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. 2017 Sep:23:52-58.
doi: 10.1016/j.ebiom.2017.07.019. Epub 2017 Jul 29.

Vorinostat Renders the Replication-Competent Latent Reservoir of Human Immunodeficiency Virus (HIV) Vulnerable to Clearance by CD8 T Cells

Julia A Sung  1 , Katherine Sholtis  1 , Jennifer Kirchherr  1 , Joann D Kuruc  1 , Cynthia L Gay  1 , Jeffrey L Nordstrom  2 , Catherine M Bollard  3 , Nancie M Archin  1 , David M Margolis  4
Affiliations

Vorinostat Renders the Replication-Competent Latent Reservoir of Human Immunodeficiency Virus (HIV) Vulnerable to Clearance by CD8 T Cells

Julia A Sung et al. EBioMedicine. 2017 Sep.

Abstract

Latently human immunodeficiency virus (HIV)-infected cells are transcriptionally quiescent and invisible to clearance by the immune system. To demonstrate that the latency reversing agent vorinostat (VOR) induces a window of vulnerability in the latent HIV reservoir, defined as the triggering of viral antigen production sufficient in quantity and duration to allow for recognition and clearance of persisting infection, we developed a latency clearance assay (LCA). The LCA is a quantitative viral outgrowth assay (QVOA) that includes the addition of immune effectors capable of clearing cells expressing viral antigen. Here we show a reduction in the recovery of replication-competent virus from VOR exposed resting CD4 T cells following addition of immune effectors for a discrete period.

Take home message: VOR exposure leads to sufficient production of viral protein on the cell surface, creating a window of vulnerability within this latent reservoir in antiretroviral therapy (ART)-suppressed HIV-infected individuals that allows the clearance of latently infected cells by an array of effector mechanisms.

Keywords: Eradication; HIV; Immune effector; Latency; Vorinostat.

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Figures

Fig. 1
Fig. 1
Latency clearance assay overview.
Fig. 2
Fig. 2
VOR induces antigen detectable via TCR:MHC interactions. Resting CD4 T cells are exposed to 6 h of 335 nM of VOR, and then the LCA is performed (A) with or without unmodified, autologous CD8 cells at an E:T ratio of 1:10 or 1:1, (B) with unmodified, autologous CD8 cells or ex- vivo expanded HIV specific T cells (HXTC) at an E:T ratio of 1:10, or (C) with or without effectors in the presence or absence of MHC-I blockade (C). Results are shown as % of plated wells HIV Gag p24 positive ((#positive wells × 100) / total #plated wells). *Indicates p < 0.01 by Kruskal-Wallis with Dunn's posttest correction for multiple comparisons. **Indicates p < 0.05 by Fishers exact test compared to cultures without CD8 cells.
Fig. 3
Fig. 3
HIV-specific responses of participants. PBMCs from 13 of the participants who had sufficient cells cryopreserved and stored for analysis from the same leukapheresis product used in the latency clearance assay were thawed and resting overnight. Cells were counted and plated in triplicate at 400 k cells/well on an IFNγ coated plate and stimulated with 2 μg/ml of the indicated Clade B consensus peptide pool (Gag, Pol, or Nef or media alone for the Mock control). After 16 h of culture, Elispot plates were washed and incubated with secondary anti-IFNγ antibody for 24 h, after which plates were developed and spots counted. Results are shown as SFU per million PBMCs. A threshold of > 4 × Mock and > 50 SFU/106 were considered positive (dotted line). Detectable but negative responses by this threshold are shaded in gray.
Fig. 4
Fig. 4
Addition of HIV DART molecules to CD8 T cells further reduces virus recovery from VOR exposed resting CD4 T cells. Resting CD4 T cells are exposed to 6 h of 335 nM of VOR then co-cultured with CD8 T cells at an E:T of 1:10, an E:T ratio selected to minimize CD8 T cell effect, and a cocktail of HIV DART molecules (a 1:1 mixture of 7B2xCD3 and A32xCD3) or a control DART molecule with an irrelevant antigen specific arm in place of the HIV-specific arm. CD8 T cells are depleted from co-culture in all systems after 24 h prior to addition of allogeneic target cells to amplify residual virus. Results are shown as % of plated wells p24 positive (#positive wells × 100) / (total #plated wells). *Indicates p < 0.01 by Friedman test for nonparametric paired samples with Dunn's posttest correction for multiple comparisons.
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