David M. Margolis

Depletion of latent HIV-1 infection in vivo: a proof-of-concept study

BACKGROUND Persistent infection in resting CD4+ T cells prevents eradication of HIV-1. Since the chromatin remodeling enzyme histone deacetylase 1 (HDAC1) maintains latency of integrated HIV, we tested the ability of the HDAC inhibitor valproic acid to deplete persistent, latent infection in resting CD4+ T cells. PROCEDURES We did a proof-of-concept study in four volunteers infected with HIV and on highly-active antiretroviral therapy (HAART). After intensifying the effect of HAART with subcutaneous enfuvirtide 90 mug twice daily for 4-6 weeks to prevent the spread of HIV, we added oral valproic acid 500-750 mg twice daily to their treatment regimen for 3 months. We quantified latent infection of resting CD4+ T cells before and after augmented treatment by limiting-dilution culture of resting CD4+ T cells after ex-vivo activation. FINDINGS The frequency of resting cell infection was stable before addition of enfuvirtide and valproic acid, but declined thereafter. This decline was significant in three of four patients (mean reduction 75%, range 68% to >84%). Patients had slight reactions to enfuvirtide at the injection site, but otherwise tolerated treatment well. INTERPRETATION Combination therapy with an HDAC inhibitor and intensified HAART safely accelerates clearance of HIV from resting CD4+ T cells in vivo, suggesting a new and practical approach to eliminate HIV infection in this persistent reservoir. This finding, though not definitive, suggests that new approaches will allow the cure of HIV in the future.

The Human Factors YY1 and LSF Repress the Human Immunodeficiency Virus Type 1 Long Terminal Repeat via Recruitment of Histone Deacetylase 1

ABSTRACT Enigmatic mechanisms restore the resting state in activated lymphocytes following human immunodeficiency virus type 1 (HIV-1) infection, rarely allowing persistent nonproductive infection. We detail a mechanism whereby cellular factors could establish virological latency. The transcription factors YY1 and LSF cooperate in repression of transcription from the HIV-1 long terminal repeat (LTR). LSF recruits YY1 to the LTR via the zinc fingers of YY1. The first two zinc fingers were observed to be sufficient for this interaction in vitro. A mutant of LSF incapable of binding DNA blocked repression. Like other transcriptional repressors, YY1 can function via recruitment of histone deacetylase (HDAC). We find that HDAC1 copurifies with the LTR-binding YY1-LSF repressor complex, the domain of YY1 that interacts with HDAC1 is required to repress the HIV-1 promoter, expression of HDAC1 augments repression of the LTR by YY1, and the deacetylase inhibitor trichostatin A blocks repression mediated by YY1. This novel link between HDAC recruitment and inhibition of HIV-1 expression by YY1 and LSF, in the natural context of a viral promoter integrated into chromosomal DNA, is the first demonstration of a molecular mechanism of repression of HIV-1. YY1 and LSF may establish transcriptional and virological latency of HIV, a state that has recently been recognized in vivo and has significant implications for the long-term treatment of AIDS.

Coaxing HIV-1 from resting CD4 T cells: histone deacetylase inhibition allows latent viral expression.

Background: Histone deacetylase (HDAC), a host mediator of gene repression, inhibits HIV gene expression and virus production and may contribute to quiescence of HIV within resting CD4 T cells. Objectives: To test the ability of valproic acid (VPA), an inhibitor of HDAC in clinical use, to induce expression of HIV from resting CD4 T cells. Methods: Chromatin immunoprecipitation measured the capability of VPA to deacetylate the HIV promoter, a remodeling of chromatin linked to gene expression. The effect of VPA on resting CD4 T cell phenotype was measured by flow cytometric analysis, and its effect on de novo HIV infection of peripheral blood mononuclear cells was measured ex vivo. Outgrowth of HIV from resting CD4 T cells of aviremic, HIV-infected donors treated with highly active antiretroviral therapy was compared in limiting-dilution cultures after mitogen stimulation or exposure to VPA. Results: VPA induced acetylation at the integrated HIV proviral promoter, but CD4 cells exposed to VPA did not become activated or more permissive for de novo HIV infection. VPA induced outgrowth of HIV from the resting CD4 cells of aviremic patients at concentrations achievable in vivo as frequently as did mitogen stimulation. Conclusions: With advances in antiretroviral therapy, HIV infection might be cleared by intensive time-limited treatment coupled with practical strategies that disrupt latency without enhancing new infection. HDAC inhibitors are capable of inducing expression of quiescent provirus, without fully activating cells or enhancing de novo infection, and may be useful in future clinical protocols that seek to eradicate HIV infection.

