Secondary Logo

Share this article on:

Bryostatin-1 for latent virus reactivation in HIV-infected patients on antiretroviral therapy

Gutiérrez, Carolina; Serrano-Villar, Sergio; Madrid-Elena, Nadia; Pérez-Elías, Maria J.; Martín, Maria Elena; Barbas, Coral; Ruipérez, Javier; Muñoz, Eduardo; Muñoz-Fernández, Maria Angeles; Castor, Trevor; Moreno, Santiago

doi: 10.1097/QAD.0000000000001064
CLINICAL SCIENCE

Objective: The protein kinase C (PKC) agonist bryostatin-1 has shown significant ex-vivo potency to revert HIV-1 latency, compared with other latency reversing agents (LRA). The safety of this candidate LRA remains to be proven in treated HIV-1-infected patients.

Methods: In this pilot, double-blind phase I clinical-trial (NCT 02269605), we included aviraemic HIV-1-infected patients on triple antiretroviral therapy to evaluate the effects of two different single doses of bryostatin-1 (10 or 20 μg/m2) compared with placebo.

Results: Twelve patients were included, four in each arm. Bryostatin-1 was well tolerated in all participants. Two patients in the 20 μg/m2 arm developed grade 1 headache and myalgia. No detectable increases of cell-associated unspliced (CA-US) HIV-1-RNA were observed in any study arm, nor differences in HIV-1 mRNA dynamics between arms (P = 0.44). The frequency of samples with low-level viraemia did not differ between arms and low-level viraemia did not correlate with CA-US HIV-1-RNA levels (P = 0.676). No changes were detected on protein kinase C (PKC) activity and in biomarkers of inflammation (sCD14+ and interleukin-6) in any study arm. After the single dose of bryostatin-1, plasma concentrations were under detection limits in all the patients in the 10 μg/m2 arm, and below 50 pg/ml (0.05 nmol/l) in those in the 20 μg/m2 arm.

Conclusion: Bryostatin-1 was safe at the single doses administered. However, the drug did not show any effect on PKC activity or on the transcription of latent HIV, probably due to low plasma concentrations. This study will inform next trials aimed at assessing higher doses, multiple dosing schedules or combination studies with synergistic drugs.

aDepartment of Infectious Diseases

bDepartment of Biochemistry Research, Ramón y Cajal University Hospital, Alcalá University and IRYCIS

cCEMBIO, San Pablo CEU University, Madrid

dDepartment of Cell Biology, Physiology and Immunology, Maimonides Biomedical Research Institute of Córdoba, Reina Sofía University Hospital, University of Córdoba

eMolecular ImmunoBiology Laboratory, Gregorio Marañón University Hospital, IISGM, CIBER-BBN, Madrid, Spain

fAphios Corporation, Boston, Massachusetts, USA.

Correspondence to Santiago Moreno, Department of Infectious Diseases, Hospital Universitario Ramón y Cajal, Carretera de Colmenar, Km. 9.100, 28034 Madrid, Spain. Tel: +0034 913 368 710; fax: +0034 913 368 792; e-mail: smguillen@salud.madrid.org

Received 13 January, 2016

Revised 11 February, 2016

Accepted 11 February, 2016

Back to Top | Article Outline

Introduction

Combination antiretroviral therapy (cART) is among the greatest successes of modern medicine, as it has shifted the prognosis of HIV-infected individuals from months to decades. The persistence of latent HIV-infected reservoirs remains, however, the last major barrier to virus cure [1–4]. The elimination of the small subset of latently infected memory CD4+ T cells is now among the greatest challenges of HIV research.

There is emerging consensus that reactivation of the latent reservoirs ('shock’), in combination with interventions that would enhance the ability of the host to clear these infected cells (’kill’) will be necessary for effective eradication of the virus [5]. Although T-cell activation effectively reverses latency, toxicity due to cytokine release precludes its clinical use. So, the field has focused the attention on latency reversing agent (LRA) that do not induce polyclonal T-cell activation and cytokine release [6]. Given the low frequency of latently infected resting CD4+ T cells, in-vitro models of latency have played a central role in the search for compounds that reactivate HIV [6]. Many putative LRAs have been identified using these models, which identified hystone deacetilase (HDAC) inhibitors as promising LRAs. Seminal work by Archin et al. using one of these compounds, vorinostat [7] and subsequently by Rasmussen et al. using panobinostat [8] have shown that HDAC inhibitors can disrupt HIV latency in vivo. The magnitude of this effect, however, seems to be modest, even after repeated doses [9], and importantly, no LRA has been shown to reduce the size of the latent reservoir in HIV-infected individuals. Given the multifaceted mechanisms involved in HIV latency, HDAC inhibitors are now seen as drugs to be used as a part of a combination therapy targeting other pathways of HIV latency.

