Santseharay Ramirez

Highly efficient full-length hepatitis C virus genotype 1 (strain TN) infectious culture system

Chronic infection with hepatitis C virus (HCV) is an important cause of end stage liver disease worldwide. In the United States, most HCV-related disease is associated with genotype 1 infection, which remains difficult to treat. Drug and vaccine development was hampered by inability to culture patient isolates representing HCV genotypes 1–7 and subtypes; only a recombinant 2a genome (strain JFH1) spontaneously replicated in vitro. Recently, we identified three mutations F1464L/A1672S/D2979G (LSG) in the nonstructural (NS) proteins, essential for development of full-length HCV 2a (J6) and 2b (J8) culture systems in Huh7.5 cells. Here, we developed a highly efficient genotype 1a (strain TN) full-length culture system. We initially found that the LSG substitutions conferred viability to an intergenotypic recombinant composed of TN 5′ untranslated region (5′UTR)-NS5A and JFH1 NS5B-3′UTR; recovered viruses acquired two adaptive mutations located in NS3 and NS4B. Introduction of these changes into a replication-deficient TN full-length genome, harboring LSG, permitted efficient HCV production. Additional identified NS4B and NS5B mutations fully adapted the TN full-length virus. Thus, a TN genome with 8 changes (designated TN cell-culture derived, TNcc) replicated efficiently and released infectious particles of ∼5 log10 focus-forming units per mL; passaged TNcc did not require additional changes. IFN-α and directly acting antivirals targeting the HCV protease, NS5A, and NS5B, each inhibited full-length TN infection dose-dependently. Given the unique importance of genotype 1 for pathogenesis, this infectious 1a culture system represents an important advance in HCV research. The approach used and the mutations identified might permit culture development for other HCV isolates, thus facilitating vaccine development and personalized treatment.

Robust full-length hepatitis C virus genotype 2a and 2b infectious cultures using mutations identified by a systematic approach applicable to patient strains

Hepatitis C virus (HCV) infection is a leading cause of chronic liver diseases worldwide, but treatment options are limited. Basic HCV research required for vaccine and drug development has been hampered by inability to culture patient isolates, and to date only the JFH1 (genotype 2a) recombinant replicates spontaneously in hepatoma cells and releases infectious virus. A JFH1 chimera with the 5′ end through NS2 from another genotype 2a strain, J6, had enhanced infectivity. However, the full-length J6 clone (J6CF), which we previously found to be fully functional in vivo, was replication incompetent in vitro. Through a systematic approach of culturing J6 with minimal JFH1 sequences, we identified three mutations in NS3, NS4A, and NS5B that permitted full-length J6 propagation and adaptation with infectivity titers comparable to JFH1-based systems. The most efficient recombinant, J6cc, had six adaptive mutations and did not accumulate additional changes following viral passage. We demonstrated that HCV NS3/NS4A protease-, NS5A- and NS5B polymerase-directed drugs respectively inhibited full-length J6 infection dose dependently. Importantly, the three J6-derived mutations enabled culture adaptation of the genetically divergent isolate J8 (genotype 2b), which differed from the J6 nucleotide sequence by 24%. The most efficient recombinant, J8cc, had nine adaptive mutations and was genetically stable after viral passage. The availability of these robust JFH1-independent genotype 2a and 2b culture systems represents an important advance, and the approach used might permit culture development of other isolates, with implications for improved individualized treatments of HCV patients and for development of broadly efficient vaccines.

Virology and Pathogenesis of Hepatitis C Virus Recurrence

1 In hepatitis C virus (HCV)–infected patients undergoing liver transplantation (LT), the virus infects the liver graft immediately after transplantation. The main source of HCV infection is circulating virions. Nevertheless, some data suggest that HCV present in extrahepatic compartments may contribute to HCV infection in some cases of hepatitis C recurrence. 2 Studies on early kinetics have shown that HCV replication starts a few hours after transplantation and that HCV‐RNA concentrations increase a few hours or days after the procedure, suggesting that HCV has an enormous ability to adapt to the new environment. 3 The quasispecies population may change significantly after transplantation, most likely because of the need to adapt to a new environment. There are no conclusive data supporting the role of HCV quasispecies composition and disease outcomes. 4 Persistence of HCV infection is the rule after transplantation. This is due to immunosuppression and to the immune exhaustion of the previously exposed immune system. 5 In general, HCV is not thought to be directly cytopathic. Thus, it is believed that the immune response against HCV causes liver damage. However, understanding the mechanisms of liver damage in HCV‐infected LT recipients is extremely complex because of the existence of a human leukocyte antigen–mismatched organ, the preexisting virus‐specific T cells that may be dysfunctional and/or tolerized, and the immunosuppression. 6 Despite the possible effect of immune‐mediated liver damage, it is clear that strong immunosuppression is associated with severe forms of hepatitis C recurrence (cholestatic hepatitis, fibrosing cholestatic hepatitis, and accelerated fibrosis progression). Thus, in the absence of a strong anti‐HCV immune response, HCV is able to directly (HCV proteins) or indirectly (cytokines) produce liver damage. 7 The activation of stellate cells and accelerated deposition of fibrosis are the final consequences of HCV infection in the graft. There are several mechanisms that may act synergistically to activate and perpetuate stellate cell activation in the setting of LT: ischemia‐reperfusion damage, old donor age, HCV proteins, cholestasis, rejection, infection with other viruses (cytomegalovirus), and immune‐mediated injury.

