Alexandre Soulier

Human Serum Facilitates Hepatitis C Virus Infection, and Neutralizing Responses Inversely Correlate with Viral Replication Kinetics at the Acute Phase of Hepatitis C Virus Infection

ABSTRACT The factors leading to spontaneous clearance of hepatitis C virus (HCV) or to viral persistence are elusive. Understanding virus-host interactions that enable acute HCV clearance is key to the development of more effective therapeutic and prophylactic strategies. Here, using a sensitive neutralization assay based on infectious HCV pseudoparticles (HCVpp), we have studied the kinetics of humoral responses in a cohort of acute-phase patients infected during a single nosocomial outbreak in a hemodialysis center. The 17 patients were monitored for the spontaneous outcome of HCV infection for 6 months before a treatment decision was made. Blood samples were taken frequently (15 ± 4 per patient). Phylogenetic analysis of the predominant virus(es) revealed infection by only one of two genotype 1b strains. While all patients seroconverted, their sera induced two opposing effects in HCVpp infection assays: inhibition and facilitation. Furthermore, the ability of sera to facilitate or inhibit infection correlated with the presence of either infecting HCV strain and divided the patients into two groups. In group 1, the progressive emergence of a relatively strong neutralizing response correlated with a fluctuating decrease in high initial viremia, leading to control of viral replication. Patients in group 2 failed to reduce viremia within the acute phase, and no neutralizing responses were detected despite seroconversion. Strikingly, sera of group 2, as well as naïve sera, facilitated infection by HCVpp displaying HCV glycoproteins from different genotypes and strains, including those retrieved from patients. These results provide new insights into the mechanisms of viral persistence and immune control of viremia.

Alpha Interferon Inhibits Hepatitis C Virus Replication in Primary Human Hepatocytes Infected In Vitro

ABSTRACT Chronic hepatitis C is a common cause of liver disease, the complications of which include cirrhosis and hepatocellular carcinoma. Treatment of chronic hepatitis C is based on the use of alpha interferon (IFN-α). Recently, indirect evidence based on mathematical modeling of hepatitis C virus (HCV) dynamics during human IFN-α therapy suggested that the major initial effect of IFN-α is to block HCV virion production or release. Here, we used primary cultures of healthy, uninfected human hepatocytes to show that: (i) healthy human hepatocytes can be infected in vitro and support HCV genome replication, (ii) hepatocyte treatment with IFN-α results in expression of IFN-α-induced genes, and (iii) IFN-α inhibits HCV replication in infected human hepatocytes. These results show that IFN-α acts primarily through its nonspecific antiviral effects and suggest that primary cultures of human hepatocytes may provide a good model to study intrinsic HCV resistance to IFN-α.

IL28B polymorphisms predict reduction of HCV RNA from the first day of therapy in chronic hepatitis C

BACKGROUND & AIMS Single nucleotide polymorphisms (SNPs) associated with IL28B influence the outcome of peginterferon-α/ribavirin therapy of chronic hepatitis C virus (HCV) infection. We analyzed the kinetics of HCV RNA during therapy as a function of IL28B SNPs. METHODS IL28B SNPs rs8099917, rs12979860, and rs12980275 were genotyped in 242 HCV treatment-naïve Caucasian patients (67% genotype 1, 28% genotype 2 or 3) receiving peginterferon-α2a (180 μg weekly) and ribavirin (1000-1200 mg daily) with serial HCV-RNA quantifications. Associations between IL28B polymorphisms and early viral kinetics were assessed, accounting for relevant covariates. RESULTS In the multivariate analyses for genotype 1 patients, the T allele of rs12979860 (T(rs12979860)) was an independent risk factor for a less pronounced first phase HCV RNA decline (log(10) 0.89IU/ml among T carriers vs. 2.06 among others, adjusted p < 0.001) and lower rapid (15% vs. 38%, adjusted p = 0.007) and sustained viral response rates (48% vs. 66%, adjusted p < 0.001). In univariate analyses, T(rs12979860) was also associated with a reduced second phase decline (p = 0.002), but this association was no longer significant after adjustment for the first phase decline (adjusted p = 0.8). In genotype 2/3 patients, T(rs12979860) was associated with a reduced first phase decline (adjusted p = 0.04), but not with a second phase decline. CONCLUSIONS Polymorphisms in IL28B are strongly associated with the first phase viral decline during peginterferon-α/ribavirin therapy of chronic HCV infection, irrespective of HCV genotype.

