ResearchOnline@JCU This file is part of the following work: Nugraha, Roni (2018) Characterisation of oyster allergens for improved diagnosis of mollusc allergy. PhD Thesis, James Cook University. Access to this file is available from: https://doi.org/10.25903/5d96a07003465 Copyright © 2018 Roni Nugraha. The author has certified to JCU that they have made a reasonable effort to gain permission and acknowledge the owners of any third party copyright material included in this document. If you believe that this is not the case, please email [email protected] Characterisation of Oyster Allergens for Improved Diagnosis of Mollusc Allergy Thesis submitted by Roni Nugraha B.Sc., M.Sc. For the degree of Doctor of Philosophy in the College of Public Health, Medical and Veterinary Sciences James Cook University Townsville, Australia October 2018 Acknowledgements First and foremost, I would like to express my sincere gratitude to my advisor and guide Prof. Andreas Lopata for bringing me in to the world of allergy. Without his guidance, I would not be able to finish this PhD and this thesis would never have been written. Andreas is not only the source of research inspiration, but his guidance also has profound effects on my personal life. As a lecturer at a university, I learned a lot from Andreas how to be a good researcher and mentor for the students. Secondly, I would like also to thank my co-supervisors, Prof. Kyall Zenger for introducing me to the field of bioinformatics and Dr. Sandip Kamath for his assistance and technical expertise during my experiments. Moreover, I would like to thank Prof. Jennifer Rolland and Prof. Robyn O’Hehir of the Monash University and Alfred Hospital for providing serum samples. Thirdly, I am very fortunate and proud to be a member of a very amazing research group, Molecular Allergy Research Laboratory: Dr. Fiona Baird, Dr. Elecia Johnston, Dr. Shruti Saptarshi, Dr. Michael Sharp, Dr. Martina Koeberl, Dr. Tiange Liu, Mr. Thimo Ruethers, Mrs. Thu Le, Mr. Kunal Pratap and Mr. Shaymaviswanathan Karnaneedi. Thank you for becoming my second family in Australia. Lastly, but not the least, I would like to thank my wife for the endless support, the smile and the patience; my kids, Yasmin and Zakiy and also my mother for always calling my name in her prays. ii Statement of the Contributions of Others Nature of Contribution Name and Affiliation Assistance • Prof. Andreas Lopata, James Cook Project plan and University development • Prof. Kyall Zenger • Dr. Sandip D Kamath Intellectual • Prof. Andreas Lopata, James Cook support University Editorial support • Dr. Sandip D Kamath • Dr. Aya Taki Grant proposal • Prof. Andreas L Lopata, James Cook writing University • ARC Future Fellowship for Prof. Andreas Lopata Research • National Health and Medical Research Council for Prof. Andreas Lopata • HDR Enhancement Scheme, College of Conference Public Health, Medical and Veterinary travel • ASI ICI Bursary Financial • JCU Science Research Festival support • James Cook University Postgraduate Scholarship Stipend • College Scholarship, College of Public Health, Medical and Veterinary Sciences, James Cook University • Doctoral Completion Grant, College of Write-up Public Health, Medical and Veterinary support Sciences, James Cook University • Prof. Jennifer Rolland, Monash Human blood Data University sample collection • Prof. Robyn O’Hehir, Monash University collection and The Alfred Hospital Recombinant • Dr. Sandip Kamath, James Cook Technical protein assay University support Mass • Dr. Martina Koeberl, National spectrometry Measurement Institute iii Publications arising from thesis-Published 1) Thimo Ruethers, Aya C Taki, Elecia B Johnston, Roni Nugraha, Thu TK Le, Tanja Kalic, Thomas R McLean, Sandip D Kamath, Andreas L Lopata. 2018. Seafood allergy: A comprehensive review of fish and shellfish allergens. Molecular immunology. 100:28-57. Impact Factor: 3.16 2) Roni Nugraha, Sandip D Kamath, Elecia Johnston, Kyall R Zenger, Jennifer M Rolland, Robyn E O'Hehir, Andreas L Lopata. 2018. Rapid and comprehensive discovery of unreported shellfish allergens using large-scale transcriptomic and proteomic resources. Journal of Allergy and Clinical Immunology. 141(4): 1501-1504. e8. Impact Factor: 13.258 3) Sandip D Kamath, Roni Nugraha, Andreas L Lopata. 2017. Recent advances in Diagnosis and Management of Shellfish Allergy. Food Allergy: Molecular and Clinical Practice. CRC Press: 122-154. Invited book chapter. Publications arising from thesis – Submitted 1) Roni Nugraha, Sandip D Kamath, Shaymaviswanathan Karnaneedi, Elecia Johnston, Andreas L Lopata. 