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

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 剖面分析与DNA条码技术相结合，能够获取多个 方法：表面受体定量、配对克隆型鉴定和基因分型 现在可以在相对常规的技术中与转录组一起进行测量。延续这一点 单细胞基因组学技术的快速发展，现在有可能探测表位- 以高通量方式识别单个抗原特异性细胞。这样做的目的是 建议将DNA条形码技术应用于建立能够评估B和T的试剂 与其他单细胞细胞读数一起对HIV表位具有细胞特异性，并具有高通量 举止。能够快速分辨表位特异性反应的技术的发展 将解决艾滋病毒研究中的几个需求：(I)它将极大地加速发现新的 广泛中和抗艾滋病毒抗体；(Ii)它将允许全面分析T细胞 艾滋病毒表位，允许更快速地识别与保护性免疫相关的表位； (3)它将表征艾滋病毒感染细胞的转录状态，并准确地评估 产生性感染、潜伏感染和未感染旁观者细胞之间的差异。在 现有拨款(即之前的资助期)，我们开发了新的方法来获得配对 抗原特异性B细胞的克隆型鉴定和转录组数据，包括发育 一种新的生物信息学算法的验证和基准测试 疫苗诱导的B细胞免疫球蛋白基因配对序列的重建。在这里，我们 扩展我们先前的工作，以纳入更多信息：HIV表位的抗原特异性。 我们将利用DNA条码技术开发能够解析表位的试剂- 高通量的HIV特异性B和T细胞。具体来说，我们将申请 天然HIV三聚体和gp120单体DNA条形码加速B细胞鉴定 产生中和抗体。我们还将开发基于DNA条码四聚体的 大规模剖析HIV特异性T细胞反应表位解析的技术。最后，我们将 以我们同时量化病毒基因组和宿主细胞转录组数据的能力为基础 单个细胞，并开发方法学来区分转录本和潜伏感染， 生产性感染、未感染的旁观者细胞在HIV感染中。这些技术将 广泛适用于艾滋病毒研究，并将提供高通量手段来识别 在几个先进的艾滋病毒疫苗平台中的保护相关性。

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.