The liver flukes Fasciola hepatica and F. gigantica cause infectious disease in ruminants and humans. The geographical range of these two parasite species (temperate and tropical respectively) ensures that infection can occur worldwide. Although anthelmintic treatment is effective against disease, emerging drug resistant strains leads to the development of a vaccine. However, despite several decades of research, there is no commercial vaccine available. The main challenge at present is to produce recombinant proteins in an immunologically active form using recombinant DNA technology. This is an essential step in Fasciola vaccine production. Cysteine proteases are probably the most important facilitators of virulence in flukes and are produced by all stages of the fluke life-cycle. Two classes of cysteine protease are found in the excretory and secretory material of liver flukes- these are cathepsin L and cathepsin B. As such, the major aims of this thesis were to investigate the expression and purification of Fasciola recombinant cysteine proteins, and characterisation by SDS-PAGE and immunoblotting using monoclonal and polyclonal antibodies. These studies demonstrate the production of functionally active cathepsin proteins in S. cerevisiae BJ3505 cells which will lead to vaccine candidate analysis. The second aim of this thesis was to determine the protective efficacy of stage specific target antigens against experimental infection. In addressing this issue, the protective efficacy of single and multivalent recombinant protein vaccinations of adult stage F. hepatica cathepsin L5, immature F. gigantica cathepsin L1g and juvenile F. hepatica cathepsin B were analysed in Sprague Dawley rats against F. hepatica infection. This study demonstrates that juvenile fluke target antigen-cathepsin B induces better immune protection than adult fluke antigen-cathepsin L5. Cocktails of juvenile and adult stage fluke recombinant proteins (cathepsin B and L5) elicited the highest protective immunity against experimental infection and this combination showed not only reduction in fluke recovery and size of flukes, but also marked diminution in the intensity of liver lesions in vaccinated rats. In order to assess the immunogenic property of an early infective stage fluke secreting cysteine protease as a vaccine candidate, DNA vaccination vectors encoding cathepsin B were analysed in BALB/c mice. In this study, the ability of four DNA vaccination strategies such as secretory, chemokine-activating, lymph node targeting vectors encoding cathepsin B were assessed by antibody titre, antibody avidity, western blotting and ELIPSOT assay. The results have further validated the immunoprophylactic potential of a cathepsin B vaccine against F. hepatica. In this study, we have expressed and attained high yields of F. gigantica cathepsin L1g from E. coli BL21, and compared this to a yeast-expressed system. This protease was over-expressed and formed insoluble inclusion bodies that were subsequently solubilised with urea or guanidine hydrochloride. In order to purify the urea-solubilised protein, step-wise urea gradient chromatography was used. For refolding of solubilised protein, a dilution and dialysis procedure was utilised. Proteolytic activity was confirmed by gelatin SDS-PAGE analysis. In conclusion, the determination of the immune potential of recombinant stage specific antigens allows the development of effective vaccines against Fasciola infection.
| Identifer | oai:union.ndltd.org:ADTP/210454 |
| Date | January 2008 |
| Creators | Jayaraj, Ramamoorthi, Jayaraj@menzies.edu.au |
| Publisher | RMIT University. Applied Science |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://www.rmit.edu.au/help/disclaimer, Copyright Ramamoorthi Jayaraj |
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