Jean-Claude Tardy

HIV-1 Load Comparison Using Four Commercial Real-Time Assays

ABSTRACT The HIV-1 RNA viral load is commonly used for the monitoring of disease progression and antiretroviral treatment of HIV-1-infected patients. Since the misestimating of values could lead to inappropriate therapeutical management, the comparative performances, especially the ability to span the genetic diversity of HIV-1, of available automated real-time assays need to be evaluated. We conducted a prospective study with 74 consenting patients enrolled between March 2007 and November 2008. A blood sample was obtained at the time of diagnosis of HIV seropositivity and blindly tested for HIV-1 RNA by at least 4 commercial tests: the Abbott m2000 RealTime HIV-1, bioMérieux NucliSens EasyQ HIV-1, version 1.2 (v1.2), and Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) v1.0 and v2.0 assays. The means of difference were null between CAP/CTM v2.0 and Abbott for CRF02_AG subtypes but positive in favor of CAP/CTM v2.0 for genotype B and negative in favor of NucliSens for all genotypes. The standard deviation (SD) of difference ranged from 0.3 to 0.59, depending on the considered couples of assays. Reliabilities of these four tests, appreciated by the standard deviation of difference between the measurement and the estimated “true” viral load and by the coefficient of reliability, were significantly different (P < 10−4) among each other. Significant differences were also observed within each group of HIV-1 genotype. The global disparity was higher for CRF02_AG than for B subtypes. This study indicates a risk of viral load misestimating or discrepancies between techniques, depending on the HIV-1 subtype, and speaks in favor of using the same assay for the monitoring of HIV-1-infected patients.

Detection and Characterization of Hepatitis C Virus RNA in Seminal Plasma and Spermatozoon Fractions of Semen from Patients Attempting Medically Assisted Conception

ABSTRACT To investigate the risk of transmission of hepatitis C virus (HCV) via semen in assisted reproduction techniques, semen samples from 32 men chronically infected with HCV attending a center for assisted procreation were tested for HCV RNA by a reverse transcription-PCR protocol by using a modified version of the Cobas AMPLICOR HCV assay (version 2.0; Roche Diagnostics). The sensitivity of the test was 40 copies/ml. Four of 32 seminal plasma samples (12.5%) were found to be positive for the presence of HCV RNA. The median HCV load in blood was significantly higher in patients who were found to be positive for the presence of HCV RNA in semen than in those who tested negative (P = 0.02). In one man, seven consecutive seminal plasma samples tested positive for HCV RNA, as did two consecutive motile spermatozoon fractions; the corresponding fractions obtained after migration of the spermatozoa remained negative. Despite the absence of the proven infectivity of virus in semen samples that test positive for HCV RNA, these findings highlight the fact that seminal fluid may exhibit prolonged HCV RNA excretion. The usefulness of HCV RNA detection in both seminal plasma and spermatozoon fractions before the start of a program of medically assisted reproduction in couples in whom the male partner is chronically infected with HCV would need to be evaluated prospectively with a larger population of subjects exhibiting HCV RNA in their semen.

Gene transfer of anti-gp41 antibody and CD4 immunoadhesin strongly reduces the HIV-1 load in humanized severe combined immunodeficient mice.

ObjectiveTo study the anti-HIV-1 effects of the delivery of anti-gp41 monoclonal antibody (mAb) and soluble CD4 (sCD4) immunoadhesin by genetically modified cells in HIV-1-infected, humanized severe combined immunodeficient (SCID) mice. DesignThe complementary DNA of mAb 2F5, an anti-HIV-1 gp41 antibody, and of sCD4-IgG chimeric immunoadhesin were transferred into 3T3 cells using Moloney murine leukaemia virus vectors. The cells were then incorporated into a collagen structure called the neo-organ, which allowed the continuous production of the therapeutic molecules. MethodsThe antiviral effects in vivo of 2F5 or sCD4-IgG or both compounds were evaluated in neo-organ-implanted SCID mice that were grafted with human CD4 CEM T cells and challenged with HIV-1 Lai or MN. ResultsIn SCID mice implanted with 2F5 neo-organs, antibody plasma levels reached 500–2000 ng/ml. Viral loads after HIV-1 challenge were significantly reduced in neo-organ-implanted HIV-infected mice. Although 29 × 107 and 13 × 108 HIV-1-RNA copies/ml were detected at 12 days in the controls (mice injected with Lai and MN, respectively) less than 16.5 × 103 HIV-1-RNA copies/ml were observed in all implanted mice injected with either Lai or MN. The intracellular viral load was also reduced in CD4 cells recovered from the implanted mice. Comparable antiviral effects were obtained with CD4-IgG neo-organs. ConclusionOur results confirm the anti-HIV properties of 2F5 and sCD4-IgG continuously produced in vivo after ex-vivo gene therapy in SCID mice.

