HCV Replication Research Articles

Design, Synthesis and Structure-Activity Relationship Optimization of Lycorine Derivatives for HCV Inhibition.

Lycorine is reported to be a multifunctional compound. We previously showed that lycorine is an HCV inhibitor with strong activity. Further research on the antivirus mechanism indicated that lycorine does not affect the enzymes that are indispensable to HCV replication but suppresses the expression of Hsc70 in the host cell to limit HCV replication. However, due to the cytotoxicity and apoptosis induction of lycorine, lycorine is unsafe to be a anti-HCV agent for clinical application. As a result of increasing interest, its structure was optimized for the first time and a novel series of lycorine derivatives was synthesized, all of which lost their cytotoxicity to different degrees. Structure-activity analysis of these compounds revealed that disubstitution on the free hydroxyl groups at C1 and C2 and/or degradation of the benzodioxole group would markedly reduce the cytotoxicity. Furthermore, an α, β-unsaturated ketone would improve the HCV inhibitory activity of lycorine. The C3-C4 double bond is crucial to the anti-HCV activity because hydrogenation of this double bond clearly weakened HCV inhibition.

Serum miR‐122 may serve as a biomarker for response to direct acting antivirals: effect of paritaprevir/R with dasabuvir or ombitasvir on miR‐122 in HCV‐infected subjects

Circulating microRNAs (miRNA) have been intensely investigated as biomarkers in disease and therapy. Several studies have identified miR-122 as an important regulator of HCV replication. The effect of new therapies that directly target the HCV replication life cycle on circulating microRNA levels has not been elucidated. We performed expression profiling of circulating miRNA in serum in subjects treated with HCV direct-acting antiviral agents (DAAs). Serum miRNA levels were evaluated from two studies in HCV GT1-infected treatment-naïve subjects and prior nonresponders to pegylated interferon (pegIFN) and ribavirin (RBV) who received paritaprevir/ritonavir + dasabuvir + RBV for 12 weeks, and in treatment-naïve genotype (GT)1-3-infected subjects who received paritaprevir/ritonavir + ombitasvir ± RBV for 12 weeks. Over 100 different miRNA species were detected in serum. Of these, levels of miR-122 showed the most consistent change in response to treatment across all HCV genotypes. In all subjects, miR-122 showed an average four-fold reduction between baseline and week 2, and remained below baseline through post-treatment week 12 in subjects who achieved sustained virological response. In contrast, in subjects who did not achieve SVR, miR-122 levels began to return to baseline levels after the second week of treatment. The change in miR-122 levels was similar across genotypes, and was comparable with or without RBV. This is the first report comparing expression levels of circulating miRNA in HCV GT1-3 subjects treated with IFN-free combinations of DAAs. The results suggest that serum levels of miR-122 are reduced following treatment in subjects who achieve SVR, and correlate with HCV RNA levels across genotypes.

Synthesis and antiviral evaluation of 2',3'-dideoxy-2',3'-difluoro-D-arabinofuranosyl 2,6-disubstituted purine nucleosides.

The synthesis of new 2,6-disubstituted purine 2',3'-dideoxy-2',3'-difluoro-D-arabino nucleosides is reported. Their ability to block HIV and HCV replication along with their cytotoxicity toward Huh-7 cells, human lymphocyte, CEM and Vero cells was also assessed. Among them, β-2,6-diaminopurine nucleoside 25 and guanosine derivative 27 demonstrate potent anti-HIV-1 activity (EC50 = 0.56 and 0.65 μM; EC90 = 4.2 and 3.1 μM) while displaying only moderate cytotoxicity in primary human lymphocytes.

Sofosbuvir and Ledipasvir in IBD Patients Treated With Advanced Biologics Including Ribavirin Eradicating Chronic Hepatitis C: Solataire C Trial: A Multicenter Clinical Prospective Pilot Study

