Integrated HIV DNA Levels Research Articles

Increasing contribution of integrated forms to total HIV DNA in blood during HIV disease progression from primary infection.

BackgroundIn the current context of research on HIV reservoirs, offering new insights into the persistence of HIV DNA in infected cells, which prevents viral eradication, may aid in identifying cure strategies. This study aimed to describe the establishment of stable integrated forms among total HIV DNA during primary infection (PHI) and their dynamics during the natural history of infection.MethodsTotal and integrated HIV DNA were quantified in blood from 74 PHI patients and 97 recent seroconverters (<12 months following infection, “progression cohort”). The evolution of both markers over six years was modelled (mixed-effect linear models). Their predictive values for disease progression were studied (Cox models).FindingsFor most patients during PHI, stable integrated forms were a minority among total HIV DNA (median: 12%) and became predominant thereafter (median at AIDS stage: 100%). Both total and integrated HIV DNA increased over a six-year period. Patients from the progression cohort who reached clinical AIDS during follow-up (n = 34) exhibited higher total and integrated HIV DNA levels at seroconversion and a higher percentage of integrated forms than did slower progressors (n = 63) (median: 100% vs 44%). The integrated HIV DNA load was strongly associated with the risk of developing AIDS (aRR = 2.63, p = 0.002).InterpretationThe profile of “rapid” or “slower” progression in the natural history of HIV infection appears to be determined early in the course of HIV infection. The strong predominance of unstable unintegrated forms in PHI may explain the great benefit of this early treatment, which induces a sharp decrease in total HIV DNA.FundFrench National Agency for Research on AIDS and Viral Hepatitis.

Whole Exome Sequencing of HIV-1 long-term non-progressors identifies rare variants in genes encoding innate immune sensors and signaling molecules

Common CCR5-∆32 and HLA alleles only explain a minority of the HIV long-term non-progressor (LTNP) and elite controller (EC) phenotypes. To identify rare genetic variants contributing to the slow disease progression phenotypes, we performed whole exome sequencing (WES) on seven LTNPs and four ECs. HLA and CCR5 allele status, total HIV DNA reservoir size, as well as variant-related functional differences between the ECs, LTNPs, and eleven age- and gender-matched HIV-infected non-controllers on antiretroviral therapy (NCARTs) were investigated. Several rare variants were identified in genes involved in innate immune sensing, CD4-dependent infectivity, HIV trafficking, and HIV transcription mainly within the LTNP group. ECs and LTNPs had a significantly lower HIV reservoir compared to NCARTs. Furthermore, three LTNPs with variants affecting HIV nuclear import showed integrated HIV DNA levels below detection limit after in vitro infection. HIV slow progressors with variants in the TLR and NOD2 pathways showed reduced pro-inflammatory responses compared to matched controls. Low-range plasma levels of fibronectin was observed in a LTNP harboring two FN1 variants. Taken together, this study identified rare variants in LTNPs as well as in one EC, which may contribute to understanding of HIV pathogenesis and these slow progressor phenotypes, especially in individuals without protecting CCR5-∆32 and HLA alleles.

Effect of Short-Term Antiretroviral Therapy Interruption on Levels of Integrated HIV DNA.

Analytic treatment interruption (ATI) studies are required to evaluate strategies aimed at achieving ART-free HIV remission, but the impact of ATI on the viral reservoir remains unclear. We validated a DNA size selection-based assay for measuring levels of integrated HIV DNA and applied it to assess the effects of short-term ATI on the HIV reservoir. Samples from participants from four AIDS Clinical Trials Group ATI studies were assayed for integrated HIV DNA levels. Cryopreserved peripheral blood mononuclear cells (PBMCs) were obtained for 12 participants with available samples pre-ATI and approximately 6 months after ART resumption. Four participants also had samples available during the ATI. The median duration of ATI was 12 weeks. Validation of the HIV integrated DNA size-exclusion (HIDE) assay was performed using samples spiked with unintegrated HIV DNA, HIV-infected cell lines, and participant PBMCs. The HIDE assay eliminated 99% of unintegrated HIV DNA species and strongly correlated with the established Alu-gag assay. For the majority of individuals, integrated DNA levels increased during ATI and subsequently declined upon ART resumption. There was no significant difference in the levels of integrated HIV DNA between the pre- and post-ATI time points, with a median ratio of post- to pre-ATI HIV DNA levels of 0.95. Using a new integrated HIV DNA assay, we found minimal change in the levels of integrated HIV DNA in participants who underwent an ATI, followed by 6 months of ART. This suggests that short-term ATI can be conducted without a significant impact on the levels of integrated proviral DNA in the peripheral blood.IMPORTANCE Interventions aimed at achieving sustained antiretroviral therapy (ART)-free HIV remission require treatment interruption trials to assess their efficacy. However, these trials are accompanied by safety concerns related to the expansion of the viral reservoir. We validated an assay that uses an automated DNA size-selection platform for quantifying levels of integrated HIV DNA and is less sample- and labor-intensive than current assays. Using stored samples from AIDS Clinical Trials Group studies, we found that short-term ART discontinuation had minimal impact on integrated HIV DNA levels after ART resumption, providing reassurance about the reservoir effects of short-term treatment interruption trials.

