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WO2010099169A2 - Nouveau modèle in vitro de latence du vih-1 pour le criblage d'agents de réactivation du vih-1 - Google Patents

Nouveau modèle in vitro de latence du vih-1 pour le criblage d'agents de réactivation du vih-1 Download PDF

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WO2010099169A2
WO2010099169A2 PCT/US2010/025188 US2010025188W WO2010099169A2 WO 2010099169 A2 WO2010099169 A2 WO 2010099169A2 US 2010025188 W US2010025188 W US 2010025188W WO 2010099169 A2 WO2010099169 A2 WO 2010099169A2
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cells
hiv
immunodeficiency virus
cell
latent
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WO2010099169A3 (fr
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Robert Siliciano
Hung-Chih Yang
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Johns Hopkins University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to the field of immunodeficiency viruses, particularly recombinant immunodeficiency viruses and cell lines containing the same. More specifically, the present invention provides a novel immunodeficiency virus latency model that can be used for high throughput screening to identify small molecules that can be employed to eradicate latent virus from infected individuals.
  • HAART Highly active antiretro viral therapy
  • HAART can suppress HIV-I levels in plasma to below the limit of detection of clinical assays ( ⁇ 50 copies/ml) and reduce the morbidity and mortality of HIV-I infection.
  • HAART alone fails to cure the infection.
  • HAART leaves latent integrated proviruses unaffected (1, 2).
  • Latent viral genomes reside in a small pool of infected resting memory CD4+ T-cells that constitute a stable viral reservoir. In these cells, the provirus remains transcriptionally silent as long as the host cells are in a quiescent state (3-5). This allows the virus to evade host immune surveillance and rebound quickly following discontinuation of HAART.
  • the remarkable stability of the latent viral reservoir necessitates lifelong HAART (6).
  • Novel therapies targeting the latent reservoir generally involve reactivation of latent virus (2, 7-13).
  • Expression of viral genes renders infected cells susceptible to viral cytopathic effects and immune clearance.
  • this reactivation strategy could ultimately purge latent virus from infected individuals.
  • Latent viruses respond to T-cell activation signals (10, 12, 14-17).
  • TCR T-cell reception
  • an ideal treatment should reactivate latent HIV-I, but avoid global T-cell activation.
  • an important tool would be an in vitro model that mimics the latent state of HIV-I in primary resting CD4+ T-cells and allows for high- throughput screening.
  • Some in vitro latency models have been established in transformed T-cell lines (20-22). Although useful, these cell-line models are fundamentally different from the resting CD4+ T-cells because of their proliferating nature and aberrant signaling pathways.
  • Some primary cell models have been recently developed in thymocytes or CD4+ T-cells (23-
  • the present invention relates to the field of immunodeficiency viruses, particularly recombinant immunodeficiency viruses and cell lines containing the same. More specifically, the present invention provides a novel immunodeficiency virus latency model that can be used for high throughput screening to identify small molecules that can be employed to eradicate latent virus from infected individuals.
  • the present invention provides an isolated Bcl-2-transduced cell that comprises, integrated into the genome of the cell, a transcription-competent immunodeficiency virus or a transcription-competent immunodeficiency virus-based retroviral vector.
  • the immunodeficiency virus is latent, and the expression of the latent immunodeficiency virus can be reactivated.
  • the latent immunodeficiency virus is transcriptionally inactive, but is fully transcriptionally competent.
  • the isolated cells of the present invention are transcriptionally competent, e.g., the Tat protein is functional, and the viral LTR is functional (e.g., has functional TAR and other elements necessary for transcriptional activation.
  • the subject cells are representative of cells in a reservoir of latently infected cells in an infected individual.
  • a cell comprises a stably integrated vector comprising a nucleic acid encoding the Bcl-2 gene, and an immunodeficiency virus integrated into the genome of the cell, wherein the immunodeficiency virus is latent and wherein expression of the latent immunodeficiency virus can be activated.
  • the cell is a T-cell.
  • the immunodeficiency virus is under the control of an immunodeficiency virus promoter.
  • the immunodeficiency virus may be human immunodeficiency virus (HIV) including HIV-I and HIV-2.
  • the immunodeficiency virus comprises a nucleic acid encoding a detectable marker.
  • the detectable marker is green fluorescent protein.
  • the present invention further provides an isolated primary CD4+ T-cell comprising a stably integrated vector comprising a nucleic acid encoding the Bcl-2 gene, and a recombinant human immunodeficiency virus 1 (HIV-I) integrated into the genome of the cell comprising a nucleic acid encoding green fluorescent protein under the control of an HIV-I promoter, wherein the HIV-I is latent and where in expression of the latent HIV-I can be activated.
  • the cells of the present invention are useful in screening methods for identifying agents that activate latent immunodeficiency virus.
  • Agents that activate latent immunodeficiency virus are useful, particularly in combination with established anti-HIV therapeutic agents, to reduce the reservoir of latently infected cells in an HIV-infected individual. Such agents are useful to reduce or eliminate the problem of reemergence of viremia following cessation or interruption of treatment with anti-HIV therapeutic agent(s), therefore rendering existing therapeutic agents more effective.
  • the method comprises a contacting a cell of the present invention with a candidate agent, and detecting the detectable marker, wherein the detection indicates that the candidate agent activates the latent immunodeficiency virus.
  • the present invention also provides a composition comprising an activating agent identified by the methods disclosed herein and a pharmaceutically acceptable carrier.
  • the cells of the present invention are also useful in screening methods for identifying agents that block or reduce reactivation of latent immunodeficiency virus transcription in response to T-cell activation signals. Such agents are useful to suppress reactivation of a latent immunodeficiency virus.
  • the method comprises contacting a cell of the present invention with a candidate agent and an agent that reactivates the latent immunodeficiency virus, and detecting the detectable mark, wherein the detection indicates that the candidate agent does not block reactivation of the immunodeficiency virus and wherein no detection indicates that the candidate agent blocks reactivation of the immunodeficiency virus.
  • the invention further provides methods of making a Bcl-2-transduced cell (e.g., a Bcl-2- transduced cell that harbors a latent, transcription competent immunodeficiency virus).
  • the invention further provides agents identified using a screening method of the present invention.
  • the invention further provides methods of treating an HIV infection, with the goal of eradicating the HIV from the infected individual, in contrast to suppressing the infection, as is achieved using currently applied anti-HIV therapy.
  • the method comprises administering to the subject an effective amount of a pharmaceutical composition comprising an activating agent, and administering to the subject an effective amount of one or more anti- viral agents.
  • the anti- viral agent comprises highly active antiretroviral therapy.
  • the method for treating a subject having an HIV-I infection comprises reactivating latent HIV-I in the subject by administering to a subject a therapeutically effective amount of a compound of formula (I):
  • n is an integer from 0 to 4;
  • R 1 , R 2 , and R3 can be the same or different and at each occurrence are independently selected from the group consisting of H, alkyl, substituted alkyl, amino, nitro, halogen, and -OR 4 , wherein R 4 is selected from the group consisting of H, alkyl, and substituted alkyl; and pharmaceutically acceptable salts thereof.
  • n is 1
  • Rl and R2 are each H
  • R3 is -0R4, and R4 is H
  • the compound of formula (I) has the chemical structure:
  • the methods further comprise administering to the subject a therapeutically effective amount of a compound of formula (I) in combination with highly active antiretro viral therapy (HAART).
  • HAART highly active antiretro viral therapy
  • Figure 1 depicts the generation of the Bcl-2-transduced primary CD4+ T-cells.
  • IA shows the structure of the lentiviral vector carrying the human Bcl-2 gene (EB-FLV).
  • the U3 region of 3 'LTR is deleted (3' ⁇ LTR) for self inactivation.
  • the expression of human Bcl-2 is driven by EFl ⁇ promoter, ⁇ , packaging signal; RRE, rev responsive element; cPPT, central polyp urine tract; pEFl ⁇ , EF l ⁇ promoter.
  • Figure IB illustrates the strategy to generate Bcl-2- transduced primary CD4+ T-cells. Primary CD4+ T-cells from normal donors were activated and transduced with the Bcl-2-expressing lentiviral vector.
  • Viable cells were isolated after 3 to 4 weeks of culture in the absence of T-cell receptor (TCR) stimulants or cytokines.
  • Figure 1C shows intracellular staining for Bcl-2 with FITC-conjugated anti-Bcl-2 antibody in freshly isolated CD4+ T-cells and Bcl-2-transduced cells. The Bcl-2-transduced cells were maintained in culture without cytokines for 4 weeks following transduction. Freshly isolated CD4+ T-cells stained with FITC-conjugated isotype control antibodies served as a negative control (purple).
  • Figure 2 depicts the characterization of resting Bcl-2-transduced cells.
  • Figure 2 A shows flow cytometric measurement of cell size. The forward scatter profile of Bcl-2-transduced cells coincided with that of freshly isolated resting CD4+ T-cells.
  • Figure 2B shows cell-cycle analysis in resting and activated freshly isolated CD4+ T-cells, Bcl-2-transduced cells, and latently infected Bcl-2-transduced cells. Data are plotted with DNA staining (Hoechst 33342) on the y-axis versus RNA staining (pyronin Y) on the x-axis. Cells were either left in a resting state or activated with anti-CD3 and anti-CD28 antibodies for 2 days.
  • Figure 2C depicts the expression of the activation markers CD25, CD69, and HLA-DR on resting and activated Bcl-2-transduced cells. The percentage of cells in each quadrant is indicated.
  • Figure 2D shows the levels of IL-2 and IFN- ⁇ mRNA in resting and activated freshly isolated and Bcl-2-transduced CD4+ T-cells. The levels of IL-2 and IFN- ⁇ mRNA were quantified using real-time RT-PCR and normalized to the ubiquitin mRNA levels. The fold change was relative to that observed in the freshly isolated resting CD4+ T-cells.
  • Figure 2E shows the levels of nuclear NF -KB p65 in resting and activated freshly isolated and Bcl-2-transduced CD4+ T-cells.
  • the nuclear NF -KB p65 was quantified by an ELISA-based assay and normalized to the total protein concentration of each nuclear extract. Results shown are relative OD450 values.
  • Data in Figures 2D and 2E are mean ⁇ SD of triplicate samples from 1 of 2 independent experiments, all of which produced similar results.
  • Figure 3 demonstrates that resting Bcl-2-transduced cells exhibit resistance to HIV-I infection and pheno types of TEM.
  • Figure 3 A shows the susceptibility of resting and activated freshly isolated and Bcl-2-transduced CD4+ cells to HIV-I infection. Cells were infected with reporter virus pNL4-3- ⁇ 6-drEGFP pseudotyped with HIV-1-X4 envelope. The number in each plot indicates the percentage of infected (GFP-positive) cells.
  • Figure 3B illustrates the expression of CD45RA, CD45RO, CCR7, and CD62L on resting Bcl-2-transduced cells. The percentage of cells in each quadrant is indicated.
  • Figure 4 generally illustrates the establishment of in vitro HIV-I latency in resting Bcl-2- transduced CD4+ T-cells.
  • Figure 4A shows the genome structure of the reporter virus NL4-3- ⁇ 6-drEGFP. It contains a truncated nefw ⁇ premature stop codons in the ORFs of gag, vif, vpr, and vpu that alter the indicated amino acids shown in the standard single-letter code. A portion of env was replaced with destabilized EGFP, and the signal peptide of env was mutated to allow the destabilized EGFP to remain in the cytoplasm. The red letters indicate the mutated amino acids in the signal peptide.
