2 (1580/1582) | p7 (2660/2644) | p7 (2719/2703) | NS2 (2989/2973) | NS2 (2990/2974) | NS3H (4369/4353) | NS3H (4463/4447) | NS3H (4631/4615) | NS4A (5387/5371) | NS4B (5814/5798) | NS5A (7601/7573) | NS5A (7606/7578) | NS5B (9112/9084) | |||
| Plasmid encoded | NAe | NA | A | A | T | A | T | A | T | A | G | A | G | T | T | T | A | G | C |
| Cell culture derived | |||||||||||||||||||
| Original recombinant | 1st (15) | 3.1 | – | – | – | – | – | – | C/T | – | – | – | G/a | C/t | – | – | G/a | – | – |
| Recombinant with indicated amino acid change(s) | |||||||||||||||||||
| I1425Td | 1st (15) | 3.5 | C | – | – | – | C | G/A | – | – | – | – | – | C | – | – | – | A | T |
| I1425Td | 2nd (18) | 4.4 | C | – | G | G/A | C | – | – | G | C | G/A | A/G | C | C/T | G/T | – | A | T |
| R9S, V414A, I1425T, E2412K, P2915S | 1st (11) | 3.9 | C | T/A | – | – | C | – | – | – | – | – | – | C | – | – | – | A | T |
ORF sequences of viral genomes from supernatants of passage cultures were determined by direct sequence analysis. Indicated are the numbers of viral passages and the day of the passage experiments at which supernatants were obtained, as well as the peak infectivity titers of the respective passage experiments.
Nucleotide identity at the respective position of the plasmid or of viral genomes recovered from infected cell cultures. Positions with quasispecies are written with two capital letters in the case of a 50/50 quasispecies and with a capital letter/lowercase letter in the case of a dominant/minor sequence. At the positions included in this table, coding nucleotide changes occurred at least as 50/50 quasispecies in at least one ORF. –, identity with the original plasmid sequence. Highlighted positions are engineered mutations.
Data in parentheses are the nucleotide position of the HCV sequence of the 3a(S52) plasmid/absolute nucleotide reference number in accordance with the H77 (GenBank accession no. AF009606) reference nucleotide sequence (2).
The 1st and consecutive 2nd viral passages were from the same transfection.
NA, not applicable.
Table 2.
Amino acid changes of 3a(S52) semi-FL recombinant recovered from Huh7.5 cellsa
| 3a(S52) virus | Passage (day) | Infectivity titer (log10 FFU/ml) | Amino acid identityb at the indicated amino acid positionsc in the following HCV gene: |
|||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Core (9/9) | Core (110/110) | E1 (345/345) | E2 (405/405) | E2 (414/414) | p7 (774/768) | p7 (794/788) | NS2 (884/878) | NS3H (1344/1338) | NS3H (1375/1369) | NS3H (1431/1425) | NS4A (1683/1677) | NS4B (1825/1819) | NS5A (2421/2410) | NS5A (2423/2412) | NS5B (2925/2915) | |||
| Plasmid encoded | NAe | NA | R | N | F | M | V | Y | Y | S | T | R | I | V | H | D | E | P |
| Cell culture derived | ||||||||||||||||||
| Original recombinant | 1st (15) | 3.1 | – | – | – | – | – | – | H/Y | – | – | R/q | T/i | – | – | G/d | – | – |
| Recombinant with indicated amino acid change(s) | ||||||||||||||||||
| I1425Td | 1st (15) | 3.5 | S | – | – | – | A | C/Y | – | – | – | – | T | – | – | – | K | S |
| I1425Td | 2nd (18) | 4.4 | S | – | V | V/M | A | – | – | A | A/T | Q/R | T | A/V | Q/H | – | K | S |
| R9S, V414A, I1425T, E2412K, P2915S | 1st (11) | 3.9 | S | I/N | – | – | A | – | – | – | – | – | T | – | – | – | K | S |
ORF sequences of viral genomes from supernatants of passage cultures were determined by direct sequence analysis. Indicated are the numbers of viral passages and the day of the passage experiments at which supernatants were obtained, as well as the peak infectivity titers of the respective passage experiments.
Amino acid identity at the respective position of the polyprotein encoded by the plasmid or by viral genomes recovered from infected cell cultures. Positions with quasispecies are written with two capital letters in the case of a 50/50 quasispecies and with a capital letter/lowercase letter in the case of a dominant/minor sequence. At the positions included in this table, coding nucleotide changes occurred at least as 50/50 quasispecies in at least one ORF. –, identity with the sequence of the polyprotein encoded by the plasmid. Highlighted positions are engineered mutations.
Data in parentheses are the amino acid position of the polyprotein sequence encoded by the 3a(S52) plasmid/absolute amino acid reference number in accordance with the H77 (GenBank accession no. AF009606) reference amino acid sequence (2).
The 1st and consecutive 2nd viral passage were from the same transfection.
NA, not applicable
Development of efficient semi-FL 3a(S52) and 1a(TN) infectious culture systems based on adaptive NS3H mutation I1425T.
Direct ORF sequencing of 1st-passage 3a(S52) identified three nucleotide changes, coding for Y788H (p7), I1425T (NS3H), and D2410G (NS5A), which occurred in at least an estimated 50% of viral genomes (Tables 1 and 2). In reverse genetic studies, we introduced these changes individually in 3a(S52). The 3a(S52) recombinant with the I1425T change [3a(S52)I1425T] infected most culture cells from day 35 posttransfection, reaching 3.2 log10 FFU/ml, while the other 2 recombinants did not spread (Fig. 1C). First- and 2nd-passage 3a(S52)I1425T, with peak titers of 3.5 and 4.4 log10 FFU/ml, respectively, had acquired 4 common changes coding for R9S (core), V414A (E2), E2412K (NS5A), and P2915S (NS5B) (Tables 1 and 2). The 3a(S52)I1425T recombinant with these additional changes spread to most culture cells on day 6, reaching 3.8 log10 FFU/ml (Fig. 1C). First-passage virus reached 3.9 log10 FFU/ml and did not acquire dominant coding nucleotide changes (Tables 1 and 2). This efficient JFH1-based 3a(S52) semi-FL recombinant represents the most advanced reported genotype 3a infectious culture system.
