Florence Nicot

New NS5B polymerase inhibitors for hepatitis C

Importance of the field: The current treatment of chronic hepatitis C based on the combination of pegylated interferon and ribavirin is effective in only 50% of patients. Specific targeted antiviral therapies represent a promising approach to eradicate the infection. Areas covered in this review: This review focuses on progress towards the development of the hepatitis C virus (HCV) polymerase inhibitors that have entered clinical development in recent years. What the reader will gain: Nucleos(t)ide analogues target the active site of the HCV polymerase and acts as chain terminators. They have similar activity against all genotypes and the virus has a high genetic barrier to drug resistance. Non-nucleoside inhibitors achieve polymerase inhibition by binding to one of the at least four allosteric enzyme sites. Most of them have a genotype-specific activity and they may select rapidly drug-resistant variants if HCV replication is not completely suppressed. Nonetheless, they provide additional options for addressing the needs of infected patients. Take home message: NS5B polymerase inhibitors will form an integral part of more effective anti-HCV therapy, in combination with interferon or with other directly acting antiviral agents.

Concordance between two phenotypic assays and ultradeep pyrosequencing for determining HIV-1 tropism.

ABSTRACT There have been few studies on the concordance between phenotypic assays for predicting human immunodeficiency virus type 1 (HIV-1) coreceptor usage. The sensitivity of ultradeep pyrosequencing combined with genotyping tools is similar to that of phenotypic assays for detecting minor CXCR4-using variants. We evaluated the agreement between two phenotypic assays, the Toulouse tropism test (TTT) and the Trofile assay, and ultradeep pyrosequencing for determining the tropism of HIV-1 quasispecies. The concordance between the TTT and Trofile assays was assessed for 181 samples successfully phenotyped by both assays. The TTT was 86% concordant with the standard Trofile assay and 91.7% with its enhanced-sensitivity version. The concordance between phenotypic characterization of HIV-1 tropism and ultradeep pyrosequencing genotypic prediction was further studied in selected samples. The HIV-1 tropism inferred from ultradeep pyrosequencing of 11 samples phenotyped as X4 and dualtropic and 12 phenotyped as R5-tropic agreed closely with the results of phenotyping. However, ultradeep pyrosequencing detected minor CXCR4-using variants in 3 of 12 samples phenotyped as R5-tropic. Ultradeep pyrosequencing also detected minor CXCR4-using variants that had been missed by direct sequencing in 6 of 9 samples phenotyped as X4-tropic but genotyped as R5-tropic by direct sequencing. Ultradeep pyrosequencing was 87% concordant with the Trofile and TTT phenotypic assays and was in the same range of sensitivity (0.4%) than these two phenotypic assays (0.3 to 0.5%) for detecting minor CXCR4-using variants. Ultradeep pyrosequencing provides a new way to improve the performance of genotypic prediction of HIV-1 tropism to match that of the phenotypic assays.

No evidence of occult hepatitis C virus (HCV) infection in serum of HCV antibody‐positive HCV RNA‐negative kidney‐transplant patients

Persistence of hepatitis C virus (HCV) in patients who cleared HCV is still debated. Occult HCV infection is described as the presence of detectable HCV RNA in liver or peripheral blood mononuclear cells (PBMCs) of patients with undetectable plasma HCV‐RNA by conventional PCR assays. We have assessed the persistence of HCV in 26 kidney‐transplant patients, followed up for 10.5 years (range 2–16), after HCV elimination while on hemodialysis. If HCV really did persist, arising out of the loss of immune control caused by institution of the regimen of immunosuppressive drugs after kidney transplantation, HCV reactivation would have taken place. Their immunosuppression relied on calcineurin inhibitors (100%), and/or steroids (62%), and/or antimetabolites (94%). An induction therapy, given to 22 patients, relied on rabbit antithymocyte globulin (59%) or anti‐IL2‐receptor blockers (32%). All patients had undetectable HCV RNA as ascertained by several conventional tests. At the last follow‐up, no residual HCV RNA was detected in the five liver biopsies, the 26 plasma, and in the 37 nonstimulated and 24 stimulated PBMCs tested with an ultrasensitive RT‐PCR assay (detection limit, 2 IU/ml). No biochemical or virologic relapse was seen during follow‐up. The absence of HCV relapse in formerly HCV‐infected immunocompromised patients suggests the complete eradication of HCV after its elimination while on dialysis.