Introduction: The prevalence and concomitant treatment of chronic HCV and IBD has not been elucidated. IBD and CHC of >20 years duration may have more fibrosis due to immune suppression. Treatment of IBD often requires biological therapies to achieve longer disease-free remission, which further accelerates higher HCV replication. Thus effective therapy for IBD patients requiring immunosuppressive therapy is needed. To evaluate the role and efficacy of new NS5A + NS5B inhibitors with and without RBV in treating CHC in moderate to severe IBD requiring biologic therapy Methods: 35 patients were recruited from the Kings County Hospital Medical Center, Brooklyn. Inclusion criteria CHC with IBD, Age: >18, HCV Viral Load: > 400,000IU/mL, Genotype 1. Fibrotic Score: Metavir F1 to F4. Primary End Point: SVR12 Secondary End Point: IBD activity index, sustained control of symptoms SVR 24 and complete IBD remission at 30 weeks (endoscopy at 30 weeks). The patients were divided into two groups: Group A (n=17)- RBV 1000 mg + LDV and SOF; for 8 weeks Group B (n=18): LDV and SOF; for 12 weeks All will have base line RAV and ETRAV's and SVR 24. RAVs 5A polymorphism by Quest. Viral loads 0, 7, 14 days; 4th, 8th, 12th and 24th weeks. Results: see table. Conclusion: This study demonstrates that DAA's for HCV appear to have similar efficacy and safety in the presence of active IBD therapy withTNF Alfa antagonists while keeping the IBD in uninterrupted remission.Table 1: Results

Growing our knowledge of HCV replication

Growing our knowledge of HCV replication

Longitudinal analysis of peripheral and intrahepatic NK cells in chronic HCV patients during antiviral therapy

IntroductionA strong immune response is integral to the clearance of HCV infection. NK cells are specialized cells that are able to inhibit replication of HCV in infected hepatocytes. Previous studies have correlated therapy outcome in HCV to the expression of various markers on NK cells. However, the effect of viral load reduction on NK cell function during therapy is still largely unknown, particularly in the liver. Therefore we investigated NK cell phenotype and effector function in both the peripheral and intrahepatic compartments during the course of antiviral therapy in chronic HCV patients. MethodsChronic HCV patients were treated for 24 or 48weeks with triple therapy consisting of telaprevir, pegIFN-α and ribavirin. Blood and fine needle aspiration (FNA) biopsies of the liver were collected at start and 6h, 1week and 12weeks during therapy. Flowcytometry was performed for expression of different markers (NKG2A, NKG2D, NKp46, and CD69). ResultsOur results demonstrate a highly activated phenotype of NK cells in liver compared to blood in chronic HCV patients. Six hours after start of triple therapy, no activation of intrahepatic NK cells was observed in the liver as compared to baseline. At 1week after start of triple therapy, the frequency of NK cells with the activating receptor NKp46 was increased in blood, whereas at week 12, the frequencies of the inhibitory receptor NKG2A was increased. No alterations were observed during therapy in liver NK cell phenotype. ConclusionIFN-based therapy for chronic HCV affects NK cell phenotype in peripheral blood more than in the liver.

HCV dsRNA-Activated Macrophages Inhibit HCV Replication in Hepatocytes.

Background:Macrophages play critical roles in innate immune response in the liver. Whether macrophages participate in liver innate immunity against HCV replication is poorly understoodObjectives:The aim of this study was to investigate the role of macrophages in liver innate immunity against HCV replication.Materials and Methods:Freshly isolated monocytes were purified from peripheral blood of healthy adult donors. Macrophages refer to 7-day-cultured monocytes in vitro. A hepatoma cell line (Huh7) was infected with HCV JFH-1 to generate in vitro HCV infectious system. RT-PCR was used to determine HCV RNA and mRNA levels of genes expression. ELISA was used to measure the protein level of interferon-α (IFN-α) and western blot was used to determine protein expression level of Toll-like receptor 3 (TLR3).Results:HCV dsRNA induced the expression of type I IFN (IFN-α/β) in monocyte-derived macrophages. HCV dsRNA also induced the expression of TLR3 and IFN regulatory factor-7 (IRF-7), the key regulators of the IFN signaling pathway. When HCV JFH-1-infected Huh7 cells were co-cultured with macrophages activated with HCV dsRNA or incubated in media conditioned with supernatant (SN) from HCV dsRNA-activated macrophages, HCV replication was significantly suppressed. This macrophage SN action on HCV inhibition was mediated through type I IFN, which was evidenced by the observation that antibody to type I IFN receptor could neutralize the macrophages-mediated anti-HCV effect. The role of type I IFN in macrophages-mediated anti-HCV activity is further supported by the observation that HCV dsRNA-activated macrophages SN treatment induced the expression of several IFN-stimulated genes (ISGs), ISG15, ISG56, OAS-1, OAS-2, MxA and Viperin in HCV-infected Huh7 cells.Conclusions:Macrophages may play an important role in liver innate immunity against HCV replication through a type I IFN-dependent mechanism.

