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Hepatitis C virus (HCV) strains belong to seven genotypes with numerous subtypes that respond differently to antiviral therapies. Genotype 1, and primarily subtype 1b, is the most prevalent genotype worldwide. The development of recombinant HCV infectious cell culture systems for different variants, permitted by the high replication capacity of strain JFH1 (genotype 2a), has advanced efficacy and resistance testing of antivirals. However, efficient infectious JFH1-based cell cultures of subtype 1b are limited and comprise only the 5′ untranslated region (5′UTR)-NS2, NS4A, or NS5A regions. Importantly, it has not been possible to develop efficient 1b infectious systems expressing the NS3/4A protease, an important target of direct-acting antivirals. We developed efficient infectious JFH1-based cultures with genotype 1b core-NS5A sequences of strains DH1, Con1, and J4 by using previously identified HCV cell culture adaptive substitutions A1226G, R1496L, and Q1773H. These viruses spread efficiently in Huh7.5 cells by acquiring additional adaptive substitutions, and final recombinants yielded peak supernatant infectivity titers of 4 to 5 log 10 focus-forming units (FFU)/ml. We subsequently succeeded in adapting a JFH1-based 5′UTR-NS5A DH1 recombinant to efficient growth in cell culture. We evaluated the efficacy of clinically relevant NS3/4A protease and NS5A inhibitors against the novel genotype 1b viruses, as well as against previously developed 1a viruses. The inhibitors were efficient against all tested genotype 1 viruses, with NS5A inhibitors showing half-maximal effective concentrations several orders of magnitude lower than NS3/4A protease inhibitors. In summary, the developed HCV genotype 1b culture systems represent valuable tools for assessing the efficacy of various classes of antivirals and for other virological studies requiring genotype 1b infectious viruses.

Background & Aims Inhibitors of the hepatitis C virus (HCV) NS5A protein are a key component of effective treatment regimens, but the genetic heterogeneity of HCV has limited the efficacy of these agents and mutations lead to resistance. We directly compared the efficacy of all clinically relevant NS5A inhibitors against HCV genotype 1–7 prototype isolates and resistant escape variants, and investigated the effects of pre-existing resistance-associated substitutions (RAS) on HCV escape from treatment. Methods We measured the efficacy of different concentrations of daclatasvir, ledipasvir, ombitasvir, elbasvir, ruzasvir, velpatasvir, and pibrentasvir in cultured cells infected with HCV recombinants expressing genotype 1–7 NS5A proteins with or without RAS. We engineered HCV variants that included RAS identified in escape experiments, using recombinants with or without T/Y93H and daclatasvir, or that contained RAS previously reported from patients. Results NS5A inhibitors had varying levels of efficacy against original and resistant viruses. Only velpatasvir and pibrentasvir had uniform high activity against all HCV genotypes tested. RAS hotspots in NS5A were found at amino acids 28, 30, 31, and 93. Engineered escape variants had high levels of fitness. Pibrentasvir had the highest level of efficacy against variants; viruses with RAS at amino acids 28, 30, or 31 had no apparent resistance to pibrentasvir, and HCV with RAS at amino acid 93 had a low level of resistance to this drug. However, specific combinations of RAS and deletion of amino acid 32 led to significant resistance to pibrentasvir. For the remaining NS5A inhibitors tested, RAS at amino acids 28 and 93 led to high levels of resistance. Among these inhibitors, velpatasvir was more effective against variants with RAS at amino acid 30 and some variants with RAS at amino acid 31 than the other agents. Variants with the pre-existing RAS T/Y93H acquired additional NS5A changes during escape experiments, resulting in HCV variants with specific combinations of RAS, showing high fitness and high resistance. Conclusions We performed a comprehensive comparison of the efficacy of the 7 clinically relevant inhibitors of HCV NS5A and identified variants associated with resistance to each agent. These findings could improve treatment of patients with HCV infection.