HIV dynamics linked to memory CD4+ T cell homeostasis.

The dynamics of latent HIV is linked to infection and clearance of resting memory CD4+ T cells. Infection also resides within activated, non-dividing memory cells and can be impacted by antigen-driven and homeostatic proliferation despite suppressive antiretroviral therapy (ART). We investigated whether plasma viral level (pVL) and HIV DNA dynamics could be explained by HIV’s impact on memory CD4+ T cell homeostasis. Median total, 2-LTR and integrated HIV DNA levels per μL of peripheral blood, for 8 primary (PHI) and 8 chronic HIV infected (CHI) individuals enrolled on a raltegravir (RAL) based regimen, exhibited greatest changes over the 1st year of ART. Dynamics slowed over the following 2 years so that total HIV DNA levels were equivalent to reported values for individuals after 10 years of ART. The mathematical model reproduced the multiphasic dynamics of pVL, and levels of total, 2-LTR and integrated HIV DNA in both PHI and CHI over 3 years of ART. Under these simulations, residual viremia originated from reactivated latently infected cells where most of these cells arose from clonal expansion within the resting phenotype. Since virion production from clonally expanded cells will not be affected by antiretroviral drugs, simulations of ART intensification had little impact on pVL. HIV DNA decay over the first year of ART followed the loss of activated memory cells (120 day half-life) while the 5.9 year half-life of total HIV DNA after this point mirrored the slower decay of resting memory cells. Simulations had difficulty reproducing the fast early HIV DNA dynamics, including 2-LTR levels peaking at week 12, and the later slow loss of total and 2-LTR HIV DNA, suggesting some ongoing infection. In summary, our modelling indicates that much of the dynamical behavior of HIV can be explained by its impact on memory CD4+ T cell homeostasis.

HIV persists in CCR6+CD4+ T cells from colon and blood during antiretroviral therapy.

Objectives:The objective of this article is to investigate the contribution of colon and blood CD4+ T-cell subsets expressing the chemokine receptor CCR6 to HIV persistence during antiretroviral therapy.Design:Matched sigmoid biopsies and blood samples (n = 13) as well as leukapheresis (n = 20) were collected from chronically HIV-infected individuals receiving antiretroviral therapy. Subsets of CD4+ T cells with distinct differentiation/polarization profiles were identified using surface markers as follows: memory (TM, CD45RA−), central memory (TCM; CD45RA−CCR7+), effector (TEM/TM; CD45RA−CCR7−), Th17 (CCR6+CCR4+), Th1Th17 (CCR6+CXCR3+), Th1 (CCR6−CXCR3+), and Th2 (CCR6−CCR4+).Methods:We used polychromatic flow cytometry for cell sorting, nested real-time PCR for HIV DNA quantification, ELISA and flow cytometry for HIV p24 quantification. HIV reactivation was induced by TCR triggering in the presence/absence of all-trans retinoic acid.Results:Compared with blood, the frequency of CCR6+ TM was higher in the colon. In both colon and blood compartments, CCR6+ TM were significantly enriched in HIV DNA when compared with their CCR6− counterparts (n = 13). In blood, integrated HIV DNA levels were significantly enriched in CCR6+ versus CCR6− TCM of four of five individuals and CCR6+ versus CCR6− TEM of three of five individuals. Among blood TCM, Th17 and Th1Th17 contributed the most to the pool of cells harboring integrated HIV DNA despite their reduced frequency compared with Th2, which were infected the least. HIV reactivation was induced by TCR triggering and/or retinoic acid exposure at higher levels in CCR6+ versus CCR6− TM, TCM, and TEM.Conclusion:CCR6 is a marker for colon and blood CD4+ T cells enriched for replication-competent HIV DNA. Novel eradication strategies should target HIV persistence in CCR6+CD4+ T cells from various anatomic sites.

