Core 3: Single-Cell Core

项目摘要-单电池核心 单细胞技术的最新进展彻底改变了抗体发现领域。的 本提案的单细胞核心部分将利用各种尖端技术来促进和支持 两个项目的努力。我们鉴定C.艰难梭特异性抗体 我们最近开发的LIBRA-seq技术（通过将B细胞受体与抗原特异性连接， 测序）。LIBRA-seq是用于抗体发现和抗原表征的创新平台， 特异性抗体库。与其他抗体发现方法不同，LIBRA-seq是第一个能够 同时测定B细胞受体序列和大量B抗原特异性 在单细胞水平上，细胞对抗理论上无限数量的不同抗原。因此，LIBRA-seq 提供了一个独特的机会，包括多种不同的抗原作为筛选文库的一部分，用于靶向 并有效发现针对C.很难 在我们的LIBRA-seq努力的同时，我们将专注于利用单个C。艰难抗原，我们将 并对C.艰难梭菌特异性B细胞， 荧光激活细胞分选，然后对B的配对重链-轻链进行下一代测序 细菌反应性分选的B细胞的细胞受体。我们将把这种方法称为BUG-seq。关于BUG- seq，我们将能够询问更广泛的C。艰难梭菌特异性B细胞，其延伸超出 将用于LIBRA-seq实验的特异性抗原。事实上，LIBRA-seq的努力将提供 一个有重点的方法来确定C。艰难特异性B细胞，而错误序列的努力将投下广泛的网络， 捕获不同的抗原特异性抗体。 利用LIBRA-seq和BUG-seq将提供非常重要的尖端能力 作为C.艰难梭特异性抗体。总的来说，工作 这里提出的将利用各种技术，汇集微流体，下一代测序， 蛋白质科学和免疫学，结合计算数据分析。 总之，这些努力将导致无与伦比的能力，了解抗体对C。 艰难梭菌感染，并用于鉴定针对广泛的表位的抗体治疗候选物， 抗原因此，单细胞核心的工作对于所提出的发现努力非常重要 并对C.艰难梭菌特异性抗体，这是本提案中两个项目的基本目标。

Project Summary – Single-Cell Core Recent advances in single-cell technologies have revolutionized the field of antibody discovery. The Single-Cell Core part of this proposal will utilize a variety of cutting-edge technologies to facilitate and support the efforts of the two Projects. A critical advantage of our efforts for identifying C. difficile-specific antibodies is our recently developed LIBRA-seq technology (LInking B-cell Receptor to Antigen specificity through sequencing). LIBRA-seq is an innovative platform for antibody discovery and characterization of antigen- specific antibody repertoires. Unlike other approaches for antibody discovery, LIBRA-seq is the first to enable the simultaneous determination of B cell receptor sequence and antigen specificity for a large number of B cells against a theoretically unlimited number of diverse antigens, at the single-cell level. LIBRA-seq therefore provides a unique opportunity to include multiple different antigens as part of the screening library for targeted and efficient discovery of antigen-specific antibodies against different targets on C. difficile. In parallel to our LIBRA-seq efforts that will be focused on utilizing individual C. difficile antigens, we will also work toward the identification of C. difficile-specific B cells by using whole fluorescent bacteria for fluorescent-activated cell sorting, followed by next-generation sequencing of paired heavy-light chains of the B cell receptors of the bacterium-reactive sorted B cells. We will refer to this approach as BUG-seq. With BUG- seq, we will be able to interrogate a broader repertoire of C. difficile-specific B cells that extends beyond the specific antigens that will be utilized for the LIBRA-seq experiments. Indeed, the LIBRA-seq efforts will provide a focused approach to identify C. difficile-specific B cells, while the BUG-seq efforts will cast a wide net for capturing diverse antigen-specific antibodies. The utilization of both LIBRA-seq and BUG-seq will provide highly significant cutting-edge capabilities as complementary approaches for the identification of C. difficile-specific antibodies. Overall, the work proposed here will utilize a variety of techniques, bringing together microfluidics, next-generation sequencing, protein science, and immunology, combined with computational data analysis. Together, these efforts will lead to unparalleled capabilities for understanding antibody responses to C. difficile infection, and for identification of antibody therapeutic candidates against a wide range of epitopes and antigens. The work in the Single-Cell Core is therefore highly significant for the proposed efforts for discovery and characterization of C. difficile-specific antibodies, a fundamental goal for both Projects in this proposal.