Search

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.

The dynamics of HIV reservoir accumulation off antiretroviral therapy (ART) is underexplored. Levels of integrated HIV DNA in peripheral blood mononuclear cells (PBMCs) were longitudinally monitored before and after antiviral therapy. HIV integration increased over time in both elite controllers (ECs; n = 8) and noncontrollers (NCs; n = 6) before ART, whereas integration remained stable in patients on ART (n = 4). The median annual fold change was higher in NCs than in ECs and negatively correlated with CD4/CD8 T-cell ratio. Cytotoxic T lymphocyte (CTL) function as assessed by infected CD4 T-cell elimination (ICE) and granzyme B activity did not significantly change over time in ECs, suggesting that the gradual increase in integrated HIV DNA observed in ECs was not a result of progressive loss of immune-mediated control. Also, acutely infected (n = 7) but not chronically infected (n = 6) patients exhibited a significant drop in integrated HIV DNA 12 months after ART initiation. In conclusion, in the absence of ART, integrated HIV accumulates over time both in NCs and in ECs, at variable individual rates. Starting ART early in infection leads to a greater drop in integrated HIV DNA than does initiating treatment after years of infection. The increase in integrated HIV DNA over time suggests that early treatment may be of benefit in limiting HIV reservoirs.ImportanceThe 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.

Next-generation sequencing (NGS) represents a powerful tool to unravel the genetic make-up of the HIV reservoir but limited data exist on its use in vitro. Moreover, most NGS studies do not separate integrated from unintegrated DNA even though selection pressures on these two forms should be distinct. We reasoned we could use NGS to compare infection of resting and activated CD4 T cells in vitro to address how the metabolic state affects reservoir formation and dynamics. To address these questions we obtained HIV sequences at 2, 4 and 8 days after NL4-3 infection of metabolically activated and quiescent CD4 T cells (cultured with 2 ng/mL IL-7). We compared the composition of integrated and total HIV DNA by isolating integrated HIV DNA using pulsed-field electrophoresis before performing sequencing. After a single-round infection the majority of integrated HIV DNA was intact in both resting and activated T cells. The decay of integrated intact proviruses was rapid and similar in both quiescent and activated T cells. Defective forms accumulated relative to intact ones analogously to what is observed in vivo. Massively deleted viral sequences formed more frequently in resting cells likely due to lower deoxynucleoside triphosphate (dNTP) levels and the presence of multiple restriction factors. To our surprise, the majority of these deleted sequences did not integrate into the human genome. The use of NGS to study reservoir dynamics in vitro provides a model that recapitulates important aspects of reservoir dynamics. Moreover, separating integrated from unintegrated HIV DNA is important in some clinical settings to properly study selection pressures. [ABSTRACT FROM AUTHOR]

Naïve CD44 T cells are resistant to both HIV R5 env and vesicular stomatitis virus G protein (VSV-G)-mediated fusion. However, viral particles carrying both HIV R5 env and VSV-G infect naïve cells by an unexplained mechanism. We show that VSV-G-pseudotyped virus cannot fuse to unstimulated cells because the viral particles cannot be endocytosed. However, virions carrying both HIV R5 env and VSV-G can fuse because CD4 binding allows viral uptake. Our findings reveal a unique mechanism by which R5 HIV env and VSV-G cooperate to allow entry to naïve CD4+ T cells, providing a tool to target naïve CD4+ T cells with R5 HIV to study HIV coreceptor signaling and latency. [ABSTRACT FROM AUTHOR]

Current gene transfer protocols for resting CD4+ T cells include an activation step to enhance transduction efficiency. This step is performed because it is thought that resting cells are resistant to transduction by lentiviral-based gene therapy vectors. However, activating resting cells prior to transduction alters their physiology, with foreseeable and unforeseeable negative consequences. Thus, it would be desirable to transduce resting CD4+ T cells without activation. We recently demonstrated, contrary to the prevailing belief, that wild-type human immunodeficiency virus (HIV) integrates into resting CD4+ T cells. Based on that finding, we investigated whether a commonly used, vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped lentiviral gene therapy vector could also integrate into resting CD4+ T cells. To investigate this, we inoculated resting CD4+ T cells with lentiviral particles that were pseudotyped with VSV-G or CXCR4-tropic HIV Env and assayed binding, fusion, reverse transcription, and integration. We found that the VSV-G-pseudotyped lentiviral vector failed to fuse to resting CD4+ T cells while HIV Env-pseudotyped lentiviral vectors fused, reverse transcribed, and integrated in resting cells. Our findings suggest that HIV Env could be used effectively for the delivery of therapeutic genes to resting CD4+ T cells and suggest that fusion may be the critical step restricting transduction of resting CD4+ T cells by lentiviral gene therapy vectors. [ABSTRACT FROM AUTHOR]