  • Figure 4B depicts the strategy for generating latently infected Bcl-2- transduced cells.
  • Figure 4C shows the detection of latently infected cells in the sorted GFP- negative population. The sorted GFP-negative cells were activated with anti-CD3 and anti- CD28 or PMA for 2 days and then analyzed by flow cytometry to quantify the number of GFP- positive cells. FL2 -H, red fluorescence channel.
  • Figure 4D shows that latently infected cells contain integrated viral genomes.
  • Latently infected Bcl-2-transduced cells were left untreated (upper panel) or were pretreated with either medium alone (middle panel) or 1 ⁇ M raltegravir (lower panel) for 1 day and then activated with anti-CD3 and anti-CD28 monoclonal antibodies for 2 days. Cells were analyzed using flow cytometry.
  • Figure 4E shows that cell-cycle status of latently infected cells was determined using Hoechst 33342/pyronin Y staining for DNA/RNA. The controls for the resting and activated cells are the same as in Figure 2B. The percentage of cells in each quadrant is indicated.
  • Figure 5 illustrates the response of latently infected Bcl-2-transduced CD4+ T-cells to small molecules and cytokines known to reactivate latent HIV-I.
  • Latently infected, Bcl-2- transduced cells were treated with known small-molecule activators (Figure 5A) or cytokines ( Figure 5B). Bars represent the percentage of GFP-positive cells normalized to the response to anti-CD3 plus anti-CD28 antibodies. Data are mean ⁇ SD of triplicate samples from 1 of 2 independent experiments.
  • the concentrations of small molecules and cytokines used for the experiments are as follows: phytohemagglutinin (PHA) (1 ⁇ g/ml), PMA (10 ng/ml), ionomycin (1 ⁇ M), prostratin (1 ⁇ M), DPP (1 ⁇ M), TsA (200 nM), VA (5 mM), HMBA (5 mM), IL-2 (100 U/ml), IL-Ib (5 ng/ml), IL-4 (3 ng/ml), IL-6 (5 ng/ml), TNF- ⁇ (10 ng/ml), IL-7 (10 ng/ml), and IL-12 (10 ng/ml).
  • PHA phytohemagglutinin
  • PMA 10 ng/ml
  • ionomycin 1 ⁇ M
  • prostratin (1 ⁇ M)
  • DPP 1 ⁇ M
  • TsA 200 nM
  • VA 5 mM
  • HMBA mM
  • IL-2 100 U/ml
  • Figure 6 shows that the screening of small-molecule libraries identified 5HN as a candidate activator.
  • Figure 6A provides a summary of screening results from the Johns Hopkins Drug Library (JHDL). The results were expressed as the percentage of GFP-positive cells after normalization to the response to anti-CD3 plus anti-CD28. For simplicity, only 500 drugs including the hits PMA and 5HN are shown.
  • Figure 6B shows the chemical structure of 5HN.
  • Figure 6C illustrates the effects of 5HN, PMA, and anti-CD3 plus anti-CD28 on the size of latently infected resting CD4+ T-cells. Cell size was measured by flow cytometry using the forward scatter.
  • Figure 6D shows the effect of 5HN on the transcription of HIV-I .
  • Latently infected Bcl-2-transduced cells were left unstimulated or were stimulated with the indicated concentrations of 5HN or anti-CD3 plus anti- CD28 antibodies.
  • the levels of viral mRNA were quantified using real-time RT-PCR and were normalized to the ⁇ -actin mRNA levels. The fold change is shown relative to that observed in the unstimulated samples. Data are mean ⁇ SD of triplicate samples from 1 of 2 independent experiments, all of which produced similar results.
  • Figure 7 demonstrates that 5HN does not activate CD4+ T-cells.
  • Figure 7A shows the effects of 5HN on the expression of activation markers in primary resting CD4+ T-cells.
  • Figure 7C depicts the effect of 5HN on the susceptibility of freshly isolated resting CD4+ T-cells to HIV-I infection. Cells were incubated with medium alone, 5HN, or anti-CD3/anti-CD28 antibodies for 3 days and were then infected with reporter virus NL4-3- ⁇ E-GFP. The number in each plot indicates the percentage of GFP-positive cells quantified by flow cytometry.
  • Figure 7D illustrates the effects of 5HN on the proliferation of latently infected cells. Cell proliferation was determined using Hoechst 33342/pyronin Y staining for DNA/ RNA. The controls for the resting and activated cells are the same as in Figure 2B. The percentage of cells in each quadrant is indicated.
  • Figure 8 demonstrates that 5HN activates latent HIV-I via reactive oxygen species
  • FIG. 8A shows ROS generation in primary resting CD4+ T-cells. Cells were incubated with DHRl 23 prior to 5HN treatment. DHRl 23 conversion, an indicator of intracellular ROS, was evaluated by flow cytometry.
  • Figure 8B demonstrates that 5HN activates NF -KB in primary CD4+ T-cells. Cells were left untreated, treated with 5HN, or stimulated with anti-CD3/anti-CD28 antibodies. The nuclear NF -KB p65 was quantified and normalized to the total protein concentration of each sample. Results are relative OD450 values.
  • Figure 8C shows the effects of 5HN on I ⁇ B ⁇ transcription.
  • Figure 8E shows that NF- ⁇ B is involved in the reactivation of latent HIV-I by 5HN.
  • Bcl-2-transduced cells latently infected with NL4-3- ⁇ 6- drEGFP or m ⁇ 2-NL4-3- ⁇ 6-drEGFP were treated with 5HN.
  • GFP-positive cells were measured using flow cytometry and normalized to the maximal percentage of GFP-positive cells in each population following treatment with PMA plus ionomycin.
  • Data in Figures 8B-8E are mean ⁇ SD of triplicate samples from 1 of 2 independent experiments, all of which produced similar results.
  • Figure 9 demonstrates that 5HN activates latent HIV-I independent of protein kinase c
  • FIG. 9A shows the effect of a PKC inhibitor on the reactivation of latent HIV-I by 5HN.
  • Latently infected Bcl-2-transduced cells were incubated with PKC inhibitor G ⁇ 6983 1 hour prior to the treatment with each activator. Cells were collected and analyzed for GFP-positive cells after 2 days of incubation. The results were normalized to the effect of anti-CD3 plus anti-CD28 co-stimulation. Data are mean ⁇ SD of triplicate samples from 1 of 2 independent experiments, all of which produced similar results.
  • Figure 9B shows the effect of cyclosporine A (CsA) on the reactivation of latent HIV-I by 5HN.
  • CsA cyclosporine A
  • Latently infected Bcl-2-transduced cells were incubated with CsA 1 hour prior to the treatment with each activator. Cells were collected and analyzed for GFP-positive cells after 2 days of incubation. The results were normalized to the effect of PMA treatment. Data are mean ⁇ SD of triplicate samples from 1 of 3 independent experiments.
  • Figure 10 shows the longevity and the stable expression of Bcl-2 of Bcl-2-transduced cells.
  • Figure 1OA illustrates the viability of primary CD4+ T-cells following transduction of Bcl-2. Freshly isolated CD4+ T-cells were activated with anti-CD3 and anti-CD28 antibodies and were grown in T-cell growth factors-enriched medium. Three days following transduction, cells were maintained in the medium without any supplemental cytokines. The number of viable cells was measured weekly using tryptan blue exclusion method.
  • Figure 1OB shows the viability of Bcl-2-transduced cells. The Bcl-2-transduced cells were generated and expanded as described in herein. They were then maintained in the medium without any supplemental cytokines. The number of viable cells was counted weekly.
  • Figure 1OC shows the expression levels of Bcl-2 in freshly isolated resting CD4+ T-cells and Bcl-2-transduced cells.
  • the Bcl-2- transduced samples were collected weekly from the same cell population as in Figure 1OB.
  • the Bcl-2-expression levels were determined using intracellular staining.
  • the Bcl-2 expression levels of freshly isolated CD4+ T-cells were also measured.
  • freshly isolated resting CD4+ T-cells were stained with an isotype-control antibody conjugated with FITC.
  • Figure 11 shows additional characteristics of resting Bcl-2-transduced cells.
  • Figure HA shows the morphology of resting Bcl-2-transduced cells, freshly isolated resting CD4+ T-cells, and activated primary CD4+ T-cells. Cells were stained with Hema 3* Wright-Giemsa Staining system (Fisher Healthcare, USA). Micrographs were taken with 40Ox power. Scale bar: lO ⁇ m.
  • Figure 11 B illustrates the susceptibility of activated and resting freshly isolated and Bcl-2- transduced CD4+ T-cells to HIV-I infection.
  • FIG. 12 depicts the establishment of in vitro HIV-I latency in the Bcl-2-transduced
  • FIG. 12A shows the strategy for generating latently infected Bcl-2-transduced cells by enriching the HIV-I -infected cells.
  • Figure 12B depicts the detection of latently infected cells in the sorted GFP-negative population from the enrichment strategy.
  • the sorted GFP-negative cells were activated with 2.5 ⁇ g anti-CD3 plus l ⁇ g anti-CD28 monoclonal antibodies or IOng/ml PMA plus l ⁇ M ionomycin for 2 days and then analyzed by flow cytometry to quantify the number of GFP-positive cells. The number in each plot indicates the frequency of GFP(+) cells.
  • Figure 13 demonstrates that latent HIV-I can be activated by a variety of activators from latently infected Bcl-2-transduced cells.
  • Latently infected cells were treated with small molecules (Figure 13A) or cytokines ( Figure 13B). The number in each plot indicates the frequency of GFP (+) cells.
  • Flow cytometric plots shown here came from the same experiments in Figures 4A and B. Each flow cytometric plot represents one of triplicate samples in an experiment.
  • Figure 14 shows the effects of 5HN on reactivation of latent HIV-I and cell viability.
  • the latently infected Bcl-2-transduced cells were treated with 5HN or anti- CD3 plus anti-CD28 antibodies and analyzed for percentage of the GFP-positive cells using flow cytometry. The number in each plot indicates the frequency of GFP (+) cells.
  • Figure 14B shows the dose-response curve for the reactivation of latent HIV-I in latently infected Bcl-2- transduced cells by 5HN. The effect of 5HN was determined by quantifying the GFP-positive cells using flow cytometry and was normalized to the maximal effect of 5HN.
  • Data are means ⁇ SD of triplicate samples of an representative experiment from three independent experiments.
  • Figure 14C shows the percentage of apoptosis in Bcl-2-transduced cells and freshly isolated CD4+ T-cells receiving indicated concentrations of 5HN. The fraction of apoptotic cells were determined using annexin V-PE and 7-AAD (7-amino-actinomycin) and analyzed by flow cytometry.
  • Data are means ⁇ SD of triplicate samples of a representative experiment from two independent experiments.
  • Figure 15 shows the effects of 5HN on the reactivation of latent HIV-I in J-LaT-cells.
  • Figure 15 A depicts the dose-response curve of 5HN in reactivation of latent HIV-I in J-LaT- cells. The effect of 5HN was determined by quantifying the GFP-positive cells using flow cytometry. The result was normalized to the effect of PMA.
  • Figure 15B illustrates the effects of 5HN on the transcription of viral genes in J-LaT-cells. The J-LaT-cells were left unstimulated or stimulated with the indicated concentrations of 5HN or PMA.
