Core 3: Single-Cell Core

核心3：单细胞核心

• 批准号: 10625690
• 负责人: Ivelin Georgiev
• 金额: $ 22.88万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-03 至 2028-02-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10625690
• 关键词: Antibodies; Antibody Repertoire; Antibody Response; Antigen Targeting; Antigens; B-Cell Antigen Receptor; B-Lymphocytes; Back; Bacteria; Cell Separation; Cells; Clinical; Clostridium difficile; Core Protein; Coronavirus; Data; Data Analyses; Epitopes; Goals; HIV-1; Hepatitis C; Human; Immunology; Individual; Infection; Influenza; Libraries; Light; Link; Microfluidics; Peripheral Blood Mononuclear Cell; Production; Proteins; Sampling; Science; Sorting; Specificity; Techniques; Technology; Therapeutic antibodies; Vaccines; Work; clinically significant; experimental study; innovation; next generation sequencing; pathogen; screening; single cell technology; therapeutic candidate

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.

项目摘要--单细胞核心 单细胞技术的最新进展使抗体发现领域发生了革命性的变化。这个 这项提议的单细胞核心部分将利用各种尖端技术来促进和支持 这两个项目的努力。我们努力识别艰难梭菌特异性抗体的一个关键优势是 我们最近开发的Libra-seq技术(通过以下方式将B细胞受体与抗原特异性联系起来 测序)。Libra-seq是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 同时测定大量B细胞的B细胞受体序列和抗原特异性 在单细胞水平上，细胞对抗理论上无限数量的不同抗原。天秤座--因此 提供了一个独特的机会，可以将多种不同的抗原作为筛选文库的一部分 并有效地发现针对艰难梭菌不同靶点的抗原特异性抗体。 在我们专注于利用艰难梭菌单个抗原的Libra-seq努力的同时，我们将 也致力于通过使用完整的荧光细菌来鉴定艰难梭菌特异性B细胞 荧光激活细胞分选，随后对B细胞的重轻链配对进行下一代测序 细菌反应性分选B细胞的细胞受体。我们将这种方法称为bug-seq。带着窃听器- SEQ，我们将能够询问更广泛的艰难梭菌特异性B细胞，这些B细胞超出了 将用于Libra-Seq实验的特定抗原。事实上，Libra-seq的努力将提供 一种有重点的方法来识别艰难梭菌特有的B细胞，而细菌序列的努力将为 捕获各种抗原特异性抗体。 Libra-seq和bug-seq的利用将提供非常重要的尖端能力 作为鉴定艰难梭菌特异性抗体的补充方法。总的来说，这项工作 这里提出的将利用各种技术，将微流控、下一代测序、 蛋白质科学和免疫学，结合计算数据分析。 总之，这些努力将带来无与伦比的能力，以了解C。 艰难梭菌感染，并鉴定针对多种表位和 抗原。因此，单细胞核心的工作对拟议的发现努力具有重要意义 以及艰难梭菌特异性抗体的鉴定，这是这项提案中两个项目的基本目标。