CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

rCD4s from infected individuals on suppressive ART. We also determined the effect of LRA treatment on proinflammatory cytokine production. To place our results in broader context and bridge laboratory measures of drug efficacy with clinical trial outcomes, we used a mathematical model to predict the in vivo dynamics of viral load following LRA treatment. Results: We found that PKC agonists synergize significantly with JQ1 and with HDAC inhibitors to induce intracellular HIV-1 mRNA ex vivo . Combinations of PKC agonists with JQ1 or an HDAC inhibitor also caused significant virus release – in some instances exceeding that seen with maximal T cell activation. These combinations did not induce the release of proinflammatory cytokines. Using our ex vivo measurements of virus production in response to LRAs, we then predicted in vivo changes in viral load following LRA treatment. In a realistic clinical scenario, the viral load would be expected to decay immediately after LRA activity ceases. In the most conservative scenario considered by our model, plasma viral loads of over 100 copies/mL are predicted for all treatments we investigated that contain a PKC agonist. Conclusions: Using multiple assays for latency reversal, we have completed the first ex vivo comparative study to identify highly effective LRA combinations. We demonstrated that select PKC agonist–containing combinations reverse latency at levels approaching those seen with maximal T cell activation. We demonstrate that this degree of latency reversal can be achieved without inducing functional T cell activation. Finally, we offer a mathematical framework to predict in vivo responses to LRAs using ex vivo measurements that can inform the design of future eradication clinical trials.

WEDNESDAY, FEBRUARY 25, 2015 Session P-F8 Poster Session

Poster Hall

2:30 pm– 4:00 pm Latency Models and Assays 420 Investigating Mechanisms of HIV Persistence Using Droplet Digital PCR Approaches Elizabeth M. Anderson 1 ; Robert Gorelick 2 ; Shawn Hill 1 ; Catherine A. Rhem 5 ; Mary Kearney 1 ; JohnW. Mellors 3 ; John M. Coffin 4 ; Mike Piatak 2 ; Frank Maldarelli 1 1 National Cancer Institute at Frederick, Frederick, MD, US; 2 Leidos Biomedical Research, Inc, Frederick, MD, US; 3 University of Pittsburgh, Pittsburgh, PA, US; 4 Tufts University, Boston, MA, US; 5 National Institute of Allergy and Infectious Diseases (NIAID), Bethesda, MD, US Background: HIV persists at low levels in blood during antiretroviral therapy yet mechanisms of HIV persistence remain poorly characterized. In addition, the transcriptional activity of persistent proviruses is not understood and sensitive quantitative assays are needed to investigate this process. Droplet digital PCR (ddPCR) has been used to detect retrovirus sequences, especially HIV DNA, with single targets of 60-80 nucleotides, however optimal primer-probe combinations to detect the various HIV RNA transcripts have not been defined. We have developed new quantitative assays to explore mechanisms of HIV persistence by measuring its transcriptional activity. Methods: Primer-probe sets designed to span the TAR region (all transcripts), R-U5 junction (elongated transcripts) and p24 of the gag gene (unspliced transcripts) were used to investigate the utility of ddPCR to detect multiple RNA species as well as DNA. RNA transcripts were generated for optimization and to serve as a standard. HIV-1 RNA extracted from virions in cell culture and from patient plasma during rebound viremia were reverse transcribed and assayed. DNA from uninfected CEM and latently infected ACH2 cells were tested. Finally, HIV-1 DNA extracted from PBMCs from patients (N=3) who had ongoing active HIV replication (viral RNA 58,100 to 584,204 copies/mL) was assayed with all HIV primer sets and a CCR5 set to measure total cell DNA. Results: ddPCR accurately quantified RNA transcripts (mean 1.1-fold difference) and HIV-1 RNA isolated from virions derived from cell culture (mean 1.3-fold difference) based on spec. HIV-1 RNA from patient plasma was detected with all primer sets and showed a 2.6 to 1 ratio of TAR to gag, agreeing with the expected 2 to 1 ratio in virions. HIV-1 DNA was undetectable (< 1 copy per 9,000 cells) in the uninfected CEM cells showing no non-specific priming while CCR5 DNA was quantified accurately (1.3-fold difference). For DNA isolated from ACH2 cells, which harbor a single integrated provirus, HIV targets and CCR5 were detected (mean 1.4-fold and 1.1-fold difference). Furthermore, we detected CCR5 DNA (mean 13,520 copies) and HIV-1 DNA for all patients with a mean LTR to gag ratio of 1.9 to 1. Conclusions: ddPCR approaches can distinguish HIV RNA transcription profiles in HIV infected cells, with a broad dynamic range (1-1x10 5 copies) suitable for single cell analyses. Application of these technologies will permit detailed and precise analysis of the mechanisms of HIV persistence in patients undergoing cART. 421LB High-Throughput Single-Cell Quantification of HDACi-Based HIV Reservoir Reactivation Robert W. Yucha 1 ; Emily Hanhauser 2 ; Kristen S. Hobbs 2 ; Helen Bae 3 ; Fatih Inci 4 ; Hadi Shafiee 2 ; ShuqiWang 4 ; Daniel Kuritzkes 1 ; Utkan Demirci 4 ;Timothy J. Henrich 1 1 Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA, US; 2 Brigham and Women’s Hospital, Cambridge, MA, US; 3 Harvard University, Cambridge, MA, US; 4 Stanford University, Palo Alto, CA, US Background: Reactivation of latent viral reservoirs is on the forefront of HIV-1 curative strategies. However, little is known about HIV-1 reactivation on a single cell level, such as if reactivation leads to significant increases in the number of transcriptionally active cells, or if increases in cell-associated HIV-1 RNA arise from a smaller, active portion of the reservoir. We therefore designed and implemented a novel microfluidic assay to directly measure the number of cells that produce unspliced (us)RNA from HIV-1-infected individuals on suppressive ART before and after HDAC inhibitor and T cell receptor (TCR) stimulation ex vivo . Methods: Our approach involves high throughput microscale droplet encapsulation of single total or resting CD4+ T-cells (15-45,000) followed by intra-droplet lysis, PCR amplification of HIV-1 usRNA, microscopic or fluorescent flow-based detection, and downstream DNA characterization from isolated droplets. Cells from 4 patients with varied intracellular DNA and RNA levels were stimulated with romidepsin (50nM) or anti-CD3/CD28 antibodies (100ng/mL). We also compared changes in the number of usRNA-producing cells with total usRNA from bulk-cell extracts Results: The number of usRNA producing resting CD4+ T cells by either manual or automated fluorescent enumeration obtained from 3/4 patients were 4-5 fold higher after T-cell receptor stimulation and 4-7 fold higher after stimulation with romidepsin. However, qPCR demonstrated only a 1.5-10 fold increase in the total amount of usRNA from bulk cell extracts in samples stimulated with either anti-CD3/CD28 or romidepsin. These results may suggest a concordant increase of the number of stimulated cells and usRNA copies per cell. Interestingly, a single patient on suppressive ART with very high caRNA (900,000/10 6 CD4+T-cells) showed only a 1.5-2 fold increase in usRNA producing cells and a decrease (0.3 fold) in usRNA copies per cell post-stimulation. Conclusions: Our results suggest that latent CD4+ cell activation results in a higher number of cells that produce cell-associated RNA, which correlate approximately with increases in bulk transcriptional activity in samples from 3/4 patients. This study highlights the importance of direct single-cell analysis to fully understand the impact of reactivating agents on latent reservoirs; ongoing studies will further elucidate the relationship between cellular activation and HIV-1 transcription within bulk and single cell environments.

Poster Abstracts

298

CROI 2015