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An In-Depth Comparison of Latency-Reversing Agent Combinations in Various In Vitro and Ex Vivo HIV-1 Latency Models Identified Bryostatin-1+JQ1 and Ingenol-B+JQ1 to Potently Reactivate Viral Gene Expression.

Darcis G, Kula A, Bouchat S, Fujinaga K, Corazza F, Ait-Ammar A, Delacourt N, Melard A, Kabeya K, Vanhulle C, Van Driessche B, Gatot JS, Cherrier T, Pianowski LF, Gama L, Schwartz C, Vila J, Burny A, Clumeck N, Moutschen M, De Wit S, Peterlin BM, Rouzioux C, Rohr O, Van Lint C - PLoS Pathog. (2015)

Bottom Line: Mechanistically, combined treatments led to higher activations of P-TEFb and NF-κB than the corresponding individual drug treatments.The potent effects of these two combination treatments were already detected 24 hours post-stimulation.These results constitute the first demonstration of LRA combinations exhibiting such a potent effect and represent a proof-of-concept for the co-administration of two different types of LRAs as a potential strategy to reduce the size of the latent HIV-1 reservoirs.

View Article: PubMed Central - PubMed

Affiliation: Service of Molecular Virology, Institut de Biologie et de Médecine Moléculaires (IBMM), Université Libre de Bruxelles (ULB), Gosselies, Belgium; Service des Maladies Infectieuses, Université de Liège, Centre Hospitalier Universitaire (CHU) de Liège, Domaine Universitaire du Sart-Tilman, Liège, Belgium.

ABSTRACT
The persistence of latently infected cells in patients under combinatory antiretroviral therapy (cART) is a major hurdle to HIV-1 eradication. Strategies to purge these reservoirs are needed and activation of viral gene expression in latently infected cells is one promising strategy. Bromodomain and Extraterminal (BET) bromodomain inhibitors (BETi) are compounds able to reactivate latent proviruses in a positive transcription elongation factor b (P-TEFb)-dependent manner. In this study, we tested the reactivation potential of protein kinase C (PKC) agonists (prostratin, bryostatin-1 and ingenol-B), which are known to activate NF-κB signaling pathway as well as P-TEFb, used alone or in combination with P-TEFb-releasing agents (HMBA and BETi (JQ1, I-BET, I-BET151)). Using in vitro HIV-1 post-integration latency model cell lines of T-lymphoid and myeloid lineages, we demonstrated that PKC agonists and P-TEFb-releasing agents alone acted as potent latency-reversing agents (LRAs) and that their combinations led to synergistic activation of HIV-1 expression at the viral mRNA and protein levels. Mechanistically, combined treatments led to higher activations of P-TEFb and NF-κB than the corresponding individual drug treatments. Importantly, we observed in ex vivo cultures of CD8+-depleted PBMCs from 35 cART-treated HIV-1+ aviremic patients that the percentage of reactivated cultures following combinatory bryostatin-1+JQ1 treatment was identical to the percentage observed with anti-CD3+anti-CD28 antibodies positive control stimulation. Remarkably, in ex vivo cultures of resting CD4+ T cells isolated from 15 HIV-1+ cART-treated aviremic patients, the combinations bryostatin-1+JQ1 and ingenol-B+JQ1 released infectious viruses to levels similar to that obtained with the positive control stimulation. The potent effects of these two combination treatments were already detected 24 hours post-stimulation. These results constitute the first demonstration of LRA combinations exhibiting such a potent effect and represent a proof-of-concept for the co-administration of two different types of LRAs as a potential strategy to reduce the size of the latent HIV-1 reservoirs.

No MeSH data available.