Resting CD4+ T cells restrict human immunodeficiency virus (HIV) infection at or before reverse transcription, resulting in slower kinetics of reverse transcription. In a previous study, we showed that, despite this restriction at reverse transcription, HIV integration occurs in resting CD4+ T cells, albeit with slower kinetics. In that study, the resting T cells were a mixture of memory and naïve cells. Here we asked whether the more quiescent naïve cell subset could be directly infected by HIV and, if so, whether the level of integration in naïve cells was comparable to that in memory cells. We found that HIV integrates in the naïve subset of resting CD4+ T cells without prior activation of the cells. The level of integration (proviruses/cell) in naïve cells was lower than that in memory cells. This difference between naïve and memory cells was observed whether we inoculated the cells with R5 or X4 HIV and could not be explained solely by differences in coreceptor expression. The presence of endogenous dendritic cells did not change the number of proviruses/cell in memory or naïve cells, and deoxynucleoside pools were equally limiting. Our results instead indicate the existence of a novel restriction point in naïve T cells at viral fusion that results in reduced levels of fusion to naïve CD4+ T cells. We conclude that HIV can integrate into both naïve and memory cells directly. Our data further support our hypothesis that integrated proviral infection of resting T cells can be established without T-cell activation. [ABSTRACT FROM AUTHOR]

Resting CD4+ T cells are the best-defined reservoir of latent human immunodeficiency virus type 1 (HIV-1) infection, but how the reservoir is formed is unclear. Understanding how the reservoir of latently infected cells forms is critical because it is a major barrier to curing HIV infection. The system described here may provide an in vitro model of latent HIV-1 infection in resting CD4+ T cells. We demonstrated that HIV-1 integrates into the genomes of in vitro-inoculated resting CD4+ T cells that have not received activating stimuli and have not entered cell cycle stage G1b. A percentage of the resting CD4+ T cells that contain integrated DNA produce virus upon stimulation, i.e., are latently infected. Our results show that latent HIV-1 infection occurs in unstimulated resting CD4+ T cells and suggest a new route for HIV-1 reservoir formation. [ABSTRACT FROM AUTHOR]

Resting CD4+ T cells restrict human immunodeficiency virus (HIV) infection at a step prior to integration. Despite this restriction, we showed previously that HIV integration occurs in resting CD4+ T cells in vitro, albeit at lower levels than in activated CD4+ T cells. Here we show that addition of deoxynucleosides enhanced integration and 2LTR formation in resting CD4+ T cells but that the kinetics were still significantly delayed compared to those of activated T cells. Thus, deoxynucleoside addition partially overcomes the restriction to HIV infection in resting CD4+ T cells. [ABSTRACT FROM AUTHOR]

Cell-associated HIV unspliced RNA is an important marker of the viral reservoir. HIV gag RNA-specific assays are frequently used to monitor reservoir activation. Because HIV preferentially integrates into actively transcribed genes, some of the transcripts detected by these assays may not represent genuine HIV RNA but rather chimeric host-HIV readthrough transcripts. Here, we demonstrate that in HIV-infected patients on suppressive combination antiretroviral therapy, such host-derived transcripts do not significantly contribute to the HIV gag RNA level., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: A major goal in HIV eradication research is characterizing the reservoir cells that harbor HIV in the presence of antiretroviral therapy (ART), which reseed viremia after treatment is stopped. In general, it is assumed that the reservoir consists of CD4(+) T cells that express no viral proteins. However, recent findings suggest that this may be an overly simplistic view and that the cells that contribute to the reservoir may be a diverse population that includes both CD4(+) and CD4(-) cells. In this study, we directly infected resting CD4(+) T cells and used fluorescence-activated cell sorting (FACS) and fiber-optic array scanning technology (FAST) to identify and image cells expressing HIV Gag. We found that Gag expression from integrated proviruses occurred in resting cells that lacked surface CD4, likely resulting from Nef- and Env-mediated receptor internalization. We also extended our approach to detect cells expressing HIV proteins in patients suppressed on ART. We found evidence that rare Gag(+) cells persist during ART and that these cells are often negative for CD4. We propose that these double-negative α/β T cells that express HIV protein may be a component of the long-lived reservoir., Importance: A reservoir of infected cells persists in HIV-infected patients during antiretroviral therapy (ART) that leads to rebound of virus if treatment is stopped. In this study, we used flow cytometry and cell imaging to characterize protein expression in HIV-infected resting cells. HIV Gag protein can be directly detected in infected resting cells and occurs with simultaneous loss of CD4, consistent with the expression of additional viral proteins, such as Env and Nef. Gag(+) CD4(-) cells can also be detected in suppressed patients, suggesting that a subset of infected cells express proteins during ART. Understanding the regulation of viral protein expression during ART will be key to designing effective strategies to eradicate HIV reservoirs., (Copyright © 2016 DeMaster et al.)