Serum concentrations of ribavirin and pegylated interferon and viral responses in patients infected with HIV and HCV.

The hepatitis C virus (HCV) infects a substantial proportion of patients infected with human immunodeficiency virus (HIV). Patients infected with both HCV and HIV respond poorly to anti‐HCV treatment with pegylated interferon alpha and ribavirin. But few data are available on the influence of ribavirin and interferon concentrations on treatment outcome for these patients. This study investigated the relationship between the serum pegylated interferon and ribavirin concentrations 3 and 6 months after treatment initiation, and treatment outcome in 35 HCV‐HIV coinfected patients. The pegylated interferon and ribavirin concentrations at months 3 and 6 were similar. The pegylated interferon concentrations at 3 months in responders and nonresponders were similar. However, responders tended to have higher ribavirin concentrations (2,322 ng/ml) than nonresponders (1,833 ng/ml; P = 0.08). Responders infected with HCV genotype 1 or 4 had higher ribavirin concentrations (2,672 ng/ml) than did similarly infected nonresponders (1,758 ng/ml; P = 0.04). ROC curve analysis showed that a ribavirin concentration of 2,300 ng/ml was the best threshold for predicting a nonresponse (ROC area = 0.80 ± 0.12). Thus ribavirin concentrations influence treatment outcome in HIV patients infected with HCV genotype 1 or 4. Monitoring ribavirin concentrations could help adapt ribavirin concentrations and improve the sustained virological response. J. Med. Virol. 80:1523–1529, 2008.

HIV-1 subtype B-infected MSM may have driven the spread of transmitted resistant strains in France in 2007–12: impact on susceptibility to first-line strategies

BACKGROUND Our study describes the prevalence of transmitted drug resistance (TDR) among 1318 French patients diagnosed at the time of primary HIV-1 infection (PHI) in 2007-12. METHODS HIV-1 resistance-associated mutations (RAMs) were characterized using both the 2009 WHO list of mutations and the French ANRS algorithm. A genotypic susceptibility score was estimated for each first-line recommended ART combination. RESULTS Patients were mainly MSM (72.6%). Non-B variants were identified in 33.7% of patients. The proportion of TDR was estimated as 11.7% (95% CI 10.0-13.5). The prevalences of PI-, NRTI-, first-generation NNRTI and etravirine/rilpivirine-associated RAMs were 2.5%, 5.2%, 3.9% and 3.2%, respectively. Single, dual and triple class resistance was found in 9.6%, 1.0% and 1.1% of cases, respectively. Additionally, 5/331 strains isolated in 2010-12 had integrase inhibitor (II)-related RAMs (isolated E157Q mutation in all cases). TDR was more common among MSM than in other groups (12.9% versus 8.6%, P = 0.034), and in case of B versus non-B subtype infections (13.6% versus 7.9%, P = 0.002). The proportions of fully active combinations were ≥99.2%, ≥97.3% and ≥95.3% in cases of PI-, II- and NNRTI-based regimens, respectively. In 2010-12, the proportion of fully active efavirenz-based ART was lower in cases of subtype B versus non-B infection (P = 0.021). CONCLUSIONS Compared with our previous studies, the proportion of NRTI- and first-generation NNRTI-related TDR has continued to decline in French seroconverters. However, subtype B-infected MSM could drive the spread of resistant HIV strains. Finally, we suggest preferring PI- or II- to NNRTI-based combinations to treat PHI patients.