Protein kinase C (PKC) plays an important part in transcriptional activation in resting CD4+ T cells. PKC activation induces reactivation of latent HIV-1 by targeting multiple regulatory elements on the HIV-1 long-terminal repeat, through the NF-κB and AP-1 signalling pathways [6]. Bryostatin-1 is isolated from the marine invertebrate Bugula neritina [10,11]. Bryostatin-1 exhibits excellent potential as a therapeutic agent that acts through modulation of PKC signal transduction, with an initial up-regulation followed by a sustained down-regulation [12]. As an antineoplastic agent, bryostatin-1 has been evaluated in several phase I and II clinical trials for various cancers [13–18]. Bryostatin-mediated HIV-1 reactivation involves activation of PKC via the adenosine monophospate (AMP)-activated protein kinase (AMPK) pathway [19] and does not induce T-cell activation in primary human CD4+ T cells [20].

In-vitro studies carried out by our group demonstrated that bryostatin-1 synergizes with HDAC inhibitors such as valproic acid to antagonize HIV-1 latency, and down-regulates the expression of the HIV-1 receptors CD4+ and CXCR4, preventing HIV-1-induced cytotoxicity [12,21].

More recently, we have shown that bryostatin-1 also reactivates latent HIV-1 in astrocytes [21]. Bryostatin-1 has attracted renewed attention after the recent study by Bullen et al. where a modified viral outgrowth ex-vivo assay was used to compare the efficacy of different leading LRA candidates [22]. None of the LRA tested, including several HDAC inhibitors, induced outgrowth of HIV-1 from the latent reservoir of ART-treated patients, and only bryostatin-1 caused significant increases of cell-associated unspliced (CA-US) HIV-1 RNA in patient cells. In a subsequent study from the same group, only the PKC agonists bryostatin-1 and prostratin induced significant production and release of HIV-1 virions from patient cells. In addition, PKC agonists caused little or no cytokine production [23].

Unfortunately, so far it is difficult to predict which dosing schedule of bryostatin-1 should be used in ART-treated HIV-infected patients to achieve such steady-state concentrations (C ss). As shown by Siliciano's group [22,23], bryostatin-1 concentrations of 10 nmol/l (1150 pg/ml) are required ex vivo to induce intracellular HIV-1 mRNA transcription from resting CD4+ T cells when used as a single agent. Whether this will be reproduced in vivo remains to be proven, and the safety in HIV-infected patients and potential interactions with ART is unexplored.

To inform future trials with bryostatin-1 in the field of HIV cure, we assessed the safety and efficacy of two single doses (10 and 20 μg/m2) of bryostatin-1 in long-term-treated HIV-infected patients. The primary outcome was safety and tolerability. Secondary outcomes were changes from baseline in CA-US HIV-1 RNA, PKC activation, low-level plasma HIV-1 RNA, markers of early immune activation and bryostatin-1 plasma concentrations at different time points postinfusion.

Back to Top | Article Outline

Methods

Study design and participants

We performed a pilot, double-blind phase I clinical trial to evaluate the safety of two different single doses of bryostatin-1 (10 or 20 μg/m2) in long-term treated HIV-infected patients.

The study was conducted at University Hospital Ramón y Cajal in Madrid, Spain between September 2014 and January 2015. Bryostatin-1 was provided by Aphios Corporation (Woburn, Massachusetts, USA).

Inclusion criteria were HIV-1 infection, at least 2 years receiving triple ART, viral load less than 50 copies/ml during at least 2 years also, and CD4+ T-cell count greater than 350 cells/μl. We excluded HIV-1 patients with hepatitis C virus co-infection and patients with recent use of immunomodulatory agents or valproic acid.

Back to Top | Article Outline

Ethics statement

The research protocol was approved by the Spanish Agency for Medications and Health products (AEMPS) and the local Ethics Committee (Eudra CT:2010-02247-30; ceic.hrc@salud.madrid.org). All participants signed an informed consent prior to initiation of study procedures (clinicaltrials.gov: NCT 02269605).

