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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 JQ1 induce HIV-1 recovery in resting CD4+ T cells from cART-treated HIV+ aviremic patients.Ex vivo cultures of resting CD4+ T cells purified from blood of 15 patients were mock-treated or treated with anti-CD3+anti-CD28 antibodies, JQ1(0.25μM), prostratin (0.5μM), bryostatin-1 (5nM) or ing-B (10nM) alone or in combination as indicated. Six days post-treatment, concentrations of 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 resting CD4+ T cells. Values representing higher viral production after a combinatory treatment than after the corresponding single drug treatments are shown in grey. Values representing reactivation of viral production observed exclusively after combined treatment 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.g005: PKC agonists and JQ1 induce HIV-1 recovery in resting CD4+ T cells from cART-treated HIV+ aviremic patients.Ex vivo cultures of resting CD4+ T cells purified from blood of 15 patients were mock-treated or treated with anti-CD3+anti-CD28 antibodies, JQ1(0.25μM), prostratin (0.5μM), bryostatin-1 (5nM) or ing-B (10nM) alone or in combination as indicated. Six days post-treatment, concentrations of 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 resting CD4+ T cells. Values representing higher viral production after a combinatory treatment than after the corresponding single drug treatments are shown in grey. Values representing reactivation of viral production observed exclusively after combined treatment are shown in black. ‘I’ indicates below the 150 HIV-1 RNA copies/ml limit of detection. ‘//’ indicates not tested conditions.

Mentions: Among the cell types present in CD8+-depleted PBMCs, latently infected resting CD4+ T cells represent the major reservoir of HIV infection. We therefore evaluated the reactivation potential of individual and combined treatments in this cell type. Fig 3A and Fig 4 showed that PKC agonist+JQ1 co-treatments led to (i) the highest percentages of reactivated patient cell cultures and (ii) important increases in extracellular viral RNA compared to other PKC agonist+BETi/HMBA co-treatments in CD8+-depleted PBMCs purified from blood of cART-treated HIV+ aviremic patients. Therefore, we evaluated in resting CD4+ T cells the reactivation potentials of JQ1 alone or combined with either prostratin or bryostatin-1 or another PKC agonist, ing-B. Indeed, while we were performing this study, ing-B has been reported to potently reactivate latent HIV-1 [25,26] in vitro and we decided to evaluate here its potential ex vivo. We isolated HLA-DR-, CD69-, CD25- CD4+ T cells from blood of 15 cART-treated HIV+ aviremic patients. Ex vivo cell cultures were mock-treated, treated with anti-CD3+anti-CD28 antibodies or with the compounds of interest (Fig 5 and S1 Table). Cell-associated total HIV-1 DNA and extracellular viral RNA were quantified. We detected HIV-1 RNA in 3 out of 15 patient’s cultures (20%) in mock-treated conditions. Prostratin, bryostatin-1, ing-B, and JQ1 reactivated latent HIV-1 in 60%, 53%, 53%, and 40% of the cultures, respectively (Fig 5). All individual treatments produced increases in mean HIV-1 RNA copies/ml levels that were higher than the 150 copies/ml threshold (Fig 3B). Notably, the combination bryostatin-1+JQ1 and ing-B+JQ1 produced higher increases in mean viral RNA levels relative to the individual treatments. These bryostatin-1+JQ1 and ing-B+JQ1-induced increases reached 1127 and 1547 HIV-1 RNA copies/ml, respectively (Fig 5). Importantly, the very high levels of reactivated virus following those treatments were statistically non-inferior (p>0.05) to the level obtained after anti-CD3+anti-CD28 stimulation. In contrast, we did not observe any benefit of combining prostratin with JQ1. All the combined treatments did not increase the frequency of reactivated patient cell cultures relative to the individual treatments, but synergistically increased HIV-1 recovery in most of the cultures (Fig 5, see black and grey boxes). For instance, the ing-B+JQ1 co-treatment led to reactivation in 46% of ex vivo cultures and, remarkably, all these cultures exhibited strong synergistic increases. Of note, we also performed individual and combined treatments using ing-B in CD8+-depleted PBMCs from cART-treated HIV+ aviremic patients and we observed strong reactivations of latent HIV-1. This part of our work concerning ing-B individual treatment of patient cell cultures will be reported in a separate study (L. Gama and colleagues, manuscript in preparation).


