Pantxika Bellecave

Low-frequency drug-resistant HIV-1 and risk of virological failure to first-line NNRTI-based ART: a multicohort European case–control study using centralized ultrasensitive 454 pyrosequencing

Objectives It is still debated if pre-existing minority drug-resistant HIV-1 variants (MVs) affect the virological outcomes of first-line NNRTI-containing ART. Methods This Europe-wide case–control study included ART-naive subjects infected with drug-susceptible HIV-1 as revealed by population sequencing, who achieved virological suppression on first-line ART including one NNRTI. Cases experienced virological failure and controls were subjects from the same cohort whose viraemia remained suppressed at a matched time since initiation of ART. Blinded, centralized 454 pyrosequencing with parallel bioinformatic analysis in two laboratories was used to identify MVs in the 1%–25% frequency range. ORs of virological failure according to MV detection were estimated by logistic regression. Results Two hundred and sixty samples (76 cases and 184 controls), mostly subtype B (73.5%), were used for the analysis. Identical MVs were detected in the two laboratories. 31.6% of cases and 16.8% of controls harboured pre-existing MVs. Detection of at least one MV versus no MVs was associated with an increased risk of virological failure (OR = 2.75, 95% CI = 1.35–5.60, P = 0.005); similar associations were observed for at least one MV versus no NRTI MVs (OR = 2.27, 95% CI = 0.76–6.77, P = 0.140) and at least one MV versus no NNRTI MVs (OR = 2.41, 95% CI = 1.12–5.18, P = 0.024). A dose–effect relationship between virological failure and mutational load was found. Conclusions Pre-existing MVs more than double the risk of virological failure to first-line NNRTI-based ART.

Inhibition of Hepatitis C Virus (HCV) RNA Polymerase by DNA Aptamers: Mechanism of Inhibition of In Vitro RNA Synthesis and Effect on HCV-Infected Cells

ABSTRACT We describe here the further characterization of two DNA aptamers that specifically bind to hepatitis C virus (HCV) RNA polymerase (NS5B) and inhibit its polymerase activity in vitro. Although they were obtained from the same selection procedure and contain an 11-nucleotide consensus sequence, our results indicate that aptamers 27v and 127v use different mechanisms to inhibit HCV polymerase. While aptamer 27v was able to compete with the RNA template for binding to the enzyme and blocked both the initiation and the elongation of RNA synthesis, aptamer 127v competed poorly and exclusively inhibited initiation and postinitiation events. These results illustrate the power of the selective evolution of ligands by exponential enrichment in vitro selection procedure approach to select specific short DNA aptamers able to inhibit HCV NS5B by different mechanisms. We also determined that, in addition to an in vitro inhibitory effect on RNA synthesis, aptamer 27v was able to interfere with the multiplication of HCV JFH1 in Huh7 cells. The efficient cellular entry of these short DNAs and the inhibitory effect observed on human cells infected with HCV indicate that aptamers are useful tools for the study of HCV RNA synthesis, and their use should become a very attractive and alternative approach to therapy for HCV infection.

Prevalence and Evolution of Low Frequency HIV Drug Resistance Mutations Detected by Ultra Deep Sequencing in Patients Experiencing First Line Antiretroviral Therapy Failure

