Approximately 150 million people are infected with hepatitis C virus (HCV) worldwide, and over 350,000 are estimated to die from associated chronic liver disease each year. The economical and social burden of hepatitis C is enormous, and efficient therapies and vaccines are needed. Infectious culture systems are important for HCV studies, contributing to drug and vaccine development. However, only a few HCV strains can be studied because of the lack of efficient culture systems.
HCV is an enveloped, positive-strand RNA virus from the family Flaviviridae. Its genome contains ∼9,600 nucleotides (nts) with a single open reading frame (ORF) flanked by 5′ and 3′ untranslated regions (UTRs). The polyprotein of ∼3,000 amino acids (aa) is processed into structural (Core, E1, and E2) and nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) with complex roles in the viral life cycle. HCV presents significant genetic diversity with six epidemiologically important genotypes and numerous subtypes. Viruses recovered from infected individuals are referred to as isolates or strains, and they circulate as quasispecies. Genotypes 1, 2, and 3 are the most prevalent and globally distributed. Genotype 2 is highly prevalent in West Africa as well as Asian countries such as China and Japan. In the United States, genotype 2 is the second in prevalence, with subtype 2b accounting for 10% of all infections. Genotypes 1 and 2 respond differently to standard interferon (IFN)/ribavirin (RBV) therapy, with genotype 2–infected patients achieving higher clearance rates. However, limited information is available about the sensitivity of genotype 2 viruses to direct-acting antivirals (DAAs), both in vivo and in vitro.
DAAs are expected to improve HCV clearance rates in patients with chronic hepatitis C. The subgenomic replicon systems have been of major importance for discovery, development, and preclinical testing of these compounds; however, replicons do not recapitulate the complete viral life cycle and are not suitable for testing the effect of drugs beyond viral replication. Therefore, it is essential to develop infectious culture systems for different HCV strains from the major genotypes and subtypes that reproduce all viral functions. Nevertheless, development of such systems has been a major challenge, and they are only available for a few adapted genotype 1 and 2 isolates.[7-12] Among these, only chimeric J6/JFH1(2a), JFH1(2a), J6(2a), and TN(1a) could release high-titer infectious virus particles in cell culture.
In this study, we developed efficient full-length cell-culture systems for three genotype 2b isolates. After long-term culture, viruses reached high infectivity titers by acquiring specific cell-culture-adaptive mutations that were then used to generate highly efficient molecular recombinants. These systems permitted analysis of sensitivity to frontline HCV DAAs, in comparison with previously developed 1a and 2a full-length viruses. We found differential sensitivity to NS3/NS4A protease, NS5A, and NS5B inhibitors, both at the genotype and isolate level. Importantly, we also identified front-line drugs that are efficient against all viruses, thus possibly overcoming HCV genetic diversity.
Full-length HCV culture systems that represent all viral genotypes and important subtypes will benefit drug and vaccine development for this important human pathogen. However, thus far, only adapted recombinants of JFH1(2a), J6(2a), and TN(1a) strains yielded high-titer cultures. In the present study, we developed efficient full-length cell-culture systems for genotype 2b strains DH8, J8, and DH10, of which DH8 and J8 yielded high infectivity titers. The main characteristics of the most efficient recombinants developed for isolates DH8, J8, and DH10 are summarized in Table 4. Recombinants were adapted to growth in Huh7.5 cells, and a defined set of mutations could adapt two strains, proving a cross-isolate effect. Efficient cell culture of DH10 was achieved by determining only the patient-derived ORF sequence, which was inserted into a cassette containing the 5′ and 3′ UTR from J8. These findings might facilitate the development of HCV culture systems for other genotype 2b isolates, as well as for other genotypes and subtypes.
Table 4. Major Characteristics of Developed Genotype 2b Recombinants
|J8cc-HT||4.7||4.3||—||M292L||L612M||F772C||W864R||G1154Aa, A1208Ta, N1217Y, L||S||Q1763R, I1968V||E2263V, I2440T||H2922R, G|
|DH10cc||3.6||3.0||—||G351S||—||Y792N||A992V||L||S||I1824V, N1931S, V1951A||D2434N||G|
We tested, for the first time, lead HCV inhibitors against full-length genotype 2b viruses. Our results reveal a differential activity of these drugs toward full-length genotype 1a, 2a, and 2b viruses. Genotype 2b is less sensitive to most PIs, and the efficacy of daclatasvir against genotypes 2a and 2b is largely influenced by HCV variability at the isolate level. We have demonstrated that not only NIs (sofosbuvir and mericitabine) are active against non–genotype 1 viruses, but also the NNI, BI201127, possesses activity against full-length 1a, 2a, and 2b viruses, suggesting unique pangenotypic properties among NNIs.