Related in: MedlinePlus

PKC agonists and compounds releasing active P-TEFb induce HIV-1 recovery in CD8+-depleted PBMCs from cART-treated HIV+ aviremic patients.Ex vivo cultures of CD8+-depleted PBMCs purified from blood of 24 patients were mock-treated or treated with anti-CD3+anti-CD28 antibodies, prostratin (0.5μM), bryostatin-1 (5nM), JQ1 (0.25μM), I-BET (0.25μM), I-BET151 (0.25μM) or HMBA (5mM) alone or in combination as indicated. Six days post-treatment, concentrations of genomic viral RNA in culture supernatants were determined and the values were expressed as HIV-1 RNA copies/ml. Total HIV-1 DNA was expressed as HIV-1 DNA copies/106 CD8+-depleted PBMCs. Values representing higher viral production after the combined treatment than after the single drug treatments are shown in grey. Values representing reactivation of the virus observed exclusively after combined treatments are shown in black. ‘I’ indicates below the 150 HIV-1 RNA copies/ml limit of detection. ‘//’ indicates not tested conditions.
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ppat.1005063.g004: PKC agonists and compounds releasing active P-TEFb induce HIV-1 recovery in CD8+-depleted PBMCs from cART-treated HIV+ aviremic patients.Ex vivo cultures of CD8+-depleted PBMCs purified from blood of 24 patients were mock-treated or treated with anti-CD3+anti-CD28 antibodies, prostratin (0.5μM), bryostatin-1 (5nM), JQ1 (0.25μM), I-BET (0.25μM), I-BET151 (0.25μM) or HMBA (5mM) alone or in combination as indicated. Six days post-treatment, concentrations of genomic viral RNA in culture supernatants were determined and the values were expressed as HIV-1 RNA copies/ml. Total HIV-1 DNA was expressed as HIV-1 DNA copies/106 CD8+-depleted PBMCs. Values representing higher viral production after the combined treatment than after the single drug treatments are shown in grey. Values representing reactivation of the virus observed exclusively after combined treatments are shown in black. ‘I’ indicates below the 150 HIV-1 RNA copies/ml limit of detection. ‘//’ indicates not tested conditions.

Mentions: In vitro models for HIV-1 latency do not necessarily recapitulate the events occurring during viral latency ex vivo [44]. Above we showed high potential in viral reactivation of treatments combining PKC agonists and P-TEFb-releasing agents in vitro in HIV-1 latency models of lymphoid and myeloid cell origins. We next determined whether the combined treatments also correlated with HIV-1 recovery in ex vivo cultures of CD8+-depleted PBMCs and resting CD4+ T cells isolated from cART-treated aviremic HIV-1+ patients. Firstly, we evaluated cellular viability in CD8+-depleted PBMCs cultures purified from blood of 5 uninfected donors following drug treatments (S5 Fig). BETi, HMBA and bryostatin-1 did not alter cellular metabolic activity at any concentration tested. However, we observed a dose-dependent decrease in metabolic activity in prostratin-treated CD8+-depleted PBMCs with a metabolic activity of 25% for the above used prostratin concentration (2.5μM). Therefore, we chose for further experiments a dose of prostratin (0.5μM) presenting a cellular metabolic activity of 60%. In order to evaluate HIV-1 recovery, CD8+-depleted PBMCs purified from blood of 24 cART-treated HIV+ aviremic patients were mock-treated, treated with anti-CD3+anti-CD28 antibodies as a positive control for global T cell activation or with indicated LARs (Fig 3A, Fig 4 and S1 Table). Cell-associated total HIV-1 DNA and extracellular viral RNA were quantified. The number of proviruses seeded in each well was estimated from the number of HIV-1 DNA copies/106 cells determined by qPCR assuming negligible unintegrated HIV-1 DNA in patients after long-term cART [8]. Measurements of extracellular HIV RNA represents a surrogate marker for virions release from treated cell cultures. Indeed, Mellors’ group has highlighted the necessity to measure viral production and not only unspliced cellular HIV-1 RNA following HIV-1 reactivation since these authors did not observe significant correlation between these two methods after treatment with an other LRA, the HDACi, SAHA [45]. We assumed that extracellular virions would accumulate over the course of several days; therefore, the duration of culture was extended to 6 days to maximize assay sensitivity.


