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PLANT-MADE ORAL VACCINES: EVALUATION OF CAPSULES
Title
PLANT-MADE ORAL VACCINES: EVALUATION OF CAPSULES.
Creator
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.
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Date Issued
2011
Identifier
CFH0003861, ucf:44689
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFH0003861
EXPRESSION OF HEPATITIS C VIRAL NON-STRUCTURAL 3 ANTIGEN IN TRANSGENIC CHLOROPLASTS
Title
EXPRESSION OF HEPATITIS C VIRAL NON-STRUCTURAL 3 ANTIGEN IN TRANSGENIC CHLOROPLASTS.
Creator
Bhati, Anubhuti, Daniell, Henry, University of Central Florida
Abstract / Description
Hepatitis C viral infection is the major cause of acute hepatitis and chronic liver disease and remains the leading cause of liver transplants (NIH). An estimated 180 million people are infected globally (WHO). There is no vaccine available to prevent hepatitis C. The treatment with antiviral drugs is expensive, accompanied with various side effects and is limited only to those at risk of developing advanced liver disease. The treatment is also effective in only about 30% to 50% of treated...
Show moreHepatitis C viral infection is the major cause of acute hepatitis and chronic liver disease and remains the leading cause of liver transplants (NIH). An estimated 180 million people are infected globally (WHO). There is no vaccine available to prevent hepatitis C. The treatment with antiviral drugs is expensive, accompanied with various side effects and is limited only to those at risk of developing advanced liver disease. The treatment is also effective in only about 30% to 50% of treated patients and still a high percentage of patients are resistant to therapy. Therefore, there is an urgent need for the development of effective vaccine antigens and an efficacious HCV vaccine. The non-structural 3 protein of the hepatitis C virus is a multifunctional protein of the virus required for virus polyprotein processing and replication. Vaccine antigen production via chloroplast transformation system usually results in high expression levels and eliminates the possibility of contamination with vector sequences,human or animal pathogens. The HCV NS3 antigen was expressed in the chloroplast of Nicotiana tabacum var. Petit havana and LAMD-609. The 1.9kb NS3 gene was cloned into a chloroplast expression vector, pLD-Ct containing the 16S rRNA promoter, aadA gene coding for the spectinomycin selectable marker, psbA 5' untranslated region to enhance translation in the light and 3' untranslated region for transcript stability and trnI & trnA homologous flanking sequences for site specific integration into the chloroplast genome. Chloroplast integration of the NS3 gene was first confirmed by PCR. Southern blot analysis further confirmed site-specific gene integration and homoplasmy. The NS3 protein was detected in transgenic chloroplasts by immunoblot analysis. The NS3 protein was further quantified by ELISA. Maximum expression levels of NS3 up to 2% in the total soluble protein were observed even in old leaves, upon 3-day continuous illumination. These results demonstrate successful expression of the HCV non-structural 3 antigen in transgenic tobacco chloroplasts. Animal studies to test the immunogenecity of the chloroplast derived HCV NS3 will be performed using chloroplast derived NS3 antigen.
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Date Issued
2005
Identifier
CFE0000495, ucf:46368
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0000495
Expression of Lipase from Mycobacterium tuberculosis in Nicotiana tobacum and Lactuca sativa Chloroplasts
Title
Expression of Lipase from Mycobacterium tuberculosis in Nicotiana tobacum and Lactuca sativa Chloroplasts.
Creator
Lloyd, Bethany, Daniell, Henry, Kolattukudy, Pappachan, Self, William, University of Central Florida
Abstract / Description
Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (M. tuberculosis), is a global threat and the leading cause of death among individuals infected with HIV. TB treatment requires multi-drug cocktails, due to the increasing rates of drug resistance of the bacterium. With multi-drug cocktails, strains have been documented to be resistant to all major drugs in the fight against TB. Since the strains are drug resistant, it calls for an increasing need for vaccine and treatment...
