Els Demecheleer

Diversity of the Human Immunodeficiency Virus Type 1 (HIV-1) env Sequence after Vertical Transmission in Mother-Child Pairs Infected with HIV-1 Subtype A

ABSTRACT Although several virologic and immunologic factors associated with an increased risk of perinatal human immunodeficiency virus type 1 (HIV-1) transmission have been described, the mechanism of mother-to-child transmission is still unclear. More specifically, the question of whether selective pressures influence the transmission remains unanswered. The aim of this study was to assess the genetic diversity of the transmitted virus after in utero transmission and after peripartum transmission and to compare the viral heterogeneity in the child with the viral heterogeneity in the mother. To allow a very accurate characterization of the viral heterogeneity in a single sample, limiting-dilution sequencing of a 1,016-bp fragment of the env gene was performed. Thirteen children were tested, including 6 with in utero infections and 7 with peripartum infections. Samples were taken the day after birth and at the ages of 6 and 14 weeks. A homogeneous virus population was seen in six (46.2%) infants, of whom two were infected in utero and four were infected peripartum. A more heterogeneous virus population was detected in seven infants (53.8%), four infected in utero and three infected peripartum. The phylogenetic trees of the mother-child pairs presented a whole range of different tree topologies and showed infection of the child by one or more maternal variants. In conclusion, after HIV-1 transmission from mother to child a heterogeneous virus population was detected in approximately one-half of the children examined. Heterogeneous virus populations were found after peripartum infection as well as after in utero infection. Phylogenetic tree topologies argue against selection processes as the major mechanism driving mother-to-child transmission but support the hypothesis that virus variability is mainly driven by the inoculum level and/or exposure time.

CXCR4-using HIV type 1 variants are more commonly found in peripheral blood mononuclear cell DNA than in plasma RNA.

Objective:To compare the distribution of R5-like and X4-like HIV-1envelope sequences in plasma and peripheral blood mononuclear cell (PBMC). Methods:Clonal sequencing of the HIV-1 glycoprotein 120 region was performed on PBMC DNA and plasma RNA of 11 HIV-1 subtype B-infected patients with high probability of carrying X4 virus. Coreceptor use was predicted using the position-specific scoring matrix (PSSM). Results:A total of 330 and 427 clonal envelope sequences were obtained from PBMC and plasma, respectively. PSSM interpretation revealed the presence of a mixture of predicted X4 and R5 sequences in 10 patients and pure R5 sequences in 1. The X4 sequences were significantly more represented in PBMC (with an average of 52.2% of the clonal proviral sequences scored X4) compared with plasma (19.7% X4 sequences) (P < 0.0001). At the single patient level, the higher representation of X4 sequences in PBMC reached statistical significance (P < 0.002) in 6 individuals. Conclusions:Mixtures of X4 and R5 sequences with highly divergent PSSM scores are present in both plasma and PBMC, but a shift toward a more abundant representation of X4-like PSSM scores in PBMC-derived DNA was apparent. Additional studies are needed to evaluate the clinical importance of these findings with regard to tropism prediction and the use of CCR5 anatagonists.

Drug-resistant variants that evolve during nonsuppressive therapy persist in HIV-1-infected peripheral blood mononuclear cells after long-term highly active antiretroviral therapy.

Abstract:The aim of this study was to determine whether drug-resistant virus persists in peripheral blood mononuclear cells (PBMCs) after long-term suppression of virus replication. Proviral DNA was extracted from the PBMCs of 11 patients on long-term highly active antiretroviral therapy (HAART). Genotyping of the reverse transcriptase (RT) and protease gene of several proviral variants was performed using limiting dilution polymerase chain reaction and single-copy sequencing. All patients were on successful HAART for a mean period of 59 months but had a history of suboptimal therapy and genotypic drug resistance before. Comparison of the amino acid sequence of the RT and protease gene in the different proviral variants, with that of the plasma virus isolated before HAART treatment, revealed that the different drug-resistant viral variants that evolved during the process of gradually building up resistance were still detectable in the PBMCs in 10 of the 11 patients tested. The proportion of resistant variants was found to correlate with the time that the resistant variants had been able to replicate. These data clearly show that virus variants that are able to replicate for a certain period enter the latent reservoir and remain archived in the PBMCs for a very long period.

