scholarly journals Designing and Interpreting Limiting Dilution Assays: General Principles and Applications to the Latent Reservoir for Human Immunodeficiency Virus-1

2015 ◽  
Vol 2 (4) ◽  
Author(s):  
Daniel I. S. Rosenbloom ◽  
Oliver Elliott ◽  
Alison L. Hill ◽  
Timothy J. Henrich ◽  
Janet M. Siliciano ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Latent Infection ◽  
Latent Reservoir ◽  
Online Tool ◽  
Infectious Disease Research ◽  
Immunodeficiency Virus ◽  
Standard Assay ◽  
Assay Design ◽  
Hiv 1 ◽  
Limiting Dilution

Abstract Limiting dilution assays are widely used in infectious disease research. These assays are crucial for current human immunodeficiency virus (HIV)-1 cure research in particular. In this study, we offer new tools to help investigators design and analyze dilution assays based on their specific research needs. Limiting dilution assays are commonly used to measure the extent of infection, and in the context of HIV they represent an essential tool for studying latency and potential curative strategies. Yet standard assay designs may not discern whether an intervention reduces an already miniscule latent infection. This review addresses challenges arising in this setting and in the general use of dilution assays. We illustrate the major statistical method for estimating frequency of infectious units from assay results, and we offer an online tool for computing this estimate. We recommend a procedure for customizing assay design to achieve desired sensitivity and precision goals, subject to experimental constraints. We consider experiments in which no viral outgrowth is observed and explain how using alternatives to viral outgrowth may make measurement of HIV latency more efficient. Finally, we discuss how biological complications, such as probabilistic growth of small infections, alter interpretations of experimental results.

scholarly journals Designing and interpreting limiting dilution assays: general principles and applications to the latent reservoir for HIV-1

2015 ◽  
Author(s):  
Daniel I S Rosenbloom ◽  
Oliver Elliott ◽  
Alison L Hill ◽  
Timothy J Henrich ◽  
Janet M Siliciano ◽  
...  
Keyword(s):  
Statistical Method ◽  
Latent Reservoir ◽  
Hiv Latency ◽  
Online Tool ◽  
Viral Growth ◽  
Alternative Measures ◽  
Infected Cells ◽  
Assay Design ◽  
Hiv 1 ◽  
Limiting Dilution

Limiting dilution assays are commonly used to measure the extent of infection, and in the context of HIV they represent an essential tool for studying latency and potential curative strategies. To assist investigators using dilution assays, we illustrate the major statistical method for estimating the frequency of infected cells (or other infectious units) from assay results, and we offer an online tool for computing this estimate. We then recommend a procedure for optimizing assay design to achieve any desired set of sensitivity and precision goals, subject to experimental constraints. We discuss challenges involved in interpreting experiments in which no viral growth is observed and explain how using alternative measures for viral outgrowth may make measurement of HIV latency more efficient. Finally, we discuss how biological complications -- such as probabilistic growth of a small infection in culture -- alter interpretations of experimental results.

scholarly journals Rational Design of Drugs That Induce Human Immunodeficiency Virus Replication

2003 ◽  
Vol 77 (19) ◽  
pp. 10227-10236 ◽  
Author(s):  
Dean H. Hamer ◽  
Sven Bocklandt ◽  
Louise McHugh ◽  
Tae-Wook Chun ◽  
Peter M. Blumberg ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
T Cell Activation ◽  
Rational Design ◽  
Latent Reservoir ◽  
Viral Gene ◽  
Human Immunodeficiency Virus Replication ◽  
Immunodeficiency Virus ◽  
Latently Infected ◽  
Hiv 1

ABSTRACT Drugs that induce human immunodeficiency virus type 1 (HIV-1) replication could be used in combination with highly active antiretroviral therapy (HAART) to reduce the size of the latent reservoir that is in part responsible for viral persistence. Protein kinase C (PKC) is a logical target for such drugs because it activates HIV-1 transcription through multiple mechanisms. Here we show that HIV-1 gene expression can be induced by potent synthetic analogues of the lipid second messenger diacylglycerol (DAG) synthesized on a five-member ring platform that reduces the entropy of binding relative to that of the more flexible DAG template. By varying the alkyl side chains of these synthetic DAG lactones, it was possible to maximize their potency and ability to render latently infected T cells sensitive to killing by an anti-HIV-1 immunotoxin while minimizing the side effects of CD4 and CXCR4 downregulation and tumor necrosis factor alpha upregulation. The two lead compounds, LMC03 and LMC07, regulated a series of PKC-sensitive genes involved in T-cell activation and induced viral gene expression in peripheral blood mononuclear cells from HIV-1-infected individuals. These studies demonstrate the potential for the rational design of agents that, in conjunction with HAART and HIV-specific toxins, can be used to decrease or eliminate the pool of latently infected reservoirs by forcing viral expression.