2018. Conservation analysis of B-cell allergen epitopes to predict clinical cross-reactivity between shellfish and inhalant invertebrate allergens. Frontiers in Immunology. Publications arising from thesis – In Preparation 2) Roni Nugraha, Sandip D Kamath, Thimo Ruethers, Shaymaviswanathan Karnaneedi, Elecia Johnston, Andreas L Lopata. 2018. Effects of extraction buffers on the solubility and immunoreactivity of the Pacific Oyster (Crassostrea gigas) allergens. Food Chemistry. 3) Roni Nugraha, Sandip D Kamath, Thimo Ruethers, Shaymaviswanathan Karnaneedi, Elecia Johnston, Andreas L Lopata. Recombinant expression, purification and immunological characterisation of the major allergen of the pacific oyster (Crassostrea gigas) – Tropomyosin. Molecular Nutrition and Food Research. iv Abstract Discovery and characterisation of allergens in various food and inhalant sources is central to our understanding of the molecular mechanisms of allergic reactions. Allergen characterization is the most important underlying factor for better patient management with improved diagnostics, and the design and development of novel immunotherapeutics. Of the ‘Big eight’ allergen food groups, shellfish presents a unique challenge in terms of allergen discovery due to the large number and diversity of consumed species, leading to heterogeneity of allergen structure and cross-reactivity among various sources. The group of ‘shellfish’ comprises of two invertebrate phyla; arthropods and molluscs. Although all shellfish are invertebrate animals, these two groups are very distinct in evolutionary terms and subsequently contain different molecular repertoires of allergenic proteins. Co-sensitisation of patients with crustacean and mollusc allergy is often described, however, the current diagnostic approaches to manage patients is not based on sufficient molecular knowledge of these shellfish allergens. Consequently, mollusc allergy is clinically underreported and allergens are ill-defined. To date, only five mollusc allergens are listed in the WHO/International Union of Immunological Societies (IUIS) Allergen Nomenclature SubCommittee database, all of which are different tropomyosin’s (http://www.allergen.org/index.php). Additional mollusc allergens have been reported, but not yet fully characterised. A detailed review of the current status and the diagnosis of mollusc allergy is provided in Chapter 1. Current strategies for allergen identification are time- and resource- consuming, which are highly prone to missing hidden allergens present in low concentrations. Allergenic proteins are traditionally identified based on their serum-specific IgE antibody recognition. Soluble proteins derived from whole protein preparations of a suspected allergen source are screened for IgE antibody binding proteins using sera from individuals with clinically confirmed allergy. Although this approach is the current standard for allergen v identification, there are three major drawbacks. Firstly, this approach often does not detect allergenic proteins present in low abundance. Secondly, cross- reactive allergens are not easily identified due to their possible presence in unrelated allergen sources. Thirdly, the IgE recognition patterns are highly dependent on the demographics of the particular allergic patient cohort under investigation. This PhD thesis presents a comprehensive study on the improvement of allergen discovery from Pacific oyster, the most widely consumed mollusc species and immunological characterisation of the major allergen, tropomyosin, using a cohort of mollusc-sensitised patients in Australia. Furthermore, novel ways to diagnose cross-reactivity between crustacean and mollusc species were developed. Chapter 2 describes a comprehensive discovery pipeline, for allergenic proteins, that accounts for biological and molecular variability using allergenomics, high-throughput screening of genomic databases and high- resolution mass spectrometry. This methodological approach was successful in identifying 24 previously unreported allergens from over 25,000 proteins from the Pacific oyster. This is the first study to demonstrate the presence of 24 hidden allergens, also found in very different allergen sources from animals, including fish and mites, as well as plant allergens from pollen, latex and fungi. Importantly all of these allergenic proteins identified are reactive with shellfish allergic patients’ IgE antibodies. However, it was demonstrated in chapter 2 that not all allergens present in the genome and transcriptome of oyster are also detected in the extracted proteome. Allergens are often overlooked during the extraction process due to the use of inappropriate buffers which might affect the in vitro diagnostic methods that use whole proteins extracts. In Chapter 3 various buffer compositions covering a wide range of pH were evaluated to improve the detection of the unreported allergens described in