CROI 2018 Abstract eBook

Abstract eBook

Poster Abstracts

325 EXOSOMAL TAT REACTIVATES LATENT HIV-1

Conclusion: Our methods using an established HIV latency model and bulk and single cell transcriptomic analysis in LN cells shed light on biology of latency in a crucial anatomical site for HIV persistence and are valuable tools for investigating cure strategies. 327 BCL6 INHIBITION REPRESSES THF/NON-TFH HIV INFECTION AND T-CELL/ MYELOID ACTIVATION Yanhui Cai 1 , Mohamed Abdel-Mohsen 1 , Costin Tomescu 1 , Matthew Fair 1 , Livio Azzoni 1 , Emmanouil Papasavvas 1 , Fengtian Xue 2 , Jie Sun 3 , Neil D. Romberg 4 , Carole Le Coz 4 , Luis Montaner 1 1 Wistar Institute, Philadelphia, PA, USA, 2 University of Maryland, Baltimore, MD, USA, 3 Indiana University, Indianapolis, IN, USA, 4 Children’s Hospital of Philadelphia, Philadelphia, PA, USA Background: Germinal center CD4+ follicular helper T cells (Tfh) are highly susceptible to HIV infection and constitute a viral reservoir during antiretroviral therapy (ART). B cell lymphoma 6 (BCL6) is a master transcription factor for Tfh cells that if blocked can inhibit ex vivo HIV infection. BCL6-specific inhibition also reduces the frequency of lymphoid Tfh cells, germinal center formation, and pulmonary inflammatory sequelae in mice. Here we tested a novel BCL6 inhibitor to assess its antiviral effect in Tfh and non-Tfh CD4+ T cells and monocyte-derived macrophages (MDM) and its ability to modulate immune activation ex vivo. Methods: We used the inhibitor FX1 to block BCL6 activity in human CD4+ T cells, MDM and PBMC ex vivo. HIV infection was performed by spinoculation with or without FX1. HIV infection was measured by flow cytometry analysis, p24 ELISA assay and qPCR assay. HIV reactivation was assessed by stimulating PBMC from ART-suppressed individuals with phorbol-12-myristate-13-acetate plus ionomycin (PMA/IO) following by quantification of supernatant p24 via ultrasensitive p24 assay. Intracellular cytokine production was analyzed by flow cytometry after LPS stimulation for TLR4-mediated immune activation, and after GS9620 stimulation for TLR7-mediated immune activation. Statistical analysis was performed using Wilcoxon test. Results: In CD4+ T cells, FX1 treatment resulted in lower expression of HIV receptors (CD4, CXCR4 and CCR5, n=6, p<0.05) and down-regulation activation markers (HLA-DR, n=9, p<0.01). In PBMC, FX1 treatment reduced monocyte cytokine production in response to LPS (TNF-a, n=8, p<0.01; IL6, n=8, p<0.05), as well as pDC responses to GS9620 by lower TNF-a expression (n=7, p<0.05). FX1 treatment reduced ex vivo HIV infection of CD4+ T cells (n=6, p<0.05), tonsillar Tfh (n=3) / non-Tfh CD4+ T cells (n=5), and MDM (n=4), as well as multi-spliced HIV RNA production (n=6, p<0.05). As expected, FX1 treatment repressed HIV reactivation by PMA/IO (n=2), as evidenced by lower p24 production. Conclusion: Our data indicate that BCL6 inhibition may reduce HIV replication beyond ART by: 1) limiting TLR-4- and TLR-7- mediated inflammatory responses, 2) inhibiting HIV replication in lymphoid CD4+ Tfh cells, and 3) restricting de novo viral infection in CD4+ T cells and macrophages. Our data suggest that BCL6-specific inhibition should be explored in vivo as a foundation for a reduction in HIV persistence in ART-suppressed subjects.

