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Antiretroviral drugs do not interfere with bryostatin-mediated HIV-1 latency reversal
Antiviral Research 123 (2015) 163–171
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Antiretroviral drugs do not interfere with bryostatin-mediated HIV-1 latency reversal Marta Martínez-Bonet a, Maria Isabel Clemente a,b, Susana Álvarez a, Laura Díaz a,c, Dolores García-Alonso a, Eduardo Muñoz d, Santiago Moreno e, Maria Ángeles Muñoz-Fernández a,⇑ a Laboratorio de InmunoBiología Molecular, Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón (IiSGM), and Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), C/ Dr. Esquerdo 46, 28007 Madrid, Spain b Unidad de cultivos celulares, Instituto de Investigación Sanitaria Gregorio Marañón (IiSGM), C/ Dr. Esquerdo 46, 28007 Madrid, Spain c Unidad de Citometria y Sorter, Instituto de Investigación Sanitaria Gregorio Marañón (IiSGM), C/ Dr. Esquerdo 46, 28007 Madrid, Spain d Departamento de Inmunología, Facultad de Medicina, Universidad de Córdoba, Avda. Menéndez Pidal, s/n, 14004 Córdoba, Spain e Servicio de Enfermedades Infecciosas, Hospital Universitario Ramón y Cajal and IRYCIS, Ctra. de Colmenar Viejo, km. 9,100, 28034 Madrid, Spain
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Article history: Received 25 May 2015 Revised 25 September 2015 Accepted 28 September 2015 Available online 30 September 2015 Keywords: Bryostatin-1 Maraviroc Atripla HIV-reactivation HIV-latency
a b s t r a c t Although an effective combination of antiretroviral therapy (cART) controls HIV-1 viraemia in infected patients, viral latency established soon after infection hinders HIV-1 eradication. It has been shown that bryostatin-1 (BRY) inhibits HIV-infection in vitro and reactivates the latent virus through the protein kinase C-NF-jB pathway. We determined the in vitro potential effect of BRY in combination with currently used antiretroviral drugs. BRY alone or in combination with maraviroc (MVC)/Atripla (ATP) was tested for its capacity to reactivate latent virus and inhibit new infections. JLTRG-R5 cells and two latent HIV-1-infected cell lines, J89GFP and THP89GFP, were used as latency models. To quantify HIV infection, the reporter cell line TZM-bl was used. We found that BRY reactivates HIV-1 even in combination with MVC or ATP. Antiretroviral combinations with BRY do not interfere with BRY activity (i.e., the reactivation of latently infected cells) or with the antiviral activity of antiretroviral drugs. In addition, BRY-mediated down-modulation of surface CD4 and CXCR4 was not affected when it was used in combination with other antiretrovirals, and no hyperactivation or high-proliferation effects were observed in primary T cells. Moreover, the BRY treatment was able to reactivate HIV-1 in CD4+ T cells from HIV-1-infected patients under cART. Thus, we propose the use of BRY to purge the viral reservoir and recommend its combination with current antiretroviral treatments. Ó 2015 Published by Elsevier B.V.
1. Introduction Abbreviations: AIDS, acquired immunodeficiency syndrome; ATP, AtriplaÒ; BRY, bryostatin-1; cART, combination of antiretroviral therapy; DMSO, dimethyl sulfoxide; EFV, efavirenz; EGFP, enhanced green fluorescent protein; ELISA, enzymelinked immunosorbent assay; FBS, fetal bovine serum; FDA, food and drug administration; FTC, emtricitabine; GFP, green fluorescent protein; HDACi, histone deacetylase inhibitors; HIV-1, human immunodeficiency virus-1; IL-2, interleukin2; iMFI, integrated mean fluorescence intensity; LRAs, latency reversal agents; LTR, long terminal repeat; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MVC, maraviroc; PBMCs, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PHA, phytohemagglutinin; PKC, protein kinase C; SEM, standard error of the mean; TFV DF, tenofovir disoproxil fumarate; TNF, tumor necrosis factor. ⇑ Corresponding author. E-mail addresses: [email protected] (M. Martínez-Bonet), maribel. [email protected] (M.I. Clemente), [email protected] (S. Álvarez), [email protected] (L. Díaz), [email protected] (D. García-Alonso), [email protected] (E. Muñoz), [email protected] (S. Moreno), [email protected], [email protected] (M.Á. MuñozFernández). http://dx.doi.org/10.1016/j.antiviral.2015.09.014 0166-3542/Ó 2015 Published by Elsevier B.V.
An effective combination of antiretroviral therapy (cART) successfully controls HIV-1 viraemia in most HIV-1 infected patients. Although an undetectable viral load is achieved in most treated patients, HIV-1 establishes a long-term infection in a small pool of memory CD4+ T cells, which contains an integrated but transcriptionally silent provirus (Alexaki et al., 2008; Chun et al., 1997; Shen and Siliciano, 2008). Moreover, cells belonging to the monocyte/macrophage lineage represent one of the persistent major reservoirs of the virus because they maintain a low level of HIV-1 replication (Kumar et al., 2014). The origin and clinical implications of persistent low levels of viraemia are uncertain. Some studies postulate that it might be the result of the release of virus from latently infected cells (Joos et al., 2008; Kieffer et al., 2004), whereas other studies suggest that it could arise from ongoing viral replication (Chun et al., 2010, 2005; Havlir et al., 2003; Sharkey et al., 2000).
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Recently, several strategies have been developed to purge HIV-1 reservoirs, and the use of therapies with small molecules targeting HIV-1 reservoirs is one of the major challenges in the fight against AIDS. Clinical trials with cART intensification have failed to eliminate viral replication, which further supports the hypothesis that low levels of viraemia are produced by latently infected cells (Buzon et al., 2010; Gandhi et al., 2010; Gutierrez et al., 2011; McMahon et al., 2010; Vallejo et al., 2012). Therefore, it might be possible to reactivate the latent virus with a therapeutic approach known as ‘‘shock and kill” (Katlama et al., 2013). A wide range of small molecules, such as disulfiram (Xing et al., 2011), HDAC inhibitors (HDACi); (Archin et al., 2012; Shirakawa et al., 2013; Siliciano et al., 2007) and protein kinase C (PKC) activators such as ingenols (Warrilow et al., 2006), prostratin (Kulkosky et al., 2001), 1,2-diacylglycerol analogs (Hamer et al., 2003), and bryostatin-1 (BRY); (del Real et al., 2004; Mehla et al., 2010), have been proposed as agents to reactivate HIV-1 and eradicate the pool of latently HIV-infected CD4+ T cells (Kulkosky and Bray, 2006). Several clinical trials are currently ongoing to evaluate the effectiveness of these compounds to hit HIV-1 reservoirs. The results of a clinical trial with disulfiram did not show a reduction in the size of the latent reservoir (Spivak et al., 2014). However, an ex vivo approach that evaluated the effectiveness of potential candidates, including BRY and HDACi, revealed that BRY was the best candidate to reactivate HIV-1 from latency (Bullen et al., 2014). Therefore, PKC activators, alone or in combination with HDACi, are of particular relevance for purging HIV-1 reservoirs in patients. For instance, the synergistic effect of BRY with HDACi to antagonize the HIV-1 latency has been shown in vitro (Perez et al., 2010). Before clinical trials can be conducted on ART intensification with BRY to assess its impact on the size of the HIV-1 latent reservoirs, the potential effect of BRY in combination with antiretrovirals should be determined in vitro. In this study, maraviroc (MVC) as a monotherapy regimen and a fixed-dose combination of emtricitabine, tenofovir and efavirenz (AtriplaÒ, ATP) as a full cART regimen were tested in combination with BRY at two levels. Using different in vitro models of latency and an ex vivo model, we demonstrated the viral reactivation from latent reservoirs and the inhibition of acute infection.
anti-human HLA-DR-ECD were obtained from BD Biosciences Pharmingen (San Diego, CA, USA). BRY was obtained from Sigma–Aldrich (St. Louis, MO, USA), maraviroc (MVC) was obtained as a clinical formulation in a 150 mg tablet (Selzentry, Pfizer Labs, UK), and AtriplaÒ (ATP) was obtained as a clinical formulation in a 1100-mg tablet (600 mg EFV, 200 mg FTC, 300 mg TFV DF; Gilead Sciences, CA, USA). MVC and ATP were freshly dissolved in distilled water with DMSO (Sigma–Aldrich, St. Louis, MO, USA) and sterilefiltered. ATP is not a chemical entity per se, so the theoretical molecular weight of 1198.44 g/mol was calculated based on the molecular weight of each component and the relative proportion of the single drugs. Hence, 1 mM of ATP corresponds to 2.07 mM of EFV, 0.87 mM of FTC, and 0.49 mM of TFV DF. The concentration of DMSO in cell cultures was less than 0.001%. TNF-a was purchased from R&D Systems (Minneapolis, Minn.).