Back to Top | Article Outline

Study procedures

Twelve individuals were randomized to receive a single dose (10 or 20 μg/m2) of bryostatin-1 or placebo. Study participants were randomly assigned to the 10 μg/m2 active arm, 20 μg/m2 active arm or placebo arm, using a telephone randomization system. Patients and investigators were blinded to study allocation until statistical analysis was completed.

The dose regimen was selected based on clinical safety of previous clinical trials in cancer and Alzheimer diseases [13–18]. Note that the optimal dose rate delivery based on protein kinase of bryostatin-1 has not been established because of lack of reliable assays to quantify its serum levels. For example, the choice of the 24-h infusion was to produce a prolonged inhibition of PKC within tumour cells [24], which is the opposite of what we plan to achieve. In our study, bryostatin-1 was administered as a single intravenous dose during 1 h. Blood samples were collected at baseline, 15, 30 and 60 min during bryostatin-1 infusion, and 2, 4, 8, 12, 24, 48 and 72 h postinfusion.

For safety reasons, study participants were medically monitored during the first 12 h in the Clinical Trials Unit at our hospital. Physical and laboratory examinations were performed at 24, 48 and 72 h visits. Laboratory parameters included liver function tests (ALT, AST, alkaline phospatase, bilirubin), renal function (creatinine, estimated glomerular filtration rate, sodium, potassium, urinalysis), as well as amylase, glucose, and total proteins. Blood cell count were also performed at the same time points. On days 7, 14 and 21, we assessed patient-reported adverse events by telephone interview and on day 28 we performed the last visit of the trial (safety visit).

The primary outcome was safety and tolerability. Safety features were evaluated through the changes of vital signs, physical examinations, EKG, laboratory test results (haematology, biochemistry and urinalysis tests), as well as the occurrence of adverse events from baseline to day 28.

Secondary outcomes were changes from baseline in CA-US HIV-1 RNA, PKC activation, low-level plasma HIV-1 RNA, markers of early immune activation and bryostatin-1 plasma concentrations at different time points postinfusion.

Analytical control (biochemistry, haematology, conventional plasma HIV RNA with a limit detection of 50 copies/ml, CD4+ and CD8+ T-cell counts) was performed at baseline, at 48 h and at 28 days post bryostatin-1 infusion.

Back to Top | Article Outline

Measurements

Laboratory analysis for safety

Biochemistry, complete blood count, CD4+ and CD8+ T-cell counts and conventional plasma HIV-RNA with a limit detection of 50 copies/ml was performed at baseline, at 48 h and at 28 days postbryostatin-1 infusion.

Back to Top | Article Outline

Cell associated unspliced-HIV-1 RNA

RNA was extracted from stored PBMCs using RNeasy Mini Kit Qiagen (Hilden, Germany). For CA-US HIV RNA seminested real-time PCR, the eluted cellular RNA was directly subjected to two rounds of PCR amplification as previously described [25]. As external standards, synthetic run of RNA transcripts, corresponding to the HIV-1 gag region, were used. The RNA standards were a kind gift from Sharon R. Lewin, Burnet Institute, Melbourne, Australia. Serial dilutions of standards between 1 to 4.4 × 1011 input copies were made.

HIV-1 RNA copy numbers were standardized to cellular equivalents using RNA concentration (assuming that 1 ng RNA corresponds to 1000 cells [26], which has been shown to correlate with levels of glyceraldehyde phosphate dehydrogenase (GAPDH) RNA [27]. PCR results are expressed in CA-US HIV-1 RNA copies per million PBMCs.

Back to Top | Article Outline

Protein kinase C activity quantification

Resting CD4+ T cells were isolated from total PBMCs by a negative selection of CD3+/CD4+/HLA-DR/CD25 cells using magnetic beads according to the manufacturer's recommendation (Miltenye Biotec, S.L. Bergisch Gladbach, Germany). At 4, 24, 48 and 72 h postinfusion, we quantified PKC activity in resting CD4+ T cells. Active quantification of classical and novel PKC isozyme was measured by using the Signa TECT protein Kinase C Assay system (Promega Corporation, Madison, Wisconsin, USA) according to the manufacturer instructions. The final PKC activity was measured by a scintillation counter and results were expressed as enzyme activity in pmol ATP/min per μg of protein.

