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和随后的先天性和适应性免疫应答的激活中具有重要作用。DC在HIV-1感染中的作用仍然知之甚少，尽管这些细胞在开发保护性HIV疫苗的新策略中可能至关重要。该提案的目标是更好地了解HIV-1如何与DC相互作用以及这种相互作用如何影响DC协调抗病毒免疫应答的能力。尽管DC表达用于HIV-1进入的细胞表面受体，但由于在病毒基因组的逆转录完成之前发生的阻断，它们对生产性病毒复制具有抗性。然而，DC具有将病毒“传递”给CD 4 + T细胞的独特能力，从而大大增强这些细胞的感染性。如果细胞首先暴露于病毒样颗粒（VLP），HIV-1可以被诱导感染DC，VLP可以传递相关的灵长类慢病毒SIVmac的Vpx蛋白。Vpx使DC中HIV-1复制的显性限制失活，因此可以用于确定如果HIV-1可以有效感染DC，免疫应答如何受到影响。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细胞活化的共刺激分子以及I型干扰素应答，这表明病毒感染触发了这些细胞中的先天免疫信号传导途径。具体目标1将是确定HIV-1感染单核细胞来源的DC（MDDC）如何触发先天免疫信号通路。将在MDDC上进行基因表达谱分析，为待研究的途径提供生物信息学指导，然后将采用RNAi方法来鉴定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。将通过使用HIV复制抑制剂和病毒突变体的组合来确定宿主先天免疫应答被激活的HIV-1复制阶段。具体目标2将检查DC感染HIV-1对DC和T细胞培养物中病毒传播以及抗病毒和其他抗微生物T细胞应答的影响。将在MDDC和CD 4 + T细胞的共培养物中研究HIV-1（编码Vpr或Vpx-Vpr融合体，允许在DC中复制）的传播和抗原特异性T细胞应答的诱导。通过比较在允许或限制DC感染的条件下的感染，将有可能确定DC对HIV-1感染的天然抗性是否有益于宿主或病毒。总之，这些方法将为研究抗HIV免疫反应和开发更有效的疫苗策略提供新的工具。 公共卫生相关性：了解HIV感染者的免疫反应的本质是我们努力开发有效的艾滋病预防或治疗疫苗的核心。我们的建议是更好地表征HIV-1与树突状细胞的相互作用，树突状细胞构成了体内早期微生物检测系统。这些研究结果可能为诱导针对病毒的强保护性免疫应答提供新的方法。

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.