2. Materials and methods
JLTRG-R5 cells were stimulated with the indicated compounds, and a GFP-fluorescence pattern was determined 24 h later. The percentage of GFP expressing cells was used as a measure of HIV-1 LTR activation.
2.1. Cell lines and culture JLTRG-R5 (Division of AIDS, NIAID, NIH. Catalog number 11586; Dr. Olaf Kutsch), J89GFP and THP89GFP cells (kindly donated by Dr. David N Levy, NYU, USA) were maintained according to the protocol described by Kutsch et al. (2002). ACH-2 and J1.1 cells were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Dr. Thomas Folks (Clouse et al., 1989; Folks et al., 1989; Perez et al., 1991). Buffy coats from healthy subjects were obtained from the Madrid Transfusion Center. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized venous blood by a ficoll-paque density gradient (Garcia-Merino et al., 2009) and maintained in complete RPMI supplemented with 30 U/ml IL-2 and, when indicated, activated for 3 days with 1 lg/ml phytohemagglutinin (PHA; Murex Biotech, England, UK). TZM-bl (Division of AIDS, NIAID, NIH, Catalog number 8129; Dr. John C. Kappes) and HEK 293T cells (ATCC number CRL-11268, Rockefeller University, USA) were cultured according to the manufacturers’ instructions. 2.2. Reagents Anti-human CD3-PC5, anti-human CD4-PC7, anti-human CXCR4-APC, anti-human CCR5-PE, anti-human CD38-FITC and
2.3. Cell viability assays The concentration range of each compound examined in this study is in agreement with previously published results (Arberas et al., 2013; Bousquet et al., 2009; Perez et al., 2010). The toxicity of compounds was measured by an MTT assay (Sigma, St. Louis, MO, USA) according to the manufacturer’s instructions. 0.001% DMSO treated cells were included in each experiment as vehicle control (DMSO+); DMSO 10% (DMSO+++) was used as positive control of cytotoxicity. 2.4. Analysis of surface marker expression Cells were stained for 1 h at 4 °C with the corresponding conjugated antibodies in a FACS staining buffer (phosphate-buffered saline, PBS, with 2% FBS) and analyzed in a Gallios flow-cytometer (Beckman-Coulter, CA, USA). At least 20,000 CD3+ cells, 50,000 JLTRG-R5 or 20,000 J89GFP and THP89GFP cells were collected for each sample and analyzed with the Kaluza software (Beckman-Coulter, CA, USA). 2.5. HIV-1 LTR reactivation
2.6. Latent HIV-1 reactivation To determine the viral reactivation, the EGFP-fluorescence pattern was measured by flow cytometry and HIVp24Gag release measured by ELISA (INNOTESTÒ HIV-Antigen mAb, Innogenetics, Belgium). 2.7. Latent HIV-1 reactivation in CD4+ T cells from infected individuals under cART Fresh blood from HIV-1-infected individuals (n = 3) was obtained in accordance with protocols approved by the Hospital Ramón y Cajal Ethical Committee (Clinical characteristics depicted in Table S1). The participants signed an informed consent form. CD4+ T cells were isolated from PBMCs using the CD4+ T Cell Isolation Kit II (Miltenyi Biotec, Bergisch-Gladbach, Germany) and cultured under different conditions. To measure the HIV-RNA levels, the supernatants were analyzed by a robotic COBAS AmpliPrep/TaqMan system (Roche Diagnostics, Indianapolis, IN, USA). The limit of detection in this assay was 50 copies/ml.
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Fig. 1. HIV-1 reactivation. JLTRG-R5 cells were treated during 24 h with BRY (1, 10, 50, 100 nM), MVC (1, 10, 50, 100 lM) and ATP (0.3, 3, 15, 30 lM) alone (white-filled symbols) or in combination (black-filled symbols) and analyzed by flow cytometry. The results represent the integrated GFP mean fluorescence intensity (iMFI) (A). J89GFP and THP89GFP cells were stimulated with BRY (100 nM), TNF-a (3 ng/ml), 48 h later, the EGFP expression was measured by flow cytometry (B) and visualized by fluorescence microscopy (C). Histograms compare the EGFP expression in non-stimulated (black lines) and BRY treated cells (red lines) or TNF-a treated cells (blue lines). Arrows indicate syncytium formation in cell cultures. The results are representative of four independent experiments. (D) Viral reactivation in J89GFP and THP89GFP cells treated for 2 or 4 days (white and black bars, respectively) with BRY (100 nM), MVC (100 lM) and ATP (30 lM) alone or in combination was measured as EGFP expression by flow cytometry (upper panels) or p24Gag release by HIVp24Gag ELISA kit in supernatants in supernatant (lower panels). TNF-a (3 ng/ml) was used as positive control of HIV-1 reactivation. Bars represent the percentage of EGFP positive cells. The results represent the arithmetic mean ± SEM (A) or +SEM (D) of at least three independent experiments. Statistics were performed between control and singletreated cells (left panels), between BRY-treated and BRY combinations-treated cells (right panels) (A) or between control and treated cells (D). We used three significance levels: * p < 0.05; **p < 0.01; ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2.8. TZM-bl cells infection assays
2.9. Virus production
After different treatment/infection assays, TZM-bl cells were lysed using a Glo-Lysis buffer (Promega, Madison, WI, USA), and luciferase assays were performed according to the manufacturer’s instructions. Luminescence was measured using a VICTOR luminometer. All the experiments were performed three times.
X4-HIV-1NL4-3, R5-HIV-1NL(AD8) and X4/R5-HIV-189.6 viruses were obtained by transient transfection of pNL4-3, pNL(AD8) and p89.6 plasmids, respectively (NIH AIDS Research and Reference Reagent Program) in HEK 293T cells (ATCC, Manassas, VA, USA). Viral stocks were clarified by centrifugation prior to the
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evaluation of the viral titer by ELISA (Sepulveda-Crespo et al., 2014).
3. Results 3.1. Effects of bryostatin-1 combined with maraviroc and AtriplaÒ on HIV-1 LTR activation
2.10. Cell proliferation assays The proliferation of cells was examined using the BrdU Cell Proliferation Kit (Chemicon, Millipore, MA, USA) according to the manufacturer’s instructions.
2.11. Photomicroscopy Cells were photographed using a DMI 3000B Leica microscope. Images are shown at 10 magnification.
2.12. Statistics A statistical analysis was performed using SPSS software version 15.0 for Windows. Differences between the two groups (control versus different dosages of compounds or BRY-treated versus ART combined with BRY treatment) were assessed using a paired t-test using three significance levels (p < 0.05, p < 0.01 and p < 0.001).