Counterregulation of Chromatin Deacetylation and Histone Deacetylase Occupancy at the Integrated Promoter of Human Immunodeficiency Virus Type 1 (HIV-1) by the HIV-1 Repressor YY1 and HIV-1 Activator Tat

ABSTRACT Repression of human immunodeficiency virus type 1 (HIV-1) transcription may contribute to the establishment or maintenance of proviral quiescence in infected CD4+ cells. The host factors YY1 and LSF cooperatively recruit histone deacetylase 1 (HDAC1) to the HIV-1 long terminal repeat (LTR) and inhibit transcription. We demonstrate here regulation of occupancy of HDAC1 at a positioned nucleosome (nuc 1) near the transcription start site of integrated LTR. We find that expression of YY1 increases occupancy by HDAC1, decreases acetylation at nuc 1, and downregulates LTR expression. HDAC1 recruitment and histone hypoacetylation were also seen when Tat activation was inhibited by the overexpression of YY1. A YY1 mutant without an HDAC1 interaction domain and incompetent to inhibit LTR activation fails to recruit HDAC1 to LTR or decrease nuc 1 acetylation. Further, expression of a dominant-negative mutant of LSF (dnLSF), which inhibits LSF occupancy and LTR repression, results in acetylation and decreased HDAC1 occupancy at nuc 1. Conversely, exposure of cells to the histone deacetylase inhibitor trichostatin A or activation of LTR expression by HIV-1 Tat results in the displacement of HDAC1 from nuc 1, in association with increased acetylation of histone H4. Recruitment of HDAC1 to the LTR nuc 1 can counteract Tat activation and repress LTR expression. Significantly, when repression is overcome, LTR activation is associated with decreased HDAC1 occupancy. Since the persistence of integrated HIV-1 genomes despite potent suppression of viral replication is a major obstacle for current antiretroviral therapy, strategies to selectively disrupt the quiescence of chromosomal provirus may play a role in the future treatment of AIDS.

Atazanavir for the Treatment of Human Immunodeficiency Virus Infection

Atazanavir is the first once‐daily protease inhibitor for the treatment of human immunodeficiency virus type 1 infection and should be used only in combination therapy, as part of a highly active antiretroviral therapy (HAART) regimen. In addition to being the most potent protease inhibitor in vitro, atazanavir has a distinct cross‐resistance profile that does not confer resistance to other protease inhibitors. However, resistance to other protease inhibitors often confers clinically relevant resistance to atazanavir. Currently, atazanavir is not a preferred protease inhibitor for initial HAART regimens. In treatment‐naïve patients, atazanavir can be given as 400 mg/day. However, atazanavir should be pharmacologically boosted with ritonavir in treatment‐experienced patients or when coadministered with either tenofovir or efavirenz. Patients who receive atazanavir experience similar rates of adverse events compared with patients receiving comparator regimens. An exception is an increased risk of asymptomatic hyperbilirubinemia, which is due to competitive inhibition of uridine diphosphate‐glucuronosyltransferase 1A1. Although hyperbilirubinemia is a common adverse drug reaction of atazanavir therapy (22–47%), fewer than 2% of patients discontinue atazanavir therapy because of this adverse effect. Common adverse effects reported with atazanavir include infection, nausea, vomiting, diarrhea, abdominal pain, headache, peripheral neuropathy, and rash. Of significance, fewer abnormalities have been observed in plasma lipid profiles in patients treated with atazanavir compared with other protease inhibitor–containing regimens. As with other protease inhibitors, atazanavir is also a substrate and moderate inhibitor of the cytochrome P450 (CYP) system, in particular CYP3A4 and CYP2C9. Clinically significant drug interactions include (but are not limited to) antacids, proton pump inhibitors, histamine type 2 receptor antagonists, tenofovir, diltiazem, irinotecan, simvastatin, lovastatin, St. Johns wort, and warfarin. We conclude that atazanavir is a distinctively characteristic protease inhibitor owing to its in vitro potency, once‐daily dosing, distinct initial resistance pattern, and infrequent association with metabolic abnormalities.

