Modest de novo Reactivation of Single HIV-1 Proviruses in Peripheral CD4+ T Cells by Romidepsin

Kathrine Kjær,Steffen Leth,Martin Tolstrup,Paul W Denton,Ole S Søgaard,Rasmus Nymann,Christina V Konrad,Mariane H Schleimann,Jesper D Gunst,Lars Østergaard

doi:10.3389/fviro.2021.736395

Abstract

A cure for human immunodeficiency virus (HIV-1) is restricted by the continued presence of a latent reservoir of memory CD4+ T cells with proviruses integrated into their DNA despite suppressive antiretroviral therapy (ART). A predominant strategy currently pursued in HIV-1 cure-related research is the “kick and kill” approach, where latency reversal agents (LRAs) are used to reactivate transcription from integrated proviruses. The premise of this approach is that “kicking” latent virus out of hiding allows the host immune system to recognize and kill infected cells. Clinical trials investigating the efficacy of LRAs, such as romidepsin, have shown that these interventions do induce transient spikes in viral RNA in HIV-1-infected individuals. However, since these trials failed to significantly reduce viral reservoir size or significantly delay time to viral rebound during analytical treatment interruptions, it is questioned how much each individual latent provirus is actually “kicked” to produce viral transcripts and/or proteins by the LRA. Here, we developed sensitive and specific digital droplet PCR-based assays with single-provirus level resolution. Combining these assays allowed us to interrogate the level of viral RNA transcripts from single proviruses in individuals on suppressive ART with or without concomitant romidepsin treatment. Small numbers of proviruses in peripheral blood memory CD4+ T cells were triggered to become marginally transcriptionally active upon romidepsin treatment. These novel assays can be applied retrospectively and prospectively in HIV-1 cure-related clinical trials to gain crucial insights into LRA efficacy at the single provirus level.

Highlights

Human immunodeficiency virus (HIV-1) is a major part of the global disease burden causing suppression of the immune system by depletion of its central regulators, the CD4+ T cells
The assays we developed, referred to as quantitative viral transcription assay (QVTA) and single-provirus transcription assay (SPTA) quantify HIV-1 nucleic acid levels from single-proviruses seeded in 96-well plates using digital droplet PCR (ddPCR) as described above
Two common questions raised when latency reversal agents (LRAs) are used in clinical studies are: How much is each individual latent provirus “kicked” by a given intervention and what is the frequency of proviruses being activated? Here we report a pair of sensitive and specific ddPCR-based assays that can be used in tandem to answer these critical questions facing the HIV-1 cure field

Summary

Introduction

Human immunodeficiency virus (HIV-1) is a major part of the global disease burden causing suppression of the immune system by depletion of its central regulators, the CD4+ T cells. Developing a cure for HIV-1 have so far been limited by the establishment of a reservoir of latently infected memory CD4+ T cells harboring integrated HIV-1 DNA Most of these proviruses are transcriptionally silent and they ensure viral persistence despite suppressive treatment of the infection with ART [4,5,6]. Our research team have tested the HDAC inhibitors panobinostat and romidepsin as well as a TLR9 agonist as LRAs [15,16,17,18] We and another group have observed transient spikes in cell-associated unspliced HIV1 RNA copies following romidepsin infusion in HIV-1 infected individuals on suppressive ART as a marker of the “kick” [15, 17, 19]. Limited changes were found in the size of the viral reservoir after treatment with romidepsin in these studies and romidepsin treatment alone did not lead to significant delays to viral rebound during analytical treatment interruptions

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