CROI 2019 Abstract eBook
210LB NATURAL HOSTS OF SIV EMPLOY UNIQUE DNA METHYLATION PROGRAMS TO SILENCE THE CD4 GENE Joseph Mudd 1 , Stephen Lai 1 , Jacob Flynn 1 , Molly Perkins 2 , Carly Elizabeth C. Starke 1 , Maarten Leerkes 1 , Vanessa Hirsch 1 , Mark Cameron 3 , Jason Brenchley 1 1 NIH, Bethesda, MD, USA, 2 BlueBird Bio, Cambridge, MA, USA, 3 Case Western Reserve University, Cleveland, OH, USA Background: African green monkeys (AGMs) downregulate CD4 to maintain a large population of CD4-CD8aa+ virus-resistant T cells which retain CD4-helper functions. AGMs can become aviremic and apparently cured of SIV by down- regulating CD4 to completion. Thus, understanding mechanisms of HIV/SIV coreceptor control in natural hosts has important implications. Methods: To understand the mechanisms of this process, purified CD4+ T cells from four AGMs, closely-related Patas monkeys, and rhesus macaques were stimulated with SEB for 5 days and RNAseq was performed on divided cells induced to downregulate CD4 (AGM, Patas) and those that divide and maintain CD4 expression (rhesus). Results: 1,917 differentially-expressed genes (DEGs) were revealed to be common among divided, CD4-downregulated AGM and Patas T cells, yet unique from divided rhesus CD4+ T cells. Genes well-known to be regulated in natural hosts were selectively present in this dataset, including CD4, CD8A, and CXCR6 (p= 1.27e-27, 2.68e-5, 6.72e-15, respectively). Pathway analysis of DEGs revealed proteins involved in DNA methylation to be enriched in CD4-downregulated AGM and Patas T cells (p=0.013). Gene expression of the Ten-eleven translocation protein 3 (TET3), was downregulated in AGM and Patas T cells induced to downregulate CD4, but not in divided CD4+ rhesus T cells (p=1.35e-11). Unique downregulation of TET3 in CD4-downregulated AGM T cells was confirmed independently by qPCR (p= 0.006). Methylation of cytosine is associated with gene silencing, and inhibition of the DNA methylation machinery with 5-aza-2 deoxycitidine inhibited CD4 downregulation in AGM CD4+ T cells induced to divide in vitro (p=0.005), indicating CD4 can be pharmacologically manipulated in natural hosts. Single clones of CD4-CD8aa+ AGM T cells revealed higher degrees of cytosine methylation at the CD4 gene promoter (p= 0.04) and a region well-within the gene body (p= 0.0001) when compared to these same genomic regions in CD4+ AGM T cells. Conclusion: These results suggest AGMs uniquely employ epigenetic mechanisms to durably silence the CD4 gene. Targeting proteins involved in DNA methylation, such as TET3, could provide avenues for modulating SIV/HIV-1 coreceptor expression in hosts that become progressively HIV/SIV infected. 211 HIV-1 INFECTION IS ASSOCIATED WITH INCREASED USP18 AND DAMPENED TYPE 1 IFN RESPONSES Sho Sugawara , Ramy El-Diwany, Joel Blankson, David L. Thomas, Andrea Cox, Ashwin Balagopal Johns Hopkins University, Baltimore, MD, USA Background: Although HIV-1 care has greatly advanced with antiretroviral therapy (ART), people living with HIV-1 (PLWH) still suffer from adverse outcomes. We and others have reported that immune activation contributes to unfavorable outcomes in PLWH. Type 1 interferons (IFN) are a potent and broad endogenous antiviral system that contribute to immune activation in PLWH. However, the efficacy of type 1 IFN against HIV-1 is diminished. We hypothesized that HIV-1 infection results in dampened type 1 IFN responses. Methods: We treated peripheral blood mononuclear cells (PBMCs) from untreated PLWH (n=9), ART-suppressed PLWH (n=7), uninfected people who inject drugs (PWIDs, n=9), and healthy controls (n=3) with IFNα. We quantified by flow cytometry induction of phospho-STAT1 (pSTAT1), critical in type 1 IFN signaling, and upregulation of antiviral interferon-stimulated genes (ISG). We also stimulated purified CD4+ T cells with IFNα from the same subjects and measured the induction of several ISGs (MX2, ISG15, PKR, BST2) using quantitative RT-PCR (qPCR). In the same experiment, we quantified baseline expression of type 1 IFN regulators (USP18 and