CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

Conclusions: Both experiments with HDAC and HAT inhibitors demonstrate that modulating acetylation influences susceptibility of CD4+ T cells to HIV. Our experiments with tubacin suggest that microtubule acetylation may underlie enhanced HIV infection with HDAC inhibitors. We believe our preliminary results with HAT inhibitors demonstrate a proof-of-concept to decrease susceptibility of cells to HIV by directly targeting cellular processes independent of viral heterogeneity and resistance mutations. These findings are of particular significance during HIV transmission where they could potentially be exploited to further reduce the likelihood of spread between individuals. 405 Panobinostat Dosing Has Broad but Transient Immunomodulatory Effects in HIV Patients Martin Tolstrup 1 ; Christel R. Brinkmann 1 ;Thomas A. Rasmussen 1 ; Rikke Olesen 1 ; Anne Sofie Kjær 1 ; Mathias Lichterfeld 2 ; Charles Dinarello 3 ; Lars Østergaard 1 ; Ole S. Søgaard 1 1 Aarhus University Hospital, Aarhus, Denmark; 2 Ragon Institute of MIT, MGH and Harvard, Boston, MA, US; 3 University of Colorado, Denver, CO, US Background: To evaluate the immunomodulatory effects from HDACi in HIV-infected patients, we investigated a broad range of immune pathways during a latency reversal trial with panobinostat (PANO). Further, as prolonged epigenetic modulation from HDACi treatment has been raised as safety concern, we evaluated gene expression alterations up to 24 weeks after end of PANO dosing. Methods: Using flow cytometry, we investigated the impact of PANO on T cell activation (CD69, HLA-DR, CD38), T cell exhaustion (PD-1). Further, levels of activated regulatory T cells and their expression of suppressive markers (CD39 and CTLA4) were assessed. To determine broad changes in immune responsiveness to common stimuli, whole blood stimulations with LPS were performed and inflammatory responses by cytokine release were determined using luminex. Gene expression from purified PBMC’s was evaluated using affymetrix HTA 2.0 gene chip. Results: A rapid increase in proportions of both CD4 and CD8 T cells expressing CD69 were observed (p<0.01) as early as 24 hrs after first PANO dosing. This was followed by a marked increase in HLA-DR+ CD4 and CD8 T cells (p<0.01) observed at day 4. At the same time point, proportions of activated regulatory T cells increased by 40 % four days after treatment initiation (p=0.003) and MFI of the suppressive markers CD39 and CTLA4 increased by 12% (p=0.009) and 25 % (p=0.0002), respectively. LPS-induced inflammatory responses as determined by IL-1b, IL-12p40, IL-6 and TNF-a secretion were all significantly down regulated four days after dosing. Importantly, all these PANO-induced immunomodulatory effects were reversible and all markers had returned to pre-treatment levels 4 weeks after end of PANO dosing. Lastly, PANO induced significant changes in the overall gene expression pattern (fold-change >1.5, FDR-corrected p<0.05). These alterations in gene expression had regressed considerably by week 4 after end of PANO treatment and normalized entirely by week 24 post-PANO therapy. Conclusions: PANO significantly but transiently influenced T cells activation status, regulatory T cell phenotype and functional mitogen responsiveness. All measures of immune function had returned to baseline levels 4 weeks after completion of PANO and long-term follow-up revealed no sustained effect on overall gene expression. Collectively, the results suggest that PANO does not cause persistent detrimental epigenetic or immunomodulatory changes in HIV patients. 406 Multi-Dose Romidepsin in SIV-Infected RMs Reactivates Latent Virus in Absence of ART Benjamin Policicchio 1 ; Egidio Brocca-Cofano 1 ; Cuiling Xu 1 ; Dongzhu Ma 1 ; Hui Li 2 ; George Richter-Haret 1 ;Tammy Dunsmore 1 ; George M. Shaw 2 ; Ivona Pandrea 1 ; Cristian Apetrei 1 1 University of Pittsburgh, Pittsburgh, PA, US; 2 University of Pennsylvania, Philadelphia, PA, US Background: Persistent viral reservoirs represent a major obstacle in eliminating HIV-1 in infected individuals, even with ART. One of the reservoir reactivation strategies is the “flush and kill” approach, in which latent virus is reactivated with histone deacetylase inhibitors (HDACi) and eliminated through effective CTL responses. Our goals were to: 1. develop a nonhuman primate model of SIV control with conventional ART; 2. assess the HDACi romidepsin’s (RMD) ability to reactivate SIV