Development of infectious full-length cell-culture systems for HCV has been a major challenge, because molecular clones of HCV generated from patient sequences do not spontaneously replicate and spread in vitro. Approaches that used subgenomic replicon-derived mutations to adapt full-length clones have only led to culture systems with relatively low infectivity,[9, 18] possibly because replicon mutations introduce constraints in viral production.[18, 19] We recently identified three mutations in the NS3 helicase, NS4A, and NS5B, named LSG, that promoted adaptation of HCV genotype 1 and 2 full-length clones,[10, 12] and used them to adapt novel genotype 2b isolates in the present study. Similarly to J8, LSG permitted in vitro growth of DH8 and DH10. Besides LSG, additional mutations were required to produce viruses with high infectivity titers. These mutations represent unique aa that are rarely present in natural patient-derived sequences. A1951 and V1968, in NS4B, are found in less than 1% of genotype 2b sequences (LAL), and T2439 or T2440 are not present in any of the 73 deposited NS5A 2b sequences. In addition, we had previously demonstrated that changes at aa 1,931 (NS4B), 1,968 (NS4B), and 2,439 (NS5A) constitute key adaptive mutations in cell culture systems.[10, 12]
We also demonstrated that it is possible to develop functional chimeric genomes by inserting the ORF of a 2b isolate into a cassette vector with 5′/3′ UTRs of another 2b isolate, for which the UTRs were known to be functional in vitro. This finding is of major relevance for the culture of clinical isolates, because the sequence of the UTRs is technically difficult to obtain. Similar chimeric genomes have been proven functional in vivo.
The full-length cell-culture systems permit us to explore the evolutionary potential of HCV. Viruses can adapt to cell culture by acquiring different combinations of mutations and maintaining WT sequences in specific genes or domains. We generated genotype 2b viruses that did not have any changes in the NS3 protease, NS5A domain I, and NS5B finger, palm, and thumb domains (except the c-terminal portion), making them optimal tools for the study of most DAAs. Contrarily, replicon-based systems often accumulate mutations in the protease domain of NS3, potentially affecting the natural isolate sensitivity toward PIs. The importance of using viruses without cell-culture adaptive mutations in the NS3 protease domain was demonstrated for the J8 isolate, because viruses with mutations G1154A and A1208T had increased drug sensitivity, when compared to J8 viruses without these changes. Similarly, we succeeded in developing cell-culture–adapted viruses for DH8, which did not contain mutations in p7, and which could be of importance for functional or drug studies targeting this important viral protein. However, we cannot exclude that adaptive changes present in other proteins, outside the drug targets, might affect drug sensitivities of the cell-culture–adapted viruses.
We tested sensitivity of full-length viruses to selected front-line DAAs targeting NS3/4A protease, NS5A, and NS5B polymerase. Currently, there are very limited data on the activity of PIs in genotype 2 patients; small studies have suggested a benefit of telaprevir and boceprevir when added to therapy with IFN/RBV. In our in vitro systems, most PIs had higher activity against TN(1a) than against genotype 2 isolates. Among genotype 2, isolates from subtype 2b were generally less sensitive to tested PIs, in comparison with 2a isolates. These findings stress the importance of subtype determination in the clinical setting, which may be of more relevance in the era of DAA-based therapy.
Our data on PI MK-5172, highlighting its exceptional higher antiviral potency, when compared with other PIs, represent the first reported testing of this drug in HCV full-length cell-culture systems of various genotypes. Similarly to MK-5172, NS5A inhibitor daclatasvir was shown to be a highly potent HCV inhibitor, but its activity was most influenced by HCV genetic divergence at the isolate level for genotype 2b, as previously indicated for genotypes 1a and 2a.[16, 17]
NS5B inhibitors currently in phase II and III clinical trials are among the most promising anti-HCV drugs. However, they have not been extensively studied in cell-culture viruses for different genotypes and subtypes because of the lack of culture systems with genotype-specific polymerases. In the present work, we report on the effect of front-line polymerase inhibitors on full-length viruses of genotypes 1a, 2a, and 2b. As expected, NIs mericitabine and sofosbuvir were active not only against 1a, but also against 2a and 2b, which is in agreement with their apparent pan-genotypic activity in patients infected with genotypes 1, 2, and 3, in combination with IFN/RBV.[22, 23]
We investigated the activity of front-line NNIs currently in phase II (filibuvir, VX-222, and BI207127). Filibuvir has been reported to significantly reduce HCV titers in clinical studies, when used in monotherapy, in genotype 1–infected patients. Our data support the efficacy of filibuvir and VX-222 against genotype 1, but reveal limited or no activity against genotype 2 viruses. Contrarily, the NNI, BI207127, was active against all viruses. Efficacy against genotype 2 was similar to that of sofosbuvir, whereas efficacy against TN(1a) was higher for BI207127 than for sofosbuvir. This difference might be explained by the fact that NNIs target different pocket (or allosteric) sites of the RNA polymerase, which present higher genetic variability among HCV genotypes. In addition, they could be specific for genotype 1 because they have been mostly developed using Con1 replicons. Therefore, significant differences in antiviral activity against genotype 1 and non-1 viruses are expected. Among the different allosteric sites that are targeted by current advanced thumb NNIs, our results with BI207127 support that thumb pocket I is the preferable target of future NNIs with potential pangenotypic activity.
In conclusion, we have established efficient cell-culture systems for three HCV genotype 2b isolates. Approaches applied in viral adaptation should have relevance for advancing culture development for other 2b isolates and, perhaps, other genotype strains. These systems represent authentic patient-derived sequences in all the corresponding targets of the most relevant DAAs, making them optimal models for the study of antivirals. These full-length systems will, for the first time, permit the study of combination treatment with drugs targeting all structural and nonstructural proteins in genotype 2b. They will allow future studies promoting escape to current DAAs, in the context of the whole genetic background of HCV. Finally, because the viruses generated mimics all the steps of the HCV life cycle, they will permit genotype-specific functional studies of all viral proteins, thus promoting drug and vaccine development.