Feline IL-12 and IL-18 Adjuvants in FeLV DNA Vaccination Studies

Hanlon, Linda (1999) Feline IL-12 and IL-18 Adjuvants in FeLV DNA Vaccination Studies. PhD thesis, University of Glasgow.

Abstract

Cytokines are glycoproteins produced by many different cell types which have wide ranging effects on the haemopoietic and immune systems and normal homeostatic mechanisms. The advent of recombinant DNA technology and the cloning of human cytokines has facilitated the production of recombinant cytokines in sufficient quantities to allow the characterisation of their biological properties and subsequent use as novel therapeutic agents in the treatment of viral and bacterial infections, cancer and cytopenias. However, the therapeutic use of heterologous cytokines in domestic animals has been of limited success, mainly due to the variable degree of conservation between species. Therefore, the isolation and characterisation of species specific cytokines is desirable in order to facilitate further studies of the role of cytokines in diseases of domestic animals. This thesis describes the approach used to isolate and clone the feline Thl type cytokines interleukin 12 (IL-12) and interleukin 18 (IL-18), express the recombinant IL-18 protein in a mammalian expression system and investigate the potential of both D-12 and DL-18 to act as genetic adjuvants in FeLV DNA vaccination studies. IL-12 is a recently discovered heterodimeric cytokine, which serves as a pivotal regulator of T and NK cell function, stimulating proliferation, cytolytic activity and cytokine induction. IL-18 is produced predominantly by activated macrophages, acts to strongly augment IFN-gamma production by spleen cells and enhances natural killer cell activity (NK). However, perhaps the most striking property of these cytokines is their marked ability to synergistically increase IFN-gamma production, a cytokine known to be integral to the development of a functional cellular immune response, in T cells previously exposed to antigen. cDNA clones encoding the p35 and p40 subunits of IL-12 and IL-18 were isolated using RT-PCR and their sequences determined. The p35 IL-12, p40 and IL-18 cDNAs encoded predicted full length proteins of 222, 329 and 192 amino-acids, respectively. All three sequences possessed a high degree of homology with the respective cytokines of other species at both the nucleic acid and protein level. In order to evaluate the potential of IL-12, IL-18 and EFN-? DNA constructs to act as in vivo genetic adjuvants in FeLV DNA vaccination studies, these cytokines were cloned into the mammalian expression vector pCI-neo, and IL-18 protein and mRNA expression were demonstrated in an in vitro mammalian expression system using western and Northern blotting techniques. To assess the potential of a DNA vaccine to protect against feline leukaemia virus (FeLV) infection, a significant pathogen of the domestic cat, a novel FeLV DNA vaccine was constructed, consisting of two separate plasmids, expressing FeLV gag/pol and FeLV env A genes. The constructs encoding feline interleukin 12 (IL-12), interleukin 18 (IL-18) and interferon gamma (IFN-gamma) were coinoculated with the vaccine to establish if protection could be enhanced. Twenty-nine SPF cats were included in the trial. Cats in group A were immunised intramuscularly with the FeLV DNA vaccine alone, cats in groups B, C and D were inoculated with the FeLV DNA vaccine and plasmids expressing EFN-?, IL-12 or 12 and IL-18, respectively, and the control cats, in group E, were immunised with empty pCI-neo plasmid. One hundred micrograms of each DNA construct was inoculated at 0, 2 and 4 weeks and intraperitoneal challenge with FeLV-A/Glasgow-1 viral isolate was performed at 7 weeks. No detectable FeLV-specific humoral immune response was elicited following immunisation. Fifteen weeks after challenge, virus isolation (VI) revealed that 2 of the 6 cats in group A, 2 of the 5 cats in group B, 4 of the 6 cats in group C, and 3 of the 6 cats in group E, tested virus isolation positive, while all the cats in group D (vaccine, IL-12 and IL-18) tested virus isolation negative. These findings demonstrate that the combination of IL-12 and IL-18 may act as a potent vaccine adjuvant, markedly enhancing the efficacy of the novel FeLV DNA vaccine. These studies provide the basis for further investigations of the potential of IL-12 and IL-18 in the treatment of feline disease, particularly as vaccine adjuvants against other feline pathogens, such as FIV, and the examination of their wider clinical potential in immunotherapy and cancer treatment. Ultimately these cytokines may form part of a new array of therapeutic agents to treat feline disease.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: David Onions
• Keywords: Veterinary science, Animal diseases, Virology, Immunology
• Date of Award: 1999
• Depositing User: Enlighten Team
• Unique ID: glathesis:1999-76119
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 16:37
• Last Modified: 19 Nov 2019 16:37
• URI: https://theses.gla.ac.uk/id/eprint/76119