Virginie Ferré

Molecular Evolution of Hepatitis A Virus: a New Classification Based on the Complete VP1 Protein

ABSTRACT Hepatitis A virus (HAV) is a positive-stranded RNA virus in the genus Hepatovirus in the family Picornaviridae. So far, analysis of the genetic variability of HAV has been based on two discrete regions, the VP1/2A junction and the VP1 N terminus. In this report, we determined the nucleotide and deduced amino acid sequences of the complete VP1 gene of 81 strains from France, Kosovo, Mexico, Argentina, Chile, and Uruguay and compared them with the sequences of seven strains of HAV isolated elsewhere. Overall strain variation in the complete VP1 gene was found to be as high as 23.7% at the nucleotide level and 10.5% at the amino acid level. Different phylogenetic methods revealed that HAV sequences form five distinct and well-supported genetic lineages. Within these lineages, HAV sequences clustered by geographical origin only for European strains. The analysis of the complete VP1 gene allowed insight into the mode of evolution of HAV and revealed the emergence of a novel variant with a 15-amino-acid deletion located on the VP1 region where neutralization escape mutations were found. This could be the first antigenic variant of HAV so far identified.

Evaluation of the Genotypic Prediction of HIV-1 Coreceptor Use versus a Phenotypic Assay and Correlation with the Virological Response to Maraviroc: the ANRS GenoTropism Study

ABSTRACT Genotypic algorithms for prediction of HIV-1 coreceptor usage need to be evaluated in a clinical setting. We aimed at studying (i) the correlation of genotypic prediction of coreceptor use in comparison with a phenotypic assay and (ii) the relationship between genotypic prediction of coreceptor use at baseline and the virological response (VR) to a therapy including maraviroc (MVC). Antiretroviral-experienced patients were included in the MVC Expanded Access Program if they had an R5 screening result with Trofile (Monogram Biosciences). V3 loop sequences were determined at screening, and coreceptor use was predicted using 13 genotypic algorithms or combinations of algorithms. Genotypic predictions were compared to Trofile; dual or mixed (D/M) variants were considered as X4 variants. Both genotypic and phenotypic results were obtained for 189 patients at screening, with 54 isolates scored as X4 or D/M and 135 scored as R5 with Trofile. The highest sensitivity (59.3%) for detection of X4 was obtained with the Geno2pheno algorithm, with a false-positive rate set up at 10% (Geno2pheno10). In the 112 patients receiving MVC, a plasma viral RNA load of <50 copies/ml was obtained in 68% of cases at month 6. In multivariate analysis, the prediction of the X4 genotype at baseline with the Geno2pheno10 algorithm including baseline viral load and CD4 nadir was independently associated with a worse VR at months 1 and 3. The baseline weighted genotypic sensitivity score was associated with VR at month 6. There were strong arguments in favor of using genotypic coreceptor use assays for determining which patients would respond to CCR5 antagonist.

Evidence of recombination in natural populations of hepatitis A virus

Genetic analysis of selected genome regions of hepatitis A virus (HAV) suggested that distinct genotypes of HAV could be found in different geographical regions. At least seven HAV genotypes have been identified all over the world, including four human genotypes (I, II, III, and VII) and three simian strains (IV, V, and VI). Phylogenetic analysis using full-length VP1 sequences revealed that human strain 9F94 has a close genetic relation with strain SLF-88 (sub-genotype VII). Nevertheless, the same analysis using full-length VP2 or VP3 sequences revealed that strain 9F94 has a close genetic relation with strain MBB (sub-genotype IB). To test the possibility of genetic recombination, phylogenetic studies were carried out, revealing that a crossing over had taken place in the VP1 capsid protein. These findings indicate that capsid-recombination can play a significant role in shaping the genetic diversity of HAV and, as such, can have important implications for its evolution, biology, and control.

Substitution of a Nonnucleoside Reverse Transcriptase Inhibitor for a Protease Inhibitor in the Treatment of Patients with Undetectable Plasma Human Immunodeficiency Virus Type 1 RNA

Seventy-three patients infected with human immunodeficiency virus type 1 (HIV-1) were enrolled in a prospective observational study to investigate the efficacy and tolerance of substituting a nonnucleoside reverse transcriptase inhibitor (NNRTI) for a protease inhibitor (PI) in patients whose plasma viral load (pVL) was controlled by a PI regimen. After a median follow-up of 52 weeks, 63 patients (86.3%) had undetectable pVLs. The incidence of virological breakthrough at 12 months of follow-up was 6.5% (95% confidence interval [CI], 1-20) among patients who had been antiretroviral naive before receiving HAART and 19.2% (95% CI, 6-34) among patients who had been treated with antiretroviral drugs before receiving the PI regimen (P=.10).

