Ex VivoBioactivity 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. Durable blockade of viral replication by combinations of antiretroviral drugs has transformed human immunodeficiency virus type 1 (HIV-1) infection from an untreatable, lethal condition characterized by progressive immune deficiency into a chronic, manageable medical problem for the vast majority of patients with access to therapy (1). Despite the ability of antiretroviral therapy (ART) to block ongoing HIV-1 replication and allow for restoration of the circulating CD4ϩ T cell population, HIV-1 eradication does not occur with these drugs due to the presence of long-lived viral reservoirs in resting memory CD4 ϩ T cells(2-4). ART can continuously suppress viral replication for years or even decades; however, patients who stop therapy will develop viremia within a matter of weeks and progress to overt immunodeficiency if ART is not resumed (5). This rebound viremia arises from a minority of cells among the resting memory CD4 ϩ T cell population harboring unexpressed HIV-1 proviral DNA that is stably integrated into the cellular genome (6).The HIV-1 latent reservoir in patients on ART is stable over a period of many years and does not decay significantly during the life span of an infected patient (7). It is generally accepted that eradication of the virus will require elimination of this latent reservoir (8, 9). The absence of specific markers to dist...

In vitro effects of the small-molecule protein kinase C agonists on HIV latency reactivation

“…Various small-molecule PKC activators, including PEP005, prostratin and bryostatin-1, are helpful to reactivate HIV latency. PEP005 (ingenol-3-angelate), an activator of protein kinase C (PKC), induces nuclear translocation of PKCδ, exhibiting activity of clinical anti-tumor and actinic keratosis28293031, and potential for HIV latency reactivation323334. Prostratin is another PKC activator that extracted from the tropical plant, Homalanthus nutans with potent anti-tumor and cell activation properties.…”

Kinetics of HIV-1 Latency Reversal Quantified on the Single-Cell Level Using a Novel Flow-Based Technique

“…Several HDAC inhibitors (HDACis) targeting HDAC molecules have been tested for their ability to reactivate latently HIV-1-infected cells, including vorinostat, panobinostat, entinostat, and romidepsin (RMD). These HDACis proved to efficiently induce HIV-1 expression in latently infected resting CD4 ϩ T cells from HIV-1-infected individuals (8,12,13). RMD, a drug that has been used for the treatment of peripheral T-cell lymphoma, …”

HIV-1 Eradication: Early Trials (and Tribulations)

“…The results of this study indicate that no current in vitro latency model faithfully recapitulates the nature of the HIV-1 latent reservoir. Pre-clinical research on LRA efficacy and toxicity gathered from non-human primate SIV models[91, 92], humanized mouse models[93–96] and purified resting CD4 + T cells obtained from HIV-1 infected, aviremic patients[97, 98] are current strategies addressing this significant knowledge gap (see Boxes 1, 2). …”

“…However, the modest outcomes of these clinical trials have led to a re-evaluation of these agents as lead compounds. In two separate ex vivo analyses, LRAs from the protein kinase C family, bryostatin-1[35] and ingenol 3,20 dibenzoate[98], demonstrated significantly higher latency reversal potential than HDAC inhibitors tested. Bryostatin-1 also demonstrated synergy with HDAC inhibitors when used together ex vivo [97].…”