Experimental gene therapy : the transfer of Tat-inducible interferon genes protects human cells against HIV-1 challenge in vitro and in vivo in severe combined immunodeficient mice

Objectives: To evaluate in vitro and in vivo a strategy for gene therapy for AIDS based on the transfer of interferon (IFN)‐&agr;, &bgr; and ‐&ggr; genes to human cells. Design: Human U937 promonocytic cells were stably transfected with Tat‐inducible IFN expression vectors conferring an antiviral state against infection with HIV. Methods: Transfected cells were either infected by HIV‐1 in vitro or transplanted into severe combined immunodeficient (SCID) mice for an HIV challenge in vivo. Results: U937 cell lines stably carrying IFN transgenes under the positive control of the HIV‐1 Tat protein were highly resistant to HIV‐1 replication in vitro. This antiviral resistance was associated with a strong induction of IFN synthesis immediately following the viral infection. HIV‐1 proteins were found to be specifically trapped within the genetically modified cells. In contrast, all IFN‐U937 cells permitted full HIV‐2 replication. Transfected cells injected into SCID mice and challenged against HIV‐1 were strongly resistant to infection when cells were transduced with IFN‐&agr; or IFN‐&bgr; genes. However, IFN‐&ggr;‐transfected cells permitted HIV‐1 infection in vivo despite the induction of a high level of IFN‐&ggr; secretion. The quantity of proviral DNA was 105‐fold lower in IFN‐&agr;‐ or IFN‐&bgr;‐transfected U937 cells collected from these SCID mice than that in non‐transfected cells. Conclusions: Our results substantiated the validity of a strategy, based on the transfer of HIV‐1‐inducible IFN‐&agr; or IFN‐&bgr; genes, to confer antiviral resistance to human cells.

Prediction of the Virological Response to Etravirine in Clinical Practice : Comparison of Three Genotype Algorithms

The current Agence Nationale de Recherche sur le SIDA (ANRS)/International AIDS Society (IAS) algorithm predicts resistance to etravirine for viruses harboring ≥3 mutations from a list of 13 reverse transcriptase (RT) mutations. Two weighted algorithms, best correlated with fold changes to etravirine, have been described recently. A retrospective virological analysis of a major French city HIV sequences database was undertaken to assess the proportion of etravirine resistant viruses according to these three algorithms and the correlations between them. Two thousand six hundred eighty RT sequences were analyzed, including 749 from naive patients and 926 from patients previously treated with non‐nucleoside reverse transcriptase inhibitor (NNRTI). Combinations of mutations associated with etravirine resistance according to the three algorithms were found in 0%, 2.3%, and 3.6% of naive patients, and in 2.4%, 20.4%, and 19.3% of patients previously treated with NNRTIs. Concordance between the algorithms was weak (2 × 2 Kendalls tau: 0.787, 0.395, and 0.584). Most of the discordance was due to the differential weights attributed to Y181C/V, L100I, and K101P in the two weighted algorithms. It is concluded that the current ANRS/ IAS algorithm probably underestimates the proportion of viruses partially resistant to etravirine in NNRTI‐experienced patients. Improvements in algorithms are needed to take into account the partial resistance associated with some mutation patterns, and should include either additional mutations to the current list and/or differential weights for specific mutations. Surveys of naive patients should be conducted to estimate the risk of primary resistance to etravirine in a minority of cases. J. Med. Virol. 81:672–677, 2009

CD4+CD25+CD127− assessment as a surrogate phenotype for FOXP3+ regulatory T cells in HIV-1 infected viremic and aviremic subjects†

Although likely pivotal, the role of regulatory T cells (Tregs) in HIV pathogenesis remains elusive. This can be partly explained by analytical issues regarding their phenotypic identification in clinical studies. Instead of intracellular FOXP3 staining, CD4+CD25+CD127− phenotype has been proposed as an alternative to identify Tregs in clinical samples. However, its use remains controversial in viremic patients. Therefore, the objective of the present study was to assess the correlation between frequencies of CD4+CD25+CD127− and CD4+CD25+FOXP3+ lymphocytes in viremic and matched aviremic HIV‐infected patients.