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 resting CD4+ T cells from cART-treated HIV+ aviremic patients.Ex vivo cultures of resting CD4+ T cells purified from blood of 15 patients were mock-treated or treated with anti-CD3+anti-CD28 antibodies, JQ1(0.25μM), prostratin (0.5μM), bryostatin-1 (5nM) or ing-B (10nM) alone or in combination as indicated. Six days post-treatment, concentrations of 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 resting CD4+ T cells. Values representing higher viral production after a combinatory treatment than after the corresponding single drug treatments are shown in grey. Values representing reactivation of viral production observed exclusively after combined treatment 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

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

ppat.1005063.g005: PKC agonists and JQ1 induce HIV-1 recovery in resting CD4+ T cells from cART-treated HIV+ aviremic patients.Ex vivo cultures of resting CD4+ T cells purified from blood of 15 patients were mock-treated or treated with anti-CD3+anti-CD28 antibodies, JQ1(0.25μM), prostratin (0.5μM), bryostatin-1 (5nM) or ing-B (10nM) alone or in combination as indicated. Six days post-treatment, concentrations of 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 resting CD4+ T cells. Values representing higher viral production after a combinatory treatment than after the corresponding single drug treatments are shown in grey. Values representing reactivation of viral production observed exclusively after combined treatment are shown in black. ‘I’ indicates below the 150 HIV-1 RNA copies/ml limit of detection. ‘//’ indicates not tested conditions.
Mentions: Among the cell types present in CD8+-depleted PBMCs, latently infected resting CD4+ T cells represent the major reservoir of HIV infection. We therefore evaluated the reactivation potential of individual and combined treatments in this cell type. Fig 3A and Fig 4 showed that PKC agonist+JQ1 co-treatments led to (i) the highest percentages of reactivated patient cell cultures and (ii) important increases in extracellular viral RNA compared to other PKC agonist+BETi/HMBA co-treatments in CD8+-depleted PBMCs purified from blood of cART-treated HIV+ aviremic patients. Therefore, we evaluated in resting CD4+ T cells the reactivation potentials of JQ1 alone or combined with either prostratin or bryostatin-1 or another PKC agonist, ing-B. Indeed, while we were performing this study, ing-B has been reported to potently reactivate latent HIV-1 [25,26] in vitro and we decided to evaluate here its potential ex vivo. We isolated HLA-DR-, CD69-, CD25- CD4+ T cells from blood of 15 cART-treated HIV+ aviremic patients. Ex vivo cell cultures were mock-treated, treated with anti-CD3+anti-CD28 antibodies or with the compounds of interest (Fig 5 and S1 Table). Cell-associated total HIV-1 DNA and extracellular viral RNA were quantified. We detected HIV-1 RNA in 3 out of 15 patient’s cultures (20%) in mock-treated conditions. Prostratin, bryostatin-1, ing-B, and JQ1 reactivated latent HIV-1 in 60%, 53%, 53%, and 40% of the cultures, respectively (Fig 5). All individual treatments produced increases in mean HIV-1 RNA copies/ml levels that were higher than the 150 copies/ml threshold (Fig 3B). Notably, the combination bryostatin-1+JQ1 and ing-B+JQ1 produced higher increases in mean viral RNA levels relative to the individual treatments. These bryostatin-1+JQ1 and ing-B+JQ1-induced increases reached 1127 and 1547 HIV-1 RNA copies/ml, respectively (Fig 5). Importantly, the very high levels of reactivated virus following those treatments were statistically non-inferior (p>0.05) to the level obtained after anti-CD3+anti-CD28 stimulation. In contrast, we did not observe any benefit of combining prostratin with JQ1. All the combined treatments did not increase the frequency of reactivated patient cell cultures relative to the individual treatments, but synergistically increased HIV-1 recovery in most of the cultures (Fig 5, see black and grey boxes). For instance, the ing-B+JQ1 co-treatment led to reactivation in 46% of ex vivo cultures and, remarkably, all these cultures exhibited strong synergistic increases. Of note, we also performed individual and combined treatments using ing-B in CD8+-depleted PBMCs from cART-treated HIV+ aviremic patients and we observed strong reactivations of latent HIV-1. This part of our work concerning ing-B individual treatment of patient cell cultures will be reported in a separate study (L. Gama and colleagues, manuscript in preparation).

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