Safety and efficacy of faldaprevir in combination with pegylated interferon α-2b and ribavirin in Japanese patients with genotype-1 chronic hepatitis C virus infection.

AIM: We evaluated the safety and efficacy of the hepatitis C virus (HCV) NS3/4A A protease inhibitor faldaprevir plus pegylated interferon α-2b and ribavirin (PegIFNα-2b/RBV) in Japanese patients with HCV genotype-1 infection. METHODS: Treatment-naïve patients were randomized (1:1) to faldaprevir 120 mg q.d. for 12 or 24 weeks (response-guided therapy [RGT], n = 44), or faldaprevir 240 mg q.d. for 12 weeks (n = 43), each combined with PegIFNα-2b/RBV for 24 or 48 weeks (RGT). Response-guided therapy was based on early treatment success (HCV RNA <25 IU/mL at week 4 and <25 IU/mL undetected at week 8). Treatment-experienced patients received 240 mg q.d. for 24 weeks, plus PegIFNα-2b/RBV RGT (24 or 48 weeks, prior relapsers, n = 29) or PegIFNα-2b/RBV (48 weeks, 5 prior partial responders/breakthroughs, 10 prior null responders). The primary objective was safety; sustained virologic response 12 weeks post-treatment (SVR12) was a secondary end-point. RESULTS: All except one patient experienced drug-related adverse events. Adverse events led to faldaprevir discontinuation in 1 (2%), 13 (20%), and 3 (6.8%) patients on faldaprevir 120 mg, faldaprevir 240 mg 12 weeks, and faldaprevir 240 mg 24 weeks, respectively. The SVR12 rates were: 86% with faldaprevir 120 mg and 74% with faldaprevir 240 mg among treatment-naïve patients; and 86%, 60%, and 40% among prior relapsers, partial responders/breakthroughs, and null responders, respectively. CONCLUSIONS: In treatment-naïve Japanese patients, faldaprevir 120 mg q.d. plus PegIFNα-2b/RBV was better tolerated than faldaprevir 240 mg q.d. plus PegIFNα-2b/RBV, with at least comparable efficacy. In treatment-experienced patients, most prior relapsers achieved SVR12 with 24 weeks of faldaprevir 240 mg q.d. plus PegIFNα-2b/RBV. Clinicaltrials.gov NCT01579474.

Baseline hepatitis C virus (HCV) NS3 polymorphisms and their impact on treatment response in clinical studies of the HCV NS3 protease inhibitor faldaprevir.

A challenge to the treatment of chronic hepatitis C with direct-acting antivirals is the emergence of drug-resistant hepatitis C virus (HCV) variants. HCV with preexisting polymorphisms that are associated with resistance to NS3/4A protease inhibitors have been detected in patients with chronic hepatitis C. We performed a comprehensive pooled analysis from phase 1b and phase 2 clinical studies of the HCV protease inhibitor faldaprevir to assess the population frequency of baseline protease inhibitor resistance-associated NS3 polymorphisms and their impact on response to faldaprevir treatment. A total of 980 baseline NS3 sequences were obtained (543 genotype 1b and 437 genotype 1a sequences). Substitutions associated with faldaprevir resistance (at amino acid positions 155 and 168) were rare (<1% of sequences) and did not compromise treatment response: in a phase 2 study in treatment-naive patients, six patients had faldaprevir resistance-associated polymorphisms at baseline, of whom five completed faldaprevir-based treatment and all five achieved a sustained virologic response 24 weeks after the end of treatment (SVR24). Among 13 clinically relevant amino acid positions associated with HCV protease resistance, the greatest heterogeneity was seen at NS3 codons 132 and 170 in genotype 1b, and the most common baseline substitution in genotype 1a was Q80K (99/437 [23%]). The presence of the Q80K variant did not reduce response rates to faldaprevir-based treatment. Across the three phase 2 studies, there was no significant difference in SVR24 rates between patients with genotype 1a Q80K HCV and those without Q80K HCV, whether treatment experienced (17% compared to 26%; P = 0.47) or treatment naive (62% compared to 66%; P = 0.72).

Molecular determinants and dynamics of hepatitis C virus secretion.