Human Polycomb group EED protein negatively affects HIV-1 assembly and release

BackgroundThe human EED protein, a member of the superfamily of Polycomb group (PcG) proteins with WD-40 repeats, has been found to interact with three HIV-1 components, namely the structural Gag matrix protein (MA), the integrase enzyme (IN) and the Nef protein. The aim of the present study was to analyze the possible biological role of EED in HIV-1 replication, using the HIV-1-based vector HIV-Luc and EED protein expressed by DNA transfection of 293T cells.ResultsDuring the early phase of HIV-1 infection, a slight negative effect on virus infectivity occurred in EED-expressing cells, which appeared to be dependent on EED-MA interaction. At late times post infection, EED caused an important reduction of virus production, from 20- to 25-fold as determined by CAp24 immunoassay, to 10- to 80-fold based on genomic RNA levels, and this decrease was not due to a reduction of Gag protein synthesis. Coexpression of WTNef, or the non-N-myristoylated mutant NefG2A, restored virus yields to levels obtained in the absence of exogenous EED protein. This effect was not observed with mutant NefΔ57 mimicking the Nef core, or with the lipid raft-retargeted fusion protein LAT-Nef. LATAA-Nef, a mutant defective in the lipid raft addressing function, had the same anti-EED effect as WTNef. Cell fractionation and confocal imaging showed that, in the absence of Nef, EED mainly localized in membrane domains different from the lipid rafts. Upon co-expression with WTNef, NefG2A or LATAA-Nef, but not with NefΔ57 or LAT-Nef, EED was found to relocate into an insoluble fraction along with Nef protein. Electron microscopy of HIV-Luc producer cells overexpressing EED showed significant less virus budding at the cell surface compared to control cells, and ectopic assembly and clustering of nuclear pore complexes within the cytoplasm.ConclusionOur data suggested that EED exerted an antiviral activity at the late stage of HIV-1 replication, which included genomic RNA packaging and virus assembly, resulting possibly from a mistrafficking of viral genomic RNA (gRNA) or gRNA/Gag complex. Nef reversed the EED negative effect on virus production, a function which required the integrity of the Nef N-terminal domain, but not its N-myristoyl group. The antagonistic effect of Nef correlated with a cellular redistribution of both EED and Nef.

Low‐level viremia is associated with non‐B subtypes in patients infected with HIV with virological success following HAART introduction

This prospective study aimed to determine factors associated with detection of very low‐level viremia in patients infected with HIV‐1 with virological success following HAART introduction. Fifty‐seven patients, mostly (n = 51, 89%) treated with a protease inhibitor‐based regimen, were included and followed for 2 years. Viral loads were monitored by Abbott m2000 RealTime HIV‐1. Patients were classified as (i) HIV‐RNA‐negative if viral loads remained strictly undetectable (0 copies/ml), or (ii) HIV‐RNA‐positive if at least one HIV‐1 RNA could be detected in 1–49 copies/ml during follow‐up. At month 24, 44 patients (77%) were in the HIV‐RNA‐positive group, whereas 13 (23%) remained without very low‐level viremia. Univariate analysis, Kaplan–Meier curves and the Cox proportional hazard model revealed that B subtype was the only predictor of belonging to the HIV‐RNA‐negative group (HR 3.98; 95% CI 1.08–14.7). This association needs to be confirmed. Further study of the reservoir and the mechanisms of viral latency according to HIV‐subtype will also be necessary to develop new therapeutic strategies and eradicate HIV infection. J. Med. Virol. 85: 953–958, 2013.

Variants With Different Mutation Patterns Persist in the Quasispecies of Enfuvirtide-Resistant HIV-1 Population During and After Treatment In Vivo

Background:Genotypic and phenotypic resistance in 11 HIV-1-infected patients receiving enfuvirtide (ENF), as part of a salvage regimen, has been evaluated. Methods:Resistance mutations were detected by sequencing the gp41 ectodomain from plasma samples. During treatment, longitudinal samples from 1 patient were sequenced after limiting dilution of complementary DNA to isolate single genomes. Phenotypic resistance was evaluated with a new recombinant virus assay (PHENOSCRIPT; VIRalliance, Paris, France), allowing the determination of coreceptor use. Results:All patients experienced ENF failure. One to 4 mutations in the 36-to-45 gp41 region appeared during ENF therapy in all patients and disappeared after ENF removal. Mixtures of wild type and mutants unexpectedly persisted under ENF treatment, however, despite continued replication, leading to discordant results between genotypic and phenotypic data. Sequencing of isolated genomes from 1 patient confirmed that a wild-type first heptad repeat region (HR1) region was still present at the end of therapy. Several mutated variants coexisted at different time points, despite a tendency toward quasispecies reduction with time. Conclusion:Individual variability of the mutation pattern and persistence of strains without mutation in the region mainly targeted by ENF resistance probably reflect the fact that resistance to ENF may rely on regions of gp41 or gp120 other than residues 36 to 45.

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.