Dynamics of Hepatitis B Virus Resistance to Lamivudine

ABSTRACT Lamivudine was the first approved inhibitor of hepatitis B virus (HBV) reverse transcriptase (RT). Lamivudine resistance develops in 53% to 76% of patients after 3 years of treatment. We extensively characterized the dynamics of HBV quasispecies variant populations in four HBV-infected patients who developed lamivudine resistance. Virological breakthrough was preceded by 2 to 4 months by the emergence of quasispecies variants bearing amino acid substitutions at RT position 204, i.e., within the YMDD catalytic motif (rtM204V/I). Three patients had a gradual switch from a YMDD variant population at baseline to a 100% lamivudine-resistant variant population, whereas the remaining patient had a fluctuating pattern of resistance variant dynamics. Careful analysis of amino acid substitutions located outside domain C of HBV RT, including those known to partially restore replication capacities in vitro, showed that the in vivo replication of HBV variants is driven by multiple forces, including intrinsic replicative advantages conferred by mutations accumulating outside domain C and the changing environment in which these variants replicate. Our findings also suggest that individual treatment optimization will require sensitive methods capable of detecting the emergence of viral resistance before the relevant variants acquire optimal replicative capacities.

Retreatment with sofosbuvir and simeprevir of patients with hepatitis C virus genotype 1 or 4 who previously failed a daclatasvir‐containing regimen

Failure to achieve sustained virological response (SVR) with hepatitis C virus (HCV) direct‐acting antiviral‐based regimens is commonly associated with emergence of resistance‐associated variants (RAVs). To avoid cross‐resistance, recent guidelines recommend that patients who have failed on nonstructural protein 5A (NS5A) inhibitors should be retreated with sofosbuvir (SOF; NS5B inhibitor) combined with simeprevir (SIM; protease inhibitor [PI]); however, supporting evidence is lacking. This “real‐world” study comprised patients who had failed to achieve SVR on previous NS5A‐based therapy with daclatasvir (DCV) plus pegylated interferon (Peg‐IFN) and ribavirin (RBV), with (n = 3) or without (n = 13) asunaprevir (ASV; PI). All 16 patients were retreated for 12 weeks with SOF plus SIM, without RBV. Antiviral efficacy was evaluated using the primary endpoint of SVR12 (SVR 12 weeks post‐treatment); on‐treatment response was also assessed. Patients (N = 16; 13 male; mean age: 54 years [range, 43‐73]) were chronically infected with HCV genotype (GT) 1 (1a, n = 11; 1b, n = 3) or 4 (n = 2); they had advanced fibrosis or compensated cirrhosis (FibroScan, 9.6‐70 kPa; cirrhosis, n = 9); median baseline HCV‐RNA level was 1.38 × 106 IU/mL. No patient discontinued treatment because of adverse events or virological failure. All patients achieved HCV RNA below lower limit of quantification (<12 IU/mL) by end of treatment (EOT) and 10 of 16 had a rapid response (week 4). SVR12 was achieved by 14 of 16 patients; the remaining 2 relapsed by 4 weeks post‐EOT (both were GT 1a infected with cirrhosis; 1 had previously failed DCV‐ASV plus Peg‐IFN and RBV). Presence of SIM RAVs/polymorphisms (R155K and Q80K) at study baseline did not predict retreatment failure. Conclusion: Our findings support the concept of retreating NS5A inhibitor failures with SOF combined with SIM. However, the most difficult‐to‐cure patients may need more than 12 weeks of treatment and/or the addition of RBV. (Hepatology 2016;63:1809‐1816)

