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

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

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

Predicted viral load decay for the quad and RAL-combination treatments using the best fit of the SRI model (Eq (1) when <i>δ</i>₂ = <i>δ</i>_<i>M</i>2) to the data.

<p>The estimated population parameters for each treatment group where used to plot the viral load decline under the effect of RAL+RTI (red) and quad therapy (blue). The dotted blue and red lines show the analytical approximation for the second phase of decay for quad therapy and RAL-combination therapy, respectively (see equation S.14 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006478#ppat.1006478.s001" target="_blank">S1 Text</a>). The shadowed section highlights phase 1b for RAL-combination therapy. The viral load at the start of phase 2 in patients under RAL-based therapy is reduced with respect to the corresponding level in RAL-free therapy by a factor of . We fixed the values of the following parameters (see text for details): <i>V</i>_<i>I</i>(0)/<i>V</i>(0) = 0.98, <i>δ</i>_<i>M</i>1 = 0.02 day^-1, <i>k</i> = 2.6 day^-1 and <i>c</i> = 23 day^-1 based on previous studies [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006478#ppat.1006478.ref014" target="_blank">14</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006478#ppat.1006478.ref016" target="_blank">16</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006478#ppat.1006478.ref039" target="_blank">39</a>]. In addition, for RAL combination we used <i>η</i> = 0.95, <i>ε</i> = 0 and <i>ω</i> = 0.94, and for the quad therapy <i>η</i> = <i>ε</i> = 0.95 and <i>ω</i> = 0 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006478#ppat.1006478.ref014" target="_blank">14</a>]. The estimated best-fit population parameters are (estimated standard deviation in parenthesis): <i>δ</i>₁ = 0.23 (0.04) day^-1, <i>k</i>₁ = 0.017 (0.01) day^-1, <i>δ</i>₂ = 0.85 (0.07) day^-1 and <i>V</i>(0) = 4.8 (0.07).</p

Comparison of the viral load decay rates during treatment with or without RAL.

<p>Rows represent each phase of decay and columns the estimates for each treatment using two methodologies: a heuristic multi-exponential model (two columns on the left) and the SRI model in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006478#ppat.1006478.g002" target="_blank">Fig 2B</a> and Eq (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006478#ppat.1006478.e008" target="_blank">1</a></b>) (two columns on the right). The estimation of the rate for phase 1b is only applicable in the case of treatment with RAL. All values are in units of day^-1. For the SRI model, we also indicate the parameter combination defining the decay rate of each phase.</p

Schematics of the models.

<p>(A) The standard model with pre- and post-integration phases of infection. We follow two types of target cells that after infection will be short-lived, , or long-lived, . Target cells, , are infected by infectious virus, <i>V_i</i>, at rate . The infection can be blocked by the activity of RTIs with effectiveness <i>η</i>. These infected cells, <i>I</i>₁, are lost at rate <i>δ</i>₁, or can undergo provirus integration at rate <i>k</i> and become productively infected cells <i>I</i>₂. InSTIs block integration with efficacy <i>ω</i>. Cells with integrated provirus, <i>I</i>₂, are lost at rate <i>δ</i>₂. Virions are produced by these cells at rate <i>p</i> per cell and are cleared from the circulation at rate <i>c</i> per virion. Protease inhibitors block the production of infectious virus <i>V</i>_<i>Ii</i>, and lead to production of non-infectious virus <i>V</i>_<i>Ini</i>, with efficacy <i>ε</i>. The subscripts <i>I</i> and <i>M</i> are used to distinguish virions produced by short-lived and long-lived infected cells, respectively. The dynamics of long-lived cells are similar, but possibly with different rates as indicated. (B) The slow and rapid integration (SRI) model. The SRI model proposes that both short-lived cells with fast integration (<i>I</i>₁) and long-lived cells with slow integration (<i>M</i>₁) generate productively infected cells that die quickly (<i>I</i>₂) (i.e. <i>δ</i>₂ = <i>δ</i>_{<b><i>M</i>2</b>}<b>)</b>.</p

Representative individual fits for the three datasets.

<p>Blue, red and green circles represent HIV RNA measurements for the quad-based-, RAL-combination- and RAL-mono therapy, respectively. Solid black lines represent best fits from the SRI model using the mixed-effects approach. Parameter estimates for each individual are presented in Table E in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006478#ppat.1006478.s001" target="_blank">S1 Text</a>.</p

Quantification of HIV-1 from plasma and PBMC pre- and post-chemotherapy.

<p><b>A</b>) Plasma HIV-1 RNA decreased for 5 patients, increased for 3 patients, and was below the limit of detection at both time points for 1 patient. Median viral load pre-chemotherapy was 1 copy/mL versus 4 copies/mL post-chemotherapy. Open symbols indicate undetectable samples. <b>B</b>) Total HIV-1 DNA levels per 10⁶ CD4+ cells decreased in 4 patients and increased in 5 patients. Median HIV-1 DNA levels per 10⁶ CD4+ cells decreased from 355 to 228 copies per 10⁶ CD4+ cells from pre- to post-chemotherapy. <b>C</b>) 2-LTRs were detectable pre-chemotherapy in 2 of 9 patients versus detectable in 3 of 9 post-chemotherapy. Patients with undetectable 2-LTRs at both time points are not shown. Open symbols indicate undetectable samples.</p