Highly efficient infectious cell culture of three hepatitis C virus genotype 2b strains and sensitivity to lead protease, nonstructural protein 5A, and polymerase inhibitors

Hepatitis C virus (HCV) is a genetically diverse virus with multiple genotypes exhibiting remarkable differences, particularly in drug susceptibility. Drug and vaccine development will benefit from high‐titer HCV cultures mimicking the complete viral life cycle, but such systems only exist for genotypes 1a and 2a. We developed efficient culture systems for the epidemiologically important genotype 2b. Full‐length molecular clones of patient strains DH8 and DH10 were adapted to efficient growth in Huh7.5 cells by using F1468L/A1676S/D3001G (LSG) mutations. The previously developed J8cc prototype 2b recombinant was further adapted. DH8 and J8 achieved infectivity titers >4.5 log10 Focus‐Forming Units/mL. A defined set of DH8 mutations had cross‐isolate adapting potential. A chimeric genome with the DH10 polyprotein coding sequence inserted into a vector with J8 untranslated regions was viable. Importantly, we succeeded in generating DH8, J8, and DH10 viruses with authentic sequences in the regions targeted by lead direct‐acting antivirals. Nonstructural protein (NS)5B inhibitors sofosbuvir, mericitabine, and BI207127 had activity against 1a (strain TN), 2a (strains JFH1 and J6), and the 2b strains, whereas VX‐222 and filibuvir only inhibited 1a. Genotype 2b strains were least sensitive to seven lead protease inhibitors, including MK‐5172 with high overall potency. NS5A inhibitor daclatasvir was exceptionally potent, but efficacy was affected by the HCV strain. Conclusion: Highly efficient HCV full‐length 2b culture systems can be established by using consensus clones with defined mutations. Lead protease and NS5A inhibitors, as well as polymerase inhibitors sofosbuvir, mericitabine, and BI207127, show cross‐activity against full‐length 1a, 2a, and 2b viruses, but important sensitivity differences exist at the isolate level. Infectious cultures for different HCV strains will advance studies on viral biology and pathogenesis and promote individualized patient treatment. (Hepatology 2014;59:395–407)

Hypervariable Region 1 Deletion and Required Adaptive Envelope Mutations Confer Decreased Dependency on Scavenger Receptor Class B Type I and Low-Density Lipoprotein Receptor for Hepatitis C Virus

ABSTRACT Hypervariable region 1 (HVR1) of envelope protein 2 (E2) of hepatitis C virus (HCV) serves important yet undefined roles in the viral life cycle. We previously showed that the viability of HVR1-deleted JFH1-based recombinants with Core-NS2 of H77 (H77ΔHVR1, genotype 1a) and S52 (S52ΔHVR1, genotype 3a) in Huh7.5 cells was rescued by E2 substitutions N476D/S733F and an E1 substitution, A369V, respectively; HVR1-deleted J6 (J6ΔHVR1, genotype 2a) was fully viable. In single-cycle production assays, where HCV RNA was transfected into entry-deficient Huh7-derived S29 cells with low CD81 expression, we found no effect of HVR1 deletion on replication or particle release for H77 and S52. HCV pseudoparticle assays in Huh7.5 cells showed that HVR1 deletion decreased entry by 20- to 100-fold for H77, J6, and S52; N476D/S733F restored entry for H77ΔHVR1, while A369V further impaired S52ΔHVR1 entry. We investigated receptor usage by antibody blocking and receptor silencing in Huh7.5 cells, followed by inoculation of parental and HVR1-deleted HCV recombinants. Compared to parental viruses, scavenger receptor class B type I (SR-BI) dependency was decreased for H77ΔHVR1/N476D/S733F, H77N476D/S733F, S52ΔHVR1/A369V, and S52A369V, but not for J6ΔHVR1. Low-density lipoprotein receptor (LDLr) dependency was decreased for HVR1-deleted viruses, but not for H77N476D/S733F and S52A369V. Soluble LDLr neutralization revealed strong inhibition of parental HCV but limited effect against HVR1-deleted viruses. Apolipoprotein E (ApoE)-specific HCV neutralization was similar for H77, J6, and S52 viruses with and without HVR1. In conclusion, HVR1 and HVR1-related adaptive envelope mutations appeared to be involved in LDLr and SR-BI dependency, respectively. Also, LDLr served ApoE-independent but HVR1-dependent functions in HCV entry.