Improved V3 genotyping with duplicate PCR amplification for determining HIV-1 tropism

OBJECTIVES To determine whether genotyping of HIV-1 by duplicate PCR amplification of the region encoding the V3 loop is more sensitive than single PCR for detecting CXCR4-using viruses. PATIENTS AND METHODS The V3 genotypes of the HIV-1 infecting 152 patients enrolled in the multicentre GenoTropism ANRS study were determined by all the participating laboratories using a single PCR and V3 bulk sequencing. In parallel, one laboratory determined the V3 genotype using duplicate PCR and bulk sequencing of pooled amplicons. HIV tropism was predicted with the geno2pheno10 algorithm. The phenotypes of all samples were determined with the Trofile assay and the Toulouse tropism test (TTT) recombinant virus assay. RESULTS Geno2pheno10 was 56.8% sensitive and 75.9% specific when compared with the Trofile assay for detecting CXCR4-using viruses after a single PCR. Duplicate amplification and bulk sequencing of the pooled PCR amplicons increased the sensitivity to 68.2% and specificity to 79.6%. Geno2pheno10 was 64.1% sensitive and 77.0% specific when compared with the TTT assay for detecting CXCR4-using viruses after a single PCR. Duplicate amplification and sequencing of the pooled PCR amplicons increased sensitivity to 76.9% and specificity to 80.5%. CONCLUSIONS The genotypic determination of HIV-1 tropism can be improved by duplicate amplifications and sequencing the pooled PCR products. This is a good compromise between improved sensitivity and reasonable cost for the genotype-based determination of tropism.

Characterization of CXCR4-using HIV-1 during primary infection by ultra-deep pyrosequencing

OBJECTIVES R5 viruses have long been thought to account for almost all strains present in primary HIV-1 infections (PHIs), but recent studies using sensitive phenotypic assays have revealed that 3%-6.4% of subjects also harbour CXCR4-using viruses. Phenotypic assays provide only qualitative results: the presence or absence of CXCR4-using viruses. We have therefore used ultra-deep pyrosequencing (UDS) to determine the frequency of CXCR4-using viruses among HIV-1 quasispecies. METHODS We first screened 200 patients for HIV-1 tropism using a sensitive phenotypic assay during PHI and identified 11 infected with an R5X4 dual/mixed (D/M) virus population. We then used UDS of the V3 env region with the geno2pheno algorithm (false positive rate = 5.75) to identify the HIV-1 quasispecies. RESULTS CXCR4-using viruses were detected in all but 1 of the 11 patients by UDS, and accounted for 0.2%-100% of the virus populations. The frequency of CXCR4-using viruses was <20% in six subjects and 100% in four subjects. Virus populations containing 100% CXCR4-using variants during PHI persisted for at least 1-2 years after the acute phase. The CCR5 Δ32 heterozygous genotype was similarly prevalent in patients infected with D/M (27%) and R5 (15%) viruses. CONCLUSIONS UDS and the phenotype were concordant for determining HIV-1 coreceptor usage. UDS analysis indicated large differences in the percentage of CXCR4-using viruses in the HIV-1 quasispecies during PHI. Further studies should examine the impact of the proportion of CXCR4-using viruses on disease prognosis.

Analytical sensitivity of three real-time PCR assays for measuring subtype B HIV-1 RNA

BACKGROUND Lack of HIV RNA during antiretroviral therapy (ART) is regarded as a desirable outcome. Commercial assays of HIV virus load now need to detect virus RNA concentrations below 50 c/ml and several of them have claimed a limit of detection (LOD) of 20-45 c/ml. OBJECTIVES AND STUDY DESIGN We have compared the performances of three commercial assays of HIV RNA, the Abbott RealTime HIV-1, the Qiagen Artus RG HIV-1 and the Roche Cobas Ampliprep Cobas TaqMan (CAPCTM) HIV-1 vs 2.0 using replicate of specimens with HIV-1 subtype B RNA concentrations of 20-200 c/ml. RESULTS Despite fair-to-moderate agreement between the three assays, probit analysis showed that their LODs differed; they were 81, 65 and 18c/ml respectively. The CAPCTM HIV-1 vs 2.0 values were higher than those of the other two; the maximum difference was 0.26 log c/ml. By testing 20 replicate of each concentration, coefficients of variation were between 0.6% and 9.2% (Abbott RealTime HIV-1), 10.3% and 38% (Qiagen Artus RG HIV-1) and 5.2% and 13.1% (Roche CAPCTM HIV-1 vs 2.0). The three assays also differed in their reproducibility and linearity for virus loads of 50-200 c/ml. CONCLUSION The analytical performances of commercial virus load assays differ. Direct comparisons of widely used commercial assays in clinical studies could help to identify the residual viremia that is clinically relevant for effective long term therapy.

