Total Human Immunodeficiency Virus Type 1 DNA Research Articles

Factors associated with unquantifiable total HIV-1 DNA in peripheral blood in persons living with HIV: An observational study

BackgroundThe human immunodeficiency virus type 1 (HIV-1) cannot be eradicated even with suppressive antiretroviral therapy because its retrotranscribed genome integrates into the DNA of host cells, creating a long-term reservoir. Quantification of total HIV-1 DNA in peripheral blood is a biomarker of this reservoir that can predict progression of the infection, treatment response, and HIV-1-related complications. A deeper understanding of the reservoir may help develop a cures. ObjectiveThis study aimed to characterize persons living with HIV-1 (PLWH) with unquantifiable total HIV-1 DNA in blood (below the quantification threshold) and identify associated factors. MethodsWe have conducted a retrospective observational study. During the study period, all PLWH who had total leukocyte-associated HIV-1 DNA measured by quantitative PCR were included. We have isolated a population of participants with HIV-1 DNA levels below the quantification threshold (40 copies/106 leukocytes). ResultsOut of 1094 patients analysed, 62 had unquantifiable and 1032 quantifiable HIV-1 DNA levels in blood. We have found that those with unquantifiable HIV-1 DNA had a higher CD4 T cell nadir (p = 0.006) and a lower viral load zenith (p < 0.001). Multivariate analyses showed that initiation of treatment in primary infection was the only protective factor against HIV-1 DNA quantifiability, the odds of HIV-1 DNA quantifiability decreased by 82% in those treated within 30 days of infection, after controlling for other factors. ConclusionOur research highlights the importance of an early start of anti-retroviral therapy to limit the size of the HIV-1 reservoir, as receiving treatment during primary infection was found as the only protective factor against quantifiability of HIV-1 DNA in blood.

The CARMA Study: Early Infant Antiretroviral Therapy-Timing Impacts on Total HIV-1 DNA Quantitation 12 Years Later.

BackgroundStrategies aimed at antiretroviral therapy (ART)–free remission will target individuals with a limited viral reservoir. We investigated factors associated with low reservoir measured as total human immunodeficiency virus type 1 (HIV-1) DNA in peripheral blood mononuclear cells (PBMCs) in perinatal infection (PaHIV).MethodsChildren from 7 European centers in the Early Treated Perinatally HIV Infected Individuals: Improving Children’s Actual Life (EPIICAL) consortium who commenced ART aged <2 years, and remained suppressed (viral load [VL] <50 copies/mL) for >5 years were included. Total HIV-1 DNA was measured by quantitative polymerase chain reaction per million PBMCs. Factors associated with total HIV-1 DNA were analyzed using generalized additive models. Age, VL at ART initiation, and baseline CD4% effects were tested including smoothing splines to test nonlinear association.ResultsForty PaHIV, 27 (67.5%) female 21 (52.5%) Black/Black African, had total HIV-1 DNA measured; median 12 (IQR, 7.3–15.4) years after ART initiation. Eleven had total HIV-1 DNA <10 copies/106 PBMCs. HIV-1 DNA levels were positively associated with age and VL at ART initiation, baseline CD4%, and Western blot antibody score. Age at ART initiation presented a linear association (coefficient = 0.10 ± 0.001, P ≤ .001), the effect of VL (coefficient = 0.35 ± 0.1, P ≤ .001) noticeable >6 logs. The effect of CD4% (coefficient = 0.03 ± 0.01, P = .049) was not maintained >40%.ConclusionsIn this PaHIV cohort, reduced total HIV-1 DNA levels were associated with younger age and lower VL at ART initiation. The impact of early-infant treatment on reservoir size persists after a decade of suppressive therapy.

Preferential Infection of α4β7+ Memory CD4+ T Cells During Early Acute Human Immunodeficiency Virus Type 1 Infection.

