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

摘要/总结： 单细胞基因组技术正在改变现代免疫学。单细胞转录组 与DNA条形码技术相结合的分析能够获得多个 表面受体定量、配对克隆型身份和基因型 现在可以在相对常规的技术中与转录组一起测量。扩建这幢 随着单细胞基因组学技术的快速发展，现在可以探测表位， 以高通量方式检测单个抗原特异性细胞的特异性。这个目标 建议应用DNA条形码技术构建能够评估B和T的试剂 细胞对HIV表位的特异性以及其他单细胞细胞读数， 方式能够快速解析表位特异性应答的技术的发展 它将解决艾滋病毒研究中的几个需求：（i）它将大大加快发现新的 广泛中和HIV抗体;（ii）它将允许全面分析T细胞 艾滋病毒表位，允许更快速地鉴定与保护性免疫相关的表位; (iii)它将描述HIV感染细胞的转录状态， 生产性感染，潜伏感染和未感染的旁观者细胞之间的差异。在 现任赠款（即以前的资助期），我们开发了新的方法，以获得配对 抗原特异性B细胞中的克隆型同一性和转录组数据，包括发育， 一种新的生物信息学算法的验证和基准测试， 在疫苗诱导的B细胞中重建成对的免疫球蛋白基因序列。这里我们 扩展我们先前的工作，纳入额外的信息：HIV抗原表位的抗原特异性。 我们将使用DNA条形码技术开发能够解析表位的试剂- HIV特异性B和T细胞的特异性。具体来说，我们将应用 针对天然HIV三聚体和gp 120单体的DNA条形码，以加速B细胞的识别 产生中和抗体。我们还将开发基于DNA条形码四聚体的 大规模分析HIV特异性T细胞应答表位解析的技术。最后，我们将 建立在我们同时定量病毒基因组和宿主细胞转录组数据的能力上， 单细胞和开发方法来区分转录组从潜伏感染， 在HIV感染中，有效感染的和未感染的旁观者细胞。这些技术之后 广泛适用于艾滋病毒研究，并将提供高通量的手段，以确定 在几个先进的艾滋病毒疫苗平台的保护相关。

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.