Please use this identifier to cite or link to this item: https://hdl.handle.net/2440/112856
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Type: Theses
Title: Strategies for the development of recombinant porcine adenovirus-based vaccines against Hepatitis C virus
Author: Jagdale, Harshwardhan
Issue Date: 2016
School/Discipline: School of Medicine
Abstract: Hepatitis C virus (HCV) infects over 200 million people worldwide and results in persistent infection of approximately 80% of cases. Consequently, HCV is a leading global contributor to severe liver disease. Although, direct-acting antiviral agents (DAA) are available, these drugs do not prevent re-infection and cost in excess of US $80, 000 per patient. Thus, the development of an inexpensive HCV vaccine represents the most effective measure to reduce the current epidemic. Patients who recover following acute HCV infections provide valuable clues for vaccine design. These patients develop a broad and sustained cell-mediated immunity (CMI) against multiple HCV proteins, in particular the non-structural (NS) proteins. Therefore, the focus of this thesis was to develop novel vaccine strategies to elicit CMI against HCV NS proteins which may mimic the immunity that facilitates spontaneous control of acute HCV infection. Dendritic cells (DC) are crucial to initiate CMI making these cells attractive targets for any vaccine. Recombinant porcine adenoviruses (rPAV) appear capable of targeting DC as rPAV encoding influenza virus antigens have been shown to elicit both humoral and CMI. Furthermore, rPAV are not infectious nor replicate in humans making them safe. The potential of rPAV to elicit HCV-specific CMI is unknown. Therefore, I attempted to construct a cytolytic rPAV encoding a lytic protein as the cytolytic nature of viruses significantly augments immunogenicity due to the release of natural adjuvants known as danger associated molecular patters (DAMPs) from infected cells. As a prelude to this aim, the lytic activity of two cytolytic proteins, namely adenovirus death protein (ADP) from AD5 and mouse perforin (PRF) were compared following transfection of HEK293T cells with pVAX DNA encoding ADP or PRF (chapter 3). In two independent cell death assays (viz. luciferase assay and lactate dehydrogenase release assay), it was determined that PRF was more lytic, making it a suitable protein to be encoded in a lytic rPAV HCV vaccine. I then attempted to modify the rPAV shuttle DNA to facilitate cloning and expression of HCV NS antigens. Initially, the efficiency of the truncated CMV (tCMV) promoter and the full-length CMV (fCMV) promoter to drive eGFP expression was compared. Using flow cytometry and confocal microscopy, the tCMV promoter was shown to be more efficient. In addition, the rPAV shuttle DNA was modified to contain a multiple cloning site to facilitate gene insertion. Subsequently, recombinant PAV plasmids encoding PRF controlled by the tCMV or SV40 promoters were constructed, but recombinant virus was not recovered. This is most likely because PRF killed the cells prior to virus assembly. Subsequently, several experiments were conducted (Chapter 5) with a reporter virus encoding LUC and eGFP and established that rPAV production requires early passage cells (P11) cultured for 10-14 days post transfection. Based on these results, a non-lytic rPAV devoid of PRF was constructed to elicit CMI to conserved HCV NS proteins (NS4B and NS5B). Although, the construction of a cytolytic rPAV failed, the use of rPAV vector encoding NS4B and NS5B proteins is the next logical step to best augment the protective CMI. The construction of this rPAV vaccine accomplished during this thesis may lead to future preclinical studies.
Advisor: Gowans, Eric James
Wijesundara, Danushka
Grubor-Bauk, Branka
Dissertation Note: Thesis (M.Phil.) -- University of Adelaide, School of Medicine, 2016.
Keywords: Hepatitis C
adenovirus
vaccine
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: http://www.adelaide.edu.au/legals
DOI: 10.4225/55/5b21d8ca95948
Appears in Collections:Research Theses

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