  • FIG. 16 shows the effects of 5HN or anti-CD3 plus anti-CD28 antibodies on the proliferation of primary CD4+ T-cells.
  • Cells were stained with CFSE (carboxyfluorescein succinimidyl ester) and then treated with 2 ⁇ M 5HN or anti-CD3 plus anti-CD28 antibodies for 3 days. Cell division was measured by the dilution of CFSE using flow cytometry. The percentage indicated the number of cells that had undergone cellular division.
  • CFSE carboxyfluorescein succinimidyl ester
  • cytokines play a role in T-cell survival (29).
  • IL-7 controls the generation and maintenance of memory CD4+ T-cells in vivo (30, 31).
  • IL-7 also reactivates latent HIV-I (12, 32), and thus using IL-7 to promote the establishment of latency in vitro has been difficult.
  • the antiapoptotic protein Bcl-2 is a downstream effector of IL-7 signaling and plays an essential role in counteracting the proapoptotic effects of cytokine withdrawal and maintaining the survival of resting memory CD4+ T-cells (29, 33, 34).
  • the strategy for generation of an in vitro HIV-I latency model in primary CD4+ T-cells takes advantage of the prosurvival effects of Bcl-2.
  • a method for establishing latent HIV-I infection in the Bcl-2-transduced primary human CD4+ T-cells is thus provided. Libraries of small molecules were screened and a novel compound that efficiently reactivates latent HIV-I but does not induce global T-cell activation was identified.
  • the present invention thus reveals a new strategy for reactivating latent HIV-I and illustrates the application of this in vitro primary cell model in finding novel agents for the cure of HIV-I infection.
  • reactivated reactivating, and “reactivation,” respectively. Therefore, as would be clear to one skilled in the art, these terms are used interchangeably herein.
  • the disclosure of a latent immunodeficiency virus that can be activated also refers to a latent immunodeficiency virus that can be reactivated as these terms both convey to the skilled artisan a state of transcriptional activity of an immunodeficiency virus as opposed to a state of latency characterized by transcriptional inactivity or silence.
  • activated or reactivated refers to an immunodeficiency virus that, after a period of latency, becomes transcriptionally active, and in certain embodiments forms infectious viral particles.
  • activated or reactivated as used herein in the context of in vitro reactivated immunodeficiency virus in a cell, refers to an immunodeficiency virus that, after a period of latency, becomes transcriptionally active, i.e., a functional Tat protein mediates transcription from a functional immunodeficiency virus promoter (e.g., a long terminal repeat promoter).
  • a functional immunodeficiency virus promoter e.g., a long terminal repeat promoter
  • biological sample encompasses a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay.
  • the definition encompasses blood and other liquid samples of biological origin (including, but not limited to, serum, plasma, urine, saliva, stool and synovial fluid), solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof.
  • the definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations, such as CD4 " T lymphocytes, glial cells, macrophages, tumor cells, peripheral blood mononuclear cells (PBMC), and the like.
  • the terms further encompass a clinical sample, and also include cells in culture, cell supernatants, tissue samples, organs, bone marrow, and the like.
  • agent may refer to an agent that activates/reactivates latent immunodeficiency virus in a cell (in this case, a “reactivating agent”) or may refer to an agent that blocks reactivation of latent immunodeficiency virus in a cell.
  • Agents may encompass numerous chemical classes, and are generally synthetic, semi-synthetic, or naturally occurring inorganic or organic molecules. In certain embodiments, agents may be small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Agents, however, are not limited to compounds of this size.
  • Agents can also include peptides, polypeptides, antibodies, proteins (e.g., recombinant proteins), macromolecules and siRNA. Agents can also include therapeutic viral vectors, for example adenoviral vectors, as would be known to those skilled in the art. Agents may comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and may include at least an amine, carbonyl, hydroxyl or carboxyl group, and may contain at least two of the functional chemical groups. The agents may comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Agents may also comprise biomolecules including glycoproteins, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Agents may be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries.
  • Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.
  • an agent may be assessed for any cytotoxic activity it may exhibit toward control cells not infected with an immunodeficiency virus, using well-known assays, such as trypan blue dye exclusion, an MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- 2H-tetrazolium bromide]) assay, and the like.
  • Agents that do not exhibit cytotoxic activity toward control cells not infected with an immunodeficiency virus are considered suitable candidate agents.
  • a “therapeutically effective amount” as provided herein refers to an amount of an agent of the present invention, either alone or in combination with another therapeutic agent, including an anti-viral agent, necessary to provide the desired therapeutic effect, e.g., an amount that is effective to prevent, alleviate, or ameliorate symptoms of disease or prolong the survival of the subject being treated.
  • the term "therapeutically effective amount” as provided herein refers to an amount of the presently disclosed compound of formula (I), either alone or in combination with highly active antiretroviral therapy (HAART), necessary to provide the desired therapeutic effect, e.g., an amount that is effective to prevent, alleviate, or ameliorate symptoms of disease or prolong the survival of the subject being treated.
  • HAART highly active antiretroviral therapy
  • the exact amount required will vary from subject to subject, depending on age, general condition of the subject, the severity of the condition being treated, the particular compound and/or composition administered, and the like.
  • An appropriate "therapeutically effective amount” in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation.
  • host cell includes an individual cell or cell culture which can be or has been a recipient of any recombinant vector(s) or construct of the invention.
  • Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change.
  • a host cell includes cells tranfected or infected in vitro with a recombinant vector or a construct of the invention.
  • a host cell which comprises a recombinant vector or construct of the invention is a "recombinant host cell.”
  • immunodeficiency virus refers to human immunodeficiency virus- 1 (HIV-I); human immunodeficiency virus-2 (HIV-2); any of a variety of HIV subtypes and quasispecies; simian immunodeficiency virus (SIV); and feline immunodeficiency virus (FIV).
  • the latent replication competent or non-replication competent immunodeficiency virus can be human immunodeficiency virus (HIV).
  • the immunodeficiency virus can be HIV-I or HIV-2.
  • latent immunodeficiency virus in an isolated cell, the term also includes immunodeficiency virus-based retroviral vectors (e.g., a recombinant immunodeficiency virus). While human immunodeficiency virus (HIV) is exemplified in this specification, the disclosure pertains to other immunodeficiency viruses as well and is not meant to be limited to HIV.
  • immunodeficiency virus-based retroviral vectors e.g., a recombinant immunodeficiency virus.
  • HIV human immunodeficiency virus
  • the term "isolated,” in the context of an isolated cell, refers to a cell (including a Bcl-2-transduced cell) that is in an environment different from that in which the cell naturally o ccurs .
  • latent as used herein in the context of a latent immunodeficiency virus refers to a genomically integrated immunodeficiency virus that is transcriptionally silent or inactive, e.g., immunodeficiency virus transcripts are undetectable or are at background levels, in a cell comprising the latent immunodeficiency virus.
  • Optional or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
  • the phrase "the detectable marker is optionally a fluorescent protein” means that the detectable marker may comprise a fluorescent protein or may not comprise a fluorescent protein such that the description includes both the presence of the fluorescent protein and the absence of the florescent protein.
  • a "subject” means an individual and can include domesticated animals, (e.g., cats, dogs, etc.); livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.) and birds.
  • the subject is a mammal such as a primate or a human.
  • the term also includes mammals that are susceptible to infection by an immunodeficiency virus.
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse affect attributable to the disease.
  • Treatment covers any treatment of a disease in a subject, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, e.g., causing regression of the disease, e.g., to completely or partially remove symptoms of the disease.
  • treatment encompasses prevention of establishment of a systemic infection following initial contact with the virus, and prophylactic treatment of an individual not yet infected with the virus.
  • the present invention provides Bcl-2-transduced cells that comprise, integrated into the genomes of the cells, a transcription-competent immunodeficiency virus or a transcription- competent immunodeficiency virus-based retroviral vector.
  • a transcription-competent immunodeficiency virus or a transcription- competent immunodeficiency virus-based retroviral vector Under basal in vitro culture conditions, the genomically integrated HIV is latent, and the expression of the latent HIV can be reactivated.
  • Standard culture media typically involve standard culture media, a temperature of about 37°C, and 5% CO 2 .
  • Standard culture media can include, but are not limited to, RPMI 1640 medium, McCoy's 5A medium, Leibovitz's L15 medium, Eagle's minimal essential medium, Dulbecco's modified Eagle's medium, and the like.
  • the medium can be supplemented with additional components, e.g., 10 mM HEPES buffer; 2 mM L- glutamine; 100 U/ml penicillin; 100 ⁇ g/ml streptomycin; and heat-inactivated fetal bovine serum, in an amount (in volume/volume) of from about 2% to about 5% from about 5% to about 10%, from about 10% to about 15%, or from about 15% to about 20%, or higher.
  • additional components e.g., 10 mM HEPES buffer; 2 mM L- glutamine; 100 U/ml penicillin; 100 ⁇ g/ml streptomycin; and heat-inactivated fetal bovine serum, in an amount (in volume/volume) of from about 2% to about 5% from about 5% to about 10%, from about 10% to about 15%, or from about 15% to about 20%, or higher.
  • heat-inactivated fetal bovine serum can be used.
  • basal in vitro culture conditions generally exclude the presence in the medium of a facto r(s) that would activate HIV transcription and/or production of HIV virions, including the factors disclosed for reactivation of latent HIV.
  • the latent HIV can be reactivated, e.g., the latent HIV becomes transcriptionally activated.
  • Basal in vitro conditions also exclude the presence of an agent to be tested for its ability to activate HIV, inhibit HIV transcription, suppress HIV reactivation, or inhibit activation of a target cell.
  • the latent HIV can be reactivated, e.g., the latent HIV becomes transcriptionally activated.
  • Culture conditions that result in reactivation of latent HIV include contacting the Bcl-2-transduced cell for a suitable period of time with an effective amount of one or more reactivating agents.
  • reactivating agents include, but are not limited to, activators of NF- ⁇ B including, but not limited to, phytohemagglutinin (PHA), phorbol esters, e.g., tetradecatloyl phorbol acetate (TPA), and TNF- ⁇ ; exposure to an antigen for which a cell surface T-cell receptor is specific; an agent that cross-links cell-surface T-cell receptor, e.g., anti-CD3 antibody; and inhibitors of histone deacetylase, e.g., trichostatin A, sodium butyrate, and trapoxin.
  • PHA phytohemagglutinin
  • phorbol esters e.g., tetradecatloyl phorbol acetate (TPA)
  • TNF- ⁇ tetradecatloyl phorbol acetate
  • exposure to an antigen for which a cell surface T-cell receptor is specific an agent that cross-links cell-surface T-cell receptor,
  • An effective amount of a reactivating agent is an amount effective to achieve transcriptional activation of the latent immunodeficiency virus.
  • Effective amounts of exemplary reactivating agents are as follows: from about 5 nM to about 10 nM TPA; from about 0.1 ng/ml to about 10 ng/ml TNF- ⁇ ; from about 0.3 ⁇ g/ml to about 10 ⁇ /ml anti-CD3 antibody; from about 30 nM to about 1 ⁇ M TSA; PMA/TPA at from about 0.3 ng/ml to about 30 ng/ml; activating anti-CD28 antibody at from about 0.3 ⁇ g/ml to about 10 ⁇ g/ml; soluble recombinant CD 154 at from about 0.1 ⁇ g/ml to about 10 ⁇ g/ml; sodium butyrate at from about 0.1 mM to about 1 mM; and Prostratin at from about 0.003 ⁇ M to about 3 ⁇ M.