We introduced I1425T in 1a(TN), leading to viral spread to most culture cells from day 36 with infectivity titers of 3.7 log10 FFU/ml (Fig. 1D). First-, 2nd-, and 3rd-passage 1a(TN)I1425T, with peak titers of 3.9, 4.4, and 5.0 log10 FFU/ml, respectively, had 4 common changes coding for Y834C (NS2), Q1060H (NS3P), R1088K (NS3P), and R2028S (NS5A) (Tables 3 and 4). The 1a(TN)I1425T recombinant with these additional changes spread in two independent transfections to most cells on days 10 and 4, respectively, reaching 3.2 and 4.2 log10 FFU/ml, respectively (Fig. 1D). First-passage viruses reached 4.2 and 3.9 log10 FFU/ml, respectively; virus recovered from one passage did not acquire coding nucleotide changes, while virus from another passage acquired one coding change in NS4B (Tables 3 and 4). Subsequently, we found that the 1a(H77) semi-FL recombinant with the 5 mutations adapting 1a(TN), including I1425T, replicated, while a semi-FL recombinant with only I1425T did not replicate. Spread to most culture cells was observed on day 99 with a peak titer of 3 log10 FFU/ml. First-passage virus with a peak titer of 4.0 log10 FFU/ml had acquired 5 additional coding changes (see Table S6 in the supplemental material). Thus, with the semi-FL approach it was also possible to develop efficient genotype 1a culture systems.
Table 3.
Coding nucleotide changes of 1a(TN) semi-FL recombinant recovered from Huh7.5 cellsa
| 1a(TN) virus | Passage (day) | Infectivity titer (log10 FFU/ml) | Nucleotide identityb at the indicated nucleotide positionsc in the following HCV gene: |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Core (811/811) | E1 (1384/1384) | NS2 (2842/2842) | NS2 (2997/2997) | NS3P (3521/3521) | NS3P (3604/3604) | NS3H (4615/4615) | NS4A (5380/5380) | NS4B (5983/5983) | NS5A (6423/6423) | |||
| Plasmid encoded | NAf | NA | T | T | A | T | G | G | T | T | A | C |
| Cell culture derived | ||||||||||||
| Recombinant with indicated amino acid change(s) | ||||||||||||
| I1425Td | 1st (6) | 3.9 | T/c | – | G | – | C | A | C | – | – | A |
| I1425Td | 2nd (8) | 4.4 | – | – | G | C | C | A | C | – | – | A |
| I1425Td | 3rd (11) | 5.0 | C/T | G/T | G | – | C | A | C | C/T | – | A |
| Y834C, Q1060H, R1088K, I1425T, R2028S | ||||||||||||
| Expt 1e | 1st (15) | 4.2 | – | – | G | – | C | A | C | – | – | A |
| Expt 2e | 1st (10) | 3.9 | – | – | G | – | C | A | C | – | C | A |
See footnote a of Table 1.
See footnote b of Table 1.
Data in parentheses are the nucleotide position of the HCV sequence of the 1a(TN) plasmid/absolute nucleotide reference number in accordance with the H77 (GenBank accession no. AF009606) reference nucleotide sequence (2).
The 1st and consecutive 2nd and 3rd viral passages were from the same transfection.
The 1st passages were from two independent transfections.
NA, not applicable.
Table 4.
Amino acid changes of 1a(TN) semi-FL recombinant recovered from Huh7.5 cellsa
| 1a(TN) virus | Passage (day) | Infectivity titer (log10 FFU/ml) | Amino acid identityb at the indicated amino acid positionsc in the following HCV gene: |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Core (157/157) | E1 (348/348) | NS2 (834/834) | NS2 (886/886) | NS3P (1060/1060) | NS3P (1088/1088) | NS3H (1425/1425) | NS4A (1680/1680) | NS4B (1881/1881) | NS5A (2028/2028) | |||
| Plasmid encoded | NAf | NA | V | I | Y | F | Q | R | I | V | N | R |
| Cell culture derived | ||||||||||||
| Recombinant with indicated amino acid change(s) | ||||||||||||
| I1425Td | 1st (6) | 3.9 | V/a | – | C | – | H | K | T | – | – | S |
| I1425Td | 2nd (8) | 4.4 | – | – | C | L | H | K | T | – | – | S |
| I1425Td | 3rd (11) | 5.0 | A/V | S/I | C | – | H | K | T | A/V | – | S |
| Y834C, Q1060H, R1088K, I1425T, R2028S | ||||||||||||
| Expt 1e | 1st (15) | 4.2 | – | – | C | – | H | K | T | – | – | S |
| Expt 2e | 1st (10) | 3.9 | – | – | C | – | H | K | T | – | T | S |
See footnote a of Table 2.
See footnote b of Table 2.
Data in parentheses are the amino acid position of the polyprotein sequence encoded by the 1a(TN) plasmid/absolute amino acid reference number in accordance with the H77 (GenBank accession no. AF009606) reference amino acid sequence (2).
The 1st and consecutive 2nd and 3rd viral passages were from the same transfection.
The 1st passages were from two independent transfections.
NA, not applicable.
Introduction of I1425T did not improve the growth characteristics of the 1b(J4) and 4a(ED43) semi-FL recombinants following transfection of Huh7.5 cells (data not shown).
Viruses with genotype 1a-, 2a-, and 3a-specific NS3P and NS5A showed differential sensitivity to NS3P and NS5A inhibitors.
To determine the sensitivity of viruses to the NS3P inhibitor asunaprevir and the NS5A inhibitor daclatasvir, we used virus stocks of semi-FL 1a(TN), 1a(H77), and 3a(S52), which were developed in this study; of full-length 2a(J6cc), which we developed recently (27); and of reference virus 2a(J6/JFH1) (11). As has been observed for other protease inhibitors (14), 3a(S52) was the least sensitive to asunaprevir (EC50, 1,514 nM), being 3.6-fold less sensitive than 2a(J6/JFH1). The sensitivity of 2a(J6cc) to asunaprevir was comparable to that of 2a(J6/JFH1), while the two genotype 1a viruses showed 7.0- and 11.1-fold higher sensitivities (Fig. 2A and C). For daclatasvir, the two 1a viruses also showed the highest sensitivity, with EC50 values being 2.1- and 3.1-fold lower than those for 2a(J6/JFH1) (Fig. 2B and C). The 3a(S52) recombinant was 9-fold less sensitive than 2a(J6/JFH1). We previously reported the low sensitivity of 2a(J6cc), which is 36.7-fold less sensitive than 2a(J6/JFH1) (27). Daclatasvir EC50 values were comparable to those reported for J6/JFH1-based recombinants with genotype-specific NS5A (15).
Fig 2.