Novel methods of hepatitis C treatment and prevention

Despite available treatment, Hepatitis C remains one of most serious burdens to public health. Current therapy based on pegylated interferon-alpha and ribavirin has significant side effects and its effectiveness varies for different genotypes of the virus. Four novel drugs - viral protease inhibitors (telaprevir, boceprevir, simeprevir) and polymerase inhibitor - sofosbuvir have been introduced in last years for use in combination with standard-of-care treatment. For the first time interferon free therapies were approved with the use of combination of sofosbuvir+ribavirin. New therapies improve virological response rates but also increase the cost, side effects and raise the issue of drug resistance. Numerous novel anti-HCV compounds have been evaluated in advanced clinical trials including inhibitors of viral proteins (protease, polymerase and NS5A) and inhibitors of host factors involved in HCV replication (cyclophilin A, microRNA - miR-122). New interferon-free therapies reducing severe side effects are expected to enter the market within few months. At the same time efforts are undertaken to determine the host and viral factors with predictive value for HCV treatment response, enabling personalized therapy approach. The main success in this field was the discovery of interleukin IL28B polymorphism, which correlates with positive standard-of-care treatment response. An effective vaccination may be an alternative for antiviral drugs, but no anti-HCV vaccine is available currently. It is well proved that successful vaccination should induce antibody and T-cell responses specific against a range of HCV genotypes. With this aim, new subunit and genetic candidate vaccines have been evaluated in I and II phase clinical trials. This review summarizes the recent developments in the field of new drug development and vaccine studies against hepatitis C virus.

Improvement of liver function parameters in advanced HCV-associated liver cirrhosis by IFN-free antiviral therapies

Successful antiviral treatment of decompensated hepatitis B with HBV polymerase inhibitors is associated with improvement of liver function and even de-listing of patients on the transplant waiting list. To what extent liver function also improves in cirrhotic patients with chronic hepatitis C receiving novel IFN-free therapies is unknown. We here analysed 80 consecutive patients with advanced HCV associated liver cirrhosis including 34 patients (43%) with Child B/C cirrhosis and 42 patients (53%) with platelet counts of We show that (i) liver function parameters recover in the majority of patients when HCV replication is successfully blocked irrespectively of the underlying HCV genotype, (ii) that, however, liver disease still can progress in some patients despite achieving a sustained virological response and (iii) that HCV relapse after transient suppression of HCV replication is usually not associated with clinically meaningful worsening of hepatic function even though severe biochemical hepatitis flares can occur in single cases.

Host cell mTORC1 is required for HCV RNA replication

ObjectiveChronically HCV-infected orthotopic liver transplantation (OLT) recipients appear to have improved outcomes when their immunosuppressive regimen includes a mammalian target of rapamycin (mTOR) inhibitor. The mechanism underlying this observation is...

The Novel Cyclophilin Inhibitor CPI-431-32 Concurrently Blocks HCV and HIV-1 Infections via a Similar Mechanism of Action.

HCV-related liver disease is the main cause of morbidity and mortality of HCV/HIV-1 co-infected patients. Despite the recent advent of anti-HCV direct acting antivirals (DAAs), the treatment of HCV/HIV-1 co-infected patients remains a challenge, as these patients are refractory to most therapies and develop liver fibrosis, cirrhosis and liver cancer more often than HCV mono-infected patients. Until the present study, there was no suitable in vitro assay to test the inhibitory activity of drugs on HCV/HIV-1 co-infection. Here we developed a novel in vitro “co-infection” model where HCV and HIV-1 concurrently replicate in their respective main host target cells—human hepatocytes and CD4+ T-lymphocytes. Using this co-culture model, we demonstrate that cyclophilin inhibitors (CypI), including a novel cyclosporin A (CsA) analog, CPI-431-32, simultaneously inhibits replication of both HCV and HIV-1 when added pre- and post-infection. In contrast, the HIV-1 protease inhibitor nelfinavir or the HCV NS5A inhibitor daclatasvir only blocks the replication of a single virus in the “co-infection” system. CPI-431-32 efficiently inhibits HCV and HIV-1 variants, which are normally resistant to DAAs. CPI-431-32 is slightly, but consistently more efficacious than the most advanced clinically tested CypI—alisporivir (ALV)—at interrupting an established HCV/HIV-1 co-infection. The superior antiviral efficacy of CPI-431-32 over ALV correlates with its higher potency inhibition of cyclophilin A (CypA) isomerase activity and at preventing HCV NS5A-CypA and HIV-1 capsid-CypA interactions known to be vital for replication of the respective viruses. Moreover, we obtained evidence that CPI-431-32 prevents the cloaking of both the HIV-1 and HCV genomes from cellular sensors. Based on these results, CPI-431-32 has the potential, as a single agent or in combination with DAAs, to inhibit both HCV and HIV-1 infections.

