Determining the spatial-temporal and transcriptomic host response to emerging RNA viruses at a single cell resolution

National Institute Of Allergy And Infectious Diseases

Investigators

Abstract

Zoonotic disease spillovers effect human health on a global scale. Often, the pathogenesis associated with infection does not result only from direct action of the pathogen but arises through dysregulation of the immune response. Ameliorating the pathogenetic propensities of the virus and developing new antivirals or immune modulators requires detailed knowledge of how the host is responding to the infection and how the infectious agent is counteracting the host response. To accelerate this process, in vivo studies of complex host-pathogen interactions that are spatially, transcriptomically, and temporally resolved are needed. An in vivo integrated-omic approach that combines measurement of spatial and transcriptomic cell states over time with computational methods can identify important components of host responses that cannot be observed in vitro. This then allows for testable hypothesis generation in a more targeted manner to decrease the time to therapeutic discovery. Current technologies in single-cell RNA sequencing (scRNA-Seq) and high content tissue (HCT) imaging provide these types of data but are currently difficult to apply to emerging diseases due to requiring work in BSL-3/4 settings. Additionally, new computational pipelines are needed to integrate outputs of the multiple methods to create a picture of the full immune response. Molecular studies are useful to define the mechanism of how individual cellular proteins interact with a pathogen. However, how this plays out in the in vivo environment is not fully assessed beyond testing for pathogenesis in knock out models of disease. What is missing with molecular studies is the effect that these interactions have on coordinating the complex immune responses. Moving the molecular information into the infection microenvironment and characterizing how the molecular events shape and alter this environment, can bridge this gap between molecular interactions and pathogenesis. The approach is technically innovative. Through the work associated with this project, we developed a staining method, Opal-plex, that allows for spatial measurements of over 15 protein markers on highly fixed tissues samples generated in BSL3 or BSL4 settings. These new imaging methods have been propagated in published papers that enable their adoption by the wider community involved in studies of tissues from studies conducted at this level of biocontainment. In addition, we have developed methods for applying single-cell RNA sequencing methods into BSL4 settings while preserving sample quality. Ongoing work is applying these methods to the response of wild-type and mutant animals that lack a key innate immune sensor to determine how this component of the early immune response contributes to containment of mouse adapted Ebola virus.