Efficient Infectious Cell Culture Systems of the Hepatitis C Virus (HCV) Prototype Strains HCV-1 and H77

ABSTRACT The first discovered and sequenced hepatitis C virus (HCV) genome and the first in vivo infectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed efficient infectious cell culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77C in vivo infectious clones. We initially adapted a genome with the HCV-1 5′UTR-NS5A (where UTR stands for untranslated region) and the JFH1 NS5B-3′UTR (5-5A recombinant), including the genotype 2a-derived mutations F1464L/A1672S/D2979G (LSG), to grow efficiently in Huh7.5 cells, thus identifying the E2 mutation S399F. The combination of LSG/S399F and reported TNcc(1a)-adaptive mutations A1226G/Q1773H/N1927T/Y2981F/F2994S promoted adaptation of the full-length HCV-1 clone. An HCV-1 recombinant with 17 mutations (HCV1cc) replicated efficiently in Huh7.5 cells and produced supernatant infectivity titers of 104.0 focus-forming units (FFU)/ml. Eight of these mutations were identified from passaged HCV-1 viruses, and the A970T/I1312V/C2419R/A2919T mutations were essential for infectious particle production. Using CD81-deficient Huh7 cells, we further demonstrated the importance of A970T/I1312V/A2919T or A970T/C2419R/A2919T for virus assembly and that the I1312V/C2419R combination played a major role in virus release. Using a similar approach, we found that NS5B mutation F2994R, identified here from culture-adapted full-length TN viruses and a common NS3 helicase mutation (S1368P) derived from viable H77C and HCV-1 5-5A recombinants, initiated replication and culture adaptation of H77C containing LSG and TNcc(1a)-adaptive mutations. An H77C recombinant harboring 19 mutations (H77Ccc) replicated and spread efficiently after transfection and subsequent infection of naive Huh7.5 cells, reaching titers of 103.5 and 104.4 FFU/ml, respectively. IMPORTANCE Hepatitis C virus (HCV) was discovered in 1989 with the cloning of the prototype strain HCV-1 genome. In 1997, two molecular clones of H77, the other HCV prototype strain, were shown to be infectious in chimpanzees, but not in vitro. HCV research was hampered by a lack of infectious cell culture systems, which became available only in 2005 with the discovery of JFH1 (genotype 2a), a genome that could establish infection in Huh7.5 cells. Recently, we developed in vitro infectious clones for genotype 1a (TN), 2a (J6), and 2b (J8, DH8, and DH10) strains by identifying key adaptive mutations. Globally, genotype 1 is the most prevalent. Studies using HCV-1 and H77 prototype sequences have generated important knowledge on HCV. Thus, the in vitro infectious clones developed here for these 1a strains will be of particular value in advancing HCV research. Moreover, our findings open new avenues for the culture adaptation of HCV isolates of different genotypes.

Neutralization resistance of hepatitis C virus can be overcome by recombinant human monoclonal antibodies

Immunotherapy and vaccine development for hepatitis C virus (HCV) will depend on broadly reactive neutralizing antibodies (NAbs). However, studies in infectious strain JFH1‐based culture systems expressing patient‐derived Core‐NS2 proteins have suggested neutralization resistance for specific HCV strains, in particular, of genotype 2. To further examine this phenomenon, we developed a panel of HCV genotype 2 recombinants for testing of sensitivity to neutralization by chronic‐phase patient sera and lead human monoclonal antibodies (HMAbs). The novel Core‐NS2 recombinants, with patient‐derived genotype 2a (strain T9), 2b (strains DH8 and DH10), and 2c (strain S83) consensus sequences, were viable in Huh7.5 hepatoma cells without requirement for adaptive mutations, reaching HCV infectivity titers of 3.9‐4.5 log10 focus‐forming units per milliliter. In in vitro neutralization assays, we demonstrated that the novel genotype 2 viruses as well as prototype strains J6/JFH1(2a) and J8/JFH1(2b), all with authentic envelope proteins, were resistant to neutralization by genotype 2a, 2b, 2c, 2j, 2i, and 2q patient sera. However, these patient sera had high titers of HCV‐specific NAbs, because they efficiently reduced the infectivity of J6(2a) and J8(2b) with deleted hypervariable region 1. The genotype 2a, 2b, and 2c viruses, found resistant to polyclonal patient sera neutralization, were efficiently neutralized by two lead HMAbs (AR4A and HC84.26). Conclusion: Using novel 2a, 2b, and 2c cell‐culture systems, expressing authentic envelope proteins, we demonstrated resistance of HCV to patient‐derived polyclonal high‐titer NAbs. However, the same genotype 2 culture viruses were all sensitive to HMAbs recognizing conformational epitopes, indicating that neutralization resistance of HCV can be overcome by applying recombinant antibodies. These findings have important implications for HCV immunotherapy and vaccine development. (Hepatology 2013;58:1587–1597)