Naïve CD4(4) T cells are resistant to both HIV R5 env and vesicular stomatitis virus G protein (VSV-G)-mediated fusion. However, viral particles carrying both HIV R5 env and VSV-G infect naïve cells by an unexplained mechanism. We show that VSV-G-pseudotyped virus cannot fuse to unstimulated cells because the viral particles cannot be endocytosed. However, virions carrying both HIV R5 env and VSV-G can fuse because CD4 binding allows viral uptake. Our findings reveal a unique mechanism by which R5 HIV env and VSV-G cooperate to allow entry to naïve CD4(+) T cells, providing a tool to target naïve CD4(+) T cells with R5 HIV to study HIV coreceptor signaling and latency.

Resting CD4(+) T cells restrict human immunodeficiency virus (HIV) infection at or before reverse transcription, resulting in slower kinetics of reverse transcription. In a previous study, we showed that, despite this restriction at reverse transcription, HIV integration occurs in resting CD4(+) T cells, albeit with slower kinetics. In that study, the resting T cells were a mixture of memory and naïve cells. Here we asked whether the more quiescent naïve cell subset could be directly infected by HIV and, if so, whether the level of integration in naïve cells was comparable to that in memory cells. We found that HIV integrates in the naïve subset of resting CD4(+) T cells without prior activation of the cells. The level of integration (proviruses/cell) in naïve cells was lower than that in memory cells. This difference between naïve and memory cells was observed whether we inoculated the cells with R5 or X4 HIV and could not be explained solely by differences in coreceptor expression. The presence of endogenous dendritic cells did not change the number of proviruses/cell in memory or naïve cells, and deoxynucleoside pools were equally limiting. Our results instead indicate the existence of a novel restriction point in naïve T cells at viral fusion that results in reduced levels of fusion to naïve CD4(+) T cells. We conclude that HIV can integrate into both naïve and memory cells directly. Our data further support our hypothesis that integrated proviral infection of resting T cells can be established without T-cell activation.

Resting CD4+ T cells restrict human immunodeficiency virus (HIV) infection at a step prior to integration. Despite this restriction, we showed previously that HIV integration occurs in resting CD4+ T cells in vitro, albeit at lower levels than in activated CD4+ T cells. Here we show that addition of deoxynucleosides enhanced integration and 2LTR formation in resting CD4+ T cells but that the kinetics were still significantly delayed compared to those of activated T cells. Thus, deoxynucleoside addition partially overcomes the restriction to HIV infection in resting CD4+ T cells.

Resting CD4(+) T cells are the best-defined reservoir of latent human immunodeficiency virus type 1 (HIV-1) infection, but how the reservoir is formed is unclear. Understanding how the reservoir of latently infected cells forms is critical because it is a major barrier to curing HIV infection. The system described here may provide an in vitro model of latent HIV-1 infection in resting CD4(+) T cells. We demonstrated that HIV-1 integrates into the genomes of in vitro-inoculated resting CD4(+) T cells that have not received activating stimuli and have not entered cell cycle stage G(1b). A percentage of the resting CD4(+) T cells that contain integrated DNA produce virus upon stimulation, i.e., are latently infected. Our results show that latent HIV-1 infection occurs in unstimulated resting CD4(+) T cells and suggest a new route for HIV-1 reservoir formation.