Targeted Derepression of the Human Immunodeficiency Virus Type 1 Long Terminal Repeat by Pyrrole-Imidazole Polyamides

ABSTRACT The host factor LSF represses the human immunodeficiency virus type 1 long terminal repeat (LTR) by mediating recruitment of histone deacetylase. We show that pyrrole-imidazole polyamides targeted to the LTR can specifically block LSF binding both in vitro and within cells via direct access to chromatin, resulting in increased LTR expression.

Dose proportional inhibition of HIV-1 replication by mycophenolic acid and synergistic inhibition in combination with abacavir, didanosine, and tenofovir.

Mycophenolate mofetil (MMF), a therapeutically used inhibitor of inosine monophosphate dehydrogenase is hydrolyzed to its active metabolite mycophenolic acid (MPA) in vivo. MPA exhibits anti-HIV activity in vitro. We tested MPA alone and in combination with abacavir (ABC), didanosine (DDI), lamivudine (3TC) and tenofovir (TFV) against wild-type human immunodeficiency virus type-1 (HIV-1) and nucleoside reverse transcriptase inhibitor (NRTI)-resistant HIV-1. MPA (62.5-500 nM), when combined with ABC or DDI, synergistically enhanced activity against wild-type HIV and the NRTI-resistant HIV clone DRSM34. MPA also enhanced the activity of TFV against both wild-type HXB2 and TFV-resistant strain HIV(K65R), in a more than additive manner. No significant antiproliferative effect of MPA (< or =0.25 microM) alone or in the presence of ABC, DDI and TFV was observed. This indicates that the antiviral effects of MMF may be clinically achievable without fully blocking T-cell proliferation or inducing immunosuppression. These findings provide further rationale for the clinical testing of MMF in combination with ABC, DDI, and TFV.

A prospective evaluation of the effect of atazanavir on the QTc interval and QTc dispersion in HIV‐positive patients

Atazanavir (ATV), an HIV protease inhibitor (PI) that may be preferred for the treatment of HIV‐infected patients with cardiovascular comorbidities because of its favourable effects on plasma lipids, has been associated with cardiac rhythm disturbances.

Interleukin-7 induces HIV type 1 outgrowth from peripheral resting CD4+ T cells.

