Approaches for Improved Interventions Against RNA Viruses with Pandemic Potential

Abstract

RNA viruses cause significant human disease globally every year and hold serious potential to cause pandemics due to their dynamic viral evolution. Over the 20th and 21st centuries, there have been eight pandemics caused by RNA viruses, most recently the COVID-19 pandemic of 2020 and the H1N1 influenza pandemic of 2009. We currently rely on two main interventions to combat these viruses: 1) antivirals to limit severe disease during infection, and 2) vaccines to prevent infection and spread throughout the population. In the aftermaths of pandemics, genetically variable endemic viruses can persist, circulating seasonally through evasion of the variable immunity generated by current vaccines and resisting antiviral inhibition. This ongoing evolution necessitates continued updates to and improvements in our interventions to combat them more efficiently. We as researchers, thus, must develop novel approaches to improve these interventions to both mitigate overall seasonal disease and better prepare for future pandemics. Therefore, the goal of this dissertation was to develop molecular tools that can be used to 1) advance the development of antiviral inhibitors and 2) improve current vaccine platforms, to ultimately reduce disease from RNA viruses both seasonally and in the face of pandemic emergence. In Chapter 2, I review the development of reverse genetics systems for multiple negative-sense RNA viruses and how they can be used to create useful genetic tools to study questions of fundamental viral biology. I then discuss how these systems can be utilized to identify and investigate potential therapeutics to combat diseases caused by these viruses. In Chapter 3, I describe the generation of a cell-based fluorescent reporter assay that can be used to assess the inhibition of viral proteases from multiple viral families, such as coronaviruses and flaviviruses. We show that this plasmid-based system can be used to assess the inhibitory profile of antivirals against a wide range of coronaviral proteases, including those with clinically reported resistance mutations, in a low biocontainment environment. This project ultimately demonstrates the value in developing adaptable cell-based tools that can be used to both identify and assess the broad inhibition profile of antivirals and provide an indication of which antivirals may be most effective against an emergent pandemic virus to limit disease. Finally, in Chapter 4, I discuss the development of an mRNA-LNP-based improved seasonal influenza vaccine. We demonstrate that this vaccine is effective in preventing severe disease in mouse and ferret models of infection, comparable to a matched FDA-approved inactivated influenza vaccine. Importantly, we found that this vaccine induces broader cross-reactive antibodies to antigenically diverse influenza viral proteins, suggesting it may also improve widely protective influenza vaccine strategies. In sum, this work details a novel method of using mRNA-LNP-based technology to improve seasonal and broader vaccine-mediated protection against severe influenza disease.