CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

Results: Induction of IFN-I secretion in cDC depended on recognition of viral reverse transcripts by cGAS, but was largely unaffected by IFI16 or other known antimicrobial DNA sensors. RNAseq profiles obtained from single cDCs from EC infected with HIV-1 revealed the existence of distinct subpopulations of cells, characterized by differential expression of genes related to IFN signaling, immune activation, cytokine signaling and cellular metabolism, consistent with cell-intrinsic immune responses that are associated with improved stimulation of antigen-specific T cell responses. Conclusions: Cell-intrinsic immune recognition of HIV-1 by cGAS in cDC induces type I IFN responses that initiate complex changes in gene expression profiles leading to functional maturation that can enhance antiviral immunity in elite controllers. These data suggest that cell-intrinsic immune recognition of HIV in CD4 T cells and in cDC can have distinct, and partially opposing functions in HIV-1 disease pathogenesis. 186 Characterization of Interferon- α Subtypes in the LPAC Model Michael Harper 1 ; Kathrin Gibbert 2 ; Eric Lee 1 ; Kejun Guo 1 ; Stephanie Dillon 1 ; Martin McCarter 3 ; Ulf Dittmer 2 ; Cara C.Wilson 1 ; Mario Santiago 1 1 University of Colorado Anschutz Medical Campus, Denver, CO, US; 2 University of Duisburg-Essen, Essen, Germany; 3 University of Colorado Anschutz Medical Campus, Aurora, CO, US Background: IFN α can inhibit acute HIV infection but is also associated with pathogenesis. The mechanism for this dual impact of IFN α remains unknown. There are 12 IFN α proteins but only one, IFN α 2, has been used clinically. The IFN α subtypes are biologically distinct yet difficult to distinguish due to their high sequence similarity. Here we develop a method based on next-generation sequencing (NGS) to enumerate the relative abundance of different IFN α subtype transcripts. We then determined the relative potency of each IFN α subtype using the Lamina Propria Aggregate Culture model (LPAC), which recreates infection of the gut-associated lymphoid tissue (GALT), a major site of early HIV-1 replication. Methods: Primer sets for quantitative PCR and Illumina NGS were designed in highly conserved regions that encompass polymorphic sites that can be used for IFN α subtype classification. Since plasmacytoid dendritic cells (pDCs) are the major producers of IFN α and likely migrate to the GALT during acute infection, total IFN α was quantified in RNA from pDCs that were negatively selected from PBMCs and exposed to cell-free R5-tropic HIV-1 (Ba-L) for 6 hours. Moreover, IFN α subtype distribution was determined in LPAC cells ± HIV-1. To determine the potencies of individual IFN α subtypes, each IFN α subtype was added to LPAC cells once at 400pg/mL immediately post-infection with HIV-Bal normalized to 10ng p24/mL. Productive infection was assessed using intracellular p24 flow cytometry and infectious virion release by TZM.bl assay at 4dpi. Results: Negatively selected pDCs showed an average IFN α induction of >400-fold after 6hrs post HIV-1 exposure (n=5, p=0.0087). NGS analyses showed differential expression of IFN α subtypes in pDCs and LPAC cells co-incubated with HIV-1. In particular, IFN α 5, IFN α 8, and IFN α 1/13 were overrepresented in multiple ex vivo conditions. In 2 donors tested so far, IFN α 8 was the most potent in restricting productive HIV-1 infection and infectious virion release whereas IFNa16 was the least potent. Conclusions: We developed a method for quantifying IFN α transcript production and the subtype distribution in primary cells. IFN α subtypes are not equally potent at inhibiting HIV-1. IFN α 8 appears to be both highly expressed and the most potent at restricting HIV-1 in an ex vivo model of mucosal R5-tropic HIV-1 infection. The novel method presented here will be used to test if IFN α subtype expression profiles correlate with chronic immune activation in chronic HIV infection. 