The current model of hepatitis C virus (HCV) production involves the assembly of virions on or near the surface of lipid droplets, envelopment at the ER in association with components of VLDL synthesis, and egress via the secretory pathway. However, the cellular requirements for and a mechanistic understanding of HCV secretion are incomplete at best. We combined an RNA interference (RNAi) analysis of host factors for infectious HCV secretion with the development of live cell imaging of HCV core trafficking to gain a detailed understanding of HCV egress. RNAi studies identified multiple components of the secretory pathway, including ER to Golgi trafficking, lipid and protein kinases that regulate budding from the trans-Golgi network (TGN), VAMP1 vesicles and adaptor proteins, and the recycling endosome. Our results support a model wherein HCV is infectious upon envelopment at the ER and exits the cell via the secretory pathway. We next constructed infectious HCV with a tetracysteine (TC) tag insertion in core (TC-core) to monitor the dynamics of HCV core trafficking in association with its cellular cofactors. In order to isolate core protein movements associated with infectious HCV secretion, only trafficking events that required the essential HCV assembly factor NS2 were quantified. TC-core traffics to the cell periphery along microtubules and this movement can be inhibited by nocodazole. Sub-populations of TC-core localize to the Golgi and co-traffic with components of the recycling endosome. Silencing of the recycling endosome component Rab11a results in the accumulation of HCV core at the Golgi. The majority of dynamic core traffics in association with apolipoprotein E (ApoE) and VAMP1 vesicles. This study identifies many new host cofactors of HCV egress, while presenting dynamic studies of HCV core trafficking in infected cells.

Viral resistance in hepatitis C virus genotype 1-infected patients receiving the NS3 protease inhibitor Faldaprevir (BI 201335) in a phase 1b multiple-rising-dose study.

Faldaprevir (BI 201335) is a selective NS3/4A protease inhibitor under development for the treatment of chronic hepatitis C virus (HCV) infection. NS3/4A genotyping and NS3 protease phenotyping analyses were performed to monitor the emergence of resistance in patients with HCV genotype 1 infection receiving faldaprevir alone or combined with pegylated interferon alfa 2a and ribavirin (PegIFN-RBV) during a phase 1b study. Among all baseline variants, a maximum 7-fold reduction in in vitro sensitivity to faldaprevir was observed for a rare NS3 (V/I)170T polymorphism. During faldaprevir monotherapy in treatment-naive patients, virologic breakthrough was common (77%, 20/26) and was associated with the emergence of resistance mutations predominantly carrying NS3 substitutions R155K in GT1a and D168V in GT1b. D168V conferred a greater reduction in faldaprevir sensitivity (1,800-fold) than R155K (330-fold); however, D168V was generally less fit than R155K in the absence of selective drug pressure. Treatment-experienced patients treated with faldaprevir-PegIFN-RBV triple therapy showed higher viral load reductions, lower rates of breakthrough (8%, 5/62), and less frequent emergence of resistance-associated variants compared with faldaprevir monotherapy. (This study has been registered at ClinicalTrials.gov under registration no. NCT00793793.).

Antiviral effect, safety, and pharmacokinetics of five-day oral administration of Deleobuvir (BI 207127), an investigational hepatitis C virus RNA polymerase inhibitor, in patients with chronic hepatitis C.

Deleobuvir (BI 207127) is an investigational oral nonnucleoside inhibitor of hepatitis C virus (HCV) NS5B RNA polymerase. Antiviral activity, virology, pharmacokinetics, and safety were assessed in HCV genotype 1-infected patients receiving 5 days' deleobuvir monotherapy. In this double-blind phase 1b study, treatment-naive (TN; n = 15) and treatment-experienced (TE; n = 45) patients without cirrhosis received placebo or deleobuvir at 100, 200, 400, 800, or 1,200 mg every 8 h (q8h) for 5 days. Patients with cirrhosis (n = 13) received deleobuvir at 400 or 600 mg q8h for 5 days. Virologic analyses included NS5B genotyping and phenotyping of individual isolates. At day 5, patients without cirrhosis had dose-dependent median HCV RNA reductions of up to 3.8 log10 (with no placebo response); patients with cirrhosis had median HCV RNA reductions of approximately 3.0 log10. Three patients discontinued due to adverse events (AEs). The most common AEs were gastrointestinal, nervous system, and skin/cutaneous tissue disorders. Plasma exposure of deleobuvir was supraproportional at doses ≥ 400 mg q8h and approximately 2-fold higher in patients with cirrhosis than in patients without cirrhosis. No virologic breakthrough was observed. NS5B substitutions associated with deleobuvir resistance in vitro were detected in 9/59 patients; seven encoded P495 substitutions, including P495L, which conferred 120- to 310-fold-decreased sensitivity to deleobuvir. P495 variants did not persist in follow-up without selective drug pressure. Deleobuvir monotherapy was generally well tolerated and demonstrated dose-dependent antiviral activity against HCV genotype 1 over 5 days.