scholarly journals Characterization of Chemokine Receptor Utilization of Viruses in the Latent Reservoir for Human Immunodeficiency Virus Type 1

2000 ◽  
Vol 74 (17) ◽  
pp. 7824-7833 ◽  
Author(s):  
Theodore Pierson ◽  
Trevor L. Hoffman ◽  
Joel Blankson ◽  
Diana Finzi ◽  
Karen Chadwick ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Chemokine Receptor ◽  
Latent Reservoir ◽  
Immunodeficiency Virus ◽  
Latently Infected ◽  
Hiv 1

ABSTRACT Latently infected resting CD4+ T cells provide a long-term reservoir for human immunodeficiency virus type 1 (HIV-1) and are likely to represent the major barrier to virus eradication in patients on combination antiretroviral therapy. The mechanisms by which viruses enter the latent reservoir and the nature of the chemokine receptors involved have not been determined. To evaluate the phenotype of the virus in this compartment with respect to chemokine receptor utilization, full-length HIV-1 env genes were cloned from latently infected cells and assayed functionally. We demonstrate that the majority of the viruses in the latent reservoir utilize CCR5 during entry, although utilization of several other receptors, including CXCR4, was observed. No alternative coreceptors were shown to be involved in a systematic fashion. Although R5 viruses are present in the latent reservoir, CCR5 was not expressed at high levels on resting CD4+ T cells. To understand the mechanism by which R5 viruses enter latent reservoir, the ability of an R5 virus, HIV-1 Ba-L, to infect highly purified resting CD4+ T lymphocytes from uninfected donors was evaluated. Entry of Ba-L could be observed when virus was applied at a multiplicity approaching 1. However, infection was limited to a subset of cells expressing low levels of CCR5 and markers of immunologic memory. Naive cells could not be infected by an R5 virus even when challenged with a large inoculum. Direct cell fractionation studies showed that latent virus is present predominantly in resting memory cells but also at lower levels in resting naive cells. Taken together, these findings provide support for the hypothesis that the direct infection of naive T cells is not the major mechanism by which the latent infection of resting T cells is established.

scholarly journals Persistence of Wild-Type Virus and Lack of Temporal Structure in the Latent Reservoir for Human Immunodeficiency Virus Type 1 in Pediatric Patients with Extensive Antiretroviral Exposure

2002 ◽  
Vol 76 (18) ◽  
pp. 9481-9492 ◽  
Author(s):  
Christian T. Ruff ◽  
Stuart C. Ray ◽  
Patricia Kwon ◽  
Rebekah Zinn ◽  
Amanda Pendleton ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Temporal Structure ◽  
Latent Reservoir ◽  
Drug Resistant ◽  
Wild Type ◽  
Time Points ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Although highly active antiretroviral therapy (HAART) for human immunodeficiency virus type 1 (HIV-1) infection can reduce levels of HIV-1 RNA in plasma to below the limit of detection, replication-competent forms of the virus persist in all infected individuals. One form of persistence involves a stable reservoir of latent but potentially infectious virus that resides in resting memory CD4+ T cells. The mechanisms involved in maintaining this latent reservoir are incompletely understood. In the present study, we examined the dynamic characteristics of this reservoir in a cohort of children who developed drug-resistant HIV-1 as a result of extensive exposure to inadequately suppressive one- or two-drug regimens prior to the advent of HAART. We have previously shown that drug-resistant viruses selected by nonsuppressive pre-HAART regimens can enter and persist in this reservoir. We have extended these findings here by demonstrating that archival wild-type HIV-1 persists in this reservoir despite the fact that in these patients drug-resistant mutants have been favored by the selective conditions for many years. Phylogenetic analysis of replication-competent viruses persisting in resting CD4+ T cells revealed a striking lack of temporal structure in the sense that isolates obtained at later time points did not show greater sequence divergence than isolates from earlier time points. The persistence of drug-sensitive virus and the lack of temporal structure in the latent reservoir provide genetic evidence for the idea that HIV-1 can persist in a latent form free of selective pressure from antiretroviral drugs in long-lived resting memory CD4+ T cells. Although there may be other mechanisms for viral persistence, this stable pool of latently infected cells is of significant concern because of its potential to serve as a lasting source of replication-competent viruses, including the infecting wild-type form and all drug-resistant variants that have arisen subsequently.