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide1. The acute phase-infection is often sub-clinical, with clearance in only 20–30% of the cases. Furthermore, vigorous cellular immune responses are essential for viral clearance2, whereas the role of neutralizing antibodies (NAbs) remains controversial3–6. During chronic HCV infection the virus persists despite HCV-specific CD8+ T-cell responses2,7, and continuous pressure from NAbs apparently drives viral evolution and reduces viral load5. A recent study showed that clearance of a chronic HCV infection was induced after an initial strong NAb response had reduced viral load, facilitating effective cellular immune responses8. This supports the importance of NAbs in controlling HCV, thus strengthening the case for their therapeutic relevance. Several promising human monoclonal antibodies (HMAbs) were developed with neutralizing effect in vitro and in vivo9,10. These antibodies could be of great importance as potential therapeutics and as tools to study the function of HCV envelope proteins revealing potential targets for vaccine design. A major challenge in developing prophylactic and therapeutic HCV antibodies is its great diversity, with 6 epidemiologically important major genotypes and numerous subtypes11. In an infected individual the virus replicates rapidly, generating closely related quasispecies of importance for immune evasion2. Since the discovery of HCV genotype 2a strain JFH112, recombinant cell-culture systems expressing strain specific Core-NS2 proteins (Core, E1, E2, p7, and NS2) have been developed for all major HCV genotypes13–20, including a genotype 1a and 1b panel17. The isolate specific envelope proteins enable detailed cross-genotype and -subtype neutralization studies using HCV patient polyclonal antibodies. Prior studies revealed differential neutralization susceptibility and patterns of neutralization for the major genotypes but differences also occurred between subtypes13,21. Especially genotype 2 viruses showed differences on a subtype-specific level. In one study we found that a 2a isolate was difficult to neutralize, whereas a 2b isolate showed intermediate neutralization susceptibility13. In contrast, genotype 1a and 1b isolates showed intermediate susceptible to neutralization13. In another study, we reported that the genotype 2a virus without hypervariable region 1 (HVR1) did not require adaptive mutations and had significantly increased susceptibility to NAb compared to the wild-type virus21. Considering that genotypes 1 and 2 are widely distributed worldwide, and are commonly found in Europe, Japan, and USA22, further studies exploring differences in neutralization among genotype 2 viruses would be highly relevant. However, in order to make valid comparisons, several strains of each subtype should be studied. At the outset of this study, genotype 2 was represented by two Core-NS2 systems, J6/JFH1(2a) and J8/JFH1(2b), and by one full-length system, JFH1(2a)12–14. Genotype 2 is diverse with numerous subtypes (2a-2r); six subtypes were confirmed by full-length sequences (2a, 2b, 2c, 2i, 2k, and 2q)12,23–27. Subtypes 2a, 2b, and 2c are the most prevalent, and we therefore sought to develop Core-NS2 recombinants of these subtypes to investigate the neutralization potential of human polyclonal antibodies present in genotype 2 patient sera and to compare it with the neutralizing potential of two lead HMAbs, AR4A9 and HC84.2610.

The hepatitis C virus (HCV) envelope proteins E1 and E2 play a key role in host cell entry and represent important targets for vaccine and drug development. Here, we characterized HCV recombinants with chimeric E1/E2 complexes in vitro . Using genotype 1a/2a JFH1-based recombinants expressing 1a core-NS2, we exchanged E2 with functional isolate sequences of genotypes 1a (alternative isolate), 1b, and 2a. While the 1a-E2 exchange did not impact virus viability, the 2a-E2 recombinant was nonviable. After E2 exchange from three 1b isolates, long delays were observed before spread of infection. For recovered 1b-E2 recombinants, single E2 stem region amino acid changes were identified at residues 706, 707, and 710. In reverse genetic studies, these mutations increased infectivity titers by ∼100-fold, apparently without influencing particle stability or cell binding although introducing slight decrease in particle density. In addition, the 1b-E2 exchange led to a decrease in secreted core protein of 25 to 50%, which was further reduced by the E2 stem region mutations. These findings indicated that compensatory mutations permitted robust infectious virus production, without increasing assembly/release. Studies of E1/E2 heterodimerization showed no differences in intracellular E1/E2 interaction for chimeric constructs with or without E2 stem region mutations. Interestingly, the E2 stem region mutations allowed efficient entry, which was verified in 1a-E1/1b-E2 HCV pseudoparticle assays. A CD81 inhibition assay indicated that the mutations influenced a late step of the HCV entry pathway. Overall, this study identified specific amino acids in the E2 stem region of importance for HCV entry and for production of infectious virus particles.