Recombinants with NS3P and NS5A of genotypes 1a, 2a, and 3a showed differential sensitivity to the NS3P inhibitor asunaprevir and the NS5A inhibitor daclatasvir. Huh7.5 cell cultures in 96-well plates were infected with the indicated recombinants and treated with serial dilutions of asunaprevir (A) or daclatasvir (B) as described in Materials and Methods. Following incubation, for each well the number of HCV-infected cells was determined by immunostaining. The percentage of HCV-positive cells compared with the number of HCV-positive cells in untreated controls is shown. Values are means of 3 replicates with SEMs. After logarithmic transformation of x values, sigmoidal dose-responses [y = top/(1 + 10[log10 EC50 − x] · Hill slope)] were fitted to the data. (C) Median EC50 values with 95% confidence intervals as well as the median fold EC50 differences compared to the EC50 for the 2a(J6/JFH1) reference virus with 95% confidence intervals were calculated from experiments whose results are shown in panels A and B as well as additional replicate experiments, as described in Materials and Methods. P values were calculated as described in Materials and Methods. P values of <0.0001, shown in bold, were considered significant. NA, not applicable. Viruses used for treatment were 2a(J6/JFH1) 3rd-passage virus (the ORF sequence was confirmed), 1a(TN) 2nd-passage virus (Tables 3 and 4), 1a(H77) 1st-passage virus (see Table S6 in the supplemental material), 2a(J6cc) J6_LSGΔ33U+F776S/P1100L/N1931S 1st-passage virus for asunaprevir treatment (see Table 3 in reference 27) and 2a(J6cc) J6_LSGΔ33U 4th-passage virus for daclatasvir treatment (see Table 3 in reference 27), and 3a(S52) 2nd-passage virus (Tables 1 and 2).
The combination of asunaprevir and daclatasvir was effective against infection with genotype 1a, 2a, and 3a recombinants in vitro.
To evaluate the efficacy of combination treatment, cultures infected with 1a(TN), 1a(H77), 2a(J6/JFH1), 2a(J6cc), and 3a(S52) were treated with the NS3P inhibitor asunaprevir and the NS5A inhibitor daclatasvir by application of a cross-titration scheme. In nontreated cultures, viruses spread to ≥80% of cells on days 3 to 6 postinfection (Fig. 3). Under single treatment with the NS5A inhibitor, regardless of the concentration, all recombinants spread to ≥80% of cells between days 3 and 28 (Fig. 3). In contrast, single treatment with the NS3P inhibitor resulted in on-treatment virologic suppression for 1a(TN), 1a(H77), and 2a(J6cc) at the highest concentration applied. Overall, combination therapy with comparatively low fold EC50s resulted in on-treatment virologic suppression (Fig. 3). Thus, 1a(TN) was suppressed using 4- or 20-fold the EC50 of the NS3P inhibitor in combination with ≥4-fold the EC50 of the NS5A inhibitor (Fig. 3A). For 1a(H77), combination therapy was less efficient; virologic suppression was observed only in the cultures treated with 20- and 100-fold the EC50s of the NS3P and NS5A inhibitors, respectively, or with 100-fold the EC50 of the NS3P inhibitor (Fig. 3B). Comparatively low fold EC50s resulted in suppression of 2a and 3a viruses, even though overall they showed significantly (2- to 118-fold) lower sensitivity to both inhibitors in single treatments than the two 1a viruses (Fig. 2 and 3C to E). The lowest fold EC50s of the NS3P and NS5A inhibitors resulting in virologic suppression were 4 and 20, respectively, or 20 and 0.8, respectively, for 2a(J6/JFH1), 0.8 and 100, respectively, or 4 and 4, respectively, for 2a(J6cc), and 4 and 4, respectively, for 3a(S52) (Fig. 3C to E).
Fig 3.
Long-term combination treatment of recombinants with genotype 1a-, 2a-, and 3a-specific NS3P and NS5A with the protease inhibitor asunaprevir and the NS5A inhibitor daclatasvir. Cultures were infected with the recombinants designated in the subheadings and were treated with the indicated fold EC50s of daclatasvir and asunaprevir, using a cross-titration scheme, as described in Materials and Methods. Daclatasvir concentrations are shown on the y axis, while asunaprevir concentrations are shown on the x axis. Cultures whose results are shown in panel A were treated in 3 different experiments; cultures whose results are shown in panels B to E were treated in the same experiment, respectively. For cultures with viral escape, the z axis and orange bars indicate the day on which ≥80% of cells were infected. ⧫, cultures with on-treatment virologic suppression; •, not done; *, not done due to cytotoxicity caused by asunaprevir, which led to termination of the cell cultures when used at doses of ≥30,000 nM. Viruses used for treatment were 2a(J6/JFH1) 3rd-passage virus (the ORF sequence was confirmed), 1a(TN) 2nd-passage virus (Tables 3 and 4), 1a(H77) 1st-passage virus (see Table S6 in the supplemental material), 2a(J6cc) 1st-passage virus J6_LSGΔ33U+F776S/P1100L/N1931T (see Table 3 in reference 27), and 3a(S52) 2nd-passage virus (Tables 1 and 2).
To confirm that viral spread in treated cultures could have resulted from viral escape with acquisition of resistance mutations, we sequenced NS3P and NS5A domain I of viruses from selected cultures (Fig. 3; Table 5). Nontreated cultures did not acquire mutations. In cultures treated with the NS3P inhibitor, we found mutations at NS3P amino acid positions 155, 168, and 170 for 1a(TN); 155 for 1a(H77); 43, 122, 156, 158, and 168 for 2a(J6/JFH1); 72, 168, and 174 for 2a(J6cc); and 166 and 168 for 3a(S52) (Table 5) (putative resistance mutations are given with H77 relative reference position numbers). In cultures treated with the NS5A inhibitor, mutations were found at NS5A amino acid positions 28 and 30 for 1a(TN); 28, 30, 32, and 93 for 1a(H77); 28 and 140 for 2a(J6/JFH1); 24, 28, 31, 34, and 85 for 2a(J6cc); and 24 and 93 for 3a(S52) (Table 5). S24A, also found in 3a(S52) not treated with daclatasvir, could be a culture adaptive change. Under treatment with inhibitors at 0.8-fold the EC50, 3a(S52), treated with either inhibitor, and 2a(J6cc), treated with the NS3P inhibitor, did not acquire putative resistance mutations in the targeted region. Overall, in cultures showing viral spread under treatment, mutations indicative of viral escape were detected.
Table 5.
