Liu, Tiange (2017) Development of a mouse model to evaluate the degree of immunological cross-reactivity between prawns and abalone. PhD thesis, James Cook University.

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Abstract

Food allergy is one of the major health concerns in both industrial and developing countries. The most frequent pattern of food allergy is IgE-mediated hypersensitivity. As the global consumption of seafood grows, the prevalence of shellfish allergy has been increasing significantly. Unfortunately, so far, there is no cure for shellfish allergy. Many patients follow the elimination diet to protect themselves from the potential life-threatening consequence. Prawns and abalone are two of the most widely distributed shellfish species in the Asian-Pacific region and the in vitro IgE cross-reactivity between them has been reported. However, there is a lack of in vivo studies to comprehensively assess the degree of the immunological cross-reactivity between prawns and abalone and to identify the cross-reactive allergens.

Mouse models are very useful tools to study allergic diseases. Without involving patients and possible health risks, the conditions of allergen exposure can be designed on experimental animals and subsequently substantial evidence for humoral and cellular responses can be obtained for the evaluation of potential allergic reactions. Therefore, in this PhD thesis, a mouse model has been developed for the in-depth assessment of the immunological cross-reactivity between prawns and abalone.

Black tiger prawn (Penaeus monodon) has been selected for the development of this shellfish allergy model, because as one of the glorbally commercial seafood products the major allergens of Black tiger prawn (BTP) have been well characterised using immunochemical methods and mass spectroscopy. Moreover, tropomyosin is the most abundant allergen in BTP extracts, and has over 95% amino acid sequence identity with tropomyosins from other prawn species. Jade tiger abalone (Haliotis. laevigata × Haliotis. rubra) is one of the three main species of abalone farmed in Australia. The consuption but also the aquaculture facilities of Jade tiger abalone are rapidly expanding, nevertheless the allergenicity of this shellfish species is poorly understood. Therefore, Jade tiger abalone has been included in this project.

Firstly, heated extracts of Black tiger prawn (hBTP) was produced and a serial optimisation was performed to induce a good mouse model that could mimic prawn allergy. Two mouse strains C57BL/6 and BALB/c mice were employed, and different doses of intraperitoneal sensitisation and frequencies of oral challenges with hBTP were compared for the optimum production of allergen-specific IgE antibody. In Chapter 2, the best induction protocol for the prawn allergy model was demonstrated using BALB/c mice. Moreover, murine humoral and cellular reactions including serum IgE, IgG and mast cell protease-1, splenic cytokines, circulating and splenic eosinophil and basophil, as well as intestinal IgA were thoroughly analysed for the characterising this murine model.

The whole heated extracts of Black tiger prawn and Jade tiger abalone (hAbal) were prepared, and using this model, the immune responses against heterologous allergenic extracts were investigated in detail in Chapter 3. The capacity of serum IgE and IgG to bind to heterologous extracts and the production of Th2 cytokine after stimulation with heterologous extracts were very limited as demonstrated. Interestingly, murine splenocytes more frequently exposed to heterologous shellfish extracts could release more IL-5 and IL-13, indicating the increasing risk of immunological cross-reactivity in patients allergic to prawns when continues consuming abalone, and vice versa. Moreover, as compared to hBTP, hAbal was for the first time demonstrated to have a stronger sensitisation potency in experimental animals. Furthermore, to identify the allergens in hBTP and hAbal, the antibody binding proteins were detected using mouse sera. A very similar antibody binding pattern in hBTP and hAbal was illustrated in the immunoblot of patient IgE and mouse IgG. In both hBTP and hAbal, tropomyosin (TM) was demonstrated to be the predominant allergen. In addition to TM, arginine kinase was also identified to generate antibody reactive in both hBTP and hAbal. Notably, the murine hBTP-specific IgG could recognise abalone TM in vitro, indicating the serologic cross-reactivity to TM from prawns and abalone. Therefore, TM was the key allergenic protein for the prawn-abalone cross-reactivity.

The allergenic properties of prawn and abalone TMs were particularly examined in Chapter 4. The DNA of TM from Jade tiger abalone was for the first time sequenced and cloned. An amino acid sequence alignment of TMs from Black tiger prawn and Jade tiger abalone was performed, specifically for the previously identified eight IgE binding epitopes on prawn TM. The amino acid sequence identity is 63.4% and only one of eight predicted IgE binding regions showed identical primary structure. Moreover, recombinant TMs (rTMs) from BTP and Abal were expressed using E. coli system. Not only the IgE-reactivity to both rTMs was observed using patient sera, but also the allergy-like responses were successfully induced in BALB/c mice using prawn or abalone rTM. Prawn rTM displayed a similar allergenic potential as hBTP, in contrast abalone rTM elicited a lower level of serum IgE as compared to hAbal, as well as splenic IL-5 and IL-13, eosinophils and basophils.

With the availability of prawn and abalone rTMs, the potential immunological cross-reactivity to TM from prawns and abalone was assessed using a mouse model in Chapter 5. Prawn-TM-sensitised mice demonstrated a considerable level of Th2 reactions to hAbal. However, no significant responses were observed after orally challenging abalone-TM-sensitised mice with hBTP, indicating the presence of unique immunodominant fragments in abalone TM. Heterologous TM elicited very limited effector cells, suggesting the low degree of in vivo immunological cross-reactivity to TM from prawns and abalone.

In summary, this thesis has demonstrated the establishment of a suitable mouse model for shellfish allergy that revealed the limited level of immunological cross-reactivity between heated extracts of prawns and abalone. The evaluation of rTMs using human sera and the mouse model has highlighted its allergenicity and its responsibility in prawn-abalone cross-reactivity. The research presented in this PhD thesis provides important contributions to the precise diagnosis and medical advices for susceptible individuals. Future studies are warranted to identify B and T cell epitopes of the detected allergens, and using this mouse model to develop better diagnostics and immunotherapies for shellfish allergy.

Item ID:	53096
Item Type:	Thesis (PhD)
Keywords:	food allergy, mouse model establishment, Th2 responses, shellfish allergy, immune response, seafood allergy, prawns, abalone, cross-reactivity, Haliotis Laevigata, Haliotis rubra, Penaeus monodon
Related URLs:	https://researchonline.jcu.edu.au/43730/
Additional Information:	Publications arising from this thesis are available from the Related URLs field. The publications are: Chapter 1: Liu, Tiange, Navarro, Severine, and Lopata, Andreas L. (2016) Current advances of murine models for food allergy. Molecular Immunology, 70. pp. 104-117.
Date Deposited:	10 Apr 2018 23:03
FoR Codes:	11 MEDICAL AND HEALTH SCIENCES > 1107 Immunology > 110701 Allergy @ 80% 11 MEDICAL AND HEALTH SCIENCES > 1101 Medical Biochemistry and Metabolomics > 110106 Medical Biochemistry: Proteins and Peptides (incl Medical Proteomics) @ 20%
SEO Codes:	92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920108 Immune System and Allergy @ 100%
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