No description available.

No description available.

DNA vaccines offer unique promise as a means of generating immunity against infectious and malignant disease. Unfortunately a number of obstacles, including rapid degradation of naked plasmid DNA (pDNA), poor cellular uptake by antigen presenting cells (APCs) and subsequent low levels of gene expression have limited the ability of DNA vaccines to raise sufficient immune responses towards the target antigen. This thesis is focused on investigating the immunostimulatory potential of liposomal nanoparticulate (LN) formulations of pDNA (stabilized plasmid lipid particles; SPLP) and cytosine-guanine oligodeoxynucleotides (CpG-ODN; LN CpG-ODN), and examining their ability to act together as a non-viral DNA vaccine in attempt to address the shortcomings of current DNA vaccine approaches. One focus of this thesis concerns investigating the immunostimulatory activity of LN formulations of CpG-ODN and pDNA. It is shown that despite dramatic differences in pharmacokinetics and biodistribution of LN CpG-ODN following intravenous (i.v.) and subcutaneous (s.c.) administration the resultant immune response is very similar, which is concluded to be due to the intrinsic ability of APCs to sequester LN CpG- ODN. In addition, it is demonstrated that lipid encapsulation dramatically enhances the immunostimulatory potential of pDNA and it is observed that SPLP maintains immunostimulatory activity in Toll-like receptor 9 (TLR9) knock-out mice. Together theses findings highlight the need for DNA-based therapies to consider both TLR9-dependent and -independent immunostimulatory activities of pDNA when constructing non-viral vectors. Furthermore, a new role for SPLP as a non-viral gene delivery vehicle for the generation of a systemically administered genetic vaccine in the presence of LN CpG-ODN is introduced. The ability of vaccination with SPLP to act prophylactically, to protect mice from tumour challenge, and therapeutically, in a novel vaccination strategy where the antigen is expressed at the tumour site as a result of SPLP-mediated transfection, is explored, demonstrating that in the presence of LN CpG-ODN SPLP possesses potential as a non-viral delivery system for DNA-based cancer vaccines. In summary, this work represents a substantial advance in the understanding of the immunostimulatory potential of both SPLP and LN CpG-ODN and provides insight into their ability to work together as a non-viral DNA vaccine.

Hepatitis C virus (HCV) causes a significant health problem worldwide due to the lack of effective vaccines. It has been recognized that a rapid, vigorous, and broadly targeted cell-mediated immune response (Th1-like) is often associated with the clearance of HCV infections. DNA vaccines represent a promising means for HCV vaccination because they tend to induce a Th1-biased cell-mediated response in the host cell. Currently, the delivery of DNA vaccine for HCV in large animals as well as in humans is not as effective as in small animals. Nano delivery systems would be a promising approach to overcome this problem. Carbon nanotubes (CNTs) have been extensively studied for delivering drugs, proteins, peptides, and nucleic acids including plasmid DNA to cells and organs with varying degrees of success, but few of them have been applied to DNA vaccine for HCV.<p> This thesis presents a study of using functionalized CNTs (f-CNTs) to improve the efficacy of plasmid DNA vaccine delivery for HCV. First, CNTs were functionalized via 1,3-dipolar cycloaddition reaction with the appropriate amino acids and aldehydes. NMR and TEM results suggested that the CNTs were successfully functionalized and became soluble in water. Then plasmid DNAs which encode green fluorescence protein reporter gene, luciferase reporter gene, and HCV core protein, respectively, were delivered into human hepatoma cells via calcium phosphate precipitation method, f-CNT delivery system, and a combination of f-CNT and calcium phosphate method, respectively. The result showed that f-CNTs, in combination with the calcium phosphate method, significantly enhanced the gene expression in human hepatoma cells.<p> Consequently, this study concludes that the f-CNT can significantly enhance gene expression in liver cells conferred by a plasmid DNA when combined with calcium phosphate precipitation method. Even though the mechanisms of this enhancement await further investigation, the results of this thesis may have important implications in developing DNA vaccines for infectious diseases in general and for hepatitis C in particular.