scholarly journals The dynamics of the HIV-1 latent reservoir – considering the heterogeneous subpopulations

2019 ◽  
Author(s):  
Ruian Ke ◽  
Kai Deng
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiretroviral Therapy ◽  
Latent Reservoir ◽  
Major Barrier ◽  
Clinical Observations ◽  
Wide Range ◽  
Immunodeficiency Virus ◽  
Latently Infected ◽  
Ctl Escape ◽  
Hiv 1

AbstractA major barrier to finding a cure for human immunodeficiency virus type-I (HIV-1) infection is the existence and persistence of the HIV-1 latent reservoir. Although the size of the reservoir is shown to be extremely stable under effective antiretroviral therapy, multiple lines of evidence suggest that the reservoir is composed of dynamic and heterogeneous subpopulations. Quantifying the dynamics of these subpopulations and the processes that maintain the latent reservoir is crucial to the development of effective strategies to eliminate this reservoir. Here, we constructed a mathematical model to consider four latently infected subpopulations, according to their ability to proliferate and the type of virus they are infected. Our model explains a wide range of clinical observations, including variable estimates of the reservoir half-life and dynamical turnover of cytotoxic T lymphocyte (CTL) escape viruses in the reservoir. It suggests that very early treatment leads to a reservoir that is small in size and is composed of less stable latently infected cells (compared to the reservoir in chronically infected individuals). The shorter half-lives estimated from individuals treated during acute infection is likely driven by cells that are less prone to proliferate; in contrast, the remarkably consistent estimate of the long half-lives in individuals who are treated during chronic infection are driven by fast proliferating cells that are likely to be infected by CTL escape mutants. Our model shed light on the dynamics of the reservoir in the absence and presence of antiretroviral therapy. More broadly, it can be used to estimate the turnover rates of subpopulations of the reservoir as well as to design and evaluate the impact of various therapeutic interventions to purge the HIV-1 reservoir.Author summaryHuman immunodeficiency virus (HIV) infects tens of millions of people globally and causes approximately a million death each year. Current treatment for HIV infection suppresses viral load but does not eradicates the virus. A major barrier to cure HIV infection is the existence and persistence of populations of cells that are latently infected by HIV, i.e. the HIV latent reservoir. Understanding and quantifying the kinetics of the reservoir is therefore critical for developing and evaluating effective therapies to purge the reservoir. Recent studies suggested that this reservoir is heterogenous in their population dynamics; yet most previous mathematical models consider this reservoir as a homogenous population. Here we developed a model explicitly tracking the heterogenous subpopulations of the reservoir. We show that this model explains a wide range of clinical observations, and then demonstrate its utility to make quantitative predictions about varies interventions that aim to restrict or reduce the size of the reservoir.

scholarly journals A Novel Assay Allows Genotyping of the Latent Reservoir for Human Immunodeficiency Virus Type 1 in the Resting CD4+ T Cells of Viremic Patients

