CROI 2016 Abstract eBook

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Poster Abstracts


Essential Role for Vpr in Productive Infection of Dendritic Cells by HIV-1 Caitlin Miller ; Hisashi Akiyama; Luis Agosto; Andrew Henderson; Rahm Gummuluru Boston Univ Sch of Med, Boston, MA, USA

Background: Viral protein R (Vpr) is a small accessory protein that is packaged into the budding HIV-1 particle through its association with the p6 region of Gag, and is thus present at a high quantity in target cells upon infection. While numerous functions have been attributed to Vpr, very few of these have been studied in the context of HIV-1 infection of dendritic cells (DCs). In this work, we show that Vpr expression is essential at early steps of viral replication in DCs and that infections with ΔVpr viruses or viruses that encode Vpr mutants unable to interact with the DNA damage repair complex SLX4 (SLX4com), display a significant replication defect within a single round of infection. Methods: To determine the mechanism of Vpr-dependent enhancement of virus replication in DCs, we constructed a panel of replication competent and single cycle (luciferase expressing) viruses with point mutations in Vpr at F34I, W54R, Q65R and H71R, or frame shift mutation (ΔVpr). Effects of selective Vpr inactivation on integration, viral protein expression, and virus spread in infected DC cultures was determined by Alu -PCR, Western blot analysis, luciferase expression, and ELISA. Results: We observed reduced virus spread in DCs infected with viruses lacking Vpr expression (ΔVpr), or those encoding mutant Q65R- or H71R-Vpr alleles that disrupt the previously characterized association between Vpr and the Cul4a/DCAF/DDB1 ubiquitin ligase complex as well as SLX4com. Attenuated replication of HIV-1/ΔVpr in DCs was not due to a defect in provirus integration or in viral glycoprotein production. Finally, a 8-10 fold decrease in luciferase expression was observed in infections of DCs with single cycle viruses encoding ∆Vpr, Vpr-Q65R or Vpr-H71R compared to WT virus infection suggesting that Vpr interaction with the DNA damage response pathway is critical for inducing optimal levels of transcriptional activity from the viral LTR. Conclusions: Together, this data suggests that Vpr expression modulates the level of infection in a single round of replication in DCs. Contrary to previous reports in other cell types, this is not due to effects of Vpr on integration or viral glycoprotein production. Rather, Vpr affects the level of viral LTR transcriptional output through a mechanism that is mediated by the recruitment of the SLX4com and associated ubiquitin ligase machinery. These interactions are necessary for robust viral transcription during infection of DCs by HIV-1. 186 Background: Mature monocyte-derived dendritic cell (MDDC)-mediated HIV-1 transmission to CD4 + T cells (trans-infection) occurs across tight cellular junctions called virological synapses (VS). We have previously reported that anti-gp120 bNAbs inefficiently block HIV-1 transmission across VS due to steric hindrance, whereas MPER recognizing bNAbs that bind to MDDC plasma membrane efficiently inhibit this transfer. In this study, we determined if binding of newer and more potent bNAbs to plasma membrane of virus- transmitting MDDCs as well as CD4 + T cells is necessary for efficient neutralization of cell-to-cell transmission of HIV-1. Methods: HIV-1 primary isolates were incubated with LPS-matured MDDCs or used to infect activated primary CD4 + T cells. Neutralization of virus transfer from these HIV-1 laden cells was examined by measuring luciferase activity after incubating with TZM-bl reporter cell line in the absence or presence of increasing concentrations of anti-gp120 (2G12, VRC01, PGT121 and PGT126) or MPER (4E10 and 10E8) bNAbs and was compared with that of cell free virus using the Wilcoxon rank sum test. Immunofluorescence microscopy and ELISA was used to examine bNAb binding to MDDCs or CD4 + T cells. Results: Compared to the ability to block infection of TZM-bls by cell free virus, 10E8 showed similar ability to inhibit MDDC-mediated trans-infection (p>0.05), but it was significantly less potent in blocking CD4 + T cell transfer (p=0.04). PGT121 neutralized cell free virus, MDDC-mediated trans-infection, and CD4 + T cell mediated cell-to-cell virus transfer with similar efficiency. VRC01 was less efficient in blocking both MDDC and CD4 + T virus transmission as compared to cell-free virus. Binding assay revealed that both 2G12 and VRC01 displayed negligible binding to MDDC or CD4 + T cell plasma membrane. Intriguingly, PGT121 robustly bound plasma membranes of both MDDCs (p=0.0013) and CD4 + T cells (p=0.0135), while 10E8 bound only MDDC surface (p=0.002) but not CD4 + T cell surface (p>0.05). Conclusions: Ability to block cell-to-cell transmission is strongly correlated with the ability of bNAbs to bind cell surface of virus transmitting cell even in the absence of virus antigen, suggesting that binding to host cell membrane is an important parameter of determining the neutralization potential of anti-HIV-1 bNAbs. Since cell-to-cell transmission is one of the major HIV-1 transmission pathways at mucosa, it would be interesting to test these membrane-binding bNAbs as topical antivirals. 