Feasibility of Detecting Human Immunodeficiency Virus Type 1 Drug Resistance in DNA Extracted from Whole Blood or Dried Blood Spots

ABSTRACT Due to high cost, availability of human immunodeficiency virus type 1 (HIV-1) drug resistance testing in resource-poor settings is still limited. We therefore evaluated the usefulness of viral DNA extracted from either whole blood or dried blood spots (DBS). Samples were collected from 50 patients receiving therapy and 10 therapy-naïve patients. Amplification and sequencing of RNA and DNA was performed using an in-house assay. Protease (PR) and reverse transcriptase (RT) sequences of plasma viral RNA were obtained for 96.6% and 89.7%, respectively, of the 29 patients with a detectable viral load. For cellular viral DNA, useful PR and RT sequences were obtained for 96.6% and 93.1% of the whole-blood-cell samples and for 93.1% and 93.1% of the DBS samples, respectively. For the 31 patients with an undetectable viral load, PR and RT sequences were obtained for 67.7% and 61.3% of the whole-blood-cell DNA preparations and for 54.8% and 58.1% of the DBS DNA preparations, respectively. A good correlation between RNA and DNA sequences was found; most discordances were caused by the detection of mixed amino acids. Of the RT drug-resistant mutations, 13 (38.2%) were seen in RNA only, 6 (17.6%) in DNA only, and 15 (44.1%) in both. Repeated amplification and sequencing of DNA extracts revealed a lack of reproducibility for the detection of drug resistance mutations in a number of samples, indicating a possible founder effect. In conclusion, this study shows the feasibility of genotypic drug resistance testing on whole blood cells or DBS and its possible usefulness for HIV-1 subtyping or examining the overall distribution of drug resistance in a population. For individual patients, RNA sequencing was shown to be superior to DNA sequencing, especially for patients who experienced early treatment failure. The use of DNA extracted from whole blood or DBS for the detection of archived drug resistance mutations deserves further study.

Impact of triplicate testing on HIV genotypic tropism prediction in routine clinical practice

Guidelines state that the CCR5-inhibitor Maraviroc should be prescribed to patients infected with R5-tropic HIV-1 only. Therefore, viral tropism needs to be assessed phenotypically or genotypically. Preliminary clinical trial data suggest that genotypic analysis in triplicate is associated with improved prediction of virological response by increasing the detection of X4-tropic variants. Our objective was to evaluate the impact of triplicate genotypic analysis on prediction of co-receptor usage in routine clinical practice. Samples from therapy-naive and therapy-experienced patients were collected for routine tropism testing at three European clinical centres. Viral RNA was isolated from plasma and proviral DNA from peripheral blood mononuclear cells. Gp120-V3 was amplified in a triplicate nested RT-PCR procedure and sequenced. Co-receptor usage was predicted using the Geno2Pheno([coreceptor]) algorithm and analysed with a false-positive rate (FPR) of 5.75%, 10%, or an FPR of 20% and according to the current European guidelines on the clinical management of HIV-1 tropism testing. A total of 266 sequences were obtained from 101 patient samples. Discordance in tropism prediction for the triplicates was observed in ten samples using an FPR of 10%. Triplicate testing resulted in a 16.7% increase in X4-predicted samples and to reclassification from R5 to X4 tropism for four cases rendering these patients ineligible for Maraviroc treatment. In conclusion, triplicate genotypic tropism testing increases X4 tropism detection in individual cases, which may prove to be pivotal when CCR5-inhibitor therapy is applied.

Comparison of phenotypic and genotypic tropism determination in triple-class-experienced HIV patients eligible for maraviroc treatment

Background Determination of HIV-1 tropism is a pre-requisite to the use of CCR5 antagonists. This study evaluated the potential of population genotypic tropism tests (GTTs) in clinical practice, and the correlation with phenotypic tropism tests (PTTs) in patients accessing routine HIV care. Methods Forty-nine consecutive plasma samples for which an original TrofileTM assay was performed were obtained from triple-class-experienced patients in need of a therapy change. Viral tropism was defined as the consensus of three or more tropism calls obtained from the combination of two independent population PTT assays (Trofile Biosciences, San Francisco, CA, USA, and Virco, Beerse, Belgium), population GTTs and GTTs based on ultra-deep sequencing. If no consensus was reached, a clonal PTT was performed in order to finalize the tropism call. This two-step approach allowed the definition of a reference tropism call. Results According to the reference tropism result, 35/49 samples were CCR5 tropic (R5) (patients eligible for maraviroc treatment) and 14/49 were assigned as non-R5 tropic. The non-R5 samples [patients not eligible for maraviroc treatment according to the FDA/European Medicines Agency (EMEA) label] group included both the CXCR4 (X4) samples and the dual and mixed CCR5/CXCR4 (R5/X4) samples. Compared with TrofileTM population PTTs, population GTTs showed a higher sensitivity (97%) and a higher negative predictive value (91%), but almost equal specificity and an equal positive predictive value. Conclusions In line with recent reports from clinical trial data, our data support the use of population genotypic tropism testing as a tool for tropism determination before the start of maraviroc.

Drug resistance is rarely the cause or consequence of long-term persistent low-level viraemia in HIV-1-infected patients on ART.

