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

描述：我们最近在应用单 B 细胞探测和克隆技术来分离能够有效和广泛中和 HIV-1 初级分离株的人类抗体以及其他技术方面取得了突破，这证明了人类 B 细胞系统能够产生针对病毒的有效免疫力。为了在这一令人鼓舞的发现和挖掘免疫引发广泛中和抗体 (bnAb) 的机制方面取得进展，我们面临着两个未解决的基本免疫学问题的挑战：必须经过免疫学选择以产生抗 HIV-1 bnAb 的初始和/或创始 (n/f) B 细胞及其 B 细胞受体 (BCR) 序列是什么，以及这些选定的 n/f BCR 如何与 HIV-1 包膜发生反应（环境）抗原？通过从感染 SHIV 的恒河猴（包括淋巴结）和感染 HIV-1 的血清转换器获得珍贵的纵向样本，我们有一个独特的机会通过识别负责 bnAb 的 n/f BCR 并确定它们对 Env 抗原的反应性来解决这些问题。除了单 B 细胞分析外，我们还率先应用深度测序技术来挖掘整个抗体库中的抗 HIV-1 bnAb。借助这些先进技术，可以对单个和大量（数百万）B 细胞进行高分辨率分析，跟踪 bnAb 反应是可行的，从而推动我们对识别 bnAb 对应的 n/f BCR 的探索。此外，我们将分离自体感染 HIV-1 Env，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：在感染 SHIV 的恒河猴和感染 HIV-1 的人类中产生 bnAb 的 n/ BCR 可以通过对纵向收集的血液和淋巴结细胞中的主题抗体组进行深度测序来识别，并且 n/f BCR 识别自体感染的 HIV-1 包膜。如果成功，该项目将描绘出复杂而漫长的 B 细胞免疫过程，揭示 HIV-1 Env 抗原启动有效抗体反应所必须采取的备受期待的初始步骤。该项目还将生成一组 bnAb 对应的 n/f BCR，这对于免疫原筛选非常有价值，并且还可能识别有可能引发这些 n/f BCR 的 HIV-1 包膜。

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.