Guillaume Besson

Clonally expanded CD4+ T cells can produce infectious HIV-1 in vivo

Significance Reservoirs of HIV-infected cells persist during antiretroviral therapy, and understanding persistence is essential to develop HIV curative strategies. During replication, HIV integrates into the host genome; most proviruses are not infectious, but some with replication-competent HIV persist. Cells with integrated HIV can proliferate, potentially expanding the reservoir, but whether cells with replication-competent HIV actually undergo expansion is unknown. HIV reactivation is often lethal to infected cells, and others have reported finding no replication-competent HIV in expanded populations. We describe a highly expanded clone containing infectious HIV that was the source of viremia for years in a patient. Clonally expanded populations can represent a long-lived reservoir of HIV. Curative strategies will require targeting this persistence mechanism. Reservoirs of infectious HIV-1 persist despite years of combination antiretroviral therapy and make curing HIV-1 infections a major challenge. Most of the proviral DNA resides in CD4+T cells. Some of these CD4+T cells are clonally expanded; most of the proviruses are defective. It is not known if any of the clonally expanded cells carry replication-competent proviruses. We report that a highly expanded CD4+ T-cell clone contains an intact provirus. The highly expanded clone produced infectious virus that was detected as persistent plasma viremia during cART in an HIV-1–infected patient who had squamous cell cancer. Cells containing the intact provirus were widely distributed and significantly enriched in cancer metastases. These results show that clonally expanded CD4+T cells can be a reservoir of infectious HIV-1.

HIV-1 DNA Decay Dynamics in Blood During More Than a Decade of Suppressive Antiretroviral Therapy

BACKGROUND Human immunodeficiency virus type 1 (HIV-1) DNA dynamics during long-term antiretroviral therapy (ART) are not defined. METHODS Blood mononuclear cells obtained during 7-12 years of effective ART were assayed for total HIV-1 DNA and 2-long terminal repeat (LTR) circles by quantitative polymerase chain reaction (qPCR). Slopes of HIV-1 DNA were estimated by participant-specific linear regressions. Plasma was assayed for residual viremia (HIV-1 RNA) by qPCR. RESULTS Thirty participants were studied. HIV-1 DNA decreased significantly from years 0-1 and 1-4 of ART with median decay slopes of -0.86 (interquartile range, -1.05, -0.59) and -0.11 (-0.17, -0.06) log10(copies/10(6) CD4+ T-cells)/year, respectively (P < .001). Decay was not significant for years 4-7 (-0.02 [-0.06, 0.02]; P = .09) or after year 7 of ART (-0.006 [-0.030, 0.015]; P = .17). All participants had detectable HIV-1 DNA after 10 years (median 439 copies/10(6) CD4+ T-cells; range: 7-2074). Pre-ART HIV-1 DNA levels were positively associated with pre-ART HIV-1 RNA levels (Spearman = 0.71, P < .001) and with HIV-1 DNA at years 4, 7, and 10 on ART (Spearman ≥ 0.75, P < .001). No associations were found (P ≥ .25) between HIV-1 DNA slopes or levels and % activated CD8+ T-cells (average during years 1-4) or residual viremia (n = 18). 2-LTR circles were detected pre-ART in 20/29 and in 8/30 participants at last follow-up. CONCLUSIONS Decay of HIV-1 DNA in blood is rapid in the first year after ART initiation (86% decline), slows during years 1-4 (23% decline/year), and subsequently plateaus. HIV-1 DNA decay is not associated with the levels of CD8+ T-cell activation or persistent viremia. The determinants of stable HIV-1 DNA persistence require further elucidation. Clinical Trials Registration. NCT00001137.

Protective properties of non-nucleoside reverse transcriptase inhibitor (MC1220) incorporated into liposome against intravaginal challenge of Rhesus Macaques with RT-SHIV

In the absence of an effective vaccine against HIV, it is urgent to develop an effective alternative such as a microbicide. Single and repeated applications of MC1220 microbicide were evaluated in macaques. First, animals were given a single application of 0.5% or 1.5% MC1220-containing liposomal gel. A second group were treated with 0.5% MC1220 once a day for 4 days. The control groups were treated by liposomal gel alone. Thirty minutes after the last application, animals were challenged with RT-SHIV. In the first protocol, 2 of 4 animals treated by 0.5% of the MC1220 and 2 of 5 treated by 1.5% were protected. In the second protocol, 3 of 5 treated animals were protected and 5 of 5 controls were infected. The RNA viral load at necropsy was significantly lower (p=0.05) in treated-infected animals than in controls. In both protocols, the number of CD4+ T cells was lower at viremia peak in infected than in protected animals.