Mutations in viral genomes under single or combination treatment with HCV NS3P inhibitor asunaprevir and NS5A inhibitor daclatasvira
| Virus and fold EC50 for NS3P/NS5A inhibitor | Day | Nucleotide identityb at the indicated positionsc in the following HCV gene: |
||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| NS3P |
NS5A domain I |
|||||||||||||||||||||||||
| 3546/1069/43d | 3634/1098/72 | 3784/1148/122d | 3883/1181/155d | 3886/1182/156d | 3891/1184/158d | 3915/1192/166 | 3921/1194/168d | 3922/1194/168d | 3923/1194/168d | 3928/1196/170d | 3940/1200/174 | 6265/1975/3 | 6327/1996/24 | 6340/2000/28d | 6345/2002/30d | 6346/2002/30d | 6347/2002/30d | 6350/2003/31d | 6352/2004/32d | 6357/2006/34 | 6511/2057/85 | 6534/2065/93d | 6535/2065/93d | 6675/2112/140 | ||
| 1a(TN) | T | T | G | G | C | G | G | G | A | C | T | A | C | A | T | C | A | A | G | C | A | G | T | A | T | |
| 0/0 | 8 | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – |
| 0/100 | 15 | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | C | – | – | – | – | – | – | – |
| 0.8/100 | 28 | – | – | – | G/a | – | – | – | – | – | – | T/c | – | – | – | – | – | – | C | – | – | – | – | – | – | – |
| 20/0 | 29 | – | – | – | – | – | – | – | – | C | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – |
| 20/0.8 | 43 | – | – | – | – | – | – | – | C | – | – | – | – | – | – | C/t | C/a | – | – | – | – | – | – | – | – | – |
| Amino acid change | NAg | NA | NA | R-Ke | NA | NA | NA | D-He | D-Ae | NA | I-Te | NA | NA | NA | M-Te | Q-Ke | NA | Q-He | NA | NA | NA | NA | NA | NA | NA | |
| 1a(H77) | T | T | G | G | C | G | G | G | A | C | T | A | C | A | T | C | A | A | G | C | A | G | T | A | T | |
| 0/0 | 6 | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – |
| 0/100 | 10 | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | C/a/t | – | – | – | – | T/c | – | – |
| 4/100 | 24 | – | – | – | A | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | G | – |
| 20/0 | 15 | – | – | – | A | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – |
| 20/20 | 34 | – | – | – | A | – | – | – | – | – | – | – | – | – | – | T/c | – | A/g | – | – | T/C | – | – | – | – | – |
| Amino acid change | NA | NA | NA | R-Ke | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | M-Te | NA | Q-Re | Q-He | NA | P-Le | NA | NA | Y-He | Y-Ce | NA | |
| 2a(J6/JFH1) | T | C | A | G | C | G | T | G | A | T | T | C | C | A | T | A | A | G | G | C | C | A | T | A | A | |
| 0/0 | 6 | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – |
| 0/100 | 8 | – | – | – | – | – | – | – | – | – | – | – | – | – | – | C | – | – | – | – | – | – | – | – | – | – |
| 0.8/100 | 22 | C/T | – | – | – | – | – | – | – | – | G/T | – | – | – | – | C | – | – | – | – | – | – | – | – | – | – |
| 20/0 | 31 | G | – | G | – | – | A | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – |
| 4/4 | 41 | – | – | – | – | G | – | – | – | – | – | – | – | – | – | C | – | – | – | – | – | – | – | – | – | G |
| Amino acid change | F-L/Ve | NA | K-Re | NA | A-Ge | V-M | NA | NA | NA | D-Ee | NA | NA | NA | NA | F-S | NA | NA | NA | NA | NA | NA | NA | NA | NA | I-V | |
| 2a(J6cc) | T | C | A | G | C | G | T | G | A | T | T | C | C | A | T | A | A | G | G | C | C | A | T | A | G | |
| 0/0 | 6 | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – |
| 0/100 | 29 | – | – | – | – | – | – | – | – | – | – | – | – | – | – | C | – | – | – | – | – | C/a | A/g | – | – | – |
| 0.8/20 | 38 | – | – | – | – | – | – | – | – | – | – | – | – | – | – | C | – | – | – | G/a | – | – | – | – | – | – |
| 4/0 | 17 | – | – | – | – | – | – | – | – | – | G/a | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – |
| 4/0.8 | 43 | – | T/c | – | – | – | – | – | – | – | A | – | C/g | – | G/A | – | – | – | – | – | – | – | – | – | – | – |
| Amino acid change | NA | T-M | NA | NA | NA | NA | NA | NA | NA | D-Ee | NA | T-R | NA | T-A | F-S | NA | NA | NA | M-I | NA | L-I | Q-R | NA | NA | NA | |
| 3a(S52) | T | T | C | G | C | G | G | C | A | G | T | C | A | T | T | G | C | A | C | C | C | A | T | A | T | |
| 0/0 | 6 | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – |
| 0/100 | 10 | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | C/A | – | – |
| 0.8/100 | 13 | – | – | – | – | – | – | – | – | – | – | – | – | – | G/T | – | – | – | – | – | – | – | – | C | – | – |
| 4/0 | 13 | – | – | – | – | – | – | A/G | – | G/A | – | – | – | A/g | T/g | – | – | – | – | – | – | – | – | – | – | – |
| 4/0.8 | 20 | – | – | – | – | – | – | – | – | G | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – |
| Amino acid change | NA | NA | NA | NA | NA | NA | A-T | NA | Q-R | NA | NA | NA | D-Gf | S-A | NA | NA | NA | NA | NA | NA | NA | NA | Y-H/Ne | NA | NA | |
NS3P and NS5A domain I were sequenced for 1a(TN), 1a(H77), 2a(J6/JFH1), 2a(J6cc), and 3a(S52) viruses in supernatants from the peak of infection from cultures treated with various concentrations of the NS3P and NS5A inhibitors (Fig. 3). For each virus, the fold EC50 and the day postinfection at which the supernatant was obtained are indicated.
For each recombinant, the original nucleotide, found at the respective position of the plasmid, is given. For each culture virus treated with the specified fold EC50 of inhibitors, the nucleotide found at the respective position is given. –, identity with the original plasmid sequence. Positions with quasispecies are written with two capital letters in the case of a 50/50 quasispecies and with a capital letter/lowercase letter(s) in the case of a dominant/minor sequence. At the positions included in this table, coding nucleotide changes occurred at least as a minor quasispecies in at least one sequenced culture virus. For the coding nucleotide changes observed, the resulting amino acid change is given.
The positions are indicated by absolute nucleotide reference numbers in accordance with the H77 (GenBank accession no. AF009606) reference nucleotide sequence/absolute amino acid reference numbers in accordance with the H77 (GenBank accession no. AF009606) reference amino acid sequence/relative amino acid reference numbers in accordance with the H77 (GenBank accession no. AF009606) reference amino acid sequence (2).
Changes at these amino acid positions were described to confer resistance to genotype 1 isolates.
The mutated amino acid was described to confer resistance to genotype 1 isolates.
D3G adapted the JFH1-based recombinant with S52-specific NS5A (15).
NA, not applicable.
Asunaprevir and daclatasvir show synergism at higher drug concentrations.
To determine the type of interaction between the studied inhibitors, we carried out synergy assays using the method of Chou and Talalay (25, 26). The 1a(TN) (Fig. 4A and B), 2a(J6/JFH1) (Fig. 4C and D), and 3a(S52) (Fig. 4E and F) viruses were treated with asunaprevir and daclatasvir singly or in combination using 2-fold dilution series with concentration ranges based on EC50 values previously determined for the respective drugs and viruses (Fig. 2). For combination treatments, a fixed concentration ratio, also determined by use of the EC50 values, was used. Inhibition percentages were determined and entered as fractional effect (Fa) values into CompuSyn software as described in Materials and Methods. Fa values were plotted against the concentration of single drugs or drug combinations; representative dose-effect curves for single and combination treatments are shown in Fig. 4A, C, and E. Linear correlation coefficients (r values) for all curves shown were >0.95, indicating a high goodness of fit. Further, combination index (CI) values were calculated, and values between 0 and 2 were plotted against Fa values; representative Fa-CI plots are shown in Fig. 4B, D, and F. CI values at key Fa values as well as CI values for the actual experimental data points are shown as insets in Fig. 4B, D, and F. In general, CI values below 1 indicate synergism, while CI values above 1 indicate antagonism.