To investigate the efficacy of BRY in combination with cART, JLTRG-R5 cells were treated for 24 h with the maximum safety concentration range of BRY (1–100 nM), MVC (1–100 lM) and/or ATP (0.3–30 lM; Fig. S1A). BRY antagonized HIV-1 latency very efficiently at concentrations of up to 50 nM (Fig. 1A; left panel). The combination of BRY with both antiretrovirals tested did not diminish the effects of BRY on LTR activation (Fig. 1A; right panel). The triple combination of BRY-MVC-ATP at the highest concentration appeared to increase the levels of GFP iMFI (MFI per percentage of positive cells) by approximately 50% compared with the BRY-treatment alone. The surface expression of CD4 and CXCR4 were analyzed 24 h post-treatment with BRY alone or in combination with antiretrovirals, and a significant decrease in both expressions was observed, even at low doses of BRY, compared with the control of JLTRG-R5 cells (Fig. S2A and B). Both MVC and BRY-treated cells showed an increase in the CCR5 surface expression (Fig. S2C; left panel). BRY-ATP-treated cells presented a CCR5 expression profile similar to BRY-treated cells, whereas the BRY-MVC treatment and the triple combination of BRY-MVC-ATP resulted in an increase of
Fig. 2. HIV-1 infection inhibition. Reporter TZM-bl cells were pretreated for 1 h with BRY (10, 50, 100 nM), MVC (10, 50, 100 lM) and ATP (3, 15, 30 lM) alone or in combination and infected with (A) X4-HIV-1NL4.3, (B) R5-HIV-1NL(AD8) or (C) X4/R5-HIV-189.6 (40, 20 or 40 ng HIVp24Gag/106 cells, respectively). 3 h after infection, virus was removed and cells were treated again with the compounds at the same concentration range. Luciferase activity was quantified 48 h post-infection. The results are expressed as the percentage of infectivity relative to that of the control (Ct) infected cells (black bar) normalized to 100%. White bar indicates the background luminescence values of non-infected cells. Values are the means + SEM of three independent infections, each measured in duplicate. Statistics were performed between Ct infected cells and treated infected cells (grey bars). We used two significance levels: *p < 0.05; **p < 0.001.
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approximately 2- to 3-fold, respectively, when compared with BRY-treated cells (Fig. S2C; right panel). 3.2. Virus reactivation in HIV-1 latently infected lymphocytic and monocytic cells HIV-latent reservoirs are established in T-cells and monocyte/ macrophages in the body, and these cells harbor the infection for the entire life of the individual. Therefore, we investigated whether BRY could reactivate latent HIV-infection in both T-cells and monocytic cells. After a treatment with BRY, latently infected J89GFP and THP89GFP cells were reactivated with evidence of EGFP expression within 48 h compared with the negative control (Fig. 1B and C). TNF-a was used as a positive control in this experiment leading to the formation of syncytia in J89GFP cells (Fig. 1B and C). The effect of BRY, measured by EGFP expression (Fig. 1D, upper panels) or HIVp24Gag release on cell supernatants (Fig. 1D, lower panels), was also observed even combined with MVC or ATP at days 2 and 4 after the treatment. 3.3. The combination of bryostatin-1 with maraviroc and AtriplaÒ does not modify the antiviral effects of the drugs To determine whether BRY interfered with the effects of the antiretrovirals, several drug combinations were tested against X4-HIV-1NL4.3 (Fig. 2A), R5-HIV-1NL(AD8) (Fig. 2B) or X4/R5-HIV189.6 infection in the reporter TZM-bl cells (Fig. 2C). TZM-bl viability was assessed after 72 h of treatment (Fig. S1B). As expected, MVC completely inhibited the R5 virus infection and partially inhibited (approximately 40%) the infectivity of the dual strain. In contrast, ATP decreased the luciferase values to similar background levels, regardless of the HIV-1 tropism. Because BRY is able to down-modulate CD4, the infection with the tested virus was reduced approximately 50% when compared with non-treated and HIV-infected control cells. Neither MVC nor ATP lost their antiviral activity when combined with BRY. Moreover, the triple combination of drugs showed the maximum HIV-inhibition rate for all HIV-1 strains. 3.4. Viral reactivation and inhibition of reactivated virus THP89GFP cells reached almost complete reactivation levels when treated with BRY; thus, we investigated whether these cells produced infectious viral particles after BRY stimulation by simultaneously analyzing the viral reactivation and the inhibition of reactivated virus resulting from the drug-combination. Towards this goal, we developed a co-culture model of BRY-treated THP89GFP cells (Fig. 3A) or their supernatants (Fig. 3B) with TZM-bl cells pre-treated with MVC (100 lM) or/and ATP (30 lM). Both conditions significantly increased the luciferase expression versus the non-treated TZM-bl cells. When the reporter cells were pre-treated with MVC or ATP, new infections of reactivated X4/R5HIV-189.6 were inhibited 50% or 80%, respectively. The partial infection inhibition with MVC is likely because 89.6 is a dual-tropic strain (Fig. 3). 3.5. Effects of bryostatin-1 combined with maraviroc and AtriplaÒ on T cell phenotype We studied how BRY and antiretroviral combinations may affect the T-cell phenotype and activation. Flow cytometry assays were performed on non-stimulated or PHA-stimulated PBMCs from healthy subjects after exposure to the highest non-toxic concentration of each compound separately or in combination (Fig. S1C). Both non-activated and activated cells showed a significant decrease in surface CD4 expression (Fig. 4A) when treated
Fig. 3. Viral reactivation and inhibition of new infections with reactivated HIV-1. THP89GFP cells were stimulated with 100 nM BRY; 24 h later, washed cells (A) or their supernatants (B) were added to MVC (100 lM) or/and ATP (30 lM) pretreated TZM-bl cells. Luciferase activity was assessed 48 h post-infection. The results are expressed as the percentage of infectivity relative to that of the BRYtreated THP89GFP cells in co-culture with non-treated TZM-bl cells (black bar) normalized to 100%. The white bar indicates the background luminescence values of non-treated THP89GFP cells in co-culture with non-treated TZM-bl cells. Values are the means + SEM of three independent infections. We used three significance levels: *p < 0.05; **p < 0.01; ***p < 0.001.
with BRY, BRY-ATP or BRY-MVC-ATP. Moreover, ATP-treated, non-activated cells decreased their CD4 expression up to 25%. Only the BRY treatment caused a significant decrease in the CXCR4 expression (Fig. 4B). However, its combination with antiretrovirals also negatively modulated the CXCR4 expression on both PBMCs and CD4+ T cell surfaces. As described by Arberas et al. (2013), MVC, alone or in combination, increased the CCR5 surface expression on activated cells (Fig. 4C). The effects on T-cell activation after in vitro BRY-MVC-ATP exposure were analyzed by measuring the MFI of CD38 and HLA-DR activation markers on PBMCs and CD4+ T lymphocytes (Fig. 4D and E). The BRY treatment led to an induction of CD38 and HLA-DR expression in non-stimulated T cells, but not in PHA-stimulated cells. However, ATP appeared to slightly affect the expression profile of both markers in resting PBMCs but not in CD4+ T cells. No proliferation was observed in CD4+ T cells after 24 h of the BRY treatment, but it was detected after 6 days of treatment (Fig. S3). Our results indicate that BRY, alone or in combination with MVC or ATP, only induces a low level of activation in human primary T cells.
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Fig. 4. Effects on T cell phenotype. Isolated PBMCs from healthy subjects were treated either with BRY (50 nM), MVC (100 lM) and ATP (30 lM) alone or in combination for 24 h. The expression of cell surface receptors CD4, CXCR4, CCR5, CD38 and HLA-DR was analyzed by flow cytometry in resting (white bars) or PHA activated (black bars); PBMCs (left panels) and CD4+ T cells (CD3+CD4+, right panels). The results represent the percentage of MFI relative to that of control cells (Ct) normalized to 100% of cell surface receptors CD4 (A), CXCR4 (B) CCR5 (C) CD38 (D) and HLA-DR (E). The results represent the arithmetic mean + SEM of at least three independent experiments. Statistics were performed between control and treated cells. We used two significance levels: *p < 0.05; **p < 0.001.