Arginine 112 is involved in HCV translation modulation by NS5A domain I

HCV NS5A has three domains, which have multiple roles in the viral life cycle. We previously found that NS5A is able to down-regulate HCV RNA translation through a mechanism requiring the polyU/UC region within the viral 3′UTR to which NS5A binds. In this study, we further investigated the role of domain I in modulating viral translation. Using a series of deletion and substitution mutants, we identified a number of positively charged residues that played a role in this modulatory effect, most prominently R112. The R112A mutation negated the ability of domain I and full-length NS5A to modulate viral translation. Additionally, the R112A mutation impeded domain I binding to the polyU/UC RNA, suggesting a mechanism for this down-regulatory effect. Finally, the R112A mutation rendered HCV replication deficient. These results collectively point to a crucial role for the R112 residue of NS5A in the modulation of HCV life cycle by NS5A.

K312 and E446 are involved in HCV RNA translation modulation by NS5A domains II and III

HCV NS5A plays a critical role in the HCV life cycle. We previously demonstrated that NS5A down-regulates viral translation through a mechanism requiring the polyU/UC region of the viral 3′UTR and that each of the three domains is capable of carrying out this function individually. In this study, we mapped the regions and amino acid residues within domains II and III involved in the modulation of viral translation. Using a series of deletion and amino acid substitution mutants, we found that K312 and E446 play important roles in the modulation of viral translation by NS5A domains II and III, respectively. In the context of full-length NS5A, mutations of K312 and E446 alone or in combination again abrogate translation down-regulation. In a transient replication assay using HCV subgenomic replicons, the K312A mutation alone does not affect HCV replication throughout a 96-h time course. While the E446A mutation can increase HCV replication at early time points (4–24h), the K312A and E446A double mutation can enhance viral replication at 24–96h, suggesting both residues are involved. Our results shed more light on the functions of NS5A.

Modelling hepatitis C therapy--predicting effects of treatment.

Mathematically modelling changes in HCV RNA levels measured in patients who receive antiviral therapy has yielded many insights into the pathogenesis and effects of treatment on the virus. By determining how rapidly HCV is cleared when viral replication is interrupted by a therapy, one can deduce how rapidly the virus is produced in patients before treatment. This knowledge, coupled with estimates of the HCV mutation rate, enables one to estimate the frequency with which drug resistant variants arise. Modelling HCV also permits the deduction of the effectiveness of an antiviral agent at blocking HCV replication from the magnitude of the initial viral decline. One can also estimate the lifespan of an HCV-infected cell from the slope of the subsequent viral decline and determine the duration of therapy needed to cure infection. The original understanding of HCV RNA decline under interferon-based therapies obtained by modelling needed to be revised in order to interpret the HCV RNA decline kinetics seen when using direct-acting antiviral agents (DAAs). There also exist unresolved issues involving understanding therapies with combinations of DAAs, such as the presence of detectable HCV RNA at the end of therapy in patients who nonetheless have a sustained virologic response.

Design, synthesis and evaluation of novel anti-HCV molecules that deliver intracellularly three highly potent NS5A inhibitors.

The design and synthesis of new non-symmetrical NS5A inhibitors with sulfur containing amino acids is reported along with their ability to block HCV replication in an HCV 1b replicon system. These compounds display EC50 values in the picomolar range with a large therapeutic index (>10(6)). Moreover, cellular pharmacology studies show that our preferred compounds intracellularly deliver three potent NS5A inhibitors.