Human monoclonal antibodies (HMAbs) with neutralizing capabilities constitute potential immune-based treatments or prophylaxis against hepatitis C virus (HCV). However, lack of cell culture-derived HCV (HCVcc) harboring authentic envelope proteins (E1/E2) has hindered neutralization investigations across genotypes, subtypes, and isolates. We investigated the breadth of neutralization of 10 HMAbs with therapeutic potential against a panel of 16 JFH1-based HCVcc-expressing patient-derived Core-NS2 from genotypes 1a (strains H77, TN, and DH6), 1b (J4, DH1, and DH5), 2a (J6, JFH1, and T9), 2b (J8, DH8, and DH10), 2c (S83), and 3a (S52, DBN, and DH11). Virus stocks used for in vitro neutralization analysis contained authentic E1/E2, with the exception of full-length JFH1 that acquired the N417S substitution in E2. The 50% inhibition concentration (IC50) for each HMAb against the HCVcc panel was determined by dose-response neutralization assays in Huh7.5 cells with antibody concentrations ranging from 0.0012 to 100 μg/mL. Interestingly, IC50 values against the different HCVcc's exhibited large variations among the HMAbs, and only three HMAbs (HC-1AM, HC84.24, and AR4A) neutralized all 16 HCVcc recombinants. Furthermore, the IC50 values for a given HMAb varied greatly with the HCVcc strain, which supports the use of a diverse virus panel. In cooperation analyses, HMAbs HC84.24, AR3A, and, especially HC84.26, demonstrated synergistic effects towards the majority of the HCVcc's when combined individually with AR4A. Conclusion: Through a neutralization analysis of 10 clinically relevant HMAbs against 16 JFH1-based Core-NS2 recombinants from genotypes 1a, 1b, 2a, 2b, 2c, and 3a, we identified at least three HMAbs with potent and broad neutralization potential. The neutralization synergism obtained when pooling the most potent HMAbs could have significant implications for developing novel strategies to treat and control HCV. (Hepatology 2014;60:1551–1562)

A challenge for hepatitis C virus (HCV) vaccine development is defining conserved epitopes that induce protective antibodies against this highly diverse virus. An envelope glycoprotein (E2) segment located at amino acids (aa) 412 to 423 contains highly conserved neutralizing epitopes. While polyclonal antibodies to aa 412 to 423 from HCV-infected individuals confirmed broad neutralization, conflicting findings have been reported on polyclonal antibodies to an adjacent region, aa 434 to 446, that may or may not interfere with neutralization by antibodies to aa 412 to 423. To define the interplay between these antibodies, we isolated human monoclonal antibodies (HMAbs) to aa 412 to 423, designated HC33-related HMAbs (HC33 HMAbs), and characterized their interactions with other HMAbs to aa 434 to 446. A subset of the HC33 HMAbs neutralized genotype 1 to 6 infectious cell culture-derived HCV virions (HCVcc) with various activities. Although nonneutralizing HC33 HMAbs were isolated, they had lower binding affinities than neutralizing HC33 HMAbs. These antibodies could be converted to neutralizing antibodies by affinity maturation. Unidirectional competition for binding to E2 was observed between HC33 HMAbs and HMAbs to aa 434 to 446. When HMAbs to aa 434 to 446, which mediated neutralization, were combined with neutralizing HC33 HMAbs, biphasic patterns in neutralization were observed. A modest degree of antagonism was observed at lower concentrations, and a modest degree of synergism was observed at higher concentrations. However, the overall effect was additive neutralization. A similar pattern was observed when these antibodies were combined to block E2 binding to the HCV coreceptor, CD81. These findings demonstrate that both of these E2 regions participate in epitopes mediating virus neutralization and that the antibodies to aa 412 to 423 and aa 434 to 446 do not hinder their respective virus-neutralizing activities.