Phylogeography, risk factors and genetic history of hepatitis C virus in Gabon, central Africa.

Background The epidemiological and molecular characteristics of hepatitis C virus (HCV) infection in the general population have been poorly investigated in Africa. The aim of this study was to determine the prevalence, genotype distribution and epidemic history of HCV in the Gabonese general population. Methods/Principal Findings A total of 4042 sera collected from adults in 220 villages in all nine administrative areas of the country were screened for antibodies to HCV. HCV NS5B region sequencing was performed for molecular characterization and population genetic analyses. Of 4042 tested sera, 455 (11.2%) were positive. The seroprevalence of HCV varied significantly by administrative area, with the highest rate in Ogooué-Lolo province (20.4%) and the lowest in Ogooué-Maritine province (3.7%). History of parenteral injections, past hospital admission and age over 55 years were independent risk factors for HCV infection (p<0.0001). Phylogenetic analyses showed that 91.9% of the strains were genotype 4 (HCV-4), 5.7% genotype 1 and 2.2% genotype 2. HCV-4 strains were highly heterogeneous, with more than eight subtypes; subtype 4e predominated (57.3%). Coalescence analyses indicated that subtype 4e was the oldest, with an estimated most recent common ancestor of 1702 [95% CI, 1418–1884]. The epidemic profile indicated that it spread exponentially during the first part of the 20th century, probably by iatrogenic transmission. Conclusions/Significance These results confirm the endemicity of HCV subtype 4e in Gabon and show that its spread is due to a cohort effect, with previous, possibly iatrogenic events. More extensive epidemiological studies are needed to better characterize the route of transmission and the dissemination of HCV in Gabon.

New Insights into Prevalence, Genetic Diversity, and Proviral Load of Human T-Cell Leukemia Virus Types 1 and 2 in Pregnant Women in Gabon in Equatorial Central Africa

ABSTRACT Human T-cell leukemia virus type 1 (HTLV-1) is highly endemic in areas of central Africa; mother-to-child transmission and sexual transmission are considered to be the predominant routes. To determine the prevalence and subtypes of HTLV-1/2 in pregnant women in Gabon, we conducted an epidemiological survey in the five main cities of the country. In 907 samples, the HTLV-1 seroprevalence was 2.1%, which is lower than that previously reported. Only one case of HTLV-2 infection was found. The HTLV-1 seroprevalence increased with age and differed between regions (P ≤ 0.05), with the highest prevalence (5%) in the southeastern region. A wide range of HTLV-1 proviral loads was observed among the infected women. The level of the proviral load was correlated with a high HTLV-1 antibody titer (P ≤ 0.02). Sequencing of HTLV-1 env and long terminal repeat fragments showed that all but one strain belonged to the central African subtype B; the outlier was of cosmopolitan subtype A. The new strains of subtype B exhibited wide genetic diversity, but there was no evidence of clustering of specific genomes within geographical regions of the country. Some strains were closely related to simian T-cell leukemia virus type 1 strains of great apes, suggesting that in these areas some HTLV-1 strains could arise from relatively recent interspecies transmission. The sole HTLV-2 strain belonged to subtype B. In this study we showed that the prevalence of HTLV-1 in the southeast is one of the highest in the world for pregnant women.

Short-Course Raltegravir Intensification Does Not Increase 2 Long Terminal Repeat Episomal HIV-1 DNA in Patients on Effective Antiretroviral Therapy