  • Non-limiting examples of effective amounts of exemplary reactivating agents are as follows: 10 nM TPA; 10 ng/ml TNF- ⁇ ; 5 ⁇ g/ml anti-CD3 antibody; and 400 nM TSA.
  • Other non-limiting examples of activating agents include PMA, Prostratin, IL-2, histone deacetylase inhibitors (trichostatin A, sodium butyrate), recombinant soluble CD 154, activating or superantagonistic anti-CD28 antibody, and agents identified using the methods described herein.
  • Whether transcription is reactivated can be determined using any known method, including, but not limited to, detecting production of a detectable marker operably linked to an HIV promoter; and detecting production of a viral protein under transcriptional control of an HIV promoter.
  • the detectable marker is a fluorescent protein, it can be detected using florescent detection techniques, which are known in the art and are described in greater detail below.
  • Other methods for detecting expressed proteins can also be used to detect the detectable marker. For example, antibody staining and/or enzymatic detection techniques, or any other detection technique known in the art for detecting expressed proteins can be used to detect the detectable marker.
  • the detectable marker is a fluorescent protein
  • detection of the markers is by flow cytometry, using a fluorescence activated cell sorter (FACS).
  • FACS fluorescence activated cell sorter
  • Detection of the markers can also be by plate based fluorometry.
  • a single well plate based format, a 96-well plate based format, a 384-well plate based format, or any other well plate based format can be used. In 384-well plates from approximately 10,000 to approximately 200,000 cells per well in a total volume of 80-100 ⁇ l can be used.
  • Detection of the markers can also be made by flow cytometry, or fluorometry, or fluorescence microscopy (automated) on live cells or on fixed cells.
  • EGFP For fluorometric analysis, EGFP requires no co factors, stains, or other agents for detection besides a light source.
  • Ideal excitation for EGFP as given by the manufacturer (Clontech, Palo Alto, CaI.), is at 488 nm, and ideal emission is at 508 nm. As would be clear to one skilled in the art, however, other wave lengths of light can be used. For example, excitation at 435 nm and emission at 530 nm, or other suitable parameters can be used.
  • ideal excitation and emission spectra are 557 nm and 579 nm respectively.
  • Flow-cytometric analysis can be performed with a FACStar Plus, a FACScan, a LSR, an ARIA and CellQuest software (BD Biosciences, San Jose, CaI.), or with equivalent hardware and software.
  • a Bcl-2 -transduced cell can also be photographed in culture using a Nikon TE300 inverted microscope and Hoffman optics (Modulation Contrast, Inc., Greenvale, N. Y.) at IOOX by using a SenSys:140E B&W cooled charge-coupled device camera (Photometries, Inc., Arlington, Ariz.), or another equivalent system.
  • a Piston green fluorescent protein set can be used (Chroma, Inc., Rockingham, Vt.).
  • the plates holding cells can be screened at the beginning of the cell culture (0 h) to account for the influence of compound auto fluorescence.
  • detectable fluorescence in the wavelength spectrum of the reporter marker is attributable to compound autofluorescence.
  • Autofluorescence in the wavelength spectrum of the indicator marker is indicated by an increase in the level of the indicator marker fluorescence spectrum intensity that is characteristic for the constitutive expression of the indicator marker in the cells.
  • Recombinant immunodeficiency virus may also be referred to as an "immunodeficiency virus-derived vector,” “immunodeficiency virus-based vector,” or “immunodeficiency virus-based retroviral vector.”
  • the recombinant immunodeficiency virus-based vector is generated using standard recombinant DNA methods, and comprises a detectable marker for viral expression (transcription). The Examples describe specific embodiments of immunodeficiency virus including those incorporated destabilized EGFP, and those containing mutations into particular HIV genes.
  • the latent immunodeficiency virus is a wild-type immunodeficiency virus.
  • HIV genome sequences are known in the art for a variety of HIV-I and HIV-2 strains, and can be found in GenBank under various accession numbers, including AJ203647, AAAJ302646; AF133821, NC001802, L36874, and NC001722.
  • SIV genome sequences are known in the art for a variety of SIV strains, and can be found in GenBank under various accession numbers, including AF334679, and NCOOl 549. Any of a variety of strains and quasispecies can be used.
  • the transcription-competent immunodeficiency virus is recombinant, e.g., the immunodeficiency virus comprises heterologous (non-immunodeficiency virus) sequences.
  • the recombinant immunodeficiency virus is in a vector.
  • Suitable vectors include, but are not limited to, plasmid vectors; Semliki forest virus vectors; vaccinia virus vectors; adenoviral vectors; and the like. Many such vectors are available commercially.
  • the immunodeficiency polynucleotide is inserted into a vector, typically by means of DNA ligase attachment to a cleaved restriction enzyme site in the vector.
  • the recombinant immunodeficiency virus comprises a nucleotide sequence that encodes a detectable marker protein.
  • Suitable detectable marker proteins include, but are not limited to, fluorescent proteins e.g. a green fluorescent protein (GFP) (including enhanced GFP (EGFP), e.g., available from Clontech); a fluorescent protein from an Anthozoa species (as described in, e.g., Matz et al., 17 NAT. BIOTECH. 969-73 (1999)); beta-galactosidase; luciferase; and the like.
  • the nucleotide sequence encoding the detectable marker is operably linked to a promoter.
  • the promoter is an immunodeficiency virus promoter
  • the detectable marker provides a read-out for transcriptional activity of the immunodeficiency virus.
  • a recombinant vector need not include the entire immunodeficiency virus genome.
  • a recombinant vector includes at least the long terminal repeat (LTR) from the immunodeficiency virus, a nucleotide sequence encoding the Tat protein, and a nucleotide sequence encoding a detectable marker, where both the Tat-encoding sequence and the detectable marker-encoding sequence are operably linked to the viral LTR, e.g., are under transcriptional control of the viral LTR.
  • LTR long terminal repeat
  • the recombinant vector may further include other elements, such as sequences necessary for propagation of the vector, such as an origin of replication for replication in a bacterial of eukaryotic cell; sequences encoding a selectable marker for selection of bacterial cells that contain the vector, such as antibiotic resistance genes (e.g., ampicillin resistance; and the like). Such elements are well known to those skilled in the art.
  • the recombinant immunodeficiency virus is an HIV- derived vector referred to as LTR-Tat-IRES-GFP, where both Tat and GFP coding sequences are under transcriptional control of the HIV long terminal repeat (LTR). Both open reading frames are translated from a single mRNA due to the presence of an internal ribosome entry site (IRES) derived from the encephalomyocarditis virus.
  • IRES elements are known in the art, and any such IRES can be used in a recombinant immunodeficiency virus-based vector.
  • Naturally occurring IRES sequences are known in the art and include, but are not limited to, IRES sequences derived from mengovirus, bovine viral diarrhea virus (BVDV), hepatitis C virus (HCV; e.g., nucleotides 1202-1812 of the nucleotide sequence provided under GenBank Accession number AJ242654), GTX, Cyr ⁇ la, Cyr ⁇ lb, polio virus, the immunoglobulin heavy-chain-binding protein (BiP), immunoglobulin heavy chain, a picornavirus, murine encephalomyocarditis virus, poliovirus, and foot and mouth disease virus (e.g., nucleotide numbers 600-1058 of the nucleotide sequence provided under GenBank Accession No.
  • the present invention provides methods for making an isolated cell that comprises, integrated into the genome of the cell, (1) a vector comprising the Bcl-2 gene (e.g., the human Bcl-2 gene in a lentiviral vector); and (2) a transcription-competent immunodeficiency virus (or a transcription-competent immunodeficiency virus-based vector), such that under basal in vitro culture conditions the immunodeficiency virus is latent, and expression of the latent immunodeficiency virus can be reactivated.
  • a vector comprising the Bcl-2 gene e.g., the human Bcl-2 gene in a lentiviral vector
  • a transcription-competent immunodeficiency virus or a transcription-competent immunodeficiency virus-based vector
  • the method generally involves further introducing into such cells in vitro a recombinant, transcription-competent immunodeficiency virus (or a transcription-competent immunodeficiency virus-based vector) that comprises a nucleotide sequence encoding a selectable marker operably linked to an immunodeficiency virus LTR promoter; and selecting a cell from the population that comprises the recombinant immunodeficiency virus integrated into the genome of the cell, and that does not produce the detectable marker.
  • a recombinant, transcription-competent immunodeficiency virus or a transcription-competent immunodeficiency virus-based vector
  • a cell from the population that comprises the recombinant immunodeficiency virus integrated into the genome of the cell, and that does not produce the detectable marker.
  • the recombinant immunodeficiency virus is introduced into cells using any known means, including, but not limited to, electroporation, calcium phosphate precipitation, infection (where the recombinant immunodeficiency virus is packaged into a viral particle or a help virus is used), and the like.
  • the detectable marker is a fluorescent protein, and detection of the marker is by flow cytometry, using a fluorescence activated cell sorter (FACS).
  • FACS fluorescence activated cell sorter
  • the selection step involves selecting a population of cells that, under basal in vitro culture conditions, does not fluoresce or that has low fluorescence, such that the relative fluorescence units are from 10° to about 10 1 . This first selected population may include both uninfected cells and cells that are have latent HIV.
  • the first selected population may be subjected to at least one additional selection.
  • a second selection may be achieved by contacting the first selected population for a suitable period of time with an agent that reactivates the latent HIV.
  • a proportion of the first selected population will exhibit no or low levels of detectable marker, and a proportion will exhibit higher levels of detectable marker.
  • cells that exhibit fluorescence in a range of from about 10 2 to about 10 3 or higher, relative fluorescence units are selected, and are a second selected population of cells.
  • the second selected population of cells may then be subjected to a third selection step and so forth.
  • a third selection step may involve maintaining the second selected population for a suitable period of time under basal in vitro culture conditions, and selecting a population that exhibits no or low fluorescence such that the relative fluorescence units are from 10° to about 10 1 , which population is a third selected population.
  • any of the first, second, or third selected populations may be subjected to cloning, e.g., limiting dilution cloning.
  • Cells may be plated in individual wells of a multi-well plate at a density of one cell per well.
  • the present invention further provides screening methods for identifying an agent that activates a latent immunodeficiency virus. As more fully described in the Examples section, the methods generally involve contacting a Bcl-2 -transduced cell that comprises a transcription- competent latent immunodeficiency virus (or a transcription-competent recombinant immunodeficiency virus-based vector) integrated into the genome of the cell with a test agent; and determining whether the latent immunodeficiency virus is activated. The present invention further provides screening methods for identifying an agent that blocks reactivation of latent immunodeficiency virus in response to a T-cell activation signal.
  • the methods generally involve contacting a Bcl-2-transduced cell that comprises a transcription- competent latent immunodeficiency virus (or a transcription-competent recombinant immunodeficiency virus-based vector) integrated into the genome of the cell with a test agent and an agent that activates T-cells; and determining whether the latent immunodeficiency virus is activated.
  • the screening methods of the present invention usually include one or more controls.
  • a test sample includes a test agent, and a control sample has all the components of the test sample except for the test agent.
