Development of multi-modal single-cell technology to dissect epitope specificity to HIV

开发多模式单细胞技术来剖析 HIV 表位特异性

• 批准号: 10415029
• 负责人: Steven Edward Bosinger
• 金额: $ 66.04万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-26 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10415029
• 关键词: Address; Algorithms; Antibodies; Antigens; B-Lymphocytes; Bar Codes; Benchmarking; Bioinformatics; Biological Assay; Cells; Clinical Research; Communities; DNA; Data; Development; Dissection; Epigenetic Process; Epitopes; Funding; Gene Expression; Genetic Transcription; Genomics; Genotype; Goals; Grant; HIV; HIV Envelope Protein gp120; HIV Infections; HIV vaccine; Immune response; Immune system; Immunity; Immunoglobulin Genes; Immunology; Individual; Measures; Mediating; Membrane Proteins; Methodology; Methods; Modality; Modernization; Molecular Structure; Oligonucleotides; Patients; Reagent; Research; Resolution; Series; Specificity; Structure; Surface; System; T cell response; T-Cell Immunologic Specificity; T-Lymphocyte; Technology; Vaccinee; Vaccines; Validation; Viral; Viral Genome; Virus; Work; antigen-specific T cells; base; immune function; in vivo; monomer; multimodality; neutralizing antibody; new technology; novel; protein expression; receptor; response; single cell technology; single-cell RNA sequencing; technology development; tool; transcriptome; transcriptomics; vaccine platform

ABSTRACT/SUMMARY: Single-cell genomic technology is transforming modern immunology. Single-cell transcriptomic profiling combined with DNA bar-coding technology is capable of acquiring information on multiple modalities simultaneously; surface receptor quantitation, paired clonotype identity, and genotype can now be measured alongside the transcriptome in relatively routine technology. Extending this rapid technological development in single-cell genomics, it is now possible to probe epitope- specificity of individual antigen-specific cells in a high-throughput fashion. The goal of this proposal is to apply DNA bar-coding technology to build reagents capable of assessing B and T cell specificity to HIV epitopes alongside other single-cell cell readouts, and in a high throughput manner. The development of technology capable of rapid resolution of epitope specific responses would address several needs in HIV research: (i) it would greatly accelerate the discovery of novel broadly neutralizing antibodies against HIV; (ii) it would allow comprehensively profiling of T cell HIV epitopes, allowing more rapid identification of epitopes associated with protective immunity; (iii) it would characterize transcriptional states of HIV-infected cells and accurately assess differences between productively-infected, latently-infected and uninfected bystander cells. In the incumbent grant (i.e. prior funding period), we developed novel methodology to obtain paired clonotype identity and transcriptome data in antigen-specific B cells, including development, validation and benchmarking of a novel bioinformatics algorithm capable of accurately reconstructing paired immunoglobulin gene sequences in vaccine-elicited B cells. Here, we extend our prior work to incorporate additional information: antigen specificity for HIV epitopes. We will use DNA bar-coding technology to develop reagents capable of resolving epitope- specificity of HIV-specific B and T cells in a high throughput fashion. Specifically, we will apply DNA-bar codes to native HIV trimers and gp120 monomers to accelerate identification of B cells producing neutralizing antibodies. We will also develop DNA bar-coded tetramer-based technology to massively profile HIV-specific T cell responses epitope resolution. Lastly, we will build on our ability to simultaneous quantify viral genomes and host cell transcriptome data in single cells and develop methodology to differentiate the transcriptomes from latently-infected, productively-infected, and uninfected bystander cells in HIV infection. These technologies would be broadly applicable to HIV research and would provide high throughput means to identify correlates of protection in several advanced HIV vaccine platforms.

摘要/摘要： 单细胞基因组技术正在改变现代免疫学。单细胞转录组 分析与DNA棒编码技术结合使用，能够获取有关多个的信息 同时的方式；表面受体定量，配对的克隆型身份和基因型 现在可以与相对常规技术中的转录组一起测量。扩展此 单细胞基因组学的快速技术发展，现在可以探测表位 - 以高通量方式的单个抗原特异性细胞的特异性。目标的目标 建议是应用DNA棒编码技术来构建能够评估B和T的试剂 与其他单细胞读数以及高吞吐量以及高吞吐量的细胞特异性 方式。能够快速解决表位特定响应的技术的发展 将解决艾滋病毒研究中的几种需求：（i）它将大大加速新颖的发现 广泛中和抗艾滋病毒的抗体； （ii）它将允许全面分析T细胞 HIV表位，可以更快地识别与保护性免疫相关的表位； （iii）它将表征HIV感染细胞的转录状态并准确评估 有效感染，潜在感染和未感染的旁观者细胞之间的差异。在 现任赠款（即先前的资金期），我们开发了新的方法来获得配对 抗原特异性B细胞中的克隆型身份和转录组数据，包括发育， 一种能够准确的生物信息学算法的验证和基准测试 重建疫苗吸收的B细胞中配对的免疫球蛋白基因序列。在这里，我们 扩展我们先前的工作以纳入其他信息：艾滋病毒表位的抗原特异性。 我们将使用DNA条编码技术来开发能够解决表位的试剂 - HIV特异性B和T细胞的特异性以高通量方式。具体来说，我们将申请 DNA-bar代码为天然HIV三聚体和GP120单体加速鉴定B细胞 产生中和抗体。我们还将开发基于DNA栏编码的四聚体 大规模介绍HIV特异性T细胞反应的技术表位分辨率。最后，我们会的 基于我们同时量化病毒基因组和宿主细胞转录组数据的能力 单细胞并开发方法来区分转录组与潜在感染的， 艾滋病毒感染中有效地感染了且未感染的旁观者细胞。这些技术会 广泛适用于艾滋病毒研究，将提供高通量的手段来识别 几个先进的HIV疫苗平台中的保护相关。