Date of Award
12-20-2024
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Influenza A virus (IAV) poses substantial epidemiological burdens around the world, with an estimated annual global mortality rate in the hundreds of thousands. Its rapid rate of mutation gives it a propensity for causing seasonal epidemics and occasional pandemics. As an airborne virus, IAV is most commonly transmitted through droplets and aerosols. Antibodies play a crucial role in the immune defense against IAV infection. By understanding the mechanisms of antibody-mediated defense and the transmission dynamics of IAV, we can better inform strategies for prevention, vaccination, and treatment, ultimately mitigating its impact on public health. The work encompassed in this dissertation seeks to use confocal fluorescence microscopy towards two aims. The first is to examine the effects of modulating antigen density and orientation on the ability of antibodies to leverage bivalent binding. The second is to use virus-like particles (VLPs) as a safer alternative to infectious virus to compare and evaluate various methods of aerosolization and collection of virions for aerosol research. Hemagglutinin (HA) is the most abundant protein on the IAV surface and serves as its receptor binding protein. The binding affinity between HA and sialic acid is very low; only through hundreds of interactions can a virion effectively bind to a host cell. In this way, IAV leverages multivalent binding to compensate for the low affinity. However, high HA densities also leave the virus more vulnerable to recognition and subsequent neutralization by antibodies, which can also attach with multiple binding sites. The tradeoffs between greater receptor binding and the risk of antibody neutralization remain unclear, particularly for a pleiomorphic virus like IAV that can vary greatly in its morphology. To investigate how viral surface features affect the ability of antibodies to bind bivalently, we developed fluorescence-based assays to measure dissociation kinetics and steady-state binding of antibodies to intact virions. Studying two antibodies that target the HA head domain (S139/1 and C05), we found that bivalent attachment can offset weak monovalent affinity through crosslinking. By varying HA density in engineered viruses and synthetic nanoparticles, we observed that bivalent binding remains effective down to one-tenth the typical physiological HA density. Additionally, by using structure-guided modeling and an antibody that tilts HA, we identified HA anchor flexibility as another factor influencing antibody binding. Studying the transmission of airborne pathogens has become increasingly critical due to recent global pandemics and ongoing threats. VLPs are non-infectious particles that mimic the viral structure, providing a safer alternative for researching aerosolized virus. The application of VLPs for testing bioaerosol instruments has been limited, with significant variations in the efficiencies of different aerosolization and collection techniques. We aimed to evaluate various aerosolization and sampling methods using influenza VLPs as a model. Our findings indicate that medical nebulizers are most effective at preserving hemagglutinin and neuraminidase antigenicity, while the liquid spot sampler (LSS) yields the highest concentration of VLPs in collected samples and best preserves the morphology of filamentous virions. Separately, expressed and purified VLPs are also used in the design of a high-sensitivity lateral flow assay. Together, these findings contribute to a deeper understanding of antibody interactions and airborne viral transmission.
Language
English (en)
Chair
Michael Vahey
Committee Members
Amit Pathak; Jacco Boon; Matthew Bersi; Rohit Pappu; Srikanth Singamaneni