TO THE EDITOR—Controversy exists about whether human immunodeficiency virus type 1 (HIV-1) replication persists in patients receiving effective antiretroviral therapy. Residual viremia can often be detected in such patients by sensitive quantitative polymerase chain reaction assays, but the source of this viremia is unknown [1]. Possibilities include ongoing cycles of HIV-1 replication, long-lived HIV-1–infected cells that continuously or intermittently produce virus, or both. If residual plasma viremia is derived from ongoing HIV-1 replication, intensification of an effective regimen with another potent antiretroviral drug should lower viremia. Such treatment intensification studies have been conducted with different classes of antiretroviral agents (nucleoside reverse-transcriptase inhibitors, nonnucleoside reverse-transcriptase inhibitors, protease inhibitors, and the integrase inhibitor raltegravir), and none has shown reduction in residual viremia [2–6]. However, Buzon et al [7] showed transient increases in episomal (2-Long Terminal Repeat [2-LTR] circle) HIV-1 DNA levels in 13 of 45 patients after raltegravir intensification, suggesting raltegravir blocked ongoing HIV-1 replication without affecting plasma viremia. Moreover, Yukl et al [5] showed reductions in unspliced HIV-1 RNA levels in CD4+ T cells from the terminal ileum in 5 of 7 patients after raltegravir intensification. To provide additional insight into the effects of raltegravir intensification, we analyzed total HIV-1 DNA and 2-LTR circle levels in peripheral blood mononuclear cells (PBMCs) obtained before and after 28 days of raltegravir intensification from a study of 10 patients, published by McMahon et al [4] in Clinical Infectious Diseases, that showed no effect of raltegravir on residual viremia. Total cellular DNA was extracted (Dynabeads) from 5 × 106 PBMCs and assayed for total HIV-1 DNA and 2-LTR HIV-1 DNA circle levels by quantitative, real-time polymerase chain reaction (limit of detection, 15 copies per 106 PBMCs for both assays). HIV-1 DNA was detected in all subjects, ranging from 154 to 6,879 copies per 106 PBMCs and showing no consistent change between baseline (before intensification) and 28 days after intensification (Table 1). Levels of 2-LTR circles were detected in 3 of 10 patients at baseline, but only 2 still tested positive after intensification (range, 19–638 copies per 106 PBMCs). There was no increase in 2-LTR circles in these 2 patients after raltegravir intensification. There was also no change in the ratio of 2-LTR circles to total HIV-1 DNA level after intensification. Finally, none of the 7 patients who were negative for 2-LTR circles at baseline become positive after intensification. Table 1. Quantification of HIV-1 DNA and 2 Long Terminal Repeat Circles in Peripheral Blood Mononuclear Cells These results do not support an effect of raltegravir intensification on 2-LTR HIV-1 DNA circles, which is contrary to the report by Buzon et al [7] that showed an increase in 2-LTR circles in 29% of patients 2–4 weeks after raltegravir intensification. Differences in patient characteristics, sampling time, antiretroviral regimens, duration of HIV-1 RNA suppression, and sample size may be responsible for the discrepancies between the 2 studies. Nevertheless, most evidence reported to date, including the results presented here, refute ongoing HIV-1 replication in patients receiving effective antiretroviral therapy as the source of residual viremia [2, 5, 6]. Additional studies are needed, however, to assess the impact of treatment intensification on HIV-1 replication in tissue compartments.

One-Step, Multiplex, Real-Time PCR Assay with Molecular Beacon Probes for Simultaneous Detection, Differentiation, and Quantification of Human T-Cell Leukemia Virus Types 1, 2, and 3

ABSTRACT A single-tube, multiplex, real-time PCR assay with molecular beacons was established in which various probes were used for the simultaneous detection, differentiation, and quantification of human T-cell leukemia virus types 1, 2, and 3 (HTLV-1, HTLV-2, and HTLV-3, respectively) and of simian T-cell leukemia virus types 1 and 3 (STLV-1 and STLV-3, respectively). The quantitative amplification of the standards with MT4 (HTLV-1) and C19 (HTLV-2) cell lines and a molecular clone of HTLV-3 was linear, with the simplex and multiplex methods having similar efficiencies. A maximum difference of 0.9 (mean, 0.4; range, 0.0 to 0.9) was found between threshold cycle values in single and multiplex reactions. The efficiency with each probe in the multiplex reaction was close to 100%, indicating strong linear amplification. The albumin gene was used to standardize the copy number. Comparable results for the detection and quantification of HTLV-1 were obtained with our new methods and with other real-time PCR methods described previously. With our new multiplex assay, however, we were able to detect and quantify HTLV-2 and -3 and STLV-1 and -3 in clinical specimens, with an excellent dynamic range of 106 to 100 copies per assay, which the other assays could not do. Thus, it will be possible to determine a wide range of HTLV types in both standard and clinical samples, with a detection of 1 to 10 HTLV copies in samples containing at least 100 cells. Furthermore, our system can provide evidence for multiple infections with the three HTLV types, with separate proviral load results. Our new method also could be used for epidemiological studies in Africa and in countries where HTLVs and STLVs are endemic.