Fig 4.
Synergy quantification for combination treatment of genotype 1a, 2a, and 3a recombinants with asunaprevir and daclatasvir. Huh7.5 cell cultures in 96-well plates were infected with 1a(TN) (A, B), 2a(J6/JFH1) (C, D), and 3a(S52) (E, F) and treated with 2-fold dilutions of asunaprevir and daclatasvir either singly or in combination, as described in Materials and Methods. Following incubation, the number of HCV-infected cells in each well was determined by immunostaining. The percentage of HCV-positive cells compared with the number of HCV-positive untreated controls was calculated and inhibition percentages were entered in CompuSyn software as Fa values with 0.01 < Fa < 0.99. For each treatment experiment, 5 to 7 data points were entered. Dose-effect curves (A, C, E) were calculated using CompuSyn, plotting the entered Fa values against the entered dose values. For combination treatment, the combined treatment doses were entered. Each data point represents the mean of 3 to 4 replicates; r values for all curves were >0.95. CI values were calculated using CompuSyn, and CI values with 0 < CI < 2 were plotted against Fa values, generating Fa-CI plots (B, D, F). The black tables in panels B, D, and F show calculated CI values at key Fa values (representing 25, 50, 75, and 90% inhibition) derived from the plotted curve. The blue tables in panels B, D, and F show calculated CI values at the Fa values obtained for the actual experimental points. In these tables, to the far right, the fold EC50 of the asunaprevir and daclatasvir combination treatment which resulted in the respective Fa and CI values is given. The verbal categories for the CI values reported are listed in Materials and Methods. Viruses used for treatment were 2a(J6/JFH1) 3rd-passage virus (the ORF sequence was confirmed), 1a(TN) 2nd-passage virus (Tables 3 and 4), and 3a(S52) 2nd-passage virus (Tables 1 and 2).
For 1a(TN), the Fa-CI plot indicated synergism in the higher dose range at Fa values of at least 0.6, with moderate synergism determined at an Fa of 0.75 and synergism determined at an Fa of 0.9 (Fig. 4B). CI values for the 3 actual experimental points in this range indicated slight to moderate synergism or synergism. A nearly additive effect was predicted in an intermediate dose range (resulting in Fa values of 0.3 to 0.55), while antagonism was predicted at lower doses (resulting in Fa values of ≤0.25). The 3 actual experimental points in this lower dose range indicated nearly additive to antagonistic effects (Fig. 4B).
For 2a(J6/JFH1), overall similar effects were observed (Fig. 4D). However, the Fa-CI plot indicated synergism at Fa values of ≥0.8, with moderate synergism at an Fa value of 0.9. CI values for the 2 actual experimental points in this range indicated moderate synergism and synergism. Nearly additive effects were predicated at Fa values of 0.3 to 0.75; CI values for the 3 actual experimental points in this range indicated moderate antagonism and synergism. At lower Fa values, antagonism was predicted (Fig. 4D).
For 3a(S52), synergism was predicted at an Fa of at least 0.95 (Fig. 4F). CI values for the 2 actual experimental points in this range indicated moderate synergism and synergism. For Fa values of 0.8 to 0.95, additive effects were predicted, while at lower Fa values, antagonism was predicted. The 4 actual experimental points in the latter range indicated antagonistic to strongly antagonistic effects (Fig. 4F).
Overall, for the tested viruses, synergism was predicted in the high dose range, while additive to antagonistic effects were found at lower doses. The fold EC50s required to elicit synergistic effects were comparatively low for 1a(TN), intermediate for 2a(J6/JFH1), and relatively high for 3a(S52).
DISCUSSION
Here we report on the development of efficient semi-FL genotype 1a and 3a HCV recombinants relying only on JFH1 NS3H, NS5B, and the 3′ UTR, thus expressing genotype-specific NS3P and NS5A, major targets of DAAs (3). While 1a recombinants showed the greatest sensitivity to NS3P and NS5A inhibitors, combination treatment with EC50-adjusted concentrations resulted in on-treatment virologic suppression of all treated genotype 1a, 2a, and 3a recombinants. Finally, we found that the studied inhibitors had synergistic effects at higher drug concentrations, which were also observed in vivo.
To implement newly developed DAAs in efficient HCV treatment regimens, genotype-specific drug efficacies, genetic correlates of viral resistance, and efficient drug combinations need to be determined. To achieve these determinations in a timely and systematic manner, culture systems that express genotype-specific target proteins are required. Replicons recapitulate only the partial viral life cycle, and several mutations selected in replicons (8, 9) were not permissive in the context of the complete viral life cycle in vitro and in vivo (13, 28, 29). Here we developed genotype 1a and 3a semi-FL recombinants, which allowed study of combination treatment with lead compound NS3P and NS5A inhibitors in the context of the complete viral life cycle. It should be noted that the semi-FL approach was recently used to also develop an efficient 2b recombinant (30). While full-length culture systems have been developed for genotype 1a, 2a, and 2b strains (12, 27, 31, 32), the 3a semi-FL system reported here extends a previously developed JFH1-based 5′ UTR-NS2 recombinant (22) to also include the NS3P, NS4A, NS4B, and NS5A regions. Thus, this is the most advanced 3a culture system reported and represents an important step toward developing full-length culture systems for this clinically important genotype.
To demonstrate the applicability of the developed systems, we chose to study combination treatment with lead compound NS3P inhibitor asunaprevir and NS5A inhibitor daclatasvir, recently shown to be effective in genotype 1-infected patients (6, 7). In this study, these inhibitors showed the greatest efficacy against genotype 1, for which most initial DAAs were designed (Fig. 2). In line with these observations, initial macrocyclic protease inhibitors such as asunaprevir were shown to bind genotype 2 and 3 proteases less efficiently than genotype 1 proteases, resulting in comparatively high EC50s in cell culture-based assays (Fig. 2) (4, 33, 34). Further reverse genetic studies could identify determinants contributing to the differential sensitivity of the treated viruses. At amino acid positions known to confer resistance of genotype 1 isolates to NS3P inhibitors (3), sequence variation among the recombinants treated in this study was found at positions 36, 80, 168, and 170 (alignments are found in reference 14). Variation at residue 80 was implicated in the reduced sensitivity of genotype 2 (35). The low sensitivity of 3a(S52) is supposed to be due to genotype 3a-specific Q168 (14, 33, 35). At positions associated with genotype 1 resistance to the NS5A inhibitor, sequence variation was found among treated recombinants at positions 28, 30, 31, 37, 54, and 58 (alignments are found in reference 15). Variations at amino acids 31 and 92 proved responsible for the low sensitivity of 2a(J6) and 3a(S52) NS5A recombinants, respectively (15).