3.6. HIV-1 induction in CD4+ T cells from infected individuals under cART Our next goal was to study the effects of BRY on CD4+ T cells isolated from three HIV-1 infected individuals on suppressive cART. Purified CD4+ T cells were cultivated in the presence of ATP and treated or not treated with BRY (50 nM). After 2 days, the levels of HIV-1-virion-associated RNA were evaluated. BRY was exceptionally potent, inducing high levels of HIV-1 RNA from two of the three patients (Fig. 5A). When the experiment was continued up to day 6 for the third patient, we observed that the levels of HIV-1 RNA in the supernatant of BRY-treated cultures increased
3-fold when compared with non-treated cultures (Fig. 5B). This finding suggests that the activity of BRY observed in immortal cell line models of HIV-1 latency may translate to potent viral reactivation from latently infected primary cells from HIV-1 patients. 4. Discussion The persistent low levels of HIV-1 viraemia, the limitations of current ARTs, and the lack of a valid anti-HIV-1 vaccine candidate highlight the need for new therapies to eradicate HIV-1. Nevertheless, the difficulty to obtain primary cells latently infected with HIV makes it an almost impossible challenge. Hence, in this study, we
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Fig. 5. Viral induction from human CD4+ T cells from fully suppressed HIV-1 positive donors. CD4+ T cells were purified from PBMCs of three HIV-1 infected patients under suppressive cART. After 2 (A) or 6 (B) days of treatment with BRY at 50 nM, HIV-1 RNA was quantified in the cell supernatants using the COBAS AmpliPrep/TaqMan HIV-1 test. Non-treated BRY cells, BRY, white bars; BRY treated cells, +BRY, black bars.
employed JLTRG-R5, J89GFP and THP89GFP cells as a postintegration HIV latency model to explore the impact of MVC and/ or ATP in combination with BRY. Although J89GFP and THP89GFP cell line models are not frequently used to assess latency reversal agents (LRA) (Butera et al., 1991; Duh et al., 1989; Folks et al., 1987, 1988; Jordan et al., 2003, 2001; Perez et al., 1991), their exclusive characteristics make them an experimentally tractable and relevant model to study postintegration HIV latency and reactivation (Kutsch et al., 2002). Nevertheless, to confirm the results obtained in these cell lines, we have performed the same reactivation experiments in another two other well-established latency-models, such as ACH-2 and J1.1 cell lines. We assessed the cell toxicity and the HIV-1 reactivation and the obtained data were in concordance to that exposed in the article (Fig. S4). Numerous researchers have studied the effects of BRY on HIV-1 latency and its effects in CD4 and CXCR4 down-modulation (Mehla et al., 2010; Perez et al., 2010). To simultaneously analyze the viral reactivation and the inhibition of reactivated virus caused by the drug combination, we performed co-culture experiments with THP89GFP cells and TZM-bl cells. Moreover, we also evaluated the cellular activation and proliferation as well as the CD4, CXCR4, and CCR5 surface expression levels in human primary T cells. BRY is attractive because it is an FDA-approved drug, and it has already been tested in human trials for cancer and Alzheimer’s disease. Consequently, data on the pharmacokinetics and toxicity of BRY in humans are available (Irie et al., 2012). Phase II clinical trials have reported that 2 lg/ml of BRY in blood (20-fold the concentration of this study) was reasonably well tolerated and have demonstrated that lower doses of BRY (50 nM) are able to reactivate latent HIV-1 (Madhusudan et al., 2003). BRY has been remarked as the best effective single agent that disrupts the latent reservoir between a battery of LRAs (Bullen et al., 2014; Spina et al., 2013). On the other hand, BRY notably induced activation in nonstimulated T cells and cell proliferation after 6 days of treatment. Although these effects are considerably lower than that observed in PHA-stimulated cells, the in vivo consequences of this cellular activation could be responsible for inflammatory responses. In order to avoid this, further studies should be done with BRY pulses instead of continuous treatment, to achieve minimal cellular activation, as observed by Wei et al. (2014) in primary cells after 4-h pulse of RMD. Another approach could be the combination of different LRAs in order to test whether lower doses of these drugs could induce similar levels of latent HIV stimulation with lower levels of T cell activation. In addition to PKC agonists, other LRAs, such as HCACi, have been proposed as candidates for the so-called ‘‘shock and kill”
strategy to purge HIV-1 reservoirs. Due to the molecular complexity of the viral latency, it is likely that the synergistic activity of drugs targeting different pathways will be needed to effectively reactivate HIV-1 from latency in different reservoirs. Indeed, synergy in latency reversion between prostratin (Reuse et al., 2009) or BRY (Perez et al., 2010) and classical HDACi has been previously described. Moreover, our group has observed a synergistic profile in HIV-1 reactivation when BRY was combined with novel promising HDACi, such as panobinostat and romidepsin. Our results show a slight but not statistically significant synergy between BRY, MVC and ATP in HIV-1 reactivation (Fig. 1). In agreement with these results, we have previously reported that MVC can activate NF-jB and subsequently reverse latent HIV-1 in resting CD4+ T-cells from ART suppressed HIV-1-infected patients (Madrid-Elena et al., 2014). This finding could be of clinical relevance because it is thought that a reactivation therapy will be achieved by alternative cycles of immunoactivation therapy plus ART intensification followed by standard cycles of cART. Thus, adding MVC in the ART intensification cocktail could enhance the anti-latency activity of BRY-1 even at lower doses of this drug. Despite its limitation, our model allowed us to assess in a single system both the viral reactivation and the inhibition of new infections, which provides important information. This model appears to be an accurate reporter model for assessing the infectivity of reactivated virus. Likewise, BRY significantly reactivated latent virus from CD4+ T cells isolated from HIV-infected individuals, and this reactivation was even stronger than the one obtained with prostratin or its analogs (Beans et al., 2013) in similar studies. This work demonstrated that the combination of ART with BRY does not interfere with BRY activity or the antiretroviral activity. Our in vitro findings could be corroborated in vivo in the NCT02269605 clinical trial. This trial is a Phase I trial designed to evaluate two different doses of BRY on HIV-1 latency and reservoir in HIV-1 infected patients receiving antiretroviral treatment. Overall, these data support the notion that reactivation conducted under the cover of cART could lead to the selective killing of latently infected cells and the ‘‘purging” of the latent viral reservoir.
Conflicts of interest The authors declare that there are no conflicts of interest.
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Acknowledgments We thank the Center of Transfusion of Madrid for the buffy coats and the Spanish HIV HGM BioBank, which is supported by the Instituto de Salud Carlos III (ISCIII), and is funded by the RD12/0017/00XX project as part of the Plan Nacional R+D+I and cofounded by the ISCIII-Subdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (FEDER). We are grateful to Dr. Rafael Samaniego of the Instituto de Investigación Biomédica Gragorio Marañón Microscopy Unit for his technical assistance. This work was supported by the grant number RD12/0017/0037 as part of the Plan Nacional R+D+I and co-financed by the ISCIIISubdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (FEDER), PT13/0010/0028, Fondo Investigación Sanitaria (grant number PI13/02016), ‘‘Fundación para la Investigación y la Prevención del Sida en España” (FIPSE), Comunidad de Madrid (grant number S-2010/BMD-2332), CYTED 214RT0482. CIBER-BBN is an initiative funded by the VI National R&D&I Plan 2008–2011, Iniciativa Ingenio 2010, the Consolider Program, and CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. MMB was supported by FI10/00141 (ISCIII).
