Strategies of lentivirus persistence

描述（由申请人提供）：慢病毒不断进化和逃避免疫控制的能力是开发有效的艾滋病毒疫苗的主要障碍。拟议的研究将使用表征良好的马传染性贫血病毒（EIAV）模型来确定在慢病毒疾病进展过程中进化和选择免疫逃逸变异的重要病毒因子。病毒在体内的终身持久性是其逃避免疫识别和消除的能力以及其复制能力的功能，即其复制能力。这些研究的总体目标是确定疾病进展过程中宿主免疫环境的变化是否可重复地选择复制能力改变的病毒变体。具体而言，我们提出验证在疾病进展过程中Rev和SU的遗传变化有助于逃避广泛中和抗体和CTL的假设，以牺牲病毒复制能力为代价。第一个目的是确定在EIAV疾病的顺序阶段占主导地位的env/rev基因型是否在复制表型上有所不同。将在生长动力学和生长竞争试验中测试含有代表疾病各阶段的显性env/rev基因型的传染性克隆的繁殖能力，并估计变异对的相对适合度。第二个目标将确定Rev和SU变异对复制能力和免疫逃避的影响。Rev的复制表型将被量化为核输出活性，并用于推断基于对CTL杀伤敏感性的免疫逃避表型。SU复制和免疫逃避表型将分别作为传染性和对中和抗体的敏感性来测量。这些值以及在Specific Aim 1中获得的复制适合度评分将用于统计模型中，以确定在疾病进展过程中，哪些env/rev表型影响病毒载量的变化。第三个具体目标将使用基于eiav的假病毒来鉴定免疫逃避和复制表型的特定分子决定因素。这将确定从广泛中和抗体中逃脱与复制能力之间是否存在遗传联系。在第四个目标中，体内竞争分析将直接测试第二个目标中开发的统计模型的预测。将含有在免疫逃避和/或复制表型上不同的env/rev基因型的病毒接种到具有与EIAV相关的不同免疫环境的马身上，包括：免疫缺陷、幼稚和恢复期。感染后，将通过定量RT-PCR和序列分析确定优势基因型。这些结果将确定免疫逃避与复制表型之间的关系，并确定SU和Rev表型在体内不同免疫环境下EIAV选择中的各自作用。这项详细的综合分析将确定免疫逃逸的关键病毒决定因素，并确定限制病毒从广泛反应性免疫反应中逃逸的新疫苗靶点。

DESCRIPTION (provided by applicant): The ability of lentiviruses to continually evolve and escape immune control is the central impediment in developing an effective vaccine for HIV. The proposed studies will use the well-characterized equine infectious anemia virus (EIAV) model to identify virus factors important in the evolution and selection of immune escape variants during progression of lentiviral disease. The lifelong persistence of virus in vivo is a function of its ability to evade immune recognition and elimination as well as its ability to replicate, i.e. its replicative capacity. The overall goal of these studies is to determine if changes in the host immune environment during progression of disease reproducibly select for virus variants with altered replicative capacity. Specifically, we propose test the hypothesis that genetic changes in Rev and SU during progression of disease contribute to evasion of broadly neutralizing antibody and CTL at a cost in virus replicative capacity. The first aim will determine if env/rev genotypes that predominate at sequential stages of EIAV disease differ in replication phenotype. Infectious clones containing dominant env/rev genotypes representative of each stage of disease will be tested for replicative capacity in growth kinetic and growth competition assays, and the relative fitness of pairs of variants will be estimated. The second aim will determine the impact of variation in Rev and SU on replicative capacity and immune evasion. The replication phenotype of Rev will be quantified as nuclear export activity, and used to infer an immune evasion phenotype based on sensitivity to CTL killing. The SU replication and immune evasion phenotypes will be measured as infectivity and sensitivity to neutralizing antibody, respectively. These values, together with the replication fitness score obtained in Specific Aim 1, will be used in statistical models to determine which env/rev phenotypes affect changes in virus load during progression of disease. The third specific aim will use EIAV-based pseudovirus to identify specific molecular determinants of immune evasion and replication phenotypes. This will establish if there is a genetic link between escape from broadly neutralizing antibody and replicative capacity. In the fourth aim, in vivo competition assays will directly test the predictions of the statistical model developed in the second aim. Viruses containing env/rev genotypes that differ in immune evasion and/or replication phenotype will be inoculated into horses that have distinct immune environments with respect to EIAV, including: immunodeficient, naove, and convalescent. Following infection, dominant genotypes will be identified by quantitative RT-PCR and sequence analysis. These results will identify the relationship between immune evasion and replication phenotype and determine the respective roles of SU and Rev phenotype in EIAV selection under different immune environments in vivo. This detailed, integrative analyses will identify critical virus determinants of immune evasion and identify new vaccine targets that limit virus escape from broadly reactive immune responses. PUBLIC HEALTH RELEVANCE: Persistent virus infections are the etiological agents of AIDS and some cancers. The proposed studies will identify the genetic mechanisms that enable lentiviruses to modify their replication and thereby escape elimination by the host immune system. This strategy allows the virus to persist in vivo and, over time, acquire new mutations that leads to progression of clinical disease. The results of this study will identify new vaccine strategies for HIV-1 and persistent viruses that inhibit immune escape and prevent progression to AIDS and cancer.