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Title: Liposome-mediated antigen delivery: formulation and optimisation
Author: McNeil, Sarah E.
ISNI:       0000 0001 3388 8569
Awarding Body: Aston University
Current Institution: Aston University
Date of Award: 2005
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Live conventional vaccines are generally effective at provoking protective immunity against the infectious agent. However, there are many disadvantages regarding their adverse side effects and overall safety profile. Alternative vaccine strategies such as subunit and plasmid-based vaccines, using recombinant technology, are much safer, yet less effective. Therefore, the immunogenicity of such vaccines could be enhanced by utilising delivery and/or adjuvant systems, to provoke the appropriate immune responses. The role of liposomal systems within plasmid-based delivery was examined, looking at the effects of varying liposomal composition and method of preparation on the physical characteristics, transfection efficiency in vitro and immunogenicity in vivo. Compared to naked DNA alone, entrapment of plasmid DNA within liposomal vesicles results in complete protection from degradation by intracellular enzymes, with the DNA maintaining full structure and function. For liposome-mediated gene delivery small cationic lipids have been shown to be potent candidates, acquiring a net positive charge, which effectively interact with the anionic charges of the DNA to generate high incorporation values. In contrast, neutral liposomes are much larger aggregated structures with lower incorporation of DNA. The method of preparation was shown to effect the spatial location of plasmid DNA to liposomal systems. The dehydration-rehydration procedure (DRV) carried out in the presence of DNA effectively entraps the plasmid with little effect on liposome size and surface charge. Alternatively, upon addition of plasmid DNA the measured vesicle size of small unilamellar vesicles (SUV) or 'empty' (water containing) DRV increases due to the formation of SUV-DNA or DRV-DNA complexes, with the majority of the DNA localised on the surface of the liposomes. When applied in vitro, transfection efficiency of SUV-DNA complexes was greater than DRV(DNA). Transfection efficiency of SUVDNA complexes varied depending on the cationic lipid present within the lipid bilayer, with DC-Chol showing most efficiency. Furthermore, these DC-Chol cationic liposomes were formulated in combination with two different 'helper' lipids, the fusogenic lipid dioleoyl phosphatidylethanolamine (DOPE) or the stabilising lipid Cho!. The manner in which complexes form, the resultant structure and their transfection efficiency in vitro varied depending on the combining effects of both the type of 'helper' lipid incorporated within the lipid bilayer and total lipid to DNA charge ratio, with the overall structural size playing a significant role in promoting transfection. Transfection efficiency in vitro was significantly reduced when complexes were stabilised by the inclusion of phosphatidylcholines, with both the phospholipid head group and the alkyl-chain length influencing transfection efficiency. The production of DRV vesicles incorporating DNA were also produced in the range of IOO-200nm by the addition of a disaccharide (i.e. sucrose), prior to freeze-drying during the dehydration-rehydration procedure. In this instance, with an increase in sucrose/lipid mass ratio, the z-average diameter of liposomes decreased, while the percentage plasmid DNA, pRc/CMV HBS, entrapment remained relatively high (92%). Despite this, these small DRV(DNA) were found to be poor transfecting agents in vitro. After an initial screening process in vitro, a select few liposomal systems were subcutaneously administered in vivo. For all the liposomal formulations tested there was no induction of a humoral immune response, as no antibody titres were detected against the encoded antigen. However, SUV-DNA complexes composed of PC:Choi:DC-Chol (16:8:4 Ilmole/ml) and the production of small modified DRV(DNA) by the addition of sucrose generated sufficiently high levels of cell-mediated immunity. With regard to protein antigen delivery and adjuvanticity, the association with liposomal systems significantly enhances the immunogenicity of the fusion protein, Ag8SB-ESAT -6, a promising tuberculosis vaccine antigen. Several factors were shown to influence adjuvanticity of these liposomal systems. For example, the inclusion of the immunomodulator, TDB, effectively enhanced immunity against tuberculosis by increasing the adjuvanticity of these liposomal systems. Such immune responses were prolonged and most effective when these liposomal systems were either neutral or possessed a net positive charge rather than a negative charge and when the protein antigen was entrapped within these vesicles rather than surface complexed. Therefore, the overall protection against infection by tuberculosis was enhanced, presumably as a result of these liposomes forming depots, whereby the protein antigen is released slowly and at a controlled rate, maintaining therapeutic levels of the antigen in vivo to exert its therapeutic effect.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Pharmacy ; Biological Sciences