in controller RMs; 3. assess the effect of CTLs on viral control; 4. develop monitoring capabilities for reactivation studies. Methods: Four RMs were IV-infected with the SIVsmmFTq infectious molecular clone. All RMs received ART (PMPA; FTC; L-870812) for 9 months from 65 days post-infection. ART was halted and RMD was administered in 3 rounds at 35 day intervals to three RMs, followed by CD8 + cell depletion with a CD8 depleting antibody (M-T807R1). Plasma viral load was monitored with a single copy assay. Cell-associated RNA and DNA were monitored by qPCR. PBMC histone acetylation, IFN- γ production by CTLs and changes in levels of T cells and their immune activation/proliferation status were assessed by flow cytometry. Results: SIVsmmFTq RMs receiving conventional ART controlled virus replication to <10 copies/ml, without viral blips. At ART cessation, the virus transiently rebounded (up to 10 6 copies/ml), followed by control to undetectable levels (<10 copies/ml), suggesting effective immune control in early treated RMs. RMD administration resulted in significant virus rebounds (up to 10 4 copies/ml) followed by gradual viral decline. RMD was well-tolerated and resulted in a massive surge in T cell activation and a transient decrease of T cells during the first week post treatment. CD8 + cell depletion resulted in a robust viral rebound (up to 10 7 copies/ml) that was controlled upon CD8 + T cell recovery. Conclusions: We developed a new RMmodel of virus control with conventional ART and demonstrated that RMD can reactivate SIV in vivo . The levels of virus replication, timing of the virus rebound and rapid control of virus replication after RMD administration suggest the reactivated virus is replication-competent and RMD does not persistently alter CTL function. CD8 + cell depletion resulted in higher viral rebound compared to RMD administration, suggesting that RMD did not completely ablate CTL function. Altogether, our results show HDACis can effectively reverse SIV latency. 407 Suberanilohydroxamic Acid (SAHA)-Induced Histone Modifications in the HIV Promoter in a Human, Primary CD4 T Cell Model of Latency Brian Reardon 1 ; Amey Mukim 2 ; Savitha Deshmukh 2 ; Christopher H.Woelk 3 ; Douglas D. Richman 1 ; Celsa A. Spina 1 1 University of California San Diego, La Jolla, CA, US; 2 VA San Diego Healthcare System, La Jolla, CA, US; 3 University of Southampton, Southampton, United Kingdom Background: Latently infected CD4 T cells are a major obstacle to HIV eradication with combined antiretroviral therapy (cART). Histone deacetylation at the HIV promoter leads to silencing of viral genes during latency. Use of histone deacetylase inhibitors (HDACi), in the presence of cART, to reactivate viral transcription has been proposed as a means to deplete cellular reservoirs. However, HDACi-induced histone modifications in the promoter of integrated HIV, in association with viral transcription, have not been fully examined in latently infected human primary CD4 T cells. Here, we use a primary T cell model of latency to investigate whether the HDACi SAHA induces histone modifications at the HIV promoter that correlate with HIV activation. Methods: As described previously (Spina et al. PLoS Pathog. 2013), resting primary CD4 T cells with latent HIV (NL4-3) infection were derived from co-culture with acutely infected, autologous T cells and isolated by flow cytometry. Latently infected cells, from 5 different donors, were treated with 1.2 uM SAHA, anti-CD3/CD28, or vehicle control for 24 hours, in the presence of 0.5uM raltegravir. Induced HIV activation was measured by Tat RNA expression (droplet digital PCR). Histone modifications in the nuc-0 region of the provirus LTR, associated with activation (H3K9 acetylation, H3K4 methylation) or repression (H3K27 methylation), were measured with ChIP-RT-qPCR. Results: In 3 of 4 SAHA treated donor samples, Tat RNA expression was increased (~1.5 - 2 fold vs. controls); 1 donor sample was not measured due to insufficient RNA recovery. In 4 of 5 SAHA-treated donor samples, histone markers of transcriptional activation were increased and a histone marker of transcriptional repression was decreased compared to control treatment. However, no individual or combination of histone modifications was correlated with Tat expression across donors. Work is ongoing to determine whether examination of histone modifications at earlier time points may produce more definitive results.

Poster Abstracts

292

CROI 2015