Monitoring Integration over Time Supports a Role for Cytotoxic T Lymphocytes and Ongoing Replication as Determinants of Reservoir Size.

The establishment of a latent reservoir represents a barrier to cure among HIV-infected individuals. The dynamics of HIV reservoir accumulation over time in patients before antiviral therapy is underexplored, in large part because it is difficult to accurately and reproducibly measure the size of HIV reservoir in this setting. In our study, we compared the dynamics of integrated HIV DNA over time in ECs and NCs before and after ART was initiated. We found that integrated HIV DNA levels progressively increase over time in the absence of ART, but with a higher, albeit variable, rate in NCs compared to ECs. In addition, integrated HIV DNA declines more dramatically when ART is initiated in acute rather than chronic HIV infection, suggesting important differences between acute and chronic infection. Our study highlights the role of HIV replication and CTL control in reservoir accumulation in sanctuary sites and why ART appears to be more effective in acute infection.

Concurrent measures of total and integrated HIV DNA monitor reservoirs and ongoing replication in eradication trials.

Interest in targeting HIV reservoirs is fueling trials that may decrease reservoir size and/or induce viral replication. Therefore, we aimed to develop strategies to sensitively measure changes in these parameters in patients on and off antiretroviral therapy (ART). Achieving these goals may help evaluate the effects of future clinical trials. To determine the relationship between measurements of total and integrated HIV DNA and their role as markers of reservoir size and ongoing replication, these parameters were measured during the first year of ART, during long-term effective ART, and during a clinical trial aimed at targeting reservoirs. Total and integrated HIV DNA were measured in patient samples using quantitative PCR techniques. CD4(+)T cell counts and plasma viremia were also monitored. Unintegrated HIV DNA became undetectable during the first year of ART. Total and integrated HIV DNA levels were generally equal in well controlled patients on ART, and low-level plasma viremia correlated best with integration measures. Finally, patients who controlled plasma viremia (<400 copies/ml) during interferon-α monotherapy exhibited a decrease in the level of integrated but not total HIV DNA and a rise in the ratio of total to integrated HIV DNA over time. Our findings suggest that appearance of unintegrated HIV DNA reflects residual HIV expression and de-novo reverse transcription, providing insight into the mechanism by which interferon-α reduces the HIV reservoir. We conclude that concurrent measurements of total and integrated HIV DNA provide information regarding reservoir size and ongoing replication in trials targeting HIV.

Integrated HIV DNA accumulates prior to treatment while episomal HIV DNA records ongoing transmission afterwards

We investigated the dynamics of HIV RNA and HIV DNA levels after the commencement of raltegravir-based antiretroviral therapy (ART) in primary (PHI) and chronically HIV-infected (CHI) individuals (the PINT study). We recruited 8 PHI and 8 CHI ART-naive individuals who commenced a 1-year combination regimen of Truvada and the integrase inhibitor raltegravir. Nonlinear mixed effects modelling was used to determine multiphasic decay of plasma HIV RNA levels (pVL), as well as dynamics of total, episomal [2-long terminal repeats (LTR)] and integrated HIV DNA in CD4 T cells from peripheral blood. Although pVL decreased faster through first and second phase for PHI individuals there was no difference in the final level reaching a mean of 9 copies/ml by week 16 that was maintained thereafter. Total HIV DNA and integrated HIV DNA levels from CHI patients were significantly higher than from PHI patients. However, at no time did 2-LTR levels differ between groups. Of note, 2-LTR circles exhibited an initial increase peaking at week 3 followed by biphasic decay with a half-life of 29 days. Second phase integrated HIV DNA levels were significantly correlated with duration of infection and consistent with this form of infection occurring at approximately 100 000 integration events per day in the absence of ART, achieving its 50% level 2 years after infection. Integrated HIV DNA levels accumulate with duration of untreated HIV infection. The relatively short half-life and high levels of 2-LTR circles after 1 year support continued HIV transmission during ART.