Xiaoli Tang , Huafei Lu, Stacey Chapman, Bharat Ramratnam Brown University, Providence, RI, USA

Background: Highly active antiretroviral therapy (HAART) can suppress but not eradicate HIV-1. Clinical grade Latency Reversal Agents (LRAs) can reactivate latent HIV-1 in vitro but have not reproducibly reduced HIV-1 burden in vivo Methods: Here, we describe the latency reversing potency of HIV-1 Tat formulated as an exosomal preparation. The backbone of Tat was altered to include both an N-terminus membrane signal and a C-terminus nuclear localization signal. To facilitate CD4+ cellular delivery, we further modified the construct by addition of a CD4+ receptor targeting moiety (Interleukin -16 C-terminal 20 amino acid domain) fused to the N-terminus of lysosome- associated membrane protein 2 variant b (Lamp2b). The resulting construct (pExo-Tat) was engineered into the backbone of both lentivirus and adeno- associated virus allowing the generation of cell lines that permanently produced exosomes harboring Tat (Exo-Tat). Results: Exo-Tat was 3-fold more potent than LRAs (HDACi, Disulfiram) in activating virus using cell line models of HIV-1 latency or promoter activity. We next isolated rCD4+ T lymphocytes from 14 HIV-1 infected individuals on suppressive HAART (7-20 years). Exo Tat was incubated with rCD4+ T cells and viral reactivation was assessed by quantifying cell associated HIV RNA or p24 antigen levels in fresh uninfected cells co-cultured with supernatants of Exo-Tat treated patient rCD4+T. Exo-Tat activated HIV as measured by intracellular HIV-1 RNA (14/14) and serial p24 levels in culture media (3/3). We next compared the latency reversing potency of Exo-Tat with that of LRAs panobinostat and disulfiram. While these two LRAs did not consistently reactivate latent HIV-1 from rCD4+ T cells, both synergistically enhanced the potency of Exo-Tat by up to 4-fold. Exo-Tat had no effect on rCD4+T cell apoptosis, immune activation status or cytokine expression as measured by flow cytometry and ELISA. Conclusion: Our findings identify exosomal Tat as a new class of biologic product with potential utility in the combinatorial targeting of latent HIV-1. Lesley R. de Armas , Kyle Russell, Sion Williams, Robert K. Suter, Stefano Rinaldi, Li Pan, Suresh Pallikkuth, Rajendra Pahwa, Ramzi T. Younis, Savita Pahwa University of Miami, Miami, FL, USA Background: HIV eradication is hindered by the existence of latent HIV reservoirs in CD4 T cells. T follicular helper (Tfh) cells are a subset of CD4 T cells present in lymph nodes (LN) which harbor high frequencies of HIV-infected cells. Here, we used a primary cell model of HIV latency and the dual reporter virus, HIV DuoFluo (provided by E. Verdin, UCSF) to investigate latency establishment in CD4 and Tfh cells. Methods: Tonsils from HIV-neg donors (n=8) were obtained as a source of LN CD4 T cells. Purified CD4 were activated with anti-CD3/CD28 beads for 3d prior to infection with HIV DuoFluo, containing GFP under HIV-LTR promoter and mKO2 under EF1α promoter. Live, uninfected (U, GFP-mKO2-), latent (L, GFP-mKO2+), and productive (P, GFP+mKO2+/-) infected cells were analyzed 3d later for surface expression of CD4, CXCR5, and PD1 and were sorted in bulk for gene expression (GE) analysis of 96 genes involved in T cell function and metabolism by Fluidigm Biomark assay. Single-cell sorting of U, L, and P cells (62 cells per group) was performed using cells from one tonsil donor for 3’ whole transcriptome amplification using BD Precise platform. Limma and FDR correction were applied to determine significant differences in GE. Results: Tfh (PD1+CXCR5+) were more permissive to latent infection compared to Non-Tfh (PD1-CXCR5-) cells by comparing ratios of L: P (Mean: 0.4 vs. 1.0, respectively, p=0.02 student’s t test). Biomark analysis of Tfh cells showed differential GE of 36 genes between P and U and 8 unique genes between P and L cells (ANOVA, p<0.05). 0 genes were exclusively differentially expressed (DEGs) between L and U, though IL21 was downregulated in P and L Tfh compared to U which may affect overall function of Tfh. P had reduced expression of genes involved in transcriptional regulation and metabolism compared to U, and L had higher expression of TNF superfamily members and restriction factor, APOBEC3G compared to P. In single cell analysis, U and L overlapped with 0 DEGs while P showed 202 DEGs compared to U and 155 compared to L (121 overlapped between the 2 comparisons). FCGR2A, the proposed marker of latent cells in blood was not detectable in sorted single cells suggesting differences between blood and LN markers of HIV latency.

Poster Abstracts

326 SINGLE-CELL TRANSCRIPTOMICS TO EVALUATE HIV LATENCY ESTABLISHMENT IN CD4 T CELLS

CROI 2018 114