For daclatasvir, EC50 values were ∼3 log10 units lower than EC50 values for asunaprevir. However, in long-term treatment experiments, for daclatasvir, despite its high potency, the barrier to resistance appeared to be comparatively low, highlighting the need for combination therapy. Daclatasvir resistance mutations might be easier to acquire, and/or viruses with these mutations might be characterized by higher fitness as well as a higher level of resistance. Thus, in genotype 1a replicons, many daclatasvir resistance mutations conferred >10,000-fold resistance (36), while most asunaprevir resistance mutations conferred <100-fold resistance (37). We observed that recombinants of different HCV genotypes and isolates differed somewhat in their capacity to escape EC50-adjusted equipotent drug concentrations. For example, at 20 times the EC50 of asunaprevir, all 2a(J6cc) cultures showed on-treatment virologic suppression, while most 1a(H77) cultures showed viral escape (Fig. 3). A reason for such differences might be that the capacity of different recombinants to acquire resistance mutations depended on the sequence context. Further, the effect of acquired resistance mutations might also depend on the sequence context. Such observations were previously made in in vitro as well as in vivo studies (15, 38).
Recently, a sustained viral response was achieved by combination of the BMS NS3P and NS5A inhibitors in genotype 1-infected patients (6, 7). Here, we showed that combination treatment with EC50-adjusted concentrations resulted in on-treatment virologic suppression of genotype 1a, 2a, and 3a recombinants in a concentration-dependent manner (Fig. 3). Using 60 mg daclatasvir per day, the peak (maximum) plasma concentration (Cmax) reported in patients was ∼2,025 nM; thus, calculated Cmax/EC50 values are 32,885 and 50,625 for 1a(TN) and 1a(H77), respectively; 15,794 and 431 for 2a(J6/JFH1) and 2a(J6cc), respectively; and 1,748 for 3a(S52) (Fig. 2) (39; http://www.natap.org/2010/AASLD/AASLD_64.htm). Using 200 mg asunaprevir twice daily, the Cmax value reported in patients was ∼118 nM. Thus, Cmax/EC50 values were 2 and 3.2 for 1a(TN) and 1a(H77), respectively; 0.3 for 2a(J6/JFH1) as well as 2a(J6cc); and 0.1 for 3a(S52) (Fig. 2) (39; http://www.natap.org/2010/AASLD/AASLD_64.htm). Given the efficient distribution of these drugs to the liver, for daclatasvir, liver concentrations of >100 times the EC50 should easily be achievable for the tested genotype 1a, 2a, and 3a viruses (Fig. 2) (5). For asunaprevir, with a reported liver/plasma ratio of ≥40, liver concentrations can be expected to be at least 80-fold greater than the EC50 of the tested 1a viruses but only approximately 10-fold greater than the EC50 of the tested 2a viruses and 3-fold greater than the EC50 of 3a(S52) (Fig. 2) (4). Our in vitro data indicate that asunaprevir, used at these fold EC50s in combination with daclatasvir, contributed to suppression of infection even with 2a and 3a viruses (Fig. 3). Further, our synergy studies indicate that at these fold EC50s, synergistic effects could occur in combination treatment (Fig. 4). It should, however, be noted that in vivo effective drug concentrations could be lower due to drug serum protein binding and that in vivo EC50 values might differ from in vitro EC50 values, which might limit the value of these calculations. Also, given the suboptimal efficacy of especially initial protease inhibitors against genotypes 2a and 3a, these drugs might eventually not be used for treatment of patients infected with these genotypes. We believe that the developed genotype 1a, 2a, and 3a recombinants could play an important role for testing of the efficacy of newer inhibitors with increased potency and broad activity also against genotypes 2a and 3a and combinations of these (3, 34). However, due to differences between the developed in vitro assay with frequent cell splitting and the situation in the human liver, clinical studies will eventually have to show if treatment combinations with promising in vitro efficacy also result in a sustained viral response in patients.
Mutations for resistance to asunaprevir and daclatasvir were studied for genotype 1 in patients and replicons (3, 6, 7, 36, 37, 40). In contrast, little is known about genotype 2 and 3 resistance mutations. In this study, 1a viruses acquired mutations at positions described to confer resistance to genotype 1a, while 2a and 3a viruses acquired additional mutations (Table 5) (3, 36, 37). Changes at the following positions were previously not implicated in genotype 1 resistance: amino acids 72, 166, and 174 in NS3P and amino acids 24, 34, 85, and 140 in NS5A. Future studies will be required to systematically identify correlates of resistance of non-genotype 1 isolates.
In conclusion, we developed genotype 1a and 3a semi-FL recombinants and used them, together with previously developed genotype 2a recombinants, to study a combination of BMS NS3P and NS5A inhibitors in the context of the complete viral life cycle in vitro. Infection with 1a, 2a, and 3a recombinants with differential sensitivity to BMS NS3P and NS5A inhibitors was suppressed by combination treatment with the inhibitors at EC50-adjusted concentrations. This argues for individualized treatment regimens, requiring detailed knowledge on determinants of resistance. Our findings also highlight the benefit of combining antivirals with different properties such as daclatasvir with high potency but a low barrier to resistance and asunaprevir with a lower potency but a higher barrier to resistance. We further found that the studied inhibitors had synergistic effects at drug concentrations reported in vivo. Eventually, it will be important to identify drugs with a high potency, preferably against all genotypes, and a high barrier to resistance, which is apparently the case for nucleoside polymerase HCV inhibitors. The developed 1a and 3a semi-FL recombinants appear to be valuable tools for preclinical studies of drug combinations. Thus, various combinations of protease and NS5A inhibitors could be evaluated. Further, the developed recombinants will aid with the development of antivirals targeting additional HCV proteins, such as NS4A and NS4B. They further enable novel functional studies of genotype-specific genome regions.
Supplementary Material
ACKNOWLEDGMENTS
We thank Nathalie Uzcategui and Anna-Louise Sørensen for laboratory assistance, Steen Ladelund for statistical advice, Jens Ole Nielsen and Ove Andersen for support (Copenhagen University Hospital, Hvidovre, Denmark), Charles Rice (Rockefeller University) and Robert Purcell (National Institutes of Health) for reagents, and CTL Europe GmbH for customized software.
This study was supported by grants from Copenhagen University Hospital, Hvidovre, Denmark (to J.M.G., T.K.H.S., and S.B.N.S.), the Region H Foundation (to J.M.G. and J.B.), The Lundbeck Foundation (to J.M.G., T.K.H.S., and J.B.), The Novo Nordisk Foundation (to J.M.G., Y.-P.L, and J.B.), The Danish Council for Independent Research, Medical Science (to J.B.), The A. P. Møller and Chastine Mc-Kinney Møller Foundation (to J.M.G., T.K.H.S., and J.B.), and The Danish Cancer Society (to J.M.G. and J.B.) and Ph.D. stipends from the Faculty of Health Sciences, University of Copenhagen (to T.K.H.S. and S.B.N.S.).