Clinical utility of hepatitis C virus core antigen quantification in patients with chronic hepatitis C

BACKGROUND Hepatitis C virus (HCV) core antigen has been proposed as a surrogate marker of HCV replication. HCV core antigen detection and quantification can thus theoretically be used instead of nucleic acid testing (NAT) to diagnose infection and manage antiviral therapy, with several advantages compared to HCV RNA assays. HCV core antigen can now be easily detected and quantified by means of a chemiluminescent microparticle immunoassay on the Abbott Architect device. OBJECTIVE The aim of the present study was to evaluate the clinical performance of the Architect HCV Ag assay for the detection and quantification of HCV core antigen in patients with chronic HCV genotype 1-6 infections. RESULTS The Architect HCV Ag assay had a specificity of 100% (95%CI: 97.8-100%). HCV core antigen levels were not influenced by the HCV genotype. The lower limit of detection of 3fmol/L corresponded to approximately 1000IU/mL of HCV RNA, regardless of the real-time PCR assay used. HCV core antigen and HCV RNA levels correlated for HCV genotype 1-4 (r=0.89 and r=0.88 for the m2000 and the CAP/CTM v2.0 assay, respectively). CONCLUSIONS The Architect HCV Ag assay is highly specific and easy to perform. It represents a valuable screening, diagnostic and monitoring tool, especially in the era of new all-oral, interferon-free antiviral strategies that do not require high analytical sensitivity.

Retreatment with sofosbuvir and simeprevir of patients with HCV GT1 or 4 who previously failed a daclatasvir‐containing regimen

Failure to achieve sustained virological response (SVR) with hepatitis C virus (HCV) direct‐acting antiviral‐based regimens is commonly associated with emergence of resistance‐associated variants (RAVs). To avoid cross‐resistance, recent guidelines recommend that patients who have failed on nonstructural protein 5A (NS5A) inhibitors should be retreated with sofosbuvir (SOF; NS5B inhibitor) combined with simeprevir (SIM; protease inhibitor [PI]); however, supporting evidence is lacking. This “real‐world” study comprised patients who had failed to achieve SVR on previous NS5A‐based therapy with daclatasvir (DCV) plus pegylated interferon (Peg‐IFN) and ribavirin (RBV), with (n = 3) or without (n = 13) asunaprevir (ASV; PI). All 16 patients were retreated for 12 weeks with SOF plus SIM, without RBV. Antiviral efficacy was evaluated using the primary endpoint of SVR12 (SVR 12 weeks post‐treatment); on‐treatment response was also assessed. Patients (N = 16; 13 male; mean age: 54 years [range, 43‐73]) were chronically infected with HCV genotype (GT) 1 (1a, n = 11; 1b, n = 3) or 4 (n = 2); they had advanced fibrosis or compensated cirrhosis (FibroScan, 9.6‐70 kPa; cirrhosis, n = 9); median baseline HCV‐RNA level was 1.38 × 106 IU/mL. No patient discontinued treatment because of adverse events or virological failure. All patients achieved HCV RNA below lower limit of quantification (<12 IU/mL) by end of treatment (EOT) and 10 of 16 had a rapid response (week 4). SVR12 was achieved by 14 of 16 patients; the remaining 2 relapsed by 4 weeks post‐EOT (both were GT 1a infected with cirrhosis; 1 had previously failed DCV‐ASV plus Peg‐IFN and RBV). Presence of SIM RAVs/polymorphisms (R155K and Q80K) at study baseline did not predict retreatment failure. Conclusion: Our findings support the concept of retreating NS5A inhibitor failures with SOF combined with SIM. However, the most difficult‐to‐cure patients may need more than 12 weeks of treatment and/or the addition of RBV. (Hepatology 2016;63:1809‐1816)

