Mouse Model of HIV-1 Infection and Drug Addiction

描述（由申请人提供）：这是一项修订后的第II阶段CEBRA申请，是我们第I阶段项目的竞争性更新和扩展，旨在采用免疫活性的常规小鼠开发CNS中HIV-1感染的新模型。在第一阶段，我们首次证明了HIV-1可以在几种类型的原代鼠细胞中复制，我们构建了EcoHIV，一种能够进入小鼠淋巴细胞，复制和传播的嵌合HIV-1。重要的是，自首次提交以来，我们发现静脉接种EcoHIV的小鼠在3个月内以高频率持续感染，如血清转化和脾脏和腹腔巨噬细胞中存在病毒所示。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，以及脑损伤，其中一种包含类似于其他逆转录病毒神经病的多核巨细胞的结构。我们相信，这些结果使我们更接近所提出的模型的原理证明。我们根据这些发现和评审员的建议修改了申请，总体目标是研究小鼠中HIV-1感染和神经系统的发病机制。我们计划确定HIV-1神经发病机制在这个模型和人类疾病之间的相似性，并利用它们来详细研究HIV-1复制动力学和病毒包膜在脑疾病的发生和发展中的作用。初步研究吗啡在该模型中的作用。具体目标是：目的：1）研究EcoHIV感染和病毒进入免疫功能正常的近交系小鼠脑内的特征; 2）建立EcoHIV小鼠模型中HAD样神经发病机制的实验范式; 3）研究HIV-1 gp 120结构域对EcoHIV小鼠模型神经发病机制的作用; 4）研究HIV-1介导的EcoHIV-1毒力对小鼠神经发病机制的影响; 5）（探索性目的）：探索吗啡对小鼠体内HIV-1复制的调节作用。我们将研究EcoHIV复制在体外和体内使用DNA和RNA PCR，ELISA和免疫组织化学结合激光捕获显微切割。将通过组织切片显微镜检查、流式细胞术以及通过IHC、Elisa、RNA和真实的时间PCR以及蛋白质阵列评价脑中蛋白质和基因表达的变化来对病理变化进行评分。该计划将开发和测试能够在任何常规小鼠中复制和诱发疾病的嵌合HIV-1物种，并符合CEBRA高影响/高风险研究的标准。该模型对研究HIV-1的神经发病机制、成瘾和药物滥用的影响以及开发治疗和疫苗具有重要价值。

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.