Characterization of Antibody-Dependent Cellular Cytotoxicity in HIV Infection

描述（由申请人提供）：了解控制感染或提供灭菌免疫的免疫反应的基础仍然是寻找有效的艾滋病毒疫苗或免疫疗法的主要目标。候选疫苗诱导的针对表面包膜糖蛋白的抗体（Abs）不能中和广泛的原代病毒分离物。因此，引起细胞毒性细胞反应一直是最近疫苗试验的主要目标。然而，这种方法未能成功地在已感染艾滋病毒的疫苗接种者体内遏制病毒复制。抗体依赖性细胞毒性（ADCC）已被证明介导对致病性猴免疫缺陷病毒攻击的绝育免疫[Hessel 2007]。在ADCC中，携带Fc的抗体结合覆盖在被感染CD4+靶T细胞上的病毒表位和携带Fc受体的效应细胞，最常见的是自然杀伤细胞（NKs），结合Ab并使用穿孔素递送颗粒酶，诱导靶细胞凋亡。我们希望研究感染患者的ADCC，以了解自然感染获得的最佳反应的大小和特征。首先，我们将使用我们实验室开发的颗粒酶B细胞毒性测定法比较一组患者血清介导的ADCC。基于这些发现，我们将选择ADCC最多的患者血清，生成单克隆抗体（mab），并根据表位特异性、亲和力、效力、宽度、IgG同型和Fc型对单克隆抗体进行表征。我们还将评估ADCC是否与经典中和不同。最后，我们将使用显微镜检查突触之间的效应器，抗体和目标。这项研究的结果将提供对介导ADCC的抗体特征的深入了解，这可能是设计HIV疫苗或免疫疗法的重要目标。假设：抗体依赖性细胞毒性（ADCC）是一种功能，已被证明介导对慢病毒感染的保护。我们假设血清中ADCC活性的变化是由hiv特异性抗体的数量、特异性和亚类决定的。目的1：表征ADCC中hiv感染者血清的效力。目的2：表征具有ADCC活性的抗体的特异性和广度。目的3：利用固定和活细胞激光扫描共聚焦显微镜（LSCM）、透射电子显微镜（TEM）、冷冻电子显微镜（cryo-EM）和断层扫描技术表征靶效应突触的结构和功能。

DESCRIPTION (provided by applicant): Understanding the basis of an immune response that controls infection or provides sterilizing immunity remains a major goal in the search for effectiv vaccines or immunotherapies for HIV. Antibodies (Abs) induced by candidate vaccines to the surface envelope glycoprotein have not neutralized a broad array of primary virus isolates. For this reason, eliciting a cytotoxic cellular response has been the primary goal in most recent vaccine trials. However, this approach has not been successful in containing viral replication in vaccinees that have become HIV-infected. Antibody-dependant cellular cytotoxicity (ADCC) has been shown to mediate sterilizing immunity against challenge with pathogenic simian immunodeficiency virus [Hessel 2007]. In ADCC, Fc-bearing Abs bind viral epitopes coating an infected CD4+ target T cell and an Fc receptor bearing effector, most commonly natural killer cells (NKs), bind the Ab and use perforin to deliver granzymes which induce apoptosis in the target. We want to study ADCC in infected patients to understand the magnitude and characteristics of the best responses achieved by natural infection. First, we will compare ADCC mediated by the sera of a cohort of patients using a granzyme B cytotoxicity assay developed in our lab. Based on these findings, we will select the sera of patients with the most ADCC, generate monoclonal Abs (mAbs), and characterize the mAbs based on epitope specificity, affinity, potency, breadth, IgG isotype, and Fc type. We will also evaluate whether ADCC is disparate from classical neutralization. Finally, we will use microscopy to examine the synapse between effectors, Abs, and targets. The outcome of this research will provide insight into the characteristics of Abs that mediate ADCC that are likely important goals in the design of HIV vaccines or immunotherapies. Hypothesis: Antibody-dependent cellular cytotoxicity (ADCC) is a function that has been shown to mediate protection from lentiviral infection. We hypothesize that variations in ADCC activity of sera are dictated by the amount, specificity, and subclass of HIV-specific antibodies. Aim 1: Characterize the potency of sera of HIV-infected individuals in ADCC. Aim 2: Characterize the specificity and breadth of antibodies with ADCC activity. Aim 3: Characterize the structure and function of the target-effector synapse using both fixed and live cell laser scanning confocal microscopy (LSCM), transmission electron microscopy (TEM) and cryo-electron microscopy (cryo-EM) and tomography.