In-vitro model to recapitulate the natural HIV-1 latency

描述(申请人提供)：潜伏的HIV-1宿主已成为根除病毒的主要障碍。消除这种宿主的目标当然需要开发除目前的抗逆转录病毒疗法(ART)之外的新的治疗方法。要开发出这样的病毒，必须清楚地了解潜伏的HIV-1在体内是如何形成的。已经发展了几个主要的细胞模型来研究病毒从潜伏期重新激活的机制。然而，很明显，当前的细胞模型都不能很好地概括 体内控制前病毒潜伏期的机制。因此，开发一种新的模拟自然前病毒潜伏期的体外模型将是重要的。此R03应用程序的目标是开发一种HIV-1潜伏期的细胞模型，具有当前模型中缺失的高级特征，如添加细胞毒性T细胞(CTL)压力、使用原发HIV毒株等。基本假设是，将这些特征包含到细胞模型中将密切模拟体内发生的前病毒潜伏期。在该模型中，我们将用CD4-CD3q嵌合抗原受体基因修饰的CD8T细胞(即CD4CAR)作为HIV特异性CTL，并用分子克隆衍生的HIV野生型原代毒株HIVJRCSF感染靶细胞。在具体目标1中，我们将建立一种原代细胞模型，通过感染HIVJRCSF激活的CD4T细胞并将其与CD4CAR+CD8T细胞共培养来产生潜伏感染细胞(LICs)。然后，我们将对这些LIC进行表征，以验证它们与体内的LIC有多接近。一旦这个模型被开发出来，我们将追求我们的长期目标，在后续的研究中找到在LIC上表达的独特的细胞表面标记。接下来将设计新的方法，通过这些标记物在体内消除LIC。在特定的目标2中，我们将确定非诱导前驱病毒是否也在这个模型中产生。如果是这样的话，我们将对这些病毒进行表征，以阐明它们在体内形成的机制。我们期望在这个项目期间，我们将开发预期的前驱潜伏期细胞模式，作为在未来研究中产生关键新信息的重要工具。

DESCRIPTION (provided by applicant): The latent HIV-1 reservoir has become a major obstacle to virus eradication. The goal of eliminating this reservoir certainly requires the development of new therapeutic approaches other than the current antiretroviral therapy (ART). To develop one, a clear understanding of how latent HIV-1 is formed in vivo is necessary. Several primary cell models have been developed to study the mechanisms of viral reactivation from latency. However, it is evident that none of the current cell models closely recapitulates the mechanisms governing proviral latency in vivo. Therefore, developing a new in-vitro model which mimics natural proviral latency would be important. The objective of this R03 application is to develop a cell model of HIV-1 latency with advanced features that are missing in the current models, like the addition of cytotoxic T cell (CTL) pressure, the use of primary HIV strain, etc. The underlying hypothesis is that the inclusion of these features into a cell model wil closely mimic proviral latency that occurs in vivo. We will use CD8 T cells gene-modified with CD4-CD3q chimeric antigen receptor (namely, CD4CAR) as HIV-specific CTLs and the molecular clone-derived primary wild-type HIV strain HIVJRCSF to infect target cells in the model. In Specific Aim 1, we will develop a primary cell model to generate latently infected cells (LICs) by infecting activated CD4 T cells with HIVJRCSF and coculturing them with CD4CAR+CD8 T cells. Then we will characterize these LICs to verify how closely they represent their in-vivo counterparts. Once this model is developed, we will pursue our long-term goal to find unique cell surface markers expressed on LICs in subsequent studies. This will be followed by designing new approaches to target LICs through these markers for elimination in vivo. In Specific Aim 2, we will determine if noninduced proviruses are also generated in this model. If so, we will characterize these viruses to elucidate the mechanisms of their formation in vivo. It is our expectation that during this project period, we will develop the intended cell mode of proviral latency as an important tool for generating critical new information in future studies.