Modulation of Dendritic Cells against HIV-1

描述（由申请人提供）：目前，没有艾滋病毒/艾滋病疫苗。抗逆转录病毒疗法（ART）由于病毒库的存在和抗逆转录病毒耐药菌株的发展而不能从感染者身上根除艾滋病毒。树突状细胞（dc）与HIV储存库的发展和传播有关。现有数据表明，HIV颗粒被DC-SIGN捕获并在ph较轻的核内体中传递，以逃避溶酶体的降解。内化的颗粒随后通过trans传递到t细胞。一些实验室报告使用siRNA对抗DC-SIGN来减少dc中的HIV感染。但是参与dc内HIV运输的细胞内分子尚不清楚。四联蛋白如CD9、CD63和CD81在内体中与HIV-1颗粒共定位，但它们在这种环境中的确切作用尚未完全阐明。因此，本项目的目标是通过启动HIV-DNA疫苗来调节dc，并评估DC-SIGN、CD63、CD81和CD9在dc细胞内运输中的作用。核心假设是，由于DC-SIGN、CD63、CD81和CD9参与了HIV的捕获、内化和向易感T细胞的传播，因此对这些分子的RNAi干扰将有助于阐明它们的作用。中心假设将由三个具体目标来检验。目的1将评估用HIV-DNA疫苗启动对病毒捕获和破坏的DCs的调节。目的2将评估DC-SIGN的RNAi干扰对HIV病毒向t细胞传播的影响。目标3将评估CD9、CD63和CD81在dc内HIV内部贩运中的作用。该方法是通过转染HIV DNA启动dc，并通过RNAi干扰敲低DC-SIGN、CD9、CD63和CD81的表达，以阐明它们在t细胞内化、细胞内定位和转感染中的确切作用。我们将使用免疫荧光共聚焦显微镜，电子显微镜，gp120 Env ELISA, Western blot， RT PCR和共培养分析来获得我们的结果。我们预计HIV- DNA引物将激活dc通过抗原加工和递呈途径捕获和加工HIV。我们也期望RNAi沉默四跨蛋白会将HIV转移到溶酶体降解。我们预计这一战略将防止艾滋病毒库的产生和传播。这些结果可能对理解艾滋病毒的发病机制和传播产生深远而重要的影响。

DESCRIPTION (provided by applicant): Currently, a vaccine for HIV/AIDS is not available. The antiretroviral therapies (ART) fail to eradicate HIV from infected individuals because of the existence of viral reservoirs and development of ART-resistant strains. Dendritic cells (DCs) are implicated in the development and spread of HIV reservoirs. Available data suggest that HIV particles are captured by DC-SIGN and routed in endosomes with milder pHs to evade lysosomal degradation. Internalized particles are subsequently transmitted to T-cells in trans. A few labs have reported using siRNA against DC-SIGN to diminish HIV infection in DCs. But the intracellular molecules participating in trafficking of HIV within DCs are not clear. Tetraspanins such as CD9, CD63 and CD81 colocalize with HIV-1 particles in endosomes, but their exact role in this milieu has not been fully elucidated. Therefore, the goals of this project are to modulate DCs by priming with an HIV-DNA vaccine and evaluate roles of DC-SIGN, CD63, CD81, and CD9 in intracellular trafficking within DCs. The central hypothesis is that, since DC-SIGN, CD63, CD81 and CD9 participate during capture, internalization and transmission of HIV to susceptible T cells, RNAi interference of these molecules will help to elucidate their roles. The central hypothesis will be tested by three specific aims. Aim 1 will evaluate modulation of DCs with HIV-DNA vaccine priming on capture and destruction of virus. Aim 2 will evaluate RNAi interference of DC-SIGN on transmission of HIV virions to T-cells. Aim 3 will evaluate the roles of CD9, CD63 and CD81 in internal trafficking of HIV within DCs. The approach is to prime DCs by HIV DNA transfection, and knock down expressions of DC-SIGN, CD9, CD63 and CD81 by RNAi interference to elucidate their exact roles in, internalization, intracellular localization and trans infection of T-cells. We will employ immunofluorescent confocal microscopy, electron microscopy, gp120 Env ELISA, Western blot, RT PCR and coculture assays to obtain our results. We expect that HIV- DNA priming will activate DCs to capture and process HIV via antigen processing and presentation pathways. It is also expected that RNAi silencing of tetraspanins will divert HIV to lysosomal degradation. We anticipate that this strategy will prevent creation and transmission of HIV reservoirs. These outcomes could have a profound and significant impact on understanding the pathogenesis and spread of HIV.