Dengue virus nonstructural protein 3 redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis.

Dengue virus (DENV) modifies cellular membranes to establish its sites of replication. Although the 3D architecture of these structures has recently been described, little is known about the cellular pathways required for their formation and expansion. In this report, we examine the host requirements for DENV replication using a focused RNAi analysis combined with validation studies using pharmacological inhibitors. This approach identified three cellular pathways required for DENV replication: autophagy, actin polymerization, and fatty acid biosynthesis. Further characterization of the viral modulation of fatty acid biosynthesis revealed that a key enzyme in this pathway, fatty acid synthase (FASN), is relocalized to sites of DENV replication. DENV nonstructural protein 3 (NS3) is responsible for FASN recruitment, inasmuch as (i) NS3 expressed in the absence of other viral proteins colocalizes with FASN and (ii) NS3 interacts with FASN in a two-hybrid assay. There is an associated increase in the rate of fatty acid biosynthesis in DENV-infected cells, and de novo synthesized lipids preferentially cofractionate with DENV RNA. Finally, purified recombinant NS3 stimulates the activity of FASN in vitro. Taken together, these experiments suggest that DENV co-opts the fatty acid biosynthetic pathway to establish its replication complexes. This study provides mechanistic insight into DENV membrane remodeling and highlights the potential for the development of therapeutics that inhibit DENV replication by targeting the fatty acid biosynthetic pathway.

Hepatitis C virus stimulates the phosphatidylinositol 4-kinase III alpha-dependent phosphatidylinositol 4-phosphate production that is essential for its replication.

Phosphatidylinositol 4-kinase III alpha (PI4KA) is an essential cofactor of hepatitis C virus (HCV) replication. We initiated this study to determine whether HCV directly engages PI4KA to establish its replication. PI4KA kinase activity was found to be absolutely required for HCV replication using a small interfering RNA transcomplementation assay. Moreover, HCV infection or subgenomic HCV replicons produced a dramatic increase in phosphatidylinositol 4-phosphate (PI4P) accumulation throughout the cytoplasm, which partially colocalized with the endoplasmic reticulum. In contrast, the majority of PI4P accumulated at the Golgi bodies in uninfected cells. The increase in PI4P was not observed after infection with UV-inactivated HCV and did not reflect changes in PI4KA protein or RNA abundance. In an analysis of U2OS cell lines with inducible expression of the HCV polyprotein or individual viral proteins, viral polyprotein expression resulted in enhanced cytoplasmic PI4P production. Increased PI4P accumulation following HCV protein expression was precluded by silencing the expression of PI4KA, but not the related PI4KB. Silencing PI4KA also resulted in aberrant agglomeration of viral replicase proteins, including NS5A, NS5B, and NS3. NS5A alone, but not other viral proteins, stimulated PI4P production in vivo and enhanced PI4KA kinase activity in vitro. Lastly, PI4KA coimmunoprecipitated with NS5A from infected Huh-7.5 cells and from dually transfected 293T cells. In sum, these results suggest that HCV NS5A modulation of PI4KA-dependent PI4P production influences replication complex formation.

Roles for endocytic trafficking and phosphatidylinositol 4-kinase III alpha in hepatitis C virus replication.

