CROI 2016 Abstract eBook

Abstract Listing

Poster Abstracts

stimulated genes (ISG) that restrict virus replication. As NF-kB also stimulates transcription of the HIV-1 LTR promoter, we hypothesized that viral RNA PAMP might induce a transcriptional cascade that enhances HIV expression in latently infected cells. We are studying how novel host factors, including RNA-binding proteins and chromatin-modifying complexes, modulate HIV reactivation by viral RNA PAMP. Methods: To test this hypothesis, we transfected J-Lat (H2) CD4+ reporter T cell lines containing an integrated HIV-1 sequence, 5’ LTR promoter and a GFP reporter gene, with viral RNA PAMP: short chain poly-I:C, long chain poly-I:C (PIC-H), and Sendai virus RNA, in comparison to latency-reactivating drugs SAHA (a chromatin modifier) and prostratin (an NF-kB and PKC activator). Transcription factors and novel host proteins thought to be involved were targeted by RNA interference (mRNA depletion) using siRNA. Results: In J-Lat cells, HIV-1 transcription was activated by viral RNA PAMP, with PIC-H exhibiting a 10-fold increase. PIC-H concurrently induces IFN-beta. Expression of cell stress marker MICB was also induced, raising the possibility of detection of newly-reactivated HIV by natural killer (NK) cells. Interestingly, latency-reversing drugs prostratin and SAHA not only reactivate latent HIV, but significantly induced IFN-alpha/beta, and ISG56, MXA, and antiretroviral gene MXB. We are also studying novel negative regulators of latent HIV reactivation. Targeting RNA binding proteins PKR, NF90, hnRNP A1, and RNase H2 with siRNA resulted in a 2- to 5-fold increase in HIV-1 transcription in cells transfected with PIC-H or Sendai virus RNA. Knockdown of PKR or NF90 also enhanced HIV-1 reporter expression by prostratin more than 2-fold. Conclusions: Viral RNA PAMP can induce latent HIV-1 reactivation in a T cell reporter model, a process regulated by RNA-binding host factors. We are currently exploring the molecular mechanisms involved, and seeking to identify RNA species bound to host factors using next-generation sequencing. These innate antiviral immune pathways might be leveraged in new antiviral therapies to target and eradicate latent HIV reservoirs. 372 CSF1R Antagonists Sensitize HIV-Infected Macrophages to Death Francesc Cunyat ; Jennifer N. Rainho; Mario Stevenson Univ of Miami, Miami, FL, USA Background: HIV can persist in the face of antiviral suppression and there is a need to develop new strategies to eradicate the viral reservoir in infected individuals. HIV activates the colony-stimulating factor 1 receptor (CSF1R or MCSF-R) by phosphorylation to turn monocyte-derived macrophages (MDM) resistant to apoptotic pathways driven by TNF- related apoptosis-inducing ligand (TRAIL). In this study we assessed if CSF1R antagonists sensitize HIV-infected MDM to cell death. Methods: The ability to block the phosphorylation of CSF1R in MDM by CSF1R antagonists was quantified by ELISA after priming the cells with soluble CSF1 in the presence of active or inactive compounds. HIV-infected MDMwere treated with CSF1R antagonists in the presence of soluble TRAIL, stained for intracellular gag p24 and analyzed by flow cytometry to assess the amount of infected cells at the end of the cultures. Reporter GFP-HIV was also used to infect MDM and monitor the infection rates by GFP expression. Quantitative PCR and reverse transcriptase assays were performed from extracted cell-associated DNA and cell-free supernatants, respectively, to quantify the effect of the compounds on the absolute cell loss and the viral output. Treatment effects with active compounds were compared to inactive compounds or DMSO by two-way ANOVA. Results: The active CSF1R antagonists PLX3397 and PLX5622 avoid the activation of CSF1R by inhibiting its phosphorylation in human MDM. Inactivation of CSF1R in cultures of infected cells correlated with a 33-38% reduction of the viral output and 45-60%myeloid cell loss, which was unaffected when treated with a non-nucleoside reverse transcriptase inhibitor, nevirapine, or an inactive CSF1R antagonist. Intracellular gag p24+ cells were specifically sensitized to apoptosis after 4 days of treatment, showing a 25-38% infection decrease. Infections with reporter HIV showed also a 50% decrease in the amount of productively infected GFP + -cells after a 5-day treatment. Conclusions: The CSF1R antagonists PLX3397 and PLX5622 reduce the percentage of HIV-infected MDM and the overall viral production in vitro . The promising results suggest that compounds targeting CSF1R could potentially purge highly resistant HIV-infected myeloid cells by restoring their apoptotic sensitivity to TRAIL and impact the size of the macrophage viral reservoir in HIV-infected individuals. 