2005 ◽  
Vol 79 (8) ◽  
pp. 5185-5202 ◽  
Author(s):  
Daphne Monie ◽  
Rachel P. Simmons ◽  
Richard E. Nettles ◽  
Tara L. Kieffer ◽  
Yan Zhou ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Latent Reservoir ◽  
Drug Resistant ◽  
Plasma Virus ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT A latent reservoir for human immunodeficiency virus type 1 (HIV-1) consisting of integrated provirus in resting memory CD4+ T cells prevents viral eradication in patients on highly active antiretroviral therapy (HAART). It is difficult to analyze the nature and dynamics of this reservoir in untreated patients and in patients failing therapy, because it is obscured by an excess of unintegrated viral DNA that constitutes the majority of viral species in resting CD4+ T cells from viremic patients. Therefore, we developed a novel culture assay that stimulates virus production from latent, integrated HIV-1 in resting CD4+ T cells in the presence of antiretroviral drugs that prevent the replication of unintegrated virus. Following activation, resting CD4+ T cells with integrated HIV-1 DNA produced virus particles for several days, with peak production at day 5. Using this assay, HIV-1 pol sequences from the resting CD4+ T cells of viremic patients were found to be genetically distinct from contemporaneous plasma virus. Despite the predominance of a relatively homogeneous population of drug-resistant viruses in the plasma of patients failing HAART, resting CD4+ T cells harbored a diverse array of wild-type and archival drug-resistant viruses that were less fit than plasma virus in the context of current therapy. These results provide the first direct evidence that resting CD4+ T cells serve as a stable reservoir for HIV-1 even in the setting of high levels of viremia. The ability to analyze archival species in viremic patients may have clinical utility in detecting drug-resistant variants not present in the plasma.

scholarly journals Nonstructured Treatment Interruptions Are Associated With Higher Human Immunodeficiency Virus Reservoir Size Measured by Intact Proviral DNA Assay in People Who Inject Drugs

2020 ◽  
Author(s):  
Gregory D Kirk ◽  
Jacqueline Astemborski ◽  
Shruti H Mehta ◽  
Kristen D Ritter ◽  
Gregory M Laird ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Latent Reservoir ◽  
Persons Who Inject Drugs ◽  
Major Barrier ◽  
Dna Assay ◽  
Proviral Dna ◽  
Hiv Rna ◽  
Treatment Interruptions ◽  
Immunodeficiency Virus ◽  
Hiv 1

Abstract The latent reservoir for human immunodeficiency virus type 1 (HIV-1) in CD4+ T cells is a major barrier to cure. HIV-1–infected persons who inject drugs (PWID) often struggle to maintain suppression of viremia and experience nonstructured treatment interruptions (NTIs). The effects of injecting drugs or NTIs on the reservoir are unclear. Using the intact proviral DNA assay, we found no apparent effect of heroin or cocaine use on reservoir size. However, we found significantly larger reservoirs in those with frequent NTIs or a shorter interval from last detectable HIV RNA measurement. These results have important implications for inclusion of PWID in HIV-1 cure studies.

scholarly journals Benzodiazepines Drive Alteration of Chromatin at the Integrated HIV-1 LTR

Viruses ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 191
Author(s):  
Weam Elbezanti ◽  
Angel Lin ◽  
Alexis Schirling ◽  
Alexandria Jackson ◽  
Matthew Marshall ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Chromatin Immunoprecipitation ◽  
Molecular Mechanisms ◽  
Transcriptional Silencing ◽  
Latent Reservoir ◽  
Prior Work ◽  
Immunodeficiency Virus ◽  
Runx Proteins ◽  
Hiv 1

Antiretroviral therapy (ART) lowers human immunodeficiency virus type 1 (HIV-1) viral load to undetectable levels, but does not eliminate the latent reservoir. One of the factors controlling the latent reservoir is transcriptional silencing of the integrated HIV-1 long terminal repeat (LTR). The molecular mechanisms that control HIV-1 transcription are not completely understood. We have previously shown that RUNX1, a host transcription factor, may play a role in the establishment and maintenance of HIV-1 latency. Prior work has demonstrated that inhibition of RUNX1 by the benzodiazepine (BDZ) Ro5-3335 synergizes with suberanilohydroxamic acid (SAHA) to activate HIV-1 transcription. In this current work, we examine the effect of RUNX1 inhibition on the chromatin state of the integrated HIV-1 LTR. Using chromatin immunoprecipitation (ChIP), we found that Ro5-3335 significantly increased the occupancy of STAT5 at the HIV-1 LTR. We also screened other BDZs for their ability to regulate HIV-1 transcription and demonstrate their ability to increase transcription and alter chromatin at the LTR without negatively affecting Tat activity. These findings shed further light on the mechanism by which RUNX proteins control HIV-1 transcription and suggest that BDZ compounds might be useful in activating HIV-1 transcription through STAT5 recruitment to the HIV-1 LTR.

scholarly journals Determinants of the Establishment of Human Immunodeficiency Virus Type 1 Latency

