Spatial-Temporal Dissection of Stratified Host Tissue Responses to Severe acute respiratory syndrome-related coronaviruses in situ to Understand Intra-host Pathogenesis

项目总结/摘要 独特的宿主-病原体相互作用和适应是真核生物之间协同进化的基石 和它们的病毒病原体。鉴于RNA病毒在宿主体内的快速复制和突变率， 了解这些宿主内部在其天然组织环境中的相互作用对于开发下一代 抗病毒药物和疫苗。持续的COVID-19大流行继续阻碍SARS-CoV-2的根除 关注变体（VoC），即使是刺突蛋白的微小变化也会影响细胞进入，抗体 中和、疫苗效力和免疫应答，从而导致免疫逃逸。为什么这些变种， 沿着其他严重急性呼吸综合征相关冠状病毒（SARSr-CoV）， 宿主致病机制，以及它们是如何实现的？是否存在不同的宿主因素或反应， 影响组织特异性向性？这些问题需要通过对照实验来回答 方法解剖和deconvolute的共同进化的病毒-宿主相互作用在原位。这项建议旨在 结合联合收割机强大的反向遗传学系统，下一代组织成像平台和强大的动物模型 系统地确定宿主的免疫反应、病毒逃避策略以及免疫反应之间和免疫反应之间的关系， 宿主内病毒动力学我们建议使用这些强大的方法，首先确定宿主组织 人类COVID-19尸检和非人类灵长类动物尸检中对个体VoC的反应。接下来我们 寻求将联合收割机空间谱系追踪与标记的SARS-CoV-2 VoC和其他使用ACE 2的SARS-CoV相结合 用于进入人源化ACE 2-K18小鼠模型。为了概括体内宿主内病毒变异，我们将 工程改造这些病毒以包括编码独特条形码的短肽序列。每组条形码 对应于特定的病毒株或变体。用这些条形码化的合并物感染人源化ACE 2小鼠 病毒，结合抗体或基于杂交的空间读数和宿主的单细胞表征 免疫反应，将使系统水平的宿主内病毒的研究方法， 变化和竞争动态。这项工作所产生的空间框架和概念进步 适用于各种各样的其他生物系统和疾病，从而为更好地- 设计的治疗方法和快速反应，以了解，控制并最终根除新的生物 威胁

PROJECT SUMMARY/ABSTRACT Distinctive host-pathogen interactions and adaptations are the cornerstones of co-evolution between eukaryotes and their viral pathogens. Given the rapid replication and mutation rates of RNA viruses within their host, understanding these intra-host interactions in their native tissue context is central to developing next-generation anti-viral and vaccines. The ongoing COVID-19 pandemic continues to thwart eradication due to SARS-CoV-2 Variants of Concern (VoC), where even minor changes to the spike protein can affect cellular entry, antibody neutralization, vaccine efficacy and immune responses, thus leading to immune escape. Why do these variants, along with other Severe acute respiratory syndrome–related coronaviruses (SARSr-CoVs), differ in their host pathogenesis, and how do they achieve that? Are there differential host factors or responses that influence tissue-specific tropism? These questions need to be answered with controlled experimental approaches to dissect and deconvolute the coevolutionary viral-host interactions in situ. This proposal seeks to combine powerful reverse genetics systems, next-generation tissue imaging platforms and robust animal models towards the systematic determination of host immune responses, virus evasion strategies and both inter- and intra-host viral dynamics. We propose to use these powerful methodologies to first determine host tissue responses to individual VoCs within human COVID-19 autopsies and non-human primate necropsies. Next, we seek to combine spatial-lineage tracing with tagged SARS-CoV-2 VoCs and other SARSr-CoVs that use ACE2 for entry in the humanized ACE2-K18 mouse model. To recapitulate intra-host viral variation in vivo, we will engineer these viruses to include a short sequence of peptides encoding unique barcodes. Each set of barcodes corresponds to a specific virus strain or variant. Infection of humanized ACE2 mice with a pool of these barcoded viruses, coupled with antibody or hybridization-based spatial readouts and single-cell characterization of host immune responses, will enable a methodical approach towards the systems-level investigation of intra-host viral variation and competition dynamics. The spatial framework and conceptual advances resulting from this work are applicable to a broad myriad of other biological systems and diseases, thus paving the way to better- designed therapeutics and rapid responses to understand, control and eventually eradicate new biological threats.