1. IL-2 + IL-7, Scriptaid, Valproic acid, and Prostratin shows the most promise as a novel therapeutic regiment.
Our ideal model that any new drug regimen should have much better effect in HIV gene expression in comparison with apoptosis, we look at the ratio of activation versus apoptosis, figure 7. Valproic acid at 100 g/ml had the highest effect with the ratio of percentage internal p24 + T cells to percentage of active caspase 3 + T cells at 12 followed by Scriptaid 100 g/ml at a ratio of more than 9. In addition, both IL-2 + IL-7 alongside Prostratin had a ratio of greater than 5 representing a possible higher benefit to risk profile.
Activation of Latently Infected Thymocytes with Histone Deacetylase Inhibitors
Histone Deacetylase Inhibitors demonstrated same toxicity to the thymocytes as IL-2 + IL-7 or Prostratin
Fig. 5. Activation of HIV-1 p24 Gene Expression. Various immune modulators include HDACI, cytokines IL-2 + IL-7 at 30 units/ml along with prostratin 1.25 nmol were added after the thymocytes were infected with NL4-3 at a MOI of 0.1 Efavirenz was used as the NNRTI at 10 M and continual throughout the experiment.
In accordance of our ideal model of immune activation of latently infected HIV-1 T cells without inducing significantly more cell death, we measure the Caspase 3 activity of the thymocytes which represented apoptosis secondary to drug toxicity. As seen in figure 6, among all days of stimulation and drugs tested, there were no significant difference in any of the drugs in the measured apoptosis.
Alvaro Galvis, Viet Hoang and David Camerini, Center for Virus Research, Center for Immunology and
Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697
Background
The current treatment for HIV-1, highly active antiretroviral therapy (HAART), offers long-term suppression of HIV replication and reduces the levels of plasma virus. However, HAART is unable to eradicate HIV infection due to the persistence of latently infected cells. Studies on HIV-1 dynamics in vivo have shown that cells expressing high levels of viral RNA are rapidly cleared while cells expressing low levels persist. Treatment of patients with HAART results in the accumulation of such long-lived latently infected cells. The ability of HIV-1 to enter and exit from latency is therefore one of the major features of the virus life cycle that prevents the clearance of the virus from infected individuals and limits the effectiveness of current antiviral therapy.
Rationale
Previous studies have shown that treatment of latently infected cells with HDAC inhibitors (HDACI) reactivated viral gene expression. A small clinical study with HIV patients taking the anti-depressant valproic acid (an HDAC inhibitor) lead to a decrease in latent viral reservoir by 60%. To further explore the HDAC mechanism of latency our study focused on testing several HDACI in a fetal thymic organ culture model.
Histone Deacetylase Inhibitors Demonstrated Similar Thymocyte Toxicity as IL-2 + IL-7 or Prostratin
An ideal agent for purging the reservoir of latently HIV-1 infected cells would induce HIV-1 gene expression without inducing cell death. We therefore measured the Caspase 3 activity of the treated thymocytes which represented apoptosis secondary to drug toxicity. As seen in figure 2, among all days of stimulation and drugs tested, there were no significant differences in the measured apoptosis.
Establishment of Latency
Many mechanisms of latency has been proposed including:
-Absence of NFkB and NFAT
-Restriction in the levels of HIV Tat
-Reduction in the levels of P-TEFb
However, establishment of specific restrictive chromatin structure at the HIV-LTR, mediated in part by histone deacetylases (HDAC), may be a primary event leading to the restriction of Tat levels during the establishment of latency.
Methods:
Cell Isolation and Culture
To isolate a population of latently infected T cells, thymocytes were isolated and treated with collagenase B and DNase. Thymocytes were subsequently depleted of CD8+ and CD11b+ cells and infected with X4 HIV-1, at a multiplicity of infection, (MOI) of 0.3, then incubated with Nevirapine or Efavirenz for 21 days. They were subsequently incubated with HDACI or IL-2 + IL-7 or CD3 & CD28 MAbs, or left untreated for seven days. HIV-1 replication was measured using an enzyme linked immunosorbent assay (ELISA) for the viral capsid protein, p24, with concurrent measurement of internal p24 expression via cell staining.
Flow Cytometry
Fluorescein isothiocyanate (FITC), phycoerythrin (PE) or peridinin chlorophyll protein (PerCP) conjugated human CD4 and CD8 monoclonal antibodies were used to detect immature and mature thymocytes (Caltag, South San Francisco, CA). FITC-labeled anti-HIV-1 capsid protein (p24) MAb (Beckman Coulter) and anti-active caspase-3 MAb for the detection of apoptosis were used following cell permeabilization. Live cells were gated by low angle versus 900 lightscatter and isotype control MAb were used to define positive staining.
Results
To delineate whether HDACI represent a viable approach for eradication of the HIV reservoir we incubated latently infected cells with Trichostatin (300 nM), Nullscript (100 g/ml), Scriptaid (100 g/ml), Valproic acid (50 and 100 g/ml) compared to IL-2 + IL-7 and unstimulated cell (figure 1). At day two of stimulation, IL-2 + IL-7, Scriptaid and Valproic Acid increased internal p24 expression by HIV (p < 0.05). Moreover, at day four of stimulation Scriptaid and Nullscript had the most significant induction of HIV gene expression with a p < In addition, IL-2 + IL-7, Valproic Acid at both concentrations and Trichostatin induced significantly increased HIV gene expression (p < 0.05). At day seven of stimulation, however, only Trichostatin treated cells had significantly higher HIV gene expression (p < 0.05).
IL-2 + IL-7, Scriptaid, Valproic Acid, and Prostratin Show the Most Promise as Novel Therapeutic Regimens.
Since an ideal agent for purging the reservoir of latently HIV-1 infected cells would induce HIV-1 gene expression without inducing cell death, we calculated the ratio of HIV activation versus apoptosis (figure 3). Valproic Acid at 100 g/ml had the highest effect with a ratio of percent internal p24+ cells to active caspase 3+ cells of 12 followed by Scriptaid with a ratio of more than 9. In addition, both IL-2 + IL-7 and Prostratin had a ratio of greater than 5.
Conclusions and Future Directions
All HDACI had a better ratio of activation to apoptosis than IL-2 + IL7 stimulation.
Valproic Acid and Scriptaid, showed potential as therapeutic agents to reactivate latent virus in HAART patients.
Our data suggest that latency is tied directly to a restrictive chromatin structure that is controlled by histone deacetylases.
Further exploration of this mechanism will be accomplished with ChIP assays with our FTOC model of latency.
In future experiments, we will use a replication defective virus that is tagged with GFP.
This will assist us in keeping the FOTC alive for an extended period of time while minimizing the activation of apoptosis.
Acknowledgements
Medical Scientist Training Program. University of California, Irvine.
Center for Virus Research. University of California, Irvine.