Single-Cell Technologies and Single-Cycle Viruses as Tools to Understand and Prevent Severe Influenza Disease

Abstract

Influenza viruses are a leading cause of acute respiratory disease worldwide, with billions of infections causing significant morbidity and mortality each year. While seasonal influenza virus infection typically causes mild illness in most individuals, the very young and the very old are prone to further complications stemming from severe influenza disease. In contrast, young adults are disproportionately burdened with severe illness after infection with pandemic influenza viruses. Thus, everyone is at risk of experiencing severe influenza disease. We do not understand, however, why some individuals experience mild vs severe disease as the mechanisms and interactions that dictate influenza disease progression are incompletely defined. Unfortunately, current influenza vaccines and antivirals are not sufficient to prevent severe influenza disease: antivirals can only limit disease when treatment is initiated before progression to severe illness, and seasonal influenza vaccines offer narrow, strain-specific protection. Therefore, there is an urgent need to both better understand the dynamics underlying influenza disease pathogenesis and generate enhanced therapeutics to prevent it. In Chapter 2, I review the history of reverse genetics systems for orthomyxoviruses and how these systems have been leveraged to generate manipulated influenza viruses. I then discuss how these genetically altered influenza viruses have been used to probe the influenza virus-host interactions that occur during severe influenza disease in vivo. In Chapter 3, I use a host of genetics-based tools, including a manipulated influenza virus, lineage tracing, and single-cell RNA sequencing, to uncover the influenza virus-host relationships that underlie severe influenza disease in a mouse model of infection. This work demonstrated that diverse gene expression patterns with the potential to influence disease outcomes exist in a single homogenous population of respiratory epithelial cells both before and during influenza virus infection. Finally, in Chapter 4, we generated a manipulated influenza virus to serve as an inactivated influenza vaccine booster. We found this vaccine boosted quantities of broadly acting antibodies that are not generally elicited by typical seasonal influenza vaccines and provided protection from both morbidity and mortality associated with lethal challenge of a drifted influenza virus in a mouse model of infection.