Hepatitis C virus (HCV) reorganizes cellular membranes to establish sites of replication. The required host pathways and the mechanism of cellular membrane reorganization are poorly characterized. Therefore, we interrogated a customized small interfering RNA (siRNA) library that targets 140 host membrane-trafficking genes to identify genes required for both HCV subgenomic replication and infectious virus production. We identified 7 host cofactors of viral replication, including Cdc42 and Rock2 (actin polymerization), EEA1 and Rab5A (early endosomes), Rab7L1, and PI3-kinase C2gamma and PI4-kinase IIIalpha (phospholipid metabolism). Studies of drug inhibitors indicate actin polymerization and phospholipid kinase activity are required for HCV replication. We found extensive co-localization of the HCV replicase markers NS5A and double-stranded RNA with Rab5A and partial co-localization with Rab7L1. PI4K-IIIalpha co-localized with NS5A and double-stranded RNA in addition to being present in detergent-resistant membranes containing NS5A. In a comparison of type II and type III PI4-kinases, PI4Ks were not required for HCV entry, and only PI4K-IIIalpha was required for HCV replication. Although PI4K-IIIalpha siRNAs decreased HCV replication and virus production by almost 100%, they had no effect on initial HCV RNA translation, suggesting that PI4K-IIIalpha functions at a posttranslational stage. Electron microscopy identified the presence of membranous webs, which are thought to be the site of HCV replication, in HCV-infected cells. Pretreatment with PI4K-IIIalpha siRNAs greatly reduced the accumulation of these membranous web structures in HCV-infected cells. We propose that PI4K-IIIalpha plays an essential role in membrane alterations leading to the formation of HCV replication complexes.

RNA interference and single particle tracking analysis of hepatitis C virus endocytosis.

Hepatitis C virus (HCV) enters hepatocytes following a complex set of receptor interactions, culminating in internalization via clathrin-mediated endocytosis. However, aside from receptors, little is known about the cellular molecular requirements for infectious HCV entry. Therefore, we analyzed a siRNA library that targets 140 cellular membrane trafficking genes to identify host genes required for infectious HCV production and HCV pseudoparticle entry. This approach identified 16 host cofactors of HCV entry that function primarily in clathrin-mediated endocytosis, including components of the clathrin endocytosis machinery, actin polymerization, receptor internalization and sorting, and endosomal acidification. We next developed single particle tracking analysis of highly infectious fluorescent HCV particles to examine the co-trafficking of HCV virions with cellular cofactors of endocytosis. We observe multiple, sequential interactions of HCV virions with the actin cytoskeleton, including retraction along filopodia, actin nucleation during internalization, and migration of internalized particles along actin stress fibers. HCV co-localizes with clathrin and the ubiquitin ligase c-Cbl prior to internalization. Entering HCV particles are associated with the receptor molecules CD81 and the tight junction protein, claudin-1; however, HCV-claudin-1 interactions were not restricted to Huh-7.5 cell-cell junctions. Surprisingly, HCV internalization generally occurred outside of Huh-7.5 cell-cell junctions, which may reflect the poorly polarized nature of current HCV cell culture models. Following internalization, HCV particles transport with GFP-Rab5a positive endosomes, which is consistent with trafficking to the early endosome. This study presents technical advances for imaging HCV entry, in addition to identifying new host cofactors of HCV infection, some of which may be antiviral targets.

Resistance Analyses of HCV NS3/4A Protease and NS5B Polymerase from Clinical Studies of Deleobuvir and Faldaprevir.

