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Title: Feline IL-12 and IL-18 as adjuvants to a DNA vaccine for FeLV
Author: O'Donovan, Lucy
ISNI:       0000 0001 3452 7140
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2002
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The Th1 cytokines interleukin 12 (IL-12) and interleukin 18 (IL-18) are important mediators of the cell-mediated immune response. Separately, both cytokines act on T and natural killer (NK) cells and stimulate the production of interferon gamma (IFNgamma). These molecules can also act synergistically on their effector cells to produce a dramatic IFNgamma response. IL-12 is important in the differentiation and proliferation of T helper type 1 (Th1) cells, while IL-18 increases Th1 proliferation and IL-12-driven Th1 development. The role of these interleukins in the cellular immune pathway has led to their use as adjuvants to vaccines that require strong cell mediated immunity. A previous vaccination trial investigated the use of various feline cytokine constructs in their role as adjuvants to a DNA vaccine to feline leukaemia virus (FeLV) [Hanlon et al. 2001]. This study demonstrated that cytokine plasmid constructs encoding IL-12 and IL-18, together elicited complete protection from viraemia and significant protection of animals from latent proviral infection. In vitro analysis of these cytokine constructs prior to use in vivo, failed to detect IL-18 protein expression by Western blot analysis. In addition, suitable assays for measurement of in vitro bioactivity of these constructs were not available at that time. The preliminary work for this project focused on the development of improved feline IL-12 and IL-18 constructs and demonstration of in vitro expression and bioactivity. IL-12 may be administered using separate plasmids encoding the subunits p35 and p40, which can potentially lead to several problems. Firstly cells must take up both plasmids in order to produce bioactive IL-12 heterodimer. Also the overproduction of p40 subunit is a potential risk, as p40 homodimer molecules can produce antagonism of the heterodimer by binding to the IL-12 receptor and rendering it inactive. A new construct was therefore developed linking the cDNA of each subunit by a synthetic linker sequence which overcomes these potential problems. The in vitro expression of this feline flexi-IL-12 construct was demonstrated by Western blot analysis. Specific in vitro bioactivity was then shown using the dose dependent production of IFNy from equine lymph node cells. IL-18 is synthesised as a biologically inactive precursor molecule pro-IL-18, which is cleaved within the cell by the pro tease caspase-1 to produce bioactive mature-IL-18. This molecule then possesses the natural signalling peptide required for extracellular secretion. Inoculation of pro-IL-18 plasmid therefore must rely on the presence of endogenous caspase-1 for protein secretion. Immunisation with mature-IL-18 produces protein that lacks a signalling peptide and is inefficiently secreted from the cell. For these reasons, a construct was produced which encoded feline mature-IL-18 fused to a synthetic signal peptide IL-lp receptor antagonist protein (ILRAP). ILRAP-IL-18 in vitro protein expression was analysed by Western blot analysis. It was found that unlike pro- and mature-IL-18 constructs, the ILRAP signal peptide facilitated secretion from the cell and further, was associated with higher relative expression than the previously used IL-18 expression construct. Secreted ILRAP-IL- 18 was also shown to be bioactive by measuring the specific dose dependent production of human IFNyfrom KG-1 cells. In addition, the feline IL-18 receptor was amplified from the total RNA of MYA-1 cells.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available