HIV infection and innate defense mechanisms in dendritic cells

异性性传播是世界范围内HIV-1感染的主要方式。因此，了解粘膜中的早期先天免疫应答对于设计预防感染的新策略至关重要。 令人信服的证据表明，在HIV-1感染后的最初几天和几周内发生的免疫事件是形成HIV-1/艾滋病病程的关键决定因素。先天性免疫反应限制HIV-1感染失败的机制目前正在深入研究中。这个项目的目标是使用系统生物学方法来剖析针对HIV-1的先天免疫应答中的早期事件。我们的提案（项目4）将使用原代DC-T细胞系统来检验HIV-1通过干扰树突状细胞（DC）功能，特别是在这些细胞中诱导I型干扰素（IFN）来操纵人类先天免疫应答动力学的假设。 我们推测HIV-1延迟DC成熟，并且一种或多种HIV-1蛋白编码IFN拮抗剂。在具体目标#1中，我们将确定HIV-1感染和IFN/模式识别应答信号传导对彼此的相互影响。在具体目标#2中，我们将评估新鉴定的细胞限制因子在病毒感染背景下在DC中的作用。我们将评估病毒复制和从树突状细胞转移到T淋巴细胞的效率，DC活化的模式和IFN/PRR信号通路。我们将使用慢病毒转导系统来下调或过表达选定的宿主因子（50-100），以测试它们在髓样DC谱系和T淋巴细胞中功能获得或丧失的影响。在具体目标#3中，我们将使用一系列不同亚型的原代病毒分离物、缺失辅助基因的HIV-1全长分子克隆来鉴定DC成熟和IFN产生的推定病毒抑制剂。我们将通过将单个辅助和调节HIV-1基因插入重组纽卡斯尔病病毒（NDV）载体中来证实和扩展我们的发现，这些载体可诱导快速而强烈的先天免疫反应。该项目结合了安娜费尔南德斯博士的互补专业领域- Sesma和Viviana Simon博士Fernando-Sesma博士在原代人DC以及不同病毒（如流感病毒、登革热病毒（DENV）和NDV）在这些细胞中引发免疫应答方面拥有丰富的经验。Simon博士在HIV-1分子病毒学和影响HIV-1复制的宿主因素（如AP 0 BEC 3）方面具有丰富的专业知识。我们项目的结果将作为项目6中生成的数学模型的原始数据。该研究项目将确定项目1和2中鉴定的限制因子对DC和T淋巴细胞中的信号传导、DC成熟、先天免疫应答和病毒抑制的作用，这两种细胞群体与粘膜免疫最相关。

Heterosexual transmission is the dominant mode of HIV-1 acquisition woridwide. Understanding the eariy innate immune response in the mucosa is, thus, essential for devising novel strategies to prevent infection. Compelling evidence suggests that immunological events occurring during the first days and weeks after HIV-1 infection are critical determinants shaping the course of HIV-1/AIDS disease. The mechanisms that underiie the failure of innate immune responses to restrict HIV-1 infection are currently under intense investigation. The goal of this program project is to dissect the early events in the innate immune response directed at HlV-1 using a systems biology approach. Our proposal (project 4) will use a primary DC-T cell system to test the hypothesis that HIV-1 manipulates the kinetics of human innate immune responses by interfering with dendritic cell (DC) function, in particular, the induction of type I interferon (IFN) in those cells. We speculate that HIV-1 delays DC maturation and that one or more of the HIV-1 proteins encodes an IFN antagonist. In specific aim #1 we will determine the reciprocal impact of HIV-1 infection and IFN/pattern recognition response signaling on each other. In specific aim #2 we will evaluate the role of newly identified cellular restriction factors in DCs within the context of viral infection. We will assess the efficiency of viral replication and transfer from dendritic cells to T lymphocytes, the pattern of DC activation and the IFN/PRR signaling pathways. We will use lentiviral transduction systems to down-regulate or over-express selected host factors (50-100) to test the effect of their gain or loss of function in myeloid DC lineages and T lymphocytes. In specific aim #3 we will identify putative viral inhibitors of DC maturation and IFN production using a series of primary viral isolates of different subtypes, HIV-1 full-length molecular clones deleted of accessory genes. We will confirm and expand our findings by inserting single accessory and regulatory HIV-1 genes into recombinant Newcastle Disease Virus (NDV) vectors which induce rapid and strong innate immune responses. This project combines the complementary areas of expertise of Dr. Ana Fernandez- Sesma and Dr. Viviana Simon. Dr. Fernandez-Sesma has extensive experience with primary human DCs and the initiation of immune responses in those cells by different viruses, such as Influenza, Dengue (DENV) and NDV. Dr. Simon has great expertise in HIV-1 molecular virology and host factors influencing HlV-1 replication, such as AP0BEC3. The results of our project will serve as raw data forthe mathematical models generated in project 6. This research project will determine the role of the restriction factors identified in project 1 and 2 on signaling, DC maturation, innate immune responses and viral inhibition in DCs and Tlymphocytes, both primary cell populations most relevant to mucosal immunity.