Novel Assays for Measurement of Total Cell-Associated HIV-1 DNA and RNA

ABSTRACT Although a number of PCR-based quantitative assays for measuring HIV-1 persistence during suppressive antiretroviral therapy (ART) have been reported, a simple, sensitive, reproducible method is needed for application to large clinical trials. We developed novel quantitative PCR assays for cell-associated (CA) HIV-1 DNA and RNA, targeting a highly conserved region in HIV-1 pol, with sensitivities of 3 to 5 copies/1 million cells. We evaluated the performance characteristics of the assays using peripheral blood mononuclear cells (PBMCs) from 5 viremic patients and 20 patients receiving effective ART. Total and resting CD4+ T cells were isolated from a subset of patients and tested for comparison with PBMCs. The estimated standard deviations including interassay variability and intra-assay variability of the assays were modest, i.e., 0.15 and 0.10 log10 copies/106 PBMCs, respectively, for CA HIV-1 DNA and 0.40 and 0.19 log10 copies/106 PBMCs for CA HIV-1 RNA. Testing of longitudinally obtained PBMC samples showed little variation for either viremic patients (median fold differences of 0.80 and 0.88 for CA HIV-1 DNA and RNA, respectively) or virologically suppressed patients (median fold differences of 1.14 and 0.97, respectively). CA HIV-1 DNA and RNA levels were strongly correlated (r = 0.77 to 1; P = 0.0001 to 0.037) for assays performed using PBMCs from different sources (phlebotomy versus leukapheresis) or using total or resting CD4+ T cells purified by either bead selection or flow cytometric sorting. Their sensitivity, reproducibility, and broad applicability to small numbers of mononuclear cells make these assays useful for observational and interventional studies that examine longitudinal changes in the numbers of HIV-1-infected cells and their levels of transcription.

New Tools for Quantifying HIV-1 Reservoirs: Plasma RNA Single Copy Assays and Beyond

Quantification of plasma HIV-1 RNA below the limit of FDA-approved assays by a single copy quantitative PCR assays (SCA) has provided significant insights into HIV-1 persistence despite potent antiretroviral therapy as well as a means to assess the impact of therapeutic strategies, such as treatment intensification, on residual viremia. In this review, we discuss insights gained from plasma HIV-1 RNA SCA and highlight the need for additional assays to characterize better the cellular and tissue reservoirs of HIV-1. Accurate, reproducible, and sensitive assays to quantify HIV-1 reservoirs, before and after therapeutic interventions, are essential tools in the quest for a cure of HIV-1 infection.

Y chromosome and HIV DNA detection in vaginal swabs as biomarkers of semen and HIV exposure in women.

Background The inability to quantify sexual exposure to HIV limits the power of HIV prevention trials of vaccines, microbicides, and preexposure prophylaxis in women. We investigated the detection of HIV-1 and Y chromosomal (Yc) DNA in vaginal swabs from 83 participants in the HPTN 035 microbicide trial as biomarkers of HIV exposure and unprotected sexual activity. Methods One hundred forty-three vaginal swabs from 85 women were evaluated for the presence of Yc DNA (Quantifiler Duo DNA quantification kit; Applied Biosystems) and total HIV-1 DNA (single-copy in-house quantitative polymerase chain reaction assay). Y DNA detection was paired with self-reported behavioral data with regard to recent coitus (⩽1 week before collection) and condom usage (100% vs. <100% compliance). Results Yc DNA was detected in 62 (43%) of 143 swabs. For the 126 visits at which both behavioral data and swabs were collected, Yc DNA was significantly more frequent in women reporting less than 100% condom usage (odds ratio, 10.69; 95% confidence interval, 2.27–50.32; P = 0.003). Notably, 27 (33%) of 83 swabs from women reporting 100% condom usage were positive for Yc DNA. HIV DNA was only detected in swabs collected postseroconversion. Conclusions The use of Yc DNA in HIV prevention trials could reliably identify subgroups of women who have unprotected sexual activity and could provide valuable exposure-based estimates of efficacy.