HIV-1 expression within resting CD4+ T cells after multiple doses of vorinostat.

BACKGROUND: A single dose of the histone deacetylase inhibitor vorinostat (VOR) up-regulates HIV RNA expression within resting CD4(+) T cells of treated, aviremic human immunodeficiency virus (HIV)-positive participants. The ability of multiple exposures to VOR to repeatedly disrupt latency has not been directly measured, to our knowledge. METHODS: Five participants in whom resting CD4(+) T-cell-associated HIV RNA (rc-RNA) increased after a single dose of VOR agreed to receive daily VOR Monday through Wednesday for 8 weekly cycles. VOR serum levels, peripheral blood mononuclear cell histone acetylation, plasma HIV RNA single-copy assays, rc-RNA, total cellular HIV DNA, and quantitative viral outgrowth assays from resting CD4(+) T cells were assayed. RESULTS: VOR was well tolerated, with exposures within expected parameters. However, rc-RNA measured after dose 11 (second dose of cycle 4) or dose 22 (second dose of cycle 8) increased significantly in only 3 of the 5 participants, and the magnitude of the rc-RNA increase was much reduced compared with that after a single dose. Changes in histone acetylation were blunted. Results of quantitative viral outgrowth and other assays were unchanged. CONCLUSIONS: Although HIV latency is disrupted by an initial VOR dose, the effect of subsequent doses in this protocol was much reduced. We hypothesize that the global effect of VOR results in a refractory period of ≥ 24 hours. The optimal schedule for VOR administration is still to be defined.

Generation of HIV latency in humanized BLT mice.

Here we demonstrate that a combination of tenofovir, emtricitabine, and raltegravir effectively suppresses peripheral and systemic HIV replication in humanized BLT mice. We also demonstrate that antiretroviral therapy (ART)-treated humanized BLT mice harbor latently infected resting human CD4+ T cells that can be induced ex vivo to produce HIV. We observed that the levels of infected resting human CD4+ T cells present in BLT mice are within the range of those observed circulating in patients undergoing suppressive ART. These results demonstrate the potential of humanized BLT mice as an attractive model for testing the in vivo efficacy of novel HIV eradication strategies.

Valproic acid without intensified antiviral therapy has limited impact on persistent HIV infection of resting CD4+ T cells.

OBJECTIVES: Valproic acid and intensified antiretroviral therapy may deplete resting CD4+ T-cell HIV infection. We tested the ability of valproic acid to deplete resting CD4+ T-cell infection in patients receiving standard antiretroviral therapy. METHODS: Resting CD4+ T-cell infection was measured in 11 stably aviremic volunteers twice prior to, and twice after Depakote ER 1000 mg was added to standard antiretroviral therapy. Resting CD4+ T-cell infection frequency was measured by outgrowth assay. Low-level viremia was quantitated by single copy plasma HIV RNA assay. RESULTS: A decrease in resting CD4+ T-cell infection was observed in only four of the 11 patients. Levels of immune activation and HIV-specific T-cell response were low and stable. Valproic acid levels ranged from 26 to 96 microg/ml when measured near trough. Single copy assay was performed in nine patients. In three patients with depletion of resting CD4+ T-cell infection following valproic acid, single copy assay ranged from less than 1-5 copies/ml. Continuous low-level viremia was observed in three patients with stable resting CD4+ T-cell infection (24-87, 8-87, and 1-7 copies/ml respectively) in whom multiple samples were analyzed. CONCLUSION: The prospective addition of valproic acid to stable antiretroviral therapy reduced the frequency of resting CD4+ T-cell infection in a minority of volunteers. In patients in whom resting CD4+ T-cell infection depletion was observed, viremia was rarely detectable by single copy assay.

Hexamethylbisacetamide and disruption of human immunodeficiency virus type 1 latency in CD4(+) T cells.