Back to Top | Article Outline

Plasma HIV-1 RNA detection by transcription-mediated amplification

Five millilitres of plasma sample was used to assessed the presence of HIV-1 RNA collected at baseline, 15, 30 and 60 min from the beginning of the bryostatin-1 infusion and 2, 4, 8, 12, 24, 48 and 72 h after completion of the infusion by transcription-mediated amplification (TMA) detection method (Procleix Ultrio Elite Assay; Gen-Probe Incorporated, San Diego, California, USA).

Back to Top | Article Outline

Bryostatin-1 pharmacokinetics analysis

To evaluate the protein kinase of bryostatin-1, we measured drug concentrations from plasma samples before bryostatin-1 dose, 15, 30 and 60 min from the beginning of the infusion and 2, 4, 8, 12, 24, 48 and 72 h after the end of the infusion. The drug concentration was analysed by liquid chromatography (LC) mass spectrometry (MS) with a triple quadruple mass spectrometer (QqQ) in the multiple reaction-monitoring (MRM) mode, according to the procedures previously described [18].

Back to Top | Article Outline

Markers of inflammation (soluble CD14 and interleukin-6)

Soluble CD14 (sCD14) and interleukin-6 (IL-6) were quantified in cryopreserved plasma samples at baseline, 8, 24, 48 and 72 h after bryostatin-1 infusion. sCD14 levels were determined using the commercial kit Quantikine ELISA Human sCD14 (R&D Systems, Abingdon, UK) and IL-6 levels were assessed using the Quantikine HS ELISA Human IL-6 kit (R&D Systems) according to the manufacturer's instructions. All assays were run in triplicate.

Back to Top | Article Outline

Statistical analysis

No calculation of the simple size needed to detect statistically significant differences in secondary outcomes (e.g. CA-US HIV-1 RNA) was performed. The sample size is, however, comparable to previous in-vivo studies using candidate LRA designed to find statistically significant increases of HIV-1 transcription [8,9].

Qualitative variables were reported as a frequency distribution whereas quantitative variables were described as median and interquartile ranges (IQRs). We used linear mixed models with a random effect for each patient to allow for correlations caused by repeated observations to assess whether longitudinal changes in numerical outcome measures were overall significantly different from baseline. A robust variance estimator was used given the limited sample size and the deviations from normality. Interaction terms were created to assess whether these changes over time differed significantly between treatment groups. The equality of all mean changes among the three groups was tested using a Wald test for the interaction term. Continuous outcome variables were log-transformed when necessary to satisfy model assumptions.

All statistical analyses were conducted with Stata v. 13.0 (StataCorp LP, College Station, Texas, USA).

Back to Top | Article Outline

Results

Twelve patients underwent randomization and baseline procedures, and all (four in each study arm) completed the full protocol. The general characteristics of the study sample and the development of adverse events are described in Table 1. All individuals retained viral load less than 50 copies/ml and maintained more than 350 CD4+ T cells/μl throughout the study. Bryostatin-1 was well tolerated in all the participants, regardless of the dose of the drug received. No abnormalities appeared in blood cell counts or blood chemistry. Two patients in the 20 μg/m2 arm reported grade 1 headache and myalgia 48 h after bryostatin-1 infusion, which resolved spontaneously within the first 24 h without any specific treatment. One of the patients (BRY1002) developed a grade 1 rash 2 h after bryostatin-1 infusion.

Table 1

Table 1

Back to Top | Article Outline

Bryostatin-1 concentration in plasma

Given the very limited protein kinase data of bryostatin-1, we determined drug concentrations after the 10 and 20 μg/m2 single doses. As represented in Fig. 1, drug concentrations peaked at 0.5 h postinfusion and gradually decayed in the first 8 h. Noteworthy, the highest concentration achieved in one single patient in the peak 20 μg/m2 (0.05 nmol/l) was far below the threshold of 1 nmol/l (1105 pg/ml) presumably needed to reactivate HIV-1 transcription in combination with other LRAs [12], and even further below from the threshold of 10 nmol/l (11 050 pg/ml) necessary to reactivate latent HIV-1 when used as a single agent [23].

