High-throughput mapping of antigen specificity to B-cell-receptor sequence for characterizing antibody responses in HIV-vaccinated and infected individuals

项目摘要。寻找有效的HIV-1疫苗仍然是当务之急， 了解免疫系统如何识别HIV-1有助于为疫苗设计提供信息。最近，很多 目前的研究重点是了解HIV-1感染和疫苗接种的抗体反应， 已经证明在治疗和预防中是有用的，并且作为抗体特异性疫苗设计的模板。而 针对HIV-1的抗体反应是多克隆和复杂的，下一代测序（NGS）的进展 技术使我们能够以前所未有的分辨率看到这种多克隆反应，作为一个集合， 单个单克隆抗体序列。序列鉴定之后通常是功能性抗体 表征，其主要组成部分是抗原/表位特异性的作图。 标准抗体分析流水线的主要挑战是序列鉴定和 抗体的功能表征过程通常是分离的。这样才能真正做到高... 抗体-抗原特异性的通量作图，仅提供了一小部分的有限信息， 从任何给定样品中选择抗体序列。为了应对这一挑战，我们建议制定一项 对于给定的样品，该技术将使得能够从抗体序列映射到抗原特异性， 单个高通量实验。该技术，LIBRA-seq（连接B细胞受体抗原特异性 通过测序），涉及将B细胞样品与（理论上无限的） 条形码化抗原，从而能够同时回收：（i）配对的重链-轻链BCR序列 和（ii）对给定B细胞的抗原特异性。特别是，该技术开发项目将广泛关注 在两个具体目标中：在具体目标1中，我们将评估不同抗原条形码化策略的效果， 其他测定变量对LIBRA-seq准确性和性能的影响。这一目标的目标是优化天秤座- seq能够从测序实验中准确检测BCR序列和抗原特异性。在 具体目标2，我们的目标是同时定位给定HIV特异性B细胞的靶表位， 筛选具有表位敲除突变沿着野生型抗原的抗原混合物。这些 这些努力不仅将导致识别艾滋病毒特异性B细胞，而且还将提供残留水平 来自相同高通量实验的关于抗原上的特异性表位靶的信息。 最终，对于给定的感染或疫苗接种样本，LIBRA-seq技术将提供以下能力： 以在单个细胞中恢复数万至数十万B细胞的抗体序列和抗原特异性， 细胞水平。为了证明LIBRA-seq的实用性，我们将表征来自HIV-1感染的样品， 疫苗接种队列。更一般地说，LIBRA-seq将是高效和准确的B细胞免疫的不可或缺的工具。 分析，不仅对HIV-1的疫苗和抗体发现领域具有广泛影响的潜力， 而且还用于具有生物医学意义的广泛的其它病原体。

Project Summary. The search for an effective HIV-1 vaccine remains a top priority, and a deeper understanding of how the immune system recognizes HIV-1 can help inform vaccine design. Lately, much effort has focused on understanding antibody responses to HIV-1 infection and vaccination, since antibodies have proven useful in therapy and prevention, and as templates for antibody-specific vaccine design. While antibody responses to HIV-1 are polyclonal and complex, advances in next-generation sequencing (NGS) technologies enable us to see such polyclonal responses at an unprecedented resolution, as a collection of individual monoclonal antibody sequences. Sequence identification is typically followed by functional antibody characterization, a primary component of which is the mapping of antigen/epitope specificity. A major challenge with the standard antibody analysis pipeline is that the sequence identification and functional characterization processes for antibodies are generally decoupled. This prevents truly high- throughput mapping of antibody-antigen specificity, providing only limited information for a small subset of selected antibody sequences from any given sample. To address this challenge, here we propose to develop a technology that, for a given sample, will enable the mapping of antibody sequence to antigen specificity from a single high-throughput experiment. The technology, LIBRA-seq (LInking B-cell Receptor to Antigen specificity through sequencing), involves physically mixing a B-cell sample with a (theoretically unlimited) pool of barcoded antigens, thus enabling the simultaneous recovery of: (i) paired heavy-light chain BCR sequences and (ii) antigen specificity for a given B cell. In particular, this technology development project will broadly focus on two specific aims: In Specific Aim 1, we will evaluate the effect of different antigen barcoding strategies and other assay variables on LIBRA-seq accuracy and performance. The goal in this aim is to optimize the LIBRA- seq ability to accurately detect BCR sequence and antigen specificity from a sequencing experiment. In Specific Aim 2, we will aim to simultaneously map the target epitope of a given HIV-specific B cell, by screening a cocktail of antigens with epitope-knockout mutations along with the wildtype antigens. These efforts will not only lead to the identification of HIV-specific B cells, but will also provide residue-level information about the specific epitope target on the antigen from the same high-throughput experiment. Ultimately, for a given infection or vaccination sample, the LIBRA-seq technology will provide the ability to recover antibody sequence and antigen specificity for tens to hundreds of thousands of B cells at the single- cell level. To demonstrate the utility of LIBRA-seq, we will characterize samples from HIV-1 infection and vaccination cohorts. More generally, LIBRA-seq will be an integral tool for efficient and accurate B-cell analysis, with the potential for broad impact on the fields of vaccine and antibody discovery not only for HIV-1 but also for a wide range of other pathogens of biomedical significance.