Efficacy and Tolerability of a Nucleoside Reverse Transcriptase Inhibitor-sparing Combination of Lopinavir/ritonavir and Efavirenz in Hiv-1-infected Patients

Background:Recommended antiretroviral regimens include a nucleoside reverse transcriptase inhibitor (NRTI) component. Class cross-resistance and mitochondrial toxicity are recognized as problems with this class of antiretrovirals. Methods:In a pilot open-label study, 65 antiretroviral-naive and 21 experienced but nonnucleoside reverse transcriptase inhibitor-naive HIV-1-infected adults were given a combination of lopinavir/ritonavir (533.3/133.3 mg twice daily) and efavirenz (600 mg once daily) for 48 weeks. Results:At baseline, the mean viral load was 4.84 log10 copies/mL and the mean CD4 count was 311 cells/mm3. At week 24, the proportions of patients with a viral load <400 copies/mL were 78% and 93% using an intent-to-treat and on-treatment analysis, respectively. At week 48, proportions were 73% and 97%, respectively. Treatment discontinuation occurred in 21 patients during the 48-week period, with 33% of those attributable to drug-related adverse effects. A viral load >400 copies/mL at week 24 or 48 was associated with nonadherence in 3 patients and virologic failure in 1 patient. After an increase during the first 8 weeks, fasting lipid levels remained stable up to 48 weeks. Conclusion:The lopinavir/ritonavir-efavirenz combination is associated with a high rate of virologic response and should be compared with more classic NRTI-containing regimens in randomized and controlled clinical trials.

Cellular normalization of viral DNA loads on whole blood improves the clinical management of cytomegalovirus or Epstein Barr virus infections in the setting of pre-emptive therapy.

Two quantitative duplex real‐time PCR assays were developed for co‐amplification of human albumin and cytomegalovirus (CMV) or Epstein Barr virus (EBV) genes after automated extraction on whole blood, and compared two units for expressing viral DNA loads (copies per ml of blood or per 106 peripheral blood leukocytes (PBLs)) on 1,138 positive samples. Both PCRs were characterized by high sensitivity, reproducibility, and linear range. Automated extraction by a MagNA Pure LC Instrument was shown to be more efficient when peripheral blood cell count was inferior to 5 × 109 PBLs/L. Albumin co‐amplification allows the detection of PCR inhibitors and normalization of viral load according to the number of cells calculated in the sample. The two ways of expressing viral load results were highly correlated, but quantitative differences varied in relation to variations of blood cell count. As these two viruses are highly cell associated, viral loads can be underestimated in patients with leucopenia. In the setting of pre‐emptive strategies during CMV infection, the units in which results are expressed can influence clinical management, as illustrated in this article. J. Med. Virol. 81:90–98, 2009.

Long term oncostatin M treatment induces an osteocyte-like differentiation on osteosarcoma and calvaria cells

Previous in vitro studies on primary osteoblastic and osteosarcoma cells (normal and transformed osteoblasts) have shown that oncostatin M (OSM), a member of the interleukin-6 family, possesses cytostatic and pro-apoptotic effects in association with complex and poorly understood activities on osteoblast differentiation. In this study, we use rat osteosarcoma cells transduced with lentiviral particles encoding OSM (lvOSM) to stably produce this cytokine. We show that after several weeks of culture, transduced OSRGA and ROS 17/2.8 cells are growth inhibited and sensitized to apoptosis induced by the kinase inhibitor Staurosporine (Sts). Moreover, this long term OSM treatment induces (i) a decrease in osteoblastic markers, (ii) morphological changes leading to an elongated and/or stellate shape and (iii) an increase in osteocytic markers (sclerostin and/or E11), suggesting an osteocyte-like differentiation. We also show that non transformed rat calvaria cells transduced with lvOSM differentiate into stellate shaped cells expressing sclerostin, E11, Phex and functional hemichannels. Together, these results indicate that osteosarcoma cells stably producing OSM do not develop resistance to this cytokine and thus could be a valuable new tool to study the anti-cancer effect of OSM in vivo. Moreover, OSM-over-expressing osteoblastic cells differentiate into osteocyte-like cells, the major cellular contingent in bone, providing new culture conditions for this cell type which is difficult to obtain in vitro.

Nevirapine-raltegravir combination, an NRTI and PI/r sparing regimen, as maintenance antiretroviral therapy in virologically suppressed HIV-1-infected patients.

