الفهرس 
INTRODUCTION & AIM OF WORKOnly 14 pages are availabe for public view
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Abstract
According to the most recent UNAID (United Nations Programme on HIV/AIDS) global summary of the HIV (Human immunodeficiency virus) /AIDS (Acquired immunodeficiency syndrome) epidemic, there are over 33 million people currently living with HIV (”UNAIDS report on the global AIDS epidemic,” 2012). Despite nearly three decades of focused research since the discovery of HIV-1, to date there is no cure or effective prophylactic vaccine for HIV-1 infection. Although the advent of Highly Active Antiretroviral Therapy (HAART) has dramatically decreased the morbidity and mortality associated with HIV-1 infection, viral eradication is not achievable due to the persistence of latently-infected cells during treatment (Nakagawa et al., 2012). Accumulating data suggest that “non-AIDS” cardiovascular, pulmonary, renal and hepatic diseases are amplified by HIV-1 infection, and even patients with viral suppression develop premature immune senescence. These realities have created a pronounced interest in developing strategies to eradicate HIV-1 in infected individuals (Sullivan et al., 2011).
Up till 2013, the only known instance of successful HIV-1 eradication is that of the “Berlin Patient”. This individual, diagnosed with both HIV disease and acute myelogenous leukemia, underwent an allogeneic bone marrow transplant using stem cells from a donor who was
homozygous for the CCR5-Δ32 mutation. After repopulation with these HIV-1-resistant cells lacking the CCR5 viral coreceptor, the Berlin Patient has maintained undetectable viremia for multiple years and appears to be cured of HIV-1 infection. There is an overarching theme that can be distilled from this singular success: HIV-1 eradication may be achieved by rendering an individual‟s cells resistant to HIV-1 infection (Allers et al., 2011; Hutter et al., 2009). In 2013, two recent reports increased the hopes that it is possible to control the virus that causes AIDS with early treatment; so further therapy is not immediately needed. (Saez-Cirion et al., 2013) reports that 14 patients with HIV, who received antiretroviral treatment within 10 weeks of infection, had their viral loads decreased so much that scientists say they are ”functionally cured”. In March 2013, a report announced that a baby in Mississippi who was born with HIV was also shown to be ”functionally cured” because of the very early treatment. She received high doses of three antiretroviral drugs within 30 hours of her birth. There is no evidence, two years later that HIV is present in her blood (Park, 2013).
The molecular and immunologic foundations of future clinical management strategies may also be found in Elite Controllers (EC), HIV-1-infected individuals who naturally suppress HIV-1 to undetectable levels in the absence of antiretroviral therapy. In addition to undetectable viremia, elite controllers have stably low HIV-1 proviral DNA levels in peripheral blood mononuclear cells (PBMC) (Saksena, Rodes, Wang, & Soriano, 2007), suggesting they may also harbor important clues about clearance of the latent reservoir and the eradication of HIV-1 infection. Elite controllers represent a promising model for a functional cure wherethe host immune response to HIV-1 infection can be studied. An increased understanding of the viral and host factors that influence elite control is still necessary to provide information on the nature and mechanisms of control of HIV-1 replication. It is widely hoped that the knowledge gained regarding the mechanism of virus control could inform both the vaccine and HIV eradication efforts (Deeks & Walker, 2007). Some recent reports suggest that CD4+ T cells from elite controllers are unusually persistent and are refractory to HIV-1 infection and integration in vitro, indicative of intrinsic antiretroviral defense mechanisms (Chen et al., 2011; Saez-Cirion et al., 2011), However other studies reported otherwise (O’Connell, Rabi, Siliciano, & Blankson, 2011; Rabi et al., 2011) so the role of the intrinsic immunity in the elite controllers is yet to be identified. The study of host restriction factors (intrinsic immunity) and their contributions toward an effective immune response can lead to the development of novel strategies for an effective HIV-1 therapeutic vaccine.
“Restriction factors” are set of antiretroviral immune factors have been identified over the last decade that provide the host with a pre-mobilized defense against retroviral infection. This set of genes and cellular microRNAs is part of complexity intrinsic immune mechanisms in which retroviruses interact with their hosts (Malim & Bieniasz, 2012; Wolf & Goff, 2008). Emerging data suggest that these genes and microRNAs may be attractive targets for curative approaches. In direct relation to the previous statement, a number of intrinsic immune factors have been identified over the last decade, which suppresses retroviral replication in vitro. In particular, members of the apolipoprotein BmRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) gene family (Sheehy, Gaddis, Choi, & Malim, 2002; Sheehy, Gaddis, & Malim, 2003), tripartite motif-containing protein 5α (TRIM5α) (Stremlau et al., 2004), and BST-2 (tetherin/CD317) (Neil, Zang, & Bieniasz, 2008; Van Damme et al., 2008) have garnered substantial attention since they specifically inhibit HIV-1 replication in vitro, and were among the first such factors to be discovered. Hence, the precise molecular details governing each of their interactions with HIV-1 have been characterized extensively in cell culture and cell-free systems in vitro. In addition, there is a rapidly growing list of restriction factors (both genes and microRNAs) that use diverse molecular mechanisms to exert potent suppressive activity against HIV-1 in a range of cell types and tissues.