Fig. 1

Fig. 1

Back to Top | Article Outline

Effect of bryostatin-1 on HIV-1 latency

To explore whether these concentrations elicited detectable effects on HIV-1 mRNA transcription from latently infected cells, we measured CA-US HIV-1 RNA levels in PBMC. We did not observe any differences in the amount of CA-US HIV-1 RNA between groups (P = 0.44) or between time-points within groups (Fig. 2a).

Fig. 2

Fig. 2

Back to Top | Article Outline

Effect of bryostatin-1 on protein kinase C activation

Low concentrations of bryostatin-1 in vitro appear able to provide PKC activation for antagonizing HIV-1 latency minimizing the risk of toxicities. To prove this in vivo, we measured PKC activation at baseline, and post 4, 24, 48 and 72 h postbryostatin-1 infusion. We quantified the PKC activation in the three groups and we did not observe overall differences, P = 0.263 (Fig. 2b). These findings indicate that bryostatin-1 failed to activate PKC at the single, low doses administered.

Back to Top | Article Outline

Effect of bryostatin-1 on plasma HIV-1 RNA

We also determined changes in low-level plasma HIV-1 RNA using TMA methods and we did not observe any differences between arms (Fig. 3). The proportion of positive samples among patients treated with placebo, bryostatin-1 10 μg/m2 or bryostatin-1 20 μg/m2 was comparable (44, 54 and 56%, respectively, P = 0.573). To evaluate whether the presence of plasma low-level HIV-1 RNA would imply reactivation of HIV-1 mRNA transcription from the latently infected cells, we compared median CA-US HIV-1 RNA concentrations in samples with low-level viraemia; no differences were observed, indicating that positivity of the TMA assay was unrelated to the extent of HIV-1 mRNA transcription.

Fig. 3

Fig. 3

Back to Top | Article Outline

Inflammation after bryostatin-1 infusion

Although T-cell activation effectively reverses latency, toxicity due to cytokine release precludes its clinical use. We did not find between-group differences in changes in the inflammatory markers sCD14 and IL-6 (P = 0.62 and P = 0.76, respectively) (Fig. 4).

Fig. 4

Fig. 4

Back to Top | Article Outline

Discussion

We herein present the first phase I clinical trial with bryostatin-1 as a candidate LRA in ART-treated HIV-infected individuals. A single dose of 10 or 20 μg/m2 administered over 1 h was safe, well tolerated but failed to induce detectable activation of HIV-1 RNA transcription.

In our study, two of the four participants in the active arm only experienced grade 1, transient, adverse effects, including myalgias, headache and rash. Hence, we report for the first time, that bryostatin-1 is a safe agent at the selected doses in combination with ART. This is in keeping with previous data from phase I and phase II clinical trial in humans as an antineoplastic agent, in which the safety profile of bryostatin-1 administered intravenously has been reasonably good. These oncology studies provide extensive information on the safety of use of this substance [13–18], with common adverse effects. Intermittent doses were given, usually ranging from 25 to 65 μg/m2, administered as a 1 h or 24 h i.v. infusion, weekly or for 3 weeks out of 4, or over 24 h once weekly, and up to 8 weeks. The dose-limiting toxicity in oncology phase I studies was myalgia, which seems to be cumulative and dose-dependent, usually appearing after two or three cycles of treatment. Myalgia occurs at one to two days after infusion and tends to get worse with repeated administration. Importantly, no life-threatening adverse effects have been reported for bryostatin-1, even at the maximum assessed doses of 180 μg/m2 [28].

The optimal dose rate delivery based on protein kinase of bryostatin-1 has not been established. Until recently none of these studies had characterized the human protein kinase of the agent due to the lack of a sensitive and reliable assay to determine plasma levels of bryostatin-1. Thus, the different administration schedules have been selected based on the mechanism of action of bryostatin-1. For example, the choice of the 24-h infusion was to produce a prolonged inhibition of PKC within tumour cells to maximize the drug's antiproliferative effect against leukemias and lymphomas [29], which is the opposite of what we plan to achieve. Previous studies from our group suggested that high concentrations of bryostatin-1 (25 nmol/l or higher) could induce the translocation of PKC to the plasma membrane and then activate NF-κB through a pathway that involves IκBα phosphorylation and degradation [12]. In contrast low concentrations of bryostatin-1 (10 nmol/l or lower) may translocate PKCα and Ras-GRP1 to cellular localizations other than plasma membrane, and could mediate the activation of the Ras-Raf-ERK pathway and the activation of nuclear SP1 (unpublished results). Hence, low concentrations of bryostatin-1 in vitro appear able to provide PKC activation for antagonizing HIV-1 latency minimizing the risk of toxicities. This remains to be proven in vivo. To our knowledge, only one study has been able to assess the pharmacokinetics of bryostatin-1. Following multiple doses of bryostatin-1 (from 6 to 20 μg/m2 administered over 14–21 days in continuous infusion) in patients with myeloid malignancies, Smith et al., 2011 used a validated LC/MS/MS assay and detected C ss ranging roughly from 100 pg/ml (0.11 nmol/l) to 1000 pg/ml (1.1 nmol/l) [15]. Significantly, patients who received doses below 12 μg/m2, as well as some of those receiving higher doses, had exposures below the limit of quantitation of the method (50 pg/ml). Consistent with these results, we observed low plasma concentrations of bryostatin-1 with the doses used in our study.