Hepatitis C virus (HCV) causes a significant health problem worldwide due to the lack of effective vaccines. It has been recognized that a rapid, vigorous, and broadly targeted cell-mediated immune response (Th1-like) is often associated with the clearance of HCV infections. DNA vaccines represent a promising means for HCV vaccination because they tend to induce a Th1-biased cell-mediated response in the host cell. Currently, the delivery of DNA vaccine for HCV in large animals as well as in humans is not as effective as in small animals. Nano delivery systems would be a promising approach to overcome this problem. Carbon nanotubes (CNTs) have been extensively studied for delivering drugs, proteins, peptides, and nucleic acids including plasmid DNA to cells and organs with varying degrees of success, but few of them have been applied to DNA vaccine for HCV.<p> This thesis presents a study of using functionalized CNTs (f-CNTs) to improve the efficacy of plasmid DNA vaccine delivery for HCV. First, CNTs were functionalized via 1,3-dipolar cycloaddition reaction with the appropriate amino acids and aldehydes. NMR and TEM results suggested that the CNTs were successfully functionalized and became soluble in water. Then plasmid DNAs which encode green fluorescence protein reporter gene, luciferase reporter gene, and HCV core protein, respectively, were delivered into human hepatoma cells via calcium phosphate precipitation method, f-CNT delivery system, and a combination of f-CNT and calcium phosphate method, respectively. The result showed that f-CNTs, in combination with the calcium phosphate method, significantly enhanced the gene expression in human hepatoma cells.<p> Consequently, this study concludes that the f-CNT can significantly enhance gene expression in liver cells conferred by a plasmid DNA when combined with calcium phosphate precipitation method. Even though the mechanisms of this enhancement await further investigation, the results of this thesis may have important implications in developing DNA vaccines for infectious diseases in general and for hepatitis C in particular.

DNA vaccines offer unique promise as a means of generating immunity against infectious and malignant disease. Unfortunately a number of obstacles, including rapid degradation of naked plasmid DNA (pDNA), poor cellular uptake by antigen presenting cells (APCs) and subsequent low levels of gene expression have limited the ability of DNA vaccines to raise sufficient immune responses towards the target antigen. This thesis is focused on investigating the immunostimulatory potential of liposomal nanoparticulate (LN) formulations of pDNA (stabilized plasmid lipid particles; SPLP) and cytosine-guanine oligodeoxynucleotides (CpG-ODN; LN CpG-ODN), and examining their ability to act together as a non-viral DNA vaccine in attempt to address the shortcomings of current DNA vaccine approaches. One focus of this thesis concerns investigating the immunostimulatory activity of LN formulations of CpG-ODN and pDNA. It is shown that despite dramatic differences in pharmacokinetics and biodistribution of LN CpG-ODN following intravenous (i.v.) and subcutaneous (s.c.) administration the resultant immune response is very similar, which is concluded to be due to the intrinsic ability of APCs to sequester LN CpG- ODN. In addition, it is demonstrated that lipid encapsulation dramatically enhances the immunostimulatory potential of pDNA and it is observed that SPLP maintains immunostimulatory activity in Toll-like receptor 9 (TLR9) knock-out mice. Together theses findings highlight the need for DNA-based therapies to consider both TLR9-dependent and -independent immunostimulatory activities of pDNA when constructing non-viral vectors. Furthermore, a new role for SPLP as a non-viral gene delivery vehicle for the generation of a systemically administered genetic vaccine in the presence of LN CpG-ODN is introduced. The ability of vaccination with SPLP to act prophylactically, to protect mice from tumour challenge, and therapeutically, in a novel vaccination strategy where the antigen is expressed at the tumour site as a result of SPLP-mediated transfection, is explored, demonstrating that in the presence of LN CpG-ODN SPLP possesses potential as a non-viral delivery system for DNA-based cancer vaccines. In summary, this work represents a substantial advance in the understanding of the immunostimulatory potential of both SPLP and LN CpG-ODN and provides insight into their ability to work together as a non-viral DNA vaccine.