BACKGROUND & AIM: The resistance profile of anti-hepatitis C virus (HCV) agents used in combination is important to guide optimal treatment regimens. We evaluated baseline and treatment-emergent NS3/4A and NS5B amino-acid variants among HCV genotype (GT)-1a and -1b-infected patients treated with faldaprevir (HCV protease inhibitor), deleobuvir (HCV polymerase non-nucleoside inhibitor), and ribavirin in multiple clinical studies. METHODS: HCV NS3/4A and NS5B population sequencing (Sanger method) was performed on all baseline plasma samples (n = 1425 NS3; n = 1556 NS5B) and on post-baseline plasma samples from patients with virologic failure (n = 113 GT-1a; n = 221 GT-1b). Persistence and time to loss of resistance-associated variants (RAVs) was estimated using Kaplan-Meier analysis. RESULTS: Faldaprevir RAVs (NS3 R155 and D168) and deleobuvir RAVs (NS5B 495 and 496) were rare (<1%) at baseline. Virologic response to faldaprevir/deleobuvir/ribavirin was not compromised by common baseline NS3 polymorphisms (e.g. Q80K in 17.5% of GT-1a) or by NS5B A421V, present in 20% of GT-1a. In GT-1b, alanine at NS5B codon 499 (present in 15% of baseline sequences) was associated with reduced response. Treatment-emergent RAVs consolidated previous findings: NS3 R155 and D168 were key faldaprevir RAVs; NS5B A421 and P495 were key deleobuvir RAVs. Among on-treatment virologic breakthroughs, RAVs emerged in both NS3 and NS5B (>90%). Virologic relapse was associated with RAVs in both NS3 and NS5B (53% GT-1b; 52% GT-1b); some virologic relapses had NS3 RAVs only (47% GT-1a; 17% GT-1b). Median time to loss of GT-1b NS5B P495 RAVs post-treatment (5 months) was less than that of GT-1b NS3 D168 (8.5 months) and GT-1a R155 RAVs (11.5 months). CONCLUSION: Faldaprevir and deleobuvir RAVs are more prevalent among virologic failures than at baseline. Treatment response was not compromised by common NS3 polymorphisms; however, alanine at NS5B amino acid 499 at baseline (wild-type in GT-1a, polymorphism in GT-1b) may reduce response to this deleobuvir-based regimen.

Daclatasvir inhibits hepatitis C virus NS5A motility and hyper-accumulation of phosphoinositides.

Combinations of direct-acting antivirals (DAAs) against the hepatitis C virus (HCV) have the potential to revolutionize the HCV therapeutic regime. An integral component of DAA combination therapies is HCV NS5A inhibitors. It has previously been proposed that NS5A DAAs inhibit two functions of NS5A: RNA replication and virion assembly. In this study, we characterize the impact of a prototype NS5A DAA, daclatasvir (DCV), on HCV replication compartment formation. DCV impaired HCV replicase localization and NS5A motility. In order to characterize the mechanism behind altered HCV replicase localization, we examined the impact of DCV on the interaction of NS5A with its essential cellular cofactor, phosphatidylinositol-4-kinase III α (PI4KA). We observed that DCV does not inhibit PI4KA directly, nor does it impair early events of the NS5A-PI4KA interaction that can occur when NS5A is expressed alone. NS5A functions that are unaffected by DCV include PI4KA binding, as determined by co-immunoprecipitation, and a basal accumulation of the PI4KA product, PI4P. However, DCV impairs late steps in PI4KA activation that requires NS5A expressed in the context of the HCV polyprotein. These NS5A functions include hyper-stimulation of PI4P levels and appropriate replication compartment formation. The data are most consistent with a model wherein DCV inhibits conformational changes in the NS5A protein or protein complex formations that occur in the context of HCV polyprotein expression and stimulate PI4P hyper-accumulation and replication compartment formation.

Possibilities for RNA interference in developing hepatitis C virus therapeutics.

The discovery and characterization of the RNA interference (RNAi) pathway has been one of the most important scientific developments of the last 12 years. RNAi is a cellular pathway wherein small RNAs control the expression of genes by either degrading homologous RNAs or preventing the translation of RNAs with partial homology. It has impacted basic biology on two major fronts. The first is the discovery of microRNAs (miRNAs), which regulate almost every cellular process and are required for some viral infections, including hepatitis C virus (HCV). The second front is the use of small interfering RNAs (siRNAs) as the first robust tool for mammalian cellular genetics. This has led to the identification of hundreds of cellular genes that are important for HCV infection. There is now a major push to adapt RNAi technology to the clinic. In this review, we explore the impact of RNAi in understanding HCV biology, the progress in design of RNAi-based therapeutics for HCV, and remaining obstacles.

Potential roles for cellular cofactors in hepatitis C virus replication complex formation.