High-Dose Pegylated Interferon-α and Ribavirin in Nonresponder Hepatitis C Patients and Relationship With IL-28B Genotype (SYREN Trial)

BACKGROUND & AIMS In patients with chronic hepatitis C who failed to respond to standard therapy, high-dose pegylated interferon (IFN)-α and/or ribavirin could induce a stronger antiviral response and prevent treatment failure and HCV resistance when combined with direct-acting antivirals. The influence of genetic determinants in this context remains unknown. METHODS Eighty-three patients infected with HCV genotype 1 who were nonresponsive to standard therapy received pegylated IFN-α2a (360 μg once per week or 180 μg twice per week) with ribavirin (1.0-1.2 or 1.2-1.6 g/d) for up to 72 weeks. Virological responses were assessed at different time points, and the influence of the IL-28B genotype was studied. RESULTS At weeks 12 and 24, respectively, 47 (56.6%) and 50 (60.2%) patients achieved a ≥2-Log10 decrease of HCV RNA levels; 8 (9.6%) and 21 (25.3%) patients had undetectable HCV RNA after 12 and 24 weeks of treatment, respectively. Patients with a CT IL-28B genotype responded significantly better and earlier than those with a TT genotype. In multivariate analysis, the IL-28B genotype was an independent predictor of the virological responses at weeks 4, 12, and 24. CONCLUSIONS High-dose pegylated IFN-α with standard or high doses of ribavirin induces a potent antiviral response in a substantial number of patients who did not respond to standard therapy. The IL-28B genotype is an independent predictor of the antiviral response. High-dose pegylated IFN-α in combination with ribavirin and protease inhibitors appears as an attractive option for future study in this population.

Hepatitis C virus replication kinetics in chimpanzees with self-limited and chronic infections

Summary.  The availability of molecular beacon‐based, real time polymerase chain reaction (PCR) and a semi‐automated sample extraction procedure have made it possible for us to retrospectively examine HCV replication kinetics in HCV naive chimpanzees infected during the past 20 years. We compared these in 17 animals that developed chronic infection, and in 21 that developed self‐limited infection. No differences were found in infecting dose, or replication kinetics in the acute phase between these two types of infection. An unanticipated finding was the fact that 10 of 17 animals developing chronic infection partially controlled virus replication for 48 ± 48 weeks after typical acute phase viraemia, and prior to development of chronic infection. Twenty‐nine out of 30 (29/30) sera, which were negative by quantitative PCR during the downregulated period, were, however, positive by the more sensitive Genprobe isothermal transcription‐mediated amplification (TMA) assay. Thus, downregulation was not complete. Ten animals showing self‐limited infection showed complete resolution of viraemia by TMA assay. Quasispecies analysis revealed that in all, except one case, the virus reappearing after downregulation was essentially identical to that of the originally infecting virus.

Dynamic Range of Hepatitis C Virus RNA Quantification with the Cobas Ampliprep-Cobas Amplicor HCV Monitor v2.0 Assay

ABSTRACT Accurate quantification of hepatitis C virus (HCV) RNA is needed in clinical practice to decide whether to continue or stop pegylated interferon-α-ribavirin combination therapy at week 12 of treatment for patients with chronic hepatitis C. Currently the HCV RNA quantification assay most widely used worldwide is the Amplicor HCV Monitor v2.0 assay (Roche Molecular Systems, Pleasanton, Calif.). The HCV RNA extraction step can be automated in the Cobas Ampliprep device. In this work, we show that the dynamic range of HCV RNA quantification of the Cobas Ampliprep/Cobas Amplicor HCV Monitor v2.0 procedure is 600 to 200,000 HCV RNA IU/ml (2.8 to 5.3 log IU/ml), which does not cover the full range of HCV RNA levels in infected patients. Any sample containing more than 200,000 IU/ml (5.3 log IU/ml) must thus be retested after dilution for accurate quantification. These results emphasize the need for commercial HCV RNA quantification assays with a broader range of linear quantification, such as real-time PCR-based assays.