  • the method for identifying an agent that activates a latent human immunodeficiency virus involves contacting a Bcl-2-transduced cell with a test agent, which cell comprises a recombinant, genomically-integrated, latent, transcription-competent HIV that comprises a nucleotide sequence encoding a selectable marker operably linked to an HIV promoter; and determining the effect, if any, of the test agent on production of the detectable marker, wherein production of the detectable marker indicates that the test agent activates a latent HIV.
  • the subject method for identifying an agent that blocks reactivation of latent immunodeficiency virus in response to a T-cell activation signal involves contacting a Bcl-2-transduced cell with a test agent and a reactivating agent, which subject cell comprises a recombinant genomically-integrated, latent, transcription-competent HIV that comprises a nucleotide sequence encoding a selectable marker operably linked to an HIV promoter; and determining whether the latent immunodeficiency virus is activated.
  • a decrease in production of the detectable marker, compared to a control lacking the test agent, indicates that the test agent blocks activation of the latent HIV.
  • Suitable reactivating agents include those described herein.
  • Suitable periods of time for contacting a Bcl-2-transduced cell with a reactivating agent are from about 0.5 hour to about 24 hours, e.g., from about 1 hour to about 2 hours, from about 2 hours to about 4 hours, from about 4 hours to about 8 hours, from about 8 hours to about 12 hours, from about 12 hours to about 16 hours, from about 16 hours to about 20 hours, or from about 20 hours to about 24 hours.
  • Contacting a Bcl-2-transduced cell with an effective amount of a reactivating agent is typically conducted under standard culture conditions of 37°C and 5% CO 2 .
  • reagents can be included in the disclosed methods.
  • these include reagents like salts, neutral proteins, e.g., albumin, detergents, etc., that can be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions.
  • Reagents that improve the efficiency of the assay such as nuclease inhibitors, anti-microbial agents, etc., also can be used.
  • the reagents can be added in any order. Incubations can be performed at any suitable temperature, typically between 37° C and 40 0 C. Incubation periods can be selected for optimum activity, but can also be optimized to facilitate rapid high-throughput screening.
  • the present invention provides a compound of formula (I), and derivatives or analogs thereof, for reactivating latent immunodeficiency virus:
  • n is an integer from 0 to 4.
  • R 1 , R 2 , and R3 can be the same or different and at each occurrence are independently selected from the group consisting of H, alkyl, substituted alkyl, amino, nitro, halogen, and -OR 4 , wherein R 4 is selected from the group consisting of H, alkyl, and substituted alkyl; and pharmaceutically acceptable salts thereof. While the following terms in relation to compounds of Formula (I) are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter. These definitions are intended to supplement and illustrate, not preclude, the definitions that would be apparent to one of ordinary skill in the art upon review of the present disclosure.
  • R groups such as groups R 1 , R 2 , and the like
  • Ri and R 2 can be substituted alkyls, or Ri can be hydrogen and R 2 can be a substituted alkyl, and the like.
  • R or group will generally have the structure that is recognized in the art as corresponding to a group having that name, unless specified otherwise herein. For the purposes of illustration, certain representative "R" groups as set forth above are defined below.
  • alkyl refers to Ci -2 O inclusive, linear (i.e., "straight-chain"), branched, or cyclic, saturated or at least partially and in some cases fully unsaturated (i.e., alkenyl and alkynyl) hydrocarbon chains, including for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups.
  • Branched refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain.
  • Lower alkyl refers to an alkyl group having 1 to about 8 carbon atoms (i.e., a C 1-8 alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms.
  • Higher alkyl refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.
  • alkyl refers, in particular, to C 1-8 straight-chain alkyls.
  • alkyl refers, in particular, to C 1-8 branched- chain alkyls.
  • Alkyl groups can optionally be substituted (a "substituted alkyl") with one or more alkyl group substituents, which can be the same or different.
  • alkyl group substituent includes but is not limited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl.
  • alkyl chain There can be optionally inserted along the alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as "alkylaminoalkyl”), or aryl.
  • substituted alkyl includes alkyl groups, as defined herein, in which one or more atoms or functional groups of the alkyl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.
  • halo refers to fluoro, chloro, bromo, and iodo groups.
  • nitro refers to the -NO2 group.
  • amino refers to the -NH 2 group.
  • hydroxyl refers to the -OH group.
  • alkoxyl refers to an alkyl-O- group wherein alkyl is as previously described and, in some embodiments, can be represented by -OR 4 , as R 4 is define herein.
  • alkoxyl as used herein can refer to Ci -2 O inclusive, linear, branched, or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including, for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, butoxyl, t-butoxyl, and pentoxyl.
  • a ring structure for example, but not limited to a 3 -carbon, a 4-carbon, a 5-carbon, a 6-carbon, a 7-carbon, and the like, aliphatic and/or aromatic cyclic compound, including a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure, comprising a substituent R group, wherein the R group can be present or absent, and when present, one or more R groups can each be substituted on one or more available carbon atoms of the ring structure.
  • n is an integer generally having a value ranging from 0 to the number of carbon atoms on the ring available for substitution.
  • Each R group if more than one, is substituted on an available carbon of the ring structure rather than on another R group.
  • the structure above where n is 0 to 2 would comprise compound groups including, but not limited to:
  • an "analog” refers to a chemical compound in which one or more individual atoms or functional groups of a parent compound have been replaced, either with a different atom or with a different functional group.
  • thiophene is an analog of furan, in which the oxygen atom of the five-membered ring is replaced by a sulfur atom.
  • a “derivative” refers to a chemical compound which is derived from or obtained from a parent compound and contains essential elements of the parent compound but typically has one or more different functional groups.
  • Such functional groups can be added to a parent compound, for example, to improve the molecule's solubility, absorption, biological half life, and the like, or to decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, and the like.
  • An example of a derivative is an ester or amide of a parent compound having a carboxylic acid functional group.
  • the term "congener” refers to one or more chemical compounds which differ only by the number and position(s) of particular a substituent group, e.g., a halogen group, on a ring. Commonly known congeners are, for example, polychlorinated biphenyls, which differ only by the number and position of chlorine atoms on a biphenyl ring structure.
  • Ri and R 2 are each H; n is 1; and R3 is hydroxyl, i.e., R3 is -OR 4 , wherein R 4 is H.
  • the compound of Formula (I) is 5-hydroxynaphthalene-l,4-dione, which can be represented by the following chemical structure:
  • the methods of the present invention are suitable for treating individuals who have an immunodeficiency virus infection; who are at risk of contracting an immunodeficiency virus infection; and who were treated for an immunodeficiency virus infection, but who relapsed.
  • individuals include, but are not limited to, individuals with healthy, intact immune systems, but who are at risk for becoming HIV-infected ("at-risk" individuals).
  • At-risk individuals include, but are not limited to, individuals who have a greater likelihood than the general population of becoming HIV-infected.
  • the present invention provides methods for treating an immunodeficiency virus infection in an individual and methods for reducing the reservoir of latent immunodeficiency virus in an individual.
  • the methods generally involve administering to an individual in need thereof an effective amount of a reactivating agent that activates latent immunodeficiency virus.
  • the agent is administered as part of a combination therapy with at least one other anti-viral therapeutic agent.
  • the invention further provides methods for blocking reactivation of latent immunodeficiency virus in an individual.
  • the methods generally involve administering to an individual in need thereof an effective amount of an agent of the present invention that blocks activation of latent immunodeficiency virus.
  • the agent is administered as part of a combination therapy with at least one other anti-viral therapeutic agent.
  • An effective amount of a reactivating agent that reactivates latent HIV is an amount that reactivates latent HIV and reduces the reservoir of latent HIV in an individual by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.
  • a "reduction in the reservoir of latent HIV" (also referred to as “reservoir of latently infected cells”) refers to a reduction in the number of cells in the individual that harbor a latent HIV infection. Whether the reservoir of latently infected cells is reduced can be determined using any known method, including the method described in Blankson et al. (2000) J. Infect. Disease 182(6): 1636-1642.
  • an effective amount of a reactivating agent that reactivates latent HIV is an amount that kills 10 1 , 10 2 , 5xlO 2 , 10 3 , 5xlO 3 , 10 4 , 5xlO 4 , 10 5 , or more, cells in an individual, which cells harbor latent HIV.
  • methods for determining whether the methods of the present invention are effective in treating an immunodeficiency virus infection include any known test for indicia of immunodeficiency virus infection, including, but not limited to, measuring viral load, e.g., by measuring the amount of immunodeficiency virus in a biological sample, e.g., using a polymerase chain reaction (PCR) with primers specific for an immunodeficiency virus polynucleotide sequence; detecting and/or measuring a polypeptide encoded by an immunodeficiency virus, e.g., p24, gpl20, reverse transciptase, using, e.g., an immunological assay with an antibody specific for the polypeptide; and measuring CD4 cell count in the individual.
  • measuring viral load e.g., by measuring the amount of immunodeficiency virus in a biological sample, e.g., using a polymerase chain reaction (PCR) with primers specific for an immunodeficiency virus polynucleo
  • compositions comprise an agent of the present invention, for example, a reactivating agent.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the agent (e.g., a reactivating agent) is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Water may be a carrier when the pharmaceutical composition is administered orally.
  • Saline and aqueous dextrose may be carriers when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions may be employed as liquid carriers for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried slim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the pharmaceutical composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • compositions of the present invention can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • a pharmaceutical composition comprises an effective amount of a reactivating agent together with a suitable amount of a pharmaceutically acceptable carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • an agent of the present invention can be administered with compounds that facilitate uptake of the agent by target cells or otherwise enhance transport of an agent to a particular site for action.
  • Absorption promoters, detergents and chemical irritants e.g., keratino lytic agents
  • keratino lytic agents can enhance transmission of an agent into a target tissue (e.g., through the skin).
  • Suitable agents for use in the methods of the present invention for mucosal/nasal delivery are also described in Chang, et al., Nasal Drug Delivery, “Treatise on Controlled Drug Delivery", Ch. 9 and Tables 3-4B thereof, (Marcel Dekker, 1992).
  • Suitable agents which are known to enhance absorption of drugs through skin are described in Sloan, Use of Solubility Parameters from Regular Solution Theory to Describe Partitioning-Driven Processes, Ch. 5, "Prodrugs: Topical and Ocular Drug Delivery” (Marcel Dekker, 1992), and at places elsewhere in the text. All of these references are incorporated herein for the sole purpose of illustrating the level of knowledge and skill in the art concerning drug delivery techniques.
  • colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • compositions of the present invention may be administered by any particular route of administration including, but not limited to oral, parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracelebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intraosseous, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, iontophoretic means, or transdermal means.
  • routes of administration including, but not limited to oral, parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabron
  • the pharmaceutical compositions disclosed herein may be used alone or in concert with other therapeutic agents at appropriate dosages defined by routine testing in order to obtain optimal efficacy while minimizing any potential toxicity.
  • the dosage regimen utilizing a pharmaceutical composition of the present invention may be selected in accordance with a variety of factors including type, species, age, weight, sex, medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular pharmaceutical composition employed.
  • a physician of ordinary skill can readily determine and prescribe the effective amount of the pharmaceutical composition (and potentially other agents including therapeutic agents) required to prevent, counter, or arrest the progress of the condition.
  • Optimal precision in achieving concentrations of the therapeutic regimen within the range that yields maximum efficacy with minimal toxicity may require a regimen based on the kinetics of the pharmaceutical composition's availability to one or more target sites. Distribution, equilibrium, and elimination of a pharmaceutical composition may be considered when determining the optimal concentration for a treatment regimen.
