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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

Compounds releasing active P-TEFb and PKC agonists act synergistically to increase HIV-1 production.J-Lat 9.2 (panels A and C) and U1 (panels B and D) cell lines were mock-treated or treated with two doses of JQ1, I-BET, I-BET151, HMBA alone or in combination with three doses of either bryostatin-1 or prostratin as indicated. At 24 hours post-treatment, viral production was estimated by measuring CA-p24 antigen concentration in culture supernatants. The mock-treated value was arbitrarily set at a value of 1. Means and standard errors of the means from duplicate samples are indicated. One representative experiment from three is represented. For each combinatory treatment, the fold-synergy was calculated by dividing the effect observed after co-treatments by the sum of the effects obtained after the individual treatments.
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ppat.1005063.g001: Compounds releasing active P-TEFb and PKC agonists act synergistically to increase HIV-1 production.J-Lat 9.2 (panels A and C) and U1 (panels B and D) cell lines were mock-treated or treated with two doses of JQ1, I-BET, I-BET151, HMBA alone or in combination with three doses of either bryostatin-1 or prostratin as indicated. At 24 hours post-treatment, viral production was estimated by measuring CA-p24 antigen concentration in culture supernatants. The mock-treated value was arbitrarily set at a value of 1. Means and standard errors of the means from duplicate samples are indicated. One representative experiment from three is represented. For each combinatory treatment, the fold-synergy was calculated by dividing the effect observed after co-treatments by the sum of the effects obtained after the individual treatments.

Mentions: To assess whether compounds releasing active P-TEFb (JQ1, I-BET, I-BET151 and HMBA) synergize with PKC agonists (prostratin and bryostatin-1) in reactivating HIV from latency, we first determined their optimal concentrations in terms of both their HIV-1 reactivation potential and their cellular toxicity (S1 Fig). We measured induction of HIV-1 p24 capsid protein production by p24-enzyme-linked immunosorbent assay (ELISA) and we estimated the cell viability by tetrazolium salt-based assay which assesses cell metabolic activity in two well-studied HIV-1 latency cellular models, the T-lymphoid J-Lat 9.2 and promonocytic U1 cell lines. Increasing doses of compounds of interest augmented p24 antigen level in the cell supernatants at 24 hours post-treatment in a dose-dependent manner and caused a decrease in metabolic activity of less than 30% (S1 Fig). Based on our reactivation potential and cell viability data we selected two concentrations of BETi/HMBA and three concentrations of PKC agonists for combinations studies (Fig 1). Two compounds synergize when their combination produces higher effect than the sum of effects arising from separate treatments. We observed a synergistic activation of virus production in the J-Lat 9.2 cell line (Fig 1A and 1C). Bryostatin-1 at 5nM and 10nM produced synergistic increases in p24 production with each dose of BETi/HMBA (Fig 1A). Prostratin at 1.25μM and 2.5μM concentrations also synergized with each dose of BETi/HMBA and the fold-synergy was even higher than the one observed for bryostatin-1+BETi/HMBA combinations (compare Fig 1C to 1A). We also observed high synergistic activations of virus production in the promonocytic U1 cell line (Fig 1B and 1D). Interestingly, synergy in U1 cells was even higher than the one observed in J-Lat 9.2 cell line. Importantly, combined treatments of 10nM bryostatin-1 or 2.5μM prostratin with 0.5μM BETi and 5mM HMBA were the most potent among the tested combinations and produced in most cases the highest synergies in both cellular models. Of note, the same concentrations also produced synergistic activation of virus production in another HIV-1 latency model of promyelocytic origin, the OM10.1 cell line (S2 Fig). The OM10.1 cell line constitutes a more physiological model of HIV latency than the U1 monocytic cell line, since the latter contains HIV proviruses harboring mutations in tat gene. Therefore, we chose these drug concentrations for the next reactivation studies.


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)

Compounds releasing active P-TEFb and PKC agonists act synergistically to increase HIV-1 production.J-Lat 9.2 (panels A and C) and U1 (panels B and D) cell lines were mock-treated or treated with two doses of JQ1, I-BET, I-BET151, HMBA alone or in combination with three doses of either bryostatin-1 or prostratin as indicated. At 24 hours post-treatment, viral production was estimated by measuring CA-p24 antigen concentration in culture supernatants. The mock-treated value was arbitrarily set at a value of 1. Means and standard errors of the means from duplicate samples are indicated. One representative experiment from three is represented. For each combinatory treatment, the fold-synergy was calculated by dividing the effect observed after co-treatments by the sum of the effects obtained after the individual treatments.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1005063.g001: Compounds releasing active P-TEFb and PKC agonists act synergistically to increase HIV-1 production.J-Lat 9.2 (panels A and C) and U1 (panels B and D) cell lines were mock-treated or treated with two doses of JQ1, I-BET, I-BET151, HMBA alone or in combination with three doses of either bryostatin-1 or prostratin as indicated. At 24 hours post-treatment, viral production was estimated by measuring CA-p24 antigen concentration in culture supernatants. The mock-treated value was arbitrarily set at a value of 1. Means and standard errors of the means from duplicate samples are indicated. One representative experiment from three is represented. For each combinatory treatment, the fold-synergy was calculated by dividing the effect observed after co-treatments by the sum of the effects obtained after the individual treatments.
Mentions: To assess whether compounds releasing active P-TEFb (JQ1, I-BET, I-BET151 and HMBA) synergize with PKC agonists (prostratin and bryostatin-1) in reactivating HIV from latency, we first determined their optimal concentrations in terms of both their HIV-1 reactivation potential and their cellular toxicity (S1 Fig). We measured induction of HIV-1 p24 capsid protein production by p24-enzyme-linked immunosorbent assay (ELISA) and we estimated the cell viability by tetrazolium salt-based assay which assesses cell metabolic activity in two well-studied HIV-1 latency cellular models, the T-lymphoid J-Lat 9.2 and promonocytic U1 cell lines. Increasing doses of compounds of interest augmented p24 antigen level in the cell supernatants at 24 hours post-treatment in a dose-dependent manner and caused a decrease in metabolic activity of less than 30% (S1 Fig). Based on our reactivation potential and cell viability data we selected two concentrations of BETi/HMBA and three concentrations of PKC agonists for combinations studies (Fig 1). Two compounds synergize when their combination produces higher effect than the sum of effects arising from separate treatments. We observed a synergistic activation of virus production in the J-Lat 9.2 cell line (Fig 1A and 1C). Bryostatin-1 at 5nM and 10nM produced synergistic increases in p24 production with each dose of BETi/HMBA (Fig 1A). Prostratin at 1.25μM and 2.5μM concentrations also synergized with each dose of BETi/HMBA and the fold-synergy was even higher than the one observed for bryostatin-1+BETi/HMBA combinations (compare Fig 1C to 1A). We also observed high synergistic activations of virus production in the promonocytic U1 cell line (Fig 1B and 1D). Interestingly, synergy in U1 cells was even higher than the one observed in J-Lat 9.2 cell line. Importantly, combined treatments of 10nM bryostatin-1 or 2.5μM prostratin with 0.5μM BETi and 5mM HMBA were the most potent among the tested combinations and produced in most cases the highest synergies in both cellular models. Of note, the same concentrations also produced synergistic activation of virus production in another HIV-1 latency model of promyelocytic origin, the OM10.1 cell line (S2 Fig). The OM10.1 cell line constitutes a more physiological model of HIV latency than the U1 monocytic cell line, since the latter contains HIV proviruses harboring mutations in tat gene. Therefore, we chose these drug concentrations for the next reactivation studies.

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