57704 reactivates latent HIV-1 via the PI3K/Akt signaling pathway.

<p>(A) Latent HIV-1 reactivation activity of prostratin in the absence (control) or presence of inhibitors of calcineurin (1 µM cyclosporin A), JNK (1 µM SP00125), PI3K (20 nM wortmannin), Akt (500 nM Akt Inhibitor IV) and PKC (500 nM Go6983). (B) Latent HIV-1 reactivation activity of 57704 in the absence (control) or presence of inhibitors of p38 MAPK (50 nM SB203580), calcineurin, JNK, PI3K, Akt, PKC and NF-κB (20 µM NF- κB activation inhibitor). (C) Western blot analysis of phosphorylated Akt in J89GFP treated with 5 µM 57704. (D) Latent HIV-1 reactivation activity of 57704 in the absence (control) or presence of a pan-inhibitor of PI3K (15 µM LY294002) or inhibitors targeting the p110α (2 µM PI-103), p110δ (5 µM IC87114), p110γ (2 µM AS-605240) or p110β (2 µM TGX-221) isoforms. (E) PI3K isoform transcript levels in cells treated with 5 µM 57704 or 5 µM 57704 SAHA.(F) Inhibition of the PI3K p110α, β, δ and γ isoforms by 100 nM wortmannin alone or in combination with 5 µM 57704 or 5 µM 57704 SAHA.</p

Cytotoxicity of 57704 and SAHA in different cells.

1<p>Data represent the mean ± standard deviation from 3 replicate experiments.</p>2<p>Data represent the mean from 2 independent replicate experiments.</p

Latent HIV-1 reactivation activity of 57704 and T cell activation in CD8+-depleted MNC.

<p>(A) Quantitation of cell-free HIV-1 RNA in the culture supernatants of CD8+-depleted blood MNC incubated with 57704 or SAHA. (B) Surface expression of CD69, CD38 and HLA-DR after 24 h in purified T cells following exposure to 10 µg/mL PHA, 57704 or SAHA. (C) Surface expression of CD69, CD38 and HLA-DR after 5 days in purified T cells following exposure to 10 µg/mL PHA, 57704 or SAHA.</p

Latent HIV-1 reactivation activity of 57704, alone and in combination with other inducing agents, in the U1 and ACH2 cell line models of virus latency.

<p>HIV-1 expression was assessed by quantitative PCR analysis of early and late viral RNA transcripts. Where appropriate, TNFα was used as an activation control. (A) Latent HIV-1 reactivation of activity 57704 in the J89GFP cell line. An EC₅₀ of 10.3±1.2 µM was determined for 57704 in this cell line. (B) Latent HIV-1 reactivation activity of 57704 in the ACH2 cell line. (C) Latent HIV-1 reactivation of activity 57704 in the U1 cell line. (D) Graph of activation of latent HIV-1 early and late gene transcript expression in the ACH2 and U1 cell lines. EC₅₀ values were determined in Sigma Plot by nonlinear regression, as described previously. (E) Latent HIV-1 reactivation of 5 µM 57704 in combination with 1 µM prostratin in the U1 cell line. (F) Latent HIV-1 reactivation of 5 µM 57704 in combination with 5 µM SAHA in the U1 cell line. (G) Latent HIV-1 reactivation of 5 µM 57704 in combination with 2.5 µM DSF in the U1 cell line. (H) Latent HIV-1 reactivation of 5 µM 57704 in combination with 20 µM BIX-01294 in the U1 cell line. Data represent the mean ± standard deviation from 3 replicate experiments.</p

In vitro activity of 57704 and SAHA against class I HDAC isoforms.

<p>Data represent the mean ± standard deviation from 3 replicate experiments.</p

Cell number changes during cell culture.

Cell numbers at the conclusion of each week are normalized relative to the total number of cells that were seeded in the flask at the beginning of each week.</p

Characteristics of study subjects.

<p>Characteristics of study subjects.</p

<i>Ex vivo</i> activation of CD4⁺ T-cells from donors on suppressive ART can lead to sustained production of infectious HIV-1 from a subset of infected cells

<div><p>The fate of HIV-infected cells after reversal of proviral latency is not well characterized. Simonetti, <i>et al</i>. recently showed that CD4⁺ T-cells containing intact proviruses can clonally expand <i>in vivo</i> and produce low-level infectious viremia. We hypothesized that reversal of HIV latency by activation of CD4⁺ T-cells can lead to the expansion of a subset of virus-producing cells rather than their elimination. We established an <i>ex vivo</i> cell culture system involving stimulation of CD4⁺ T-cells from donors on suppressive antiretroviral therapy (ART) with PMA/ionomycin (day 1–7), followed by rest (day 7–21), and then repeat stimulation (day 21–28), always in the presence of high concentrations of raltegravir and efavirenz to effectively block new cycles of viral replication. HIV DNA and virion RNA in the supernatant were quantified by qPCR. Single genome sequencing (SGS) of p6-PR-RT was performed to genetically characterize proviruses and virion-associated genomic RNA. The replication-competence of the virions produced was determined by the viral outgrowth assay (VOA) and SGS of co-culture supernatants from multiple time points. Experiments were performed with purified CD4⁺ T-cells from five consecutively recruited donors who had been on suppressive ART for > 2 years. In all experiments, HIV RNA levels in supernatant increased following initial stimulation, decreased or remained stable during the rest period, and increased again with repeat stimulation. HIV DNA levels did not show a consistent pattern of change. SGS of proviruses revealed diverse outcomes of infected cell populations, ranging from their apparent elimination to persistence and expansion. Importantly, a subset of infected cells expanded and produced infectious virus continuously after stimulation. These findings underscore the complexity of eliminating reservoirs of HIV-infected cells and highlight the need for new strategies to kill HIV-infected cells before they can proliferate.</p></div