The correlation of neutralizing antibodies to treatment outcome in patients with chronic hepatitis C virus (HCV) infection has not been established. The aim of this study was to determine whether neutralizing antibodies could be used as an outcome predictor in patients with chronic HCV, genotype 1, infection treated with pegylated interferon-α and ribavirin. Thirty-nine patients with chronic hepatitis C, genotype 1a or 1b, with either sustained virologic response (n = 23) or non-sustained virologic response (n = 16) were enrolled. Samples taken prior to treatment were tested for their ability to neutralize 6 different HCV genotype 1 cell culture recombinants (1a: H77/JFH1, TN/JFH1, DH6/JFH1; 1b: J4/JFH1, DH1/JFH1, DH5/JFH1). The results were expressed as the highest dilution yielding 50% neutralization (NAb50-titer). We observed no genotype or subtype specific differences in NAb50-titers between patients with chronic HCV infection with and without sustained virologic response when tested against any of the included culture viruses. However, NAb50-titers varied significantly with a mean reciprocal NAb50-titer of 800 (range: 100-6400) against DH6/JFH1 compared to a mean NAb50-titer of 50 (range

The majority of broadly neutralizing antibodies to hepatitis C virus (HCV) are against conformational epitopes on the E2 glycoprotein. Many of them recognize overlapping epitopes in a cluster, designated as antigenic domain B, that contains residues G530 and D535. To gain information on other regions that will be relevant for vaccine design, we employed yeast surface display of antibodies that bound to genotype 1a H77C E2 mutant proteins containing a substitution either at Y632A (to avoid selecting non-neutralizing antibodies) or D535A. A panel of nine human monoclonal antibodies (HMAbs) was isolated and designated as HC-84-related antibodies. Each HMAb neutralized cell culture infectious HCV (HCVcc) with genotypes 1–6 envelope proteins with varying profiles, and each inhibited E2 binding to the viral receptor CD81. Five of these antibodies neutralized representative genotypes 1–6 HCVcc. Epitope mapping identified a cluster of overlapping epitopes that included nine contact residues in two E2 regions encompassing aa418–446 and aa611–616. Effect on virus entry was measured using H77C HCV retroviral pseudoparticles, HCVpp, bearing an alanine substitution at each of the contact residues. Seven of ten mutant HCVpp showed over 90% reduction compared to wild-type HCVpp and two others showed approximately 80% reduction. Interestingly, four of these antibodies bound to a linear E2 synthetic peptide encompassing aa434–446. This region on E2 has been proposed to elicit non-neutralizing antibodies in humans that interfere with neutralizing antibodies directed at an adjacent E2 region from aa410–425. The isolation of four HC-84 HMAbs binding to the peptide, aa434–446, proves that some antibodies to this region are to highly conserved epitopes mediating broad virus neutralization. Indeed, when HCVcc were passaged in the presence of each of these antibodies, virus escape was not observed. Thus, the cluster of HC-84 epitopes, designated as antigenic domain D, is relevant for vaccine design for this highly diverse virus., Author Summary Hepatitis C virus (HCV) is a highly diverse virus and a significant challenge for vaccine development is to identify protective epitopes conserved in the majority of viral genotypes and subtypes. This problem is compounded by the fact that the envelope E1E2 proteins, the targets for neutralizing antibody response, are two of the most variable proteins of the virus. Modified E2 antigens were constructed that are not bound by antibodies to previously recognized clusters of highly immunogenic epitopes on E2. Their employment as screening antigens has led to the isolation of a novel panel of human monoclonal antibodies to HCV E2. Functional and biochemical studies revealed that these antibodies bind and neutralize HCV of different genotypes and subtypes. Several of these antibodies neutralized cell culture infectious HCV with genotypes 1–6 envelope proteins. Furthermore, when virus was passaged in culture in the presence of each of these antibodies, virus escape was not observed. Thus, these epitopes are relevant in vaccine design for this virus.

