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

B 细胞受体序列抗原特异性的高通量图谱，用于表征 HIV 疫苗接种者和感染者的抗体反应

• 批准号: 10081501
• 负责人: Ivelin Georgiev
• 金额: $ 87.9万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-02 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10081501
• 关键词: Address; Affect; Antibodies; Antibody Repertoire; Antibody Response; Antibody Therapy; Antigen Targeting; Antigens; Atlases; B-Lymphocyte Epitopes; B-Lymphocytes; Bar Codes; Benchmarking; Binding Sites; Biological Assay; Cell Line; Cells; Characteristics; Collection; Complex; DNA; Epitope Mapping; Epitopes; Event; Generations; Goals; HIV; HIV Infections; HIV Vaccine Trials Network; HIV-1; HIV-1 vaccine; Human; Immune system; Immunodominant Epitopes; Individual; Infection; Knock-out; Lead; Libraries; Light; Link; Maps; Measures; Monoclonal Antibodies; Mutation; Peptides; Performance; Polysaccharides; Prevention; Process; Receptors, Antigen, B-Cell; Recovery; Resolution; Sampling; Sequence Determination; Specificity; Technology; Time; Vaccinated; Vaccination; Vaccine Design; Vaccines; Validation; analysis pipeline; antigen binding; cell determination; co-infection; cohort; experimental study; next generation sequencing; novel; pathogen; prevent; response; screening; technology development; tool; vaccine candidate

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.

项目摘要。寻找有效的HIV-1疫苗仍然是重中之重，并且更深入 了解免疫系统如何识别HIV-1可以帮助疫苗设计。最近，很多 努力集中在理解对HIV-1感染和疫苗接种的抗体反应，因为抗体 事实证明，在治疗和预防方面有用，并作为抗体特异性疫苗设计的模板。尽管 对HIV-1的抗体反应是多克隆和复杂的，下一代测序的进步（NGS） 技术使我们能够以前所未有的分辨率看到这种多克隆响应 单个单克隆抗体序列。序列鉴定通常是功能抗体 表征，其主要成分是抗原/表位特异性的映射。 标准抗体分析管道的主要挑战是序列识别和 通常将抗体的功能表征过程解耦。这可以防止真正的高 抗体 - 抗原特异性的吞吐量映射，仅提供有限的信息 从任何给定样品中选择的抗体序列。为了应对这一挑战，我们在这里建议开发一个 对于给定样品，将使抗体序列映射到抗原特异性的技术 单高通量实验。该技术，天秤座（将B细胞受体链接到抗原特异性 通过测序），涉及将B细胞样品与A（理论无限的）池进行物理混合 条形码抗原，因此可以同时恢复：（i）配对的重型链链BCR序列 （ii）给定B细胞的抗原特异性。特别是，这个技术开发项目将广泛关注 在两个具体目标上：在特定目标1中，我们将评估不同抗原条形码策略的效果和 关于天秤座准确性和性能的其他测定变量。此目的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 特定目标2，我们将通过 筛选抗原的鸡尾酒，以及野生型抗原。这些 努力不仅会导致识别HIV特异性B细胞，还将提供残基级 来自相同高通量实验的抗原上特定表位目标的信息。 最终，对于给定的感染或疫苗接种样本，天秤座技术将提供能力 恢复抗体序列和抗原特异性，以数十万到数十万个B细胞 细胞水平。为了证明Libra-Seq的实用性，我们将表征HIV-1感染中的样本，并 疫苗接种队列。更一般地，Libra-Seq将是有效且准确的B细胞的组成工具 分析，可能对HIV-1的疫苗和抗体发现产生广泛影响 但也有许多其他生物医学意义的病原体。