BackgroundEstablishment of persistent human immunodeficiency virus type 1 (HIV-1) reservoirs occurs early in infection, and biomarkers of infected CD4+ T cells during acute infection are poorly defined. CD4+ T cells expressing the gut homing integrin complex α4β7 are associated with HIV-1 acquisition, and are rapidly depleted from the periphery and gastrointestinal mucosa during acute HIV-1 infection.MethodsIntegrated HIV-1 DNA was quantified in peripheral blood mononuclear cells obtained from acutely (Fiebig I–III) and chronically infected individuals by sorting memory CD4+ T-cell subsets lacking or expressing high levels of integrin β7 (β7negative and β7high, respectively). HIV-1 DNA was also assessed after 8 months of combination antiretroviral therapy (cART) initiated in Fiebig II/III individuals. Activation marker and chemokine receptor expression was determined for β7-defined subsets at acute infection and in uninfected controls.ResultsIn Fiebig I, memory CD4+ T cells harboring integrated HIV-1 DNA were rare in both β7high and β7negative subsets, with no significant difference in HIV-1 DNA copies. In Fiebig stages II/III and in chronically infected individuals, β7high cells were enriched in integrated and total HIV-1 DNA compared to β7negative cells. During suppressive cART, integrated HIV-1 DNA copies decreased in both β7negative and β7high subsets, which did not differ in DNA copies. In Fiebig II/III, integrated HIV-1 DNA in β7high cells was correlated with their activation.Conclusionsβ7high memory CD4+ T cells are preferential targets during early HIV-1 infection, which may be due to the increased activation of these cells.

Naive CD4+ T Cells Harbor a Large Inducible Reservoir of Latent, Replication-competent Human Immunodeficiency Virus Type 1.

The latent human immunodeficiency virus type 1 (HIV-1) reservoir represents a major barrier to a cure. Based on the levels of HIV-1 DNA in naive (TN) vs resting memory CD4+ T cells, it is widely hypothesized that this reservoir resides primarily within memory cells. Here, we compared virus production from TN and central memory (TCM) CD4+ T cells isolated from HIV-1-infected individuals on suppressive therapy. CD4+ TN and TCM cells were purified from the blood of 7 HIV-1-infected individuals. We quantified total HIV-1 DNA in the CD4+ TN and TCM cells. Extracellular virion-associated HIV-1 RNA or viral outgrowth assays were used to assess latency reversal following treatment with anti-CD3/CD28 monoclonal antibodies (mAbs), phytohaemagglutinin/interleukin-2, phorbol 12-myristate 13-acetate/ionomycin, prostratin, panobinostat, or romidepsin. HIV-1 DNA was significantly higher in TCM compared to TN cells (2179 vs 684 copies/106 cells, respectively). Following exposure to anti-CD3/CD28 mAbs, virion-associated HIV-1 RNA levels were similar between TCM and TN cells (15 135 vs 18 290 copies/mL, respectively). In 4/7 donors, virus production was higher for TN cells independent of the latency reversing agent used. Replication-competent virus was recovered from both TN and TCM cells. Although the frequency of HIV-1 infection is lower in TN compared to TCM cells, as much virus is produced from the TN population after latency reversal. This finding suggests that quantifying HIV-1 DNA alone may not predict the size of the inducible latent reservoir and that TN cells may be an important reservoir of latent HIV-1.

No Effect of Pegylated Interferon-α on Total HIV-1 DNA Load in HIV-1/HCV Coinfected Patients.

Pegylated interferon-alpha (pIFN-α) is suggested to lower human immunodeficiency virus type-1 (HIV-1) DNA load in antiretroviral therapy (ART)-treated patients. We studied kinetics of HIV-1 DNA levels in 40 HIV-1/hepatitis C virus (HCV) coinfected patients, treated with pIFN-α for HCV and categorized into 3 groups according to start of ART: chronic HIV-1 infection (n = 22), acute HIV-1 infection (n = 8), no-ART (n = 10). Total HIV-1 DNA levels in 247 peripheral blood mononuclear cell samples were stable before, during, and after pIFN-α treatment in all groups. Our results question the benefit of pIFN-α as an immunotherapeutic agent for reducing the HIV-1 reservoir.