No description available.

No description available.

DNA vaccines offer unique promise as a means of generating immunity against infectious and malignant disease. Unfortunately a number of obstacles, including rapid degradation of naked plasmid DNA (pDNA), poor cellular uptake by antigen presenting cells (APCs) and subsequent low levels of gene expression have limited the ability of DNA vaccines to raise sufficient immune responses towards the target antigen. This thesis is focused on investigating the immunostimulatory potential of liposomal nanoparticulate (LN) formulations of pDNA (stabilized plasmid lipid particles; SPLP) and cytosine-guanine oligodeoxynucleotides (CpG-ODN; LN CpG-ODN), and examining their ability to act together as a non-viral DNA vaccine in attempt to address the shortcomings of current DNA vaccine approaches. One focus of this thesis concerns investigating the immunostimulatory activity of LN formulations of CpG-ODN and pDNA. It is shown that despite dramatic differences in pharmacokinetics and biodistribution of LN CpG-ODN following intravenous (i.v.) and subcutaneous (s.c.) administration the resultant immune response is very similar, which is concluded to be due to the intrinsic ability of APCs to sequester LN CpG- ODN. In addition, it is demonstrated that lipid encapsulation dramatically enhances the immunostimulatory potential of pDNA and it is observed that SPLP maintains immunostimulatory activity in Toll-like receptor 9 (TLR9) knock-out mice. Together theses findings highlight the need for DNA-based therapies to consider both TLR9-dependent and -independent immunostimulatory activities of pDNA when constructing non-viral vectors. Furthermore, a new role for SPLP as a non-viral gene delivery vehicle for the generation of a systemically administered genetic vaccine in the presence of LN CpG-ODN is introduced. The ability of vaccination with SPLP to act prophylactically, to protect mice from tumour challenge, and therapeutically, in a novel vaccination strategy where the antigen is expressed at the tumour site as a result of SPLP-mediated transfection, is explored, demonstrating that in the presence of LN CpG-ODN SPLP possesses potential as a non-viral delivery system for DNA-based cancer vaccines. In summary, this work represents a substantial advance in the understanding of the immunostimulatory potential of both SPLP and LN CpG-ODN and provides insight into their ability to work together as a non-viral DNA vaccine. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

This thesis details various techniques and considerations for the purification of complex biomacromolecules.   Initially an α-mannosidase from babaco fruit was purified using anion exchange-, lectin affinity- and size exclusion chromatography.  The enzyme was approximately 260-280 kDa in size with an apparent an unusual octagonal stoichiometry and displayed properties similar to other known plant α-mannosidases.   Mucins were fractionated by ion exchange and size exclusion chromatography to assess the properties that govern the mucin surface coating interactions in biomaterial research.  Commercially available mucins, of bovine and porcine origin, as wells as crude human mucin were tested. All showed to consist of a population of molecules which differ in size, charge and composition.   The third part of the thesis concerns different aspects of plasmid DNA purification processes. A two-step method for analysis of plasmid DNA consisting of size exclusion followed by thiophilic adsorption chromatography was evaluated. It allowed determination of the supercoiled plasmid DNA concentration in all process steps without requirement for extensive sample preparation. This method was shown to be fully comparable in terms of accuracy to capillary gel electrophoresis, considered as the industry standard. Purification of plasmid DNA generally involves bacterial cell alkaline lysis, which creates a solution with flocculate material which needs to be removed prior to further processing. The addition of ammonium hydrogen carbonate to the suspension was evaluated to clarify the solution. The released carbon dioxide and ammonium lifts the flocculate to the surface and allows draining of a clear solution. The method is fully scalable, does not affect the plasmid DNA quality and requires no special equipment. Thiophilic adsorption chromatography was evaluated for simplification of an existing commercial large scale purification process and was shown to increase both product purity and yields of several tested plasmids. Also, implementation of this step significantly reduced overall production process time.