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Antiretroviral drugs do not interfere with bryostatin-mediated HIV-1 latency reversal Marta Martínez-Bonet a, Maria Isabel Clemente a,b, Susana Álvarez a, Laura Díaz a,c, Dolores García-Alonso a, Eduardo Muñoz d, Santiago Moreno e, Maria Ángeles Muñoz-Fernández a,⇑ a Laboratorio de InmunoBiología Molecular, Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón (IiSGM), and Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), C/ Dr. Esquerdo 46, 28007 Madrid, Spain b Unidad de cultivos celulares, Instituto de Investigación Sanitaria Gregorio Marañón (IiSGM), C/ Dr. Esquerdo 46, 28007 Madrid, Spain c Unidad de Citometria y Sorter, Instituto de Investigación Sanitaria Gregorio Marañón (IiSGM), C/ Dr. Esquerdo 46, 28007 Madrid, Spain d Departamento de Inmunología, Facultad de Medicina, Universidad de Córdoba, Avda. Menéndez Pidal, s/n, 14004 Córdoba, Spain e Servicio de Enfermedades Infecciosas, Hospital Universitario Ramón y Cajal and IRYCIS, Ctra. de Colmenar Viejo, km. 9,100, 28034 Madrid, Spain
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Article history: Received 25 May 2015 Revised 25 September 2015 Accepted 28 September 2015 Available online 30 September 2015 Keywords: Bryostatin-1 Maraviroc Atripla HIV-reactivation HIV-latency
a b s t r a c t Although an effective combination of antiretroviral therapy (cART) controls HIV-1 viraemia in infected patients, viral latency established soon after infection hinders HIV-1 eradication. It has been shown that bryostatin-1 (BRY) inhibits HIV-infection in vitro and reactivates the latent virus through the protein kinase C-NF-jB pathway. We determined the in vitro potential effect of BRY in combination with currently used antiretroviral drugs. BRY alone or in combination with maraviroc (MVC)/Atripla (ATP) was tested for its capacity to reactivate latent virus and inhibit new infections. JLTRG-R5 cells and two latent HIV-1-infected cell lines, J89GFP and THP89GFP, were used as latency models. To quantify HIV infection, the reporter cell line TZM-bl was used. We found that BRY reactivates HIV-1 even in combination with MVC or ATP. Antiretroviral combinations with BRY do not interfere with BRY activity (i.e., the reactivation of latently infected cells) or with the antiviral activity of antiretroviral drugs. In addition, BRY-mediated down-modulation of surface CD4 and CXCR4 was not affected when it was used in combination with other antiretrovirals, and no hyperactivation or high-proliferation effects were observed in primary T cells. Moreover, the BRY treatment was able to reactivate HIV-1 in CD4+ T cells from HIV-1-infected patients under cART. Thus, we propose the use of BRY to purge the viral reservoir and recommend its combination with current antiretroviral treatments. Ó 2015 Published by Elsevier B.V.
1. Introduction Abbreviations: AIDS, acquired immunodeficiency syndrome; ATP, AtriplaÒ; BRY, bryostatin-1; cART, combination of antiretroviral therapy; DMSO, dimethyl sulfoxide; EFV, efavirenz; EGFP, enhanced green fluorescent protein; ELISA, enzymelinked immunosorbent assay; FBS, fetal bovine serum; FDA, food and drug administration; FTC, emtricitabine; GFP, green fluorescent protein; HDACi, histone deacetylase inhibitors; HIV-1, human immunodeficiency virus-1; IL-2, interleukin2; iMFI, integrated mean fluorescence intensity; LRAs, latency reversal agents; LTR, long terminal repeat; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MVC, maraviroc; PBMCs, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PHA, phytohemagglutinin; PKC, protein kinase C; SEM, standard error of the mean; TFV DF, tenofovir disoproxil fumarate; TNF, tumor necrosis factor. ⇑ Corresponding author. E-mail addresses: [email protected] (M. Martínez-Bonet), maribel. [email protected] (M.I. Clemente), [email protected] (S. Álvarez), [email protected] (L. Díaz), [email protected] (D. García-Alonso), [email protected] (E. Muñoz), [email protected] (S. Moreno), [email protected], [email protected] (M.Á. MuñozFernández). http://dx.doi.org/10.1016/j.antiviral.2015.09.014 0166-3542/Ó 2015 Published by Elsevier B.V.
An effective combination of antiretroviral therapy (cART) successfully controls HIV-1 viraemia in most HIV-1 infected patients. Although an undetectable viral load is achieved in most treated patients, HIV-1 establishes a long-term infection in a small pool of memory CD4+ T cells, which contains an integrated but transcriptionally silent provirus (Alexaki et al., 2008; Chun et al., 1997; Shen and Siliciano, 2008). Moreover, cells belonging to the monocyte/macrophage lineage represent one of the persistent major reservoirs of the virus because they maintain a low level of HIV-1 replication (Kumar et al., 2014). The origin and clinical implications of persistent low levels of viraemia are uncertain. Some studies postulate that it might be the result of the release of virus from latently infected cells (Joos et al., 2008; Kieffer et al., 2004), whereas other studies suggest that it could arise from ongoing viral replication (Chun et al., 2010, 2005; Havlir et al., 2003; Sharkey et al., 2000).
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Recently, several strategies have been developed to purge HIV-1 reservoirs, and the use of therapies with small molecules targeting HIV-1 reservoirs is one of the major challenges in the fight against AIDS. Clinical trials with cART intensification have failed to eliminate viral replication, which further supports the hypothesis that low levels of viraemia are produced by latently infected cells (Buzon et al., 2010; Gandhi et al., 2010; Gutierrez et al., 2011; McMahon et al., 2010; Vallejo et al., 2012). Therefore, it might be possible to reactivate the latent virus with a therapeutic approach known as ‘‘shock and kill” (Katlama et al., 2013). A wide range of small molecules, such as disulfiram (Xing et al., 2011), HDAC inhibitors (HDACi); (Archin et al., 2012; Shirakawa et al., 2013; Siliciano et al., 2007) and protein kinase C (PKC) activators such as ingenols (Warrilow et al., 2006), prostratin (Kulkosky et al., 2001), 1,2-diacylglycerol analogs (Hamer et al., 2003), and bryostatin-1 (BRY); (del Real et al., 2004; Mehla et al., 2010), have been proposed as agents to reactivate HIV-1 and eradicate the pool of latently HIV-infected CD4+ T cells (Kulkosky and Bray, 2006). Several clinical trials are currently ongoing to evaluate the effectiveness of these compounds to hit HIV-1 reservoirs. The results of a clinical trial with disulfiram did not show a reduction in the size of the latent reservoir (Spivak et al., 2014). However, an ex vivo approach that evaluated the effectiveness of potential candidates, including BRY and HDACi, revealed that BRY was the best candidate to reactivate HIV-1 from latency (Bullen et al., 2014). Therefore, PKC activators, alone or in combination with HDACi, are of particular relevance for purging HIV-1 reservoirs in patients. For instance, the synergistic effect of BRY with HDACi to antagonize the HIV-1 latency has been shown in vitro (Perez et al., 2010). Before clinical trials can be conducted on ART intensification with BRY to assess its impact on the size of the HIV-1 latent reservoirs, the potential effect of BRY in combination with antiretrovirals should be determined in vitro. In this study, maraviroc (MVC) as a monotherapy regimen and a fixed-dose combination of emtricitabine, tenofovir and efavirenz (AtriplaÒ, ATP) as a full cART regimen were tested in combination with BRY at two levels. Using different in vitro models of latency and an ex vivo model, we demonstrated the viral reactivation from latent reservoirs and the inhibition of acute infection.
anti-human HLA-DR-ECD were obtained from BD Biosciences Pharmingen (San Diego, CA, USA). BRY was obtained from Sigma–Aldrich (St. Louis, MO, USA), maraviroc (MVC) was obtained as a clinical formulation in a 150 mg tablet (Selzentry, Pfizer Labs, UK), and AtriplaÒ (ATP) was obtained as a clinical formulation in a 1100-mg tablet (600 mg EFV, 200 mg FTC, 300 mg TFV DF; Gilead Sciences, CA, USA). MVC and ATP were freshly dissolved in distilled water with DMSO (Sigma–Aldrich, St. Louis, MO, USA) and sterilefiltered. ATP is not a chemical entity per se, so the theoretical molecular weight of 1198.44 g/mol was calculated based on the molecular weight of each component and the relative proportion of the single drugs. Hence, 1 mM of ATP corresponds to 2.07 mM of EFV, 0.87 mM of FTC, and 0.49 mM of TFV DF. The concentration of DMSO in cell cultures was less than 0.001%. TNF-a was purchased from R&D Systems (Minneapolis, Minn.).
