Development of a Yeast Biosynthetic Platform for Rapid Production and Engineering of an Influenza Virus-like Particle Vaccine

Abstract

Although vaccination is the primary means of preventing influenza virus infection, current influenza vaccines are only moderately effective and do not provide protection against strains with pandemic potential. To overcome these limitations, new vaccine approaches are desperately needed. Virus-like particles (VLP) have recently emerged as attractive influenza vaccine candidates due to their immunogenicity, safety, and tunability. Despite their potential, engineering influenza VLP to elicit long-lasting protection against diverse influenza strains remains challenging. In this dissertation we develop a novel platform for the rapid synthesis, engineering, and testing of influenza VLP vaccine candidates in order to accelerate their development as next generation influenza vaccines. Forward progress in the development of influenza VLP vaccines has been impeded by their manufacture in higher eukaryotic hosts, which are cumbersome to genetically manipulate. To accelerate VLP synthesis, we investigated if the lower eukaryote Saccharomyces cerevisiae could support the production of influenza VLP. We show that, while S. cerevisiae can successfully express the critical influenza hemagglutinin (HA) and M1 proteins from diverse influenza subtypes, HA does not efficiently traffic to the plasma membrane where VLP formation takes place. Notably, we demonstrate that fusion of a fluorescent protein to the C-terminus of HA results in a dramatic increase in plasma membrane localization. Furthermore, following cell wall removal of cells expressing M1 and this fluorescent HA, we discovered the supernatant contained fluorescent nanoparticles possessing functional properties of influenza virus including the ability to agglutinate red blood cells and undergo endocytosis in a sialic acid dependent manner. Thus, we demonstrate for the first time the capacity of S. cerevisiae to support influenza VLP production, significantly enhancing the speed at which influenza VLP can be manufactured. To facilitate the rapid engineering of these yeast VLP strains, high-throughput protein detection methods are necessary for screening and strain characterization. To fill this need, we developed yeast intracellular staining (yICS), a technique that enables fluorescent antibodies to access intracellular compartments of yeast cells while maintaining their cellular integrity for flow cytometry. We demonstrate that yICS expedites identification of rare, high-producing yeast clones and allows simultaneous detection of multiple intracellular proteins. Along with the yeast influenza VLP system, these approaches combine to enable rapid prototyping of influenza VLP vaccine candidates. To support the comprehensive testing of VLP vaccines, we next validated high-dimensional mass cytometry (CyTOF) for in-depth immune characterization. As a proof of concept, we applied CyTOF to study the early stages of influenza infection in the mouse lung. This technology enabled quantitative tracking of 24 cellular subsets over the course of infection including their expression of activation markers, cytokines, and influenza viral proteins, making this study the most comprehensive single-cell analysis of the influenza immune landscape to date. Our investigation provides a high-resolution view of the early inflammatory response followed by the initiation of the adaptive response. Specifically, we show the importance of effector T cells in shaping the lung cytokine environment and provide new insights into the susceptibility of immune cells to viral infection and replication in vivo. Overall, this dissertation develops novel tools that synergistically combine to form a rapid platform for designing, building, and testing influenza VLP that not only can accelerate development of universal influenza vaccines but is also generally applicable for viral vaccine development, biologics synthesis, and gaining a systematic understanding of their impact on the immune system.