Dissecting the pharmaceutical and immunological aspects of Myrmecia pilosula (jack jumper) ant venom immunotherapy

Myrmecia pilosula (Jack Jumper) is an endemic Australian ant whose sting is a frequent cause of insect allergy in parts of South-Eastern and South-Western Australia, causing severe anaphylaxis in approximately 3% of the population. The venom of Myrmecia pilosula ant contains IgE-binding components frequently responsible for the severe anaphylactic reactions in humans. A treatment modality based on purified M. pilosula ant venom extract has been developed by members of the Tasmanian Jack Jumper Allergy Program. The treatment, known as Jack Jumper Ant Venom Immunotherapy (JJA VIT), was proven to reduce the risk of severe anaphylaxis in sensitized patients and improve patients' Quality of Life. However, the current treatment is associated with frequent adverse reactions and long treatment duration. As the principal Pharmacist and Quality Manager responsible for the manufacture of JJA VIT products, it is my primary interest to continuously improve the quality, safety and efficacy of this important life-saving treatment, which is uniquely Australian. My review of allergic reactions to the venom of stinging ants (as detailed in Chapter 1 and Chapter 2) illustrated the burden and impact of M. pilosula venom allergy in Australia and highlights aspects of M. pilosula venom and JJA VIT that warrant further scientific investigations, which consequently shaped the objectives and research questions of this PhD thesis. This research has two general objectives that were aimed to advance this treatment modality, and I have performed several interconnected studies to answer my research questions (Chapter 3). My first research objective was to improve the quality of the JJA VIT produced. In Chapter 4 I explored the intrinsic and extrinsic factors that could influence batch-to-batch consistency and quality of pharmaceutical grade Jack Jumper ant venom (JJAV) extracts in the form of Active Pharmaceutical Ingredients, particularly with respect to their IgE-binding components and activities. In this analysis, I found that components of the venom with molecular weight of >20 kDa are significantly affected by elevated temperature above 40¬¨‚àûC. Notably, these venom components are capable of binding to IgE and they were of unknown identity, and their identities are revealed in Chapter 5. I analysed the proteome and allergenome of JJAV separated using a combination of various gel electrophoresis and liquid chromatography techniques. To help divulge the identity of novel JJAV components capable of binding IgE, I employed a tandem Mass Spectrometry technique. From this study, I identified 17 novel JJAV proteins, including two glycoproteins, and confirmed the presence of four known Myr p and pilosulin peptides in JJAV. Most of the newly identified IgE-binding proteins were enzymes, including phospholipase A2, hyaluronidase, arginine kinase, and dipeptidyl peptidase IV. My second research objective was to improve the safety and efficacy of JJA VIT. For this purpose, I analysed the response of subjects undergoing JJA VIT with respect to their IgE-binding recognition to JJAV components pre-treatment and I correlated this information with treatment tolerability and efficacy. I subsequently linked this clinical data with the various JJAV components identified via tandem Mass Spectrometry and report my results in Chapter 5. In this study, I established correlations between recognition of certain IgE-binding bands with JJAV-specific IgE titre by ImmunoCAP, intradermal test threshold, and treatment-related issues. Finally, driven by the relative difficulty in obtaining pharmaceutical grade JJAV extracts and the recent increase in demand to treat patients with JJAV allergy within Tasmania and interstate, I explored the safety and efficacy of treatment with low-dose JJA VIT using Advax‚Äöv묢 adjuvant. In order to enable a clinical trial using this novel combination product, I performed fundamental pharmaceutical and immunological studies. In Chapter 6 I report the physicochemical and microbiological stability and murine immunogenicity of low-dose JJA VIT in combination with Advax adjuvant. I observed that JJA VIT formulated with Advax is both physicochemically and microbiologically stable for at least 2 days when stored at 4 and 25¬¨‚àûC, with a trend for an increase in allergenic potency observed beyond 2 days of storage. Importantly, JJA VIT formulated with Advax significantly increased the production of JJAV-specific IgG, consistent with a JJAV antigen-sparing effect of the adjuvant, which supports the use of Advax adjuvant with JJA VIT in future clinical trials. Overall, my PhD project has advanced our knowledge on the pharmaceutical and immunological aspects of JJA VIT and provides a robust platform to enhance the quality, safety and efficacy of this life-saving treatment modality.