2. Materials and methods
JLTRG-R5 cells were stimulated with the indicated compounds, and a GFP-fluorescence pattern was determined 24 h later. The percentage of GFP expressing cells was used as a measure of HIV-1 LTR activation.
2.1. Cell lines and culture JLTRG-R5 (Division of AIDS, NIAID, NIH. Catalog number 11586; Dr. Olaf Kutsch), J89GFP and THP89GFP cells (kindly donated by Dr. David N Levy, NYU, USA) were maintained according to the protocol described by Kutsch et al. (2002). ACH-2 and J1.1 cells were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Dr. Thomas Folks (Clouse et al., 1989; Folks et al., 1989; Perez et al., 1991). Buffy coats from healthy subjects were obtained from the Madrid Transfusion Center. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized venous blood by a ficoll-paque density gradient (Garcia-Merino et al., 2009) and maintained in complete RPMI supplemented with 30 U/ml IL-2 and, when indicated, activated for 3 days with 1 lg/ml phytohemagglutinin (PHA; Murex Biotech, England, UK). TZM-bl (Division of AIDS, NIAID, NIH, Catalog number 8129; Dr. John C. Kappes) and HEK 293T cells (ATCC number CRL-11268, Rockefeller University, USA) were cultured according to the manufacturers’ instructions. 2.2. Reagents Anti-human CD3-PC5, anti-human CD4-PC7, anti-human CXCR4-APC, anti-human CCR5-PE, anti-human CD38-FITC and
2.3. Cell viability assays The concentration range of each compound examined in this study is in agreement with previously published results (Arberas et al., 2013; Bousquet et al., 2009; Perez et al., 2010). The toxicity of compounds was measured by an MTT assay (Sigma, St. Louis, MO, USA) according to the manufacturer’s instructions. 0.001% DMSO treated cells were included in each experiment as vehicle control (DMSO+); DMSO 10% (DMSO+++) was used as positive control of cytotoxicity. 2.4. Analysis of surface marker expression Cells were stained for 1 h at 4 °C with the corresponding conjugated antibodies in a FACS staining buffer (phosphate-buffered saline, PBS, with 2% FBS) and analyzed in a Gallios flow-cytometer (Beckman-Coulter, CA, USA). At least 20,000 CD3+ cells, 50,000 JLTRG-R5 or 20,000 J89GFP and THP89GFP cells were collected for each sample and analyzed with the Kaluza software (Beckman-Coulter, CA, USA). 2.5. HIV-1 LTR reactivation
2.6. Latent HIV-1 reactivation To determine the viral reactivation, the EGFP-fluorescence pattern was measured by flow cytometry and HIVp24Gag release measured by ELISA (INNOTESTÒ HIV-Antigen mAb, Innogenetics, Belgium). 2.7. Latent HIV-1 reactivation in CD4+ T cells from infected individuals under cART Fresh blood from HIV-1-infected individuals (n = 3) was obtained in accordance with protocols approved by the Hospital Ramón y Cajal Ethical Committee (Clinical characteristics depicted in Table S1). The participants signed an informed consent form. CD4+ T cells were isolated from PBMCs using the CD4+ T Cell Isolation Kit II (Miltenyi Biotec, Bergisch-Gladbach, Germany) and cultured under different conditions. To measure the HIV-RNA levels, the supernatants were analyzed by a robotic COBAS AmpliPrep/TaqMan system (Roche Diagnostics, Indianapolis, IN, USA). The limit of detection in this assay was 50 copies/ml.
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Fig. 1. HIV-1 reactivation. JLTRG-R5 cells were treated during 24 h with BRY (1, 10, 50, 100 nM), MVC (1, 10, 50, 100 lM) and ATP (0.3, 3, 15, 30 lM) alone (white-filled symbols) or in combination (black-filled symbols) and analyzed by flow cytometry. The results represent the integrated GFP mean fluorescence intensity (iMFI) (A). J89GFP and THP89GFP cells were stimulated with BRY (100 nM), TNF-a (3 ng/ml), 48 h later, the EGFP expression was measured by flow cytometry (B) and visualized by fluorescence microscopy (C). Histograms compare the EGFP expression in non-stimulated (black lines) and BRY treated cells (red lines) or TNF-a treated cells (blue lines). Arrows indicate syncytium formation in cell cultures. The results are representative of four independent experiments. (D) Viral reactivation in J89GFP and THP89GFP cells treated for 2 or 4 days (white and black bars, respectively) with BRY (100 nM), MVC (100 lM) and ATP (30 lM) alone or in combination was measured as EGFP expression by flow cytometry (upper panels) or p24Gag release by HIVp24Gag ELISA kit in supernatants in supernatant (lower panels). TNF-a (3 ng/ml) was used as positive control of HIV-1 reactivation. Bars represent the percentage of EGFP positive cells. The results represent the arithmetic mean ± SEM (A) or +SEM (D) of at least three independent experiments. Statistics were performed between control and singletreated cells (left panels), between BRY-treated and BRY combinations-treated cells (right panels) (A) or between control and treated cells (D). We used three significance levels: * p < 0.05; **p < 0.01; ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2.8. TZM-bl cells infection assays
2.9. Virus production
After different treatment/infection assays, TZM-bl cells were lysed using a Glo-Lysis buffer (Promega, Madison, WI, USA), and luciferase assays were performed according to the manufacturer’s instructions. Luminescence was measured using a VICTOR luminometer. All the experiments were performed three times.
X4-HIV-1NL4-3, R5-HIV-1NL(AD8) and X4/R5-HIV-189.6 viruses were obtained by transient transfection of pNL4-3, pNL(AD8) and p89.6 plasmids, respectively (NIH AIDS Research and Reference Reagent Program) in HEK 293T cells (ATCC, Manassas, VA, USA). Viral stocks were clarified by centrifugation prior to the
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evaluation of the viral titer by ELISA (Sepulveda-Crespo et al., 2014).
3. Results 3.1. Effects of bryostatin-1 combined with maraviroc and AtriplaÒ on HIV-1 LTR activation
2.10. Cell proliferation assays The proliferation of cells was examined using the BrdU Cell Proliferation Kit (Chemicon, Millipore, MA, USA) according to the manufacturer’s instructions.
2.11. Photomicroscopy Cells were photographed using a DMI 3000B Leica microscope. Images are shown at 10 magnification.
2.12. Statistics A statistical analysis was performed using SPSS software version 15.0 for Windows. Differences between the two groups (control versus different dosages of compounds or BRY-treated versus ART combined with BRY treatment) were assessed using a paired t-test using three significance levels (p < 0.05, p < 0.01 and p < 0.001).
