Efficient delivery of vaccines and therapeutic agents in vivo is a critical aspect for ensuring a desired immunological or biological response is achieved. This work focuses on developing effective delivery systems for vaccines and therapeutics to achieve biological potency while maintaining patient friendly administration. Traditional vaccines are administered via parenteral injection, which requires skilled personnel for administration and does not elicit strong mucosal immune responses. An alternative approach is to develop an oral vaccine; however, this requires antigens to be protected during transit through the gastrointestinal tract and be transported across specialized intestinal sampling cells called M cells. These M cells are extremely rare, making them an important target for oral vaccines. The protein invasin, from Yersinia psuedotuberculosis, naturally binds [mathematical symbols] integrin, a receptor found exclusively on M cells within the gastrointestinal tract. Therefore, we generated combinatorial libraries of invasin, followed by a directed evolution and high-throughput screening strategy to identify invasin variants with increased affinity towards [mathematical symbols] integrin. This process led to the creation of an invasin variant exhibiting a nine-fold decrease in EC₅₀, which could be used for targeted oral vaccine systems. In order to test for increased vaccine efficacy due to the engineered invasin ligand, we developed a polymeric microparticle delivery system. These microparticles were formulated to encapsulate the model antigen ovalbumin and be decorated with the invasin targeting ligands. To measure physiological trafficking and intestinal retention, novel fluorescent nanocrystals were loaded into particles conjugated to invasin. These nanocrystals served as a contrast agent for in vivo imaging in mice. While these particles were unsuccessful in generating an antibody response toward ovalbumin when administered to mice, a response directed to the targeting ligand itself was observed. These findings provide insights for further optimizing a delivery system for oral vaccination. In addition to developing an oral vaccine delivery system, we created a high concentration therapeutic protein formulation, suitable for low-volume subcutaneous administration. By adding crowding agents, we were able to generate reversible protein nanoclusters with low viscosity. These nanoclusters were found to revert to monomer upon dilution and pharmacokinetic profiles similar to solutions. / text
| Identifer | oai:union.ndltd.org:UTEXAS/oai:repositories.lib.utexas.edu:2152/23391 |
| Date | 03 March 2014 |
| Creators | Khan, Tarik Ali |
| Source Sets | University of Texas |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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