BACKGROUND: Novel therapeutic approaches are needed to attack persistent proviral human immunodeficiency type 1 (HIV-1) infection. Hexamethylbisacetamide (HMBA), a hybrid bipolar compound, induces expression of the HIV-1 promoter in the long terminal repeat (LTR) region in a Tat-independent manner but mimics the effect of Tat, overcoming barriers to LTR expression and increasing the processivity of LTR transcription complexes. METHODS: We studied alterations in cellular factors and their LTR occupancy induced by HMBA in models of latent HIV-1 infection. We measured the induction of viral outgrowth by HMBA in resting CD4(+) T cells from aviremic HIV-1-infected donors. RESULTS: HMBA induced outgrowth of HIV-1 from resting CD4(+) T cells recovered from aviremic patients treated with antiretroviral therapy (ART). HMBA triggered cyclin-dependent kinase 9 (CDK9) recruitment to the LTR, a key factor in the induction of efficient HIV-1 expression, via an unexpected interaction with the transcription factor Sp1. The availability of Sp1 and Sp1 DNA binding sites were necessary for HMBA-induced CDK9 recruitment and LTR expression. HMBA signaling via both protein kinase C mu and phosphatidylinositol 3-kinase appeared to contribute to LTR induction. CONCLUSIONS: The novel mechanism through which HMBA disrupts latent HIV-1 infection involves 2 cellular kinases that may be therapeutically exploited to induce expression of persistent proviral HIV-1.

Attacking HIV provirus: therapeutic strategies to disrupt persistent infection.

The therapeutic armamentarium for human immunodeficiency virus type 1 (HIV-1) infection continues to expand. New targets such as entry and integration have recently been successfully exploited. However, HIV-infected patients in need of treatment are currently committed to lifelong suppressive therapy. The persistence of integrated HIV DNA genomes capable of producing virus is a fundamental obstacle to the eradication or cure of HIV infection. Rational molecular or pharmacologic strategies to eliminate persistent HIV proviral genomes are an unaddressed therapeutic need. Coupled with potent antiretroviral therapy, treatments that could efficiently deplete the persistent DNA reservoir of HIV could radically alter treatment paradigms. Prior attempts to target persistent proviral infection deployed intensive antiretroviral therapy (ART) in combination with global inducers of T-cell activation. Initial trials of this approach were unsuccessful. Non-specific T-cell activation may induce high-level viral replication above a level that can be fully contained by ART, while increasing the susceptibility of uninfected cells. Selective targeting of HIV provirus via agents that induce the expression of quiescent HIV, but have limited effects on the uninfected host cell is an alternate approach to attack latent HIV. Recent studies define the role of repressive chromatin structure in maintaining HIV quiescence, and suggest that mechanisms that remodel chromatin about the HIV promoter are a possible therapeutic target. Other studies have uncovered specific factors that may act to induce or maintain latency by limiting the efficiency of HIV gene expression. Attempts to deplete latent HIV using drugs that alter chromatin structure have entered clinical study.

Attacking latent HIV provirus: from mechanism to therapeutic strategies.

PURPOSE OF REVIEW: The persistence of small population integrated proviral HIV genomes capable of expressing HIV within long-lived CD4 T cells is a fundamental obstacle to the eradication or cure of HIV infection. As potent antiretroviral therapy by itself appears to be an impractical approach to the eradication of this quiescent reservoir of HIV infection, new approaches are required. RECENT FINDINGS: Initial studies failed to demonstrate that simultaneous, intensive antiretroviral therapy in combination with global inducers of CD4 T-cell activation could eradicate HIV infection. Global T-cell activation may induce viral replication and increase the number of susceptible uninfected target cells beyond the threshold that can be contained by current antiretroviral therapy. Future advances in antiretroviral therapy may, however, change this equation. An alternative approach to overcoming HIV latency is to develop agents capable of inducing the expression of quiescent HIV without enhancing de novo infection. More selective, targeted approaches may avoid the undesirable consequences of viral induction via signals that result in parallel T-cell activation. Such approaches using the cytokine IL 7, the novel protein kinase agonist prostratin, and inhibitors of the chromatin remodeling enzyme histone deacetylase have recently entered advanced preclinical and clinical testing. SUMMARY: Many obstacles to the eradication of HIV infection exist. Encouraging advances in practical, targeted approaches to the major reservoir of persistence within resting CD4 T cells are, however, beginning to enter clinical testing.

Delayed spread and reduction in virus titer after anterior chamber inoculation of a recombinant of HSV-1 expressing IL-16.