MicroRNAs (miRNA) are a class of small non-protein-coding RNAs that typically regulate gene expression by binding to protein-coding regions of mRNA, thereby repressing translation. MicroRNAs modulate the replication and propagation of several viruses including hepatitis C virus (Jopling, 2012), primate foamy virus (Lecellier et al., 2005), and influenza A virus (L. Song, Liu, Gao, Jiang, & Huang, 2010), and host microRNAs have recently been implicated in the transcriptional repression of HIV-1 in latently-infected resting CD4+ T cells. Human miRNA-28, miRNA-125b, miRNA-150, miRNA-223, and miRNA-382 target the 3‟ UTR of HIV-1 transcripts, interfering with HIV-1 accessory gene expression potentially shifting productive infection into latency in resting CD4+ T lymphocytes (Huang et al., 2007). The difference in expression level of several anti-HIV-1 miRNAs in monocytes and macrophages correlates with cellular permissibility to HIV-1 infection in
vitro (X. Wang et al., 2009). Two new studies have attempted to examine the role of miRNA in elite control, but are inconclusive due to lack of appropriate patient comparator groups (e.g. HAART-suppressed individuals to correct for the effects of viremia on miRNA) (Witwer, Watson, Blankson, & Clements, 2012). In next chapter, we will review each of those restriction factors (genes and microRNAs) in details.
Most of the restriction factors are known (at least in vitro) to be interferon-stimulated genes. Interferon-α treatment potently suppresses HIV-1 in vitro and in vivo patients. Induction of interferon-α (IFN-α) expression is a critical first step in the defense against a range of viral infections (Isaacs & Lindenmann, 1957). The antiretroviral activity of the IFN-α cytokine was demonstrated in vitro almost immediately after the discovery of HIV-1, and includes inhibition of HIV-1 reverse transcription, viral assembly and virion release (Poli, Orenstein, Kinter, Folks, & Fauci, 1989). IFN-α has been reported to suppress HIV-1 viremia in chronically infected individuals, often reducing plasma viral load by well over 2 logs (Asmuth et al., 2010; Pillai et al., 2012). However, the mechanism underlying the antiretroviral potency of IFN-α remains to be elucidated. Despite observations from microarray experiments indicating that a vast number of genes are induced by IFN-α treatment in vitro, it is likely that only a small fraction of these induced genes are relevant to the IFN-α-mediated control of HIV-1 in vivo. By scrutinizing the molecular effects of exogenous IFN-α treatment in HIV-1-infected individuals, it may be possible to identify novel antiretroviral effector mechanisms that underlie the suppressive capacity of IFN-α in vivo. Our group recently published data suggesting that the BST-2
INTRODUCTION & AIM OF WORK
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restriction factor play critical roles in the interferon-mediated suppression of HIV-1 viremia in chronically infected individuals (Pillai et al., 2012). Recent data suggest that type I interferon modulates cellular microRNA profile as an antiviral mechanism against hepatitis C virus (HCV) (Jopling, 2012). The relevance of microRNA to the IFN-α-mediated suppression of HIV-1 remains to be addressed, and is part of the focus of this proposal.
The overall aim of the work is:
Identification of host cell-intrinsic immune factors (microRNAs and/or restriction factors genes) that can be modulated to suppress HIV-1 in vivo.
In order to achieve this aim, we measured the expression profile of both restriction factors genes and microRNA in two different cohorts of individuals.
Aim (1):
Effects of interferon-α treatment on anti-HIV-1 cell-intrinsic immunity (Both anti-HIV-1 restriction factors genes and cellular microRNAs) in HIV/HCV co-infected patients in vivo.
We hypothesize that particular cellular genes and/or microRNAs consistently modulated in vivo in response to interferon-α treatment, and one or more of these identified interferon-modulated genes and/or microRNAs exhibit suppressive activity against HIV-1 replication in vivo.
Identification of interferon-α-modulated microRNA and host restriction factors may lead to novel antiretroviral strategies. By characterizing IFN-α effects on miRNA profile, we may be able to identify particular miRNA variants that regulate the expression of host restriction factors. This critical information may allow us to specifically enhance intrinsic immune pressure in the human host while avoiding the nonspecific, toxic cascade typically associated with IFN-α treatment.
To address this hypothesis in aim 1, we measured the expression profiling of a well established 34 anti-HIV-1 restriction factors and 754 microRNAs in a longitudinally collected, cryopreserved specimens from antiretroviral therapy (ART)-naïve HIV/HCV-coinfected individuals in the Swiss HIV Cohort Study (SHCS) who underwent IFN-α/ribavirin combination (IFN-α/riba) therapy for HCV disease. Our group, identified from the literature, 34 established host restriction factors genes meet two inclusion criteria: those restriction factor have an evidence of direct inhibition of HIV-1 in vitro in replication assays, Also the gene must be expressed in human peripheral blood cells, since this is the cell type we are using in our study. We created a custom real-time PCR array to measure the expression profile for those 34 genes and call it the Cummilative Restriction array or “CuRe” array. We used commercially available real-time PCR arrays to measure the expression of 754 different cellular microRNAs.
Aim (2):
Identification of the role of anti-HIV-1 intrinsic immunity (Both microRNAs and anti-HIV-1 restriction factors) in the natural HIV-1 viral control in the “elite controllers” individuals in vivo.
We hypothesize that cellular restriction of HIV-1 replication by host microRNAs and restriction factors plays a significant role in the control of HIV-1 in ”elite controllers”. To address this hypothesis, we prospectively collected blood samples from individuals enrolled in the UCSF SCOPE Cohort to perform a cross-sectional comprehensive analysis of host anti-HIV restriction factors and miRNA expression (using the above CuRe array, and the microRNA commercial arrays) in CD4+ T cells between elite controllers, HIV-1-infected untreated (viremic non-controllers) individuals, HIV-1- infected treated (aviremic) individuals, and uninfected controls to identify a unique signature pattern of restriction factors and miRNA expression in peripheral CD4+ T cells that is specific to elite controllers. In collaboration with a biostatistician at the UCSF Clinical & Translational Science Institute, we identified particular gene and microRNA variants that are over- and/or under-expressed in elite controllers with respect to the other sampled patient groups.