2009 ◽  
Vol 83 (7) ◽  
pp. 3078-3093 ◽  
Author(s):  
Alexandra Duverger ◽  
Jennifer Jones ◽  
Jori May ◽  
Frederic Bibollet-Ruche ◽  
Frederic A. Wagner ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Gene Regulation ◽  
Human Immunodeficiency Virus Type ◽  
Latent Infection ◽  
Transcriptional Interference ◽  
Cellular Gene ◽  
Primary Mechanism ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Recent research has emphasized the notion that human immunodeficiency virus type 1 (HIV-1) latency is controlled by a restrictive histone code at, or DNA methylation of, the integrated viral promoter (long terminal repeat [LTR]). The present concept of HIV-1 latency has essentially been patterned from the principles of cellular gene regulation. Here we introduce an experimental system that allows for the qualitative and quantitative kinetic study of latency establishment and maintenance at the population level. In this system, we find no evidence that HIV-1 latency establishment is the consequence of downregulation of initial active infection followed by the establishment of a restrictive histone code at the viral LTR. Latent infection was established following integration of the virus in the absence of viral gene expression (silent integration) and was a function of the NF-κB activation level in the host cell at the time of infection. In the absence of a role for epigenetic regulation, we demonstrate that transcriptional interference, a mechanism that has recently been suggested to add to the stabilization of HIV-1 latency, is the primary mechanism to govern latency maintenance. These findings provide direct experimental evidence that the high number of viral integration events (>90%) found in actively expressed genes of CD4+ memory T cells from highly active antiretroviral therapy-suppressed patients represent indeed latent infection events and that transcriptional interference may be the primary mechanism to control HIV-1 latency in vivo. HIV-1 latency may thus not be governed by the principles of cellular gene regulation, and therapeutic strategies to deplete the pool of latently HIV-1-infected cells should be reconsidered.

scholarly journals Noninferiority of Simplified Dolutegravir Monotherapy Compared to Continued Combination Antiretroviral Therapy That Was Initiated During Primary Human Immunodeficiency Virus Infection: A Randomized, Controlled, Multisite, Open-label, Noninferiority Trial

2019 ◽  
Vol 69 (9) ◽  
pp. 1489-1497 ◽  
Author(s):  
Dominique L Braun ◽  
Teja Turk ◽  
Fabian Tschumi ◽  
Christina Grube ◽  
Benjamin Hampel ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiretroviral Therapy ◽  
Pilot Study ◽  
Latent Reservoir ◽  
Combination Antiretroviral Therapy ◽  
Open Label ◽  
Once Daily ◽  
Noninferiority Trial ◽  
Immunodeficiency Virus ◽  
Hiv 1

Abstract Background Patients who start combination antiretroviral therapy (cART) during primary human immunodeficiency virus type 1 (HIV-1) infection show a smaller HIV-1 latent reservoir, less immune activation, and less viral diversity compared to patients who start cART during chronic infection. We conducted a pilot study to determine whether these properties would allow sustained virological suppression after simplification of cART to dolutegravir monotherapy. Methods EARLY-SIMPLIFIED is a randomized, open-label, noninferiority trial. Patients who started cART <180 days after a documented primary HIV-1 infection and had an HIV-1 RNA <50 copies/mL plasma for at least 48 weeks were randomized (2:1) to monotherapy with dolutegravir 50 mg once daily or to continuation of cART. The primary efficacy endpoint was the proportion of patients with <50 HIV-1 RNA copies/mL on or before week 48; noninferiority margin 10%. Results Of the 101 patients randomized, 68 were assigned to simplification to dolutegravir monotherapy and 33 to continuation of cART. At week 48 in the per-protocol population, 67/67 (100%) had virological response in the dolutegravir monotherapy group vs 32/32 (100%) in the cART group (difference, 0.00%; 95% confidence interval, –100%, 4.76%). This showed noninferiority of the dolutegravir monotherapy at the prespecified level. Conclusion In this pilot study consisting of patients who initiated cART during primary HIV-1 infection and had <50 HIV-1 RNA copies/mL for at least 48 weeks, monotherapy with once-daily dolutegravir was noninferior to cART. Our results suggest that future simplification studies should use a stratification according to time of HIV infection and start of first cART. Clinical Trials Registration NCT02551523.

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