Using Pharmacokinetic and Viral Kinetic Modeling To Estimate the Antiviral Effectiveness of Telaprevir, Boceprevir, and Pegylated Interferon during Triple Therapy in Treatment-Experienced Hepatitis C Virus-Infected Cirrhotic Patients

ABSTRACT Triple therapy combining a protease inhibitor (PI) (telaprevir or boceprevir), pegylated interferon (PEG-IFN), and ribavirin (RBV) has dramatically increased the chance of eradicating hepatitis C virus (HCV). However, the efficacy of this treatment remains suboptimal in cirrhotic treatment-experienced patients. Here, we aimed to better understand the origin of this impaired response by estimating the antiviral effectiveness of each drug. Fifteen HCV genotype 1-infected patients with compensated cirrhosis, who were nonresponders to prior PEG-IFN/RBV therapy, were enrolled in a nonrandomized study. HCV RNA and concentrations of PIs, PEG-IFN, and RBV were frequently assessed in the first 12 weeks of treatment and were analyzed using a pharmacokinetic/viral kinetic model. The two PIs achieved similar levels of molar concentrations (P = 0.5), but there was a significant difference in the 50% effective concentrations (EC50) (P = 0.008), leading to greater effectiveness for telaprevir than for boceprevir in blocking viral production (99.8% versus 99.0%, respectively, P = 0.002). In all patients, the antiviral effectiveness of PEG-IFN was modest (43.4%), and there was no significant contribution of RBV exposure to the total antiviral effectiveness. The second phase of viral decline, which is attributed to the loss rate of infected cells, was slow (0.19 day−1) and was higher in patients who subsequently eradicated HCV (P = 0.03). The two PIs achieved high levels of antiviral effectiveness. However, the suboptimal antiviral effectiveness of PEG-IFN/RBV and the low loss of infected cells suggest that a longer treatment duration might be needed in cirrhotic treatment-experienced patients and that a future IFN-free regimen may be particularly beneficial in these patients.

Virological failure of patients on maraviroc-based antiretroviral therapy

OBJECTIVES Virological failure (VF) in patients on maraviroc-based treatment has been associated with altered HIV tropism and resistance to maraviroc. This multicentre study aimed to characterize VF in patients treated with maraviroc. METHODS We analysed 27 patients whose treatment failed between 2008 and 2011. They had been screened for HIV tropism before maraviroc initiation using population-based V3 genotyping. HIV-1 tropism and resistance of R5 viruses to maraviroc at VF and at baseline were determined retrospectively using an ultrasensitive recombinant virus assay (RVA). RESULTS Viruses from 27 patients given maraviroc on the basis of the R5 genotype were characterized at the time of treatment failure. The RVA indicated that 12 patients harboured CXCR4-using viruses and 15 (56%) had pure R5 viruses at failure. One-third of those harbouring CXCR4-using viruses (4/12) were infected with R5X4/X4 viruses according to the RVA before maraviroc initiation. We analysed the phenotypic resistance to maraviroc of four patients harbouring R5 viruses at failure; two harboured viruses whose maximum percentage inhibition was reduced by 65%-90%, while the other two were infected with susceptible viruses. All patients had effective concentrations of drugs. CONCLUSIONS Half of the maraviroc-treated patients who experienced VF harboured CXCR4-using viruses at failure, one-third of them were detected by a phenotypic method before maraviroc initiation. Phenotypic assessment of R5 virus resistance to CCR5 antagonists at failure could help optimize antiretroviral therapy.