187 HIV Vpu Inhibits NF-kB Activity but Does Not InterfereWith Interferon Regulatory Factor 3 Lara Manganaro ; Elisa de Castro; Ana Maestre; Adolfo Garcia-Sastre; Ana Fernandez-Sesma;Viviana A. Simon Icahn School of Medicine at Mount Sinai, New York, NY, US Background: HIV accessory proteins have been shown to antagonize a number of different host innate restriction mechanisms. In particular, HIV Vpu counteracts the inhibitory effect of Tetherin on particle release and also hampers Tetherin mediated activation of the NF- κ B pathway. The role of HIV Vpu in regulating the interferon response to infection by degradation of the Interferon Regulatory Factor 3 (IRF3) has, however, been subject of conflicting reports. We therefore, systematically investigated the expression of IRF3 in primary human CD4+ T cells and macrophages infected with different HIV strains. In addition we also tested the ability of Vpu to interfere with innate immune signaling pathways like NF- κ B and IRF3 pathway. Methods: Primary CD4+ T cells and primary macrophages from several healthy donors were infected with different viruses and the levels of IRF3 were determined by both western blot analysis and intracellular staining. To assess the ability of HIV Vpu to interfere with innate immune pathways, we transfected Vpu into 293T together with different NF-kB or IRF3 reporter plasmids. We used TNF-alpha and transfection of IKKb to selectively activate NF- κ B and overexpression of TBK1 to induce IRF3. Results: Here we report that HIV Vpu variants from different viruses fail to degrade IRF3 in infected primary T lymphocytes and macrophages. We first analyzed by Western Blot the levels of IRF3 in uninfected cells or infected with a HIV wild-type virus or HIV lacking Vpu. We did not observe any changes in IRF3 expression levels. We also used an intracellular staining approach to directly compare the levels of IRF3 at a single cell level in the infected and uninfected cell population. We found that the percentage of IRF3 positive cells in the HIV infected population versus the uninfected was comparable. In addition, we observed that HIV NL4.3 Vpu had no effect on IRF3 dependent gene expression in reporter assays while it is able to down-modulated NF- κ B dependent transcription downstream IKKb activation. Moreover all tested HIV-1 Vpus retained the ability to down-modulated NF- κ B activity. Conclusions: Using different approaches we show that HIV Vpu does not affect the of IRF3 protein expression levels in primary cells during infection. Moreover, we found that Vpu is able to downregulate NFKB but IRF3 dependent transcription. Taken together, these results suggest that HIV Vpu regulates antiviral innate response by only acting on the NF- κ B pathway. 188 HIV-1 Exploits CD169 to Evade IFN α -Induced Antiviral State in Myeloid Cells Hisashi Akiyama 1 ; Nora Ramirez 1 ; Gregory Gibson 2 ; SimonWatkins 2 ; Zandrea Ambrose 2 ; Rahm Gummuluru 1 1 Boston University School of Medicine, Boston, MA, US; 2 University of Pittsburgh School of Medicine, Pittsburgh, PA, US; 3 University of Pittsburgh School of Medicine, Pittsburgh, PA, US Background: A hallmark of HIV-1 infection in vivo is chronic immune activation concomitant with type I interferon (IFN-I) production. Although IFN-Is induce an antiviral state in many cell types, HIV-1 can replicate even in the presence of IFN-Is in vivo. We have recently identified the IFN-I-inducible protein CD169 as an HIV-1 receptor on myeloid dendritic cells that can mediate robust infection of CD4 + T cells in trans. Since CD169 expression is also induced by IFN-Is on macrophages, we hypothesized that CD169 induced by IFN-I could facilitate productive HIV-1 infection in macrophages in cis and thus offset antiviral effects of IFN-Is. Methods: To investigate the effect of IFN-Is on HIV-1 replication in myeloid cells, a monocyte cell line, THP-1 or primary monocyte-derived macrophages (MDMs) were treated with IFN α for 48 hours and infected with HIV∆env-luc reporter virus pseudotyped with HIV-1 Lai (HIV/Lai) or VSV-G (HIV/G) glycoproteins, or replication competent HIV-luc. HIV-1 fusion was measured by the conventional Vpr-BlaM assay. To investigate if CD169 + myeloid cells are productively infected in vivo, lymph nodes (LNs) from pigtailed macaques chronically infected with RT-SHIV mne027 were stained for p27 gag and CD169. Results: As reported previously, HIV/G infection was severely attenuated in IFN-treated-THP-1. Surprisingly, however, replication of HIV/Lai was enhanced in IFN-treated-THP-1 than in untreated THP-1. We found that HIV/Lai fusion was greatly enhanced in IFN-treated-THP-1, while that of HIV/G was severely attenuated. This enhanced fusion and infection depended on CD169 since pretreatment with α CD169 blocking antibody abrogated the enhancement of virus fusion and replication in IFN-treated-THP-1. IFN α treatment of MDMs

Poster Abstracts

198

CROI 2015