The Depsipeptide Romidepsin Reverses HIV-1 Latency In Vivo

Abstract

Pharmacologically-induced activation of replication competent proviruses from latency in the presence of antiretroviral treatment (ART) has been proposed as a step towards curing HIV-1 infection. However, until now, approaches to reverse HIV-1 latency in humans have yielded mixed results. Here, we report a proof-of-concept phase Ib/IIa trial where 6 aviremic HIV-1 infected adults received intravenous 5 mg/m2 romidepsin (Celgene) once weekly for 3 weeks while maintaining ART. Lymphocyte histone H3 acetylation, a cellular measure of the pharmacodynamic response to romidepsin, increased rapidly (maximum fold range: 3.7–7.7 relative to baseline) within the first hours following each romidepsin administration. Concurrently, HIV-1 transcription quantified as copies of cell-associated un-spliced HIV-1 RNA increased significantly from baseline during treatment (range of fold-increase: 2.4–5.0; p = 0.03). Plasma HIV-1 RNA increased from <20 copies/mL at baseline to readily quantifiable levels at multiple post-infusion time-points in 5 of 6 patients (range 46–103 copies/mL following the second infusion, p = 0.04). Importantly, romidepsin did not decrease the number of HIV-specific T cells or inhibit T cell cytokine production. Adverse events (all grade 1–2) were consistent with the known side effects of romidepsin. In conclusion, romidepsin safely induced HIV-1 transcription resulting in plasma HIV-1 RNA that was readily detected with standard commercial assays demonstrating that significant reversal of HIV-1 latency in vivo is possible without blunting T cell-mediated immune responses. These finding have major implications for future trials aiming to eradicate the HIV-1 reservoir.

Trial registration: clinicaltrials.gov NTC02092116.

Trial registration: ClinicalTrials.gov NCT02092116.

Fig 1. Flow diagram.

The flow diagram shows information about the method of recruitment and the number of patients undergoing romidepsin treatment.

Fig 2. Romidepsin induced HIV-1 transcription and presence of extracellular viral RNA.

Panel (A) shows mean (SEM) levels of H3 acetylation measured by flow cytometry in lymphocytes. (B) shows mean (SEM) change from baseline in the level of CA US HIV-1 RNA. The first sample time point after each of the 3 romidepsin doses is approximately ½ hour after end of the 4 hour infusion. (C) shows individual levels of plasma HIV-1 RNA, determined using the Roche Cobas Taqman assay, while panel (D) shows mean plasma HIV-1 RNA data for all 6 participants determined using a Transcription-Mediated Amplication assay. (E) is an overlay of H3 acetylation and CA US HIV-1 RNA data presented in Panels A and B. (F) is an overlay of CA US HIV-1-RNA (B) and plasma HIV-1 RNA (E). SEM, standard error of mean; CA US HIV-1-RNA: cell-associated unspliced HIV-1 RNA; LoQ, limit of quantitation; TMA, Transcription-Mediated Amplication. Statistical comparisons were performed using Wilcoxon matched-pairs signed-ranks test, Asterisk indicate p<0.05.

Fig 3. The size of the HIV-1 reservoir was unchanged by romidepsin.

Panels (A) to (E) show measures of the size of the viral reservoir as well as in the inducible HIV-1 reservoir during romidepsin treatment. (A) Absolute levels of total HIV-1 DNA per 10⁶ CD4⁺ T cells. (B) Fold changes in total HIV-1 DNA per 10⁶ CD4⁺ T cells. (C) Absolute levels of 2-LTR HIV-1 DNA per 10⁶ CD4⁺ T cells. (D) Frequency of cells with multiply spliced HIV RNA upon maximal cellular activation with PMA/ionomycin as measured using a tat/rev induced limiting dilution assay (TILDA). (E) shows results from a quantitative viral outgrowth assay (qVOA) which was used to assess the frequency of resting CD4⁺ cells carrying inducible replication competent proviruses at baseline and day 56. LoQ, limit of quantitation; Lod, limit of detection; IUBM, infectious units per million.

Fig 4. Romidepsin increased T cell activation and reduced the proportion of lymphocytes expressing PD-1.

Flow cytometric characterization of CD4⁺ / CD8⁺ T cell subsets and activation status. Relative proportions of both CD4⁺ (A) and CD8⁺ (B) T cell memory subsets are shown in the first two panels. Next, the proportion of CD4⁺ (C) and CD8⁺ (D) T cells expressing the early activation marker CD69. (E) Percentage of CD4⁺ and (F) CD8⁺ T cells co-expressing the late activation markers HLA-DR and CD38. (G, H) Show the effect of romidepsin on the proportion of CD4⁺ and CD8⁺ T cells expressing the exhaustion marker PD-1. CM, central memory; EM, effector memory TD; terminally differentiated. Statistical comparisons were performed using Wilcoxon matched-pairs signed-ranks test, Asterisk indicate p<0.05.

Fig 5. HIV-specific T cell responses were preserved during romidepsin treatment.

Flow cytometric characterization of HIV-gag-specific CD8⁺ and CD4⁺ T cells within the memory subsets at baseline (Base, n = 6), on treatment (ROMI, n = 5) and at follow-up (Post, n = 6). Proportion of EM (A) and TD (B) CD8⁺ T cells producing only IFNγ or both IFNγ and TNFα. Horizontal bars show median values. (C, D) Median fluorescence intensity (MFI) for IFNγ and TNFα for HIV-specific EM CD8⁺ T cells and (E, F) TD CD8⁺ T cells identified in Panels A-B. (G), Proportion of polyfunctional memory EM CD4⁺ T cells producing IFNγ, TNFα and IL-2. (H) Expression levels (MFI) for each cytokine (i.e. IL-2, TNFα and IFNγ) examined on the polyfunctional HIV-specific EM CD4⁺ T cells identified in panel (G) TD, terminally differentiated; EM, effector memory. Statistical comparisons were performed using Wilcoxon matched-pairs signed-ranks test, Asterisk indicate p<0.05.