Limits...
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 JQ1 induce HIV-1 recovery in CD8+-depleted PBMCs from cART-treated HIV+ aviremic patients in the presence of cART.Ex vivo cultures of CD8+-depleted PBMCs purified from blood of 11 patients were mock-treated or treated with anti-CD3+anti-CD28 antibodies, JQ1 (0.25μM), bryostatin-1 (5nM) or ing-B (10nM) alone or in combination as indicated. Concentrations of viral RNA in culture supernatants were determined one day, three days or six days post-treatment. 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 a combinatory treatment than after the corresponding single drug treatments are shown in grey. Values representing reactivation of virus production observed exclusively after combined treatments are shown in black. ‘I’ indicates below the 15 HIV-1 RNA copies/ml limit of detection, ‘//’ indicates not tested conditions.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4520688&req=5

ppat.1005063.g006: PKC agonists and JQ1 induce HIV-1 recovery in CD8+-depleted PBMCs from cART-treated HIV+ aviremic patients in the presence of cART.Ex vivo cultures of CD8+-depleted PBMCs purified from blood of 11 patients were mock-treated or treated with anti-CD3+anti-CD28 antibodies, JQ1 (0.25μM), bryostatin-1 (5nM) or ing-B (10nM) alone or in combination as indicated. Concentrations of viral RNA in culture supernatants were determined one day, three days or six days post-treatment. 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 a combinatory treatment than after the corresponding single drug treatments are shown in grey. Values representing reactivation of virus production observed exclusively after combined treatments are shown in black. ‘I’ indicates below the 15 HIV-1 RNA copies/ml limit of detection, ‘//’ indicates not tested conditions.

Mentions: At day 1 (Fig 6 and S1 Table), the combination bryostatin-1+JQ1 was the only condition that caused viral reactivation in the cell cultures from 7 patients tested (100% of reactivation). Surprisingly, this combined treatment exceeded the effect observed after anti-CD3+anti-CD28 stimulation (p = 0.0256) (Fig 3C). The combination ing-B+JQ1 reactivated latent HIV-1 in 83% of cultures, a result identical to that observed with ing-B alone and that exceeded the percentage obtained after stimulation with the positive control (57%) (Fig 6). Importantly, this ing-B+JQ1 combination resulted in a higher mean level of extracellular HIV-1 RNA than the mean level obtained with ing-B alone (mean of 292 HIV-1 RNA copies/ml and of 160 HIV-1 RNA copies/ml, respectively (Fig 3C)).


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 JQ1 induce HIV-1 recovery in CD8+-depleted PBMCs from cART-treated HIV+ aviremic patients in the presence of cART.Ex vivo cultures of CD8+-depleted PBMCs purified from blood of 11 patients were mock-treated or treated with anti-CD3+anti-CD28 antibodies, JQ1 (0.25μM), bryostatin-1 (5nM) or ing-B (10nM) alone or in combination as indicated. Concentrations of viral RNA in culture supernatants were determined one day, three days or six days post-treatment. 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 a combinatory treatment than after the corresponding single drug treatments are shown in grey. Values representing reactivation of virus production observed exclusively after combined treatments are shown in black. ‘I’ indicates below the 15 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.g006: PKC agonists and JQ1 induce HIV-1 recovery in CD8+-depleted PBMCs from cART-treated HIV+ aviremic patients in the presence of cART.Ex vivo cultures of CD8+-depleted PBMCs purified from blood of 11 patients were mock-treated or treated with anti-CD3+anti-CD28 antibodies, JQ1 (0.25μM), bryostatin-1 (5nM) or ing-B (10nM) alone or in combination as indicated. Concentrations of viral RNA in culture supernatants were determined one day, three days or six days post-treatment. 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 a combinatory treatment than after the corresponding single drug treatments are shown in grey. Values representing reactivation of virus production observed exclusively after combined treatments are shown in black. ‘I’ indicates below the 15 HIV-1 RNA copies/ml limit of detection, ‘//’ indicates not tested conditions.
Mentions: At day 1 (Fig 6 and S1 Table), the combination bryostatin-1+JQ1 was the only condition that caused viral reactivation in the cell cultures from 7 patients tested (100% of reactivation). Surprisingly, this combined treatment exceeded the effect observed after anti-CD3+anti-CD28 stimulation (p = 0.0256) (Fig 3C). The combination ing-B+JQ1 reactivated latent HIV-1 in 83% of cultures, a result identical to that observed with ing-B alone and that exceeded the percentage obtained after stimulation with the positive control (57%) (Fig 6). Importantly, this ing-B+JQ1 combination resulted in a higher mean level of extracellular HIV-1 RNA than the mean level obtained with ing-B alone (mean of 292 HIV-1 RNA copies/ml and of 160 HIV-1 RNA copies/ml, respectively (Fig 3C)).

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