Modulation of Dendritic Cells against HIV-1

描述(申请人提供)：目前尚无艾滋病毒/艾滋病疫苗。抗逆转录病毒疗法(ART)无法从感染者身上根除艾滋病毒，因为存在病毒库和产生抗逆转录病毒株。树突状细胞(DC)与HIV宿主的发展和扩散有关。现有数据表明，艾滋病毒颗粒被DC-SIGN捕获，并通过具有较温和的PHS的内噬体路由，以避免溶酶体的降解。内化的颗粒随后以反式形式传递给T细胞。一些实验室报告使用针对DC-SIGN的siRNA来减少DC中的HIV感染。但参与DC内艾滋病毒贩运的细胞内分子尚不清楚。Tetraspanins如CD9、CD63和CD81与HIV-1颗粒在内体中共定位，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使用HIV-DNA疫苗来调节DC，并评估DC-SIGN、CD63、CD81和CD9在DC内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，这些分子的RNAi干扰将有助于阐明它们的作用。核心假设将通过三个具体目标进行检验。目的1评价HIV-DNA疫苗对DC捕获和销毁病毒的调节作用。目的2评价DC-SIGN对HIV病毒粒子向T细胞传播的RNAi干扰作用。AIM 3将评估CD9、CD63和CD81在DC内艾滋病毒内部贩运中的作用。方法是通过HIV DNA转染来激活DC，并通过RNAi干扰来下调DC-SIGN、CD9、CD63和CD81的表达，以阐明它们在T细胞的内化、细胞内定位和反式感染中的确切作用。我们将使用免疫荧光共聚焦显微镜、电子显微镜、gp120Env-EL ISA、Western印迹、RT-PCR和共培养实验来获得我们的结果。我们期望HIV-DNA启动将激活DC通过抗原处理和提呈途径捕获和处理HIV。还预计，Tetraspanins的RNAi沉默将使HIV转移到溶酶体降解。我们预计，这一战略将防止艾滋病毒宿主的形成和传播。这些结果可能会对理解艾滋病毒的发病机制和传播产生深远而重大的影响。

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.