Footnotes
Published ahead of print 28 December 2012
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.02164-12.
REFERENCES
- 1. Gottwein JM, Bukh J. 2008. Cutting the Gordian knot—development and biological relevance of hepatitis C virus cell culture systems. Adv. Virus Res. 71:51–133 [DOI] [PubMed] [Google Scholar]
- 2. Simmonds P, Bukh J, Combet C, Deleage G, Enomoto N, Feinstone S, Halfon P, Inchauspe G, Kuiken C, Maertens G, Mizokami M, Murphy DG, Okamoto H, Pawlotsky JM, Penin F, Sablon E, Shin IT, Stuyver LJ, Thiel HJ, Viazov S, Weiner AJ, Widell A. 2005. Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. Hepatology 42:962–973 [DOI] [PubMed] [Google Scholar]
- 3. Sarrazin C, Hezode C, Zeuzem S, Pawlotsky JM. 2012. Antiviral strategies in hepatitis C virus infection. J. Hepatol. 56(Suppl 1):S88–S100 [DOI] [PubMed] [Google Scholar]
- 4. McPhee F, Sheaffer AK, Friborg J, Hernandez D, Falk P, Zhai G, Levine S, Chaniewski S, Yu F, Barry D, Chen C, Lee MS, Mosure K, Sun LQ, Sinz M, Meanwell NA, Colonno RJ, Knipe J, Scola P. 2012. Preclinical profile and characterization of the hepatitis C virus NS3 protease inhibitor asunaprevir (BMS-650032). Antimicrob. Agents Chemother. 56:5387–5396 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Gao M, Nettles RE, Belema M, Snyder LB, Nguyen VN, Fridell RA, Serrano-Wu MH, Langley DR, Sun JH, O'Boyle DR, Lemm JA, Wang C, Knipe JO, Chien C, Colonno RJ, Grasela DM, Meanwell NA, Hamann LG. 2010. Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature 465:96–100 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. Lok AS, Gardiner DF, Lawitz E, Martorell C, Everson GT, Ghalib R, Reindollar R, Rustgi V, McPhee F, Wind-Rotolo M, Persson A, Zhu K, Dimitrova DI, Eley T, Guo T, Grasela DM, Pasquinelli C. 2012. Preliminary study of two antiviral agents for hepatitis C genotype 1. N. Engl. J. Med. 366:216–224 [DOI] [PubMed] [Google Scholar]
- 7. Chayama K, Takahashi S, Toyota J, Karino Y, Ikeda K, Ishikawa H, Watanabe H, McPhee F, Hughes E, Kumada H. 2012. Dual therapy with the nonstructural protein 5A inhibitor, daclatasvir, and the nonstructural protein 3 protease inhibitor, asunaprevir, in hepatitis C virus genotype 1b-infected null responders. Hepatology 55:742–748 [DOI] [PubMed] [Google Scholar]
- 8. Lohmann V, Korner F, Koch J, Herian U, Theilmann L, Bartenschlager R. 1999. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285:110–113 [DOI] [PubMed] [Google Scholar]
- 9. Saeed M, Scheel TK, Gottwein JM, Marukian S, Dustin LB, Bukh J, Rice CM. 2012. Efficient replication of genotype 3a and 4a hepatitis C virus replicons in human hepatoma cells. Antimicrob. Agents Chemother. 56:5365–5373 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Wakita T, Pietschmann T, Kato T, Date T, Miyamoto M, Zhao Z, Murthy K, Habermann A, Krausslich HG, Mizokami M, Bartenschlager R, Liang TJ. 2005. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat. Med. 11:791–796 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Lindenbach BD, Evans MJ, Syder AJ, Wolk B, Tellinghuisen TL, Liu CC, Maruyama T, Hynes RO, Burton DR, McKeating JA, Rice CM. 2005. Complete replication of hepatitis C virus in cell culture. Science 309:623–626 [DOI] [PubMed] [Google Scholar]
- 12. Yi M, Villanueva RA, Thomas DL, Wakita T, Lemon SM. 2006. Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells. Proc. Natl. Acad. Sci. U. S. A. 103:2310–2315 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. Pietschmann T, Zayas M, Meuleman P, Long G, Appel N, Koutsoudakis G, Kallis S, Leroux-Roels G, Lohmann V, Bartenschlager R. 2009. Production of infectious genotype 1b virus particles in cell culture and impairment by replication enhancing mutations. PLoS Pathog. 5:e1000475 doi:10.1371/journal.ppat.1000475 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Gottwein JM, Scheel TK, Jensen TB, Ghanem L, Bukh J. 2011. Differential efficacy of protease inhibitors against HCV genotypes 2a, 3a, 5a, and 6a NS3/4A protease recombinant viruses. Gastroenterology 141:1067–1079 [DOI] [PubMed] [Google Scholar]
- 15. Scheel TK, Gottwein JM, Mikkelsen LS, Jensen TB, Bukh J. 2011. Recombinant HCV variants with NS5A from genotypes 1–7 have different sensitivities to an NS5A inhibitor but not interferon-alpha. Gastroenterology 140:1032–1042 [DOI] [PubMed] [Google Scholar]
- 16. Murayama A, Date T, Morikawa K, Akazawa D, Miyamoto M, Kaga M, Ishii K, Suzuki T, Kato T, Mizokami M, Wakita T. 2007. The NS3 helicase and NS5B-to-3′X regions are important for efficient hepatitis C virus strain JFH-1 replication in Huh7 cells. J. Virol. 81:8030–8040 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. Sakai A, Takikawa S, Thimme R, Meunier JC, Spangenberg HC, Govindarajan S, Farci P, Emerson SU, Chisari FV, Purcell RH, Bukh J. 2007. In vivo study of the HC-TN strain of hepatitis C virus recovered from a patient with fulminant hepatitis: RNA transcripts of a molecular clone (pHC-TN) are infectious in chimpanzees but not in Huh7.5 cells. J. Virol. 81:7208–7219 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18. Yanagi M, Purcell RH, Emerson SU, Bukh J. 1997. Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee. Proc. Natl. Acad. Sci. U. S. A. 94:8738–8743 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19. Yanagi M, St Claire M, Shapiro M, Emerson SU, Purcell RH, Bukh J. 1998. Transcripts of a chimeric cDNA clone of hepatitis C virus genotype 1b are infectious in vivo. Virology 244:161–172 [DOI] [PubMed] [Google Scholar]
- 20. Yanagi M, Purcell RH, Emerson SU, Bukh J. 1999. Hepatitis C virus: an infectious molecular clone of a second major genotype (2a) and lack of viability of intertypic 1a and 2a chimeras. Virology 262:250–263 [DOI] [PubMed] [Google Scholar]