  • the dosages of a pharmaceutical composition disclosed herein may be adjusted when combined to achieve desired effects.
  • dosages of the pharmaceutical composition and various therapeutic agents may be independently optimized and combined to achieve a synergistic result wherein the pathology is reduced more than it would be if either were used alone.
  • toxicity and therapeutic efficacy of a pharmaceutical composition disclosed herein may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effect (the term "therapeutic effect," as used herein, may include the reactivation of latent immunodeficiency virus and/or the effect of a therapeutic agent used in combination with a reactivating agent) is the therapeutic index and it may be expressed as the ratio LD50/ED50.
  • Pharmaceutical compositions exhibiting large therapeutic indices are preferred except when cytotoxicity of the composition is the activity or therapeutic outcome that is desired.
  • compositions that exhibit toxic side effects may be used, a delivery system can target such compositions to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • a delivery system can target such compositions to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the pharmaceutical compositions of the present invention may be administered in a manner that maximizes efficacy and minimizes toxicity.
  • Data obtained from cell culture assays and animal studies may be used in formulating a range of dosages for use in humans.
  • the dosages of such compositions lie preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose may be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (the concentration of the test composition that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information may be used to accurately determine useful doses in humans.
  • Levels in plasma may be measured, for example, by high performance liquid chromatography.
  • the dosage administration of the compositions of the present invention may be optimized using a pharmacokinetic/pharmacodynamic modeling system. For example, one or more dosage regimens may be chosen and a pharmacokinetic/pharmacodynamic model may be used to determine the pharmacokinetic/pharmacodynamic profile of one or more dosage regimens. Next, one of the dosage regimens for administration may be selected which achieves the desired pharmacokinetic/pharmacodynamic response based on the particular pharmacokinetic/pharmacodynamic profile. See WO 00/67776, which is entirely expressly incorporated herein by reference.
  • the compositions may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily.
  • the daily dosage of the compositions may be varied over a wide range from about 0.1 ng to about 1 ,000 mg per patient, per day. The range may more particularly be from about 0.001 ng/kg to 10 mg/kg of body weight per day, about 0.1-100 ⁇ g, about 1.0-50 ⁇ g or about 1.0-20 mg per day for adults (at about 60 kg).
  • the daily dosage of the pharmaceutical compositions may be varied over a wide range from about 0.1 ng to about 1000 mg per adult human per day.
  • the compositions may be provided in the form of tablets containing from about 0.1 ng to about 1000 mg of the composition or 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 15.0, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, or 1000 milligrams of the composition for the symptomatic adjustment of the dosage to the patient to be treated.
  • An effective amount of the composition is ordinarily supplied at a dosage level of from about 0.1 ng/kg to about 20 mg/kg of body weight per day.
  • the range is from about 0.2 ng/kg to about 10 mg/kg of body weight per day. In another embodiment, the range is from about 0.5 ng/kg to about 10 mg/kg of body weight per day.
  • the compositions may be administered on a regimen of about 1 to about 10 times per day.
  • Doses of a composition of the present invention can optionally include 0.0001 ⁇ g to 1,000 mg/kg/administration, or 0.001 ⁇ g to 100.0 mg/kg/admini strati on, from 0.01 ⁇ g to 10 mg/kg/administration, from 0.1 ⁇ g to 10 mg/kg/administration, including, but not limited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
  • treatment of humans or animals can be provided as a onetime or periodic dosage of a composition of the present invention 0.1 ng to 100 mg/kg such as 0.0001, 0.001, 0.01, 0.1 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively or additionally, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively or additionally, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
  • compositions of the present invention may be administered at least once a week over the course of several weeks.
  • the pharmaceutical compositions are administered at least once a week over several weeks to several months.
  • the pharmaceutical compositions are administered once a week over four to eight weeks.
  • the pharmaceutical compositions are administered once a week over four weeks.
  • compositions may be administered at least once a day for about 2 days, at least once a day for about 3 days, at least once a day for about 4 days, at least once a day for about 5 days, at least once a day for about 6 days, at least once a day for about 7 days, at least once a day for about 8 days, at least once a day for about 9 days, at least once a day for about 10 days, at least once a day for about 11 days, at least once a day for about 12 days, at least once a day for about 13 days, at least once a day for about 14 days, at least once a day for about 15 days, at least once a day for about 16 days, at least once a day for about 17 days, at least once a day for about 18 days, at least once a day for about 19 days, at least once a day for about 20 days, at least once a day for about 21 days, at least once a day for about 22 days, at least once a day for about 23 days, at least once a day for
  • compositions may be administered about once every day, about once every 2 days, about once every 3 days, about once every 4 days, about once every 5 days, about once every 6 days, about once every 7 days, about once every 8 days, about once every 9 days, about once every 10 days, about once every 11 days, about once every 12 days, about once every 13 days, about once every 14 days, about once every 15 days, about once every 16 days, about once every 17 days, about once every 18 days, about once every 19 days, about once every 20 days, about once every 21 days, about once every 22 days, about once every 23 days, about once every 24 days, about once every 25 days, about once every 26 days, about once every 27 days, about once every 28 days, about once every 29 days, about once every 30 days, or about once every 31 days.
  • compositions of the present invention may alternatively be administered about once every week, about once every 2 weeks, about once every 3 weeks, about once every 4 weeks, about once every 5 weeks, about once every 6 weeks, about once every 7 weeks, about once every 8 weeks, about once every 9 weeks, about once every 10 weeks, about once every 11 weeks, about once every 12 weeks, about once every 13 weeks, about once every 14 weeks, about once every 15 weeks, about once every 16 weeks, about once every 17 weeks, about once every 18 weeks, about once every 19 weeks, about once every 20 weeks.
  • compositions of the present invention may be administered about once every month, about once every 2 months, about once every 3 months, about once every 4 months, about once every 5 months, about once every 6 months, about once every 7 months, about once every 8 months, about once every 9 months, about once every 10 months, about once every 11 months, or about once every 12 months.
  • compositions may be administered at least once a week for about 2 weeks, at least once a week for about 3 weeks, at least once a week for about 4 weeks, at least once a week for about 5 weeks, at least once a week for about 6 weeks, at least once a week for about 7 weeks, at least once a week for about 8 weeks, at least once a week for about 9 weeks, at least once a week for about 10 weeks, at least once a week for about 11 weeks, at least once a week for about 12 weeks, at least once a week for about 13 weeks, at least once a week for about 14 weeks, at least once a week for about 15 weeks, at least once a week for about 16 weeks, at least once a week for about 17 weeks, at least once a week for about 18 weeks, at least once a week for about 19 weeks, or at least once a week for about 20 weeks.
  • compositions may be administered at least once a week for about 1 month, at least once a week for about 2 months, at least once a week for about 3 months, at least once a week for about 4 months, at least once a week for about 5 months, at least once a week for about 6 months, at least once a week for about 7 months, at least once a week for about 8 months, at least once a week for about 9 months, at least once a week for about 10 months, at least once a week for about 11 months, or at least once a week for about 12 months.
  • compositions of the present invention can be combined with one or more therapeutic agents, particularly, anti-viral therapies like HAART.
  • the compositions of the present invention and other therapeutic agents can be administered simultaneously or sequentially by the same or different routes of administration.
  • the determination of the identity and amount of therapeutic agent(s) for use in the methods of the present invention can be readily made by ordinarily skilled medical practitioners using standard techniques known in the art.
  • an agent of the present invention e.g., a reactivating agent
  • a therapeutic agent that inhibits one or more immunodeficiency virus functions, which functions include, but are not limited to, viral replication; viral protease activity; viral reverse transcriptase activity; viral entry into a cell; viral integrase activity; activity of one or more of Rev, Tat, Nef, Vpr, Vpu, and Vif; and the like.
  • the therapeutic agent is HAART.
  • therapeutic agents that can be administered in combination therapy with one or more reactivating agents include, but are not limited to, anti-inflammatory, anti- viral, anti-fungal, anti-mycobacterial, antibiotic, amoebicidal, trichomonocidal, analgesic, anti- neoplastic, anti-hypertensives, anti-microbial and/or steroid drugs, to treat antiviral infections.
  • patients are treated with a reactivating agent in combination with one or more of the following; ⁇ -lactam antibiotics, tetracyclines, chloramphenicol, neomycin, gramicidin, bacitracin, sulfonamides, nitrofurazone, nalidixic acid, cortisone, hydrocortisone, betamethasone, dexamethasone, fluocortolone, prednisolone, triamcinolone, indomethacin, sulindac, acyclovir, amantadine, rimantadine, recombinant soluble CD4 (rsCD4), anti-receptor antibodies (e.g., for rhinoviruses), nevirapine, cidofovir (VistideTM), trisodium phosphonoformate (FoscarnetTM), famcyclovir, pencyclovir, valacyclovir, nucleic acid/replication
  • the agents of the present invention may be combined with other therapeutic agents including, but not limited to, immunomodulatory agents, anti-inflammatory agents (e.g., adrenocorticoids, corticosteroids (e.g., beclomethasone, budesonide, flunisolide, fluticasone, triamcinolone, methlyprednisolone, prednisolone, prednisone, hydrocortisone), glucocorticoids, steroids, non-steriodal anti-inflammatory drugs (e.g., aspirin, ibuprofen, diclofenac, and COX-2 inhibitors), and leukotreine antagonists (e.g., montelukast, methyl xanthines, zafirlukast, and zileuton), ⁇ 2-agonists (e.g., albuterol, biterol, fenoterol, isoetharie, metaproter
  • anti-inflammatory agents e.g., ad
  • an agent of the present invention in combination with a second therapeutic agent may be administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, at about 1 hour apart, at about 1 to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours part.
  • two or more therapies are administered within the same patent visit.
  • an agent of the present invention e.g., a reactivating agent
  • one or more other therapies are cyclically administered. Cycling therapy involves the administration of a first therapy (e.g., a first reactivating agent) for a period of time, followed by the administration of a second therapy (e.g.
  • the administration of the combination therapy of the present invention may be repeated and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months.
  • reaction conditions e.g., component concentrations, desired solvents, solvent mixtures, temperatures, pressures and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions.
  • Monoclonal anti-CD3 (catalog no. 555336) and anti-CD28 (catalog no. 555725) antibodies were from BD Biosciences.
  • VA, HMBA, PMA, 5HN, NAC, TsA, and DHRl 23 were from Sigma- Aldrich.
  • Prostratin and DPP were from LC Laboratories.
  • IL-Ib, IL-2, IL-4, IL-6, IL-7, IL- 12, and TNF- ⁇ were from R&D Systems.
  • PDTC was from EMD.
  • Fredericamycin A was a gift from Ben Shen (University of Wisconsin Madison, Madison, Wisconsin, USA).
  • J-Lat full-length clone 10.6 was obtained through NIH AIDS Research and Reference Reagent Program (contributed by Eric Verdin). 5HN was dissolved in DMSO at a concentration of 10 mM and then was aliquotted and stored at -20 0 C for up to 2 months.
  • the lentiviral vector FURW was a gift from Linzhao Cheng (Johns Hopkins University School of Medicine).
  • Bcl-2 cDNA was a gift from M. Hardwick (Johns Hopkins University School of Medicine).
  • Bcl-2 was first amplified by PCR from cDNA using the primers containing the Kpnl and EcoRl recognition sites and then subcloned downstream of EFl ⁇ promoter in the vector pEFl/Myc-His-A (Invitrogen).