Over 130 million people worldwide are chronically infected with hepatitis C virus (HCV). New antiviral treatment strategies are needed due to limitations with current therapy. The identification of cellular cofactors of infection has the potential to broadly expand our therapeutic targets. We recently reported an RNA interference screen of host membrane trafficking genes in HCV infection and replication and identified several cellular co-factors for viral replication. Phosphatidylinositol 4-kinase III alpha (PI4K-IIIalpha) was found to be essential for HCV replication. PI4K-IIIalpha co-localized with viral replication markers. Silencing of PI4K-IIIalpha by siRNAs prior to HCV infection prevented rearrangement of intracellular membranes associated with viral replication complexes, termed the membranous web. Our data suggest that PI4K-IIIalpha is involved in establishing HCV replication complexes, however the mechanism is unknown. From our analysis, along with several other studies that have identified cellular cofactors for HCV replication, we propose that PI4K-IIIalpha may nucleate replication complex formation by facilitating the interaction of viral and/or cellular proteins with cellular membrane-associated phospholipids.

Cervical keratinocytes containing stably replicating extrachromosomal HPV-16 are refractory to transformation by oncogenic H-Ras.

Ras expression in human epithelial cells with integrated HPV genomes has been shown to cause tumorigenic transformation. The effects of Ras in cells representing early stage HPV-associated disease (i.e., when HPV is extrachromosomal and the oncogenes are under control of native promoters) have not been examined. Here, we used human cervical keratinocyte cell lines containing stably replicating extrachromosomal HPV-16 and present the novel finding that these cells resist transformation by oncogenic H-Ras. Ras expression consistently diminished anchorage-independent growth (AI), reduced E6 and E7 expression, and caused p53 induction in these cells. Conversely, AI was enhanced or maintained in Ras-transduced cervical cells that were immortalized with a 16E6/E7 retrovirus, and minimal effects on E6 and E7 expression were observed. Ras expression with either episomal HPV-16 or LXSN-E6/E7 was insufficient for tumorigenic growth suggesting that other events are needed for tumorigenic transformation. In conclusion, our results indicate that Ras-mediated transformation depends on the context of HPV oncogene expression and that this is an important point to address when developing HPV tumor models.

Propagation of infectious human papillomavirus type 16 by using an adenovirus and Cre/LoxP mechanism.

Human papillomavirus type 16 (HPV16) infection is a major risk factor for the development of squamous cell cancers of the cervix and of the head and neck. A major barrier to understanding the progression from initial infection to cancer has been the lack of in vitro models that allow infection, replication, and persistence of the viral genome as an episome in differentiated epithelial cells. To overcome this barrier, we designed an adenoviral delivery vector that contained a full HPV16 genome flanked by LoxP homologous recombination sites and a fluorescent reporter that was expressed only after the HPV genome was excised by Cre recombinase. This system delivered circular HPV16 genomes to cervical epithelial cells and well differentiated human airway epithelia. After delivery, the HPV16 genome replicated and persisted as an episome in cervical keratinocytes. These cells developed an immortalized phenotype and a dysplastic epithelial appearance. Moreover, induction of differentiation led to the expression of late genes and production of infectious HPV16 virions. This work provides a means of introducing biologically active HPV genomes into epithelial cells, which are normally difficult to transfect. These methods allow the study of HPV genome replication and gene expression in the earliest stages of HPV genome establishment, and they may provide a means to study nononcogenic HPV viral types.

Telomerase activation in cervical keratinocytes containing stably replicating human papillomavirus type 16 episomes.

Retroviral transduction and expression of the human papillomavirus type 16 (HPV-16) E6 gene has been shown to activate telomerase in human cervical and foreskin keratinocytes. There still remains some controversy, however, as to whether expression of E6 in the context of the whole HPV-16 genome can activate telomerase. In this study, we have generated human cervical keratinocyte clones that contain stably replicating HPV-16 episomes. Interestingly, the majority of the clones exhibited low or no telomerase activity at early passage and this was associated with low transcript levels of the reverse transcriptase component of telomerase, hTERT. The HPV-16-containing clones became immortal without a crisis and, at later passage, exhibited elevated levels of telomerase and higher levels of hTERT without any apparent increase in HPV-16 copy number, E6 transcript levels, or ability to degrade p53. These results indicate that HPV-16 by itself does not necessarily cause telomerase activation in cervical keratinocytes, but rather, supports a model in which HPV-16 facilitates telomerase activation in conjunction with other viral or cellular changes over time.