PURPOSE: The timing of T-cell infiltration of the hypothalamus is crucial in the prevention of bilateral retinitis in mice inoculated with HSV-1 through the anterior chamber (AC). In H129-infected mice, T-cells are recruited to the suprachiasmatic nuclei of the hypothalamus too late to protect infected mice from development of bilateral retinitis. The purpose of these studies was to determine whether alteration of T-cell recruitment to the hypothalamus would affect the timing and pattern of virus spread after AC inoculation. METHODS: A recombinant of the H129 strain of HSV-1 expressing IL-16, a cytokine with lymphocytic and monocytic chemoattractant properties, was constructed, and mice were inoculated in the AC with H129wt, H129wt and H129/IL-16, or H129wt and H129/pGal10 (a recombinant virus containing vector only). RESULTS: AC inoculation of BALB/c mice with H129wt and H129/IL-16 resulted in a delay of virus spread to the hypothalamus and the contralateral retina, and this delay correlated with decreased virus titers in infected tissues, compared with mice infected with H129wt or mice infected with H129wt and H129/pGal10. Although the number of infiltrating T-cells in the brains of mice infected with H129wt, H129wt and H129/IL-16, or H129wt and H129/pGal10 was similar, more Mac-1-positive cells were detected early (postinoculation day 2) in the injected eyes of mice infected with H129wt and H129/IL-16 than in mice infected with H129wt and/or H129wt and H129/pGal10. CONCLUSIONS: These results suggest that early recruitment of Mac-1-positive cells to the injected eye may play a role in delaying virus spread in mice infected with H129wt and the IL-16-expressing recombinant virus. IL-16 delivery vectors could be exploited to prevent or delay HSV-1 infection of the hypothalamus, allowing development of the antiviral immune response and subsequent inhibition of virus spread into the optic nerve and retina.

Infiltration of T-lymphocytes in the brain after anterior chamber inoculation of a neurovirulent and neuroinvasive strain of HSV-1.

Following anterior chamber (AC) inoculation of BALB/c mice with the KOS strain of herpes simplex virus type 1 (HSV-1), or with H129, a neuroinvasive and neurovirulent strain of HSV-1, both strains of virus spread from the injected eye through the brain to cause retinitis. However, KOS-infected mice develop retinitis in the uninoculated eye only, whereas H129-infected mice develop bilateral retinitis. Previous studies have shown that infiltrating T-cells in the suprachiasmatic nuclei (SCN) of the hypothalamus of KOS-infected mice concomitant with or before virus protect KOS-infected mice from ipsilateral retinitis. To determine the timing of T cell infiltration and cytokine production in the brain of H129-infected mice, adjacent, frozen sections of the brain were immunostained for virus, T-cells, IL-2, TNF-alpha or IFN-gamma. T-cells infiltrated the brains of H129-infected mice and cytokines were produced in infected tissues. However, virus spread to the optic nerve and retina of both the inoculated and uninoculated eye before T-cells and cytokines were detected in the SCN of H129-infected mice. These results suggest that infiltrating T-cells in the SCN of H129-infected mice may arrive too late to prevent the spread of virus into the optic nerves and retinas and thus prevent development of bilateral retinitis in infected mice.

Rapid spread of a neurovirulent strain of HSV-1 through the CNS of BALB/c mice following anterior chamber inoculation.

Following uniocular anterior chamber (AC) inoculation of BALB/c mice with the KOS strain of herpes simplex virus type 1 (HSV-1), virus spreads from the injected eye to the ipsilateral suprachiasmatic nucleus (SCN) in the central nervous system (CNS) to infect the optic nerve and retina of the contralateral eye, and mice develop retinitis in that eye only. In contrast, after AC inoculation of BALB/c mice with the H129 strain of HSV-1, mice develop bilateral retinitis. The pathway(s) by which H129 spreads to cause bilateral retinitis is not known. To determine the route and timing of H129 spread after AC inoculation, BALB/c mice were injected in the AC of the right eye with 5 x 10(3) PFU of H129. Brains from 30 mice were sectioned on a brain matrix and the amount of virus in the brain and eyes was determined by plaque assay. Frozen sections were prepared from the eyes, brain, and trigeminal ganglia of an additional 30 mice, and HSV-1 antigen was detected by immunohistochemistry. After AC inoculation, H129 follows a pathway similar to KOS in the CNS, but H129 appears to spread more rapidly than KOS within the CNS. Unlike KOS, H129 is able to infect brain stem nuclei and H129-infected mice developed neurological impairments in addition to bilateral retinitis. The results of these studies suggest that the ability of H129 to spread rapidly in the CNS allows early virus infection of retino-recipient nuclei proximal to the contralateral and ipsilateral optic nerves. Early infection of retino-recipient nuclei, such as the SCN may allow virus to spread into the retinas before a virus-specific immune response can be induced.