Dendritic Cell Restriction and Innate Immune Sensing of HIV Infection

描述(由申请人提供)：树突状细胞(DC)在病原体的早期检测以及随后的先天和获得性免疫反应的激活中具有重要作用。树突状细胞在HIV-1感染中的作用仍然知之甚少，尽管这些细胞很可能在开发保护性艾滋病毒疫苗接种的新策略中至关重要。这项建议的目标是更好地从根本上了解HIV-1如何与DC相互作用，以及这种相互作用如何影响DC协调抗病毒免疫反应的能力。尽管树突状细胞表达HIV-1进入细胞表面的受体，但由于在病毒基因组反转录完成之前发生了阻断，它们对有效的病毒复制具有抵抗力。然而，DC拥有一种独特的能力，可以将病毒传递给CD4+T细胞，从而极大地增强这些细胞的传染性。如果细胞首先接触到病毒样颗粒(VLP)，这些病毒样颗粒(VLP)可以传递相关灵长类慢病毒SIVmac的VPX蛋白，则HIV-1可以被诱骗感染DC。VPX使DC中HIV-1复制的主要限制失活，因此可以用来确定如果HIV-1能够有效感染DC，免疫反应是如何受到影响的。在初步研究中，我们发现感染HIV-1的MDDC(使用含有VPX的SIVmac VLP)上调参与T细胞激活的共刺激分子以及I型干扰素反应，表明病毒感染在这些细胞中触发了一条先天免疫信号通路。具体目标1将确定HIV-1感染单核细胞来源的树突状细胞(MDDC)如何触发先天性免疫信号通路。基因表达谱将在MDDC上进行，为将要研究的途径提供生物信息学指南，然后将使用RNAi方法来识别参与诱导共刺激分子和其他先天免疫基因的宿主细胞分子。宿主先天免疫反应被激活的HIV-1复制阶段将通过使用HIV复制抑制剂和病毒突变体的组合来确定。具体目标2将研究DC感染HIV-1对DC和T细胞培养中病毒传播的影响，以及对抗病毒和其他抗微生物T细胞反应的影响。HIV-1(编码VPR或允许在DC中复制的VPX-VPR融合)的传播和抗原特异性T细胞反应的诱导将在MDDC和CD4+T细胞的共同培养中进行研究。通过比较允许或限制DC感染的条件下的感染情况，可以确定DC对HIV-1感染的天然抵抗力是对宿主有利还是对病毒有利。总之，这些方法将为研究抗艾滋病毒免疫反应和开发更有效的疫苗战略提供新的工具。 公共卫生相关性：了解艾滋病毒感染者的免疫反应的性质是我们努力开发有效的艾滋病预防或治疗性疫苗的核心。我们的建议是更好地表征HIV-1与树突状细胞的相互作用，树突状细胞构成了人体内早期的微生物检测系统。这些研究结果可能为诱导针对病毒的强大保护性免疫反应提供新的方法。

DESCRIPTION (provided by applicant): Dendritic cells (DCs) have essential roles in early detection of pathogens and in subsequent activation of both innate and adaptive immune responses. The role of DCs in infection with HIV-1 remains poorly understood, even though these cells are likely to be of critical importance in developing new strategies for protective HIV vaccination. The goal of this proposal is to gain a better fundamental understanding of how HIV-1 interacts with DCs and how this interaction affects the ability of the DCs to coordinate an anti-viral immune response. Even though DCs express cell surface receptors for HIV-1 entry, they are resistant to productive viral replication due to a block that occurs before reverse transcription of the viral genome can be completed. However, DCs have a unique ability to "hand-off" the virus to CD4+ T cells, and thus greatly enhance the infectivity of these cells. HIV-1 can be coaxed into infecting DCs if the cells are first exposed to virus-like particles (VLPs) that deliver the Vpx protein of the related primate lentivirus, SIVmac. Vpx inactivates a dominant restriction for HIV-1 replication in DCs and can therefore be used to determine how the immune response is affected if HIV-1 can productively infect DCs. In preliminary studies, we have found that MDDC infected with HIV-1 (using SIVmac VLPs containing Vpx) up-regulate co-stimulatory molecules involved in the activation of T cells as well as a type I interferon response, suggesting that viral infection triggers an innate immune signaling pathway in these cells. Specific Aim 1 will be to determine how HIV-1 infection of monocyte-derived DC (MDDC) triggers innate immune signaling pathways. Gene expression profiling will be performed on MDDC to provide a bioinformatics guide for the pathways to be investigated, and RNAi approaches will then be employed to identify host cell molecules involved in induction of costimulatory molecules and other innate immunity genes. The stage of HIV-1 replication at which the host innate immune response is activated will be determined by using a combination of HIV replication inhibitors and viral mutants. Specific Aim 2 will examine the consequence of DC infection with HIV-1 on the spread of virus in cultures of DC and T cells and on anti-viral and other anti-microbial T cell responses. The spread of HIV-1 (encoding Vpr or a Vpx-Vpr fusion that permits replication in DC) and the induction of antigen-specific T cell responses will be investigated in co-cultures of MDDC and CD4+ T cells. By comparing infection under conditions that permit or restrict infection of DCs, it will be possible to determine if natural resistance of DC to infection with HIV-1 is beneficial to the host or to the virus. Together, these approaches will provide new tools for studying anti-HIV immune responses and for developing more effective vaccine strategies. PUBLIC HEALTH RELEVANCE: Understanding the nature of the immune response in the HIV-infected individual lies at the heart of our efforts to develop effective prophylactic or therapeutic vaccines for AIDS. Our proposal is to better characterize the interaction of HIV-1 with dendritic cells, which make up the early microbial detection system in the body. Results of these studies may provide new approaches for inducing strong protective immune responses against the virus.