SOCS genes). We confirmed our findings in vitro by infecting primary CD4+ T cells with a GFP-tagged HIV-1, sorting GFP+ cells, and measuring baseline expression of type 1 IFN regulators. To validate our results, we interrogated an RNAseq dataset of CD4+ T cells from PLWH who were treated with pegylated-IFNα (PEG-IFN). Results: We found that untreated PLWH had diminished induction of pSTAT1 and ISGs compared to uninfected PWID and healthy controls (p<0.05 for all); ART-suppressed PLWH had intermediate induction. Among three type 1 IFN regulators, baseline USP18 levels were best correlated with MX2 induction (r=- 0.73; p<0.05; Figure) and with other ISGs. After infection of CD4 + T cells with
GFP-tagged HIV-1, GFP+ CD4+ T cells had elevated levels of USP18 compared to GFP- CD4+ T cells (p<0.05). In PLWH who received PEG-IFN in vivo, baseline USP18 levels in activated CD4+ T cells were strongly associated and inversely correlated with MX2 induction (r=-0.77; p<0.05) and with the subsequent reduction in plasma HIV-1 RNA levels (r=-0.69; p<0.05). Conclusion: Our data are the first from PLWH to support that USP18 upregulation facilitates HIV-1 evasion of endogenous antiviral control. USP18 has been reported to inhibit type 1 IFN responses in other viral infections, and could be exploited as a molecular target to control HIV-1.
212 SINGLE HOUSING OF MACAQUES INCREASES THE IMMUNE IMPACT OF SIV INFECTION Selena Guerrero-Martin 1 , Kirstin McGee 1 , Leah H. Rubin 2 , Suzanne E. Queen 2 , Erin Shirk 1 , Ming Li 1 , Brandon Bullock 1 , David Graham 1 , Chistine Zink 1 , Lucio Gama 2 , Robert J. Adams 1 , Janice E. Clements 2 , Joseph Mankowski 2 , Kelly A. Metcalf Pate 2 1 Johns Hopkins University, Baltimore, MD, USA, 2 Johns Hopkins University School of Medicine, Baltimore, MD, USA Background: Simian immunodeficiency virus (SIV)-infected macaques are an essential animal model for the study of HIV infection, especially in the quest for an effective cure or vaccine. Macaques are a social species, yet are often singly housed for infectious disease research studies. Singly housed uninfected macaques show signs of stress, including decline in CD4+ T cell count and other changes in their immune response. SIV also causes perturbations to the immune response, as reflected most prominently by the decline in CD4+ T cell counts that is commonly used to monitor disease progression, yet the effect of single housing on the progression of SIV infection has yet to be explored. In the context of SIV and HIV, stress has previously been demonstrated to result in lower CD4+ T cell counts, more T cell activation, higher viral loads and increased mortality. We therefore hypothesized that singly housed SIV-infected macaques would demonstrate a greater impact on the immune system and less control of viral replication compared with singly housed SIV-infected macaques. Methods: We compared retrospective data on lymphocyte subset counts, T cell activation and viral loads from 35 singly and 41 socially housed SIV-infected pigtailed macaques (Macaca nemestrina) for three pre-infection timepoints and two post-infection timepoints during acute infection using linear mixed effects regression modeling. Results: Singly housed macaques demonstrated a more profound decline in the number of circulating CD4+ T cells (P = 0.0012), CD8+ T cells (P = 0.0003) and total lymphocytes (P < 0.0001) throughout acute infection compared to socially housed macaques, with the magnitude of CD4+ T cell decline in socially housed animals more closely mirroring that seen in HIV-infected patients during acute infection. We additionally observed a greater percentage of circulating activated CD69+ CD4+ T cells (P < 0.0001) and CD69+ CD8+ T cells (P < 0.0001) in singly housed macaques. Singly housed macaques furthermore had higher viral loads in the plasma (P < 0.001) and cerebral spinal fluid (P < 0.001) throughout acute infection compared to socially housed macaques, and greater variability in plasma viral load data (P < 0.0001).
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