Show moreTuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (M. tuberculosis), is a global threat and the leading cause of death among individuals infected with HIV. TB treatment requires multi-drug cocktails, due to the increasing rates of drug resistance of the bacterium. With multi-drug cocktails, strains have been documented to be resistant to all major drugs in the fight against TB. Since the strains are drug resistant, it calls for an increasing need for vaccine and treatment development for the purpose of preventing and managing the disease. The most widely distributed vaccine against TB is Bacillus Calmette-Gue(&)#180;rin (BCG). Apart from being ineffective in certain individuals, BCG offers only a limited timeframe of protection, is unable to serve as a booster for extending this timeframe and due to the intradermal route of administration requires costly refrigeration and syringes.LipY protein, a M. tuberculosis cell wall lipase, may play a potential role as not only a drug target but a potential vaccine antigen. LipY is known to be up-regulated during both active infection and dormancy. In a previous study, sera from TB patients had shown an IgG and IgM response against it. In this study transplastomic Lactuca sativa and Nicotiana tabacum plants were generated by transforming the chloroplasts through the particle delivery system with pLsDv-LipY and pLD-LipY vectors respectively. The vectors were flanked by the native trnI and trnA gene sequence to facilitate homologous recombination into the chloroplast genome. The vector also contained the 16S rRNA promoter, the selectable marker gene, aadA for specitinomycin resistance, the rbcL untranslated region, the LsPpsbA (PpsbA in N. tabacum) promoter, and LsTpsbA (tpsbA in N. tabacum) untranslated region. Site specific integration of the LipY gene into the chloroplast genome was confirmed by PCR. Homoplasmy of transplastomic plants was confirmed by Southern blot analysis. These plants showed normal growth and were fertile, producing seeds. Once germinated, these seeds did not show Mendelian segregation of the transgene. Immunoblot analysis was performed to analyze the expression of the LipY protein. A 40kDa protein was produced in E.coli, and a 25kDa protein was produced in chloroplasts; a cleaved product in chloroplasts is still valuable as an antigen for vaccine production. Future studies will include testing this chloroplast derived antigen in animal models for vaccine development. ?
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Date Issued
2012
Identifier
CFE0004502, ucf:49289
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004502
Expression and functional evaluation of exendin 4 fused to cholera toxin B subunit in tobacco chloroplasts to treat type 2 diabetes
Title
Expression and functional evaluation of exendin 4 fused to cholera toxin B subunit in tobacco chloroplasts to treat type 2 diabetes.
Creator
Nityanandam, Ramya, Daniell, Henry, Naser, Saleh, Siddiqi, Shadab, University of Central Florida
Abstract / Description
The prevalence of type 2 diabetes has been steadily increasing around the globe. Glucagon like peptide (GLP-1), a powerful incretin increases insulin secretion in a glucose dependent manner. But GLP-1 is subjected to rapid enzymatic degradation (half-life: 2 min in circulation). The commercially available GLP-1 analog, exenatide has a longer half life with potent insulinotropic effects (about 2.4 hr) which requires cold storage and daily subcutaneous injections. In this study, exendin 4 (EX4)...
Show moreThe prevalence of type 2 diabetes has been steadily increasing around the globe. Glucagon like peptide (GLP-1), a powerful incretin increases insulin secretion in a glucose dependent manner. But GLP-1 is subjected to rapid enzymatic degradation (half-life: 2 min in circulation). The commercially available GLP-1 analog, exenatide has a longer half life with potent insulinotropic effects (about 2.4 hr) which requires cold storage and daily subcutaneous injections. In this study, exendin 4 (EX4), lizard derived GLP-1R agonist, was expressed as cholera toxin B subunit (CTB)-fusion protein in chloroplasts of tobacco to facilitate transmucosal delivery in the gut by utilizing the ability of CTB pentamer to bind the GM1 receptors on the intestinal epithelium and to bioencapsulate EX4 within plant cells to confer protection in the digestive system. The LAMD tobacco leaves were bombarded with chloroplast vectors expressing modified EX4. The transgene integration was confirmed by PCR analysis and Southern blot analysis. Densitometric analysis revealed expression level of the protein varied from 9-13% of the total leaf protein depending on the developmental stage and time of harvest. The pentameric structure and functionality of CTB-EX4 fusion protein was confirmed by CTB-GM1 binding assay. The effect of transplastomic protein on insulin secretion was tested in ?-TC6, a mouse pancreatic cell line. The plant derived CTB-EX4, partially purified with anti-CTB antibody conjugated protein A beads, showed the increase of insulin ~ 2.5 fold increase when compared to untreated cells. The transplastomic protein showed a linear increase in insulin secretion comparable to the commercially available EX4. The current cost of treatment with EX4 varies between $1800-$2200, annually. Production of functional EX4 in plants should facilitate low cost orally deliverable form of this drug for treatment of type 2 diabetes.
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Date Issued
2011
Identifier
CFE0004485, ucf:49306
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004485