BACKGROUND The introduction of highly sensitive HIV-1 viral load assays with a lower quantification limit of 20 copies/ml uncovered that in a number of patients on ART, the viral load systematically fluctuates around or slightly above the detection limit of the assays. This study aimed to analyse the presence or occurrence of drug resistance mutations in HIV-1-infected patients during long-term persistent low-level viraemia (PLLV) under ART. METHODS A retrospective study was carried out in which baseline and on-therapy presence of drug resistance mutations in the HIV-1 protease and reverse transcriptase genes were analysed in patients with PLLV between 20 and 250 copies/ml. For all available plasma samples collected during PLLV, resistance analysis was attempted with an ultrasensitive amplification and sequencing protocol. RESULTS Resistance analysis was successful for 154 samples collected longitudinally from 23 patients over a median period of 4.7 years (IQR 3.3-5.7). Twenty of these patients were on a boosted protease inhibitor (PI)-based regimen (87%). Single drug resistance mutations were detected in isolated samples of 4 patients, 2 of the 3 patients who initiated a non-nucleoside reverse transcriptase inhibitor-based regimen and 2 of the 20 on a PI-based regimen. Only one of the detected mutations decreased susceptibility to the therapy regimen taken at the time of sample collection. Drug resistance mutations were not found in the three patients who developed virological failure (viral load >250 copies/ml) during the study. CONCLUSIONS Long episodes of PLLV in patients on boosted PI-based regimens rarely result in the selection of new drug-resistant variants.

Markers associated with persisting low-level viraemia under antiretroviral therapy in HIV-1 infection

OBJECTIVES To identify host and viral characteristics associated with long-term persisting low-level viraemia (PLLV) under antiretroviral therapy (ART). PATIENTS AND METHODS Seventy-one ART-treated patients with long-term PLLV (20-250 copies/mL) and 102 control patients with systematically undetectable viral load (VL) were selected retrospectively from ART-treated patients followed at the Ghent HIV reference centre. Host and viral characteristics were compared using univariate and multivariate analyses. RESULTS Higher plasma VL at therapy initiation (OR 3.52; 95% CI 1.86-6.65; P < 0.001), therapy re-initiation after an interruption (OR 3.94; 95% CI 1.70-9.16; P = 0.001), male gender (OR 4.28; 95% CI 1.40-13.00; P = 0.011), a protease inhibitor-based regimen (OR 2.90; 95% CI 1.20-6.97; P = 0.017) and predicted CCR5 co-receptor tropism (OR 2.53; 95% CI 1.05-6.11; P = 0.039) were independently associated with PLLV. CONCLUSIONS VL at ART initiation, therapy history, gender, ART regimen and co-receptor tropism were independently associated with PLLV. Gender, therapy history, co-receptor tropism and VL at ART initiation could be valuable predictive markers to identify patients at risk for PLLV.

Longitudinal sequencing of HIV-1 infected patients with low-level viremia for years while on ART shows no indications for genetic evolution of the virus

HIV-infected patients on antiretroviral therapy (ART) may present low-level viremia (LLV) above the detection level of current viral load assays. In many cases LLV is persistent but does not result in overt treatment failure or selection of drug resistant viral variants. To elucidate whether LLV reflects active virus replication, we extensively sequenced pol and env genes of the viral populations present before and during LLV in 18 patients and searched for indications of genetic evolution. Maximum likelihood phylogenetic trees were inspected for temporal structure both visually and by linear regression analysis of root-to-tip and pairwise distances. Viral coreceptor tropism was assessed at different time points before and during LLV. In none of the patients consistent indications for genetic evolution were found over a median period of 4.8 years of LLV. As such these findings could not provide evidence that active virus replication is the main driver of LLV.

Quantification of total HIV-1 DNA in buffy coat cells, feasibility and potential added value for clinical follow-up of HIV-1 infected patients on ART

BACKGROUND Successfully treated HIV-1 infected patients have a sustained undetectable viral RNA load. In these cases the total HIV-1 DNA load may constitute a valuable tool to further follow the overall viral burden. The value of this marker outside of cure research has been rarely studied. OBJECTIVES To develop a quantitative (q)PCR for total HIV-1 DNA quantification in buffy coat cells and to evaluate the value of this parameter in clinical follow-up. STUDY DESIGN A qPCR using primers and a probe in the conserved HIV-1 LTR region was adapted for use on DNA extracted from buffy coat cells. Sensitivity, accuracy and reproducibility were evaluated using 8E5 cells and samples from naive and treatment experienced patients. The clinical value of DNA load analysis was assessed by testing 119 longitudinal samples from 9 patients before and after ART initiation and 249 cross sectional samples from therapy-experienced patients. RESULTS Inter- and intra-assay coefficients of variability were 5.56 and 5.94 (%CV). HIV-1 DNA was detected in 249 of the 263 (94.7%) patients on ART for at least 5 months (median: 53 months; IQR: 28-84 months). The HIV-1 DNA load varied between 0.60 and 3.37 copies/106 blood cells and showed significant correlation with the pre-ART CD4+ T-cell count nadir and peak viral RNA load. ART initiation resulted in a slow and limited decline of the total HIV-1 DNA concentration. CONCLUSIONS Quantification of total HIV-1 DNA from buffy coat cells is feasible, sensitive and reliable. Although determination of the on-therapy HIV-1 DNA load may be informative, regular testing has limited clinical value because of the very slow evolution.