Although previous trials aimed at achieving HIV-1 reactivation have focused on HDAC inhibitors, we have assessed in the present study, for the first time, a candidate LRA with a distinctly different mechanism compared with previous trials [7–9,30,31], namely, PKC activation. The main limitation of our present clinical trial appears to be that the assessed doses were insufficient to induce PKC activation and, hence, evaluating higher doses may be warranted in future studies to prove its efficacy.

We herein have demonstrated the safety and tolerability of single doses of bryostatin-1. The road is paved for next trials in humans aimed at inducing HIV-1 transcription from the latent reservoirs. Next trials of bryostatin-1 for HIV curative interventions are warranted to optimize the drug's reactivation potential and to explore its effects on the latent HIV reservoir when administered at multiple doses. As repeated doses of single LRA or in combination with other LRAs is ultimately envisioned as the treatment regimen for HIV cure purposes, multiple ascending dosing schedules of bryostatin-1 need to be assessed. A phase I/II study with single higher doses or a multiple ascending doses study to assess the safety, tolerability and efficacy of bryostatin-1 to reactivate latent HIV-1 reservoirs in blood and gut-associated lymphoid tissue might be the next step forward. Bryostatin-1 is also of extreme interest to be considered as a part of combination regimens with immune enhancement treatments as a part of a shock and kill strategy [5]. There is a wide consensus that successful latency disruption might require targeting different pathways involved in the maintenance of HIV quiescence. As mentioned, HDAC inhibitors and PKC agonistics have shown in-vitro [32,33] and ex-vivo [23] synergistic activity with bryostatin-1 and in consequence, might be an attractive combination. On the other hand however, HDAC inhibitors have already been shown to induce HIV-1 reactivation from latently infected cells in vivo [7,8]. This remains to be proven for bryostatin-1, despite the exceptional potency shown ex vivo [22,23]. The proof of concept that bryostatin-1 can disrupt HIV-1 latency in vivo still needs to be demonstrated in clinical studies.

In summary, our findings will inform next trials with bryostatin-1 designed to activate and eliminate the latent HIV-1 reservoir. Bryostatin-1 has proved to be safe and well tolerated in single doses and is a promising agent to be considered as a part of combination strategies aimed at curing HIV-1 infection.

Back to Top | Article Outline

Acknowledgements

The authors thank Ms Ester Dominguez for technical assistance. We thank Dr Sharon Lewin and Dr Ajantha Solomon for their assistance with the establishment of the cell-associated unspliced RNA measurement. This work was supported in part by the RD12/0017/0017 and RD12/0017/0037 projects as part of the Plan Nacional R + D + I and co-funded by Instituto de Salud Carlos III (ISCIII) – Subdirección General de Evaluación and Fondo Europeo de Desarrollo Regional (FEDER).

Author contributions – conceived and designed the clinical trial: C.G., S.M and T.C.; conceived and designed the experiments: C.G, N.M-E, and S.M.; performed the experiments: M.E.M, C.B, J.R., E.M. and M.A.M-F; managed study participant recruitment and follow-up: S.S., M.J.P-E. and S.M.; analysed the data: C.G., S.S-V, N.M., M.E.M., C.B., M.A.M-F and S.M.; contributed reagents/materials/analysis tools: C.G., S.S., N.M-E, M.E.M-F and C.B.; contributed to the writing of the manuscript: C.G., S.S-V and S.M. All the authors contributed to the writing of the final version and reviewed the manuscript for approval.

