Broadly Neutralizing Monoclonal Antibodies Against HIV-1

描述（由申请人提供）：该项目的长期目标是鉴定广泛识别HIV-1包膜糖蛋白（Env）并在体外阻断感染的新型单克隆抗体（mab），以指导疫苗开发。这一目标将在没有抗逆转录病毒治疗的情况下控制感染的HIV-1感染者的当地队列中实现（JAIDS, 50:403- 8,2009）。这些个体中大约13%有广泛中和抗体，为我们的研究提供了研究人群。我们方法的一个关键要素是开发一种新的测定方法来普查env特异性记忆B细胞克隆（BMem），这使得快速和直接克隆这些细胞表达的抗体的全长分子克隆成为可能（PNAS, 106:3952- 7,2009）。这种方法允许使用表达在细胞表面的天然寡聚HIV-1包膜糖蛋白（Env）和直接中和假病毒进行克隆鉴定。env反应克隆用于生产单克隆抗体，用于评估表位特异性和中和广度。该信息将用于验证这样的假设，即中和广度是由由一个或极少数中和特异性组成的单克隆或少克隆反应决定的，而不是由一系列中和特异性组成的多克隆反应。有两个具体目标。目的1-从具有持续广泛中和抗体反应的NVS中开发env特异性BMem的克隆特异性谱-抗env反应的克隆特异性谱将通过限制稀释分析、单抗分离和表位定位来确定，以识别广泛中和的单抗。目的-2-比较血浆抗体和单克隆抗体之间的中和广度，以确定重建循环抗体库中和谱所需的单克隆抗体数量。单克隆抗体的中和宽度将使用标准化的假病毒测定来确定。该数据将用于确定正在进行的广泛中和抗体反应的克隆性。这一目的将检验这样一个假设，即中和广度是由单克隆或少克隆反应决定的，这些反应由一个或很少的中和特异性组成，而不是由一系列中和特异性组成的多克隆反应。

DESCRIPTION (provided by applicant): The long-term goal of this project is to identify novel monoclonal antibodies (mAbs) that broadly recognize the HIV-1 envelope glycoprotein (Env) and block infection in vitro to guide vaccine development. This goal will be pursued in local cohorts of HIV-1 infected individuals who control their infections in the absence of anti-retroviral therapy (JAIDS, 50:403-8, 2009). Approximately 13% of these individuals have circulating broadly neutralizing antibodies providing the study population for our studies. A key element of our approach is the development of a new assay to census Env-specific memory B cell clones (BMem) that allows the rapid and direct cloning of full-length molecular clones of the antibodies expressed by these cells (PNAS, 106:3952-7, 2009). This method permits clone identification using native oligomeric HIV-1 envelope glycoproteins (Env) expressed on cell surfaces and by direct neutralization of pseudoviruses. Env-reactive clones are used to produce mAbs that will be evaluated for epitope specificity and neutralization breadth. This information will be used to test the hypothesis that neutralization breadth is determined by monoclonal or pauciclonal responses comprised of one or a very few neutralizing specificities as opposed to a polyclonal response comprised of a mosaic of neutralizing specificities. There are two specific aims. Aim 1- To develop clonal specificity profiles of Env-specific BMem from NVS who have ongoing broadly neutralizing antibody responses- Clonal specificity profiles of anti-Env responses will be determined by limiting dilution analysis, mAb isolation, and epitope mapping to identify broadly neutralizing mAbs. Aim-2- To compare neutralization breadth between plasma antibodies and mAbs representing a full clonal profile of BMem to determine the number of mAbs that must be pooled to reconstruct the neutralization profile of the circulating antibody pool. Neutralization breadth of mAbs will be determined using standardized pseudovirus assays. This data will be used to determine the clonality of an ongoing broadly neutralizing antibody response. This aim will test the hypothesis that neutralization breadth is determined by monoclonal or pauciclonal responses comprised of one or a very few neutralizing specificities as opposed to a polyclonal response comprised of a mosaic of neutralizing specificities. PUBLIC HEALTH RELEVANCE: This research is relevant to public health in that it seeks to understand how people who are infected with the AIDS virus make protective immune responses. This information will help guide the development of a vaccine against the AIDS virus.