Redefining the viral reservoirs that prevent HIV-1 eradication.

This Perspective proposes definitions for key terms in the field of HIV-1 latency and eradication. In the context of eradication, a reservoir is a cell type that allows persistence of replication-competent HIV-1 on a timescale of years in patients on optimal antiretroviral therapy. Reservoirs act as a barrier to eradication in the patient population in which cure attempts will likely be made. Halting viral replication is essential to eradication, and definitions and criteria for assessing whether this goal has been achieved are proposed. The cell types that may serve as reservoirs for HIV-1 are discussed. Currently, only latently infected resting CD4(+) T cells fit the proposed definition of a reservoir, and more evidence is necessary to demonstrate that other cell types, including hematopoietic stem cells and macrophages, fit this definition. Further research is urgently required on potential reservoirs in the gut-associated lymphoid tissue and the central nervous system.

Rapid quantification of the latent reservoir for HIV-1 using a viral outgrowth assay.

HIV-1 persists in infected individuals in a stable pool of resting CD4(+) T cells as a latent but replication-competent provirus. This latent reservoir is the major barrier to the eradication of HIV-1. Clinical trials are currently underway investigating the effects of latency-disrupting compounds on the persistence of the latent reservoir in infected individuals. To accurately assess the effects of such compounds, accurate assays to measure the frequency of latently infected cells are essential. The development of a simpler assay for the latent reservoir has been identified as a major AIDS research priority. We report here the development and validation of a rapid viral outgrowth assay that quantifies the frequency of cells that can release replication-competent virus following cellular activation. This new assay utilizes bead and column-based purification of resting CD4(+) T cells from the peripheral blood of HIV-1 infected patients rather than cell sorting to obtain comparable resting CD4(+) T cell purity. This new assay also utilizes the MOLT-4/CCR5 cell line for viral expansion, producing statistically comparable measurements of the frequency of latent HIV-1 infection. Finally, this new assay employs a novel quantitative RT-PCR specific for polyadenylated HIV-1 RNA for virus detection, which we demonstrate is a more sensitive and cost-effective method to detect HIV-1 replication than expensive commercial ELISA detection methods. The reductions in both labor and cost make this assay suitable for quantifying the frequency of latently infected cells in clinical trials of HIV-1 eradication strategies.

Challenges in detecting HIV persistence during potentially curative interventions: a study of the Berlin patient.

There is intense interest in developing curative interventions for HIV. How such a cure will be quantified and defined is not known. We applied a series of measurements of HIV persistence to the study of an HIV-infected adult who has exhibited evidence of cure after allogeneic hematopoietic stem cell transplant from a homozygous CCR5Δ32 donor. Samples from blood, spinal fluid, lymph node, and gut were analyzed in multiple laboratories using different approaches. No HIV DNA or RNA was detected in peripheral blood mononuclear cells (PBMC), spinal fluid, lymph node, or terminal ileum, and no replication-competent virus could be cultured from PBMCs. However, HIV RNA was detected in plasma (2 laboratories) and HIV DNA was detected in the rectum (1 laboratory) at levels considerably lower than those expected in ART-suppressed patients. It was not possible to obtain sequence data from plasma or gut, while an X4 sequence from PBMC did not match the pre-transplant sequence. HIV antibody levels were readily detectable but declined over time; T cell responses were largely absent. The occasional, low-level PCR signals raise the possibility that some HIV nucleic acid might persist, although they could also be false positives. Since HIV levels in well-treated individuals are near the limits of detection of current assays, more sensitive assays need to be developed and validated. The absence of recrudescent HIV replication and waning HIV-specific immune responses five years after withdrawal of treatment provide proof of a clinical cure.

A pilot study assessing the safety and latency-reversing activity of disulfiram in HIV-1-infected adults on antiretroviral therapy.