Back to Top | Article Outline

Conflicts of interest

There are no conflicts of interest.

Back to Top | Article Outline

References

1. Chun TW, Stuyver L, Mizell SB, Ehler LA, Mican JA, Baseler M, et al. Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. PNAS 1997; 94:13193–13197.
2. Chun TW, Carruth L, Finzi D, Shen X, DiGiuseppe JA, Taylor H, et al. Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature 1997; 387:183–188.
3. Finzi D, Hermankova M, Pierson T, Carruth LM, Buck C, Chaisson RE, et al. Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science 1997; 278:1295–1300.
4. Blankson JN, Persaud D, Siliciano RF. The challenge of viral reservoirs in HIV-1 infection. Annu Rev Med 2002; 53:557–593.
5. Eisele E, Siliciano RF. Redefining the viral reservoirs that prevent HIV-1 eradication. Immunity 2012; 37:377–388.
6. Xing S, Siliciano RF. Targeting HIV latency: pharmacologic strategies toward eradication. Drug Discov Today 2013; 18 (11–12):541–551.
7. Archin NM, Liberty L, Kashuba D, Choudhary SK, Kuruc JD, Crooks M, et al. Administration of vorinostat disrupts HIV-1 latency in patients on antiretroviral therapy. Nature 2012; 487:482–485.
8. Rasmussen T, Tolstrup M, Brinkmann CR, Olesen R, Erikstrup C, Solomon A, et al. Panobinostat, a histone deacetylase inhibitor, for latent-virus reactivation in HIV-infected patients on suppressive antiretroviral therapy: a phase 1/2, single group, clinical trial. Lancet HIV 2014; 1:e1413-21.
9. Elliott JH, Wightman F, Solomon A, Ghneim K, Ahlers J, Cameron MJ, et al. Activation of HIV transcription with short-course vorinostat in HIV-infected patients on suppressive antiretroviral therapy. PLoS Pathog 2014; 10:e1004473.
10. Zhang X, Zhang R, Zhao Cai H, Gush K, Kerr RG, et al. Preclinical pharmacology of the natural product anticancer agent bryostatin 1, an activator of protein kinase C. Cancer Res 1996; 56:802–808.
11. Amador ML, Jimeno J, Paz-Ares L, Cortes-Funes H, Hidalgo M. Progress in the development and acquisition of anticancer agents from marine sources. Ann Oncol 2003; 14:1607–1615.
12. Pérez M, de Vinuesa AG, Sanchez-Duffhues G, Marquez N, Bellido ML, Muñoz-Fernandez MA, et al. Bryostatin-1 synergizes with histone deacetylase inhibitors to reactivate HIV-1 from latency. Curr HIV Res 2010; 8:418–429.
13. Plimack ER, Tan T, Wong Y-N, von Mehren MM, Malizzia L, Roethke SK, et al. A phase I study of temsirolimus and bryostatin-1 in patients with metastatic renal cell carcinoma and soft tissue sarcoma. Oncologist 2014; 19:354–355.
14. Morgan RJ, Leong L, Chow W, Gandara D, Frankel P, Garcia A, et al. Phase II trial of bryostatin-1 in combination with cisplatin in patients with recurrent or persistent epithelial ovarian cancer: a California cancer consortium study. Invest New Drugs 2012; 30:723–728.
15. Smith BD, Jones RJ, Cho E, Kowalski J, Karp JE, Gore SD, et al. Differentiation therapy in poor risk myeloid malignancies: results of a dose finding study of the combination bryostatin-1 and GM-CSF. Leuk Res 2011; 35:87–94.
16. Pavlick AC, Wu J, Roberts J, Rosenthal M, Hamilton A, Wadler S, et al. Phase I study of bryostatin-1, a protein kinase C modulator, preceding cisplatin in patients with refractory nonhematologic tumors. Cancer Chemother Pharmacol 2009; 64:803–810.
17. Ajani J, Jiang Y, Faust J, Chang BB, Ho L, Yao JC, et al. A multicenter phase II study of sequential paclitaxel and bryostatin-1 (NSC 339555) in patients with untreated, advanced gastric or gastroesophageal junction adenocarcinoma. Invest New Drugs 2006; 24:353–357.