To investigate the efficacy of BRY in combination with cART, JLTRG-R5 cells were treated for 24 h with the maximum safety concentration range of BRY (1–100 nM), MVC (1–100 lM) and/or ATP (0.3–30 lM; Fig. S1A). BRY antagonized HIV-1 latency very efficiently at concentrations of up to 50 nM (Fig. 1A; left panel). The combination of BRY with both antiretrovirals tested did not diminish the effects of BRY on LTR activation (Fig. 1A; right panel). The triple combination of BRY-MVC-ATP at the highest concentration appeared to increase the levels of GFP iMFI (MFI per percentage of positive cells) by approximately 50% compared with the BRY-treatment alone. The surface expression of CD4 and CXCR4 were analyzed 24 h post-treatment with BRY alone or in combination with antiretrovirals, and a significant decrease in both expressions was observed, even at low doses of BRY, compared with the control of JLTRG-R5 cells (Fig. S2A and B). Both MVC and BRY-treated cells showed an increase in the CCR5 surface expression (Fig. S2C; left panel). BRY-ATP-treated cells presented a CCR5 expression profile similar to BRY-treated cells, whereas the BRY-MVC treatment and the triple combination of BRY-MVC-ATP resulted in an increase of
Fig. 2. HIV-1 infection inhibition. Reporter TZM-bl cells were pretreated for 1 h with BRY (10, 50, 100 nM), MVC (10, 50, 100 lM) and ATP (3, 15, 30 lM) alone or in combination and infected with (A) X4-HIV-1NL4.3, (B) R5-HIV-1NL(AD8) or (C) X4/R5-HIV-189.6 (40, 20 or 40 ng HIVp24Gag/106 cells, respectively). 3 h after infection, virus was removed and cells were treated again with the compounds at the same concentration range. Luciferase activity was quantified 48 h post-infection. The results are expressed as the percentage of infectivity relative to that of the control (Ct) infected cells (black bar) normalized to 100%. White bar indicates the background luminescence values of non-infected cells. Values are the means + SEM of three independent infections, each measured in duplicate. Statistics were performed between Ct infected cells and treated infected cells (grey bars). We used two significance levels: *p < 0.05; **p < 0.001.
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approximately 2- to 3-fold, respectively, when compared with BRY-treated cells (Fig. S2C; right panel). 3.2. Virus reactivation in HIV-1 latently infected lymphocytic and monocytic cells HIV-latent reservoirs are established in T-cells and monocyte/ macrophages in the body, and these cells harbor the infection for the entire life of the individual. Therefore, we investigated whether BRY could reactivate latent HIV-infection in both T-cells and monocytic cells. After a treatment with BRY, latently infected J89GFP and THP89GFP cells were reactivated with evidence of EGFP expression within 48 h compared with the negative control (Fig. 1B and C). TNF-a was used as a positive control in this experiment leading to the formation of syncytia in J89GFP cells (Fig. 1B and C). The effect of BRY, measured by EGFP expression (Fig. 1D, upper panels) or HIVp24Gag release on cell supernatants (Fig. 1D, lower panels), was also observed even combined with MVC or ATP at days 2 and 4 after the treatment. 3.3. The combination of bryostatin-1 with maraviroc and AtriplaÒ does not modify the antiviral effects of the drugs To determine whether BRY interfered with the effects of the antiretrovirals, several drug combinations were tested against X4-HIV-1NL4.3 (Fig. 2A), R5-HIV-1NL(AD8) (Fig. 2B) or X4/R5-HIV189.6 infection in the reporter TZM-bl cells (Fig. 2C). TZM-bl viability was assessed after 72 h of treatment (Fig. S1B). As expected, MVC completely inhibited the R5 virus infection and partially inhibited (approximately 40%) the infectivity of the dual strain. In contrast, ATP decreased the luciferase values to similar background levels, regardless of the HIV-1 tropism. Because BRY is able to down-modulate CD4, the infection with the tested virus was reduced approximately 50% when compared with non-treated and HIV-infected control cells. Neither MVC nor ATP lost their antiviral activity when combined with BRY. Moreover, the triple combination of drugs showed the maximum HIV-inhibition rate for all HIV-1 strains. 3.4. Viral reactivation and inhibition of reactivated virus THP89GFP cells reached almost complete reactivation levels when treated with BRY; thus, we investigated whether these cells produced infectious viral particles after BRY stimulation by simultaneously analyzing the viral reactivation and the inhibition of reactivated virus resulting from the drug-combination. Towards this goal, we developed a co-culture model of BRY-treated THP89GFP cells (Fig. 3A) or their supernatants (Fig. 3B) with TZM-bl cells pre-treated with MVC (100 lM) or/and ATP (30 lM). Both conditions significantly increased the luciferase expression versus the non-treated TZM-bl cells. When the reporter cells were pre-treated with MVC or ATP, new infections of reactivated X4/R5HIV-189.6 were inhibited 50% or 80%, respectively. The partial infection inhibition with MVC is likely because 89.6 is a dual-tropic strain (Fig. 3). 3.5. Effects of bryostatin-1 combined with maraviroc and AtriplaÒ on T cell phenotype We studied how BRY and antiretroviral combinations may affect the T-cell phenotype and activation. Flow cytometry assays were performed on non-stimulated or PHA-stimulated PBMCs from healthy subjects after exposure to the highest non-toxic concentration of each compound separately or in combination (Fig. S1C). Both non-activated and activated cells showed a significant decrease in surface CD4 expression (Fig. 4A) when treated
Fig. 3. Viral reactivation and inhibition of new infections with reactivated HIV-1. THP89GFP cells were stimulated with 100 nM BRY; 24 h later, washed cells (A) or their supernatants (B) were added to MVC (100 lM) or/and ATP (30 lM) pretreated TZM-bl cells. Luciferase activity was assessed 48 h post-infection. The results are expressed as the percentage of infectivity relative to that of the BRYtreated THP89GFP cells in co-culture with non-treated TZM-bl cells (black bar) normalized to 100%. The white bar indicates the background luminescence values of non-treated THP89GFP cells in co-culture with non-treated TZM-bl cells. Values are the means + SEM of three independent infections. We used three significance levels: *p < 0.05; **p < 0.01; ***p < 0.001.
with BRY, BRY-ATP or BRY-MVC-ATP. Moreover, ATP-treated, non-activated cells decreased their CD4 expression up to 25%. Only the BRY treatment caused a significant decrease in the CXCR4 expression (Fig. 4B). However, its combination with antiretrovirals also negatively modulated the CXCR4 expression on both PBMCs and CD4+ T cell surfaces. As described by Arberas et al. (2013), MVC, alone or in combination, increased the CCR5 surface expression on activated cells (Fig. 4C). The effects on T-cell activation after in vitro BRY-MVC-ATP exposure were analyzed by measuring the MFI of CD38 and HLA-DR activation markers on PBMCs and CD4+ T lymphocytes (Fig. 4D and E). The BRY treatment led to an induction of CD38 and HLA-DR expression in non-stimulated T cells, but not in PHA-stimulated cells. However, ATP appeared to slightly affect the expression profile of both markers in resting PBMCs but not in CD4+ T cells. No proliferation was observed in CD4+ T cells after 24 h of the BRY treatment, but it was detected after 6 days of treatment (Fig. S3). Our results indicate that BRY, alone or in combination with MVC or ATP, only induces a low level of activation in human primary T cells.
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Fig. 4. Effects on T cell phenotype. Isolated PBMCs from healthy subjects were treated either with BRY (50 nM), MVC (100 lM) and ATP (30 lM) alone or in combination for 24 h. The expression of cell surface receptors CD4, CXCR4, CCR5, CD38 and HLA-DR was analyzed by flow cytometry in resting (white bars) or PHA activated (black bars); PBMCs (left panels) and CD4+ T cells (CD3+CD4+, right panels). The results represent the percentage of MFI relative to that of control cells (Ct) normalized to 100% of cell surface receptors CD4 (A), CXCR4 (B) CCR5 (C) CD38 (D) and HLA-DR (E). The results represent the arithmetic mean + SEM of at least three independent experiments. Statistics were performed between control and treated cells. We used two significance levels: *p < 0.05; **p < 0.001.
