Dissecting Polyclonal Sera to Reveal Correlates of Productive Immune Responses to HIV

项目摘要/摘要 用于HIV疫苗开发的合理的、基于结构的免疫原设计已经开始交付 临床前非人类灵长类动物(NHP)和临床研究(如IAVI)都取得了令人振奋的结果 G001第一阶段试验[NCT03547245]。此外，单细胞RNAseq和电子的研究进展 基于显微镜的多克隆抗体图谱(EMPEM)使疫苗和 病原体诱导的抗体反应以前所未有的分辨率，进一步使合理的疫苗成为可能 设计。EMPEM和单细胞B细胞分析提供了根本不同的数据格式，如果 适当的整合，可以以前所未有的细节照亮免疫反应。我们最近延长了 可区分多克隆抗体表位的原始阴性染色EMPEM方法 以高分辨率低温EM为基础的EMPEM获得分子细节的特异性和方法角度 表位-副表位界面。在最好的情况下，我们可以为 从结构数据中确定互补决定区(CDR)，识别VH/V1基因用途，并查询 抗体序列数据库，可直接生成单抗。因此，我们可以做空 省去了使用常规方法产生表位特异性单抗的繁琐工作 方法：研究方法。这些分子细节对于分析牧羊式免疫或抛光免疫至关重要。 目前正在尝试的艾滋病毒疫苗开发战略，其中免疫原是 旨在诱导成熟的抗体，具有CDR的特定特征，类似于广为人知的 中和抗体(BNAbs)。在此续期申请中，我们建议延长决议， 吞吐量和我们的EMPEM方法的实用性。此外，由于大多数病原体都会进入人体 通过粘膜表面，将探索(疫苗诱导的)粘膜抗体的分析。数据 将提供与令人兴奋的HIV组合的生产性免疫反应的新关联 具有表位聚焦或生殖系靶向特性的免疫原，将有助于下选择和 在正在采取的各种方法中确定优先顺序。

Project Summary/Abstract Rational, structure-based immunogen design for HIV vaccine development has began to deliver promising results in both, pre-clinical non-human primate (NHP) and clinical studies such as the IAVI G001 phase I trial [NCT03547245]. Moreover, advances in single cell RNAseq and electron microscopy-based polyclonal antibody mapping (EMPEM) have enabled analysis of both vaccine and pathogen-induced antibody responses at unprecedented resolution, further enabling rational vaccine design. EMPEM and single cell B cell analysis deliver fundamentally different data formats that if properly integrated, can illuminate immune responses at unprecedented detail. We recently extended the original negative stain EMPEM method, capable of distinguishing polyclonal antibody epitope specificities and angle of approach, to high resolution cryoEM-based EMPEM to yield molecular details of epitope-paratope interfaces. In the best cases we can derive some sequence information for the complementarity determining regions (CDRs) from structural data, identify Vh/Vl gene usage, and query databases of antibody sequences to directly generate monoclonal antibodies. As such, we can short cut the laborious efforts to generate epitope specific monoclonal antibodies using conventional methods. These molecular details are critical for analyzing shepherding or polishing immunization strategies that are now being attempted for HIV vaccine development, wherein immunogens are designed to elicit and mature antibodies with specific features in CDRs that resemble known broadly neutralizing antibodies (bnAbs). In this renewal application we propose to extend the resolution, throughput, and utility of our EMPEM approach. Moreover, since most pathogens will enter the body through mucosal surfaces, analysis of (vaccine-induced) mucosal antibodies will be explored. The data generated will provide new correlates of productive immune responses to the portfolio of exciting HIV immunogens with epitope focusing or germline targeting properties that will help down select and prioritize amongst the various approaches being pursued.