Objectives Clinical relevance of low-frequency HIV-1 variants carrying drug resistance associated mutations (DRMs) is still unclear. We aimed to study the prevalence of low-frequency DRMs, detected by Ultra-Deep Sequencing (UDS) before antiretroviral therapy (ART) and at virological failure (VF), in HIV-1 infected patients experiencing VF on first-line ART. Methods Twenty-nine ART-naive patients followed up in the ANRS-CO3 Aquitaine Cohort, having initiated ART between 2000 and 2009 and experiencing VF (2 plasma viral loads (VL) >500 copies/ml or one VL >1000 copies/ml) were included. Reverse transcriptase and protease DRMs were identified using Sanger sequencing (SS) and UDS at baseline (before ART initiation) and VF. Results Additional low-frequency variants with PI-, NNRTI- and NRTI-DRMs were found by UDS at baseline and VF, significantly increasing the number of detected DRMs by 1.35 fold (p<0.0001) compared to SS. These low-frequency DRMs modified ARV susceptibility predictions to the prescribed treatment for 1 patient at baseline, in whom low-frequency DRM was found at high frequency at VF, and 6 patients at VF. DRMs found at VF were rarely detected as low-frequency DRMs prior to treatment. The rare low-frequency NNRTI- and NRTI-DRMs detected at baseline that correlated with the prescribed treatment were most often found at high-frequency at VF. Conclusion Low frequency DRMs detected before ART initiation and at VF in patients experiencing VF on first-line ART can increase the overall burden of resistance to PI, NRTI and NNRTI.

High prevalence of antiretroviral drug resistance among HIV-1-untreated patients in Guinea-Conakry and in Niger.

BACKGROUND The aim of the study was to assess the prevalence of antiretroviral drug resistance mutations in HIV-1 from recently diagnosed and untreated patients living in Conakry, Guinea-Conakry and in Niamey, Niger. METHODS The study was performed in two countries of Western Africa - Guinea-Conakry and Niger - using the same survey method in both sites. All newly HIV-1 diagnosed patients, naive of antiretroviral drugs, were consecutively included during September 2009 in each of the two sites. Protease and reverse transcriptase sequencing was performed using the ANRS procedures. Drug resistance mutations were identified according to the 2009 update surveillance drug resistance mutations. RESULTS In Conakry, 99 patients were included, most of whom (89%) were infected with CRF02_AG recombinant virus. Resistance analysis among the 93 samples showed that ≥1 drug resistance mutation was observed in 8 samples, leading to a prevalence of primary resistance of 8.6% (95% CI 2.91-14.29%). In Niamey, 96 patients were included; a high diversity in HIV-1 subtypes was observed with 47 (51%) patients infected with CRF02_AG. Resistance analysis performed among the 92 samples with successful genotypic resistance test showed that ≥1 drug resistance mutation was observed in 6 samples, leading to a prevalence of primary resistance of 6.5% (95% CI 1.50-11.50%). CONCLUSIONS We reported the first antiretroviral drug resistance survey studies in antiretroviral-naive patients living in Guinea-Conakry and in Niger. The prevalence of resistance was between 6% and 9% in both sites, which is higher than most of the other countries from Western Africa region.

HIV-1 Dynamics and Coreceptor Usage in Maraviroc-Treated Patients with Ongoing Replication

ABSTRACT There is evidence that HIV-1 evolution under maraviroc (MVC) pressure can lead to the selection of either X4-tropic variants and/or R5-tropic, MVC-resistant isolates. However, the viral dynamics of HIV-1 variants in patients with virological failure (VF) on MVC-containing regimens remain poorly studied. Here, we investigated the V3 loop evolution of HIV-1 on MVC in relation to coreceptor usage and the nature of HIV-1 quasispecies before MVC therapy using bulk population sequences and ultradeep sequencing. The majority of patients had no detectable minority X4 variant at baseline. The evolution of tropism was followed up until VF and showed three possibilities for viral evolution in these patients: emergence of preexisting X4 variants, de novo selection of R5 variants presenting V3 loop mutations, or replication of R5 variants without selection of known mutations.

Detection of low-frequency HIV type 1 reverse transcriptase drug resistance mutations by ultradeep sequencing in naive HIV type 1-infected individuals.

Genotypic resistance testing is recommended to evaluate the susceptibility of HIV to antiretroviral drugs. These tests are based on bulk population sequencing and thus consider only variants representing more than 20% of the viral population, whereas next generation sequencing methods allow detection below this threshold. We aimed to evaluate the potential use of ultradeep pyrosequencing (UDPS) for genotypic resistance testing in clinical routine at the University Hospital of Bordeaux, France. We performed UDPS on reverse transcriptase (RT) from 47 HIV-1 individuals, naive of antiretroviral treatment and for whom genotypic resistance testing was requested for clinical management in 2011-2012. In 8.5% of the patients, only low-frequency variants harboring RT drug resistance mutations were detected raising the question of their clinical significance. Rilpivirine-associated resistance mutations were detected in 19.1% of our population study. To conclude, UDPS could become a routine tool for the evaluation of HIV-infected patients in hospital laboratories.

