Ex Vivo Bioactivity and HIV-1 Latency Reversal by Ingenol Dibenzoate and Panobinostat in Resting CD4(+) T Cells from Aviremic Patients

Abstract

The human immunodeficiency virus type 1 (HIV-1) latent reservoir in resting CD4(+) T cells represents a major barrier to viral eradication. Small compounds capable of latency reversal have not demonstrated uniform responses across in vitro HIV-1 latency cell models. Characterizing compounds that demonstrate latency-reversing activity in resting CD4(+) T cells from aviremic patients ex vivo will help inform pilot clinical trials aimed at HIV-1 eradication. We have optimized a rapid ex vivo assay using resting CD4(+) T cells from aviremic HIV-1(+) patients to evaluate both the bioactivity and latency-reversing potential of candidate latency-reversing agents (LRAs). Using this assay, we characterize the properties of two candidate compounds from promising LRA classes, ingenol 3,20-dibenzoate (a protein kinase C agonist) and panobinostat (a histone deacetylase inhibitor), in cells from HIV-1(+) antiretroviral therapy (ART)-treated aviremic participants, including the effects on cellular activation and cytotoxicity. Ingenol induced viral release at levels similar to those of the positive control (CD3/28 receptor stimulation) in cells from a majority of participants and represents an exciting LRA candidate, as it combines a robust viral reactivation potential with a low toxicity profile. At concentrations that blocked histone deacetylation, panobinostat displayed a wide range of potency among participant samples and consistently induced significant levels of apoptosis. The protein kinase C agonist ingenol 3,20-dibenzoate demonstrated significant promise in a rapid ex vivo assay using resting CD4(+) T cells from treated HIV-1-positive patients to measure latent HIV-1 reactivation.

FIG 1

Quantification of HIV-1 viral release into culture supernatant after 48 h under four different conditions reveals that ingenol 3,20-dibenzoate has potency similar to that for CD3/28 stimulation. Aliquots of 5 million resting CD4⁺ T cells from aviremic patients on ART were exposed to CD3/28 antibody-coated beads (positive control), medium and DMSO (negative control), 100 nM ingenol 3,20-dibenzoate, or 100 nM panobinostat for 48 h. Quantitative PCR was performed on culture supernatant to detect HIV-1 mRNA release. CD3/28 antibody stimulation resulted in widely ranging, although consistently detectable, HIV-1 mRNA release in all experiments. One HIV-1-uninfected donor (A008) was included to ensure the specificity of the assay, and HIV-1 mRNA was undetectable under all conditions. Ingenol demonstrated potency similar to that of the positive control with regard to viral release (ingenol mean of 811 copies/ml compared to 3,868 copies/ml for CD3/28; ratio of means of 4.77 with P = 0.10), while panobinostat exposure led to viral release in two of six experiments (mean, 266 copies/ml).

FIG 2

Resting CD4⁺ T cells from aviremic patients exposed to 100 nM ingenol 3,20-dibenzoate demonstrated an increase in the mean intensity of fluorescence (MIF) of CD69 to levels similar to those for CD3/28 antibody exposure (ingenol mean of 336 compared to 577 for CD3/28; ratio of means of 1.7 with P = 0.239). Panobinostat at 100 nM induced no change in CD69 fluorescence compared to medium alone (MIF 4.27 versus 1.39; P = 0.063). (A) Representative flow cytometry histograms for the four conditions tested from a single experiment. The solid gray histogram represents the MFI of the isotype control, and the black outline histogram represents the MFI for each condition. (B) Results from six independent experiments.

FIG 3

A 48-h exposure to 100 nM panobinostat leads to a mean 3.57-fold increase in intracellular acetylated histone 3 (Acetyl H3) compared to that for the negative control. (A) Representative flow cytometry histograms from a single experiment measuring the mean florescence intensity (MFI) of intracellular acetylated histone 3 under four different conditions. The solid gray histogram represents the MFI of the isotype control, and the black outline histogram represents the MFI for each condition. (B) Panobinostat, a histone deacetylase inhibitor (HDACi), at 100 nM increased acetylation at histone 3 an average of 3.57-fold in six independent experiments compared to that for medium and DMSO alone (negative control; P < 0.0001). Ingenol 3,20-dibenzoate at 100 nM did not result in any significant change in acetyl H3 from baseline.

FIG 4

A 48-h exposure to 100 nM panobinostat resulted in a significantly higher percentage of resting CD4⁺ T cells expressing activated caspase 3, an early marker of apoptosis, than for the negative control (30.75% compared to 12.62%; P < 0.0001). (A) Representative panel of flow cytometry histograms from a single experiment demonstrating a 3-fold increase in activated caspase 3 expression in cells exposed to panobinostat compared to that for the medium-alone condition. The horizontal bars indicate the percentages of cells expressing activated caspase 3. FITC, fluorescein isothiocyanate. (B) Panobinostat led to increased expression of activated caspase 3 in six independent experiments compared to that for the negative control. Cells exposed to 100 nM ingenol 3,20-dibenzoate and CD3/28 antibody stimulation demonstrated modest but statistically significant decreases in activated caspase 3 expression compared to that for medium alone (6.03% for ingenol and 7.18% for CD3/28; P = 0.0001 and P = 0.001, respectively). (C) Cell viability measured by forward (FSC) and side scatter (SSC) gating for each condition is decreased in the panobinostat condition but not for ingenol or anti-CD3/28 exposed cells.