Antigen expression through the Chloroplast Transformation Technology (CTT) produces bioencapsulated subunit-vaccines, capable of eliciting immune responses when delivered orally. Considerable challenges to effective plant-based vaccines are the normalization of dosage and preservation of accumulated antigen, which is complicated by variable high water content and protease activity. This study critically examines the efficacy of lyophilization in dehydrating plant-tissues and preserving plant-derived antigens with vaccine potential. Lyophilization was optimized through gravimetric analysis using lettuce expressing Protective Antigen (PA) of Bacillus anthracis (LS-HPAG) and the human autoantigen Proinsulin (Pins) fused to Cholera toxin subunit B (LS-CTB-Pins). Lyophilization for 48-hours was sufficient treatment to reduce lettuce to 4.57% of its original weight, which retained .058% water content in the bound state; these levels corresponded with oven-dried controls while antigen was stabilized for over a year of storage at room temperature. A simulated gastric fluid assay was applied to evaluate stability of plant derived antigens during digestion. It was observed that lettuce plant cells conferred protection through antigen bioencapsulation for up to an hour under enzymatic digestive conditions. LS-HPAG immunogenicity was then demonstrated through the induction of a PA-specific IgG response by through oral boosting of C57/BL6 test mice. Survival during toxin challenge demonstrated a protective immune response if 40% of animal immunized by plant-derived PA. Lastly, the inclusion of excipient and adjuvant additives will be considered and utilized for the development of prototype vaccine capsule formulations.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:honorstheses1990-2015-2168 |
| Date | 01 May 2011 |
| Creators | New, James Stewart |
| Publisher | STARS |
| Source Sets | University of Central Florida |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | HIM 1990-2015 |
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