To the Editor: We confirm that interleukin (IL)-7 is a viable clinical candidate for use in inducing expression of latent HIV in resting CD4 cells obtained from highly active antiretroviral therapy (HAART)-treated, aviremic, HIVinfected adults. Thus far, no one with HIV infection has been cured, despite the availability of potent antiretroviral agents. Nevertheless, cure of infection is the ultimate goal of any antimicrobial therapy. Although eradication of HIV infection is currently regarded as unattainable, advances in the understanding of HIV pathogenesis have led many to reevaluate the obstacles to the clearance of virus. Eradication of HIV seems to be prevented by several factors, chief among them the presence of a stable population of long-lived quiescently infected CD4 T cells. Suppression of plasma viremia is achieved with the use of HAART; however, cessation of therapy uniformly results in the return of virus, thought to be primarily derived from these persistently infected resting memory T cells. The generation of latency occurs after HIV infection of a transcriptionally active cell, which predominantly results in productive infection and cell death. If cellular transcription ceases before either viral or immunologic cytopathic effects, however, the virus can become dormant. New treatment paradigms are needed to target and clear persistent infection in this latent viral reservoir. Such strategies must neither reduce immune function nor propagate new infection. Using a SCID-hu mouse model, Scripture-Adams et al demonstrated that IL-7 stimulation of primary human T cells and thymocytes induced substantial expression of latent HIV while having minimal effects on cell phenotype. Latently infected T cells in this model system are derived from naive CD4 cells, whereas most latently infected cells in HIV-infected individuals are resting memory CD4 cells. As reported by Pierson et al, latent infection is up to 30fold more frequent in the memory T-cell population. Because phase 1 studies of the administration of IL-7 to HAART-treated HIV-infected patients are now being planned, we sought to test whether IL-7 allowed the recovery of HIV from peripheral cells of HIV-infected patients in whom viremia is suppressed by HAART. Resting memory CD4 cells would be expected to represent most of the latently infected cells in the peripheral blood of such patients. We measured the frequency of viral recovery after the exposure of peripheral resting CD4 cells obtained from HAART-treated, aviremic, HIV-infected adults to this cytokine compared with nonspecific mitogen activation. Four HIV-infected volunteers in whom the plasma levels of HIV-1 RNA (Roche Amplicor, Branchburg, NJ) had been durably suppressed (>6 months) to <50 copies/mL were studied after receiving institution review board–approved informed consent. Resting CD4 T cells obtained by leukophoresis were isolated on 6 occasions as described by Chun and colleagues, with modifications as described. Resting CD4 cells were cultured in a limiting dilution format in 4 replicate dilutions of 5.0 to 0.625 million cells per well. These cells were activated with phytohemagglutinin (PHA) and irradiated seronegative feeder peripheral blood mononuclear cells (PBMCs) or exposed to 10 ng/mL of IL-7 (R&D Systems, Minneapolis, MN) for 72 hours. CD8-depleted, PHA-activated, seronegative donor PBMCs (up to 1.5 million per well) were then added to these limiting dilution cultures. Cultures were grown in 20 U/mL of IL-2 (Chiron, Emeryville, CA), fresh activated seronegative donor PBMCs were added weekly, and p24 was assayed in culture supernatant. Infectious units per million resting cells were estimated by a maximum likelihood. Cells that express the -chain of the IL-7 receptor, CD127, have the potential to respond to the IL-7 treatment. Analysis of the isolated resting T cells (CD3/CD4/human leukocyte antigen– D-related [HLA-DR]−) determined that 40% to 70% were CD127. Fifty percent to 60% of these cells could be identified as naive because of the presence of phenotypic markers (CD45RA, CCR7, and CD62L). Therefore, the IL-7 receptor– positive cells within the resting T cells were approximately equally divided between the naive and memory T-cell populations. As expected, receptor levels were downregulated 10-fold after exposure to IL-7 (data not shown). HIV was recovered from patients’ resting cells in every instance after maximal activation with PHA, irradiated allogeneic PBMCs, and IL-2 (Table 1). HIV was recovered in 5 of 6 experiments after exposure to IL-7 and IL-2. HIV was not recovered in 1 of 2 experiments in 1 patient (A) in whom infectious units per million resting cells (IUPM) calculated from outgrowth after PHA activation was low. Frequency of viral outgrowth (IUPM) was higher after activation than after exposure to IL-7 in 4 of 6 experiments. HIV was more frequently recovered from the resting cells of subject A after PHA activation on a single occasion, however, and more frequently after exposure to IL-7 in another experiment. Because of the rarity of latent HIV and the few experiments performed so far, a comparison of the frequency with which activation of IL-7 results in the outgrowth of latent HIV cannot yet be made. Given the infrequency of latent infection in naive cells and the frequency with which cells without naive markers displayed IL-7 receptor, it is possible that IL-7 induced viral expression from naive as well as memory cells.

Improvement in insulin sensitivity and dyslipidemia in protease inhibitor-treated adult male patients after switch to atazanavir/ritonavir.

Background Treatment of human immunodeficiency virus (HIV) with protease inhibitors (PIs) is associated with insulin resistance, triglyceride-rich dyslipidemia, and fat redistribution. Atazanavir (ATV), a potent once-daily PI, has been recognized for its convenience to patients, and some studies describe improved lipid metabolism. However, its effects on insulin sensitivity have not been elucidated. We conducted this study to test the hypothesis that ATV improves insulin resistance and dyslipidemia. Methods We prospectively studied 9 HIV-infected men with dyslipidemia (median age, 53 years; baseline triglyceride level, >200 mg/dL) on stable PI-containing antiretroviral therapy who elected to change PI therapy to ritonavir-boosted ATV therapy, dose of 300/100 mg. We measured insulin resistance at baseline and after 12 weeks of therapy using a hyperinsulinemic euglycemic clamp (insulin dose, 200 mU/m2 minute). Fasting lipid profiles and body composition (whole-body dual energy x-ray absorptiometry) were also measured at baseline and after 12 weeks. Results All 9 patients completed the study and maintained undetectable viral loads (<50 copies/mL) and stable CD4 counts. After 12 weeks, insulin sensitivity significantly improved (+28%; P = 0.008) in all patients. Triglyceride levels also improved. Conclusions Using the gold-standard euglycemic clamp, ritonavir-boosted ATV therapy improved PI-induced insulin resistance among dyslipidemic HIV-infected men on PI-based antiretroviral therapy. These findings were not attributable to a change in body weight and provide further evidence for ATVs unique metabolic profile among the PIs.