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

产品说明：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主要分离物的人抗体方面取得的突破，沿着其他人，已经证明了人B细胞系统产生针对病毒的有效免疫力的能力。为了在这一令人鼓舞的发现上取得进展，并发掘通过免疫可以引起广泛中和抗体（bnAb）的机制，我们面临着两个未解决的基本免疫学问题的挑战：必须通过免疫学选择以产生抗HIV-1 bnAb的初始和/或建立者（n/f）B细胞及其B细胞受体（BCR）序列是什么，这些选择的n/f BCR如何与HIV-1包膜（Env）抗原反应？在获得来自SHIV感染的恒河猴（包括淋巴结）和HIV-1感染的血清转换者的珍贵纵向样本后，我们有一个独特的机会通过鉴定负责bnAb的n/f BCR并确定其对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整个抗体库的抗HIV-1 bnAb。借助于这些提供单个和大量（数百万）B细胞的高分辨率分析的先进技术，追踪bnAb应答是可行的，从而促进了我们对鉴定bnAb对应的n/f BCR的探索。此外，我们将分离自体感染的HIV-1 Env，并研究其与目标n/f BCR的反应性。我们将检验以下假设：在SHIV感染的恒河猴和HIV-1感染的人中产生bnAb的n/BCR可以通过对纵向采集的血液和淋巴结细胞中的受试者抗体组进行深度测序来鉴定，并且n/f BCR识别自体感染的HIV-1 Env。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。该项目还将产生一组bnAb对应的n/f BCR，这对免疫原筛查非常宝贵，也可能鉴定出有可能引发这些n/f BCR的HIV-1 Env。

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.