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

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.