Mouse Model of HIV-1 Infection and Drug Addiction

描述（由申请人提供）：这是一项修订的II期CEBRA申请，是我们I期项目的竞争性更新和扩展，目的是利用免疫功能正常的常规小鼠开发一种新的CNS HIV-1感染模型。在第一阶段，我们首次证明HIV-1可以在几种类型的小鼠原代细胞中复制，并构建了EcoHIV，一种能够在小鼠淋巴细胞中进入、复制和传播的嵌合HIV-1。重要的是，自第一次提交以来，我们发现静脉注射EcoHIV的小鼠持续感染的频率很高，这表明在3个月的时间内血清转化和病毒在脾脏和腹膜巨噬细胞中的存在。一些动物表现出病毒渗入大脑的证据，脑组织中与炎症相关的基因表达升高，脑损伤，其中一个包含类似于其他逆转录病毒神经病变的多核巨细胞的结构。我们相信，这些结果使我们更接近于提出的模型的原理证明。根据这些发现和审稿人的建议，我们对申请进行了修改，目的是研究HIV-1感染和小鼠神经系统的发病机制。我们计划确定该模型中HIV-1神经发病机制与人类疾病之间的相似之处，并利用它们详细研究HIV-1复制动力学和病毒包膜在脑部疾病的发生和进展中的作用。本文还提出了吗啡在该模型中的作用的初步研究。具体目的是：1)研究免疫功能正常的近交系小鼠的全身EcoHIV感染和病毒进入大脑的特征；2)在EcoHIV小鼠模型中建立had样神经发病机制的实验范式；3)在EcoHIV小鼠模型中研究特异性HIV-1 gp120结构域在神经发病机制中的作用；4)研究HIV-1介导的小鼠神经发病机制与EcoHIV-1毒力的关系；5)（探索性目的）：探讨吗啡对小鼠HIV-1复制的调节作用。我们将利用DNA和RNA PCR、Elisa和免疫组织化学结合激光捕获显微解剖来研究EcoHIV在体外和体内的复制。病理变化将通过组织切片显微镜，流式细胞术，以及通过免疫组化，Elisa， RNA和实时pcr以及蛋白质阵列评估大脑中蛋白质和基因表达的变化来评分。该项目将开发和测试嵌合HIV-1物种，这些物种能够在任何常规小鼠中复制和诱导疾病，并符合CEBRA的高影响/高风险研究标准。这一模型将对研究HIV-1的神经发病机制、成瘾和滥用药物的影响以及治疗和疫苗的开发有价值。

DESCRIPTION (provided by applicant): This is a revised Stage II CEBRA application, a competing renewal and extension of our Stage I program to develop a new model for HIV-1 infection in the CNS employing immunocompetent, conventional mice. In Stage I we demonstrated for the first time that there HIV-1 can replicate in several types of primary murine cells and we constructed EcoHIV, a chimeric HIV-1 capable of entry, replication, and spread in mouse lymphocytes. Importantly, since the first submission, we found that mice inoculated i.v. with EcoHIV become persistently infected at high frequency as indicated by seroconversion and presence of virus in the spleen and peritoneal macrophages over a 3 month period. Some animals showed evidence of virus infiltration into the brain, elevated expression of genes associated with inflammation in brain tissue, and brain lesions one of which contained structures that resembled multinucleated giant cells seen other retroviral neuropathies. We believe that these results bring us closer to the proof-of-principle of the proposed model. We have revised the application in light of these findings and the Reviewers' suggestions with an overall goal of investigating HIV-1 infection and pathogenesis of the nervous system in mice. We plan to identify the similarities between HIV-1 neuropathogenesis in this model and human disease and to employ them to investigate in detail the roles of HIV-1 replication dynamics and viral envelope in initiation and progression of brain disease. Initial studies on the effect of morphine in this model are also proposed. The Specific Aims are: 1) To characterize systemic EcoHIV infection and virus entry into the brain in immunocompetent inbred mice; 2) To establish an experimental paradigm of HAD-like neuropathogenesis in the EcoHIV mouse model; 3) To investigate contribution of specific HIV-1 gp120 domains to neuropathogenesis in the EcoHIV mouse model; 4) To investigate HIV-1 mediated neuropathogenesis in the mouse as a function of EcoHIV-1 virulence; 5) (Exploratory Aim): To explore modulation of HIV-1 replication in mice by morphine. We shall investigate EcoHIV replication in vitro and in vivo using DNA- and RNA PCR, Elisa, and immunohistochemistry coupled with laser capture microdissection. Pathological changes will be scored by microscopy of tissue sections, by flow cytometry, and by evaluation of changes in protein and gene expression in the brain by IHC, Elisa, RNA- and real time-PCR, and protein arrays. This Program will develop and test chimeric HIV-1 species competent to replicate and induce disease in any conventional mouse and meets the CEBRA criteria as high impact/high risk research. This model will be valuable for studies of HIV-1 neuropathogenesis, impact of addiction and drugs of abuse, as well as development of therapies and vaccines.