Hepatitis C virus (HCV) is an important cause of chronic liver disease. Several highly diverse HCV genotypes exist with potential key functional differences. The HCV NS5A protein was associated with response to interferon (IFN)-α based therapy, and is a primary target of currently developed directly-acting antiviral compounds. NS5A is important for replication and virus production, but has not been studied for most HCV genotypes. We studied the function of NS5A using infectious NS5A genotype 1–7 cell culture systems, and through reverse genetics demonstrated a universal importance of the amphipathic alpha-helix, domain I and II and the low-complexity sequence (LCS) I for HCV replication; the replicon-enhancing LCSI mutation S225P attenuated all genotypes. Mutation of conserved prolines in LCSII led to minor reductions in virus production for the JFH1(genotype 2a) NS5A recombinant, but had greater effects on other isolates; replication was highly attenuated for ED43(4a) and QC69(7a) recombinants. Deletion of the conserved residues 414-428 in domain III reduced virus production for most recombinants but not JFH1(2a). Reduced virus production was linked to attenuated replication in all cases, but ED43(4a) and SA13(5a) also displayed impaired particle assembly. Compared to the original H77C(1a) NS5A recombinant, the changes in LCSII and domain III reduced the amounts of NS5A present. For H77C(1a) and TN(1a) NS5A recombinants, we observed a genetic linkage between NS5A and p7, since introduced changes in NS5A led to changes in p7 and vice versa. Finally, NS5A function depended on genotype-specific residues in domain I, as changing genotype 2a-specific residues to genotype 1a sequence and vice versa led to highly attenuated mutants. In conclusion, this study identified NS5A genetic elements essential for all major HCV genotypes in infectious cell culture systems. Genotype- or isolate- specific NS5A functional differences were identified, which will be important for understanding of HCV NS5A function and therapeutic targeting., Author Summary Hepatitis C virus (HCV) is a major public health burden and leads to chronic liver disease, including liver cirrhosis and liver cancer. Understanding the biological functions of the virus is crucial to the development of a vaccine and to improve current therapy through development of directly-acting antiviral compounds. The NS5A protein is a promising antiviral target, but much remains to be understood about its role in the viral life cycle. Great diversity among the seven major HCV genotypes poses challenges for broadly active inhibitors. Here we used infectious cell culture systems for NS5A of the seven major HCV genotypes, and demonstrated that all genotypes depended on the NS5A amphipathic alpha-helix, domain I, low-complexity sequence (LCS) I and domain II for viral replication. Interestingly, effects on replication and virus production by changes in LCSII and domain III varied greatly among NS5A isolates. Furthermore, we found that genotype 2 had evolved genotype-specific residues in domain I of importance for viral function. Thus, the highly diverse sequence of the NS5A protein reflected functional differences between HCV genotypes and isolates. Such differences will be important to consider in understanding HCV biology and for future development of antiviral compounds.

Hepatitis C virus (HCV) is an important cause of chronic liver disease, and interferon-based therapy cures only 40 to 80% of patients, depending on HCV genotype. Research was accelerated by genotype 2a (strain JFH1) infectious cell culture systems. We previously developed viable JFH1-based recombinants encoding the structural proteins (core, E1, E2), p7, and NS2 of prototype isolates of the seven major HCV genotypes; most recombinants required adaptive mutations. To enable genotype-, subtype-, and isolate-specific studies, we developed efficient core-NS2 recombinants from additional genotype 1a (HC-TN and DH6), 1b (DH1 and DH5), and 3a (DBN) isolates, using previously identified adaptive mutations. Introduction of mutations from isolates of the same subtype either led to immediate efficient virus production or accelerated culture adaptation. The DH6 and DH5 recombinants without introduced mutations did not adapt to culture. Universal adaptive effects of mutations in NS3 (Q1247L, I1312V, K1398Q, R1408W, and Q1496L) and NS5A (V2418L) were investigated for JFH1-based genotype 1 to 5 core-NS2 recombinants; several mutations conferred adaptation to H77C (1a), J4 (1b), S52 (3a), and SA13 (5a) but not to ED43 (4a). The mutations permitting robust virus production in Huh7.5 cells had no apparent effect on viral replication but allowed efficient assembly of intracellular infectious HCV for adapted novel or previously developed recombinants. In conclusion, previously identified mutations permitted development of novel HCV core-NS2 genotype recombinants. Mutations adapting several recombinants to culture were identified, but no mutations were universally adaptive across genotypes. This work provides tools for analysis of HCV genotype specificity and may promote the understanding of genotype-specific patterns in HCV disease and control.