PTU-113 Osteopontin is involved in chronic HBV infection and enhances HBV replication and HBSAG secretion

Introduction Hepatitis B virus (HBV) requires host cellular machinery such as cyclophilins to support its ongoing propagation (Phillips et al , Gastroenterology 2014) these host proteins represent ideal candidates for therapeutic interventions as they are generally expected to have a lower frequency of drug-resistance and antiviral efficacy across genotypes. We have also recently described a role for the host protein Osteopontin (OPN), a pro-fibrogenic downstream effector of the Hedgehog pathway, in enhancing HCV replication (Choi et al , Clinical Sciences 2014). Whilst increased levels of blood OPN have similarly been reported in chronic Hepatitis B (CHB), its role in viral replication remains unknown. This study aimed to evaluate the role of OPN in HBV replication, HBsAg secretion and HBV-driven liver injury. Method Stably (HepG2215), transiently (HUH-7) transfected and infected (HepaRG) cell lines, producing full HBV virions and HBsAg particles were cultured over 72 h in the presence/absence of several concentrations of recombinant OPN (recOPN). In addition, secreted OPN was neutralised using OPN-specific aptamers. Cells and supernatants were harvested at baseline, 24, 48 and 72 h. Intracellular OPN mRNA and HBV-DNA levels were quantitated by Real-Time qPCR. HBsAg levels were measured by ELISA. OPN levels were also measured by ELISA in cell culture supernatants and in sera of controls and HBeAg (+) CHB patients who were either treatment naive or treated with potent antiviral agents. In addition, expression of OPN was assessed in explanted livers from healthy and HBV-cirrhotic patients. Results Serum levels and intrahepatic expression of OPN were significantly increased in CHB patients compared to controls (up to 7 fold; p In vitro , mRNA and protein levels of OPN were highly correlated with intracellular HBV-DNA (r = 0.858; p Conclusion These data confirm that OPN is upregulated in the blood and livers of CHB patients. We also show that OPN directly augments HBV replication, thus identifying a novel therapeutic target. Disclosure of interest None Declared.

P097: HCV replication activity is modified by mTorinhibitors in vitro which is mediated by PML

P097: HCV replication activity is modified by mTorinhibitors in vitro which is mediated by PML

Altered effector functions of NK cells in chronic hepatitis C are associated with IFNL3 polymorphism.

Interferon α-mediated effector functions of NK cells may contribute to the control of HCV replication and the pathogenesis of liver disease. The single-nucleotide polymorphism rs12979860 near IFNL3 (previously known as IL28B) is important in response to IFN-α treatment and in spontaneous resolution of acute hepatitis C. The role of the IFNL3 polymorphism in NK cell function is unclear. Thus, we investigated the role of IFNL3 polymorphism in type I IFN-dependent regulation of NK cell functions in patients with cHC and healthy control subjects. We demonstrated a marked polarization of NK cells toward cytotoxicity in response to IFN-α stimulation in patients with hepatitis C. That TRAIL up-regulation was present, particularly in patients with the IFNL3-TT allele, was supported by a shift in the pSTAT-1:pSTAT-4 ratios toward pSTAT-1. In patients bearing the IFNL3-TT allele, NK cell effector function correlated with liver disease activity. In contrast, higher cytokine production of NK cells was observed in healthy individuals with the IFNL3-CC genotype, which may support spontaneous HCV clearance in acute infection. Overall, these findings show that the role of NK cells may differ in chronic infection vs. early antiviral defense and that the IFNL3 genotype differentially influences NK cell function.

LRRFIP1 Inhibits Hepatitis C Virus Replication by Inducing Type I Interferon in Hepatocytes.

Background:Hepatitis C virus infection is one of the leading causes of end stage liver diseases. The innate immune response slows down viral replication by activating cytokines such as type I interferon (IFN-α/β), which trigger the synthesis of antiviral proteins and modulate the adaptive immune system. Recently, leucine-rich repeat (in Flightless I) interacting protein-1 (LRRFIP1) was reported contributing to the production of interferon-β in macrophages.Objectives:The aim of this study was to assess the role of LRRFIP1 in induction of IFN-β and inhibition of HCV infection in hepatocytes.Materials and Methods:Induction of IFN-β by LRRFIP1 in Huh7 and Huh7.5.1 was determined by real-time PCR and western blotting in vitro. Inhibition of HCV replication by LRRFIP1 overexpression in hepatocytes was assessed.Results:LRRFIP1 increased the expression of IFN-β in hepatocytes with or without HCV infection. Induction of IFN-β by LRRFIP1 was enhanced with the presence of hepatitis C virus. Overexpression of LRRFIP1 in hepatocytes inhibited HCV replication. However, HCV infection did not regulate intracellular expression of LRRFIP1.Conclusions:LRRFIP1 and its mediated production of type I interferon play a role in controlling HCV infection. The findings of this study provide new target for HCV treatment and contribute to development of anti-HCV drugs.

Hepatocellular carcinoma: from hepatocyte to liver cancer stem cell.

Hepatocellular carcinoma: from hepatocyte to liver cancer stem cell.