18. Zhao M, Rudek M, He P, Smith BD, Baker SD. Validation and implementation of a method for determination of bryostatin 1 in human plasma by using liquid chromatography/tandem mass spectrometry. Anal Biochem 2005; 337:143–148.
19. Mehla R, Bivalkar-Mehla S, Zhang R, Handy I, Albrecht H, Giri S, et al. Bryostatin modulates latent HIV-1 infection via PKC and AMPK signaling but inhibits acute infection in a receptor independent manner. PLoS One 2010; 5:e11160.
20. Martínez-Bonet M, Clemente MI, Álvarez S, Díaz L, García-Alonso D, Muñoz E, et al. Antiretroviral drugs do not interfere with bryostatin-mediated HIV-1 latency reversal. Antiviral Res 2015; 123:163–171.
21. Díaz L, Martínez-Bonet M, Sánchez J, Fernández-Pineda A, Jiménez JL, Muñoz E, et al. Bryostatin activates HIV-1 latent expression in human astrocytes through a PKC and NF-kB-dependent mechanism. Sci Rep 2015; 5:12442.
22. Bullen CK, Laird GM, Durand CM, Siliciano JD, Siliciano RF. New ex vivo approaches distinguish effective and ineffective single agents for reversing HIV-1 latency in vivo. Nat Med 2014; 20:425–429.
23. Laird GM, Bullen CK, Rosenbloom DIS, Martin AR, Hill AL, Durand CM, et al. Ex vivo analysis identifies effective HIV-1 latency-reversing drug combinations. J Clin Invest 2015; 125:1901–1912.
24. El-Rayes BF, Gadgeel S, Shields AF, Manza S, Lorusso P, Philip PA. Phase I study of bryostatin 1 and gemcitabine. Clin Cancer Res 2006; 12:7059–7062.
25. Lewin SR, Vesanen M, Kostrikis L, Hurley A, Duran M, Zhang L, et al. Use of real-time PCR and molecular beacons to detect virus replication in human immunodeficiency virus type 1-infected individuals on prolonged effective antiretroviral therapy. J Virol 1999; 73:6099–6103.
26. Fischer M, Huber W, Kallivroussis A, Ott P, Opravil M, Lüthy R, et al. Highly sensitive methods for quantitation of human immunodeficiency virus type 1 RNA from plasma, cells, and tissues. J Clin Microbiol 1999; 37:1260–1264.
27. Yukl SA, Gianella S, Sinclair E, Epling L, Li Q, Duan L, et al. Differences in HIV burden and immune activation within the gut of HIV-positive patients receiving suppressive antiretroviral therapy. J Infect Dis 2010; 202:1553–1561.
28. Varterasian ML, Mohammad RM, Eilender DS, Hulburd K, Rodriguez DH, Pemberton PA, et al. Phase I study of bryostatin 1 in patients with relapsed non-Hodgkin's lymphoma and chronic lymphocytic leukemia. J Clin Oncol 1998; 16:56–62.
29. Jones RJ, Sharkis SJ, Miller CB, Rowinsky EK, Burke PJ, May WS. Bryostatin 1, a unique biologic response modifier: antileukemic activity in vitro. Blood 1990; 75:1319–1323.
30. Archin NM, Bateson R, Tripathy M, Crooks AM, Yang KH, Dahl NP, et al. HIV-1 expression within resting CD4 T-cells following multiple doses of vorinostat. J Infect Dis 2014; 210:728–735.
31. S⊘gaard OS, Graversen ME, Leth S, Olesen R, Brinkmann CR, Nissen SK, et al. The depsipeptide romidepsin reverses HIV-1 latency in vivo. PLoS Pathog 2015; 11:e1005142.
32. Reuse S, Calao M, Kabeya K, Guiguen A, Gatot J-S, Quivy V, et al. Synergistic activation of HIV-1 expression by deacetylase inhibitors and prostratin: implications for treatment of latent infection. PLoS One 2009; 4:e6093.
33. Martínet-Bonet M, Clemente I, Serramía MJ, Muñoz E, Moreno S, Muñoz-Fernández MA. Synergistic activation of latent HIV-1 expression by novel histone deacetylase inhibitors and bryostatin-1. Sci Rep 2015; 13:16445.
Keywords:

bryostatin-1; HIV eradication; HIV-1 reactivation; HIV-latency; protein kinase C; reservoirs

Copyright © 2016 Wolters Kluwer Health, Inc.