In vivo analysis of the effect of panobinostat on cell-associated HIV RNA and DNA levels and latent HIV infection

Abstract

Background: The latent reservoir in resting CD4(+) T cells presents a major barrier to HIV cure. Latency-reversing agents are therefore being developed with the ultimate goal of disrupting the latent state, resulting in induction of HIV expression and clearance of infected cells. Histone deacetylase inhibitors (HDACi) have received a significant amount of attention for their potential as latency-reversing agents.

Results: Here, we have investigated the in vitro and systemic in vivo effect of panobinostat, a clinically relevant HDACi, on HIV latency. We showed that panobinostat induces histone acetylation in human PBMCs. Further, we showed that panobinostat induced HIV RNA expression and allowed the outgrowth of replication-competent virus ex vivo from resting CD4(+) T cells of HIV-infected patients on suppressive antiretroviral therapy (ART). Next, we demonstrated that panobinostat induced systemic histone acetylation in vivo in the tissues of BLT humanized mice. Finally, in HIV-infected, ART-suppressed BLT mice, we evaluated the effect of panobinostat on systemic cell-associated HIV RNA and DNA levels and the total frequency of latently infected resting CD4(+) T cells. Our data indicate that panobinostat treatment resulted in systemic increases in cellular levels of histone acetylation, a key biomarker for in vivo activity. However, panobinostat did not affect the levels of cell-associated HIV RNA, HIV DNA, or latently infected resting CD4(+) T cells.

Conclusion: We have demonstrated robust levels of systemic histone acetylation after panobinostat treatment of BLT humanized mice; and we did not observe a detectable change in the levels of cell-associated HIV RNA, HIV DNA, or latently infected resting CD4(+) T cells in HIV-infected, ART-suppressed BLT mice. These results are consistent with the modest effects noted in vitro and suggest that combination therapies may be necessary to reverse latency and enable clearance. Animal models will contribute to the progress towards an HIV cure.

Keywords: BLT; HIV; Histone acetylation; Humanized mice; Latency; Panobinostat.

Fig. 1

Effect of panobinostat on histone acetylation, HIV RNA, and viral outgrowth from patient cells. a Human PBMCs (n = 3) were incubated with panobinostat or DMSO control for 6 h, and histone H3 acetylation was measured by flow cytometry. b Resting CD4⁺ T cells were isolated from leukapheresis product obtained from three HIV-infected patients on suppressive antiretroviral therapy (outlined in Table 1), then pulsed with panobinostat (20 nM) or untreated. HIV RNA levels were measured from 10 to 12 individual wells (1 × 10⁶ cells each) by quantitative real-time PCR. c Resting CD4⁺ T cells were isolated from leukapheresis product obtained from seven HIV-infected patients on suppressive antiretroviral therapy (outlined in Table 1), then incubated with panobinostat (20 nM) or untreated. Viral outgrowth was measured by QVOA. Mean and SEM plotted with comparison by unpaired t test in a; Mann–Whitney test in b; Wilcoxon matched-pairs signed rank test in c: ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Blue bars/symbols denote control; pink and red bars/symbols denote panobinostat-treated

Fig. 2

Panobinostat administration induces systemic histone acetylation. NSG/BLT mice were administered panobinostat (2 mg/kg, n = 3) or vehicle (n = 5) intraperitoneally. After 24 h, tissues were harvested, and cells were isolated and resuspended in 1 % Triton-X/PBS. Cell lysates were analyzed for histone acetylation by ELISA. a Data from individual tissues. b Data from all tissues. Median and interquartile range plotted with comparisons by Mann–Whitney test: ns p > 0.05, *p < 0.05, ****p < 0.0001. Green circles denote control; mustard squares denote panobinostat-treated

Fig. 3

Outline of panobinostat treatment of HIV-infected, ART-suppressed BLT mice. a Experimental outline. BLT mice were infected intravenously with HIV-1_JR-CSF. Starting at 2 weeks post exposure, mice were bled weekly for plasma viral load and flow cytometry analysis. Starting at 3 weeks post exposure, mice were administered antiretroviral therapy (ART) consisting of tenofovir disoproxil fumarate, emtricitabine, and raltegravir (light blue shading). After 6 weeks of ART, one group of mice was administered panobinostat at a dose of 2 mg/kg intraperitoneally (red circles, n = 9) or vehicle (blue squares, n = 4), twice a week for 2 weeks, in addition to ART (light red shading). At the end of the experiment, mice were harvested, and cells were isolated for real-time PCR analysis and flow cytometry analysis. b Plasma viral load was measured by quantitative real-time PCR (limit of detection = 750 copies/ml, dashed line), and c  %CD4⁺ T cells was measured by flow cytometry. Mean values and SEM plotted

Fig. 4

Analysis of cell-associated HIV RNA levels in the tissues of infected, suppressed, panobinostat-treated BLT mice. BLT mice were infected with HIV-1_JR-CSF, suppressed by antiretroviral therapy, and treated with panobinostat as described in Fig. 3. Cells were then isolated from peripheral blood, bone marrow, liver, lung, lymph nodes, spleen, and thymic organoid for real-time quantitative PCR analysis of cell-associated HIV RNA levels. a Data from individual tissues. b Data from all tissues. Median and interquartile range plotted with comparisons by Mann–Whitney test: ns p > 0.05. Blue circles denote control; red squares denote panobinostat-treated

Fig. 5

Analysis of HIV DNA levels in the tissues of infected, suppressed, panobinostat-treated BLT mice. BLT mice were infected with HIV-1_JR-CSF, suppressed by antiretroviral therapy, and treated with panobinostat as described in Fig. 3. Cells were then isolated from peripheral blood, bone marrow, liver, lung, lymph nodes, spleen, and thymic organoid for real-time quantitative PCR analysis of HIV DNA levels. a Data from individual tissues. b Data from all tissues. Median and interquartile range plotted with comparisons by Mann–Whitney test: ns p > 0.05. Blue circles denote control; red squares denote panobinostat-treated

Fig. 6

Analysis of panobinostat treatment on HIV latency in infected, suppressed BLT mice. BLT mice were infected with HIV-1_JR-CSF, suppressed by antiretroviral therapy, and treated with panobinostat as described in Fig. 3. Resting human CD4⁺ T cells were isolated from the pooled tissues of each mouse, and analyzed by flow cytometry (a). Numbers of latently infected cells per million resting CD4⁺ T cells were determined by quantitative viral outgrowth assay. Median and interquartile range plotted with comparisons by Mann–Whitney test: ns p > 0.05. Blue circles denote control; red squares denote panobinostat-treated