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.

HIV-1 DNA proviral load in treated and untreated HIV-1 sero-positive patients

As proviral human immunodeficiency virus type 1 (HIV-1) DNA can replenish and revive viral infection upon activation, its detection might offer significant therapeutic information, complementing the input provided by plasma RNA determination in the follow-up of infected individuals. A selected group of acutely infected subjects was studied to verify both total and 2-long terminal repeat (2-LTR) DNA proviral load during the acute phase of infection and thereafter. Patients were divided in two sex- and age-matched groups: 19 naive individuals who did not receive antiretroviral therapy during the observation period and 20 subjects treated according to current guidelines. Total and 2-LTR HIV-1 DNA proviral load, in addition to RNA viral load and CD4 cell count, were determined in peripheral blood mononuclear cells (PBMC) at baseline, 6 and 12 months after the first sampling. Total and 2-LTR HIV-1 DNA proviral load exhibited no significant variation at any time in the naive patients (total HIV-1 DNA ranging from 896 ± 731 to 715 ± 673 copies/105 PBMC and 2-LTR HIV-1 DNA ranging from 94 ± 105 to 65 ± 44 copies/105 PBMC), whereas a significant reduction in both total HIV-1 DNA (ranging from 997 ± 676 to 262 ± 174 copies/105 PBMC) and 2-LTR HIV-1 DNA proviral load (ranging from 116 ± 55 to 26 ± 35 copies/105 PBMC) was detected in highly active antiretroviral therapy (HAART) patients, together with a CD4+ T cell count increase and RNA load decrease. HAART negatively affects both the labile HIV burden and the integrated proviral DNA, at least in the initial period of successful treatment, suggesting that quantification of HIV-1 DNA proviral load may be an important parameter in monitoring HIV infection.

Dynamics of Total, Linear Nonintegrated, and Integrated HIV‐1 DNA In Vivo and In Vitro

In patients infected with human immunodeficiency virus type 1 (HIV-1), HIV-1 DNA persists during highly active antiretroviral treatment, reflecting long-lived cellular reservoirs of HIV-1. Recent studies report an association between HIV-1 DNA levels, disease progression, and treatment outcome. However, HIV-1 DNA exists as distinct molecular forms that are not distinguished by conventional assays. We analyzed HIV-1 RNA levels in plasma, CD4 cell counts, and levels of integrated and nonintegrated HIV-1 DNA in peripheral blood mononuclear cells (PBMCs) from patients with early or chronic infection before and during antiretroviral treatment. We also studied HIV-1 DNA decay in primary CD4 T cells infected in vitro. HIV-1 DNA was analyzed using an assay that is unaffected by the location of HIV-1 integration sites. HIV-1 RNA levels and total HIV-1 DNA levels decayed rapidly in patients during receipt of suppressive antiretroviral therapy. Ratios of total HIV-1 DNA levels to integrated HIV-1 DNA levels were high before initiation of therapy but diminished during therapy. Levels of linear nonintegrated HIV-1 DNA decayed rapidly in vitro (t (1/2) = 1- 4.8 days). Total HIV-1 DNA decays rapidly with suppression of virus replication in vivo. Clearance of HIV-1 DNA during the first 6 months of therapy reflects a disproportionate loss of nonintegrated HIV-1 DNA genomes, suggesting that levels of total HIV-1 DNA in PBMCs after prolonged virus suppression largely represent integrated HIV-1 genomes.

Analysis of early human immunodeficiency virus type 1 DNA synthesis by use of a new sensitive assay for quantifying integrated provirus.

A novel Alu-long terminal repeat (LTR)-based real-time nested-PCR assay was developed to quantify integrated human immunodeficiency virus type 1 (HIV-1) DNA in infected cells with both accuracy and high sensitivity (six proviruses within 50,000 cell equivalents). Parallel assays for total HIV-1 DNA and two-LTR HIV-1 DNA circles allowed the synthesis and fate of the different HIV-1 DNA species to be monitored upon a single round of viral replication.