BACKGROUND: Transcriptionally silent human immunodeficiency virus type 1 (HIV-1) DNA persists in resting memory CD4(+) T cells despite antiretroviral therapy. In a primary cell model, the antialcoholism drug disulfiram has been shown to induce HIV-1 transcription in latently infected resting memory CD4(+) T cells at concentrations achieved in vivo. METHODS: We conducted a single-arm pilot study to evaluate whether 500 mg of disulfiram administered daily for 14 days to HIV-1-infected individuals on stable suppressive antiretroviral therapy would result in reversal of HIV-1 latency with a concomitant transient increase in residual viremia or depletion of the latent reservoir in resting memory CD4(+) T cells. RESULTS: Disulfiram was safe and well tolerated. There was a high level of subject-to-subject variability in plasma disulfiram levels. The latent reservoir did not change significantly (1.16-fold change; 95% confidence interval [CI], .70- to 1.92-fold; P = .56). During disulfiram administration, residual viremia did not change significantly compared to baseline (1.53-fold; 95% CI, .88- to 2.69-fold; P = .13), although residual viremia was estimated to increase by 1.88-fold compared to baseline during the postdosing period (95% CI, 1.03- to 3.43-fold; P = .04). In a post hoc analysis, a rapid and transient increase in viremia was noted in a subset of individuals (n = 6) with immediate postdose sampling (HIV-1 RNA increase, 2.96-fold; 95% CI, 1.29- to 6.81-fold; P = .01). CONCLUSIONS: Administration of disulfiram to patients on antiretroviral therapy does not reduce the size of the latent reservoir. A possible dose-related effect on residual viremia supports future studies assessing the impact of higher doses on HIV-1 production. Disulfiram affects relevant signaling pathways and can be safely administered, supporting future studies of this drug.

A novel cell-based high-throughput screen for inhibitors of HIV-1 gene expression and budding identifies the cardiac glycosides.

OBJECTIVES: Highly active antiretroviral therapy (HAART) is the mainstay of treatment for HIV-1 infection. While current HAART regimens have been extremely effective, issues of associated toxicity, cost and resistance remain and there is a need for novel antiretroviral compounds to complement the existing therapy. We sought to develop a novel high-throughput method for identifying compounds that block later steps in the life cycle not targeted by current therapy. METHODS: We designed a high-throughput screen to identify inhibitors of post-integration steps in the HIV-1 life cycle. The screening method was applied to a library of compounds that included numerous FDA-approved small molecules. RESULTS: Among the small molecules that inhibited late stages in HIV-1 replication were members of the cardiac glycoside family. We demonstrate that cardiac glycosides potently inhibit HIV-1 gene expression, thereby reducing the production of infectious HIV-1. We demonstrate that this inhibition is dependent upon the human Na(+)/K(+)-ATPase, but independent of cardiac glycoside-induced increases in intracellular Ca(2+). CONCLUSIONS: We have validated a novel high-throughput screen to identify small molecule inhibitors of HIV-1 gene expression, virion assembly and budding. Using this screen, we have demonstrated that a number of FDA-approved compounds developed for other purposes potently inhibit HIV-1 replication, including the cardiac glycosides. Our work indicates that the entire cardiac glycoside family of drugs shows potential for antiretroviral drug development.

A novel PCR assay for quantification of HIV-1 RNA.

Current assays for quantification of HIV-1 virions rely on real-time reverse transcriptase (RT)-PCR detection of conserved regions of HIV-1 RNA and can be limited by detection of contaminating viral or plasmid DNA. We developed a novel RT-PCR assay using a reverse primer that hybridizes with the poly(A) tail of HIV-1 mRNAs, anchored by conserved viral nucleotides at the most distal region of the transcript. This assay can detect and quantify HIV-1 RNA with high specificity and sensitivity.

HIV-1 DNA is detected in bone marrow populations containing CD4+ T cells but is not found in purified CD34+ hematopoietic progenitor cells in most patients on antiretroviral therapy.

Identifying cellular reservoirs of human immunodeficiency virus type 1 (HIV-1) in patients on antiretroviral therapy (ART) is critical to finding a cure for HIV-1. In addition to resting CD4(+) T cells, CD34(+) hematopoietic progenitor cells have been proposed as another reservoir. We obtained bone marrow aspirates from 11 patients on ART who had undetectable plasma HIV-1 RNA. HIV-1 DNA was detected in CD4(+) T cells from peripheral blood in all patients and from bone marrow cellular fractions containing T cells in most patients. We did not find HIV-1 DNA in highly purified CD34(+) populations using either a sensitive real-time polymerase chain reaction assay or a coculture assay for replication-competent HIV-1.