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分离能够有效和广泛中和HIV-1原代分离物的人类抗体方面取得了突破，与其他技术一起，已经证明了人类b细胞系统产生有效免疫病毒的能力。为了在这一令人鼓舞的发现和揭示广泛中和抗体（bnAbs）可以通过免疫引起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的挑战：什么是必须通过免疫选择产生抗HIV-1 bnAbs的初始和/或初始（n/f） b细胞及其b细胞受体（BCR）序列，以及这些选择的n/f BCR如何与HIV-1包膜（Env）抗原反应？从感染shiv的恒河猴（包括淋巴结）和感染HIV-1的血清转化者身上获得宝贵的纵向样本，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。除了单个b细胞分析，我们还率先应用深度测序技术挖掘抗hiv -1抗体的整个抗体库。在这些先进技术的帮助下，可以对单个和大量（数百万）b细胞进行高分辨率分析，从而可以跟踪bnAb反应，从而使我们能够快速识别bnAb对应的n/f bcr。此外，我们将分离自身感染的HIV-1 Envs，并研究它们与感兴趣的n/f bcr的反应性。我们将验证以下假设：在感染shiv的恒河猴和感染HIV-1的人体内产生bnab的n/ BCRs可以通过纵向采集的血液和淋巴结细胞的受试者抗体组的深度测序来鉴定，并且n/f BCRs可以识别自身感染HIV-1的Envs。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HIV-1 Env抗原必须采取的初始步骤，以启动有效的抗体反应。该项目还将产生一组与bnab对应的n/f BCRs，这对免疫原筛选将是无价的，也可能识别有可能引物这些n/f BCRs的HIV-1 Envs。

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.