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Type: Thesis
Title: Suicide genes in a novel vaccination strategy for hepatitis C.
Author: Trahair, Hugh Francis Llewellyn
Issue Date: 2012
School/Discipline: School of Molecular and Biomedical Science
Abstract: Hepatitis C virus (HCV) is a blood transmitted virus which causes persistent infection of the liver greatly resulting in cirrhosis and hepatocellular carcinoma. Current treatment regimens for HCV, such as pegylated interferon-α and ribavirin in combination, fail to clear the virus in 45-80% of individuals. New therapies under development, including immunotherapies, face many challenges. One of which is the need to induce a broad, multifunctional T-cell response directed against NS3/4A (cell-mediated immunity (CMI)), which is associated with clearance of infection, to enhance the immune response in chronic non-responders to conventional treatment. These individuals tolerate high levels of circulating HCV antigens and fail to clear the virus normally. Dendritic cells are the only antigen presenting cells able to prime naive T-cells, the central aim of any vaccination strategy. DC detect viral infection by phagocytosis of exogenous viral antigens released from infected, often apoptotic/necrotic cells, process and present these in association with MHC II to CD4+ T-cells and to naive CD8+ T-cells in association with MHC I (cross-presentation). Cross-presentation is proposed to be required for immunity to viruses, as immunity would otherwise only occur if the virus infected the DC (if direct presentation were the only possible mechanism). Therefore, one potential way to enhance the CMI is to enhance cross-presentation through induction of apoptosis or necrosis in vaccine-targeted cells. The overall aim of this study was to immunise C57BL/6 mice with DNA vaccines encoding HCV immunogens and genes encoding toxic proteins termed “suicide genes”. Induction of apoptosis/necrosis in the vaccine targeted cells was predicted to release the vaccine-derived antigen and enhance cross-presentation. This thesis explored suicide gene function in vitro and testing in vivo to examine the effect on the immunogenicity of a DNA vaccine strategy. Initially, suicide genes from a range of bacterial and viral sources were screened in vitro by luciferase co-transfection experiments to determine cell-killing ability. A highly toxic gene, diphtheria toxin subunit A (DTA), moderately toxic gene, mouse mutant perforin (MMP) and poorly toxic gene, rotavirus non-structural protein 4 (NSP4) were selected and each was cloned into a dual-promoter vector, then used to transfect HEK293T and Cos-7 cells. Alamar blue and additional luciferase experiments confirmed the cytotoxic effects. Finally, the mode of cell death was determined as apoptosis for DTA, necrosis for MMP and apoptosis and necrosis for NSP4 by lactate dehydrogenase (LDH) assay and Annexin V/Propidium iodide flow cytometry. DTA was later confirmed to inhibit protein synthesis and thus was not pursued in vivo. The HCV NS3 and NS3/4A serine protease genes were cloned under a second promoter in each dual-promoter suicide gene construct and protein expression was detected by western blot analysis. These DNA vaccines were used to immunise C57BL/6 mice to compare the immunogenicity by IFN-γ ELISPOT. NS3 was selected as the more immunogenic candidate. Addition of NSP4 to the NS3 encoding vector enhanced immunogenicity slightly, although MMP did not.
Advisor: Gowans, Eric James
Miller, Darren Scott
Grubor-Bauk, Branka
Dissertation Note: Thesis (M.Phil.) -- University of Adelaide, School of Molecular and Biomedical Science, 2012
Keywords: suicide genes; DNA vaccine; HCV; hepatitis C; cross presentation; CMI T-cells; necrosis
Provenance: This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at:
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