BACKGROUND Nucleoside reverse transcriptase inhibitor (NRTI) and protease inhibitor (PI)/ritonavir sparing regimens may be useful to some HIV-infected patients. Nevirapine (NVP) and raltegravir (RAL) are both potent antiretrovirals with good long-term safety profiles. METHODS We retrospectively identified from our electronic database all patients with HIV RNA<50 copies/ml for >6 months on an NVP-containing regimen and no prior exposure to integrase strand transfer inhibitors who were switched to RAL plus NVP. Data was collected for 36 months or until discontinuation of RAL plus NVP for any reason. RESULTS A total of 39 patients (30 male) were included in this analysis. Median duration of prior antiretroviral therapy was 14 years (IQR 10-17) and median duration with plasma HIV-1 RNA<50 copies/ml prior to switch was 50 months (IQR 22-96). Switched regimens included mainly a boosted PI (n=24) or tenofovir disoproxil fumarate/emtricitabine (n=12). After switching, the percentages of patients with HIV-1 RNA<50 copies/ml were 87.2% (95% CI 76.7, 97.7) and 82.1% (95% CI 70.0, 94.1) at 6 and 12 months, respectively, in the intent-to-treat-exposed analysis, 97.1% (95% CI 91.6, 100) and 94.1% (95% CI 86.2, 100), respectively, in the per-protocol analysis. All patients with follow-up to month 24 (n=22) or month 36 (n=14) had HIV-1 RNA<50 copies/ml. One virological failure was observed (related to archived non-nucleoside reverse transcriptase inhibitor resistance mutation). During follow-up, no patient experienced Grade 3 or 4 adverse events. Median values of serum creatinine and lipids significantly improved after switch. CONCLUSIONS In patients with prolonged HIV-1 RNA<50 copies/ml, switching to NVP-RAL combination maintained virological suppression throughout 36 months. This combination deserves further evaluation in patients unable to tolerate NRTI or PI/ritonavir agents.

Salvage Therapy with Ritonavir-Boosted Amprenavir/Fosamprenavir: Virological and Immunological Response in Two Years Follow-up

Abstract Objective: To evaluate the efficacy of salvage regimens containing ritonavir-boosted amprenavir (APV/r) or fosamprenavir (FPV/r) in heavily pretreated protease inhibitor (PI)-experienced HIV-1 patients. Method: Evaluation of APV/r- or FPV/r-containing antiretroviral regimens in PI-experienced HIV-1 patients with 2 or more antiretroviral failures. Follow-up continued to 96 weeks with prospective collection of data. Results: 54 episodes (48 on APV/r and 6 on FPV/r) were considered in 45 patients who had received a median of 5 prior antiretroviral regimens (range, 2-13) including a median of 3 PIs (range, 2-4). Median time of treatment at analysis was 72 weeks (range, 12-210). At baseline, plasma viral load (pVL) and CD4 cell count was 67,000 copies/mL and 167 cell/mm3, respectively. At week 96, the median pVL was < 50 copies/mL and CD4 cell count was 519 cells/mm3. Proportion of patients with pVL below detection was 62% at week 48 and 61% at week 96. Fifteen patients stopped treatment because of virologic failure; one presented a full resistance profile to APV/r, based on the ANRS 2003 resistance algorithm. Median trough APV plasma concentration 4 weeks after treatment initiation was 1406 ng/mL (range, 452-4321); dose adaptation was required in only 7 patients. Conclusion: This study provides long-term follow-up of APV/r and FPV/r in the setting of salvage therapy, showing a high and sustained rate of virologic and immunologic response.

Despite an impaired response to IL-7, T cells from HIV-positive patients proliferate normally in response to IL-15 and its superagonist, RLI

Objective:In phase I/II trials, IL-7 immunotherapy has been shown to expand CD4+ T cells. However, expression of the IL-7 receptor &agr;-chain, CD127, is reduced on CD4+ T cells from HIV-positive patients, and defects in CD127 signaling have also been reported. To refine and improve cytokine immunotherapy, it is important to identify stimuli that can restore proliferation of CD4+ cells with defective responses to IL-7. Design:Observational study comparing viremic HIV-positive patients with HIV-negative controls. Methods:Peripheral blood mononuclear cells were cultured in the presence of 1 nmol/l IL-2, IL-7, IL-15 or RLI (an IL-15R&agr;/IL-15 fusion protein). Proliferation of different T-cell subsets was assessed by carboxyfluorescein succinimidyl ester fluorescence. Expression of CD127 on CD4+ T-cell subsets was also analyzed. Results:In HIV-positive patients, CD127 expression was correlated with CD4+ T-cell count in the (R2 = 0.36; P < 0.01) and (R2 = 0.45; P < 0.001) populations, whereas CD127 expression on cells was significantly reduced in HIV-positive individuals compared with controls (P = 0.001) independently of CD4+ T-cell count. In patients with high CD4+ T-cell counts, proliferation in response to IL-7 was significantly reduced only in cells (P < 0.05). RLI, and to a lesser extent IL-15, induced strong proliferation of cells from both HIV-positive patients and controls. Neither agent stimulated proliferation of or cells. Conclusion:In HIV-positive patients, cells are deficient in both CD127 expression and proliferation in response to IL-7. RLI and IL-15 specifically induced proliferation of cells, suggesting that they may have a unique potential to complement IL-7 immunotherapy.