Contribution of APOBEC3G/F activity to the development of low-abundance drug-resistant human immunodeficiency virus type 1 variants

Plasma drug-resistant minority human immunodeficiency virus type 1 variants (DRMVs) increase the risk of virological failure to first-line non-nucleoside reverse transcriptase inhibitor antiretroviral therapy (ART). The origin of DRMVs in ART-naive patients, however, remains unclear. In a large pan-European case-control study investigating the clinical relevance of pre-existing DRMVs using 454 pyrosequencing, the six most prevalent plasma DRMVs detected corresponded to G-to-A nucleotide mutations (V90I, V106I, V108I, E138K, M184I and M230I). Here, we evaluated if such DRMVs could have emerged from apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3G/F (APOBEC3G/F) activity. Out of 236 ART-naive subjects evaluated, APOBEC3G/F hypermutation signatures were detected in plasma viruses of 14 (5.9%) individuals. Samples with minority E138K, M184I, and M230I mutations, but not those with V90I, V106I or V108I, were significantly associated with APOBEC3G/F activity (Fishers P < 0.005), defined as the presence of > 0.5% of sample sequences with an APOBEC3G/F signature. Mutations E138K, M184I and M230I co-occurred in the same sequence as APOBEC3G/F signatures in 3/9 (33%), 5/11 (45%) and 4/8 (50%) of samples, respectively; such linkage was not found for V90I, V106I or V108I. In-frame STOP codons were observed in 1.5% of all clonal sequences; 14.8% of them co-occurred with APOBEC3G/F signatures. APOBEC3G/F-associated E138K, M184I and M230I appeared within clonal sequences containing in-frame STOP codons in 2/3 (66%), 5/5 (100%) and 4/4 (100%) of the samples. In a re-analysis of the parent case control study, the presence of APOBEC3G/F signatures was not associated with virological failure. In conclusion, the contribution of APOBEC3G/F editing to the development of DRMVs is very limited and does not affect the efficacy of non-nucleoside reverse transcriptase inhibitor ART.

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.

High decay of blood HIV reservoir when tenofovir/emtricitabine/elvitegravir/cobicistat is initiated during the acute primary HIV infection

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HIV-1 Coreceptor Usage Assessment by Ultra-Deep Pyrosequencing and Response to Maraviroc.

Background Maraviroc is an HIV entry inhibitor that alters the conformation of CCR5 and is poorly efficient in patients infected by viruses that use CXCR4 as an entry coreceptor. The goal of this study was to assess the capacity of ultra-deep pyrosequencing (UDPS) and different data analysis approaches to characterize HIV tropism at baseline and predict the therapeutic outcome on maraviroc treatment. Methods 113 patients with detectable HIV-1 RNA on HAART were treated with maraviroc. The virological response was assessed at months 1, 3 and 6. The sequence of the HIV V3 loop was determined at baseline and prediction of maraviroc response by different software and interpretation algorithms was analyzed. Results UDPS followed by analysis with the Pyrotrop software or geno2pheno algorithm provided better prediction of the response to maraviroc than Sanger sequencing. We also found that the H34Y/S substitution in the V3 loop was the strongest individual predictor of maraviroc response, stronger than substitutions at positions 11 or 25 classically used in interpretation algorithms. Conclusions UDPS is a powerful tool that can be used with confidence to predict maraviroc response in HIV-1-infected patients. Improvement of the predictive value of interpretation algorithms is possible and our results suggest that adding the H34S/Y substitution would substantially improve the performance of the 11/25/charge rule.