Characterization of Hepatitis C Virus Recombinants with Chimeric E1/E2 Envelope Proteins and Identification of Single Amino Acids in the E2 Stem Region Important for Entry

ABSTRACT 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.

Substitutions at NS3 Residue 155, 156, or 168 of Hepatitis C Virus Genotypes 2 to 6 Induce Complex Patterns of Protease Inhibitor Resistance

ABSTRACT Various protease inhibitors (PIs) currently are becoming available for treatment of hepatitis C virus (HCV). For genotype 1, substitutions at NS3 protease positions 155, 156, and 168 are the main determinants of PI resistance. For other genotypes, similar substitutions were selected during PI treatment but were not characterized systematically. To elucidate the impact of key PI resistance substitutions on genotypes 2 to 6, we engineered the substitutions R155A/E/G/H/K/Q/T, A156G/S/T/V, and D/Q168A/E/G/H/N/V into HCV recombinants expressing genotype 2 to 6 proteases. We evaluated viral fitness and sensitivity to nine PIs (telaprevir, boceprevir, simeprevir, asunaprevir, vaniprevir, faldaprevir, paritaprevir, deldeprevir, and grazoprevir) in Huh7.5 cells. We found that most variants showed decreased fitness compared to that of the original viruses. Overall, R155K, A156G/S, and D/Q168A/E/H/N/V variants showed the highest fitness; however, genotype 4 position 168 variants showed strong fitness impairment. Most variants tested were resistant to several PIs. Resistance levels varied significantly depending on the specific substitution, genotype, and PI. For telaprevir and boceprevir, specific 155 and 156, but not 168, variants proved resistant. For the remaining PIs, most genotype 2, 4, 5, and 6, but not genotype 3, variants showed various resistance levels. Overall, grazoprevir (MK-5172) had the highest efficacy against original viruses and variants. This is the first comprehensive study revealing the impact of described key PI resistance substitutions on fitness and PI resistance of HCV genotypes 2 to 6. In conclusion, the studied substitutions induced resistance to a panel of clinically relevant PIs, including the newer PIs paritaprevir, deldeprevir, and grazoprevir. We discovered complex patterns of resistance, with the impact of substitutions varying from increased sensitivity to high resistance.

Hepatitis C Virus Genotype 1 to 6 Protease Inhibitor Escape Variants: In Vitro Selection, Fitness, and Resistance Patterns in the Context of the Infectious Viral Life Cycle

ABSTRACT Hepatitis C virus (HCV) NS3 protease inhibitors (PIs) are important components of novel HCV therapy regimens. Studies of PI resistance initially focused on genotype 1. Therefore, knowledge about the determinants of PI resistance for the highly prevalent genotypes 2 to 6 remains limited. Using Huh7.5 cell culture-infectious HCV recombinants with genotype 1 to 6 NS3 protease, we identified protease positions 54, 155, and 156 as hot spots for the selection of resistance substitutions under treatment with the first licensed PIs, telaprevir and boceprevir. Treatment of a genotype 2 isolate with the newer PIs vaniprevir, faldaprevir, simeprevir, grazoprevir, paritaprevir, and deldeprevir identified positions 156 and 168 as hot spots for resistance; the Y56H substitution emerged for three newer PIs. Substitution selection also depended on the specific recombinant. The substitutions identified conferred cross-resistance to several PIs; however, most substitutions selected under telaprevir or boceprevir treatment conferred less resistance to certain newer PIs. In a single-cycle production assay, across genotypes, PI treatment primarily decreased viral replication, which was rescued by PI resistance substitutions. The substitutions identified resulted in differential effects on viral fitness, depending on the original recombinant and the substitution. Across genotypes, fitness impairment induced by resistance substitutions was due primarily to decreased replication. Most combinations of substitutions that were identified increased resistance or fitness. Combinations of resistance substitutions with fitness-compensating substitutions either rescued replication or compensated for decreased replication by increasing assembly. This comprehensive study provides insight into the selection patterns and effects of PI resistance substitutions for HCV genotypes 1 to 6 in the context of the infectious viral life cycle, which is of interest for clinical and virological HCV research.