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- Title
- PLANT-MADE ORAL VACCINES: EVALUATION OF CAPSULES.
- Creator
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New, James, Daniell, Henry, University of Central Florida
- Abstract / Description
-
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...
Show moreAntigen 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.
Show less - Date Issued
- 2011
- Identifier
- CFH0003861, ucf:44689
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0003861
- Title
- Assessment of molecular interactions via magnetic relaxation: a quest for inhibitors of the anthrax toxin.
- Creator
-
Santiesteban, Oscar, Perez Figueroa, Jesus, Liao, Yi, Yestrebsky, Cherie, Hampton, Michael, Lambert, Stephen, University of Central Florida
- Abstract / Description
-
Anthrax is severe disease caused by the gram-positive Bacillus anthracis that can affect humans with deadly consequences. The disease propagates via the release of bacterial spores that can be naturally found in animals or can be weaponized and intentionally released into the atmosphere in a terrorist attack. Once inhaled, the spores become activated and the anthrax bacterium starts to reproduce and damage healthy macrophages by the release of the anthrax toxin. The anthrax toxin is composed...
Show moreAnthrax is severe disease caused by the gram-positive Bacillus anthracis that can affect humans with deadly consequences. The disease propagates via the release of bacterial spores that can be naturally found in animals or can be weaponized and intentionally released into the atmosphere in a terrorist attack. Once inhaled, the spores become activated and the anthrax bacterium starts to reproduce and damage healthy macrophages by the release of the anthrax toxin. The anthrax toxin is composed of three virulent factors: (i) anthrax protective antigen (APA), (ii) anthrax lethal factor (ALF), and (iii) anthrax edema factor (AEF) that work in harmony to effectuate the lethality associated with the disease. Out of the two internalized factors, ALF has been identified to play a critical role in cell death. Studies in animals have shown that mice infected with an anthrax strain lacking ALF survive the infection whereas when ALF is present the survivability of the mice is eliminated. Although the current therapy for anthrax is antibiotic treatment, modern medicine faces some critical limitations when combating infections. Antibiotics have proven very efficient in eliminating the bacterial infection but they lack the ability to destroy or inhibit the toxins released by the bacteria. This is a significant problem since ALF can remain active in the body for days after the infection is eliminated with no way of inhibiting its destructive effects. The use of inhibitors of ALF is an attractive method to treat the pathogenesis of anthrax infections. Over the last decade several inhibitors of the enzymatic activity of ALF have been identified. In order to identify inhibitors of ALF a variety of screening approaches such as library screenings, Mass Spectroscopy- based screenings and scaffold-based NMR screening have been used. Results from these screening have yielded mainly small molecules that can inhibit ALF in low micromolar to nanomolar concentrations. Yet, although valuable, these results have very little significance with regards to treating ALF in a real-life scenario since pharmaceutical companies are not willing to invest in further developing these inhibitors. Furthermore, the low incidence of inhalation anthrax, the lack of a market for an ALF inhibitor, and the expenses associated with the approval process of the FDA, have hindered the motivation of pharmaceutical companies to pursuit these kind of drugs. Therefore we have screened a small-molecule library of FDA approved drugs and common molecules in order to identify currently approved FDA drugs that can also inhibit ALF (Chapter III). The screening revealed that five molecules: sulindac, fusaric acid, naproxen, ketoprofen and ibuprofen bound to either ALF or APA with sulindac binding both. Additionally, we have developed a nanoparticle-based screening method that assesses molecular interactions by magnetic relaxation changes (Chapter II). Using this assay, we were able to accurately measure the dissociation constants of different interactions between several ligands and macromolecules. Moreover, we have used computational docking studies to predict the binding site of the identified molecules on the ALF or APA (Chapter IV). These studies predicted that two molecules sulindac and fusaric acid could be potential inhibitors of ALF since they bind at the enzymatic pocket. As a result, we tested the inhibitory potential of these molecules as well as that of the metabolic derivatives of sulindac (Chapter V). Results from these studies provided conclusive evidence that fusaric acid and sulindac were both strong inhibitors of ALF. Furthermore, the metabolic derivatives of sulindac, sulindac sulfide and sulindac sulfone also inhibited ALF. Overall, taking together these results we have discovered the alternate use of a currently used drug for the treatment of ALF pathogenesis.
Show less - Date Issued
- 2012
- Identifier
- CFE0004794, ucf:49745
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004794