An In-Depth Comparison of Latency-Reversing Agent Combinations in Various In Vitro and Ex Vivo HIV-1 Latency Models Identified Bryostatin-1+JQ1 and Ingenol-B+JQ1 to Potently Reactivate Viral Gene Expression.

Darcis G, Kula A, Bouchat S, Fujinaga K, Corazza F, Ait-Ammar A, Delacourt N, Melard A, Kabeya K, Vanhulle C, Van Driessche B, Gatot JS, Cherrier T, Pianowski LF, Gama L, Schwartz C, Vila J, Burny A, Clumeck N, Moutschen M, De Wit S, Peterlin BM, Rouzioux C, Rohr O, Van Lint C - PLoS Pathog. (2015)

PKC agonists and compounds releasing active P-TEFb induce HIV-1 recovery in CD8+-depleted PBMCs from cART-treated HIV+ aviremic patients.Ex vivo cultures of CD8+-depleted PBMCs purified from blood of 24 patients were mock-treated or treated with anti-CD3+anti-CD28 antibodies, prostratin (0.5μM), bryostatin-1 (5nM), JQ1 (0.25μM), I-BET (0.25μM), I-BET151 (0.25μM) or HMBA (5mM) alone or in combination as indicated. Six days post-treatment, concentrations of genomic viral RNA in culture supernatants were determined and the values were expressed as HIV-1 RNA copies/ml. Total HIV-1 DNA was expressed as HIV-1 DNA copies/106 CD8+-depleted PBMCs. Values representing higher viral production after the combined treatment than after the single drug treatments are shown in grey. Values representing reactivation of the virus observed exclusively after combined treatments are shown in black. ‘I’ indicates below the 150 HIV-1 RNA copies/ml limit of detection. ‘//’ indicates not tested conditions.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520688&req=5

ppat.1005063.g004: PKC agonists and compounds releasing active P-TEFb induce HIV-1 recovery in CD8+-depleted PBMCs from cART-treated HIV+ aviremic patients.Ex vivo cultures of CD8+-depleted PBMCs purified from blood of 24 patients were mock-treated or treated with anti-CD3+anti-CD28 antibodies, prostratin (0.5μM), bryostatin-1 (5nM), JQ1 (0.25μM), I-BET (0.25μM), I-BET151 (0.25μM) or HMBA (5mM) alone or in combination as indicated. Six days post-treatment, concentrations of genomic viral RNA in culture supernatants were determined and the values were expressed as HIV-1 RNA copies/ml. Total HIV-1 DNA was expressed as HIV-1 DNA copies/106 CD8+-depleted PBMCs. Values representing higher viral production after the combined treatment than after the single drug treatments are shown in grey. Values representing reactivation of the virus observed exclusively after combined treatments are shown in black. ‘I’ indicates below the 150 HIV-1 RNA copies/ml limit of detection. ‘//’ indicates not tested conditions.
Mentions: In vitro models for HIV-1 latency do not necessarily recapitulate the events occurring during viral latency ex vivo [44]. Above we showed high potential in viral reactivation of treatments combining PKC agonists and P-TEFb-releasing agents in vitro in HIV-1 latency models of lymphoid and myeloid cell origins. We next determined whether the combined treatments also correlated with HIV-1 recovery in ex vivo cultures of CD8+-depleted PBMCs and resting CD4+ T cells isolated from cART-treated aviremic HIV-1+ patients. Firstly, we evaluated cellular viability in CD8+-depleted PBMCs cultures purified from blood of 5 uninfected donors following drug treatments (S5 Fig). BETi, HMBA and bryostatin-1 did not alter cellular metabolic activity at any concentration tested. However, we observed a dose-dependent decrease in metabolic activity in prostratin-treated CD8+-depleted PBMCs with a metabolic activity of 25% for the above used prostratin concentration (2.5μM). Therefore, we chose for further experiments a dose of prostratin (0.5μM) presenting a cellular metabolic activity of 60%. In order to evaluate HIV-1 recovery, CD8+-depleted PBMCs purified from blood of 24 cART-treated HIV+ aviremic patients were mock-treated, treated with anti-CD3+anti-CD28 antibodies as a positive control for global T cell activation or with indicated LARs (Fig 3A, Fig 4 and S1 Table). Cell-associated total HIV-1 DNA and extracellular viral RNA were quantified. The number of proviruses seeded in each well was estimated from the number of HIV-1 DNA copies/106 cells determined by qPCR assuming negligible unintegrated HIV-1 DNA in patients after long-term cART [8]. Measurements of extracellular HIV RNA represents a surrogate marker for virions release from treated cell cultures. Indeed, Mellors’ group has highlighted the necessity to measure viral production and not only unspliced cellular HIV-1 RNA following HIV-1 reactivation since these authors did not observe significant correlation between these two methods after treatment with an other LRA, the HDACi, SAHA [45]. We assumed that extracellular virions would accumulate over the course of several days; therefore, the duration of culture was extended to 6 days to maximize assay sensitivity.