187 Enhanced Replication of Transmitted/Founder HIV-1 in Gut CD4 T Cells Eric L. Lee 1 ; Kejun Guo 1 ; Michael S. Harper 1 ; Stephanie M. Dillon 1 ; Martin D. McCarter 1 ; Brad J. Barrett 1 ; Shilpa Iyer 2 ; Beatrice Hahn 2 ; CaraWilson 3 ; Mario L. Santiago 1 1 Univ of Colorado Anschutz Med Campus, Aurora, CO, USA; 2 Univ of Pennsylvania, Philadelphia, PA, USA; 3 Univ of Colorado Hosp, Aurora, Aurora, CO, USA Background: Sexual transmission of HIV-1 results in an extreme genetic bottleneck. Reconstruction of transmitted/founder (TF) and matched Chronic Control (CC) viruses revealed that TF strains had higher infectivity and replication capacity. However, these studies were performed in mitogen-activated PBMCs, which do not capture the T cell subset composition in mucosal compartments. Further, it remains unknown if TF HIV-1 are more efficient at killing gut CD4 T cells. We used the Lamina Propria Aggregate Culture (LPAC) model to analyze R5-tropic HIV-1 replication and CD4 T cell depletion with gut mucosal bacteria increased in abundance during HIV-1 infection (HAMB). Here, we compared the relative replication and depletion capacity of TF and CC strains in the LPAC model. Methods: HIV-1 subtype B TF and CC virus stocks from 3 patients (CH40, CH470, CH58) were prepared from 293T-transfected infectious molecular clones. Lamina propria mononuclear cells (LPMCs) isolated from human jejunums (n=10 donors) were spinoculated with either mock, TF or CC HIV-1 (200ng p24/10 6 LPMCs) for 2h at 1500xg and cultured for 6 days +/- Prevotella stercorea , a prominent HAMB species. Infectious titers were analyzed by TZM-bl assay and cellular infection by intracellular p24 staining. CD4 T cell depletion was calculated from total cell counts relative to mock. Wilcoxon t-test was used for statistical analyses. Results: TF infectious titers at 6 dpi were significantly higher than CC strains (CH40 p=0.004; CH470 p=0.002; CH58 p=0.002). The %p24+ CD4 T cells was also higher for CH40 TF vs CC strains (TF 34.8% vs CC 28.0%; p=0.027). Co-incubation with P. stercorea led to a 3.8x increase in infectious titer (p=0.0003), a 10.4x increase in %p24+ cells (p<0.0001) and marked T cell depletion (p<0.0001). With P. stercorea , TF strains produced more infectious virus than CC strains (p<0.0001). However, there were no consistent differences in CD4 T cell depletion between TF vs CC strains. Gut CD4 T cell depletion did not differ between TF and CC viruses alone. With P. stercorea , depletion was increased for CH58 TF vs CC (p=0.037), but not for CH470 (p=0.08) and CH40 (p=0.16). Conclusions: HIV-1 TF strains are more infectious than CC in gut T cells with or without a gut microbe linked to HIV-1 gut dysbiosis. TF and CC strains did not exhibit consistent differences in gut CD4 T cell killing. Our findings suggest that mucosal transmission and/or initial expansion selects for HIV-1 strains with enhanced replication in gut mucosal CD4 T cells. 188 Human Th17 Cells Are Highly Permissive to Productive HIV Infection Aaron Christensen-Quick 1 ; Mark Lafferty 2 ; Lingling Sun 2 ; Anthony DeVico 2 ; Alfredo Garzino-Demo 2 1 Univ of California San Diego, San Diego, CA, USA; 2 Inst of Human Virology, Baltimore, MD, USA Background: Human Immunodeficiency Virus (HIV) preferentially depletes IL-17-producing “T helper 17” (T h 17) cells during the acute phase of infection. The loss of T h 17 cells impairs the integrity of the gut mucosal barrier, promoting chronic immune activation - a key determinant of disease progression. The susceptibility of T h 17 cells has been attributed to high CD4, CCR5, and CXCR4 expression, but we hypothesized that intracellular factors also contribute to their preferential depletion during acute HIV infection. Methods: Primary human CD4 + T cells from HIV-negative blood donors were sorted according to their expression of CCR6 and activated in T h 17- or T h 0-polarizing conditions. Polarized cells were infected with one of four HIV isolates: HIV BAL , HIV IIIB , HIV AD17 (a transmitted-founder isolate) and HIV AMLV (a pseudotype HIV vector). Infected samples and Cell Membrane Binding Determines Ability of bNAb to Prevent HIV Cell-to-Cell Transfer Hisashi Akiyama 1 ; Oscar Gonzalez 2 ; Nora G. Ramirez 1 ; Manish Sagar 2 ; Rahm Gummuluru 1 1 Boston Univ Sch of Med, Boston, MA, USA; 2 Boston Univ, Boston, MA, USA

Poster Abstracts


CROI 2016

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