3.6. HIV-1 induction in CD4+ T cells from infected individuals under cART Our next goal was to study the effects of BRY on CD4+ T cells isolated from three HIV-1 infected individuals on suppressive cART. Purified CD4+ T cells were cultivated in the presence of ATP and treated or not treated with BRY (50 nM). After 2 days, the levels of HIV-1-virion-associated RNA were evaluated. BRY was exceptionally potent, inducing high levels of HIV-1 RNA from two of the three patients (Fig. 5A). When the experiment was continued up to day 6 for the third patient, we observed that the levels of HIV-1 RNA in the supernatant of BRY-treated cultures increased
3-fold when compared with non-treated cultures (Fig. 5B). This finding suggests that the activity of BRY observed in immortal cell line models of HIV-1 latency may translate to potent viral reactivation from latently infected primary cells from HIV-1 patients. 4. Discussion The persistent low levels of HIV-1 viraemia, the limitations of current ARTs, and the lack of a valid anti-HIV-1 vaccine candidate highlight the need for new therapies to eradicate HIV-1. Nevertheless, the difficulty to obtain primary cells latently infected with HIV makes it an almost impossible challenge. Hence, in this study, we
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Fig. 5. Viral induction from human CD4+ T cells from fully suppressed HIV-1 positive donors. CD4+ T cells were purified from PBMCs of three HIV-1 infected patients under suppressive cART. After 2 (A) or 6 (B) days of treatment with BRY at 50 nM, HIV-1 RNA was quantified in the cell supernatants using the COBAS AmpliPrep/TaqMan HIV-1 test. Non-treated BRY cells, BRY, white bars; BRY treated cells, +BRY, black bars.
employed JLTRG-R5, J89GFP and THP89GFP cells as a postintegration HIV latency model to explore the impact of MVC and/ or ATP in combination with BRY. Although J89GFP and THP89GFP cell line models are not frequently used to assess latency reversal agents (LRA) (Butera et al., 1991; Duh et al., 1989; Folks et al., 1987, 1988; Jordan et al., 2003, 2001; Perez et al., 1991), their exclusive characteristics make them an experimentally tractable and relevant model to study postintegration HIV latency and reactivation (Kutsch et al., 2002). Nevertheless, to confirm the results obtained in these cell lines, we have performed the same reactivation experiments in another two other well-established latency-models, such as ACH-2 and J1.1 cell lines. We assessed the cell toxicity and the HIV-1 reactivation and the obtained data were in concordance to that exposed in the article (Fig. S4). Numerous researchers have studied the effects of BRY on HIV-1 latency and its effects in CD4 and CXCR4 down-modulation (Mehla et al., 2010; Perez et al., 2010). To simultaneously analyze the viral reactivation and the inhibition of reactivated virus caused by the drug combination, we performed co-culture experiments with THP89GFP cells and TZM-bl cells. Moreover, we also evaluated the cellular activation and proliferation as well as the CD4, CXCR4, and CCR5 surface expression levels in human primary T cells. BRY is attractive because it is an FDA-approved drug, and it has already been tested in human trials for cancer and Alzheimer’s disease. Consequently, data on the pharmacokinetics and toxicity of BRY in humans are available (Irie et al., 2012). Phase II clinical trials have reported that 2 lg/ml of BRY in blood (20-fold the concentration of this study) was reasonably well tolerated and have demonstrated that lower doses of BRY (50 nM) are able to reactivate latent HIV-1 (Madhusudan et al., 2003). BRY has been remarked as the best effective single agent that disrupts the latent reservoir between a battery of LRAs (Bullen et al., 2014; Spina et al., 2013). On the other hand, BRY notably induced activation in nonstimulated T cells and cell proliferation after 6 days of treatment. Although these effects are considerably lower than that observed in PHA-stimulated cells, the in vivo consequences of this cellular activation could be responsible for inflammatory responses. In order to avoid this, further studies should be done with BRY pulses instead of continuous treatment, to achieve minimal cellular activation, as observed by Wei et al. (2014) in primary cells after 4-h pulse of RMD. Another approach could be the combination of different LRAs in order to test whether lower doses of these drugs could induce similar levels of latent HIV stimulation with lower levels of T cell activation. In addition to PKC agonists, other LRAs, such as HCACi, have been proposed as candidates for the so-called ‘‘shock and kill”
strategy to purge HIV-1 reservoirs. Due to the molecular complexity of the viral latency, it is likely that the synergistic activity of drugs targeting different pathways will be needed to effectively reactivate HIV-1 from latency in different reservoirs. Indeed, synergy in latency reversion between prostratin (Reuse et al., 2009) or BRY (Perez et al., 2010) and classical HDACi has been previously described. Moreover, our group has observed a synergistic profile in HIV-1 reactivation when BRY was combined with novel promising HDACi, such as panobinostat and romidepsin. Our results show a slight but not statistically significant synergy between BRY, MVC and ATP in HIV-1 reactivation (Fig. 1). In agreement with these results, we have previously reported that MVC can activate NF-jB and subsequently reverse latent HIV-1 in resting CD4+ T-cells from ART suppressed HIV-1-infected patients (Madrid-Elena et al., 2014). This finding could be of clinical relevance because it is thought that a reactivation therapy will be achieved by alternative cycles of immunoactivation therapy plus ART intensification followed by standard cycles of cART. Thus, adding MVC in the ART intensification cocktail could enhance the anti-latency activity of BRY-1 even at lower doses of this drug. Despite its limitation, our model allowed us to assess in a single system both the viral reactivation and the inhibition of new infections, which provides important information. This model appears to be an accurate reporter model for assessing the infectivity of reactivated virus. Likewise, BRY significantly reactivated latent virus from CD4+ T cells isolated from HIV-infected individuals, and this reactivation was even stronger than the one obtained with prostratin or its analogs (Beans et al., 2013) in similar studies. This work demonstrated that the combination of ART with BRY does not interfere with BRY activity or the antiretroviral activity. Our in vitro findings could be corroborated in vivo in the NCT02269605 clinical trial. This trial is a Phase I trial designed to evaluate two different doses of BRY on HIV-1 latency and reservoir in HIV-1 infected patients receiving antiretroviral treatment. Overall, these data support the notion that reactivation conducted under the cover of cART could lead to the selective killing of latently infected cells and the ‘‘purging” of the latent viral reservoir.
Conflicts of interest The authors declare that there are no conflicts of interest.
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Acknowledgments We thank the Center of Transfusion of Madrid for the buffy coats and the Spanish HIV HGM BioBank, which is supported by the Instituto de Salud Carlos III (ISCIII), and is funded by the RD12/0017/00XX project as part of the Plan Nacional R+D+I and cofounded by the ISCIII-Subdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (FEDER). We are grateful to Dr. Rafael Samaniego of the Instituto de Investigación Biomédica Gragorio Marañón Microscopy Unit for his technical assistance. This work was supported by the grant number RD12/0017/0037 as part of the Plan Nacional R+D+I and co-financed by the ISCIIISubdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (FEDER), PT13/0010/0028, Fondo Investigación Sanitaria (grant number PI13/02016), ‘‘Fundación para la Investigación y la Prevención del Sida en España” (FIPSE), Comunidad de Madrid (grant number S-2010/BMD-2332), CYTED 214RT0482. CIBER-BBN is an initiative funded by the VI National R&D&I Plan 2008–2011, Iniciativa Ingenio 2010, the Consolider Program, and CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. MMB was supported by FI10/00141 (ISCIII).
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