373 Effect of HIV-1 Nef on MHC-I After Latency Reversal in Primary CD4 T Cells Thomas Vollbrecht ; John Guatelli Univ of California San Diego, San Diego, CA, USA Background: The main obstacle to clearing HIV-1 infection is the elimination of latently infected cells. This dormant reservoir remains unaffected by ART and is capable of producing replication-competent virus upon reactivation. The accessory HIV-1 protein Nef is expressed early during that viral replication cycle and is known to down-regulate MHC‑I from the cell surface during acute infection. Our study aim is to demonstrate that latency reversal in a primary cell model using drugs of potential clinical relevance results in Nef-mediated modulation of MHC class-I and that this is associated with resistance to killing by CTL. Methods: We established a modified version of the Bosque and Planelles primary CD4 T cell latency model using replication-competent viruses and the drugs tenofovir and raltegravir to stop spreading infection in the cultures. We examined Nef-function upon viral reactivation with clinically relevant latency reversing agents (LRAs), specifically romidepsin, bryostatin-1 and prostratin. To analyze infected cells, we used an NL4-3-based clone in which the murine HSA protein (CD24), a cell surface marker, is expressed within the vpr ORF. To study the effect of Nef on MHC-I, we created a Nef-M20A mutant, which is selectively defective in the down-regulation of MHC-I. Additionally, we investigated the ability of a CTL clone to eliminate infected primary cells by flow cytometric assays. Results: Our results indicate that Nef down-regulates MHC-I from the surface of infected cells after latency reversal in a primary T cell-based in vitro model. Unexpectedly, the data also suggest that certain LRAs might up-regulate MHC-I expression on CD4 T cells, whereas others might enhance the activity of Nef. We now plan to demonstrate that CTL specific for the gag p17 epitope SL9 are able to kill these cells after latency reversal more efficiently when the downregulation of MHC-I by Nef is genetically absent (i.e., in case of the Nef-M20A mutant). Conclusions: Nef is functional with respect to the down-regulation of MHC class-I after latency reversal by drugs of potential clinical relevance. Unexpectedly, certain LRAs may affect the level of MHC-I directly as well as in concert with Nef. If we can now show that the lack of this Nef-activity sensitizes cells in which latency is reversed pharmacologically to killing by CTL, then we expect to invigorate drug discovery targeting Nef as an adjunct to latency reversal in viral eradication strategies. 374 Effect of Primary CD8 T Cells on Romidepsin and Bryostatin-Treated CD4 T Cells VictoriaWalker-Sperling ; Joel N. Blankson Johns Hopkins Univ Sch of Med, Baltimore, MD, USA Background: Latently infected CD4 T cells represent a major barrier to HIV-1 cure efforts. The current “shock and kill” strategy relies upon the use of latency reactivating agents (LRAs) to selectively induce production of HIV-1 mRNA and proteins in resting CD4 T cells to make them visible to the adaptive immune system. Previous work has shown that treating primary latently infected cells with combinations of LRAs is more effective than single drug treatments. One such combination is bryostatin and romidepsin, the latter of which has further been shown to induce reactivation in vivo. However, romidepsin and other HDAC inhibitors have been associated with impaired CTL responses. Thus, we sought to examine the stimulated, primary effector CD8 T cell response of cART-treated HIV+ individuals to latently-infected CD4 T cells cultured with the LRAs bryostatin and romidepsin (B/R). Methods: Resting, CD4 T cells from 6 cART-treated HIV+ chronic progressors were treated with the combination B/R to reactivate latently infected cells. To determine the ability of the CD8 effector response to identify and kill these cells, primary CD8 T cells that had been stimulated with overlapping Gag and Nef peptides and IL-2 for seven days were added to culture at 6 hours post-stimulation. Cell-associated and supernatant HIV mRNA from 24 hours post-stimulation was measured via qPCR to determine the efficacy of the CD8 effector response.

Poster Abstracts

141

CROI 2016