Deep Sequencing to Identify B-Cell Precursors of HIV-1 Neutralizing Antibodies

描述：我们最近在应用单个B细胞探测和克隆技术分离能够有效和广泛中和HIV-1初级分离株以及其他病毒的人类抗体方面取得了突破，证明了人类B细胞系统产生对病毒的有效免疫力的能力。为了在这一令人鼓舞的发现和揭示免疫诱导广泛中和抗体(BNAbs)的机制方面取得进展，我们面临着两个尚未解决的基本免疫学问题：什么是原始和/或创始(n/f)B细胞及其B细胞受体(BCR)序列，必须通过免疫选择才能产生抗HIV-1 bNAb，以及这些选择的n/f BCR与HIV-1包膜(Env)抗原如何反应？有了来自SHIV感染的恒河猴(包括淋巴结)和HIV-1感染的血清转换子的宝贵的纵向样本，我们有一个独特的机会来解决这些问题，通过识别负责bNAbs的n/f BCR并确定它们对Env抗原的反应性。除了单个B细胞分析外，我们还率先应用深度测序技术从整个抗体库中挖掘抗HIV-1bNAbs。在这些先进技术的帮助下，对单个和高数量(数百万)B细胞提供高分辨率分析，跟踪bNab反应是可行的，从而推动我们识别与bNab对应的n/f BCR。此外，我们将分离感染HIV-1env的自体病毒，并研究它们与感兴趣的n/f BCR的反应性。我们将测试这样的假设，即在感染SHV的恒河猴和HIV-1感染者中产生bNAbs的n/fBCR可以通过从纵向收集的血液和淋巴结细胞中对主题抗体进行深度测序来识别，并且n/f BCR识别感染HIV-1环境的自体感染。如果成功，该项目将描绘一个复杂而漫长的B细胞免疫过程，揭示人们期待已久的HIV-1环境抗原启动有效抗体反应所必须采取的初步步骤。该项目还将生成一组与bNab相对应的n/f bcr，这对免疫原筛选将是非常宝贵的，还可能确定有可能为这些n/f bcr加注的HIV-1环境病毒。

DESCRIPTION: Our recent breakthroughs in applying single B-cell probing and cloning technologies to isolate human antibodies capable of potently and broadly neutralizing HIV-1 primary isolates, along with others, have demonstrated the ability of human B-cell system to generate effective immunity against the virus. To progress on this encouraging discovery and unearth mechanisms by which broadly neutralizing antibodies (bnAbs) could be elicited by immunization, we are challenged by two unsolved and fundamental immunological questions: what are the naive and/or founder (n/f) B-cells and their B-cell receptor (BCR) sequences that must be immunologically selected to generate anti-HIV-1 bnAbs, and how do these selected n/f BCRs react with the HIV-1 envelope (Env) antigen? Having access to precious longitudinal samples from both SHIV-infected rhesus macaques (including lymph nodes) and HIV-1- infected seroconverters, we have a unique opportunity to address these questions by identifying the n/f BCRs responsible for bnAbs and determining their reactivity to the Env antigen. In addition to single B-cell analysis, we have also pioneered the application of deep sequencing technology to mine anti-HIV-1 bnAbs of the whole antibody repertoire. With the aid of these advanced technologies that offer high- resolution analysis of both single and high-number (millions) B-cells, it is feasible to track bnAb responses, thereby catapulting our quest to identiy bnAb-corresponding n/f BCRs. Furthermore, we will isolate the autologous infecting HIV-1 Envs and investigate their reactivity with the n/f BCRs of interest. We will test the hypotheses that n/ BCRs that give rise to bnAbs in SHIV-infected rhesus macaques and HIV-1-infected humans can be identified by deep sequencing of the subject antibodyome from longitudinally collected blood and lymph node cells, and that the n/f BCRs recognize the autologous infecting HIV-1 Envs. If successful, this project will delineate a complex and lengthy B-cell immunological process, revealing the much anticipated initial steps that the HIV-1 Env antigen must take to initiate an effective antibody response. This project will also generate a panel of bnAb-corresponding n/f BCRs that would be invaluable for immunogen screening and may also identify HIV-1 Envs that have the potential to prime these n/f BCRs.