Bottom Line: Mechanistically, combined treatments led to higher activations of P-TEFb and NF-κB than the corresponding individual drug treatments.The potent effects of these two combination treatments were already detected 24 hours post-stimulation.These results constitute the first demonstration of LRA combinations exhibiting such a potent effect and represent a proof-of-concept for the co-administration of two different types of LRAs as a potential strategy to reduce the size of the latent HIV-1 reservoirs.

View Article: PubMed Central - PubMed

Affiliation: Service of Molecular Virology, Institut de Biologie et de Médecine Moléculaires (IBMM), Université Libre de Bruxelles (ULB), Gosselies, Belgium; Service des Maladies Infectieuses, Université de Liège, Centre Hospitalier Universitaire (CHU) de Liège, Domaine Universitaire du Sart-Tilman, Liège, Belgium.

ABSTRACT
The persistence of latently infected cells in patients under combinatory antiretroviral therapy (cART) is a major hurdle to HIV-1 eradication. Strategies to purge these reservoirs are needed and activation of viral gene expression in latently infected cells is one promising strategy. Bromodomain and Extraterminal (BET) bromodomain inhibitors (BETi) are compounds able to reactivate latent proviruses in a positive transcription elongation factor b (P-TEFb)-dependent manner. In this study, we tested the reactivation potential of protein kinase C (PKC) agonists (prostratin, bryostatin-1 and ingenol-B), which are known to activate NF-κB signaling pathway as well as P-TEFb, used alone or in combination with P-TEFb-releasing agents (HMBA and BETi (JQ1, I-BET, I-BET151)). Using in vitro HIV-1 post-integration latency model cell lines of T-lymphoid and myeloid lineages, we demonstrated that PKC agonists and P-TEFb-releasing agents alone acted as potent latency-reversing agents (LRAs) and that their combinations led to synergistic activation of HIV-1 expression at the viral mRNA and protein levels. Mechanistically, combined treatments led to higher activations of P-TEFb and NF-κB than the corresponding individual drug treatments. Importantly, we observed in ex vivo cultures of CD8+-depleted PBMCs from 35 cART-treated HIV-1+ aviremic patients that the percentage of reactivated cultures following combinatory bryostatin-1+JQ1 treatment was identical to the percentage observed with anti-CD3+anti-CD28 antibodies positive control stimulation. Remarkably, in ex vivo cultures of resting CD4+ T cells isolated from 15 HIV-1+ cART-treated aviremic patients, the combinations bryostatin-1+JQ1 and ingenol-B+JQ1 released infectious viruses to levels similar to that obtained with the positive control stimulation. The potent effects of these two combination treatments were already detected 24 hours post-stimulation. These results constitute the first demonstration of LRA combinations exhibiting such a potent effect and represent a proof-of-concept for the co-administration of two different types of LRAs as a potential strategy to reduce the size of the latent HIV-1 reservoirs.

No MeSH data available.


Related in: MedlinePlus