  • the DNA sequence containing the EFl ⁇ promoter and Bcl-2 was then cloned into Zero-Blunt TOPO cloning vector PCR4Blunt-TOPO (Invitrogen), verified by direct sequencing, and subsequently subcloned into FURW using the Pad and Sail restriction sites.
  • the reporter viral vector pNL4-3- ⁇ 6-drEGFP was derived from the vector pNL4-3- ⁇ E- GFP, which contains an HIV-I genome with a portion of envelope replaced with EGFP (74). Instead of EGFP, pNL4-3- ⁇ 6-drEGFP contains destabilized EGFP, in which the PEST sequence cloned from pzGreen-DR (Clontech) was ligated in frame to the 3' end of EGFP.
  • the signal peptide of the envelope in pNL4-3- ⁇ 6- drEGFP was mutated using QuickChange II XL site-directed mutagenesis kit (Stratagene) with primer pairs 5 '- GGGGC ACCATGCGCCGTGGGAC ATCGATGATCTGT AGTG and 5 '-
  • the Bcl-2 lentiviral vectors were generated by co-transfecting HEK293T-cells with a plasmid encoding EB-FLV, VSV-G envelope (pVSVG), and a packaging vector pC-Help using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Supernatants were harvested after 72 hours, spun at 335 g, and filtered through a 0.22- ⁇ m membrane to clear cell debris. Virus was pelleted at 100,000 g with 10% volume of 20% sucrose in the bottom for 2 hours at 4°C.
  • the recombinant CXCR4-tropic HIV-I pseudoviruses were generated by the same procedure using a plasmid encoding the HIV-I envelope (pCXCR4), pC-Help, and pNL4-3- ⁇ 6-drEGFP.
  • CD4+ T-cells were activated by incubation in these plates with 1 ⁇ g/ml anti-CD28 monoclonal antibody, 100 U/ml IL-2, and T-cell growth factor-enriched medium.
  • the activated CD4+ T-cells were transduced with EB-FLV at the MOI (multiplicity of infection) of 5 to 10 by spinoculation of cells at 1200 g at room temperature for 2 hours.
  • the transduced cells were cultured in IL-2 and T-cell growth factor- enriched medium for another 3 days, and the medium was replaced with RPMI 1640 with 10% FBS and 1 % penicillin/streptomycin without supplemental cytokines.
  • viable cells were isolated using Ficoll-Hypaque density gradient centrifugation.
  • Bcl-2-transduced cells were expanded by activating with plate-bound anti-CD3 antibody and soluble anti-CD28 antibody. 100 U/ml IL-2 was then added every other day for 10-12 days. Usually, the number of cells increased to 5- to 10-fold.
  • activated Bcl-2-transduced cells were infected with reporter virus NL4- 3- ⁇ 6-drEGFP at an MOI of less than 0.1. The infected cells were maintained in IL-2 and T-cell growth factor-enriched medium for 3 days after infection.
  • RNA Extraction and Real-Time RT-PCR Total cellular RNA was isolated using RNeasy Mini Kit (QIAGEN). RT reactions were performed using Superscript III Reverse Transcriptase (Invitrogen) with random primers (Invitrogen).
  • IFN- ⁇ and IL-2 transcripts were measured using TaqMan Gene Expression Assays on an ABI 7300 Real-Time PCR System (Applied Biosystems assays Hs00174143_ml and Hs00174114_ml, respectively).
  • ubiquitin transcripts (GenBank accession number M26880) were also measured by real-time RT-PCR with SYBR Green PCR Master Mix (Applied Biosystems) and a pair of primers: 5'-ATTTGGGTCGCAGTTCTTG-S' and 5'-TGCCTTGACATTCTCGATGGT-S ' (76).
  • ⁇ -actin was used as an internal control because 5HN activates ubiquitin.
  • ⁇ -actin was measured using TaqMan Gene Expression Assay with the primers and probes of ACTB (part number 433762F). Control reactions with no template or without reverse transcriptase were negative.
  • NF- ⁇ B Activation Nuclear extracts were prepared using a Nuclear Extract Kit (Active Motif) according to the manufacturer's instructions. Protein concentration of nuclear extract was determined using DC-protein assay (Bio-Rad) following the manufacturer's instructions. NF- ⁇ B activation was measured by the TransAM NF- ⁇ B p65 ELISA kit (catalog no. 40097; Active Motif). This kit provides a 96-well plate with immobilized oligonucleotides containing the NF- ⁇ B-binding sites (5'-GGGACTTTCA-S ') derived from HIV-I LTR.
  • Nuclear extract was added to each well, and NF -KB that bound to the oligonucleotides was detected by an NF -KB p65 antibody and a secondary antibody conjugated with HRP (horse radish peroxidase).
  • HRP horseradish peroxidase
  • the compound libraries tested were The Spectrum Collection (MicroSource Discovery Inc.) and JHDL (Johns Hopkins Drug Library).
  • the 2000 compounds in The Spectrum Collection consist of FDA-approved compounds, natural products, and other bioactive compounds. A list of the compounds is available at the MicroSource Discovery website (www.msdiscovery.com/spectrum.html).
  • the compounds in JHDL are primarily FDA- approved compounds (55).
  • the percentage of GFP+ cells was normalized based on the response to anti-CD3 plus anti-CD28 antibodies.
  • DHR123 was dissolved in DMSO at a concentration of 10 mM and was stored at -20 0 C. Before assay, stock solutions were diluted in PBS to 100 ⁇ M and added at the indicated concentrations 30 minutes prior to 5HN treatment. Samples were collected and analyzed at 30, 60, and 120 minutes after addition of 5HN. Conversion of DHR123 to rhodamine 123 by ROS was quantified as increasing green fluorescence using a FACSCalibur (BD Biosciences).
  • HIV-I transcripts were measured by real-time RT-PCR with SYBR Green PCR Master Mix (Applied Biosystems) and the primer pairs that detect single-spliced viral transcripts (5'-GGGTCTCTCTGGTTAGACCAGATCTGAGCC-S ', and 5'- CTCCGCTTCTTCCTGC CAT) (77).
  • ⁇ -actin was measured using a TaqMan Gene Expression Assay. Control reactions with no template or without the addition of RT were negative.
  • Statistics Activation experiments were performed in triplicate cultures. Data are presented as the arithmetic mean ⁇ SD for a representative experiment drawn from 2 or 3 experiments, all of which provided similar results.
  • Example 1 Transduction of Primary Human CD4+ T-cells with Bcl-2
  • primary CD4+ T-cells were transduced with Bcl-2 using a lentiviral vector, EB-FLV, in which Bcl-2 expression is driven by the constitutively active promoter of elongation factor 1 ⁇ (EFl ⁇ ) ( Figure IA and IB).
  • EFl ⁇ elongation factor 1 ⁇
  • Freshly isolated primary human CD4+ T-cells were co-stimulated with anti-CD3 and anti-CD28 and then transduced with the Bcl-2-expressing lentiviral vector.
  • Transduced cells were maintained in the absence of TCR (T-cell receptor) stimulants and exogenous cytokines for more than 3 weeks.
  • Viable cells were then isolated using Ficoll-Hypaque density gradient centrifugation. As shown in Figure 1OA, more than 80% of the activated cells are lost during the first 3 weeks of cytokine withdrawal. As shown below, however, the remaining viable cells have greatly improved survival. Activated primary T-cells die quickly in culture without trophic cytokines (29). Therefore, only Bcl-2-transduced cells survive under the above culture conditions. Bcl-2 was overexpressed in the vast majority of viable cells present after 4 weeks of culture ( Figure 1C). In control experiments, cells transduced with the control lentiviral vector that did not express Bcl-2 died within 2 weeks.
  • Example 2 Bcl-2-Transduced Cells Reach a Quiescent State Similar to that of Freshly Isolated Primary Resting CD4+ T-cells Because resting memory CD4+ T-cells are a major reservoir for latent HIV-I in vivo, an in vitro latency model should consist of infected cells in a similarly quiescent state. After 4 weeks in culture without TCR stimulation or cytokines, surviving Bcl-2-transduced cells were characterized with regard to properties unique to primary resting T-cells. These include small cell size, absence of cell proliferation and cytokine production, and lack of activation markers. Resting Bcl-2-transduced cells exhibited small cell size ( Figure 2A) and scanty cytoplasm ( Figure 1 IA).
  • the host transcription factor NF- ⁇ B is critical for HIV-I replication (14-16, 42). Lack of NF -KB activation in resting CD4+ T-cells is a major factor in HIV-I latency (43). Therefore, the activation state of NF- ⁇ B in resting Bcl-2-transduced cells was examined by ELISA measurement of the nuclear levels of NF- ⁇ B p65. As Bcl-2-transduced cells returned to a quiescent state, nuclear levels of NF- ⁇ B fell to below those seen in freshly isolated resting CD4+ T-cells ( Figure 2E). Upon stimulation with anti-CD3 and anti-CD28, nuclear NF- ⁇ B was readily induced to high levels equivalent to those of activated primary CD4+ T-cells ( Figure 2E).
  • TCM and transition memory T-cells are 2 major cellular reservoirs for HIV-I in vivo (46). Determining whether the latently infected cells generated in the model described herein exhibit properties similar to those of TTM cells requires further investigation.
  • Example 3 Establishment of HIV-I Latency in Bcl-2-Transduced Resting CD4+ T-cells Latently infected cells are rare in vivo (47) because most infected cells die from viral cytopathic effects or host cytolytic responses before they can revert to a resting state in which HIV-I gene expression is shut off. Preliminary results confirmed the previously reported cytopathic effects of HIV-I gene products (48, 49).
  • this modified viral vector, pNL4-3- ⁇ 6- drEGFP gave dramatically improved viability of infected Bcl-2-transduced cells.
  • this approach made it possible to obtain large numbers of latently infected primary resting CD4+ T-cells that could be used to study upregulation of HIV-I gene expression.
  • the ultimate yield of latently infected cells was much lower (approximately 5- to 10-fold) when Bcl-2-transduced cells were infected with an HIV-I construct in which all the reading frames except env were open. For this reason, subsequent experiments were performed with the NL4-3 ⁇ 6-drEGFP virus.
  • Bcl-2-transduced cells were activated and then infected with pseudotyped NL4-3- ⁇ 6-drEGFP. Because the env gene is disrupted by insertion of EGFP, an X4 envelope was provided in trans to achieve single-round infection. The infection rate, based on the percentage of GFP+ cells 3 days after infection, was 5% to 10%. Following more than 4 weeks of culture in the absence of cytokines and other activating stimuli, approximately 20%- 30% of cells that initially expressed GFP became GFP negative. The GFP-negative cells were then isolated by sorting. To determine whether latency had been established, the GFP-negative cells were activated and analyzed for GFP expression by flow cytometry.
  • HIV-I activators were tested including TCR agonists, mitogens, cytokines, and small molecules alone or in combination. Representative flow cytometry plots are shown in Figure 13, A and B. The results are summarized in Figure 5, A and B. PMA, prostratin, and 12-deoxyphorbol 13-phenylacetate (DPP), known activators of PKC, all strongly activated latent HIV-I. Ionomycin, an activator of nuclear factor of activated T-cells (NFAT), reactivated a smaller fraction of latent HIV-I.