- 21. Gottwein JM, Scheel TK, Callendret B, Li YP, Eccleston HB, Engle RE, Govindarajan S, Satterfield W, Purcell RH, Walker CM, Bukh J. 2010. Novel infectious cDNA clones of hepatitis C virus genotype 3a (strain S52) and 4a (strain ED43): genetic analyses and in vivo pathogenesis studies. J. Virol. 84:5277–5293 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22. Li YP, Gottwein JM, Scheel TK, Jensen TB, Bukh J. 2011. MicroRNA-122 antagonism against hepatitis C virus genotypes 1–6 and reduced efficacy by host RNA insertion or mutations in the HCV 5′ UTR. Proc. Natl. Acad. Sci. U. S. A. 108:4991–4996 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23. Gottwein JM, Jensen TB, Mathiesen CK, Meuleman P, Serre SB, Lademann JB, Ghanem L, Scheel TK, Leroux-Roels G, Bukh J. 2011. Development and application of hepatitis C reporter viruses with genotype 1 to 7 core-nonstructural protein 2 (NS2) expressing fluorescent proteins or luciferase in modified JFH1 NS5A. J. Virol. 85:8913–8928 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24. Gottwein JM, Scheel TK, Hoegh AM, Lademann JB, Eugen-Olsen J, Lisby G, Bukh J. 2007. Robust hepatitis C genotype 3a cell culture releasing adapted intergenotypic 3a/2a (S52/JFH1) viruses. Gastroenterology 133:1614–1626 [DOI] [PubMed] [Google Scholar]
- 25. Chou TC, Talalay P. 1984. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul. 22:27–55 [DOI] [PubMed] [Google Scholar]
- 26. Chou TC. 2010. Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res. 70:440–446 [DOI] [PubMed] [Google Scholar]
- 27. Li YP, Ramirez S, Gottwein JM, Scheel TK, Mikkelsen L, Purcell RH, Bukh J. 2012. Robust full-length hepatitis C virus genotype 2a and 2b infectious cultures using mutations identified by a systematic approach applicable to patient strains. Proc. Natl. Acad. Sci. U. S. A. 109:E1101–E1110 doi:10.1073/pnas.1203829109 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28. Bukh J, Pietschmann T, Lohmann V, Krieger N, Faulk K, Engle RE, Govindarajan S, Shapiro M, St Claire M, Bartenschlager R. 2002. Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees. Proc. Natl. Acad. Sci. U. S. A. 99:14416–14421 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29. Scheel TK, Prentoe J, Carlsen TH, Mikkelsen LS, Gottwein JM, Bukh J. 2012. Analysis of functional differences between hepatitis C virus NS5A of genotypes 1–7 in infectious cell culture systems. PLoS Pathog. 8:e1002696 doi:10.1371/journal.ppat.1002696 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30. Murayama A, Kato T, Akazawa D, Sugiyama N, Date T, Masaki T, Nakamoto S, Tanaka Y, Mizokami M, Yokosuka O, Nomoto A, Wakita T. 2012. Production of infectious chimeric hepatitis C virus genotype 2b harboring minimal regions of JFH-1. J. Virol. 86:2143–2152 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31. Shimakami T, Welsch C, Yamane D, McGivern DR, Yi M, Zeuzem S, Lemon SM. 2011. Protease inhibitor-resistant hepatitis C virus mutants with reduced fitness from impaired production of infectious virus. Gastroenterology 140:667–675 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32. Date T, Kato T, Kato J, Takahashi H, Morikawa K, Akazawa D, Murayama A, Tanaka-Kaneko K, Sata T, Tanaka Y, Mizokami M, Wakita T. 2012. Novel cell culture-adapted genotype 2a hepatitis C virus infectious clone. J. Virol. 86:10805–10820 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33. Thibeault D, Bousquet C, Gingras R, Lagace L, Maurice R, White PW, Lamarre D. 2004. Sensitivity of NS3 serine proteases from hepatitis C virus genotypes 2 and 3 to the inhibitor BILN 2061. J. Virol. 78:7352–7359 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34. Summa V, Ludmerer SW, McCauley JA, Fandozzi C, Burlein C, Claudio G, Coleman PJ, DiMuzio JM, Ferrara M, Di Filippo M, Gates AT, Graham DJ, Harper S, Hazuda DJ, McHale C, Monteagudo E, Pucci V, Rowley M, Rudd MT, Soriano A, Stahlhut MW, Vacca JP, Olsen DB, Liverton NJ, Carroll SS. 2012. MK-5172, a selective inhibitor of hepatitis C virus NS3/4a protease with broad activity across genotypes and resistant variants. Antimicrob. Agents Chemother. 56:4161–4167 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35. Tong X, Guo Z, Wright-Minogue J, Xia E, Prongay A, Madison V, Qiu P, Venkatraman S, Velazquez F, Njoroge FG, Malcolm BA. 2006. Impact of naturally occurring variants of HCV protease on the binding of different classes of protease inhibitors. Biochemistry 45:1353–1361 [DOI] [PubMed] [Google Scholar]
- 36. Wang C, Huang H, Valera L, Sun JH, O'Boyle DR, II, Nower PT, Jia L, Qiu D, Huang X, Altaf A, Gao M, Fridell RA. 2012. Hepatitis C virus RNA elimination and development of resistance in replicon cells treated with BMS-790052. Antimicrob. Agents Chemother. 56:1350–1358 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37. McPhee F, Friborg J, Levine S, Chen C, Falk P, Yu F, Hernandez D, Lee MS, Chaniewski S, Sheaffer AK, Pasquinelli C. 2012. Resistance analysis of the hepatitis C virus NS3 protease inhibitor asunaprevir. Antimicrob. Agents Chemother. 56:3670–3681 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38. Sun JH, O'Boyle DR, II, Zhang Y, Wang C, Nower P, Valera L, Roberts S, Nettles RE, Fridell RA, Gao M. 2012. Impact of a baseline polymorphism on the emergence of resistance to the hepatitis C virus nonstructural protein 5A replication complex inhibitor, BMS-790052. Hepatology 55:1692–1699 [DOI] [PubMed] [Google Scholar]
- 39. Bifano M, Sevinsky H, Bedford BR, Coumbis JJ, Huang SP, Grasela DM, Eley T, Bertz RJ, Medlock M. 2010. Co-administration of BMS-790052 and BMS-650032 does not result in a clinically meaningful pharmacokinetic interaction in healthy subjects. Hepatology 52(Suppl):719A [Google Scholar]
- 40. Pelosi LA, Voss S, Liu M, Gao M, Lemm JA. 2012. Effect on hepatitis C virus replication of combinations of direct-acting antivirals, including NS5A inhibitor daclatasvir. Antimicrob. Agents Chemother. 56:5230–5239 [DOI] [PMC free article] [PubMed] [Google Scholar]
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