Title:
Molecular toolbox for characterizing, preventing, and treating human respiratory syncytial virus infections

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Author(s)
Vanover, Daryll Alexander
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Advisor(s)
Santangelo, Philip J.
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Abstract
RSV remains one of the leading causes worldwide of acute lower respiratory tract infections. Despite continued effort, no vaccine exists and prophylaxis is limited to high-risk populations due to cost and limited efficacy of broadly neutralizing antibodies. Additionally, much of the general biology surround RSV replication and assembly is incomplete. The overall goal of the work presented here was to identify: 1) new targets for RSV therapeutics by analyzing RSV filament assembly (structures critical to high RSV titers), and 2) novel prophylactics against RSV to prevent infection. First, we developed a live-cell labeling method using a lectin, a class of proteins that bind specific glycan moieties, specific for RSV G. SBA was found to not inhibit the infectivity or replication of RSV by several metrics. We observed the first live images of RSV filament formation using this labeling technique, and critically, we found that this process can occur prior to the plasma membrane (where RSV was originally thought to assemble). This technique was then utilized to find that filament formation is microtubule and, specifically, dynein mediated. We also discovered that the loading of RSV genome into extended filaments is a microtubule-mediated process. We used synthetic, modified mRNA to express engineered broadly neutralizing antibodies to prevent RSV infection. We developed the use of both secreted and membrane-anchored palivizumab that prevents RSV infection in vitro and in the lungs of mice. We showed that encoding a membrane anchor on the antibodies prevents the antibody from diffusing away from the cell and blocks viral particles from entering the cell. Finally, the membrane-anchor was adapted to a highly potent RSV-neutralizing single-domain camelid antibody. The anchored VHH prevented RSV infection in mice and represents a promising new tool in the future of RSV prophylaxis.
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Date Issued
2018-04-02
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Dissertation
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