Impact of the central polypurine tract on the kinetics of human immunodeficiency virus type 1 vector transduction.

Lentiviral vectors derived from human immunodeficiency virus type 1 (HIV-1) show great promise as gene carriers for future gene therapy. Insertion of a fragment containing the central polypurine tract (cPPT) in HIV-1 vector constructs is known to enhance transduction efficiency drastically, reportedly by facilitating the nuclear import of HIV-1 cDNA through a central DNA flap. We have studied the impact of the cPPT on the kinetics of HIV-1 vector transduction by real-time PCR. The kinetics of total HIV-1 DNA, two-long-terminal-repeat (2-LTR) circles, and, by an Alu-PCR, integrated proviral DNA were monitored. About 6 to 12 h after transduction, the total HIV-1 DNA reached a maximum level, followed by a steep decrease. The 2-LTR circles peaked after 24 to 48 h and were diluted upon cell division. Integration of HIV-1 DNA was first detected at 12 h postinfection. When HIV-1 vectors that contained the cPPT were used, DNA synthesis was similar but a threefold higher amount of 2-LTR circles was detected, confirming the impact on nuclear import. Moreover, a 10-fold increase in the amount of integrated DNA was observed in the presence of the cPPT. Only in the absence of the cPPT was a saturation in 2-LTR circle formation seen at a high multiplicity of infection, suggesting a role for the cPPT in overcoming a barrier to the nuclear import of HIV-1 DNA. A major effect of the central DNA flap on the juxtaposition of both LTRs is unlikely, since transduction with HIV-1 vectors containing ectopic cPPT fragments resulted in increased amounts of 2-LTR circles as well as integrated DNA. Inhibitors of transduction by cPPT-containing HIV vectors were also studied by real-time PCR. The reverse transcriptase inhibitor azidothymidine (AZT) and the nonnucleoside reverse transcriptase inhibitor alpha-APA clearly inhibited viral DNA synthesis, whereas integrase inhibitors such as the diketo acid L-708,906 and the pyranodipyrimidine V-165 specifically inhibited integration.

Molecular analysis of the differential restriction of human immunodeficiency virus type 1 replication in neuronal cell lines.

Human immunodeficiency virus type 1 (HIV-1) replication is restricted partially in SK-N-MC and completely in SK-N-SH neuronal cells. To investigate the molecular mechanism of this differential restriction of HIV-1 replication, cells infected with HIV-1 were analysed for their steady-state levels of: total and unintegrated HIV-1 DNA by DNA PCR, different species of HIV-1 RNA by RT-PCR, and HIV-1 p24 protein production by an ELISA procedure. We found that the kinetics of the infection were slower and there was a lower level of accumulation of HIV-1 macromolecules (total and unintegrated circular DNA, unspliced and spliced RNAs and viral proteins) in the SK-N-MC cells than in the permissive CEM cells. In SK-N-SH cells, HIV-1 DNA was only transiently detected during the first 24 h post-infection, and the unspliced RNA was detected up to 1 week post-infection. However, the HIV-1 spliced RNAs and the 2-LTR circular DNA were not detected at all during the course of infection. Both SK-N-MC and SK-N-SH cells showed higher levels of HIV-1 DNA, RNA and p24 protein when infected with an HIV-1 (amphotropic retrovirus) pseudotype, HIV-1B. However, the level of HIV-1 replication was still lower in SK-N-SH than in SK-N-MC cells. Moreover, although the kinetics of viral protein production were comparable in SK-N-MC cells infected with HIV-1B and CEM cells infected with HIV-1, the overall level of virus replication was still much lower in HIV-1B-infected SK-N-MC cells. These data suggest that the restriction of HIV-1 replication in neuronal cell lines takes place at both virus-entry and post-entry levels, and cellular factors may be involved in the differential restriction of HIV-1 replication in these cells.