  • PMA prostratin
  • DPP 12-deoxyphorbol 13-phenylacetate
  • valproic acid a known histone deacetylase (HDAC) inhibitor and HIV-I activator (13)
  • HDAC histone deacetylase
  • TsA trichostatin A
  • HMBA Hexamethylene bisacetamide
  • pTEFb positive transcription elongation factor b
  • cytokines that are known to activate latent HIV-I (12, 53) were also tested. Cytokines were used at concentrations previously reported to induce activation of latent HIV-I in various primary cell systems. IL-7 alone strongly induced reactivation of latent HIV-I as previously reported (12, 32), while TNF- ⁇ , a well-known activator of latent HIV-I in transformed T-cell lines (16, 21), activated only a small fraction of latent virus (Figure 5B). Consistent with this observation, previous studies using the resting CD4+ T-cells isolated from patients on HAART also revealed the low activity of TNF- ⁇ in reactivation of latent HIV-I (53). However, a combination of IL-2, IL-6, and TNF- ⁇ dramatically increased the reactivation of latent HIV-I in our system. This response is also similar to that reported in resting CD4+ T- cells isolated from patients on HAART (53).
  • Example 5 Screening of Small-Molecule Libraries for Compounds that Reactivate Latent HIV-I Using the system described above, a library of 2000 drugs and natural products and another set of more than 2400 compounds from the Johns Hopkins Drug Library (JHDL) were screened (55). Representative results are shown in Figure 6A. In total, 9 and 8 compounds that could activate latent HIV-I were discovered in the former and latter libraries, respectively. For further study, one of these compounds, 5-hydroxynaphthalene-l,4-dione (5HN), was selected because it was detected in both libraries and displayed the highest capacity to reactivate latent HIV-1. 5HN is a natural quinone (Figure 6B) found in the leaves, roots, and bark of the black walnut tree. 5HN was comparable to the combination of anti-CD3 and anti-CD28 antibodies in its ability to reactivate latent HIV-I ( Figure 6 A and Figure 14A).
  • the 5HN-treated latently infected cells retained the same small size as untreated cells as determined by flow cytometric analysis (Figure 6D). It was further shown that 5HN does not cause upregulation of classic T-cell activation markers such as CD69, CD25, or HLA-DR on freshly isolated resting CD4+ T-cells ( Figure 7A). Because the toxicity induced by T-cell-activating agents is likely to be due to cytokine release, whether 5HN induced production of T-cell cytokines was studied. IL-2 expression was not induced by 5HN, while IFN- ⁇ transcripts were upregulated by approximately 10-fold.
  • 5HN is a quinone that can be reduced to a semiquinone radical by enzymes such as NADPH oxidoreductase. Under aerobic conditions, the semiquinone radical then generates superoxide anion (02 " ) and hydrogen peroxide (H 2 O 2 ), which induce oxidative stress (56, 57). Because ROS can indirectly activate the host transcription factor NF- ⁇ B that can in turn activate latent HIV-I (58, 59), it was hypothesized that induction of oxidative stress by 5HN may play a role in the reactivation of latent virus.
  • DHR123 dihydrorhodamine 123
  • rhodamine 123 60
  • Production of ROS in primary CD4+ T- cells was detected within 2 hours of addition of concentrations of 5HN known to reactivate latent HIV-I ( Figure 8A). Because ROS can indirectly activate NF- ⁇ B, the activation of NF- ⁇ B was assessed next by measuring the nuclear levels of NF- ⁇ B using an ELISA-based assay.
  • 5HN activated NF- ⁇ B in a dose-dependent manner (Figure 8B).
  • the transcripts of I ⁇ B ⁇ , an NF- ⁇ B-responsive gene were also quantified, and showed that 5HN stimulates the expression of I ⁇ B ⁇ ( Figure 8C).
  • Antioxidants like N-acetyl cysteine (NAC) or pyrrolidine dithiocarbamate (PDTC) suppress the effects of ROS on the activation of NF- ⁇ B (59).
  • the 2 tandem NF- ⁇ B-binding sites were mutated in the enhancer region of 3' LTR of pNL4-3- ⁇ 6- drEGFP (m ⁇ 2-LTR- NL4-3- ⁇ 6-drEGFP).
  • the 5' LTR of integrated reporter virus is derived from the 3' LTR that contains mutated NF- ⁇ B-binding sites (61).
  • PKC ⁇ is a master regulator in TCR signaling pathways (62). Activation of PKC leads to activation of key transcription factors in the immune response, including NF -KB and activator protein 1 (AP-I). Because 5HN activates latent HIV-I via NF- ⁇ B, whether the effects of 5HN depend on PKC was examined. Using the pan PKC inhibitor G56983, it was found that inhibition of PKC completely suppressed PMA-induced activation of latent HIV-I and reduced by approximately 50% the response to anti-CD3 and anti-CD28 co-stimulation. However, it almost had no effect on the activation of latent HIV-I by 5HN.
  • the amount of total HIV-I DNA was quantified using real-time PCR with SYBR Green PCR Master Mix and a pair of primers (6F (5'-CATGTTTTCAGCATTATCAGAAGGA-S ') and 84R (5'-TGCTTGATGTCCCCCC ACTS')) that bind to a conserved region of gag (79).
  • a cell line that only harbors a single copy of integrated provirus was used.
  • the integration site is within human HPRT gene located on the X chromosome (80).
  • the frequency of integrated HIV-I in this male cell line can be viewed as 1 per human genome. To calculate the percentage of cells with integrated HIV-I, this cell line served as a standard.
  • the frequency of residual HIV can be calculated by determining the ratio of the amount of HIV in GFP(-) cells to the cells carrying a single-copy provirus, normalized to the copies of HPRT gene.
  • the copy number of HPRT was also measured using real-time PCR with a pair of primers: 5'- ACGTCTTGCTCGAGATGTGA-3 ' and 5 ' -AATCC AGC AGGTC AGC AAAG-3 ' .
  • the latently infected cells were generated from a female donor, while the cell line that harbor a single-copy integrated provirus was derived from a male individual. Therefore, the copy number of HPRT needs to be adjusted for the differential number of X chromosomes.
  • the frequency (GN) of residual HIV genome copies in the GFP(-) cells was calculated to be 0. 275% ⁇ 0.021% (per human genome equivalent).
  • IL-7 is a potent and proviral strain-specific inducer of latent HIV-I cellular reservoirs of infected individuals on virally suppressive HAART. J. Clin. Invest. 115:128-137.
  • Tumor necrosis factor alpha activates human immunodeficiency virus type 1 through induction of nuclear factor binding to the NF-kappa B sites in the long terminal repeat. Proc. Natl. Acad. Sci. U. S. A. 86:5974-5978. 17. Rabson, A.B., and Lin, H.C. 2000. NF-kappa B and HIV: linking viral and immune activation. Adv. Pharmacol. 48:161-207.
  • Interleukin-7 induces expression of latent human immunodeficiency virus type 1 with minimal effects on T-cell phenotype. J. Virol. 76:13077-13082.

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Abstract

La présente invention porte, dans un aspect, sur une cellule isolée transduite par Bcl-2 qui contient, intégré dans son génome, un virus de l'immunodéficience compétent pour la transcription ou un vecteur rétroviral à base de virus de l'immunodéficience compétent pour la transcription. Dans des conditions de culture in vitro basales, le virus de l'immunodéficience est latent, et l'expression du virus de l'immunodéficience latent peut être réactivée. La présente invention porte en outre sur des procédés de criblage pour identifier des agents capables de réactiver un virus de l'immunodéficience latent ou bloquer la réactivation du virus de l'immunodéficience latent. Sous un autre aspect, la présente invention porte sur de tels agents identifiés et sur des méthodes permettant de traiter un sujet souffrant d'une infection par le virus de l'immunodéficience.
PCT/US2010/025188 2009-02-24 2010-02-24 Nouveau modèle in vitro de latence du vih-1 pour le criblage d'agents de réactivation du vih-1 Ceased WO2010099169A2 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140370496A1 (en) * 2012-01-25 2014-12-18 Japan Science And Technology Agency Oligonucleotide for hiv detection, hiv detection kit, and hiv detection method
WO2016177833A1 (fr) 2015-05-04 2016-11-10 Bionor Immuno As Schéma posologique pour un vaccin contre le vih
EP3271020A4 (fr) * 2015-03-18 2018-10-31 Blood Systems, Inc. Inversion de la latence de rétrovirus avec une protéine galectine
WO2019067977A1 (fr) * 2017-09-29 2019-04-04 The George Washington University Utilisation d'agents senolytiques pour éliminer des réservoirs de vih persistants
WO2020264310A1 (fr) * 2019-06-27 2020-12-30 University Of Pittsburgh-Of The Commonwealth System Of Higher Education Compositions et procédés pour détecter la latence du vih, pour traiter une infection par le vih et pour inverser la latence du vih
WO2021188578A1 (fr) * 2020-03-16 2021-09-23 University Of Southern California Procédés de criblage permettant d'identifier des composés à petites molécules qui favorisent ou inhibent la croissance de cellules tumorales circulantes, et leurs utilisations

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* Cited by examiner, † Cited by third party
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EP1463822B1 (fr) * 2001-12-19 2011-10-26 The J. David Gladstone Institutes Procédé de production des lignees cellulaires a virus de l'immunodeficience latent
KR100491143B1 (ko) * 2001-12-26 2005-05-24 삼성에스디아이 주식회사 블랙매트릭스를 구비한 평판표시장치 및 그 제조방법
WO2006029029A2 (fr) * 2004-09-02 2006-03-16 The Uab Research Foundation Compositions permettant de detecter une reactivation latente du vih et procedes d'utilisation de celles-ci
WO2007121429A2 (fr) * 2006-04-17 2007-10-25 J. David Gladstone Institutes Procedes et compositions permettant l'activation synergique du vih latent

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140370496A1 (en) * 2012-01-25 2014-12-18 Japan Science And Technology Agency Oligonucleotide for hiv detection, hiv detection kit, and hiv detection method
US9617606B2 (en) * 2012-01-25 2017-04-11 Japan Science And Technology Agency Oligonucleotide for HIV detection, HIV detection kit, and HIV detection method
EP2808387B1 (fr) * 2012-01-25 2017-06-21 Japan Science And Technology Agency Oligonucléotide pour détection du vih, et kit ainsi que procédé de détection du vih
EP3271020A4 (fr) * 2015-03-18 2018-10-31 Blood Systems, Inc. Inversion de la latence de rétrovirus avec une protéine galectine
WO2016177833A1 (fr) 2015-05-04 2016-11-10 Bionor Immuno As Schéma posologique pour un vaccin contre le vih
WO2019067977A1 (fr) * 2017-09-29 2019-04-04 The George Washington University Utilisation d'agents senolytiques pour éliminer des réservoirs de vih persistants
WO2020264310A1 (fr) * 2019-06-27 2020-12-30 University Of Pittsburgh-Of The Commonwealth System Of Higher Education Compositions et procédés pour détecter la latence du vih, pour traiter une infection par le vih et pour inverser la latence du vih
US12514921B2 (en) 2019-06-27 2026-01-06 University Of Pittsburgh-Of The Commonwealth System Of Higher Education Compositions and methods for treating HIV infection and reversing HIV latency
WO2021188578A1 (fr) * 2020-03-16 2021-09-23 University Of Southern California Procédés de criblage permettant d'identifier des composés à petites molécules qui favorisent ou inhibent la croissance de cellules tumorales circulantes, et leurs utilisations

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