Strategies of lentivirus persistence

描述(由申请人提供)：慢病毒的持续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中心障碍。拟议的研究将使用具有良好特征的马传染性贫血病毒(EIAV)模型来识别在慢病毒疾病进展过程中在免疫逃逸变体的进化和选择中重要的病毒因子。病毒在体内的终生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函数，即其复制能力。这些研究的总体目标是确定在疾病发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。具体地说，我们提出了一种假设，即疾病进展过程中REV和SU的基因变化有助于逃避广谱中和抗体和CTL，但代价是病毒复制能力。第一个目标将确定在EIAV疾病连续阶段占主导地位的env/rev基因类型是否在复制表型上有所不同。含有代表疾病每个阶段的显性env/rev基因的感染性克隆将在生长动力学和生长竞争分析中测试复制能力，并将估计变异体对的相对适合性。第二个目标将确定REV和SU的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，并用于根据对CTL杀伤的敏感性来推断免疫逃避表型。SU复制和免疫逃避表型将分别被测量为传染性和对中和抗体的敏感性。这些值，连同在特定目标1中获得的复制适合度分数，将被用于统计模型，以确定在疾病进展期间，哪些env/rev表型会影响病毒载量的变化。第三个具体目标是使用基于EIAV的伪病毒来识别免疫逃避和复制表型的特定分子决定因素。这将确定逃避广谱中和抗体和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检验在第二个目标中开发的统计模型的预测。含有免疫逃避和/或复制表型不同的env/rev基因的病毒将被接种到与EIAV具有不同免疫环境的马身上，包括：免疫缺陷、NAOVE和恢复期。感染后，将通过定量RT-PCR和序列分析来鉴定优势基因类型。这些结果将确定免疫逃避与复制表型的关系，并确定在体内不同免疫环境下SU和REV表型在EIAV选择中的各自作用。这项详细的综合分析将确定免疫逃避的关键病毒决定因素，并确定限制病毒逃避广泛反应免疫反应的新疫苗靶标。 公共卫生相关性：持续的病毒感染是艾滋病和某些癌症的病原体。拟议的研究将确定使慢病毒能够修改其复制从而逃避宿主免疫系统消除的遗传机制。这种策略允许病毒在体内持续存在，并随着时间的推移获得新的突变，从而导致临床疾病的进展。这项研究的结果将确定针对HIV-1和持久性病毒的新疫苗策略，这些病毒可以抑制免疫逃逸，防止进展为艾滋病和癌症。

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.